-------
TMT neurotoxicity and CNS proteins
oculomotor nucleus
retrorubrai field
interpeduncular nucleus
interpeduncular nucleus,
nucleus
inferior colliculus
nucleus cuneiformis
interpostenor sub-
Ill
lpCn
IplP
IC
Cf
p,ms-medulla
PB parabrachial nucleus
MeV mesencephalic nucleus of the trigeminal nerve
I.c locus coerulus
PrV principal trigeminal nucleus
MoV motor nucleus of trigeminal nerve
RD nucleus raph? Jorsalis
DTg dorsal tegmc-;jl nucleus
LDTg lateral dorsal tegmental nucleus
RPo nucleus raphe pontis
MSO medial superior olive
LSO lateral superior olive
MTp medial nucleus of the trapezoid body
LTp lateral nucleus of the trapezoid body
MCN medial cerebellar nucleus
ICN interposed cerebellar nuclei
LCN lateral cerebellar nucleus
LVN lateral vestibular nucleus
MVN medial vestibular nucleus
PH nucleus prepositus hypoglossi
NTS nucleus tractus solitanus
SpV spinal nucleus of trigeminal nerve
gi gigantocellular reticular formation
pgi paragigamocellular reticular formation
KM nucleus raphe magnus
RO nucleus raphe obscurus
VII facial nucleus
den dorsal cochlear nucleus
ven ventral cochlear nucleus
LLv ventral nucleus of the lateral lemniscus
LLd dorsal nucleus of the lateral lemniscus
RTP nucleus reticularis tegmenti pontis
Ftp I-
-------
342
C. D. Balaban et al.
Ma • . r
•v.... 0«W.^
• * -
¦?. ~
HC*
Fin 5-8.
dendritic degeneration in the region (Figs 7-10).
Axonal degeneration, both in layer I and deeper
layers, became increasingly prominent in the day 5-7
period (Figs 22 -). There was no evidence of
differential sensitr i.y of the medial, intermediate or
lateral entorhinal cortices to TMT- The damage to
the layer (I neurons was reflected in relatively early,
degeneration in the perforant path, visible in the
dentate gyrus by day 4 (Figs 5-8). Furthermore,
damage to deeper neurons may be reflected in later-
developing terminal degeneration in a subset of their
identified terminal sites.41-4* which include both ter-
minal fields in the lateral aspect of nucleus accumbens
(Fig. 17), the medial aspect of the olfactory tubercle
(Fig. 16). layer I of the dorsal taenia tecti (Fig. 16).
the anterior olfactory nucleus (Fig. 16), and the
endopyriform nucleus (Figs 17-20) and a capsule of
degenerating fibers ensheathing the basolateral and
lateral amygdaloid nuclei (Fig. 20). However, ter-
minal degeneration was absent in the infralimbic
-------
TMT neurotoxicity and CNS proteins
343
J
Figs 9-12.
cortex terminal field identified by Luskin and
Price.41**
Terminal dtgentration: inirahippocampai and
corticohippocampai connections. The selectivity of
this temporospatial sequence of cell death in the
entorhinal cortex, dentate gyrus and CA fields was
apparent in the sequence of development of terminal
degeneration in the efferent targets of these cell
populations. The mossy fiber efferent projection from
the dentate gyrus to the proximal dendrites of CA3
pyramidal cells was first observed 4 days after TMT
administration in CA3a (Figs 5-7); it was obscured in
other CA3 subfields by pyramidal cell degeneration.
This degeneration pattern increased in density with
-------
344
C. D. Balaban et at.
13
14
18
Figs I3-I5.
Pigs t-15. AUu of degeneration in Long-Evans rats 4 days after TMT intoxication. Degenerating ceils
(dots) and ana terminals are charted on a series of drawings of transversa sections of the brain. The heavy
daframtion in piriform cortex (PCx) and CA3 of the hippocampus is shaded.
the development of dentate granule cell degeneration,
usually lagging about 2 days behind the appearance
of somatodendritic argyrophilia. Unfortunately, both
the collateral projections of mossy fibers to hilar
neurons and the projections of degenerating hilar
neurons to the inner third of the dentate molecular
layer"" could not be resolved due to dense somato-
dendritic degeneration in these regions.
The massive ablation of CA3b-c pyramidal cells
and layer IT and 1U neurons in the entorhinal cortex
produced dense, laminar patterns of terminal degen-
eration in both the molecular layer of the dentate
gyrus and strata onens and radiatum of the CA fields.
The commissural and associational dentate gyrus
projection fields of CA3c pyramidal cells"" were one
of the first regions to show massive terminal degener-
ation at 3-4 days postintoxication. occupying a char-
acteristic band within the proximal third of the
dentate molecular layer (Figs 3-8). The proximal
terminal geld disappeared by day 7, when a single.
-------
TMT neurotoxicity and CNS proteins
NOBV
Figs 16-19.
dense degenerating band appeared in the proximal
half of the molecular layer (Figs 19-23 ad 28). This
dense band probably represents a late-developing
component of CA3c degeneration (see Refs 62
and 63).
A second, distinct laminar pattern was produced
by TMT ablation of perforant path neurons in
laminae (I and III of the lateral, intermediate and
medial entorhinaJ cortex. The perforant path ter-
minal degeneration" was relatively light on day 4
(Figs 5-8). appearing more densely on days 5-7 when
it filled the outer half of the dentate molecular layer
(Figs 19-23). Consistent patterns of terminal degen-
eration were, also observed in CA1-3. This was
-------
34*
C. D. Balasan et at.
ClA
nc*
ai
•I»4oc'2M i OilM
20
Figs 20-23.
particularly obvious in CAI by day 4. when the
projections of degenerating branches of Schaffer col-
laterals (axons of CA3 pyramidal cells) occupied
strata oriens and radiatum and degenerating per*
forant path axons filled the stratum lacunosum to
produce a continuous band of terminal degeneration
around dendrites of pyramidal cells throughout the
septotemporal extent of the region. However, there
were septotemporal differences in the development of
analogous patterns of degeneration in CA3. For ex-
ample, near the septal pole of the region, the laminar
patterns of degeneration were identical in CAl-3. In
the temporal hippocampus, though, the terminal fields
in the strata oriens, radiatum and outer aspect of the
lacunosum molecuiare were light to absent in CA3,
leaving only a terminal band formed by mossy fibers
and a second, wider terminal region in the middle
third of the stratum lacunosum. This sharp transition
at the border of CA3 is shown in a photomicrograph
in Fig.- 28. This difference does not seem to be
-------
TMT neurotoxicity ami CNS prolans
347
Fi|» 24-26.
Tip 16-26. Alias of degeneration in Long-Evan* rats 7 day* after TMT intoxication.
correlated with dorso ventral or mediolateral differ-
ences in cell death in either the entorhina! cortex or
CA3.
Terminal degeneration: hippocampocortical con-
nections. In addition to intrahippocampal projections,
the ablated CAl-3 pyramidal cell* resulted in axonal
degeneration in target sites that included the granular
rctrosplenial cortex, the deep aspect of perirhinal
cortex and layer IV of the entorhinal cortex. The
relatively massive terminal degeneration in the retro-
splenial cortex appeared initially on days 3-4, as a
dense band of particulate debris restricted to layer II
of the granular division (Figs 5-S). In addition, it is
important to note that this terminal ftcki may also
receive a contribution from degenerating neurons in
the ventral sutriculum.** The debris was cleared,
though, by day 7, when argyrophiik neurons were
occasionally observed in layer It of both granular
and agranular retrosplenial cortices (Figs 19-23). The
relatively early, transient appearance of the retro-
splenial terminal field contrasted with the later ap-
pearance of degenerating CA3 projections to layer IV
-------
348
C. O. Bala ban tt ai.
\
\
V
I *,
i *
1
a*.
¦ ¦
BSTM
Fig. 17. Degenerating neurons in (he septum J days after TMT intoxication. This photomicrograph
displays argyrophilia in the septahippocampal nucleus (SHi). ventral (LSV), intermediate (LSO. and dorsal
(LSd) divisions of the lateral septal nucleus and the medial division of the bed nucleus of the stria
terminalis (BSTM).
of entorhinal cortex and the subiculum and CAI
projections to the subiculum and deep aspect of the
perirhinal cortex. Although prominent by day 7,
these projection* were partially obscured by debris
from intrinsic neurons.
Terminal degeneration: subcortical efferents. The
axons of degenerating pyramidal cells in the CA fields
and subiculum contributed to a relatively dense ter-
minal field in the lateral septal nucleus. Argyrophilic
terminal debris was scattered primarily in the ventral
Fig. 23. Photomicrograph of cupric-silver stained sections of the hippocampus 2 (2d). 3 (3d). 4 <4d), and
7 days (7d) after TMT intoxication. The 2d and 7d micrographs are from horizontal sections through the
temporal aspect of the hippocampus; the 3d and 4d are from transverse sections. Cellular defeneration
fields are demarcated by white triangles. Small black arrows indicate a late, developing terminal band in
(he stratum lacunosum. Black triangles <7d) indicate the degenerating mossy fiber terminal fields. Fields
CAI. CA3, and the dentate gyrus (DCH-art also labeled.
-------
Kig 28.
-------
330
C. D. Balaban tt aI.
division of (be lateral septal nucleus on day 4
(Figs 2 and 3); by day 7, though, it expanded to
include both the intermediate division and a dense
projection to the dorsal division (Figs 17 and 18). The
density of degeneration in the lateral septum
decreased caudally, and only the terminal field in the
dorsal division was prominent at the level of the
preoptic area and septofimbnal nucleus. With the
exception of the dorsal division, these terminal
regions surround somata of cells that degenerated on
day I.
The second major hippocampal output pathway
affected by TMT intoxication was the subiculo-
mammillary body projection. The dense terminal
field of axons of dorsal and ventral subiculum pyr-
amidal cells developed fully by day 4 after intoxi-
cation. forming a dense mass of fine, powder-like
debris within the posterior division of the medial
mammillary nucleus (Fig. 15). The pattern of dense
degeneration persisted even 7 days postintoxication.
but was never accompanied by degenerating neurons
in the terminal region.
Terminal degeneration was also observed in other
sues receiving inputs from ablated populations in
CA3 and the subiculum. For example, the ventral
taenia tecti and medial anterior olfactory nucleus
showed prominent terminal degeneration days 5-7
(Fig. 16), which may be attributed to CA3 and
subiculum pyramidal cell axons.*" Similarly, termi-
nal degeneration reflecting pyramidal cell death in the
ventral subiculum was observed initially on day 4 in
the bed nucleus of the stria terminals (Fig. 20),
followed by the appearance of a medial field of debris
in nucleus accumbens on day S. Finally, while the
relative sparing of the parasubiculum and pre-
subiculum are reflected in a paucity of debris in the
interior thalamic nucleus and nucleus reuniens, the
relatively dense terminal field in the lateral dorsal
nucleus (Fig. 20) probably reflects cell death in the
"postsubicular" region." Taken together, these data
indicate the specificity of TMT intoxication for pools
of hippocampal neurons and illustrate the utility of
selective silver stains for documenting both somato-
dendritic properties of degenerating neurons and
their axonal projections.
Piriform cortex mi anterior olfactory nucleus
Among areas that receive input from the olfactory
bulb, the piriform cortex was most exquisitely sensi-
tive to TMT intoxication. Other olfactorecipient
areas, including the anterior olfactory nucleus, the
entorhinal cortex (discussed above) and peri-
amygdaloid cortical regions (discussed with other
amygdaloid regions), showed less massive cellular
degeneration. Finally, neurons in the olfactory
tubercle and olfactory bulb wen immune to toxic
effects of TMT.
The piriform cortex, like CA3b-c, showed massive
degeneration initially on day 2, with heavy in-
volvement of most neurons in the pyramidal cell layer
(layer 2) throughout the extent of the cortex. By day
4, dense somatodendritic degeneration filled layer 2
of piriform cortex in a region bounded laterally by
the rhinal fissure, medially by the olfactory tubercle
and the posterolateral and anterior cortical amygda-
loid nuclei, rostrally by the anterior olfactory nu-
cleus, and caudally by the entorhinal cortex (Figs
1-6). Fewer argyrophilic neurons were found in the
piriform cortex by day 7, but the dense associations!
plexus of piriform cortex was filled with terminal
debris. Among targets of the piriform cortex.4"4
terminal degeneration was particularly striking in
layer IB of the piriform cortex, which formed one
part of a dense band of argyrophilic debris that
spanned the plexiform layer of the olfactory tubercle
and the anterior, posteromedial and posterolateral
cortical amygdaloid nuclei (Figs 16-22). Caudally.
this superficial band was continuous with a terminal
field in layer 1 of the medial, intermediate and lateral
entorhinal cortex. Rostrally, the superficial band is
continuous with superficial terminal degeneration in
the ventral aspect of the taenia tecti. In addition, the
layer 3 fiber plexus in the piriform cortex contained
less dense terminal and axonal debris, some of which
was contiguous with degenerating fibers within the
anterior limb of the anterior commissure, a deep
plexus of fibers in the olfactory tubercle that is
continuous with a terminal field in nucleus accum-
bens (Figs 16 and 17) and a terminal field in the
anterior olfactory nucleus (Fig. 16). This plexus was
also contiguous with degenerating axons in the amyg-
dala and endopiriform nucleus (Figs 17-22). It is
important to note, though, that degenerating neurons
in layer 3 of piriform cortex were not commonly
encountered, but scattered argyrophiiic neurons were
found in layer 3 and the deep posterolateral cortical
amygdaloid nucleus. However, the relatively minor
damage to layer 3 was indicated by the absence of
degenerating axons reflecting deep piriform cortex
projections to the mediodorsal thalamic nucleus.
Thus, layer 2 neurons in piriform cortex appear to be
selectively sensitive to TMT intoxication.
Argyrophilic neurons in the anterior olfactory nu-
cleus appeared during the same time frame as dying
neurons in the piriform and entorhinal cortices. The
pattern of argyrophilia appeared prominently on day
2 and developed completely by day 4. This day 2
degeneration pattern was associated with dense ter-
minal degeneration in the granular layer of the
olfactory bulb. By day 7, degenerating axon path-
ways could be traced from the anterior olfactory
nucleus and piriform cortex to this dense terminal
plexus spanning the granule cell layer, which con-
tained a small population of degenerating granule
cells.4'
Amygdala
The rat amygdala is divided into four basic regions,
an "olfactory amygdala", "a medial amygdaloid
-------
TMT neurotoxicity and CNS proteins
331
•• a "basolateral amygdaloid group" and a
•'Antral amygdaloid group".;3J# The "olfactory
amygdala" is comprised of the anterior amygdaloid
area, nucleus of the lateral olfactory tract, amygdalo-
pynform transition area, anterior cortical and post-
erolateral cortical amygdaloid nuclei and the rostral
pole of the medial amygdaloid nucleus. The "medial
amygdaloid group", on the other hand, includes the
medial and posteromedial cortical amygdaloid nuclei,
medial and intermediate divisions of the bed nucleus
of the stria terminalis. posteromedial portions of the
sublenticular substantia innominata. the bed nucleus
of the accessory olfactory tract, elements of the
basomediat nucleus and the amygdalohippocampal
transition area. The "basolateral amygdaloid group"
contains the lateral and basolateral amygdaloid nu-
clet. as well as elements of the basomedial nucleus,
and the "central amygdaloid group" Is comprised of
the central amygdaloid nucleus, adjacent dorsolateral
portions of the sublenticular substantia innominata
and both lateral and ventral divisions of the bed
nucleus of the stria terminalis.
These divisions, based on both architectonic and
hodologic data, showed differential temporal sus-
ceptibility to TMT intoxication. Argyrophilic neur-
ons in the amygdala appeared in two distinct waves,
the first coinciding with the development of degener-
ating neurons in the septum and hippocampus in the
period through day 4 and the second emerging during
days 5-7. A few scattered, argyrophilic neurons ap-
peared initially on days 2 and 3 in three structures
included in the "medial group", the medial division
of the bed nucleus of the stria terminalis. the caudal
pole of the posteromedial cortical amygdaloid nu-
cleus and the caudal aspect of the amygdalohippo-
campal area. This initial phase of degeneration devel-
oped slowly through day 4 (Fig. 7). then expanded
rostrally in the latter two regions on days 5-7, when
a second phase of degeneration was marked by the
appearance of degenerating neurons in both the
medial amygdaloid nucleus and a small band of cells
in the posteromedial aspect of the substantia innom-
inata I Figs 19-22) and the emergence of argyrophilic
neurons in other amygdaloid nuclear groups. In the
basolateral group, degenerating neurons appeared
during days 5-7 in a region spanning the caudal pole
of the basolateral nucleus and extending rostrally
along its lateral aspect and a region occupying the
central third of tha lateral amygdaloid nucleus. In the
"olfactory group", argyrophilic neurons appeared on
days 5-7 in the anterior cortical and posteromedial
cortical amygdaloid nuclei, as well as the rostral pole
of the medial amygdaloid nucleus. Finally, the "cen-
tral amygdaloid group" was relatively immune to
effects of TMT. Neurons in the central amygdaloid
nucleus were unaffected, and only a few, scattered
degenerating neurons were observed along the medial
aspect of the lateral division of the bed nucleus of the
stria terminalis during days 5-7 {Fig. 18). Thus, pools
of amygdala neurons are differentially susceptible to
TMT intoxication, with the primary effects emerging
5-7 days after toxin administration.
The patterns of terminal degeneration that can be
attributed to amygdala damage provide further evi-
dence of selectivity of TMT to particular pools of
neurons. For example, among projections of the bed
nucleus of the stria terminalis and the posteromedial
cortical amygdaloid nucleus/5 J'40 terminal degener-
ation was observed on day 7 in layers 1B and 2 of the
dorsal aspect of the lateral entorhinal cortex (Figs
22-24). the horizontal limb of the nucleus of the
diagonal band, and the ventral division of the lateral
septal nucleus (Fig. 17). By contrast, terminal regions
were not observed in thalamic or hypothalamic tar-
gets of the medial amygdaloid group, suggesting
either that these output neurons are not ablated by
TMT or that the axons were refractory to silver
staining during the time course in the study.
Endopiriform nucteia
Degenerating neurons in the endopiriform nucleus
appeared initially on day 2, and were evenly distrib-
uted in the nucleus by day 4 (Figs 2-6). This pattern
persisted on days 5-7. interspersed with degenerating
terminals. Damage to output neurons of this struc-
ture was suggested by the distribution of terminal
degeneration in deep layer 1 and superficial layer 2 of
the posterolateral cortical amygdaloid nucleus.-
Stocorttx
One of the most striking signs of selective TMT
toxicity was observed in the laminar distribution of
argyrophilic neurons in the neocortex. Initial signs of
argyrophilia in the neocortex appeared on day 2,
when punctate staining was distributed in the somata
of scattered neurons in the superficial third of lami-
nae V and VI; more rarely, degenerating cells were
observed in laminae I! and III. This pattern devel-
oped more extensively during day 3, such that degen-
erating neurons were prominent in the superficial
third of laminae V and VI on day 4, accompanied by
scattered degenerating cells in laminae II and III
(Figs 1-10). This pattern was present on day 4 in all
neocortical regions, except granular and agranular
retrosplenia! and posterior cingulate cortices (Figs
3-10). The distribution of argyrophilic neurons was
similar on day 7, with three changes. Fint. this
laminar pattern of degenerating neurons spread me-
dially to include the caudal aspect of the retrosplenial
cortex and occasional neurons in the caudal cingulate
cortex (Figs 18-24). Degeneration was restricted to
scattered lamina II and III neurons in rostral retro-
splenial regions. Second, then was an increase in the
density and depth of degenerating neurons in lamina
V of the ventral, dorsal and posterior insular cortices
(Figs 13-22). Finally, a dense pattern of terminal
- degeneration was observed in layer IV of the parietal
cortex, which was often distributed in relatively dense
patches of terminal debris separated by areas with
lighter punctate degeneration (Figs 16—20). This ter-
-------
352
C. D. Balaban et at.
minal pattern in primary somatosensory cortex71 was
probably a result of the concomitant appearance of
argyrophiiic neurons in the ventral posterolateral and
ventral posteromedial nuclei of the thalamus (Figs 20
and 21). This pattern of terminal degeneration was
not present in other koniocortical regions.
Careful inspection of cortical efferent sites revealed
the development of dense terminal degeneration in
two main regions: the pontine nuclear complex (Fig.
^) and the inferior olive (Figs 13 and 14). These
terminal regions were detected initially on day 3. and
developed completely by day 4. In the pontine nuclei,
terminal debris was concentrated in medial and lat-
eral fields, with very sparse to absent degeneration
elsewhere. Furthermore, by day 4. degenerating
neurons were apparent within these terminal fields. In
the inferior olive, relatively dense, restricted terminal
fields were observed (a) along the rostromedial aspect
of the medial accessory olive, (b) in a separate, lateral
region in the caudal medial accessory olive, (c) in a
region that shifted laterally from the medial third of
the ventral lamella of the principal olive caudally to
the lateral third of the ventral lamella at more rostral
levels (ending at the flexure), (d) in a separate region
in the lateral third of the dorsal lamella of the
principal olivary nucleus, (e) in a region spanning the
rostral aspect of the ventrolateral outgrowth and (f)
in a field in the lateral third of the dorsal accessory
olive. In addition, relatively sparse terminal degener-
ation was localized in the dorsal cap and in an
intermediolateral field in the caudal medial accessory
olive. These regions correspond to corticoolivary
projections." No degenerating neurons were ob-
served in the inferior olive at any time point. These
data. then, suggest that corticopontine and cortico-
olivary neurons are highly susceptible to TMT intox-
ication.
Thalamus
The heaviest cellular degeneration in the thalamus
appeared in the parafascicular nucleus and the ven-
tral posterolateral and ventral posteromedial nuclei
with different time courses. Somata containing
punctate argyrophilia were prominent in the para-
fascicular nucleus on day 3. and the argyrophilia inten-
sified by day 4 (Fig. 6). The same pattern of scattered,
degenerating neurons was also observed on day 7
(Fig. 21). It must be emphasized that a vast majority
of parafascicular nucleus neurons were unaffected by
TMT. which may account for the lack of appreciable
terminal degeneration in the caudatoputamen. By
contrast, the later development of degenerating neu-
rons in the ventral posterolateral and ventral post-
eromedial nuclei (Figs 20 and 21) was associated with
prominent degeneration in lamina IV of parietal
fields corresponding to somatosensory cortex (Figs
16-20). Argyrophiiic neurons in these nuclei were first
observed on day 4, when an occasional cell displayed•
punctate intrasomatic and intradendritic staining.
The pattern of degeneration developed completely by
day 7; intensely argyrophiiic neurons were scattered
in a region spanning the ventrobasal complex (Figs
19 and 20).
A number of other thalamic nuclei displayed minor
effects of TMT during the day 5-7 postintoxication
period. Scattered neurons degenerated in the anterior
pretectal nucleus, ventral lateral geniculate nucleus
and medial geniculate nucleus (Figs 20-23). In addi-
tion. a very low density of degenerating neurons was
observed in the paraventricular, centrolateral and
mediodorsal nuclei. Terminal degeneration resulting
from these regions could not be identified, probably
reflecting the minor damage in these regions.
Hypothalamus
Cellular degeneration was not prominent in the
hypothalamus. A sparse distribution of degenerating
neurons appeared by day 4 in the antenor hypo-
thalamic nucleus (Fig. 4), the compact subdivision of
the dorsomedial nucleus, the lateral aspect of the
more diffusely organized region of the dorsomedial
nucleus, and outside the ventral and lateral border of
the ventromedial nucleus (Fig. 5). This pattern ex-
panded on days 5-7 to include scattered neurons in
the lateral hypothalamic area and between the para-
ventricular and anterior hypothalamic nuclei, as well
as a linear array of ceils in the lateral mammillary
region (Figs 19-21). The relatively minor degener-
ative changes in these hypothalamic regions may
account for the fact that terminal degeneration orig-
inating from these populations could not be identified
positively.
Mesencephalon
Superior collicuius. The selective sensitivity of pop-
ulations of neurons within given structures was also
striking in the superior collicuius. Beginning on day
2, argyrophiiic multipolar neurons were observed in
a narrow band in the deep aspect of the superficial
gray layer, which completely spanned the rostro-
caudal and mediolateral dimensions of the structure
(Fig. 8). These degenerating neurons resembled the
wide-field neurons identified in Golgi studies41-41 both
morphologically and in their distribution near the
border of the underlying stratum opticum. The selec-
tive sensitivity of these wtde-field neurons was further
evidenced by the appearance of terminal degener-
ation by day 4 in only the caudal aspect of the lateral
posterior nucleus (Figs. 6 and 7), which is a documen-
ted terminal site of wide-field neurons." *4 This field
corresponded to the total extent of the retinotopically
organized, tectorecipient field reported previously in
LP.W By day 7, this wide>fiekl terminal cell region
included a less dense terminal field in the ventral
lateral geniculate nucleus (Figs 21 and 22). By con-
trast, terminal degeneration was absent in the supra-
geniculate, dorsal lateral geniculate, para bigeminal
and pretectal nuclei which receive input from the
superficial layers of the superior collicuius.41
A later phase of cellular degeneration in the col-
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TMT neurotoxicity and CNS proteins
353
liculus was apparent on day 3, with the emergence of
a degenerating lamina of neurons along the
superficial border of the intermediate gray layer and
the stratum opticum (Fig. 23). This lamina of ar-
gyrophilic multipolar ceils spanned the rostrocaudal
and mediolateral dimensions of the colliculus. Poten-
tial terminal regions of these neurons could not be
identified positively in this material.
Inferior colliculus. Degenerating neurons in the
inferior colliculus were restricted to the pericentral
and the dorsal and external cortical nuclei; no ar-
gyrophilic cells were observed in (he central nucleus.
8y 4 days after TMT intoxication, argyrophilic mul-
tipolar neurons were scattered dorsally and dor-
somedially in this region (Figs 9 and 10). During the
5-7 day survival period, a few additional degener-
ating neurons appeared in the lateral aspect of the
external cortical region (Figs 23 and 24). Although
terminal degeneration was observed in the medial
geniculate nucleus by 7 days (Figs 22 and 23). both
the paucity of knowledge of the distribution of the
terminal fields of the degenerating collicular regions
in rau'° and the observed degeneration of lamina VI
neurons in temporal cortex precludes positive
identification of the sources of terminals in the re-
gion.
Periaqueductal gray. Argyrophilic neurons in the
periaqueductal gray were found in two regions, a
group of cells dorsolateral and dorsal to nucleus
raphe dorsalis (Figs 10 and 24) and a gently curved
lamella of cells, extending dorsolateral^ from the
aqueduct toward the deep superior colliculus (Fig.
23). Degenerating neurons were prominent in the
former region by day 3; they appeared in the latter
region on day 5. In particular, the latter pattern
resembles the distribution of neurons projecting to
the gigantocellular reticular formation, pars alpha,IJ
and some degenerating axons were clear in this region
on day 7. The possible terminal sites of the former
neuronal population could not be identified.
Interstitial nucleus of the posterior commissure. A
discrete cluster of degenerating neurons in the inter-
stitial nucleus of the posterior commissure emerged
initially on day 2 and developed completely by day 4
(Fig. 7). The same pattern was observed on day 7
(Fig- 22). Axons originating from these neurons
could not be identified positively at any examined
survival time. .
Pons and medulla
Raphe nuclei. The distribution of degenerating
neurons in the raphe nuclei was confined primarily to
nucleus raphe obscurus and a central region in caudal
nucleus raphe magnus (Figs 12 and 14). This pattern
°f degeneration was very light on day 3, but devel-
oped completely by day 4. By day 7. only an occa-
sional degenerating neuron was observed in these
regions. As mentioned previously, some degenerating
neurons were also distributed along the margin of
nucleus raphe dorsalis. Terminal projections of these
regions could not be assessed with certainty.
Trigeminal nuclear complex. Argyrophilic neurons
appeared in two distinct regions in the trigeminal
nuclear complex, the mesencephalic nucleus (Figs 11
and 25) and the spinal trigeminal nucleus caudal to
the obex (Fig. IS). The degeneration was robust in
the latter region by day 4, consisting of a prominent
population of degenerating neurons in the superficial
lamina and an occasional degenerating cell in the
superficial aspect of the deeper, magnocellular region.
The superficial degenerating neurons were scattered
among a dense degenerating fiber plexus. This
pattern of degeneration continued caudally into the
dorsal horn of at least the upper cervical spinal cord;
more caudal sections were not stained in the study.
Neither the origin of the degenerating fiber plexus nor
the sites of termination of axons of degenerating
neurons could be ascertained in the material. How-
ever. this discrete, restricted distribution of TMT
toxicity in the trigeminal complex provides further
evidence of selective actions of the compound.
Pontine nuclei. Argyrophilic neurons in the pontine
nuclei were distributed in groups within a dense field
of terminal degeneration by day 4 (Fig. 9). This
pattern emerged first on day 3, when the terminal
field was less dense, and degenerating mossy fiber
terminals in the cerebellar granular layer were de-
tected during the same time frame. The density of
degenerating neurons increased during the S-7 day
postintoxication period, accompanied by relatively
sparse mossy fiber degeneration in the cerebellar
cortex and denser terminations in the cerebellar nu-
clei. There was no evidence of regional differences in
mossy fiber terminal density.
Pontine tegmentum and parabrachlal nucleus. De-
generating neurons in the pontine tegmentum and
para brachial nucleus appeared initially during the
S-7 day period. Argyrophilic somata were distributed
sparsely in both dorsal and ventral aspects of the
ptrabrachial nucleus; more rarely, a degenerating
neuron was observed in the brachium conjunct!vum
(Fig. 25). In the dorsal pontine tegmentum, affected
neurons wen scattered between the lateral and lateral
dorsal tegmental nuclei and the ventricular surface.
This region may represent a caudal extension of the
more ventral degenerating region in the peri-
aqueductal gray.
Cochlear nuclei. Somatodendritic argyrophilia in
the cochlear nuclei was restricted to the dorsal co-
chlear nucleus, where a sparse population of degener-
ating neurons appeared on days 3-7 after TMT
intoxication (Fig. 26). However, dense terminal de-
generation appeared throughout the molecular layer
of this nucleus on day 2, which spread to include the
superficial third of the cellular region by day 4 (Fig.
12). Argyrophilic neurons then appeared within the
terminal field. Terminal degeneration was absent in
the ventral cochlear nucleus, possibly accounting for
a component of the terminal degeneration in the
-------
354
C. D. Balasan (i at.
Fig. 29 Proiein-0-carboxy) metfcyltransfersse (PCM) immunocytnchemistry of the hippocampus is
shown for a control and TMT-treated (7 day survival) rat. Note the ubiquitous intraneuronal distribution
of PCM in die dentate gyrus (DG) and CA4 of the hippocampus from a control rat. By contrast, intense,
fragmented PCM-posiuve debris is observed in both dentate granule calls (g) and CA4 neurons in the
hippocampus from a TMT-ireated rat. In addition. PCM immunoreactivity is distributed in both the
neuropil of the CA fields (large arrows) and dentate hilar region and in a band in the molecular layer
of the dentate gyrus (small arrows), which corresponds to the distribution of a faint band of degenerating
terminals in Fig. 28 (7d> is also seen. Note the suppression of PCM expression of neurons in the hilus
of the dentate gyrus. -
-------
TMT neurotoxicity and CNS proteins
355
inferior colliculus (see above). Unfortunately, the
source(s) of the terminal field in the dorsal cochlear
nucleus could not be ascertained.
Nucleus tractus soiitarius, dorsal motor nucleus of X
and nucleus ambiguus. A striking pattern of TMT
toxicity was observed in the caudaJ half of nucleus
tractus soiitarius and adjacent regions of the dorsal
motor nucleus of the vagus nerve (Figs 12-15).
Degenerating multipolar neurons appeared in these
regions on day 3. A dense degenerating terminal field
surrounded these neurons by day 4. This pattern was
essentially unchanged by day 7. Both the degener-
ating neurons and the accompanying terminal field
spanned the medial and lateral divisions of the soli-
tary nucleus, occupying a swath through the central
third of the regions. However, degenerating cells were
only found laterally in the commissural region (Fig.
15). Argyrophilic neurons in the dorsal motor nucleus
of X were continuous with a small group of degener-
ating neurons along the dorsolateral margin of the
hypoglossal nucleus (Figs 14 and 15). Although the
efferent projections of all degenerating neurons could
not be ascertained, it is possible that these solitary
nucleus neurons contribute to the dense terminal field
in the central amygdaloid nucleus on day 7 (Figs 19
and 20). Finally, the presence of a few degenerating
cells embedded in a terminal field in nucleus ambi-
guus on day 7 (not illustrated) suggest that some
solitary nucleus-nucleus ambiguus fibers'5 may be
susceptible to TMT damage.
Lateral reticular nucleus. Both magnocellular and
parvocellular divisions of the lateral reticular
nucleus" contained a sparse population of degener-
ating neurons by day 4 (Figs 14 and 15). Although a
few degenerating neurons were also observed in the
subtrigeminal region by day 7, this pattern did not
change appreciably. These degenerating cells prob-
ably account for the presence of a few degenerating
axons in the restiform body (not illustrated) and may
contribute to degenerating mossy fiber projections to
5*
1
1
!
1
a
i
Fig. 30. GFAP concentration in selected brain regions 21
days after TMT intoxication. Immunoassay results are
expressed as percent of control values. Ban represent stan-
dard errors; n - 3 for TMT treatment, a - 5 for controls.
the cerebellar granular layer and cerebellar nuclei.
However, it must be emphasized that the bulk of
these terminal projections are probably a con-
sequence of a more profound cell death in the pontine
nuclei.
Other sites. A few degenerating neurons were ob-
served consistently in nucleus prepositus hypoglossi
(Fig. 13) and the medullary reticular formation by
day 4 (Fig. 13). These components were fairly minor
In addition, there was a prominent terminal field
along the dorsal and lateral aspects of the lateral
vestibular nucleus and the dorsolateral aspect of the
medial vestibular nucleus (Figs 12 and 26). This
projection did not seem to originate from the ves-
tibular nerve, and may reflect contnbutions from sites
of cell degeneration such a nucleus prepositus hypo-
glossi.
Protein-O-carboxyl methvltransferase and
glial fibrillary acidic protein as probes
for neurotoxicity
Because TMT induces a unique pattern of neu-
ronal degeneration, changes in expression of neu-
ronotypic and gliotypic proteins were examined fol-
lowing TMT intoxication to assess their utility as
probes for neurotoxic events and their sequelae. PCM
is a neuronotypic protein which is localized in all
neuronal somata. Thus, selective and specific toxic
effects of TMT serve as a basis for evaluating TMT-
induced alterations in the distribution of PCM im-
munoreactivity. Figure 28 shows the pattern of PCM
in control and TMT-treated rat hippocampal fields.
Although there was an alteration in the pattern and
degree of PCM immunoreactivity 4 days after toxin
treatment, the pattern did not mirror the degree of
degeneration shown with silver stains across hippo-
campal fields. In particular, the loss of PCM im-
munoreactivity in CA1 was more profound than in
CA4 at this time, despite the fact that TMT ablated
a much greater proportion of the cells in the latter
region. At longer survival times (e.g., 7 days), though,
the normal homogeneous distribution of intrasomatic
PCM immunoreactivity of affected neuronal popu-
lations was replaced by punctate, dense immuno-
reactive deposits which seem to be associated with
cellular debris (Fig. 29). Expression in surviving
neurons seemed to be depressed. Similar patterns
were seen in other brain regions damaged by TMT.
Another strategy for assessing toxin-induced dam-
age has been to measure changes in the expression of
the gliotypic protein GFAP.** Various forms of
neural trauma cause an increase in the expression of
GFAP by astrocytes surrounding the lesion." A
sensitive radioimmunoassay for GFAP was used to
document changes in GFAP in various brain regions
3 weeks following TMT intoxication (Fig. 30).
Significant increases were observed in pyriform and
entorhinal cortices, hippocampus, striatum, septum
and anterjor olfactory nuclei (P < 0.05, ANOVA and
Scheflfe test). The cerebellum, superior colliculus, and
-------
C. D. Balaban tt ai.
3S6
hypothalamus did not exhibit changes in GFAP
concentrations (/" > 0.05). With the exception of the
striatum, all of (he regions with marked increases in
GFAP also had severe neural degeneration. The
increased GFAP in the striatum could reflect degen-
eration of axons of passage from degenerating neo-
cortical and thalamic neurons. Furthermore, the non-
significant elevations of GFAP in the superior
colliculus and hypothalamus may reflect the fact that
the vast majority of neurons were unaffected by
TMT The expression of GFAP peaks at 3-5 weeks
following TMT and gradually declines toward con-
trol values by 12 weeks.** Thus. GFAP can be used
to document that neuronal damage has occurred to
either neurons or axons of passage in a given region.
However, the extent of GFAP expression does not
indicate which groups of neurons have been dam-
aged.
DISCUSSION
Regional selectivity of trimtthyitin
This study has described a number of novel fea-
tures of the distribution of TMT toxicity in rats.
Previously unreported degenerative changes were
identified in the septohippocampal nucleus, dorsal
intermediate and ventral divisions of the lateral septal
nucleus, septohypothalamic nucleus, anterior olfac-
tory nucleus, bed nucleus of the stria terminals,
endoptnform nucleus, parafascicular nucleus, ventral
posterolateral and ventral posteromedial thalamic
nuclei, superior colliculus. interstitial nucleus of the
posterior commissure, periaqueductal gray, inferior
colliculus. pontine nuclei, pars caudalis of the spinal
trigeminal nucleus, the caudal aspect of nucleus trac-
tus solitarius. dorsal vagal motor nucleus, dorsal
cochlear nucleus and lateral reticular nucleus. Lesser
degenerative changes wen observed in the pontine
tegmentum, parabrachial nucleus, nucleus propositus
hypoglossi, medullary reticular formation, nucleus
ambiguus, the anterior, dorsomedial and lateral
mammtliary nuclei of the hypothalamus, anterior
pretectal nucleus, ventral lateral geniculate nucleus,
medial geniculate nucleus, and the paraventricular,
mediodorsal and centrolateral thalamic nuclei. In
addition, these data refine previous reports that TMT
intoxication produces selective massive degeneration
of granule cells in the dentate gyrus, pyramidal cells
in CA fields of the hippocampus, and of neurons in
the subiculum, pyrifom cortex, entorhinal cortex,
amygdala, and neocortex,TA!*-,uwl *M* as well as less
prominent degenerative changes in the mesencephalic
trigeminal nucleus and raphe nuclei. We were
unable to confirm a previous report of cell death in
the olfactory tubercle.IUI Although we observed a
dense terminal field in layer lb of the tubercle, no
degenerating cells were present (data provided to
reviewers). Inspection of illustrations in the other
publications" indicates that this is merely a termi-
nological difference; the area described as olfactory
tubercle is piriform cortex in our nomenclature.
Although the data from this study indicate that the
central neurotoxic effects of TMT are more wide-
spread than described previously, they do not imply
a lack of selectivity of the toxin for particular popu-
lations of neurons. Specific, highly consistent patterns
of cell death were observed in each structure, indi-
cating that the effects are selective. This point is
appreciated by a simple analogy: i-adrenergic recep-
tors are distributed widely, yet actions of agonists or
antagonists are specific and selective at those sites.
Similarly, factors contributing to susceptibility to
TMT-induced cell death appear to be distributed in
distinct populations of neurons in a variety of central
structures. In this sense, the rigorous characterization
of the distribution of neuronal destruction provides
a basis for determining whether the earlier (days 1-4)
and later (days 5-7) phases of selective cell death at
distinct sites reflects single or multiple mechanisms of
action of the neurotoxicant.
The distribution of TMT-induced degeneration in
rats is consistent with the pattern of behavioral and
physiologic changes that have been characterized
collectively as "TMT syndrome".'010 As reported
previously,'the animals in our study dis-
played tremors, self-mutilation (usually of the tail
and foreiimbs). vocalization bouts and aggression in
response to both handling and other rats; as a result,
they are housed separately during the survival period.
The aggressive response to other rats was particularly
striking. As described by Brown el ai.,10 the rats,
when aroused, would rear on their hindlimbs, spar
with forepaws and vocalize continuously. The septal
damage documented in our data may contribute to
these symptoms, given the resemblance of the TMT
syndrome to sequelae of lateral septal lesions.1®-3*
Furthermore, the evolution of degenerative changes
in the somatosensory system (ventral posterolateral
and ventral posteromedial thalamic nuclei) may also
contribute to self-mutilation and the noted somato-
sensory dysfunction,1*1' ** while damage to the dorsal
cochlear nucleus, inferior colliculusand medial genie*
ulate body may contribute to the attenuation of the
auditory startle reflex in these animals.s,s* Relative
sparing of the geniculostriate system is consistent
with physiologic reports.3"1 Finally, as noted by
others,l0MJI seizures may reflect damage to the hip-
pocampus, amygdala and related circuits. However,
the data suggest that other features, such as cerebro-
cerebellar transmission, are disrupted in these ani-
mals. Thus, these data form the basis for deriving a
more complete neurobehavioral and neurophysio-
logy characterization of TMT toxicity.
Role of selective silver degeneration methods in
neuroto.xicoiogy
, This study provides further evidence of the utility
of selective stiver degeneration methods ("suppressive
silver staining techniques") for the assessment of
central effects of peripherally administered neuro-
toxins.1- Studies since the mid-1930s have documen-
-------
TMT neurotoxicity and CNS proteins
357
ted sensitivity and selectivity of these methods for
identifying anterograde (both direct and indirect
Wallerian) degeneration of axons, retrograde degen-
eraton of somata and dendrites after axotomy and
transneuronal degeneration of somata and dendrites
after deatferentation. This literature has been re-
viewed comprehensively by de Olmos el al.:t with a
discussion of relative merits and interpretational pit-
falls of each technique. Briefly, the selective silver
methods for impregnation of degenerating neurons
are basically modifications of Bielschowsky.
Gros-Bielschowsky or Rio-Hortega types of reduced
silver methods for staining neurofibrils of normal
tissue. These methods employ the common strategy
of placing free-floating, formaldehyde-fixed frozen
sections in initial silver impregnation solution,
followed treatment with an ammoniacal-silver
solution (tor examples of protocols, see Ref. 45). The
selective silver degeneration methods differ in the use
of suppressive agents to attenuate staining of normal
neurons and their processes. For the Carlsen and de
Olmos" cupric-silver protocol employed in this
study, the inclusion of cupric nitrate, pyridine and
allantoin in a sodium tetraborate-boric acid buffered,
alcoholic silver solution is an important suppressive
step. The Fink-Heimer protocol, by contrast,
employs an initial oxidation with potassium
permanganate, decoloration in an oxalic acid-
hydroquinone solution and incubation in an initial
silver solution containing uranyl nitrate/4 In particu-
lar. the Carlsen and de Olmos modification of the
cupric-silver method has proven to be a very
consistent, routine stain for visualizing degenerating
neuronal elements in our laboratory (e.g., Ref. 3).
The physicochemical basis for selective silver stain-
ing of degenerating neuronal processes is poorly
understood: indeed, the precise identity of ar-
gyrophilic elements within degenerating neurons has
not been characterized at the molecular level. Re-
duced silver stains for neurofibrils preferentially stain
either bundles of 10 nm neurofilaments or may stain
both 10 nm neurofilaments and microtubules of nor-
mal neurons, depending on the modifications of the
procedure.5* By contrast, selective silver degeneration
stains do not produce cytoskeieta! argyrophilia in
normal neurons, and the silver deposits in degener-
ating profiles do not conform to a purely cytoskektal
locus. Specifically, electron microscopic examination
of degenerating axon terminals after selective silver
impregnation with either Fink-Heimer or Eager tech-
niques suggests that argyrophilia is associated with
both axoplasm and degenerating mitochondria of
profiles showing clear ultrastructural signs of degen-
eration.11*'* The locus of argyrophilia in somata and
dendrites of degenerating neurons, though, has not
l*«n examined electron microscopically. Thus, de-
spite the fact that argyrophilic elements have not been
characterized at a molecular level, both ultra-
structural and light microscopic evidence (reviewed in
Ref. 24) support the specificity of selective silver
degeneration stains for moieties within degenerating
neuronal processes.
The major advantage of using silver degeneration
methods for neurotoxicant assessment is that they
provide a positive image of degenerating neurons and
their processes. This property has been exploited
most extensively in retrograde, anterograde and
transneuronal degeneration studies.^ This sensitivity
to these different types of degenerative changes sug-
gested the utility of these methods for neurotoxin
assessment. Previous studies of both 3-acetyl-
pyridineJ~ and TMT toxicity indicate that positive
staining of all degenerating processes permits accu-
rate assessment of the specificity of an effect to
particular anatomic classes of neurons. Simply, the
morphology and connectivity of degenerating neu-
rons provides positive identification of cells affected
by a particular agent. As a result, the vast data base
regarding neural connectivity can be applied directly
to correlate cell death with electrophysiologic and
behavioral sequelae. It is imperative, though, to
utilize a wide range of survival times after toxicant
administration to monitor both the time course of
development of argyrophilia in somata. dendrites and
axons of affected neurons, which will reflect the
progress of degenerative processes, and the relative
times of onset of argyrophilia in different populations
of cells. For example, the successive spread of ar-
gyrophilia from the somata and dendrites to the
axons, followed by disintegration of these processes,
suggests a somatofugal pattern course of cell death.
The major limitation of these methods, though, is
that results are difficult to quantify, given the facts
that normal neurons are negatively stained and that,
as noted by Grant.11 the number of argyrophilic,
retrogradely degenerating cells is typically lower than
the number of cells showing retrograde cellular reac-
tions with Nissl stains. Thus, silver degeneration
stains may best be exploited as a first-line screening
procedure for identifying the time course of toxic
effects in central neuronal populations; quantitative
assessment of effects in these regions can then be
pursued with a combination of biochemical and
morphometric strategies.
Neuronotypic and gliotypic proteins as probes in
neuroto.xicotogy
Neuronotypic and gliotypic proteins have been
used as markers for TMT-induced changes in other
studies. O'Callaghan and coworkers'" have docu-
mented declines in the concentrations of the synaptic
vesicle proteins synapsis I* and p38 proteins1111
following TMT-induced ceil Ion. The loss of these
neuronotypic proteins in hippocampus was Ant ob-
served 7 days after TMT, and peaked between 21 and
35 days possesion. There was very little loss seen at
the earliest times (i.e., days 2-7) postintoxication.
Similarly, we have observed a loss in PCM immuno-
reactivity- in hippocampus following TMT adminis-
tration. Although changes in PCM were evident by
-------
358
C. D. Balaban tt ai.
day 4, the silver degeneration strains were more
sensitive and accurate assays of damage at earlier
times. Thus, although PCM declined in degenerating
neurons, the loss of the protein could not be used to
determine (he earliest patterns of degeneration. One
advantage of immunoassays lies in the quantitative
aspect of the measurement. Although silver stains
were temporally sensitive for early degenerating
changes, they are not. by nature, quantitative. At
later time points, protein immunoassays can provide
a quantitative view on the extent of degeneration.
Thus, when both silver degeneration statns and im-
munochemical probes are used in concert, a more
complete picture of the location and extent of the
degenerative process can be gained.
There are several considerations when immuno-
cytochemical probes are used for neurotoxicity evalu-
ation. First, cells which are affected by TMT do not
undergo immediate death and proteolysis: thus
significant amounts of neuronotypic protein will re-
main in the region around the neuron. Second,
proteolytic fragments of neuronotypic proteins may
retain immunoreactivity in lira. This is consistent
with the altered distribution of PCM seen near
degenerating neurons in the hippocampus of TMT-
treated rats. Third, the time course for expression of
PCM and/or other neuronotypic proteins may be
altered in surviving neurons of a damaged structure,
reflecting repair or reaction to damage.
Another consideration associated with the use of
neuronotypic proteins as indices of neurotoxicity is
the possibility of trans-synaptic changes in protein
expression. We have recently documented that ab-
lation of the inferior olive with 3-acetylpyridine re-
sults in a loss in the cerebellar Purkinje cell expression
of caimodulin-sensitive phosphodiesterase.1 Since
only the climbing fiber input is lost by such treat-
ments. phosphodiesterase expression was apparently
under trans-synaptic regulation. Other calmodulin
binding proteins in the cerebellum were unchanged
by such treatments. Agents such as TMT cause a
widespread loss of many projection neurons, leading
to the possibility that some losses in neuronotypic
proteins may be a reflection of trans-synaptic
changes.
GFAP served as a reliable index of damage follow-
ing TMT intoxication. Areas which expressed the
highest percentage change in GFAP also had severe
degeneration of neurons. However, the striatum
stands out as a region which had very little direct
neuronal degeneration, but had a substantial increase
in GFAP. The most likely reason for this increase was
the destruction of axons of passage through the
striatum. Alternatively, TMT may directly affect the
expression of GFAP in specific astrocytic popu-
lations. One key point is that the expression of GFAP
occurs as a consequence of neuronal damage, and!
cannot discriminate whether neurons, axons, or sub-
classes of glia in a structure are degenerating. Thus,
although the increase in GFAP is further
confirmation that extensive degeneration has taken
place as a result of TMT. it cannot be used to predict
which neurons (or axons) have been affected in a
given structure.
Advantages of employing multiple probes for
neurotoxicity
Silver degeneration stains, immunocytochemistry
for neuronotypic proteins and immunoassays for
gliotypic proteins can be used in concert to produce
a more complete view of neurotoxicity caused by a
given toxicant. Silver stains, although not quan-
titative in nature, can be used to document regions
likely to show changes in neurotyptc and giiocypic
proteins. Immunocytochemistry of neuronotypic pro-
teins allows one to further demonstrate damage to
neurons. Furthermore, neuronotypic proteins such as
tyrosine hydroxylase or DARPP-3255 can be used to
quantify changes which occur in specific subclasses of
neurons. One caution is that the sensitivity of the
assay must be sufficient to document declines in these
particular regional markers. The robust response of
GFAP to injury provides further evidence of toxicant
involved neuronal damage. By using refined micro-
dissection of CNS areas, the GFAP concentrations
seemed to correlate with regions extensively damaged
by toxicants. Of note is the caution that GFAP
expression by reactive astrocytes cannot be used to
pinpoint which particular set of neurons were
destroyed. However, by combining the three methods
described in this study, we were able to correlate
neuronal loss with changes in silver staining,
neuronotypic proteins, and gliotypic proteins. Thus,
a case can be made for using all three approaches
when describing the type of damage produced by a
given neurotoxicanc.
One additional series of probes which may find
utility in neurotoxicity evaluations are cDNA/
mRNA probes. Degenerating cells are not likely to
transcribe mRNA; however, some structural neu-
ronotypic proteins will remain in situ until completely
proteolysed. Thus, in situ hybridization with a cDNA
probe for a regionally selective neuronotypic protein
could reveal whether transcription was occurring in a
given cell. Because mRNAs have short half-lives, the
disappearance of tissue-specific mRNAs may serve as
an early predictor of the onset of neurotoxicity.
Conversely, cDNA probes may also be used to detect
early stages of glial responses. It is likely that anti-
body probes, cONA probes, and sensitive silver
histologic stains will collectively improve the charac-
terizations of novel neurotoxicants. Thus, the data
gathered in this study provide a basis for the charac-
terization of TMT-induced changes in the expression
of tissue-specific mRNA.
A cknowledgtmtms—The authors wish to thank J. Kyle
Krady. Carol Hoover. Michael Viana and Robert
Brucklacher for technical asttttance. and Laura Karinch for
-------
TMT neurotoxicity and CNS protein) 359
typing the manuscript. Supported by a research grant from ronmentaj Protection Agency CR-813637 and > nr.r.rrh
Sdt°T lMt,,Ute R«"rch Founda" De^pS aS kSOOS91
lion tMLS), an Assistance Agreement wiih the U.S. Envi-
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TMT neurotoxicity and CNS proteins
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{Accepted 29 October 1987)
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Neurobehavioral Toxicology and Teratology, Vol. J, pp. 571-577, 1983. ° Ankho International Inc. Printed in the U.S.A.
Pattern Reversal and Flash
Evoked Potentials Following
Acute Triethyltin Exposure1
WILLIAM K. BOYES2 AND ROBERT S. DYER
Neurophysiology Branch, Neurotoxicology Division, Health Effects Research Laboratory
U.S. Environmental Protection Agency, Research Triangle Park, NC 27711
Received 14 April 1983
BOYES. W. K. AND R. S. DYER. Pattern reversal ttnd flash evoked potentials following nettle triethyltin exposure.
NEUROBEHAV TOXICOL TERATOL 5(5) 571-577, 1983.—Until recently, pattern reversal visual evoked potentials
(PREPs) had not been used to examine effects of neurotoxic agents, despite their clinical utility. Acute exposure to
triethyltin (TET), which produces cerebral edema and myelin splitting, increased flash evoked potential (FEP) latencies in
previous reports (17,18]. We recorded PREPs and FEPs in adult Long-Evans hooded rats before, and 24 hr after, exposure
to 0, 4. J or 6.0 mg/kg IP of TET bromide. Significant increases in latency and changes in amplitude of PREP peaks were
found in the 6 mg/kg rats. FEP peak latency increases did not meet the criterion for statistical significance. Thus, under the
conditions recorded, PREPs were better than FEPs as a measure of TET-induced neurotoxicity.
Pattern reversal evoked potential Flash evoked potential Triethyltin
THE development of more sensitive and reliable research
methodologies for the detection and characterization of
neurotoxicity is needed if neurotoxicity data are to be useful
in establishing safe exposure standards [42]. The recording
of pattern reversal visual evoked potentials (PREPs) has
grown in clinical neurological practice because of reports
that PREPs are altered in both frank and marginal cases of
several neurological disorders including multiple sclerosis
[26, 27, 37]. PREPs are also widely used in basic visual re-
search for analysis of linear and nonlinear components of the
visual system, and the generation of spatial frequency and
contrast sensitivity functions [4]. In neurotoxicological in-
vestigations, Merigan et al. [35] found increased PREP
latencies in acrylamide-exposed monkeys, and Fox and
Wright [20], using PREPs to estimate visual acuity, reported
acuity deficits in adult rats exposed neonatally to lead. Gen-
erally however, neurotoxicologists have not employed PREPs,
but have continued to record the more standard flash
evoked potentials (FEPs) because (1) the main emphasis has
been on detecting differences between exposed and control
conditions rather than on assessing visual function and (2)
adequate methodology has been lacking to record PREPs in
animals without either pharmacologic immobilization and re-
fraction, or extensive fixation training.
Two laboratories have described PREPs recorded either
from anesthetized rats [36], or from rats with an awake EEG
immediately following anesthetic [5, 28, 38, 39]. Total
avoidance of anesthesia is desirable in neurotoxicological
investigations because of potential interactions between the
effects of the anesthetic and those of the toxicant. Drug-drug
interactions are well known in the clinical literature and simi-
lar drug-neurotoxicant interactions also occur [471. For these
reasons, a procedure for recording PREPs from un-
anesthetized rats was developed [6]. This approach has also
been taken by Fox and Wright [21]. If awake rats are re-
strained so that the patterned stimulus is always in the field
of view, then accommodation is unnecessary because the
great depth of focus of the rat eye with a normal sized pupil
[22,32] allows the stimulus to be in constant focus on the
retina.
In this report, PREPs and FEPs recorded from awake rats
are compared for the ability to detect the effects of exposure
to a selected neurotoxicant. Pilot work indicated that the
amplitude of the two responses was maximal if recorded with
different stimulating and recording procedures. There is little
evidence to indicate whether a maximal response or a
threshold response is most likely to detect toxicity. The few
studies in which visual evoked potentials have been recorded
over a parametric manipulation of stimulation parameters in
a toxicological framework indicate that a maximal response
is just as or more likely to detect toxicity as is a threshold
response [8, 15, 16]. Therefore in this study, different proce-
dures were used to record PREPs and FEPs in order to
maximize response amplitudes. While this approach limits
the direct comparability of the results from each method, it
provides a better assessment of the utility of each response
"This manuscript has been reviewed by the Health Effects Research Laboratory of the U.S.E.P.A. and approved for publication. Mention of
trade names and commercial products does not constitute endorsement or recommendation for use.
'Supported by a National Research Council Research Associateship.
571
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572
BOYES AND DYER
for neurotoxicological purposes by optimizing the opportu-
nity of each procedure to detect toxicity.
Triethyltin (TET) was chosen as the test compound in a
dosage high enough to produce cerebral edema, myelin vacuo-
lation (31), and increased FEP latencies [17,18] within 24 hr
of dosing.
METHOD
Animals and Dosage
Adult male Long-Evans hooded rats obtained from
Charles River Breeding Co. were used. Body weights ranged
from 220 to 360 g at the time of testing. Rats were housed
individually in plastic cages with wood chip bedding. Purina
lab chow (5001) and tap water were available ad lib. The
testing room was maintained at 30±1°C to minimize TET-
induced hypothermia [ 18]. Once dosed, the rats remained in
the testing room until completion of the experiment. Animal
and testing room lights went off at 6 p.m. and on at 6 a.m. All
testing occurred between 10 a.m. and 4 p.m.
The rats were divided into four sets for daily testing pur-
poses. Within each set. rats were randomly assigned to one
of the three treatment groups. Saline solutions of TET
Bromide (Alfa Products, Danvers. MA) were made daily in
concentrations of 4.5 or 6.0 mg/ml. Rats were given a single 1
ml/kg (IP) injection of saline or TET solution to achieve dos-
ages for each treatment group of 0(n= 14), 4.5 (n= 13), or 6.0
(n=12) mg/kg TET (dosages are expressed as the salt, ap-
proximately 127c of the expressed dosage is TET). In our
laboratory. 6 mg/kg TET is approximately 34^ of the 24 hr
IP LD50 value (MacPhail and Farmer, personal communica-
tion).
5 urgerx
Prior to onset of the experiment, rats were fitted surgi-
cally with chronic recording electrodes. Following 0.35
ml/100 g Chloropent anesthesia (Fort Dodge Lab., Inc., Fort
Dodge, IN), 00-90 x 1/16" stainless steel screw electrodes
were threaded into the skull in locations selected to give
maximum amplitude FEPs and PREPs. While the PREP and
FEP are only somewhat different when recorded 3 or 4 mm
laterally, the PREP is slightly bigger at 4 mm, and the FEP
has been traditionally recorded at 3 mm [17,18]. For maximi-
zation of the PREP, and historical continuity of the FEP, the
two responses were recorded from slightly different loca-
tions. Electrodes were placed over the visual areas, 6 mm
posterior to bregma and 3 mm to the left of midline for the
FEP, and 4 mm to the right for the PREP. Ground and refer-
ence screw electrodes were placed 2 mm anterior and 2 mm
to the left and right of bregma, respectively. Insulated wires
were run from the electrodes to an Amphenol connector and
the entire assembly encased in dental cement. Each rat was
given 100,000 units of Penicillin G (IM) and allowed 1-2
weeks of recovery before the experiment began.
Recording Procedure
For PREP recording, the rats were restrained in a harness
(Alice King Chatham Medical Arts, Los Angeles, CA) and
placed with their eyes 7 cm in front of the TV screen of a
Grass Model VPG visual pattern generator. Black and white
bars (a square wave grating of 0.2 cycles per degree (cpd),
99% contrast, 30 cd/m2 luminance, 69° by 74° field size) were
displayed on the screen. Spatial frequencies of 0.2 cpd are
near the peak of the hooded rat's contrast sensitivity func-
tion, and well below the limits of spatial acuity [3J. The rat
PREP changes little with increasing contrast over 559r |5).
Thus, the stimuli employed were easily visible to the rats,
and provide maximum amplitude PREPs (6], The bars were
reversed at a rate of 0.5 Hz. black for white so that there was
no change in overall stimulus luminance. Pattern reversals
and line frequency (60 Hz) were nonsynchronized. For re-
cording FEPs, each rat was placed in a small chamber with
mirrors on 3 sides and the strobe lamp of a Grass PS-2
photostimulator on the fourth [13]. In this situation, each rat
was unrestrained but received a strong stimulation while fac-
ing any direction. Strobe intensity was set at 16 on the
photostimulator which corresponds to an estimated peak
power of 4.53 x 10**7 Lux as measured by an EG&G model
450-1 photometer and peak power integrator |20|. Flashes
were presented at 0.5 Hz.
Electrical activity from the rats' headsets was amplified by
10,000. bandpass filtered from 0.8 Hz-1 kHz tor the PREP
and 0.8 Hz-10 kHz for the FEP. The higher bandpass for
the FEP was necessary to avoid damping the sharp N 1 peak.
EEG was displayed on an oscilloscope and transmitted to a
PDP 11/70 computer for signal averaging and data storage.
Each sample epoch was 409.6 msec which had been A D
converted at a rate of 2500 Hz. An average of 200 trials was
taken for the PREP and 64 for the FEP. Calculations of
signal-to-noise enhancement based on the number of trials
averaged yield values of 14:1 for the PREP and 8.1 for the
FEP which should produce comparatively equal signal reso-
lution because the FEP amplitude is roughly twice the ampli-
tude of the PREP. Evoked potential peaks were manually
selected by cursor from averaged waveforms displayed on a
Tektronix 4054 graphics terminal. Peak latencies and
peak-to-peak amplitudes were computer-calculated, and in-
dividual waveforms were printed out on a Tektronix 4631
hard copy unit. Amplitudes are expressed as peak-to-peak
rather than baseline to peak because of the greater variability
of the latter measure [ 13].
For FEP recording, pupil dilation is necessary to assure
constant flash intensity at the retina. For PREPs however,
pupil dilation decreases the depth of focus [32] and disrupts
PREP waveforms [6,7], On each testing day therefore, the
PREP was recorded first, then the rats' pupils dilated with
topically applied drops of 19c atropine, and 1/2 hr later the
FEP was recorded. PREP and FEP recordings were obtained
24 hr prior to, and 24 hr after dosing.
Statistical Procedures
The statistical treatment of the data began with a series of
3 repeated measures MANOVAs: One each for FEP laten-
cies, PREP latencies and PREP amplitudes. FEP amplitudes
were not included because prior experimentation revealed
that only FEP peak latencies were changed by TET exposure
when hypothermia was controlled [18]. Each MANOVA in-
corporated all the measured latencies or amplitudes for each
evoked potential as dependent variables, and used the before
and after dosage values as the repeated measures, or "run."
factor. Only the dose-by-run effect with Wilks' statistic was
used [12,30], According to the Bonferroni correction proce-
dure, the desired experimental alpha level was divided by the
number of MANOVAs performed to determine the appro-
priate significance level for each MANOVA
(p =0.05/3=0.0167). Statistically significant MANOVAs
were followed by univariate ANOVAs for each amplitude or
latency to determine which peak differences contributed to
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EVOKED POTENTIALS AND TRIETHYLTIN
573
the significant MANOVA, again correcting probability levels
for the number of ANOVAs within each MANOVA. Signifi-
cant M ANOVAs were followed by Duncan's multiple range
test to determine which dosage groups differed from con-
trols.
RESULTS
The rats receiving 4.5 mg/kg TET did not appear to be-
have differently from controls in their cages or when han-
dled. Those receiving 6.0 mg/kg had sluggish movements,
but remained awake and apparently alert. No rats died.
Pre- and post-treatment FEP and PREP waveforms, av-
eraged across all rats in the 6.0 mg/kg group, are displayed in
Fig. I. The PREP was about half the amplitude of the FEP,
and consisted primarily of a major positive deflection with an
onset latency later than that of the FEP peaks. As such, the
waveshape of the PREP resembled that reported in
anesthetized rats 136, 38. 39]. In the awake rat PREP an
additional surface positive component, labeled P2. was
identifiable. The second positive peak was not reported by
Fox and Wright |21J in their awake rat PREPs, a discrepancy
which may be attributable to inter-laboratory methodological
differences. Although the human PREP general morphology
is also of a single late major positive component, in some
published human PREPs dual positive peaks appear [I0J. A
more detailed account of the properties of the awake rat
PREP may be found elsewhere [61. The PREP peaks iden-
tified for analysis were Nl, PI, N2, P2, and N3. with mean
peak latencies in the control group baseline recording ses-
sion of 45.5. 63.4, 78.9, 90.0, and 128.3 msec, respectively.
The FEP waveshape in awake rats is well characterized
I I8,I9|, and contains both "early" (PI, N I) and "late" (P2,
N2, P3, N3) components with mean control group latencies
•n this study of 21.8. 29.2, 47.1. 63.8, 78.9 and 141.3 msec,
respectively. In Fig. 1, the effect of TET on the group aver-
age waveforms is not striking, but slight discernable in-
creases in the PREP peak latencies were statistically signifi-
cant.
PREP latency data are presented in Fig. 2. Before dosing,
'he first four PREP peak latencies. (N I, PI, N2 and P2) were
reasonably equal across dose groups. For each of these
Peaks the 6.0 mg/kg TET exposure increased the post-dose
latency. The MANOVA including all PREP peak latencies
contained a significant dose-by-run effect, F(10,64)=2.42,
P =0.0(65. The dose-by-run latency increases were signifi-
cant for PI, F(2.36)=5.83,p =0.0064, and P2, F(2,36)= 10.24,
P =0.0003. The N3 latency also showed a significant dose-
by-run effect, F(2.36)=6.24, p =0.0047, but the nonuniform
baseline latencies across dose groups and the nonmonotonic
dose-response relationship make this result difficult to
'nterpret.
PREP peak-to-peak amplitude data are presented in Fig.
3. Although the MANOVA for PREP peak-to-peak ampli-
tudes was statistically significant. F(8,66)=4.19, p =0.0004,
none of the four individual amplitude ANOVAs reached
significance after Bonferroni correction (required
P =0.05/4=0.0125). The P1N2 amplitude was closest to
achieving significance, F(2,36)=4.74, p =0.0148. The con-
servative interpretation of these data is that some linear
combination of the individual PREP peak-to-peak ampli-
tudes significantly discriminates among the dosage groups.
FEP latency data are presented in Fig. 4. The FEP la-
tency MANOVA failed to reach the statistical significance
criterion, F( 12,60)= 1.75, p=0.0778. Inspection of Fig. 4 re-
FLASH EVOKED POTENTIAL IFEPl
N3
BEFORE
NT
Q
3
>-
P2
50 msec
St
N3 PATTERN REVERSAL EVOKED POTENTIAL iPBEP)
2
<
N2
N3
NT
N2
AFTER
SO msec
TIME
FIG. I. Group average flash (FEP) and pattern reversal evoked poten-
tial (PREP) waveforms before and 24 hr after 6 mg kg TET For graphi-
cal purposes, each waveform is a computer-calculated average of
the individual FEPs and PREPs from the same 12 rats.
veals that the latencies of the early peaks, PI. Nl and P2.
were changed in a similar direction, but less in magnitude,
than the latencies of the PREP peaks (Fig. 2). Little or no
increase in the latency of the later FEP peaks occurred. Al-
though the FEP peak N3 latency appears to be increased in
the "after" treatment group waveform in Fig. 1. the group
mean N3 latency actually showed a slight decrease (Fig. 4).
The discrepency is due to the difference between computing
a group mean waveform in which voltages are averaged at
fixed time points, the time point having the maximum aver-
age voltage being the peak, and computing group mean
latencies in which the maximum voltage point is selected for
each individual and the time values are averaged across rats.
The two calculations need not provide the same result. The
group average waveforms should be regarded as less accu-
rate and are presented only for general illustrative purposes.
DISCUSSION
This study illustrates the relative sensitivity of PREP and
FEP measurements, under the specific recording conditions
used, to TET-induced toxicity. Under these circumstances,
PREPs differentiated dosed from control rats better than
FEPs. Increased PREP peak latencies were found following
6.0 mg/kg TET, whereas FEP changes did not reach the
criterion for statistical significance. These data parallel some
reports in the clinical literature where the PREP is generally
considered more useful than the FEP [25]. Increased laten-
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574
BOYES AND DYER
Effects of Acute TET On Pattern Evoked Potential
24 h
AFTER
TREATMENT
24 h t
AFTER
TREATMENT
24 Kr
AFTER
TREATMENT
(C 90
24 hr
AFTER
TREATMENT
T|T DOSAGE, m«/k(
• CONTROL
¦ 4.5
4 6 0
AFTER
TREATMENT
FIG. 2. Pattern evoked potential peak latencies before (run 0) and after (run 1) rats
received an IP injection of TET Br. Statistically significant dose x run interactions
for each individual peak latency repeated measures ANOVA are denoted with an *.
In this and the subsequent figures, overlapping standard error bars were omitted for
clarity.
Effects of Acute TET On Pattern Evoked Potential
TIT OOtAOi.
• CONTROL
ItrOM
mST
AFTIN
triatmint
FIG. 3. Pattern reversal evoked potential peak-to-peak amplitudes
before (run 0) and after (run 1) rats received an IP injection of TET
Br. Although the repeated measures MA NOVA containing all 4
peak-to-peak amplitudes showed a statistically significant dose *
ran effect, none of the individual peak-to-peak amplitude ANOVAs
reached the criterion for significance after the probability levels
were corrected for the number of tests run. The conservative in-
terpretation of a significant MANOVA without significant individual
ANOVAs is that some linear combination of the peak-to-peak ampli-
tudes is altered by TET exposure.
cies of the major positive peak of the PREP have been re-
ported in patients with demyelinating optic nerve lesions
who had essentially normal'FEP latencies [23.26|. Other au-
thors however, find both the flash and pattern evoked po-
tentials to be useful in detecting diseases such as multiple
sclerosis and retrobulbar neuritis [19, 37. 40). Neetens a at.
[37] state that recording both the PREP and the FEP allowed
\Q09c of multiple sclerosis patients to be classified as abnor-
mal, greater than either method alone. In the present study
however. FEP data appear to add little to the information
available in the PREP.
The absence of statistically significant FEP changes
should not be taken as a failure to replicate the findings of
Dyer and Howell [17,18]. For the most part, the magnitude
and direction of FEP changes were similar in this study to
those reported previously, but the earlier experiments em-
ployed larger sample sizes and repeated post-dosage testing.
Trends toward increasing early peak latencies evident in Fig.
4 may have reached statistical significance had more rats
been tested.
The stimulation parameters in this study were chosen to
maximize the amplitude and reliability of the responses in
order to provide the most balanced comparison of the
methods. If the responses had been recorded under different
conditions, such as the FEP at other flash intensities or the
PREP at other pattern sizes, the comparative sensitivity of
the measures might have been different. However, maximiz-
ing the response amplitude has optimized detection of
neurotoxicity in previous studies [15]. The results of the
present study alone do not address the relative sensitivity of
all possible pattern and intensity related evoked potentials to
TET intoxication. In addition, stress associated with the
harness restraint procedure employed for the PREP but not
the FEP may have biased, in one direction or the other, the
relative sensitivity of the measures to TET. While we have
-------
evoked potentials and triethyltin
575
Effects of Acute TET On Flash Evoked Potential
N3 I
TET DOSAGE, mj/kg .
• CONTROL
¦ 4.5
4 80
BEFORE
24
AFTER
TREATMENT
BEFORE
BEFORE
24 hr
AFTER
TREATMENT
AFTER
TREATMENT
ui 85
BEFORE
BEFORE
BEFORE
AFTER
AFTER
AFTER
TREATMENT
TREATMENT
FIG. 4. Flash evoked potential peak latencies before (run 0) and after (run I) rats received
an IP injection of TET Br. The dose x run interaction of the repeated measures MANOVA
incorporating all six peak latencies was not statistically significant. In the face of a nonsig-
nificant MANOVA, individual peak latency ANOVAs were inappropriate.
been unable to record the PREP in awake rats without re-
straint, the FEP recorded in the harness shows little differ-
ence from that recorded in the mirrored chambers (unpub-
lished observations). This suggests that any stress related
influences were minor.
In addition to PREP peaks PI and P2, the latencies of the
early FEP peaks. PI and Nl. were increased, though not
significantly, in the 6.0 mg/kg group. The observed latency
changes are probably not a result of TET-induced
hypothermia for three reasons: (I) the animals were probably
not hypothermic when tested because they were housed at
30°C. the isothermic temperature for rats [40], (2) the
hypothermia produced by a 6 mg/kg TET injection is largely
attenuated 24 hr after treatment even in rats housed at 22°C
118], and (3) hypothermia increases the latency of all FEP
peaks, not just the early ones for which trends of increase
were found here [14].
In this study, slowing of visual information transmission
was observed. The effects of TET on sensory function have
not been studied extensively, despite the well known
neurotoxic properties of TET. Among the results of acute
TET exposure are cerebral edema [2,34], splitting of myelin
at the interperiod line, disruption of cellular energetics
[11,46], and hypothermia [11, 18, 33]. In the visual system, the
optic nerves and tract become swollen and show myelin vac-
uolation [29, 31, 44]. The optic disk of primates may become
edematous after TET treatment, but there is some question
about whether TET induces papilledema in non-primate
species [29,44]. Although the major damage sites are in CNS
white matter, edematous changes also occur in geniculate
bodies and in the heavily myelinated areas of cortex [2],
Amochaev et al. [1] reported that TET exposure increased
peak latencies of brainstem auditory evoked potentials.
The different pattern of changes produced in the PREP
and FEP may be related to the biological actions of TET.
Damage to the primary visual pathway produced by acute
TET exposure could have caused the measured alterations.
In humans, the PREP is thought to be limited to the primary
visual cortex [24], While the PREP in rats has been described
only recently [36, 38, 39], and the neurologic origin of PREP
peaks is yet to be determined, the waveform is similar to the
human PREP and appears to be restricted to the primary
visual projection areas [39]. The early FEP peaks represent
direct geniculo-cortical pathway activity [43,44]. Increased
latencies of PREP and trends of increased latency in the
early FEP peaks can be attributed to a delayed arrival of
activity at visual cortex. The later FEP peaks, which are
thought to represent the combined activity of the nonspecific
sensory system, subcortical projections and intracortical
loops [43,44], were unaffected by TET. Thus, the TET effect
is consistent with myelinopathic lesions and/or edema in the
specific visual projection pathway.
Although the PREP and FEP differential sensitivity to
TET may be related to the specific cells or neural systems
excited by pattern reversal or flash stimulation, the corticaily
recorded potentials used in this study do not allow an in-
-------
576
BOYES AND DYER
terpretation to be made concerning the particular cell types
involved. Clearly however, the neuronal activation patterns
generating the PREP peaks, and possibly those contributing
to the early FEP peaks, are altered by TET exposure, and
those generating later FEP peaks are not.
ACKNOWLEDGEMENTS
The authors thank M. Bercegeay. M. Geboff. I. Harrell and D.
Jenkins for technical assistance, and C. Rebert anil K. Muller for
helpful discussions.
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-------
The Journal of Neuroscience. April 1987. 7(4): 931-942
Quantitative Changes in the Synaptic Vesicle Proteins Synapsin I and
p38 and the Astrocyte-Specific Protein Glial Fibrillary Acidic Protein
Are Associated with Chemical-Induced Injury to the Rat Central
Nervous System
Thomas 0. Brock" and James P. O'Callaghan
Neurotoxicology Division, Health Effects Research Laboratory, United States Environmental Protection Agency, Research
Triangle Park, North Carolina 27711
Measurements of neuron-specific and glia-speclflc proteins
were used to characterize chemical-induced injury to the
rat CNS. Trimethyltin (TMT), a neurotoxicant that preferen-
tially damages neurons in limbic structures, was employed
to produce consistent, time-dependent, dose-related, cell
type-specific alterations in CNS morphology. Brain weights
and histology were used to verify the cytopathological af-
fects of TMT. Accompanying changes in 2 synaptic vesicle-
associated proteins, synapsin I and p38, and the aatrocyte-
associated protein, glial fibrillary acidic protein (GFAP), were
measured by radioimmunoassay (RIA). Immunohistochem-
istry of GFAP and incorporation of 'H-thymidlne Into GFAP-
pasitive astrocytes also were used to characterize astro-
cytic responses to TMT-induced injury. Finally, quantitative
2-dimensional PAGE was employed to detect additional pro-
teins affected by TMT. Acute administration of TMT caused
large dose- and time-dependent decreases in synapsin I
and p38 in hippocampus; the same proteins were largely
unaffected in a nonlimbie structure, the frontal cortex. Twelve
weeks after dosing, the concentrations of synapsin I and
p38 and, to a lesser extent, the absolute amount of these
proteins in hippocampus had returned to near control val-
ues, findings that are suggestive of reactive synaptogsne-
si». TMT caused large dose- and time-dependent increases
in GFAP that were not confined to hippocampus. Twelve
weeks after dosing, the amounts of GFAP in hippocampus
and frontal cortex had returned to near control values, find-
ings indicative of a transient astrocytic response to brain
injury. Immunohistochemistry of GFAP revealed widespread
astrocytic reactivity as a consequence of exposure to TMT,
• response that resulted in part from the proliferation of
Received Feb. 21, 1986; revised Sept. 22, 1986; accepted Sept 2S, 1986.
The authors wish to thank Mr. Michael E. Viana for excellent technical jj*"*'
tetc®. Ms. Donna Jenkins for preparation of the brain sections, M». Julia Davis
for photographic assistance, and Dts. Melvin E. Billingsley, Michael D. Browning,
unda J. Burdette, Paul Greengard, Reinhard Jahn, and Diane B. Miller for useful
discussions. T.O.B, received support from a National Research Council ReJearch
Assistantship. This paper has been reviewed by the Health Effects Research Lab-
oratory, U.S. Environmental Protection Agency, and approved for publication.
Correspondence should be addressed to James P. O'Callaghan, Ph.D., Neuro-
toxicology Division (MD-74B), U.S. Environmental Protection Agency, Research
Triangle Park, NC 277 U. , „
'Present address: Department of Child Development, Biological Sciences fU-
Center, University of North Carolina, School of Medicine, Building 220H,
Chapel Hill, NC 27514.
0270-6474/87/040931 -12502.00/0
astrocytes. Additional neurotypic proteins altered by TMT-
induced injury included one of the neurofilament (NF) triplet
proteins (p68) and a protein with the electrophoretic char-
acteristics of neuron-specific enolase (NSE). The data In-
dicate that measurements of neurotypic and gliotypic pro-
teins may be used to characterize the temporal and regional
patterns of neuronal and glial responses to injury.
Chemical-induced injury to the mammalian CNS is often man-
ifested by alterations in the cytoarchitecture of specific neu-
roanatomical regions. Within the affected area, the response to
chemical insult may encompass several types of neurons and
glia (Price and Griffin, 1980; Spencer and Schaumberg, 1980).
Because proteins that distinguish the diverse cell types com-
prising the mammalian nervous system have been described
(RafFetal., 1979; Schachner, 1982; Milner and Sutcliffe, 1983;
De Bias et al., 1984; Nestler and Greengard, 1984; Nestler et
al., 1984), we proposed that measurements of these nervous
system-specific proteins could be used to detect and characterize
the cellular responses to chemical-induced injury (O'Callaghan
and Miller, 1983). In support of this hypothesis, we have dem-
onstrated that the cytopathological changes caused by several
known neurotoxicants are accompanied by quantitative changes
in proteins associated with the affected cell types (O'Callaghan
et al., 1983; Miller and O'Callaghan, 1984; O'Callaghan and
Miller, 1984, 1985, 1986).
One neurotoxic chemical employed in our previous studies
was trimethyltin (TMT), an organometallic compound that pref-
erentially destroys limbic system neurons (Brown et al., 1979;
Bouldinet al., 1981; Chang and Dyer, 1983). By using TMT as
a denervation tool to alter the morphology of a limbic structure,
the hippocampus, we were able to demonstrate accompanying
changes in neuron-specific phosphoproteins (O'Callaghan and
Miller, 1984). On the basis of both radioenzymatic assays and
radioimmunoassays (RIAs), we found that acute administration
of TMT to the rat resulted in large, dose-related decreases in
hippocampal synapsin I, a synapse-specific, synaptic vesicle-
associated phosphoprotein (De Camilli et al., 1983a, b; Huttner
etal., l983;Navoneetal., 1984; Nestler etal., 1984). Consistent
with the region-selective effects of TMT, decrements in synapsin
I were not observed in the frontal cortex. These data suggested
that measurements of nervous system-specific proteins could be
used to characterize and quantify region-selective and cell type-
specific responses to CNS injury.
The objective of the present study was to characterize the
-------
932 BrocK and O'Callaglnan • Neurotypic and Glyotypic Protein After Brain Injury
Tabic 1. Time course of the effects of TMT on weights of
hippocampus and frontal cortex
Hemihippocampus
Hemifrontal cortex
Day
Treatment0
(gm)4
(gm)4
7
Saline
0.056 ± 0.004
0.054 ± 0.002
7
TMT
0.050 ± 0.001
0.055 ± 0.002
21
Saline
0.055 ± 0.002
0.054 ± 0.003
21
TMT
0.044 ± 0.001**
0.055 ± 0.003
35
Saline
0.055 ± 0.002
0.054 ± 0.003
35
TMT
0.036 ± 0.002**
0.054 ± 0.002
84
Saline
0.058 ± 0.002
0.058 ± 0.002
84
TMT
0.042 ± 0.004*
0.056 ± 0.003
" Subjects were treated with saline (0.9%) or TMT (8.0 mg/kg).
" Each value (rt - 5) represents the mean weight ± SEM.
* Significantly different from corresponding saline control, p < 0.01.
** Significantly different from corresponding saline control, p < 0.001.
time- and dose-dependent changes in both neuron-specific (neu-
rotypic) and glia-specific (gliotypic) proteins as a consequence
of chemical-induced injury of the CNS. Following the admin-
istration of TMT, the synaptic protein synapsin I and another
synaptic vesicle-localized protein, p38 (Jahn et al., 1985; Wie-
denmann and Franke, 1985), as well as the astroglia-localized
protein, glial fibrillary acidic protein (GFAP) (Bignami et al.,
1972, 1980; Eng, 1980, 1985), were quantified by RIA. Im-
munohistochemistry of GFAP was employed to evaluate pat-
terns of astrocytic response to injury. Finally, quantitative
2-dimensional PAGE was used to identify additional neurotypic
and gliotypic proteins affected as a consequence of the admin-
istration of TMT. Our results demonstrated that measurements
of neurotypic and gliotypic proteins could be used to charac-
terize the temporal and regional patterns of neuronal and glial
response to chemical-induced injury of the CNS.
A preliminary report of portions of this investigation has
previously appeared (Viana et al., 1985).
Materials and Methods
Materials. Bolton-Hunter reagent, '"I-protein A (2-10 nCi/ng; 1 Ci -
37 GBq) and }H-thymidine (6.7 Ci/mm; 1 Ci - 37 GBq) were purchased
from New England Nuclear (Boston, MA). Synapsin I and corresponding
antisera were the gifts of Drs. Jesse Chan and Eva Perdahl (Rockefeller
University). Monoclonal antibodies to p38 (c7.1, c7.2, and c7.3) were
the gift of Dr. Reinhard Jahn (Rockefeller University). Antiserum to
GFAP, GFAP immunohistochemistry kits, and rabbit anti-mouse IgG
were from Dako (Santa Barbara, CA). Monoclonal antineurofilament
protein (p68) was from Boehringer Mannheim Biochemicals (Indian-
apolis, IN). Nitrocellulose paper (0.2 mm pore size, of German man-
ufacture) was purchased from Schleicher and Schuell (Keene, NH). Oth-
er reagents and materials used in the RIAs were from the reported
sources (Goelz et al., 1981; Nairn et al., 1982; Jahn et al., 1984). All
reagents for 2-dimensional PAGE were purchased from Bio-Rad Lab-
oratories (Richmond, CA), with the following exceptions: Tris and di-
thiothreitol were from Calbiochem (San Diego, CA), Nonidet P-40 was
obtained from Particle Data Labs (Elmhurst, IL), and ampholines, pH
3.S-10, were from LKB (Gaithersburg, MD). Ovalbumin, BSA, and
molecular weight standards were purchased from Sigma Chemical Com-
pany (St. Louis, MO). Nuclear track emulsion (NTB-2) and XRP x-ray
film were obtained from Kodak (Rochester, NY). TMT hydroxide was
purchased from ICN Pharmaceuticals (Plainview, NY). All other re-
agents were of the highest analytical grade and were obtained from
various commercial sources.
Subjects. Male Long-Evans rats (200-250 gtn), purchased from Charles
River Breeding Laboratories (Wilmington, MA), were housed individ-
ually in a temperature (22°C ± 2)- and humidity (50 ± 10%)-controlled
colony room maintained on a 12 hr light : 12 hr dark cycle beginning
at 0600. Food (Purina Rat Chow) and water were continuously available.
The animals were administered TMT or its vehicle (0.9% saline) by
injection into a lateral tail vein; dosages of TMT are expressed as the
free base and were administered in a volume of 1.0 ml/kg body weight.
Dose-effect determinations (0.0-9.0 mg/kg TMT) were made 21 d post-
dosing. Time-effect determinations were made after the administration
of saline or 8.0 mg/kg TMT. All subjects were killed by decapitation.
Brain dissection and tissue preparation. Immediately after decapita-
tion, whole brains were removed from the skull and then bisected in
the midsagittal plane. The left hemibrains were prepared for histological
examination (see below) and the right hemibrains were prepared for
biochemical measurements according to the following procedures: the
hippocampus was removed by free-hand dissection and the frontal cor-
tex was obtained by coronal section (free-hand) at the level of the an-
terior commissure (bregma, 2.7 mm; see Paxinos and Watson. 1982).
Care was taken not to include olfactory nuclei or olfactory cortex with
samples of frontal cortex. The dissected brain regions were weighed,
homogenized by sonification (Kontes cell disrupter) in 10 volumes of
hot (90-95°C) SDS and then frozen at ~70°C for subsequent analysis
by RIA and 2-dimensional PAGE (see below). In one of the experiments,
subfields of dorsal hippocampus were dissected, as previously described
(O'Callaghan and Miller, 1984). In brief, five 700-MM-thick serial trans-
verse sections were prepared with a Mcllwain tissue chopper (Brinkman
Instruments, Westbury, NY). With the aid of a dissecting microscope,
individual slices were divided into areas approximating CA 1, CA3, and
fascia dentata using a modification (O'Callaghan and Miller. 1984. and
see legend to Table 2) of the procedure of Danscher et al. (1976). The
samples from a given region were then pooled, sonified in 75 m' of hot
SDS, and stored at -1Q°C before RIA.
Histology. The left hemibrains were immersion-fixed in 10% neutral-
buffered Formalin immediately after removal from the skull. The tissue
was embedded in paraffin and sectioned in the sagittal plane, beginning
at the midline. Two sequential sections were cut at thicknesses of 10
and 6 mm every 100 mm; the 10 mm sections were stained with cresyl
violet and the 6 mm sections were prepared for immunohistochemistry
and autoradiography (see below). With the aid of a 1 mm ocular grid,
estimations of hippocampal pyramidal cell loss were made by counting
the number of cells (CA1-CA4) with an intact nucleus and a prominent
nucleolus. Counts were obtained every third section (300 mm distance)
throughout the hippocampal formation. The nomenclature of Lorente
de N6 (1934) was used to describe anatomical subdivisions of the hip-
pocampal formation.
RIA of synapsin I. Synapsin I was measured by detergent-based RIA
according to the procedure of Goelz et al. (1981). Samples were nor-
malized in 1% SDS to a protein concentration of 5 mg/ml before
undergoing 1:50 dilution in RIA buffer (Goelz et al., 1981). Samples,
including standards, were assayed in quadruplicate using the microtiter
plate system as described by Nairn et al. (1982).
RIA of p38. Quantification of p38 was achieved by a modification of
the dot-immunobinding procedure of Jahn et al. (1984). In our proce-
dure, samples were applied to the nitrocellulose sheets, using a slot-blot
apparatus (Minifold II; Schleicher and Schuell) as a template. This mod-
ification resulted in faster sample application, greater reproducibility,
and a greater sample capacity per cm1 of nitrocellulose than the
original procedure (see Jahn et al., 1984). Samples containing 8.75 Mg
protein in 20 m1 sample buffer were loaded into each well. Incubation
of the nitrocellulose sheets with a mixture of monoclonal antibodies
against p38 (1:2000 each) was followed by washing in Tris-buffered
saline and additional incubation in blocking solution for 30 min (see
Jahn et al., 1984). This step was followed by incubation in rabbit anti-
mouse IgG (1:500) for 1 hr, with subsequent washing and blocking before
incubation in 1 "I-protein A. The specificity and linearity of the p38
assay have been described (Jahn et al., 1984, 1985).
RIA of GFAP. GFAP was assayed by the dot-immunobinding pro-
cedure of Jahn et al. (1984) as previously described (O'Callaghan and
Miller, 1985), with the exception that the technique for sample appli-
cation was modified as described above for p38. Each sample mfa'H
3.75 Mg protein in 20 m1 sample buffer. Antiserum to GFAP was used
at a dilution of 1:500; the specificity of the GFAP antiserum and the
linearity of the assay have been described (O'Callaghan and Miller,
1985).
Expression of RIA data. For assays of synapsin I, p38, and GFAP,
standard curves were constructed from dilutions of a «ingi» control
sample. By comparing the immunoreactivity of each sample (saline and
-------
The Journal of Neuroscience, April 1987, 7(4) 933
?3fǤiS; I
t'^ure !¦ Morphology of dorsal hippocampus (A, B) and frontal cortex (C, D) 21 d after the acute administration of saline, 0.9% (A. Q or TMT,
® 0 mg/kg (B, D). Bars, 100 mm. Note extensive loss of pyramidal cells in CA3-CA4 due to TMT (B). In contrast, neuronal loss was not obvious
in frontal cortex of TMT-treated subjects (£>).
TMT) with that of the sample used to construct the standard curve, the
relative specific activity (RSA) of each sample was obtained (e.g., see
O'Callaghan and Miller, 1984, 1985). The RSA of an individual sample
(saline and TMT) was then multiplied by a constant that resulted in a
mean RSA of 1.0 (100%) for the saline groups specified in the legends
to Figures 3-6 and Table 2. Data are expressed on the basis of both the
'otal (per tissue) and concentration (per milligram tissue protein). RSA
values for total (synapsin I, p38, or GFAP) reflect the amount of the
Protein assayed per hippocampus or frontal cortex, whereas RSA values
¦or concentration (synapsin I, p38, or GFAP) reflect the amount of the
Pfotein assayed per milligram of hippocampus or frontal cortex protein.
' hus, changes in the RSA total would be indicative of changes in the
at>solute amount of a given protein, whereas changes in the RSA con-
centration would indicate alterations in the relative abundance of a given
Protein.
Protein assay. Total protein was determined by the method of Lowry
ei al. (1951) using BSA as the standard.
Immunohistochemistry and microautoradiography. Sections (6 mm)
obtained from the left hemibrain were deparaffinized, incubated in 0.1%
trypsin for 15 min, and immunostained for GFAP according to the
'ndirect immunoperoxidase method of Sternberger et al. (1970); the
chromogen was aminoethylcarbazole. In some cases slides were coun-
tcrstained with Mayer's hematoxylin. The anii-GFAP used for immu-
nohistochemistry was the same as that employed in the RIA. Control
slides, prepared by omitting the primary antiserum or by using non-
¦rnmune rabbit serum, demonstrated that the staining for GFAP was
specific.
For autoradiography, subjects that had received saline or TMT (8
tfig/kg) 1, 2, 3, 4, or 5 d earlier were given an intraventricular injection
of 'H-thymidine (20 mCi in 20 m1 of water) as described (Billingsley et
al., 1982) and killed 1 hr later. Brains were removed and processed for
immunohistochemistry of GFAP, as described above. The slides then
were dipped in NTB-2 emulsion diluted 1:1 with 0.1% SDS and de-
veloped 1 week later by the method of Kopriwa and Leblond (1962).
The number of GFAP-positive cells (astrocytes), and the number of
GFAP-positive cells with reduced silver halide grains over their nuclei
were determined in the CA3c-CA4 regions of hippocampus. A cell was
counted as an astroycte if it had an intact nucleus with positive staining
for GFAP in the perikaryon or the cytoplasmic processes. Counts were
obtained from sections taken every 300 mm throughout the extent of the
hippocampal formation.
Two-dimensional gel electrophoresis and densitometry. Samples of
hippocampus and frontal cortex (150 Mg homogenate protein) were sub-
jected to 2-dimensional PAGE according to the procedure of O'Farrell
(1975), except that the concentration of acrylamide in the second di-
mension was 10%. After electrophoresis, gels were stained with Coomas-
sie blue R-250 and dried between sheets of dialysis membrane using
heat and reduced pressure. To identify the spots corresponding to GFAP
and NF p68, samples were subjected to 2-dimensional PAGE and were
transferred electrophoretically (Towbin et al., 1979; Burnette, 1981)
onto sheets of nitrocellulose. One sheet was assayed for GFAP as de-
scribed above and another sheet was assayed for neurofilament (NF)
p68 according to the double-antibody procedure described above for
p38. Using autoradiography to localize radioactivity bound to each
transfer, it was revealed that the antibodies to GFAP and NF p68 were
localized at positions corresponding to the electrophoretic mobility of
GFAP and NF p68, respectively (data not shown). Serum albumin,
actin, and a- and ^-tubulin were identified on the basis of comigration
-------
934 Brock and O'Callaghan > Neurotypic and Glyotypic Protein After Brain Injury
LU
2
s
3
>•
a.
O SAllNE
• TMT
LU
a3
DAYS AFTER TRIMETHYITIN (8 mg/kg)
Figure 2. Time course of hippocampal pyramidal cell loss following
the acute administration of TMT (8.0 mg/kg). Estimations of hippo-
campal pyramidal cell number were made as described in Materials and
Methods, Each value represents the mean ± SEM (n - 5). The mean
value for the combined cell counts obtained from sa/i'ne-treated subjects
(n = 20) is presented where lines are drawn to the ordinate.
with the pure proteins (see Brock and Mcllwain, 1985). To quantify
specific proteins, the stained gels were scanned with a computer-assisted,
television-based image analyzer (Darwin Instruments, Winston-Salem,
NC; see Telewski et al., 1983, for a description). The integrated optical
density (IOD) for selected proteins was determined after subtraction of
background values. The IOD for each protein spot was divided by the
IOD of ovalbumin that had been added to each sample before electro-
phoresis to control for gel-to-gel differences with respect to the degree
of staining or the amount of protein that entered each gel (Sinicropi and
Mcllwain, 1983). The IOD values for all proteins subjected to densi-
tometry were shown to be linearly related to the amounts of protein
applied to the gels over the range of densities observed for each protein.
Statistical analysis. The Statistical Analysis System (SAS; 1982) was
used for data analyses. Treatment effects were analyzed by the t statistic
or by analysis of variance (ANOVA). Duncan's Multiple Range Test
was used to make group comparisons following a significant ANOVA.
When group variances were unequal, comparisons were made by T. an
approximate t statistic with Satterthwaite's approximation for the de-
grees of freedom (1946).
Results
Brain weights. Acute administration of TMT caused dose- and
time-dependent decreases in hippocampal weight without af-
fecting the weight of frontal cortex. Time-course data for the
effects of 8.0 mg/kg of TMT are shown in Table 1. Twenty-one
days after administration of TMT, hippocampal weight was
reduced by 20%, an effect that was still evident 9 weeks later.
Histology. TMT (8 mg/kg) caused the characteristic destruc-
tion of hippocampal pyramidal cells with sparing of granule
neurons (Fig. I). Areas CA3-CA4 appeared to be the most af-
fected; however, neuron loss was noted throughout the pyrami-
dal cell layer in all sections examined. Consistent with our pre-
vious findings (O'Callaghan and Miller, 1984), the acute
administration of TMT was not associated with qualitative
changes in the morphology of the frontal cortex, a nonlimbic
region (Fig. 1). Time-course data for the effects of TMT on
pyramidal cell number are presented in Figure 2. Twenty-one
days after the administration of TMT, the number of pyramidal
cell neurons had declined to 25% of control values; no further
reductions in pyramidal cell number were observed by 84 d
postdosing.
RIA of synapsin 1 and p38 in hippocampus and frontal cortex.
The changes in hippocampal morphology 21 d after adminis-
tration of TMT were associated with dose-related decreases in
the synaptic vesicle-associated proteins synapsin I and p38 (Fig.
~ 135
TOTAL
CONCENTRATION
100
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Figure 3. EfFects of TMT on synapsin I (upper panels) and p38 (lower
panels) in hippocampus and frontal cortex. Subjects were killed 21 d
after treatment. Each value (n - 4-5) represents the mean RSA ± SEM
expressed on a total (per structure) or concentration (per mg protein)
basis; where vertical lines are not shown, the SEM is smaller than the
data point. Values obtained for hippocampus-saline (0 mg/kg) were used
to calculate a mean RSA - 1.0; see Materials and Methods for additional
details. 'Significantly different from saline control, p < 0.01.
3). These effects of TMT were evident whether the data were
expressed on a total (per hippocampus) or concentration (per
milligram hippocampus protein) basis. In order to determine
whether values for synapsin I and p38 would reflect the regional
specificity ofTMT-induced injury, the amounts of these proteins
in frontal cortex were determined. In agreement with our pre-
vious observations (O'Callaghan and Miller, 1984), TMT did
not affect values for synapsin I in frontal cortex. At the highest
dosage tested, however, TMT caused large (40-45%) decrements
in p38 (total and concentration).
The time course for the effects of TMT (8 mg/kg) on synapsin
I and p38 is shown in Figure 4. No significant changes in hip-
pocampal synapsin I and p38 were observed I, 2, 3, or 5 d
following exposure to TMT. Seven days after administration of
TMT, values (total and concentration) for hippocampal syn-
apsin I and p38 began to decline; maximal reductions were
reached 35 d after dosing. Values for total hippocampal synapsin
I and p38 increased slightly between 35 and 84 d postdosing
but still remained well below corresponding control values. Dur-
ing this period, the concentration of synapsin I and p38 returned
to near control values. The RSA (total and concentration) for
synapsin I and p38 in frontal cortex did not differ significantly
within the 12 week time course of the experiment.
RIA of GFAP in hippocampus and frontal cortex. In contrast
to the results obtained for synapsin I and p38, there were large
J 1 I I L-Cl I I I L.
0 3 6890 3 6 8 9
DOSE OF TRIMETHYLTIN (mg/kg)
CONCENTRATION
DOSE OF TRIMETHYITIN (mg/kB)
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The Journal of Neuroscience, April 1987, 7(4) 938
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Figure 4. Time course of the effects of 8.0 mg/kg of TMT on synapsin
1 (upper panels) and p38 (lower panels) in hippocampus and frontal
Co-
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Figure 5. Effects of TMT on GFAP in hippocampus and frontal cortex.
Subjects were killed 21 d after treatment. Each value i n - 4-5) represents
the mean RSA ± SEM expressed on a total (per structure) or concen-
tration (per mg protein) basis; where vertical lines are not shown, the
SEM is smaller than the data point. Values obtained for hippocampus-
saline (0 mg/kg) were used to calculate a mean RSA - 1.0. In the saline
(0 mg/kg) groups, note that the amount of GFAP in frontal cortex is
approximately half the amount of GFAP in hippocampus. See Materials
and Methods for additional details. 'Significantly different from saline
control, p < 0.01.
of TMT, values for GFAP in the hippocampus were increased
by as much as 300 and 500% when expressed on a total and
concentration basis, respectively. In frontal cortex, the corre-
sponding increases in GFAP were not as large in magnitude as
those in hippocampus; however, because control (0 mg/kg) val-
ues for GFAP in frontal cortex were roughly half those of hip-
pocampus, the increases in frontal cortex GFAP, expressed as
a percentage of control (0 mg/kg; frontal cortex), were nearly
comparable to the corresponding increases observed in hippo-
campus.
The time course of the effects of TMT (8.0 mg/kg) on GFAP
is shown in Figure 6. The temporal relationship of the TMT-
induced increases in GFAP (hippocampus and frontal cortex)
was similar to that of the decreases observed for synapsin I and
p38 (hippocampus): significant increases were not observed until
7 d postdosing, maximal effects were observed at day 35, and
values had returned to near control levels by day 84.
RIA ofp38 and GFAP in regions of dorsal hippocampus. Slices
of hippocampus were dissected into areas roughly approximat-
ing the major neuronal subfields to determine the intrahippo-
campal distribution of p38 and GFAP and the extent to which
each protein was altered by TMT in a given area (Table 2). The
values for p38 were uniformly distributed among hippocampal
subfields, a finding consistent with our previous observation
that the concentration of synapsin I did not vary among regions
of the hippocampus (O'Callaghan and Miller, 1984). Further-
more, as with its effects on synapsin I (O'Callaghan and Miller,
1984), TMT caused decreases in the content (total and concen-
tration) of p38 in ail regions of the hippocampal formation.
-------
934 Brock end O'Callaghan • Neurotypic and Glyotypic Protein After Brain Injury
more than 8 times greater than those of CA3. The effect of TMT
on GFAP also varied with respect to hippocampal areas; for
example, 13-, 15-, and 67-fold increases in concentration were
observed in fascia dentata, CA1, and CA3, respectively.
Immunohistochemistry of GFAP. The increased concentra-
tion of GFAP in all regions of the hippocampus and in frontal
cortex was suggestive of a widespread astrocytic response to
TMT-induced injury. To explore this possibility, we used the
antisera employed in the RIA to localize GFAP by immuno-
cytochemistry. Representative light micrographs of tissue ob-
tained from saline- and TMT-treated rats are shown in Figure
7. Although several investigators (Bignami and Dahl, 1976; Eng
and De Armond, 1982; Barrett et al., 1984; Mathewson and
Berry, 1985) have reported that GFAP immunoreactivity is
weak throughout the CNS of animals that have not undergone
nervous system trauma, we found that GFAP immunoreactivity
delineated stellate-shaped astrocytes in hippocampus (Fig. 7, A,
O and frontal cortex (Fig. 7E) of control subjects. GFAP im-
munoreactivity was distributed throughout all areas of the hip-
pocampal formation (Fig. 7A) and was intense in the astrocytic
processes, less so in the perikaryon, and absent in nuclei (Fig.
7Q. Twenty-one days after exposure to TMT (8.0 mg/kg), GFAP
immunoreactivity had increased in all cell layers of the hip-
pocampus (Fig. 7B)\ astrocytes proximal to neurons in CA1,
CA3, and CA4 appeared to be most intensely immunoreactive.
In comparison to immunoreactive astrocytes present in hip-
pocampus from saline-treated rats (Fig. 7C), those present in
hippocampus from TMT-treated rats were characterized by
markedly hypertrophied perikarya and processes (Fig. ID). In-
creased immunoreactivity associated with swollen astrocytes
was also seen in frontal cortex 3 weeks after administration of
TMT (Fig. 7F). By day 84 after exposure to TMT, astrocytic
reactivity had decreased in hippocampus and frontal cortex (data
not shown).
The number of GFAP-positive cells appeared to increase at
several sites within the hippocampus as a consequence of TMT-
induced injury. To verify that astrocyte mitosis had occurred,
rats were pulsed with 3H-thymidine. Maximal incorporation of
label was observed 4-5 d after administration of TMT. Astro-
Table 2. Effects of TMT on p38 and GFAP in region* of dorsal hippocampus
Relative specific activity
Region"
Treat-
ment*
p38
GFAP
Total
Concentration
Total
Concentration
Fascia dentata
Saline
1.00 ± 0.04
1.00 ± 0.03
1.00 ± 0.17
1.00 ± 0.14
TMT
0.50 ± 0.03**
0.57 ± 0.03**"
10.93 ± 1.40*
12.53 ± 1.51*
CAI
Saline
1.14 ± 0.02
0.96 ± 0.05
0.71 ± 0.28
0.60 ± 0.23
TMT
0.66 ± 0.04***
0.59 ± 0.04***
10.18 ± 1.76*
9.29 ± 1.57*
CA3
Saline
0.82 ± 0.03
0.94 ± 0.04
0.11 ± 0.05
0.13 ± 0.06
TMT
0.52 ± 0.05*"
0.61 ± 0.04**
7.32 ± 0.95**
8.70 ± 0.97**
"Slices of dorsal hippocampus were subdivided into areas designated fascia dentata, CAI, and CA3, as described
(O'Callaghan and Miller, 1984). When dissected in this manner, regions designated CAI and CA3 would each contain
a portion of CA2; subfields CA3c and CA4, as well as the molecular and granular layer of the dentate gyrus, would
comprise the region designated fascia dentata.
4 Subjects were killed 21 d after the administration of saline (0.9%) or TMT (8.0 mg/kg).
' Each value (n - 6) represents the mean RSA ± SEM expressed on a total (per hippocampus) or concentration (per mg
protein) basis. Values obtained for fascia dentata-saline were used to calculate a mean RSA - 1.00; see Materials and
Methods for additional details.
* Significantly different from saline, p < 0.005.
** Significantly diflfetent from saline, p < 0.0005.
**" Significantly different from saline, p < 0.0001.
>
HIPPOCAMPUS
CONCENTRATION
U
o SALINE
tu
>
fRONTAt CORTEX
CONCENTRATION
fRONTAl CORTEX
TOTAl
DAYS AFTER TRIME TH YITIN (8 mo/kg)
Figure 6. Time course of the effects of 8.0 mg/kg of TMT on GFAP
in hippocampus and frontal cortex. Each value (n — 5) represents the
mean RSA ± SEM.expressed on a total (per structure) or concentration
(per mg protein) basis. Values obtained for hippocampus-saline (7 d)
were used to calculate a mean RSA ~ 1.0. In the saline (0 mg/kg) groups,
note that the amount of GFAP in frontal cortex is approximately half
the amount of GFAP in hippocampus. See Materials and Methods for
additional details. 'Significantly different from saline control, p < 0.01.
Unlike the case with synapsin I and p38, the distribution of
GFAP within the hippocampus was uneven. Values for GFAP
in fascia dentata (which includes CA3c, CA4, and fascia dentata;
see Table 2) were nearly double those of area CAI and were
-------
The Journal of Neuroscience, April 1987, 7(4) 937
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Figure 7. GFAP immunohistochemisiry in sagittal sections of hippocampus and frontal cortex. Sections on the right (B, D. F) were obtained 21
d after acute administration of TMT (8.0 mg/kg); sections on the left (A, C, E) were obtained from corresponding saline controls. A. B. hippocampal
formation. Bars, 100 mm. C, D, Astrocytic immunoreactivity approximate to neurons (unstained) in CA3c-CA4. Bars, 50 mm. E. F, Frontal cortex.
Bars, 50 mm.
cyte counts obtained from subfields CA3c-CA4 5 d post-TMT
showed an increase in cell number (see legend to Fig. 8), with
several GFAP-positive cells showing reduced silver grains over
their nuclei. These 3H-labeled cells, however, represented less
than 1% of the GFAP-positive cells in the pyramidal cell layer.
In several instances, silver grains were localized to nuclei that
did not appear to be associated with GFAP-positive cells (e.g.,
Fig. 8). Tissue obtained from saline controls was devoid of silver
grains over GFAP-positive or GFAP-negative cells (data not
shown).
Two-dimensional gel electrophoresis. To determine if cell type-
associated proteins, in addition to synapsin I, p38, and GFAP,
-------
938 Brock ami O'Callaghan • Neurotypic and Glyotypic Protein After Brain Injury
Figure 8. GFAP immunohistochem-
istry in areas CA3c-CA4 of hippocam-
pus, combined with microautoradiog-
raphy of 'H-thymidine incorporation.
The subject was pulsed with ^-thy-
midine 5 d after administration of TMT
(8.0 mg/kg). Saline controls did not show
incorporation of 'H-thymidine; see
Materials and Methods for additional
details. Large arrow, Silver grains over-
lying nuclei of GFAP-positive cell.
Small arrows, Silver grains not clearly
associated with GFAP-positive cells.
Mean number of astrocytes (GFAP-
positive cells) in CA3c-CA4: saline,
96 ± 9; TMT, 150 ± 21. Less than 1%
of astrocytes in TMT group showed in-
corporation of 'H-thymidine.
were altered by TMT, we subjected homogenates of hippocam-
pus and frontal cortex to 2-dimensional PAGE. Representative
electrophoretic profiles are shown in Figure 9. Proteins relatively
low in abundance (e.g., synapsin I and p38) or outside the limits
of the pH gradient (e.g., synapsin I) were not detected. Of the
total number of hippocampus and frontal cortex proteins re-
solved, only a small percentage showed qualitative changes in
staining intensity as a consequence of the administration of
TMT. As was expected from the RIA data, 1 protein that was
consistently increased in hippocampus and frontal cortex was
identified by immunoblotting as GFAP. Two other proteins,
however, were consistently decreased in samples of hippocam-
pus and frontal cortex. One of these was positively identified as
the neuronal cytoskeletal protein NF p68 (Hoffman and Lasek,
Table 3. Effects of TMT on gliotypic and neurotypic proteins
resolved by 2-dimensional PAGE
Integrated optical density
(% saline)'
Protein"
Treatment4
Hippocampus
Frontal cortex
GFAP
Saline
100 ± 12
100 ± 13
TMT
410 ± 87"
461 ± 142*
NF 68
Saline
100 ± 12
100 ± 9
TMT
60 ± 12*
78 ± 16
NSE
Saline
100 ± 2
100 ± 7
TMT
63 ± 6***
77 ± 12
• GFAP, glial fibrillary acidic protein; NF 68, neurofilament p68; NSE, neuron-
specific enolase.
8 Subjects were lulled 21 d after the administration of saline (0.9%) or TMT (8.0
m»/lcg).
' Each value (n - 5) represents the mean ± SEM; see Materials and Methods for
additional details.
* Significantly different from corresponding saline control, p < 0.05.
" Significantly different from corresponding saline control, p < 0.001.
•** Significantly different from corresponding saline control, p < 0.0001.
1975), whereas the other (designated "NSE?") was tentatively
identifed as NSE (Picket et at., 1976; Schmechel et at.. 1978)
on the basis of its reported electrophoretic profile (Lim et at.,
1983; Heydorn et at., 1985). Computer-assisted densitometry
revealed that TMT caused a significant reduction in both NF
p68 and NSE in hippocampus, as well as the expected increase
in GFAP (>4-fold) in hippocampus and frontal cortex (Table
3).
Discussion
A single administration of TMT to the rat produces consistent
regional and cell type-specific damage of the CNS (Brown et al„
1979; Chang and Dyer, 1983; O'Callaghan and Miller, 1984).
Our results demonstrate that these cytopathological effects of
TMT are accompanied by time- and dose-dependent changes
in neurotypic and gliotypic proteins. Together with our previous
observations (O'Callaghan and Miller, 1984, 1985, 1986), these
data suggest that neurotypic and gliotypic proteins can be used
to characterize the temporal and regional patterns of neuronal
and glial responses to CNS damage.
In agreement with our previous findings (O'Callaghan and
Miller, 1984), TMT-induced damage of the hippocampus was
manifested morphologically by decrements in wet weight and
loss of pyramidal cells. On a per hippocampus (total) basis, these
effects were accompanied by a permanent decrease in the syn-
aptic vesicle-associated proteins synapsin I and p38. This re-
duction in total synapsin I and p38 did not reflect a generalized
decrease coincident with the loss of tissue, because the per-
centage decrease in these proteins was always greater than the
percentage decrease in hippocampal weight. This observation
indicates that a preferential loss of these proteins occurs as a
direct or indirect consequence of TMT-induced damage to the
hippocampus. Furthermore, because synapsin I and p38 are
present in all nerve terminals (De Camilli et al., 1983a, b; Na-
vone et al„ 1986), a reduction in the absolute amount of these
proteins (total) is suggestive of a loss of synapses due to damage
-------
The Journal of Neuroscience, April 1987, 7(4) 939
HIPPOCAMPUS
68 —
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TMT
Figure 9. Effects of TMT on proteins
resolved from 1.0% SDS homogenates
of hippocampus and frontal cortex.
Subjects were killed 21 d after treat-
ment with saline (0.9%) or TMT (8.0
mg/kg). Proteins were stained with
Coomassie blue R250. See Materials
and Methods for details concerning the
identification of individual proteins.
GFAP, glial fibrillary acidic protein; SF.
neurofilament p68; NSE?, neuron-spe-
cific enolase?: 5,1 serum albumin: al'.
a-tubulin: tfT, ^-tubulin: .-J. actin; and
o, ovalbumin (internal standard).
to intrinsic or afferent neurons of the hippocampal formation.
The possibility does exist, however, that a decrease in either the
number of synaptic vesicles per synapse or in the amount of
synapsin I and p38 per vesicle could account for our findings
(see Goelz et al., 1981).
The concentration of synapsin I and p38 in hippocampus was
also reduced in a dose- and time-dependent fashion following
the administration of TMT. Unlike alterations in total synapsin
I and p38, which reflect absolute changes, alterations in the
concentration of these proteins, in theory, may result from in-
creases in the content of other proteins, especially relatively
abundant ones (e.g., a- and ^-tubulin, GFAP). Alternatively,
changes in the concentration of synapsin I and p38 may reflect
specific protein alterations that accompany injury to the hip-
pocampus—an interpretation we favor because of the following
observations. First, quantitative analysis of specific protein con-
centration by 1- (O'Callaghan and Miller, 1984) and 2-dimen-
sional PAGE, as well as by specific RIAs not reported here (e.g.,
°t- and ^-tubulin; J. P. O'Callaghan, unpublished observations),
failed to reveal changes in most proteins as a consequence of
exposure to TMT, a finding that suggests that the changes in
specific protein concentrations (synapsin I, p38, GFAP, NF 68,
and neuron-specific enolase, NSE) did not result from changes
in the relative abundance of other hippocampal proteins. Sec-
ond, consistent with our findings, alterations in the concentra-
tion of specific brain proteins relevant to disease- (Marotta et
al., 198l;Selkoeetal., 1982), trauma- (Soifer et al., 1981; Perry
et al., 1985), or stress- (Greenberg and Lasek, 1985) induced
events occurred independently of changes in other tissue pro-
teins. Finally, changes in the concentration of GFAP in frontal
cortex were not associated with a concomitant decrease in syn-
apsin I and p38, which suggests that changes in the specific
concentration of a protein high in relative abundance (GFAP)
do not necessarily alter the concentrations of proteins low in
relative abundance (e.g., synapsin I and p38).
On the basis of our data, we cannot localize the loss of syn-
apsin 1 and p38 to a specific population of hippocampal neurons.
However, two lines of evidence support the hypothesis that the
reduced amounts of these proteins result primarily from the loss
of pyramidal cells. First, there was a close correspondence be-
tween the time course of pyramidal cell loss and that of the loss
of synapsin I and p38. Second, the greatest number of synapses
in hippocampal tissue occur between axon collaterals of pyrami-
dal cells and dendrites of other pyramidal cells (Braitenberg and
Schiiz, 1983); therefore, the degree (75% decrease) and
extent (CA1-CA4) of pyramidal cell loss caused by TMT would
be expected to result in a considerable loss of synapses and
synapse-localized proteins. The degree (60-70%) and extent
(CA1-CA4) of the decrease in synapsin I and p38 are consistent
with these expectations. It is likely that a more detailed under-
standing of the cellular targets associated with hippocampal
damage due to TMT or other injury-inducing paradigms may
be achieved through the use of monoclonal antibodies that pref-
erentially label specific cellular and subcellular elements of the
hippocampal formation. Such antibodies have recently been
described (Caceres et al., 1984; De Camilli et al., 1984; Levitt,
1984; Moskal and Schaffner, 1986).
An increase in the amount of synapsin I and p38 per hip-
pocampus and an increase in the concentration of these proteins
in hippocampus were observed between 5 and 12 weeks after
-------
940 Brock and O'Callaghan • Neurotypic and Glyotypic Protein After Brain Injury
administration of TMT. One explanation that may account for
these increases is that synaptic growth was evoked by partial
denervation of the hippocampus (Cotman and Nadler, 1978;
Cotman et al., 1981; Cotman and Nieto-Sampedro, 1984). The
following lines of evidence are consistent with this interpreta-
tion. (1) Synaptic replacement following injury to the CNS is
especially prevalent in the hippocampus (Cotman and Nadler,
1978; Cotman et al., 1981; Tsukahara, 1981). (2) The return of
synapsin I values toward control levels occurs more rapidly in
the neonate (Miller and O'Callaghan, 1984) than in the adult.
Both these findings are in agreement with the observation that
synaptic replacement occurs more readily in the immature an-
imal than in the adult (Gall and Lynch, 1980; Tsukahara, 1981;
McWilliams and Lynch, 1983). Finally, (3) destruction of hip-
pocampal pyramidal cells with kainic acid results first in the
loss of hippocampal synapses and then in their replacement 6-
8 weeks later (Nadler et al., 1980), findings consistent with those
for TMT. Although an increased synthesis of synapsin I and
p38 associated with synaptic ingrowth offers an attractive ex-
planation for the observed data, alterations in degradation or
transport of these proteins represent alternative hypotheses
equally worthy of further investigation.
Glial fibrillary acidic protein is the major protein of inter-
mediate filaments in astrocytes (Eng, 1983) and, as such, has
been proposed as a marker for assessing astrocytic response to
injury (for reviews, see De Armond et al., 1980; Eng and De
Armond, 1981; O'Callaghan and Miller, 1983; Eng, 1987). The
striking increments in GFAP content (total and concentration)
and the increased immunostaining of astrocytes that accom-
panies TMT-induced injury, together with our data showing the
effects of other toxic insults on GFAP (O'Callaghan and Miller,
1985, 1986), support the hypothesis that increases in GFAP
reflect astrocytic response to injury. Although the findings of
the present study suggest that neuronal damage initiates the
astroglial reaction, the possibility that astrocytes are directly
affected by TMT cannot be ruled out.
A feature of the GFAP response to TMT that set it apart from
the changes observed for synapsin I and p38 was its presence
in frontal cortex. One interpretation of these data is that in-
creased frontal cortex GFAP represented the spread of astrocytic
reactivity from the primary site of damage, the hippocampus
(e.g., see Mathewson and Berry, 1985); alternatively, this in-
crease may have reflected astrocytic response to TMT-induced
damage to the frontal cortex itself. We feel that the latter ex-
planation is the more plausible for several reasons. First, the
decrease in p38 (9.0 mg/kg) suggested neuronal damage in fron-
tal cortex due to TMT. Second, the temporal increases of GFAP
in hippocampus and frontal cortex were strikingly similar, which
does not support the idea of a spread of astrocytic reactivity
from a hippocampal focus. Finally, the temporal increase in
GFAP immunostaining due to TMT was similar in hippocam-
pus and frontal cortex, and the spread of reactivity in each region
did not extend to surrounding structures.
Astrocytic reactivity following a single stab wound to the CNS
declines with time to the pre traumatic resting state (Lagenaur
et al., 1982; Mathewson and Berry, 1985). The increased amounts
of GFAP due to TMT also declined with time to nearly control
values, indicating that acute chemical or physical lesions may
elicit a similar astrocytic response to injury. The transient nature
of the GFAP response to TMT- or stab-inflicted damage stands
in contrast to our observation of an apparently permanent in-
crease in GFAP in the cerebellum of the Gunn rat, an autosomal
recessive mutant characterized by life-long loss of cerebellar
Purkinje cells (see O'Callaghan and Miller, 1985). What distin-
guishes damage in the Gunn rat from that due to TMT or
stabbing is that neuronal loss in the former is a continuing event
throughout its life, while cell loss in the latter occurs as a single,
relatively short-lived episode. These findings raise the possi-
bility that continuing CNS damage is necessary to maintain an
astrocytic (GFAP) response. Related to this hypothesis are the
recent observations of Nieto-Sampedro et al. (1985) demon-
strating a transient production of astrocyte mitogenic and mor-
phogenic factors in response to a single cortical lesion. If these
factors regulate the astrocytic response to injury, then we predict
they will remain elevated in CNS tissue from chronically injured
subjects such as the Gunn rat or neurologically mutant mice
(e.g., Sommer and Schachner, 1981).
Changes in astrocytic size and appearance are widely accepted
responses to CNS trauma, but hyperplasia has been difficult to
document because of uncertainties associated with identifying
the dividing cells or the lineage of precursor cells (Cavanagh,
1970; Skoff and Vaughn, 1971; Skoff, 1980; Nathaniel and Na-
thaniel, 1981). These obstacles notwithstanding, the results of
several investigations indicate that astrocytic proliferation oc-
curs in response to CNS trauma. For example, Latov and co-
workers (1979) have demonstrated that a small portion of re-
active astrocytes (1-2% of GFAP-positive cells) in frontal cortex
incorporate 5H-thymidine after mechanical damage to this area.
Consistent with these observations are reports (Billingsley and
Mandel, 1982; Politis and Houle, 1985) showing that inhibitors
of DNA synthesis decrease the incorporation of 'H-thymidine
into cortical DNA and inhibit the proliferation of glial cells
following cortical trauma. Our data indicate that the effects of
TMT and perhaps other chemical toxicants (e.g., see O'Cal-
laghan and Miller, 1985, 1986), like trauma-induced damage of
the CNS, may also promote the division of astrocytes (GFAP-
positive cells) and possibly other glial cells (e.g., oligodendroglia;
see Arenella and Herndon, 19te 4; Ludwin, 1984).
On the basis of evidence obtained from specific RIAs, im-
munohistochemistry, and 2-dimensional PAGE, we have shown
that cell type-specific responses to CNS injury can be charac-
terized by measuring neurotypic and gliotypic proteins. The
sensitivity, specificity, and quantitative nature of this approach
should prove useful for characterizing specific responses to in-
jury (e.g., reactive synaptogenesis, astrocytic reactivity, or spe-
cific neurological disease states), as well as for assessing the
effects of putative neuronotropic or gliotropic factors on the
damaged or intact CNS.
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»,
BEHAVIORAL AND NEURAL BI0L0CY •, ¦-« (1990)
Delay-Dependent Impairment of Reversal Learning in Rats
Treated with Trimethyltin12
Philip J. Bushnell1
Neurotoxicologv Division. U.S. Environmental Protection Agency, Research Triangle
Park. North Carolina 2771 ]
Recent theories of hippocampal function focus on its role in the formation of
associations in the temporal domain. A reversal learning paradigm based on
leverpress automaintenance was developed to vary the CS-US relationship along
two independent dimensions, one temporal and one not: CS*-US delay and the
probability of reinforcement IPtRFT)) following the CS*. Eight male hooded
Long-Evans rats were trained to reverse these automaintained discriminations
repeatedly, until stable performance was achieved. The neurotoxicant trimethyl-
tin (TMT) was used to induce lesions in the CNS. including the CA3-4 region
of Ammon's Horn in dorsal hippocampus. Following iv injection of 7 mg/kg
TMT to half the rats, reversal learning was assessed under varying conditions
of delay and PlRFT). After recovery from the acute effects of TMT (1-2 weeks),
treated rats reversed normally when no delay separated the CS* and US; with
delays of 2 to 4 s. they reversed less completely within a session than did controls.
Changing P(RFT) did not affect reversal learning in either group, but reduced
response rates similarly in both groups. Morphological damage was quantified
by measuring the length of the remaining pyramidal cell line in sections of dorsal
hippocampus. The degree of behavioral impairment correlated significantly with
hippocampal damage only at nonzero CS*-US delays. These results indicate
that TMT impaired ability of rats to integrate temporal relationships between
stimulus events, and are consistent with theories of hippocampal mediation of
temporal associations. e im> Academe pr«». inc.
One of the most consistent functional effects of hippocampal damage
is disruption of working memory (Jarrard, 1985 ; Olton, 1983). As memory
1 The research described in this article has been reviewed by the Health Effects Research
Laboratory. U.S. Environmental Protection Agency, and approved for publication. Ap-
proval does not signify that the contents necessarily reflect the views and policies of the
Agency nor does mention of trade names or commercial products constitute endorsement
or recommendation for use.
' Portions of these data were presented at the Annual Meeting. Society of Toxicology. .
Dallas. TX. February 17. 1988. and to the Behavioral Toxicology Society, Savannah. GA,
May 17. 1988.
1 To whom all correspondence and reprint requests should be addressed at Neurotox-
icotogy Division. MD-74B. U.S. Environmental Protection Agency, Research Triangle
Part. NC 27711.
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BNB3292-2
PHILIP J. BUSHNELL
68 involves retention of information across time, recent theoretical accounts
69 of the role of the hippocampus in working memory focus on its ability
70 to form associations across time (Rawlins, 1985) and to categorize events
71 by their specific temporal contexts (Olton, 1986).
92 Consistent with its neuropathological effects on hippocampal mor-
93 phology (Brown, Aldridge, Street, & Verschoyle, 1979; Oyer, Deshields,
94 & Wonderlin, 1982b), trimethyltin (TMT) has been shown to disrupt
95 behaviors dependent upon temporally spaced events. Thus, response
96 patterning (curvature) on a fixed-interval schedule was reduced by TMT
97 in mice (Wenger, McMillan. & Chang, 1984b); pigeons, however, showed
98 an inconsistent pattern of change in response to TMT (McMillan. Wen-
99 ger, Brocco, Idemudia, & Chang. 1987). Intenresponse times and thus
100 the number of rewards earned on a DRL reinforcement schedule, which
101 requires withholding a response for a predetermined time period, were
102 reduced in rats treated with TMT (Wenger, McMillan, Chang, Zitaglio,
103 & Hardwick, 1985). Peele, Fanner, and Coleman (1989) conditioned an
104 aversion to the taste of saccharin (CS) by pairing its consumption with
105 an injection of lithium chloride (US). They observed that the degree of
106 aversion to saccharin decreased with increasing time between the CS
107 and US; moreover, rats pretreated with TMT showed a steeper gradient
108 across time than controls, indicating faster forgetting of the conditioning
109 event. Reduced accuracy in studies of delayed matching behavior after
110 TMT was reported in both pigeons (Idemudia, McMillan, & Chang, 1983)
111 and monkeys (Bushnell & Evans, 1984); rats showed delay-dependent
112 impairment in a spatial delayed response paradigm (Bushnell, 1988a;
113 Bushnell, Dunn, & New. 1988). Acquisition of an autoshaped lever touch
114 response by rats was impaired by TMT only when a 6-s delay was
115 interposed between lever retraction and pellet delivery (Cohen, Messing,
116 & Sparber, 1987). These effects of TMT treatment on time-dependent
117 behavioral processes resemble effects of surgical lesions of Jiippocampal
118 connections (Jarrard, 1986; Rawlins, Maxwell,-and StnderC 1985).
119 In these studies of the functional consequences of TMT intoxication,
120 TMT effects were revealed, or enhanced, by temporal delays between
121 events. However, none of these studies evaluated the specificity of the
122 temporal manipulation on the effect of TMT, leaving open the possibility
123 of other interpretations of the interactive effects of delay on TMT tox-
124 icity. The purpose of this study was to determine whether rats treated
125 with TMT showed impairment specific to integration of temporal infor-
126 mation, compared to integration of information of a nontemporal nature.
127 An automaintenance procedure was used for this purpose. In auto-
128 maintenance, a response (CR) is classically conditioned in the sense that
129 it is not required for delivery of reward (US); rather, it is elicited by a
130 contingency between a CS and reward. In a typical procedure, rats press
131 a lever (CS) whose retraction precedes food delivery (US): these presses
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IMPAIRMENT OF REVERSAL LEARNING
BNB3292-3
>•0001
132
133
134
135
136
137
138
139
140
141
142
143
144
145
146
147
148
149
150
151
152
153
154
155
156
157
158
159
160
161
162
171
172
173
174
175
176
177
178
179
180
181
K2
(CRs) are maintained by the pairing of the CS and the US (Hearst &
Jenkins. 1974; Locurto, Terrace, & Gibbon, 1981; Terrace, 1981).
In this study, two levers were repeatedly inserted into the chamber
for a brief time; the retraction of one lever reliably preceded pellet
delivery, while that of the second did not. Under these conditions, rats
emit many responses to the first lever (CS+) and few if any to the second
lever (New, Bushnell, & Dunn, 1988). Because its retraction is temporally
uncorrected with pellet delivery, this second lever may be thought of
as a CS°. When the contingencies between the levers are reversed, the
rat's behavior shifts toward the new CS" and away from the new CS°,
resulting in an automaintained reversal (Bushnell, 1988b; New et al.,
1988). Reversal rates stabilize after about 10 such reversals, after which
a reversal is completed within 30-50 trials of a 100-trial session. At this
point, the effect of chemical treatments may be assessed against a stable
baseline, permitting simultaneous quantification both of the rates of re-
sponding to each lever separately and of the shift in proportion of re-
sponses to the CS*. Note that the response rates provide a measure of
performance (i.e., motivation or "willingness" to respond), while the
proportion of total responses directed toward the CS* provides a measure
of learning (i.e., a change in behavior in response to a change in stimulus
events).
Finally, the relationship between the CS" and the US may be altered
parametrically (1) by introducing a delay between the CS* and the US
and (2) by reducing the probability that the US will follow the CS*.
These two manipulations differ in that the first involves time while the
second does not. Unpublished observations showed that both of these
manipulations affected reversal acquisition rate in this task, as well as
in an instrumental analogue of the task. For both manipulations, reducing
the strength of the CS+-US contingency slowed the rate at which a
reversal was acquired; reducing the probability of reinforcement to 0.8
also reduced asymptotic reversal accuracy.
The present study was designed to determine (1) whether rats treated
with TMT would show a delay-dependent deficit in automaintained re-
versal learning. (2) whether that deficit would be specific for temporal
degradation of the CS*-US relationship, and (3) the degree to which
damage to the dorsal hippocampus was related to the learning deficit
observed.
METHODS
Subjects. Eight adult male Long-Evans rats (Charles River, Raleigh,
NC) were housed individually in suspended plastic cages on heat-treated
shaved pine bedding, under a 12-h: 12-h L:D photoperiod with light onset
at 0600 h. All testing occurred in the light phase of the cycle. Each
animal was maintained at 350 g body wt by scheduled food (Ralston
-------
BNB3292-4
PHILIP J. BUSHNELL
9
[© • o o a i
i oioot:
[G • cjlc. 4
183 Purina, St. Louis. MO) delivery throughout the study; water was avail-
184 able ad libitum in the home cage.
185 Apparatus. Four standard rat operant conditioning chambers (Coul-
186 bourn Instruments Model E1010. Lancaster, PA), were each equipped
187 with two retractable response levers 3.4 cm in width, mounted with inside
188 edges 13 cm apart on one wall of the chamber. A cue light was mounted
189 4 cm above each lever. A food cup with a hinged, clear plastic door
190 (Campden Instruments, via Stoelting, Chicago, IL) was centered between
191 the levers. A microswitch attached to the door registered nosepokes into
192 the food cup. The levers were modified to register depressions of less
193 than 0.20 N. Each chamber was located in a sound-attenuating shell
194 within which white noise (80 db SPL, flat, measured at the opening of
195 the food cup) was provided. Reinforcers were 45-mg food pellets (Bio-
1% Serv, Frenchtown, NJ). Control of stimuli and recording of responses
197 were accomplished by computer (PDP8/a, Digital Equipment Corp.,
198 Maynard. MA) programmed with SUPERSKED software (State Sys-
199 terns, Kalamazoo, Ml).
200 Behavioral training. Rats were trained to press one lever using an
201 autoshaping protocol described elsewhere (Bushnell, 1988b). In brief, the
202 lever was extended into the operant chamber and lit by its cue light. In
203 the absence of a leverpress, the lever was retracted after 15 s and a
204 pellet was delivered immediately to the food cup. A leverpress during
205 the 15-s period caused immediate retraction of the lever and pellet de-
206 livery. Trials were separated by a variable-time 45-s intertrial interval
207 (ITI).
208 Single-lever automaintenance. When all rats reliably emitted lever
209 presses (nine 50-trial sessions), the schedule was modified such that a
210 leverpress no longer caused lever retraction, and a pellet was delivered
211 at the end of the 15-s period regardless of the rat's behavior. This au-
212 tomaintenance schedule remained in effect for five 50-trial sessions.
213 Two'lever automaintenance. The second lever (CS°) was then intro-
214 duced; it was extended and retracted on an identical but temporally
215 independent schedule. Pellet delivery was random with respect to the
216 CS° and could thus follow its retraction on some trials. The variable-
217 time 1T1 was reduced to 30 s (range, 1.6 to 99.1 s). Each animal received
218 three 50-trial sessions under these conditions.
219 Reversals. A reversal was defined as a change in the designation of
220 the CS+ and CS°. At the beginning of a reversal session, the lever which
221 was previously the CS* became the CS°, and vice versa. No cue for
222 this change was provided to the animal. The first reversal was pro-
223 grammed on the fourth day of two-lever automaintenance. Lever des-
224 ignations were not changed again for 10 sessions (500 trials). Responses
225 to the new CS* accounted for at least 90% of total responses for all
226 animals during the last 5 of these sessions. Subsequent reversals were
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IMPAIRMENT OF REVERSAL LEARNING
BNB3292-5
227 administered after fewer sessions, as the animals shifted responding with
228 increasing rapidity. After the sixth reversal, sessions were extended to
229 100 trials and the P(RFT) was reduced to 0.80. in an attempt to increase
230 response rates (Davey & Cleland, 1982). (P(RFT), or probability of re-
231 inforcement, was defined as the proportion of trials on which a pellet
232 was delivered after retraction of the CS*. Whether a particular trial was
233 rewarded was determined randomly by the data acquisition program.]
234 After 12 reversals on this schedule, all animals reversed reliably within
235 SO trials. Thereafter, reversals were administered on Tuesdays and Thurs-
236 days, with the other days serving as nonreversal control days.
7 TMT treatment and post-treatment observations. Trimethyltin hy-
8 droxide was obtained from K & K Laboratories, a subsidiary of ICN
9 Pharmaceuticals (Plainview, NY); it was sample No. 3 in O'Callaghan,
10 Niedzwiecki. and Means (1989), with a relatively high potency. On the
11 basis of previous studies in this laboratory, a dose of 7.0 mg/kg was
12 selected as having produced significant changes in neurochemical and
13 histological indices of TMT toxicity, without severe overt signs, in this
14 strain of rat. Intravenous injections of TMT (1.0 mg TMT base/ml saline)
15 (n = 4) or saline (n ® 4) followed reversal 44. Selection of subjects for
16 treatment was made randomly, without regard for baseline performance.
17 The behavior of the animals was followed for 6 months (116 sessions)
18 thereafter. Manipulations of P(RFT) and CS+-US delay (a time interval
19 programmed to intervene between retraction of the CS+ and delivery of
20 the food pellet into the food cup) occurred thereafter in 2- to 3-week (4-
21 to 6-reversal) blocks as shown in Table 1.
22 Statistical methods. Response frequencies to each lever for each sub-
23 ject obtained from each 10-trial block were averaged across replicate
24 reversals at each condition of Delay and P(RFT). Discrimination ratios
25 (DRs) were then calculated from the averaged response frequencies for
TABLE I
Schedule of Parametric Manipulations following Treatment with TMT
Block
Cumulative
week!
Reversals
Delay (s)
P(RFT)
2
3
4
5
6
7
8
9
10
3.5
5.5
7.5
9.5
11.5
13.5
15.5
18.5
20.5
22.5
45-51
52-55
56-59
60-63
64-67
68-71
72-72
76-81
82-85
86-89
0
2
4
0
0
0
0
0
2
4
0.8
0.8
0.8
0.8
1.0
0.6
1.0
0.8
0.8
0.8
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BNB3292-6
PHILIP J. BUSHNELL
J
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
74
75
76
each subject under each experimental condition as the proportion of CS+
responses to total responses per 10-trial block. The individual DR curves
for each rat were next fitted to a negative exponential function of the
form DR * A(l-10~**) (Hull, 1943), where X * the trial number at the
midpoint of each 10-trial block. A is the asymptotic value of the function
and R is a parameter whose magnitude varies directly with rate of ap-
proach to asymptote. Response frequencies to CS* and CS°, summed
across trial blocks, were subject to analysis of variance tANOVA, SAS
General Linear Model (SAS, 1985)] with Groups as a between-subject
factor and Response Type and Delay (or P(RFT)) as repeated measures.
The two parameters of the exponential equation, A and R, were tested
by two-way ANOVAs with TMT treatment as a between-subject factor
and Delay (or P(RFT)) as a repeated measure. The a level for each
ANOVA was 0.05.
Histology. At the end of the experiment, each rat was perfused trans-
cardially with saline and 10% neutral-buffered formalin under deep pen-
tobarbital anesthesia. Each brain was removed, hemisected sagittally,
mounted in paraffin, and cut into 60-/im sagittal sections from 0.5 to 4.0
mm lateral to midline. Every fifth section was stained with cresyl violet
and projected onto white paper at 27 x magnification. The length of the
projected hippocampal pyramidal cell line was determined for each of
three sections of dorsal hippocampus from each rat, taken between 1.3
and 2.3 mm lateral to the midline. These lengths (in cm) were averaged
for each rat and correlated with the degree of behavioral change pre-
viously observed in that rat, since the degree of TMT-induced hippo-
campal damage is inversely related to the length of the remaining cell
line (Dyer et al., 1982b).
RESULTS
The rats' body weights were not affected by TMT treatment, nor were
self-mutilation or hyperirritability observed. The lack of overt signs of
the "TMT syndrome" (Dyer et al., 1982a) and the range of histopath-
ological changes subsequently obtained (vide infra) indicate that this dose
of TMT produced a moderate, relatively selective lesion in
dorsal hippocampus.
Response frequencies of the treated animals dropped to an average of
40% of their pretreatment levels for about 2 weeks after dosing, and
recovered baseline within 3 weeks. Rates of response to the CS* av-
eraged 10—15/trial after recovery, or about 0.6-1.0/s, given the 15-s CS
duration (Figs. 1 and 2, top). All formal analyses were carried out after
recovery of baseline response rates. _
Rats treated with TMT responded as frequently as controls to both
levers at all three delays, but divided their responding between the CS4
and CS° less completely than controls as delay increased (Fig. 1, top).
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IMPAIRMENT OF REVERSAL LEARNING BNB3292-7
i
I
Control
Trimethyhin
1»»
io»
1
SO
ill
fw 1
o.zs-
I 10 0
Blocks of T« Trill*
>0
Fic. I. Effect of CS*-US delay and TMT on reversal behavior. Top: Mean (:zSE)
frequencies of response to the CS* (filled symbols) and the CS° (open symbols) plotted
as a function of 10-trial block and CS*-US delay. Symbols for delay: circles. 0 s; triangles.
2 s; squares. 4 s. Bottom: Mean (xSE) discrimination ratios plotted as a function of 10-
trial block and delay, derived from the data in the top panels of Fig. I. Lines represent
best-fitting exponential functions for each condition of delay (see text). Left panels, per-
formance of control rats
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BNB3292-8
PHILIP J. BUSHNELL
zmomc <1
P
VfeJ
o»»jooj
Trimethyltin
Control
i.oo-
1,00-
o.?S'
o.io-
B.IJ-
o.»
jl'tiii-!-4-
• io " o t i i i To
Blocki of Ten Trials
Fig. 2. Effect of probability of reinforcement and TMT on reversal behavior. Top:
Mean I ± SE) frequencies of response to the CS' (filled symbols) and the CS° (open symbols)
plotted as a function of 10-trial block and probability of reinforcement [PtRFT)). Symbols
for P(RFT): circles, 1.00: triangles. 0.80: squares. 0.60. Bottom: Mean (±SE) discrimination
ratios plotted as a function of 10-trial block and P(RFT), derived from the data in the top
panels of Fig. 2. Lines represent best-fitting exponential functions for each condition of
delay (see text). Left panels, performance of control rats (/> » 4); right panels, performance
of TMT-treated rats In - 4).
91 delay in two important ways. First, decreasing the P(RFT) to 0.60 re-
92 duced overall response rates to both levers in both groups, as the P(RFT)
93 main effect was significant (F(2, 12) = 4.42. p < .05) while the TMT
94 by P(RFT) interaction (F(2, 12) < 1) was not. Second, P(RFT) did not
95 interact with Response Type (F(2, 12) = 1.26), indicating that the relative
96 rates of response to the CS4 and CS° were not affected by reducing
97 P(RFT), as they had been by increasing the CS+-US delay. The three-
98 way interaction (TMT by P(RFT) by Response Type) was not significant
99 (F(2. 12) * 1.05).
100 Reflecting these differences in response to delay and P(RFT), the dis-
101 crimination ratios were also affected more by delay than by P(RFT).
102 While both groups showed a slight retardation in reversal as shown by
103 a slower approach to asymptotic DR across trials at 4 s delay than at 0
104 s delay, only the TMT-treated rats showed a delay-dependent reduction
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IMPAIRMENT OF REVERSAL LEARNING BNB3292-9
105 in asymptotic DR (Fig. 1, bottom). Changing the P(RFT) had little effect
106 on DR in either group (Fig. 2, bottom).
107 The asymptotic level of the DR reached during the reversal (parameter
108 A) declined with increasing delay for the TMT group only (Fig. 3, top
109 left). Statistically, the main effects of TMT (F(l, 6) » 6.98, p < .04)
110 and Delay (F(2, 12) * 6.59, p < .02), as well as the TMT by Delay
111 interaction (F(2, 12) » 6.12. p < .03) were all significant. In contrast,
112 A did not change significantly as a function of P(RFT) in either group
113 (Fig. 3, top right). The rate of approach to asymptote (parameter R) did
114 not differ significantly from control under any condition of Delay of
115 P(RFT) (Fig. 3, bottom).
116 Histological results showed that the pyramidal cell line in dorsal hip-
117 pocampus was shortened by 19 to 30% of control (Fig. 4). The TMT
118 mean (± SEM) of 4.17 ±0.13 mm differed significantly from the control
119 mean of 5.44 ± 0.15 mm (/(6) » 6.45, p < .001]. This degree of damage
120 is less than that reported in rats 120 days after the same dose of TMT
121 given orally (e.g.. Dyer et al., 1982b), perhaps due to differences in
1.0
—
o.» 1
0.*
¦ e»Atr»i
--O" TUT
1.00
0.(0
0.«0
0.1ft
0.10
0.0»
O.OOJ-
_
J
"S i j"
CS+.US My(HC>
0.1»t
0.10
0.0»
O.OO'
1.00 0.(0
Probability of lUinfa
o.to
Fig. 3. SummaryoCtheefTectsofTMT.CS*-USdelay.andP(RFT)ongroupiMUuret
of reversal learning. Top: A
-------
j* - v..; -1 .,
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IMPAIRMENT OF REVERSAL LEARNING
BNB3292-11
1.00
O.fO-
•tc Oa lay
MC 0* 1 ay
aac Oalay
0 70-
0 40+
1.1
4 4.3 % S.S
Length of Pyramidal Cell Line (nun)
Fig. 5. Scatterplot and regression functions relating the length of the remaining hip-
pocampal pyramidial cell line for each rat (jr-values) to A. the asymptotic value of the
discrimination ratio for each rat (v-values). determined at CS'-US delays of 0 s (circles),
2 s (mangles), or 4 s (squares). Filled symbols represent control rats, open symbols TMT-
treated rats. The lines represent the best-fitting linear regression functions for each delay
condition. Correlation coefficients for the three functions were: 0 s, 0.536 (ns); 2 s. 0.801
(p < .05); 4 s. 0.898 (p < .01).
1
Ie • o o o 1
122 exposure route. TMT potency (O'Callaghan et al., 1989). or body weight
123 between the animals in the two studies (Dyer et al., 1982a).
124 The two learning parameters (A and R) were regressed against the
125 degree of hippocampal damage at each level of Delay and P(RFT). A
126 correlated significantly with hippocampal pyramidal cell line length only
127 when it was determined at nonzero delays (Fig. 5). R did not correlate
128 significantly with hippocampal damage at any delay. Furthermore, no
129 value of P(RFT) produced a significant correlation between A or R and
130 the degree of hippocampal damage induced by TMT.
131 DISCUSSION
This study demonstrates that TMT caused a delay-dependent decre-
133 ment in automaintained reversal learning in rats, since impaired learning
134 (reduction in A, the measure of asymptotic accuracy of the DR) was
135 evident only when a delay was inserted between the CS* and the US
136 (compare bottom panels of Figs. 1 and 2). The reduction in A resulted
137 from a delay-dependent change in distribution of responding between the
138 CS* and the CS°, which was statistically evident in both groups, but
139 greatly exaggerated in the treated animals (Fig. 1, top).
148 In contrast to the effects of delay, reducing P(RFT) lowered overall
149 response rates slightly in both groups of animals, but did not alter the
150 distribution of responses between the CS* and the CS° (Fig. 2, top).
151 Thus the characteristics of the resulting learning curves did not differ
-------
BNB3292-12
PHILIP J. BUSHNELL
152 for either group under any P(RFT) condition (Fig. 2, bottom), despite
153 significant changes in performance of the task under conditions of low
154 reinforcement density. This result is not consistent with other obser-
155 vations from this laboratory, in which P(RFT) retarded reversal acqui-
156 sition in untreated rats (unpublished observations).
157 This dissociation between the effects of CS+-US delay and P(RFT)
158 on learning and performance indicates that TMT treatment unpaired
159 learning only under conditions of temporal discontiguity between critical
160 stimuli. The lack of effect of P(RFT) on distribution of responses (and
161 thus on the shape of the learning curves) shows that these animals'
162 learning was not impaired by discontiguity in a nontemporal dimension.
163 However, the lack of effect of P(RFT) on learning in the controls indicates
164 that this manipulation provided insufficient challenge to these highly
165 overtrained animals. Because learning in neither group was affected by
166 the reduction in P(RFT), it is not possible to conclude that the learning
167 impairment shown by the TMT rats was specific to
168 temporal discontiguity.
169 Nevertheless, reducing P(RFT) affected performance, as indicated by
170 overall response rates, in both treated and control animals. Because
17] performance was altered similarly in both groups, but learning was
172 changed only in the treated group, it is unlikely that motor and/or mo-
173 tivational changes could account for the change in learning in the treated
174 animals.
175 The effect of P(RFT) on response rate in this study differs from re-
176 ported effects of reduced P(RFT) on autoshaping in rats, in which lev-
177 erpress rate ("sign tracking") is increased, while responding to the food
178 cup ("goal tracking") is reduced under conditions of partial reinforce-
179 ment (Davey & Cleland, 1982). Apparently, this partial reinforcement
180 effect facilitates acquisition of the leverpress response itself, but has
181 inhibitory effects on pressing in well-conditioned animals.
182 After recovery from an acute phase of TMT intoxication (about 2
183 weeks), during which response rates were suppressed in the treated
184 animals, there was no further evidence of functional recovery in treated
185 rats. That is, the effects of CS+-US delay in treated rats were at least
186 as large 25 to 29 weeks after treatment as they were 4 to 8 weeks after
187 treatment. The long time course of this behavioral effect of TMT is
188 consistent with both histopathological (Brown et al., 1979; Dyer et aJ.,
189 1982b; Whittington, Woodruff, & Baisden, 1989) and biochemical (O'Cal-
190 laghan & Miller, 1984) changes in the CNS, which persist many weeks
191 after exposure to TMT. Relatively permanent behavioral effects are not
192 universally observed after TMT. however, as recovery of function in the
193 presence of permanent morphological damage has also been noted (An-
194 gell, Dunn. & Bushnell, 1989; Fountain, Schenk, & Annau, 1985; Paule
195 & McMillan. 1986).
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IMPAIRMENT OF REVERSAL LEARNING BNB3292-13
09OOOI
¦>. ¦
1%
197
198
199
200
201
202
203
204
205
206
207
208
209
210
219
220
221
222
223
224
225
226
227
228
229
230
231
232
233
234
235
236
237
238
239
?40
241
242
243
244
245
246
The delay-dependent learning deficit caused by TMT had two defining
features. First, within the first 100 reversal trials, it was characterized
by a change in asymptotic reversal performance (A), rather than a change
in rate at which the asymptote was reached (R). However, the delay
dependence of the effect extended beyond the session during which a
given reversal was begun. With a 2-s delay, the treated rats' asymptotes
eventually reached control levels in the next (nonreversal) session (data
not shown). With a 4-s delay, the control asymptote was not reached in
the subsequent nonreversal session. The fact that treated rats eventually
acquired reversal criterion with a 2-s delay suggests that learning was
retarded, but not prevented, when the temporal challenge was moderate,
but that increasing the mnemonic load by extending the delay to 4 s
prevented accurate discrimination. This pattern is consistent with TMT-
induced impairment of working memory, since delayed response deficits
in TMT-treated rats are also delay-dependent (Bushnell, 1988a).
Second, the primary cause of reversal impairment was not a reduction
in overall rate of response to the levers or the reduced responding to
the CS* (Figs. 1 and 2, top panels). It appeared rather to involve per-
sistent responding to the CS° at nonzero delays. Such persistence re-
sembles the response perseveration observed in animals with hippocam-
pal damage, e.g., in the radial-arm maze (Olton, Becker, & Handelman,
1979) or in studies of resistance to extinction (Jan-ard & Isaacson, 1965;
Rawlins. Feldon, & Gray. 1980; Rawlins, Feldon, Ursin, & Gray, 1985).
The delay-dependent learning impairment was evident both in the anal-
ysis of group performance, in which average response frequencies and
discrimination ratios were compared, and also in terms of performance
of individual animals. That is, regressions of individual A values against
hippocampal damage were significant only at nonzero delays (Fig. 5),
and were unrelated to changes in P(RFT). Thus evidence of delay-de-
pendent impairment based on group data converged with evidence from
individual animal data, with both analyses pointing to delay as the critical
variable in producing the efTect. The significant relationship between
hippocampal damage and A at nonzero delays supports the contention
that the hippocampus is involved in mediation of the behavior. However,
since the effects of TMT are not restricted to the hippocampus, the
role(s) of other structure(s) in this effect cannot be ruled out.
ACKNOWLEDGMENTS
1 thank D. Dunn. D. New. and J. Farmer for expert technical assistance. and D. A.
Eckerman and D. B. Peele for careful reviews of the manuscript.
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Angell. K. E.. Dunn. D. D.. Sl Bushnell. P. J. (1989, February). Effects of irimethyiiin
on radial maze learning in rats. Paper presented to the North Carolina Chapter, Society
of Toxicology.
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BNB3292-14
PHILIP J. BUSHNELL
.1
MRfid
® oo«
ctcoo<
• «
247 Brown. A. W., Aldridge. W. N., Street. B. W.. & Verschoyle, R. D. (1979). The behavioral
248 and neuropathology sequelae of intoxication by trimethyltin compounds in the rat.
249 American Journal of Pathology. 97, 59-82.
250 Bushnell. P. J. (1988a). Effects of delay, intertrial interval, delay behavior, and trimethyltin
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252 Bushnell, P. J. (1988b). Behavioral effects of acute p-xylene inhalation in rats: Autoshaping,
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255 induced in rats by trimethyltin: Episodic memory and visual discrimination. Toxicol-
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257 Bushnell. P. J., A Evans. H. L. (1984). Trimethyltin-induced memory impairment in
258 nonhuman primates. Federation Proceedings, 43, 763.
259 Cohen. C. A.. Messing. R. B.. A Sparber. S. B. (1987). Selective learning impairment of
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261 chopharmacology, 93, 301-307.
262 Davey. G. C. L.. A Cleland. G. G. (1982). The effect of partial reinforcement on the
263 acquisition and extinction of sign-tracking and goal-tracking in the rat. Bulletin of the
264 Psychonomic Society, 19, 115-118.
265 Dyer. R. S.. Walsh, T. J.. Wonderlin, W. F., A Bercegeay. M. (1982a). The trimethyltin
266 syndrome in rats. NeurobehavioraI Toxicology and Teratology, 4, 127-133.
267 Dyer. R. S.. Deshields, T. L.. A Wonderlin. W. F. (1982b). Trimethyltin-induced changes
268 in gross morphology of the hippocampus. Neurobehavioral Toxicology and Teratology,
269 4. 141-147.
270 Fountain. S. B.. Schenk. D. E.. A Annau. Z. (1985). Serial-pattem-leaming processes
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Neurotoxicology and Teratology, Vol. 10, pp. 237-244. Pergamon Press pic, 1988. Printed in the U.S.A.
0892-0362/88 $3.00 + .00
Effects of Delay, Intertrial Interval,
Delay Behavior and Trimethyltin on
Spatial Delayed Response in Rats1
PHILIP J. BUSHNELL
Neurotoxicology Division, MD-74B, Health Effects Research Laboratory
U.S. Environmental Protection Agency, Research Triangle Park, NC 27711
Received 24 August 1987
BUSHNELL, P. J. Effects of delay, intertrial interval, delay behavior and trimethyltin on spatial delayed response in rats.
NEUROTOXICOL TERATOL 10(3) 237-244, 1988.—Working memory was modeled in rats using a delayed response
task with spatial location as the discriminative cue. Rats received food for pressing 1 of 2 retractable levers in the choice
phase of a trial if that lever had been presented in the prior sample phase of that trial. When delays of 0-20 sec were
imposed between sample and choice, choice accuracy declined with increasing delay. With short intertrial intervals (ITIs),
choice accuracy decreased more at long delays than at short delays, showing that interference from previous trials impaired
memory but not discrimination. Rats emitted overt mediating responses during delay by pressing the levers in the retracted
position. However, the frequency of delay presses was low (<2/trial in all rats) and neither their frequency nor accuracy
was related to choice accuracy. Resetting the delay interval for each delay press did not significantly alter choice response
accuracy. Trimethyltin (TMT), 7 mg/kg IV, reduced the choice accuracy of one rat to chance levels at all delays; two other
rats were affected transiently. TMT reduced choice accuracy during weeks 1 and 4 postinjection, with significant effects on
the linear slope and intercept of the mean retention gradient during week 4. TMT did not affect responses to the retracted
levers during delays. TMT treatment also elevated levels of glial fibrillary acidic protein (GFAP) in the CNS, measured 4
weeks after treatment. Hippocampal GFAP correlated highly with the reduction in choice accuracy during week 1
(r=»-.903) and week 4 (r=»-.797) postTMT. GFAP levels also correlated significantly with TMT-induced changes in the
slopes and intercepts of the retention gradients for individual animals at both times after treatment. The delay and IT1 effects
suggest that the task adequately assessed working memory. The lack of effect of delay presses on choice accuracy suggests
that these overt mediating responses did not differentially affect choice responding. The preferential disruption of choice
accuracy at long delays by TMT indicates that the compound impaired the retention of spatial information over time,
supporting the view that time-dependent processes are particularly vulnerable to this lesion. This spatial delayed response task
thus provides a practical analytical tool for the characterization of memory deficits following exposure to neurotoxicants.
Working memory Cognitive behavior Spatial delayed response Spatial discrimination
Proactive interference Mediating responses Glial fibrillary acidic protein Trimethyltin Rat
1OXICOLOGISTS are becoming increasingly concerned
about cognitive impairment from exposure to neurotoxic
chemicals, since humans frequently report "confusion,"
"memory loss" or "forgetfiilness" after exposure to
neurotoxicants [1,7, 16, 53]; this concern is reflected in the
increasing emphasis on cognitive tasks for field studies of
exposed humans [20], In addition, however, methods are
needed to characterize cognitive dysfunction following
chemical intoxication in animals, to improve our ability to
predict and prevent adverse sequelae of chemical exposures
in humans [32],
The devastation of cognitive adequacy from memory loss
has become increasingly apparent tfith recent advances in
the neurobiology of memory [26, 29, 45, 52]. Conceptually,
this research has documented a multiplicity of memory
types. "Working memory" [22,37] is one such type, and is
defined as that process or series of processes by which fac-
tual information regarding specific, discrete units of experi-
ence is retained. Working memory is of particular interest to
the neurotoxicologist because it is a process which seems to
be common to humans and animals [23, 40, 56] and is sensi-
tive to CNS insults involving the hippocampus and related
structures in both humans [33,34] and animals [37,38,41]. It
therefore provides a useful model system for the
neurotoxicological study of memory.
Discrete-trial operant procedures for quantifying working
memoiy include delayed response [2, 17, 48] and delayed
matching-to-sample [11, 22, 43]. Formally, the procedures
differ in that in a delayed response task, the correct choice
response is completely specified by the predelay sample
¦The research described in this article has been reviewed by the Health Effects Research Laboratory, U.S. Environmental Protection
Agency and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency nor
does mention of trade names or commercial products constitute endorsement or recommendation for use.
237
-------
238
BUSHNELL
stimulus, while in delayed matching, the choice response is
not specified until the response opportunity is actually pro-
vided [21]. However, the low salience of visual cues for ro-
dents [10,49] makes it difficult to train these animals on the
typical delayed matching-to-sample tasks used in primates
and birds. By contrast, delayed response procedures using
position as the discriminative cue can take advantage of the
rat's proclivity to discriminate stimuli on the basis of spatial
location in an operant conditioning environment. A similar
task has been used to evaluate rats' response to anticholin-
ergic drugs and limbic system lesions [12.41].
Advantages of delayed response procedures for the tox-
icologist include the relative ease with which rats acquire
the tasks and the relatively short retention intervals (delays)
at which choice accuracy falls to chance levels. Because of
this rapid forgetting (chance performance is typically ob-
served at delays of 30 to 60 sec), it is possible to obtain
sufficient frequencies of correct and incorrect responses at
each of several retention intervals within a 1- to 2-hr session
to calculate retention gradients with high precision. By con-
trast, typical forgetting times for working memory are on the
order of minutes in a T-maze [46] to hours in the radial-arm
maze [3, 28].
A potential disadvantage of these procedure derives from
the fact that the discriminative stimulus has a specific loca-
tion, thus permitting the subject to exploit positional mediat-
ing responses to bridge the delay interval and facilitate per-
formance. For example, those dogs, rats [24] and monkeys
[17] that learned to position themselves near the location of
the correct response performed accurately at longer delays
than those that did not do so. Pigeons also may use
explicitly-trained mediating responses to bridge the delay,
and thereby increase greatly the length of the delay neces-
sary to degrade accuracy [47]. Because of the interpretive
difficulties provoked by such mediating responses, inves-
tigators have preferred either to design the task such that
mediating responses do not occur (e.g., [2]) or to utilize
matching-to-sample procedures, in which overt positional
mediating responses are not effective.
For the application of such procedures to toxicological re-
search, the important issue is not whether mediating re-
sponses occur, for some sort of mediation is probably inevit-
able, but rather the consistency with which subjects adopt
mediating strategies, and the potential for differential effects
of intoxication upon mediation strategy. However, little is
known about the efficacy of overt response mediation on
performance in rats.
The purposes of the present study were thus to validate a
spatial delayed response (SDR) task as a measure of the
construct of working memory in rats, and to demonstrate the
analytical power of the method by characterizing the effects
of a neurotoxic lesion on working memory. The psycholog-
ical validity of the task was assessed by parametric manipu-
lation of (1) the length of the retention interval, (2) the dura-
tion of the intertrial interval (ITI), and (3) the consequence of
making overt mediating responses during the intratrial delay.
The neurotoxicological efficacy of the procedure was then
evaluated by characterizing the mnemonic effects of
trimethyltin (TMT), a neurotoxicant with well-known effects
on CNS morphology [8,13], chemistry [5,35] and behavior
[27]. A single intravenous dose of 7 mg/kg TMT was selected
because dose-effect functions for TMT are steep [13,27] and
previous work in this laboratory indicated that this dose
produced robust neurochemical changes without serious
debilitation [5,35],
Finally, it was of interest to compare the effects of TMT
on SDR performance with its neurochemical effects in the
CNS, as assessed by increased levels of glial fibrillary acidic
protein (GFAP) [4,14], GFAP levels in the CNS increase
over basal levels in response to cellular injury caused a wide
variety of insults, including TMT intoxication [5.35], in
which damage to the hippocampus is particularly severe.
The severity of CNS damage caused by TMT. as indicated
by GFAP production, was thus compared to the severity of
the TMT-induced behavioral deficit, as assessed by the SDR
procedure.
METHOD
Subjects
The subjects were 16 male Long-Evans rats (Charles
River, Raleigh, NC), 120 days of age at the start of training.
They were maintained at 350 g by dietary restriction
throughout.
Apparatus
The apparatus consisted of 4 standard rat operant condi-
tioning chambers, each equipped with two retractable re-
sponse levers (Coulbourn Instruments, Lancaster, PA), 3.4
cm in width, mounted with inside edges 13 cm apart on one
wall of the chamber. A food cup with a swinging plastic door
(Campden Instruments, via Stoelting Co., Chicago, IL) was
centered between the levers. A microswitch attached to the
door registered nosepokes into the food cup. The levers were
modified to register depression of less than 0.20 N. The lev-
ers in half the chambers were designed such that, when re-
tracted, the front edge of the lever remained movable and
flush with the chamber wall. The rat could thus manipulate
the lever with its nose or paws through 2 mm gaps above and
below the lever. Responses made to these levers were re-
corded independently in the extended and retracted position.
Each chamber was located in a sound-attenuating shell
within which white noise (80 dB SPL, Flat, measured at the
opening of the food cup) was provided. Reinforcers were
45-mg pellets (Bio-Serv, Inc., Frenchtown, NJ). Control of
stimuli and recording of responses were accomplished by
computer (Digital PDP8/a, Maynard, MA) and SKED inter-
face with SUPERSKED software (State Systems, Inc..
Kalamazoo, MI).
Behavioral Training
Rats were first trained to respond for pellets to a single
retractable lever (3 sessions), and then to either of two levers
presented simultaneously (5 sessions). Each lever response
resulted in retraction of both levers and delivery of a single
food pellet. During two-lever training, preferences for par-
ticular levers were noted and corrected as necessary (by
extending only the nonpreferred lever for blocks of 10 trials)
to minimize position bias. In the next phase, a response to a
single lever (sample) were followed by its retraction and,
0.30 sec later, by extension of both levers (choice); a second
response to the sample lever then produced delivery of a
pellet. The rats required about 8-15 100-trial sessions to
reach asymptote (^95% correct responding). Delay intervals
between sample presentation and choice response opportu-
nity were gradually lengthened over the next 12 sessions. To
occupy the rats during the delay period, they were trained
during this phase to make nosepokes (NPs) into the food cup
-------
MEMORY AND TRIMETHYLTIN IN RATS
239
during the delay interval by delivering pellets on a variable
interval (VI) schedule. On this schedule, the first NP made
after the end of the VI (averaging 15 to 30 sec at different
times in training and programmed independently of the delay
interval) yielded delivery of a food pellet. To increase NP
response rates, only those NPs emitted within 2 sec after the
previous NP terminated the VI. Stable performance was ob-
tained after about 10 sessions on this schedule.
SDR Tests
Tests began with a 2-min presession pause in darkness,
with both levers retracted, and no contingencies in effect.
Illumination of the houselight signalled the beginning of the
first intertrial interval (ITI). During training, ITIs were de-
termined by a variable-time (VT) schedule averaging 30 sec.
To reduce the session length and steepen the retention gra-
dients (see below), the ITI was reduced to VT 5 sec begin-
ning 7 days before TMT treatment. Each trial was demar-
cated temporally by an increase in white noise (from 80 to 83
dB), coincident with extension of the sample lever. Right and
left samples were presented on a pseudorandom schedule
with equal probability. A response to the sample lever
caused its retraction and illumination of the food cup light,
which signalled the onset of the VI schedule for food pellets
during the delay period. Upon termination of the delay, both
levers were extended into the chamber: a response to the
lever previously extended as the sample in that trial was
followed immediately by illumination of the food cup light
and delivery of a food pellet. A NP to the food cup then
terminated the trial and reduced the white noise to 80 dB. A
response to the other lever produced a timeout period of 10
sec during which the houselight was extinguished and the
white noise was reduced to 80 dB. Eight delay values were
used, from 0 (actually 0.30 sec, the minimum retraction-
extension time of the levers) to 20 sec. Each session con-
sisted of 120 trials (15 trials at each of 8 delay values; the list
of values was sampled randomly without replacement before
being repeated) and required 30 to 50 min.
The Effect of ITI
The effect of ITI on choice accuracy was determined in all
rats by testing each rat with ITIs of VT 40 sec and VT 5 sec
on successive days, after stable performance had been
achieved on the VT 30 sec ITI. The order of testing was
counterbalanced.
Delay Presses
It was observed that all rats actively and repeatedly ex-
plored the retracted levers during the delay interval. Such
behavior was recorded as "delay presses" for the 8 rats
whose levers registered responses in the retracted position.
Delay press frequency was correlated with choice response
accuracy to determine whether high rates of these mediating
responses were associated with high choice accuracy at any
delay. Next, the distribution of delay presses was analyzed
in relation to their accuracy and the outcome of the trial.
Finally, for the rats whose delay presses could be recorded,
each delay press caused the delay interval to be reset, to
ensure that no delay presses occurred during the ensuing
delay; this contingency then remained in effect for the dura-
tion of the study. The performance of rats whose intervals
were reset was then compared to that of rats whose behavior
toward the retracted levers was not recorded.
Toxicology and Tissue Evaluation
TMT hydroxide (ICN Pharmaceuticals, Plainview, NY) 7
mg/kg (as base), or physiological saline was administered to
8 rats each by tail vein after testing on experimental day (ED)
67. SDR performance was assessed daily for 5 days prior to
treatment; it resumed for 5 days beginning 72 hr postinjec-
tion, and again for 5 days during the fourth week after treat-
ment. A variable ITI averaging 5 sec was used in all of these
tests. On day 28 postinjection (ED 88), all rats were decapi-
tated and their brains quickly removed and hemisected sagit-
tally. From one half brain, the hippocampus was removed,
homogenized in 1% SDS, and assayed for GFAP by slot-
immunobinding on a nitrocellulose matrix according to a
previously described procedure [5,36], Tissue levels of
GFAP were expressed in arbitrary units with control levels
normalized to a mean of unity.
Statistical Analysis
Behavioral data were analyzed by two-way repeated
measures analyses of variance (ANOVA) using the SAS
general linear models procedure [44], with between subject
factors for delay press reset and TMT, and within-subject
factors for ITI and delay. Main effects and interactions in-
volving repeated measures were subject to
-------
240
BUSHNELL
TABLE 1
DELAY PRESS RESPONSES BEFORE AND AFTER TMT
Proportion To
Absolute
Frequency
CR-L
CR-O
NC-L
CR-0
CR-L
NC-0
NC-L
NC-O
PreTMT Baseline
Control
Mean
72.0
0.61
0.07
0.30
0.02
SE
31.9
0.06
0.04
0.06
0.01
TMT
Mean
56.3
0.73
0.00
0.24
0.03
SE
46.1
0.08
0.00
0.08
0.03
Week I PostTMT
Control
Mean
71.3
0.66
0.04
0.21
0.08
SE
27.7
0.07
0.02
0.03
0.06
TMT
Mean
28.8
0.72
0.01
0.25
0.02
SE
11.9
0.07
0.01
0.05
0.02
Week 4 PostTMT
Control
Mean
225.8
0.73
0.04
0.20
0.04
SE
68.4
0.06
0.02
0.02
0.03
TMT
Mean
125.3
0.69
0.02
0.27
0.02
SE
67.8
0.10
0.01
0.08
0.01
Values are mean (±S.E.) frequencies and proportions of total frequencies
categorized by the LEVER (correct lever=CR-L or incorrect lever=NC-L) to which
they are directed on a given trial and by the OUTCOME of the trial (correct
outcome=CR-0 or incorrect outcome=NC-0) determined by the subsequent choice
response on that trial.
other words, 25% of all delay presses were directed to the
correct lever during the delay, yet were then followed by an
incorrect choice at the end of the delay.
Choice accuracy for the & rats whose delays were reset
for each delay press did not differ significantly from choice
accuracy for the 8 rats whose delay behavior had no conse-
quence [main effect of Reset, F(l,14)=0.07, ns; Reset x
Delay interaction, F(7,98)=1.34, ns].
Beginning with the first week after TMT, the performance
of one treated animal (SI4) fell to chance levels at all delays;
subsequent analysis of this rat's GFAP (see below) showed
that it was the most severely affected by TMT. Because of
this qualitatively different response, S14's data were ex-
cluded from further group analyses.
During the first week following TMT administration. SDR
performance was significantly reduced in the treated group
(n=7) compared to controls (Fig, 2). The main effect of TMT
was significant, F(l,13)=5.11, p<0.0417, as was the main
effect of Delay, F(7,91) =*49.58, p <0.0001; the TMT x
Delay interaction was not significant, F(7,91)=2.81,p<0.135.
Linear regression analysis of accuracy versus delay for each
subject (excluding S14) showed that the TMT-treated group
did not differ from control, either in slope (control means:
SE®-1.223:0.21; TMT mean±SE=<-1.76±0.19) or in
y-intercept (control«97.07±0.95; TMT=93.21±2.01).
By the fourth week after treatment, the performance of 2
treated animals (S4 and S6) had recovered, while S14 contin-
100
O ITI» 40"
• ITI" 5'
i
s
3
I
Q
to
H
e
Delay (seconds)
FIG. 1. Effect of changing of the ITI on the retention gradient. Long
ITIs (Random Time 40', open circles) improved performance differ-
entially at long delays compared to short ITIs (Random Time 5",
closed circles). Values are means+SEM.
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MEMORY AND TRIMETHYLTIN IN RATS
241
100
2 90
o
u
o
(0
b.
3
o
u
<
C
(A
+l
4>
2
so
70
60
SO
• Salln* (n - 8)
O TMT (n ¦ 7)
0 1 2 4 8 12
Delay (seconds)
18
20
FIG. 2. Effect of trimethyltin (TMT) hydroxide, 7 mg/kg IV, on the
retention gradient during the first week after treatment. Values are
means±SEM for controls (closed circles, n»8) and experimentals
excluding S14 (n=7; see text).
100
90
o
<
Gj
tn
+i
e
<0
0)
a
80
70
80
SO
• Salln* (n » 8)
O TMT (n » 5)
A S«4
V S#8
_l I I U
0 1 2 4 8 12
Delay (seconds)
18
20
FIG. 3. Effect of trimethyltin (TMT) hydroxide, 7 mg/kg IV, on the
retention gradient during week 4 after treatment. Values are
means mS EM for all controls (closed circles, n=8) and experi-
mentals whose GFAP levels remained elevated after TMT (n =5). The
performance of 2 treated animals whose GFAP levels were not ele-
vated by TMT are shown by the dotted lines at the top of the figure.
ued to score at chance accuracy at all delays. Subsequent
biochemical analysis (see below) showed that S4 and .'6 had
GFAP levels within the range of the controls. Comparison of
the SDR performance of the remaining experimental rats
(n=5) with control (Fig. 3) showed significant divergence of
the retention gradients, as shown by significant effects of
TMT, F(l, 11)= 11.48, p<0.0061, Delay, F(7,77)-45.82,
p<0.0001, and the TMT x Delay interaction, F(7,77)«3.89,
p<0.028. Post hoc paired /-tests showed that the TMT-
treated rats performed less accurately than controls at all
delays except 0 and 1 sec. Linear regression analysis as for
week 1 data showed significant changes in both slope [con-
trol (n»8): -1.05±0.17; TMT (n-5): -1.75±0.25; f(ll) =
2.32, p<0.05] and y-intercept [control: 96.82 ±0.94; TMT:
88.93 ±3.00, f(ll)-2.51,p<0.05].
GFAP levels in hippocampus were significantly,
r( 14)=4.89, p <0.001, elevated by TMT exposure, to 189.5
±16.7% of control (100.0±7.5%) at sacrifice at the end of
week 4 postii\jection. The behavioral and biochemical effects
of TMT were compared by correlating separately the change
in choice accuracy during weeks 1 and 4 after TMT
(AA=accuracy averaged across all delays posttreatment -
accuracy during a 5-day pretreatment baseline) for each
animal with its GFAP level at sacrifice. (All TMT-treated
rats were included in this analysis.) The correlation between
AA during week 1 postiqjection and hippocampal GFAP
(Fig. 4, left panel) was highly significant, r(14)«-0.903,
p<0.0001, and did not improve, r(14)--.797, p<0.00l,
when week 4 accuracy scores were used in place of week 1
accuracy scores (Fig. 4, right panel).
To characterize further the relationship between GFAP
elevation and the behavioral changes induced by TMT,
slopes and intercepts of the delay gradients were calculated
by linear regression analysis for each subject before and after
treatment with TMT or saline. Difference scores (AS and AI)
for the change in slope and intercept were then computed for
weeks 1 and 4 after TMT, and regressed against GFAP.
Since S14's performance was too variable to show a gra-
dient, its data were not included in calculating the regression
equations; S4 and S6 were, however, included. Significant
negative correlations between GFAP and AS, and GFAP and
Al, were obtained for both weeks of testing. Regression pa-
rameters for the AS analyses were: for Week 1, AS--0.0125
(GFAP)+0.0156, r(13)--.6914, p<0.01; for Week 4,
AS-~0.147(GFAP)+0.0203, r( 13)-0.6093, p<0.03. Param-
eters for the AI analyses were: for Week 1, AI-
= -0.0602(GFAP)+0.0599, r(13)=.7179, p<0.0l; for Week
4, AI--0.0691(GFAP)+0.0716, r(13)=.6461, p<0.01.
Finally, the frequency but not the distribution of delay
press responses were reduced by TMT (Table 1). However,
the reduction in frequency in the TMT group was not statis-
tically significant, since it was due to suppressed responding
in just one of the 4 animals.
DISCUSSION
The results of the present studies illustrate the validity of
the spatial delayed response (SDR) procedure as a means (1)
to quantify working memory and (2) to characterize the ef-
fects of toxicant-induced CNS damage on working memory
in rats. Validation of this technique was carried out in both
psychological and toxicologicai domains, first by demon-
strating that the task quantified the construct of interest
(working memory), and second by demonstrating that a
known limbic system neurotoxicant impaired working mem-
ory processes. Third, it was shown that this memory im-
pairment occurred in proportion to an increase in GFAP, a
biochemical marker of CNS damage.
The delay-dependent decrement in choice accuracy 4
weeks after TMT (Fig. 3) suggests that the treatment in-
-------
242
BUSHNELL
20
Week 1
r=> -0.903
\#
• \
1 1
Week 4
a
•
•
\° r= -0.797
°\
-
° \
1
SI4 \
, . 1 ^
1.0 2.0 3.0 *.0 0 1.0 2.0 3.0 4.0
Hlppocampal GFAP Level (arbitrary units)
FIG. 4. Correlation between biochemical and behavioral effects of trimethyltin
hydroxide (TMT). The change in overall choice accuracy (postTMT score minus
baseline score) is plotted for controls (closed circles) and TMT-treated animals
(open circles) against GFAP levels in hippocampus during week I (left panel.
r=-.903) and during week 4 postTMT (right panel, r=-,797), S14 was a treated
animal whose accuracy fell to chance at all delays; his data were included in these
analyses (see text).
creased the rats1 sensitivity to temporal delays. This effect is
consistent with other findings of time-dependent behavioral
changes after TMT. on long-delay flavor aversion condition-
ing [42], passive avoidance conditioning [25,50), DRL
schedules [30,54], autoshaping with delayed reinforcement
[9,31] and delayed matching-to-sample in monkeys [6,15].
However, this report provides the first clear evidence of
delay-dependent effects of TMT on working memory in rats.
Similar to the effects of TMT, shortening the ITI de-
graded choice accuracy more at long delays than at short
delays (Fig. 1). This effect showed that the rate at which
trials were delivered affected response accuracy. Such
changes are thought to result from interference from residual
attributes of previous trial(s) [19, 39, 55]. This proactive in-
interference (PI) here affected the retention of information
over time, as indicated by the significantly greater decrease
in accuracy across delays (slope) at the short ITI compared
to the long ITI [21]. This effect is consistent with reports of a
direct relationship between accuracy of memory and length
of the ITI, which may [ 18,55] or may not [ 19,39] be reflected
in changes in retention per se.
In contrast to the significant change in slope of the reten-
tion gradient caused by shortening the ITI, the 0-delay inter-
cepts of the two curves were indistinguishable (Fig. t).
The intercept of a retention gradient may be interpreted to
reflect the accuracy of encoding the information to be re-
tained over the delay [21]. In this case, shortening the ITI did
not interfere with stimulus encoding.
The fact that both TMT and shortening the ITI steepened
the retention gradient in these animals suggests the
possibility that the treated animals were more sensitive to PI.
The effects of TMT were not evaluated at different ITIs in
this study; however, unpublished results of a replicate study
showed that TMT treatment did not interact with ITI dura-
tion, suggesting that the TMT-induced decrement in reten-
tion was not due to increased sensitivity to PI.
TMT was used as a positive control neurotoxicant be-
cause of its well-known effects on CNS morphology [8.131.
chemistry [5,35] and behavior (reviewed in [27]), including
working memory [6, 15, 51], Both the biochemical and be-
havioral responses to TMT were somewhat variable, due in
part to the steep dose-effect functions it generates for these
endpoints [13,27]. This broad range of response, from com-
plete loss of discrimination capacity and large increase in
GFAP (S14), to behavior and biochemistry indistinguishable
from control (S4 and S6), was obtained after a single IV dose
of 7 mg/kg TMT. [The recovery of S4 and S6 in fact resulted
in an increase in choice accuracy over baseline during week
4 postTMT (Fig. 4). This improvement paralleled that of con-
trols, and likely reflects the gradual upward shift in baseline
which occurs with extended training in this task.] The
remaining 5 treated animals formed a group with intermediate
degrees of change on both measures. Clearly this dose of TMT
lies at or near its EDM for both endpoints (Fig. 4).
The behavioral response to TMT during week I after
treatment, considered as the group mean response for all
TMT-treated rats excluding S14 (Fig. 2), did not permit dif-
ferentiation between encoding and retention deficits in work-
ing memory. That is, the curves are statistically parallel, yet
the groups responded identically at zero delay. Regression
analysis revealed changes in neither slope nor intercept at
this time. The effect of TMT during week 1 may thus be
conservatively interpreted as a degradation in overall accu-
racy, without clear effects on either encoding or retention.
The 5 moderately-affected rats showed a reduction in
overall accuracy during week 4 after TMT (Fig. 3) similar to
that seen during week 1 after TMT. In addition, regression
analysis of the retention gradients showed both a signifi-
cantly steeper slope and reduced intercept in the TMT group
compared to controls. Thus, the responses of these 5 animals
to TMT during week 4 can be interpreted as a decrement in
encoding with an additional decrease in the retention of in-
formation over time. The change in the quality of the behav-
ioral impairment from week I to 4 is not inconsistent with the
-------
MEMORY AND TRIMETHYLTIN IN RATS
243
long time course of the action of TMT on the CNS [13,27]. It
may be important that the change in retention did not appear
until week 4, when biochemical and morphological changes
in the CNS are more pronounced.
The correlation of the TMT-induced change in overall
choice accuracy with hippocampal GFAP, a biochemical
marker of CNS insult (Fig. 4), demonstrates the close rela-
tionship between hippocampal damage and impaired per-
formance on problems requiring discrimination of spatial
cues and their retention over time [12,38,41]. It is intriguing
that the correlation between the change in overall accuracy,
AA, and week 4 GFAP was better using week 1 AA than using
week 4 AA. This difference suggests that the biochemical
and behavioral responses caused by TMT followed different
time courses. It appeared here that the biochemical change
emerged more slowly, since the immediate behavioral re-
sponse was more highly correlated with the later increase in
GFAP.
Regression analyses of the changes in slope, AS, and
intercept, AI, showed that the increase in hippocampal
GFAP level predicted changes in these two parameters
equally well, with hippocampal GFAP accounting for about
50% of the variance in AS and AI at both time points after
TMT. This result suggests that both encoding and retention
are equally related to hippocampal GFAP, and thus
presumably to the functional integrity of the hippocampus.
However, it must also be noted that TMT causes widespread
damage to the CNS [8,35], and that this evidence alone does
not implicate any particular structure in the behavioral im-
pairment observed.
The animals' use of mediating responses during the delay
posed a potential interpretive difficulty for the SDR task.
However, the class of mediating responses represented by
delay presses was not closely related to choice accuracy.
First, the frequency of delay presses predicted neither over-
all choice accuracy nor accuracy at any selected delay. Sec-
ond, while all rats emitted delay presses with high (^90%)
accuracy, such accuracy during the delay was not reflected
in choice accuracy after the delay (Table 1). Third, causing
each delay press to reset the delay interval did not signifi-
cantly affect choice accuracy. Finally, neither the frequency
nor the distribution of delay presses was affected signifi-
cantly by TMT (Table 1), despite clear and reliable disrup-
tion of choice accuracy by the compound (Figs. 2-4). It must
be concluded that delay presses did not contribute to the
accuracy of performance on this task, and that the disruption
of memory by TMT occurred by some means not involving
delay presses.
In conclusion, this study demonstrates the efficacy of the
SDR procedure for the study of working memory in rats. The
task represents a powerful tool for the behavioral analysis of
memory impairment due to chemical intoxication, as it per-
mits quantification of, and differentiation between, two
working memory processes. It is relatively easy to train and
can be administered in daily tests of 1 to 2 hr. It thus pro-
vides a quantitative method, practical for the toxicologist to
implement, for analyzing an important human cognitive
process in a widely-used experimental animal. It should
facilitate identification and characterization of chemicals
with potentially detrimental effects on human cognitive
health.
ACKNOWLEDGEMENTS
I thank Dusty Dunn for expert animal handling and assistance in
software development for the behavioral procedures, Dr. J. P.
O'Catlaghan for the TMT dosing parameters and GFAP analyses,
and Drs. D. A. Eckerman, R. C. MacPhail, D. B. Peele and M.
-Stanton for advice and reviews of the manuscript.
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Neurobehavioral Toxicology and Teratology, Vol. 5, pp. 443-459, 1983. 0 Ankho International Inc. Printed in the U.S.A.
A Time-Course Study of Trimethyltin
Induced Neuropathology in Rats
LOUIS W. CHANG
Departments of Pathology and Pharmacology/Toxicology
University of Arkansas for Medical Sciences, Little Rock, AR 72205
AND
ROBERT S. DYER
Division of Neurotoxicology, EPA Health Effects Laboratory
Research Triangle Park, NC 27711
Received 4 May 1983
CHANG, L. W. AND R. S. DYER. A time-course study of trimethyltin induced neuropathology in rats. NEUROBEHAV
TOXICOL TERATOL 5(4)443-459, 1983.—The extent and time course of trimethyltin (TMT) induced lesions in the rat
nervous system was studied. Adult Long-Evans rats were treated with a single dose of 6.0 mg TMT/kg b.w. and were
sacrificed at 8 hours. 3, 15. 30 or 60 days following exposure. Histopathological examination of the brains revealed that
neurons in the pyriform/entorhinal cortices and in the olfactory tubercle were extremely sensitive to TMT toxicity showing
significant necrotic changes 3 days after exposure. Extensive neuronal loss and astrogliosis were observed 15 days after
intoxication. In the hippocampal formation, the granule cells of fascia dentata showed early changes; however, evidence of
toxic destruction in this area was not extensive and subsided considerably by the 3rd week of intoxication. On the other
hand, destruction of the pyramidal neurons in Ammon's horn became more pronounced throughout the experimental
period, resulting in extensive cell loss in this structure. It is interesting to note that the CA, neurons in the anteromedial
(septal) portion of the Ammon's horn appeared to be more vulnerable than those located more laterally (temporally), while
the reverse pattern was observed for the CA,., neurons. Large mesencephalic trigeminal neurons in the brain stem also
showed early chromatolytic changes. Since total normal morphology of these neurons was observed at later posttreatment
time without apparent neuronal loss in these areas, total recovery of these nerve cells could be postulated.
Trimethyltin (TMT) Neuropathology TMT induced lesions
ORGANOTIN compounds have been used as stabilizers of
plastic, as chemosterilants, and as fungicides [15]. Recent
investigations have demonstrated that organotin com-
pounds, such as trimethyltin (TMT) and triethyltin (TET),
are potent neurotoxicantt producing rapid and extensive
damage to the central nervous system [12], TMT compounds
are especially toxic to neurons, producing neuronal degen-
eration and necrosis in the limbic system, including the hip-
pocampal formation, pyriform and entorhinal cortices, olfac-
tory tubercle, and amygdaloid nucleus [1, 2, 5, 7, 8, 10],
Most of the previous studies have focused on the
neurotoxic effects of TMT at a fixed time foBowing treat-
ment. The present investigation was designed to document
the progression of pathological changes in the central nerv-
ous system at various time points after administration of
TMT.
METHOD
Young adult male Long-Evans hooded rats (60-80 days
old) were used in the experiment. Animals were exposed to
trimethyltin chloride via oral intubation in a dose of 6.0 mg
TMT/kg b.w. (or 7.5 mg TMT-CI/kg b.w.). Animals were
then sacrificed in groups of five animals at 8 hours, 3 days, 15
days, 30 days, and 60 days following exposure. The saline
vehicle was administered to the control animals.
At sacrifice, animals were anesthetized and perfused
intracardially with saline solution followed by 2.5% buffered
glutaraldehyde. Brains were then removed surgically and
further immersion-fixed in a solution of 10% buffered for-
malin for at least 48 hours before tissue processing. After
dehydration in graded ethanols and embedding in Paraplast.
tissues were cut at 6 n and stained with hematoxylin-eosin
(H & E) for general histopathological examination. For
demonstration of astroglial cells and thin fibers, Holzer's
method was employed [3].
For comparative purposes, the extent of neuropathologi-
cal involvement in specific loci at each time point was rated
between - to + + + + . All sections compared were from ap-
proximately the same brain levels (Diagram 1) and were
rated by the same investigator under blind conditions.
RESULTS
Pathological lesions were observed in the hippocampal
formation (Ammon's horn and dentate gyrus), pyriform and
443
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444
CHANG AND DYER
FIG. I. Hippocampal formation, control. The fascia dentata (fd) is composed of small
granule cells. The Ammon's horn is composed of pyramidal neurons and is subdivided into
fields CA,,„ CA„,bl CA*. and CA4. H & E. x 100.
C —
B
DIAGRAM I. Approximate levels of sectioning for histopathology
comparison. A-B, parasagittal section through midline of olfactory
bubl; C-D, coronal section through the infundibulum of the
hypophysis at the base of the brain.
entorhinal cortices, olfactory tubercle, and brain stem. The
pathological changes observed were consistent over several
animals and were believed to be representative lesions in-
duced by TMT. The time-course of lesion development and
progression in each specific locus may be described as fol-
lows:
Hippocampal Formation
The hippocampal formation is composed of two main
types of cells; the small granule cells of the dentate gyri
(fascia dentata) and the larger pyramidal neurons of the
Ammon's horn. The Ammon's hom pyramidal cell layer is
further divided into fields of CA, to CA«. In our description,
the classification and terminology of Lorente de No [13] will
be followed (Fig. 1).
Although no pathological lesions were observed 8 hours
after intoxication, cellular necrosis was observed among the
granule cells in the fascia dentata 3 days after treatment
(Figs. 2 and 3). Neuronal changes in Ammon's horn were
minimal at this time. While no significant change was found
among the pyramidal neurons in the CA3,.b, neuronal swel-
ling in CAj,. (Fig. 4) and isolated neuronal necrosis in CA,,;.
and in CA4 (Fig. 5) were observed.
Between 15 and 30 days after intoxication, some neuronal
necrosis in the fascia dentata was still evident (Fig. 6). De-
struction of pyramidal neurons was very prominent at this
time, with extensive cell loss in field CA,,2 and CA,c of the
Ammon's horn (Figs. 7-9) resulting in significant thinning
(Fig. 10) and gliosis (Fig. 11) of CA,j. This change was
especially notable in the lateral (temporal) portion of Am-
mon's horn. Extensive destruction of CAjc and CA« (Figs. 7
and 12) with proliferation of astroglial cells (Figs. 12 and 13)
was also evident at this time point. It is of interest to note
that the anteromedial (septal) portion of CA1a.„ (as seen in
medial parasagittal sections) appeared to be more vulnerable
(Fig. 14) than more lateral (temporal) portions (as seen in
coronal sections) (Figs. 7 and 15). Although no significant
lesion was observed in the subiculum at this time period,
proliferation of gemistocytic (plump) astrocytes (Figs. 16 and
17) was observed in this area.
-------
trimethyltin induced neuropathology
445
FIG. 2. Fascia dentata. TMT. 3 days. Scattered neuronal necrosis among the granule cells
was observed. Swelling and necrotic changes in some of the neurons in CA, were also
evident. H & E. x250.
FIG. 3. Fascia dentata. TMT. 3 days. Higher magnification demonstrates pyknosis
(chromatin condensation) and karyorrhexis (nuclear fragmentation) of the necrotic granule
cells. H & E. x450.
By day 60, most of the necrotic activity in the hippocam-
pal formation had subsided. Although the fascia dentata re-
sumed its normal appearance, total absence of the septal
portion of CA3a.i, and severe thinning in other portions of
Ammon's horn were observed.
Pyriform and Entorhinal Cortices
Both the pyriform and entorhinal cortex are composed of
a layer of small neurons, usually 4-5 cells thick (Fig. 18).
Since the overall pathological response to TMT toxicity was
similar in these two cortical areas, they will be described
together.
Isolated neuronal necrosis was evident as early as 3 days
after the intoxication (Fig. 19). By days 15 and 30. extensive
neuronal destruction had taken place (Figs. 20 and 21). By 60
days after intoxication, severe cell loss and thinning of the
cortical layer were observed (Fig. 22).
Olfactory Tubercle
The olfactory tubercle in rats is composed of a layer of
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446
CHANG AND DYER
FIG. 4. Hippocampal formation, TMT, 3 days. Swelling of the neurons in field CAV was
observed. H & E. x450.
FIG. 3. Hippocampal formation, TMT, 3 days. Necrotic changes (-») in some of the CA,
neurons were evident, fd, fascia dentata. H & E. x450.
small neurons lining the basal portion of the brain (Figs. 23
and 24).
Only isolated neuronal necrosis was observed in this area
of the brain 3 days after the intoxication. By day 15 however,
extensive destruction of these neurons occurred (Fig. 25)
resulting in severe cell loss at later periods following intoxi-
cation.
Brain Stem
Neuronal changes in the brain stem occurred as early as 3
days after intoxication. Extensive chromatolysis occurred in
many large brain stem neurons, particularly those in the
mesencephalic trigeminal nuclei. Many of these neurons ac-
quired an eosinophilic cytoplasm and complete loss of their
basophilic Nissl substance (Figs. 26-28). These chromatoly-
tic changes were transient, and the brain stem returned to
normal appearance by the 15th day following intoxication.
The overall progression of pathological lesions with time
is summarized in Table 1. Since the extent of lesion may
vary, particularly in the hippocampus [4], depending on the
level and plane of section, all the comparisons of hippocam-
-------
TRIMETHYLTIN INDUCED NEUROPATHOLOGY
447
FIG. 6. Fascia dentata. TMT. 15 days. Scattered neuronal necrosis was still evident among
the granule cells. H & E. x450.
FIG. 7, Hippocampal formation, TMT, 15 days. Extensive destruction of the pyramidal
neurons, particularly those in fields CA,.j, CA*, and CA„ were observed leading to exten-
sive neuronal loss in these areas of the Ammon's horn. Coronal section. H & E. x 100.
pal lesions were made with parasagittal sections at approx-
imately the same level of cut. Moreover, since the dorsal
hippocampus is the major hippocampal structure in rodents,
only lesions in the dorsal hippocampus were evaluated and
compared.
DISCUSSION
Trimethyltin (TMT) compounds have been shown to be
extremely neurotoxic inducing pathological changes in the
central nervous system of rats and mice [1, 2, 5-10]. Al-
though the primary pathological changes were found in the
limbic system (hippocampal formation, pyriform and
entorhinal cortices, and olfactory tubercle), extra-limbic le-
sions including those in the spinal cord, spinal ganglia, and
brain stem have been observed in mice [6,9]. In addition to
exploring further the distribution of pathology in the brain of
rats poisoned with TMT, the present study also demon-
strates development and progression of the lesion.
Within the hippocampal formation, it appeared that al-
though the granule cells had an earlier onset of lesion devel-
opment, the toxic impact on these cells subsided with time
-------
448
CHANG AND DYER
• .fKs'te,«>' • vi*Vi'.V '.^Eg
• *• • • • A •„ i •
•« - * JV'anN
FIG. 8. Ammon's horn, field CA,.„ TMT. 15 days. Significant thinning of CA,.2 occurred as
a result of extensive neuronal loss. H & E. x250.
FIG. 9. Ammon's horn, field CA,.,, TMT, 15 days. Extensive neuronal necrosis (-~) was
observed among the pyramidal cells in this portion of the Ammon's horn. H & E. x450.
while the damage in the Ammon's horn pyramidal neurons
was more extensive at later periods following intoxication.
Among the Amnion* horn pyramidal cells, the sensitivity
appeared to be CA* > CA3i,b a CA,,t. It is interesting to
note that within Ammon's horn, a particular cell field does
not display uniform susceptibility along its septo-temporal
axis. For example, CAS showed earlier and more prominent
degeneration in more septally located segments, while the
CAt,j neurons appeared to be more affected in temporally
located segments. The precise reason for this pattern of de-
generation is still uncertain and deserves further investiga-
tion.
Under the present conditions, injury to the granule cells
of the fascia dentata appeared limited. Continued damage to
the pyramidal neurons of Ammon's horn proceeded
throughout the experimental period, leading to severe dam-
age and thinning as well as extensive astrogliosis of the area.
Pathologic damage similar in extent to that noted in the hip-
pocampus was observed in neurons of the pynform and
entorhinal cortices as well as those of the olfactory tuber-
-------
trimethyltin induced neuropathology
449
~P .4 ^ w
^ ¦ - i. ^ J. ^ •-' "-v*l-> '
•i * 1 ., N*:• in
^r *
10 <
FIG. 10. Ammcm's horn, field CA,.2, TMT, 15 days. Because of the extensive neuronal loss
in the CA, .. some portion of this cell layer appeared to be only one cell thick (-•). H & E.
x450.
FIG. 11. Amnion's horn, field CA,.,. TMT, 15 days. Proliferation of astroglial fibers (astrog-
liosis) (-•) was demonstrated with a glial stain. Holzer's stain. x450.
cle. The observation of damage to neurons in the pyriform
and entorhinal cortex and olfactory tubercle, areas impli-
cated in olfaction as well as many other functions, suggests
the possibility that the sense of smell might be disturbed after
TMT exposure. Since other authors have reported early
changes in auditory and visual function after TMT [11. 14,
16], our findings add support to the idea that damage to
sensory systems may be a prominent feature of TMT toxic-
ity. Obviously, TMT's effects are not restricted to sensory
areas, and the basis of its selectivity is not known.
Damage to brain stem neurons has been observed in mice
after TMT administration [6]. Neurons in the mesencephalic
trigeminal nuclei and median raphe nuclei were reported to
be most prominently affected. At light microscopic level,
these neurons acquired a chromatolytic appearance with ec-
centric nuclei and Nissl negative cytoplasm. Vacuolar
changes of the cytoplasm also occurred. Electron micro-
scopic study revealed extensive intracellular edema of these
nerve cells with dilation and destruction of the cytoplasmic
membranes [6]. In the present study, similar histopathologi-
-------
450
CHANG AND DYER
FIG. 12. Ammon's horn, fields CAnc and CA4, TMT, 15 days. Extensive destruction of
neurons in fields CAlc and CA, together with astrogliosis in the hilus area were observed. H
& E. x 250.
FIG. 13. Ammon's horn, field CAV. TMT, 15 days. Glial stain (Holzer) demonstrated
extensive proliferation of astrocytic fibers (astrogliosis) in the hilus area of the hippocampus.
Holzer stain. x250.
-------
trimethyltin induced neuropathology
FIG. 14. Hippocampal formation. TMT. 30 days. Medial parasagittal section demonstrated
severe cell loss in the field CA, of the anteromedial (septal) portion of the hippocampus. H &
E. x100.
FIG. 15. Hippocampal formation. TMT, 30 days. Coronal section demonstrated extensive
destruction of neurons in CA, , and CAV- Neurons in field CA1a.b at the lateral (temporal)
portion of the hippocampus appeared to be relatively spared. H & E. x 100.
-------
452
CHANG AND DYEf
FIG. 16. Subiculum, TMT. 30 days. No remarkable neuronal destruction was observed in
the subiculum of the brain. However, proliferation of large gemistocytic (plump or stuffed)
astrocytes (-*) in this area were evident. H & E. x 100.
FIG. 17. Subiculum, TMT, 30 days. Proliferation of gemistocytic (plump) astrocytes in this
area of the brain. These astrocytes appeared to be large with hypertrophic and highly
eosinophilic cytoplasm. H & E. x450.
-------
trimethyltin induced neuropathology
' f <«
( • •
• * . «• «" *¦* ' ' .•
•T • » ' • **•
• ¦ • I » •
FIG. 18. Pyriform conex. control. The normal pyriform cortex is composed of a 4-5 cell
thick layer of small neurons. H & E. x2J0.
FIG. 19. Pyriform cortex, TMT, 3 days. Isolated neuronal necrosis (-») was observed. H &
E. x450.
-------
454
CHANG AND DYEF
FIG. 20. Pyriform cortex. TMT, 15 days. Extensive neuronal destruction occurred leadino
to a significant reduction of cortical neurons and gliosis. A thin neuronal layer (-.) still could
be identified. H & E. x450.
FIG. 21. Pyriform cortex, TMT, 30 days. Extensive neuronal loss and gliosis were observed
in this area of the brain. H & E. x450.
-------
TRIMETHYLTIN induced neuropathology
F!G. 22. Pynform conex. TMT. 60 days. Marked thinning and destruction of the pyriform
cortex were evident. H & E. x 100.
| 23
FIG. 23. Olfactory tubercle, control. The olfactory tubercle is composed of a 6-8 cell thick
layer of small neurons. H & E. x 100.
-------
456
CHANG AND DYE
FIG. 24. Olfactory tubercle, control. Small granule cell-like neurons of the olfactory cortex
in rat. H & E. x450.
FIG. 25. Olfactory tubercle, TMT. 15 days. Extensive destruction and gliosis of the olfac-
tory tubercle. The general cytoarchitecture of this cortex was totally obliterated by neuronal
loss and by the proliferation of astroglial cells. H & E. x250.
-------
TRIMETHYLTIN induced neuropathology
FIG. 26. Mesencephalic trigeminal nucleus, brain stem, control. The mesencephalic trigem-
inal nucleus in the brain stem is composed of large ganglion-like neurons with large, round
nuclei and prominent Nissl substances. H & E. x450.
FIG. 27. Mesencephalic trigeminal nucleus (MTN), brain stem, TMT, 3 days. Marked
chromatolytic changes were observed in the MTN neurons. H & E. x 100.
-------
458
CHANG AND DYER
FIG. 28. Mesencephalic trigeminal nucleus, brain stem, TMT. 3 days. Many neurons ap-
peared to be chromatolytic with total loss of Nissl substance. Microvacuoles within the
cytoplasm of some of these neurons (-•) also provided a foamy appearance to these nerve
cells. H & E. X450.
TABLE 1
TIME COURSE STUDY OF TMT HISTOPATHOLOGY (L-E RATS)
(6.0 mg TMT/kg B.W )
Hippocampus*
CA, j CA„.„ CAm f.d. p.c./e.c. o.t. b.s.
8 hours
3 days
15 days
30 days
60 days
+ +
(cell loss
& gliosis)
+ + +
(extensive
cell loss
& gliosis)
+•+ +
(extensive
cell loss
+ +
(cell loss)
(cell loss)
+ + + +
(extensive
(cell loss)
+ + +
(cell loss
& gliosis)
+ + + +
(extensive
cell loss
& gliosis)
+ + + +
(extensive
cell loss)
+ + +
(gliosis)
+ + + +
(extensive
cell loss
& gliosis)
++ + + -
(extensive
cell loss)
+ +
(gliosis)
+ 4- +
(extensive
cell loss
& gliosis)
+ + +
(cell loss)
•Comparison based on medial parasagittal sections of dorsal hippocampus (anteromedial septal portion) at approx-
imately the same levels of the brain. Extent and severity of lesions were rated from - to + + + + , with - being no
observable lesion and + + + + being very extensive involvement,
f.d., fascia dentata; o.t., olfactory tubercle; p.c./e.c., pyriform/entorhinal cortices; s.c., spinal cord; b.s., brain stem.
cal changes were observed in the large brain stem neurons,
particularly those in the mesencephalic trigeminal nuclei.
However, vacuolar degeneration of these neurons was min-
imal and the chromatolytic condition appeared to be only
transient. By two weeks after intoxication, most of the
neurons in this area appeared to have normal appearance.
Since there was no apparent reduction of neuronal number in
these areas, neuronal recovery from early changes could be
postulated. It has been indicated in our earlier study that
these neuronal changes were largely a result of neuronal
edema and were potentially reversible.
In addition to providing a time course for lesion progres-
sion in certain affected areas of the brain as a result of TMT
exposure, the present investigation also revealed that the
-------
trimethyltin induced neuropathology
459
toxic effects of TMT varied depending on the neurons in-
volved. While only transient changes (e.g.. chromatolytic
changes in the brain stem neurons) or limited damage (e.g.,
granule cell layer of fascia dentata) occurred in some brain
structures, extensive and progressive destruction occurred
in other areas of the brain (e.g.. Ammon's horn, pyriform/en-
torhinal cortices and olfactory tubercle). Further investiga-
tion on the basis for the varied response of these neurons to
TMT may elucidate the pathogenetic mechanism of this po-
tent neurotoxicant. It must be emphasized that the present
observations are based only on one strain of rat. Since there
are strain and species differences in TMT toxicity [10], data
comparisons should be exercised with caution.
ACKNOWLEDGMENT
The authors wish to express their thanks to Ms. Patty Webb for
her able technical assistance and Mrs. Laurie McDonald for her
excellent help in the preparation of the manuscript. The information
in this document has been funded wholly or in part by the United
States Environmental Protection Agency under assistance agree-
ment number R 809360-01 to Dr. L. W. Chang. It has been reviewed
by the Health Effects Research Laboratory. U.S. Environmental
Protection Agency, and approved for publication. Mention of trade
names or commercial products does not constitute endorsement or
recommendation for use.
REFERENCES
1. Bouldin. T. W.. N. D. Goines. C. R. Bagnell and M. R. King-
man. Pathogenesis of trimethyltin neuronal activity, ultrastruc-
tura) and cytochemical observations. Am J Pathol 104: 237-249,
1981.
2. Brown. A. W.. W. N. Aldridge. B. W. Street and R. D. Ver-
schoyle. The behavioral and neuropathologic sequelae of in-
toxication by trimethyltin compounds in the rat. Am J Ptithol 97:
59-81. 1979.
3. Chang. L. W. A Color Atlas fur Applied Histochemistry,
Springfield. IL: Charles C. Thomas. 1979.
4. Chang. L. W. and R. S. Oyer. Topographical development of
lesions in the hippocampus of rat intoxicated with trimethyltin.
J Netiropathol Exp Neurol 42: 316, 1983.
5. Chang, L. W., T. M. Tiemeyer, 0. R. Wengerand D. E. McMil-
lan. Neuropathology of mouse hippocampus in acute trimethyl-
tin intoxication. Neurobehav Toxicol Teratol 4: 149-156, 1982.
6. Chang, L. W., T. M. Tiemeyer. G. R. Wengerand D. E. McMil-
lan. Neuropathology of trimethyltin intoxication. III. Changes
in the brain stem neurons. Environ Res 30: 399-411, 1983.
7. Chang, L. W.. T. M, Tiemeyer. G. R. Wenger. D. E. McMillan
and K. R. Reuht. Neuropathology of trimethyltin intoxication.
I. Light microscopy study. Environ Res 29s 435-444, 1982.
8. Chang. L. W., T. M. Tiemeyer. G. R. Wenger, D. E. McMillan
and K. R. Reuhl. Neuropathology of trimethyltin intoxication.
II. Electron microscopy study of the hippocampus. Environ Res
29: 445-458, 1982.
9. Chang, L. W., G. R. Wenger and D. E. McMillan.
Neuropathology of trimethyltin intoxication. IV. Changes in the
spinal cord. Environ Res, in press. 1983.
10. Chang, L. W.. G. R. Wenger. D. E. McMillan and R. S. Dyer.
Species and strain comparison of acute neurotoxic effects of
trimethyltin in mice and rats. Neurobehav Toxicol Terutol 5:
337-350, 1983.
11. Dyer, R. S., W. E. Howell and W. F. Wonderlin. Visual system
dysfunction following acute trimethyltin exposure in rats.
Neurobehav Toxicol Teratol 4: 191-196, 1982.
12. Dyer, R. S., T. J. Walsh. H. S. Swartzwelder and M. J. Wagner
(eds.) Neurotoxicology of the alkyltins. Neurobehav Toxicol
Teratol 4: 125-278. 1982.
13. Lorente de N6, R. Studies on the structure of the cerebral cor-
tex. II. Continuation of the study of the ammonic system. J
Psychol Neurol 46: 113-177, 1934.
14. Reiter, L. W., P. H. Ruppert and K. F. Dean. Trimethyltin
(TMT) disrupts acoustic startle responding in adult rats. To-
xicologist 3: 168, no. 672, 1983.
15. Smith. P. and L. Smith. Organotin compounds and applications.
Chem. Br. 11: 208-226, 1975.
16. Young, J. S. and L. D. Fechter. Trimethyltin disruption of re-
flex inhibition indicates an ototoxic effect. Toxicoloi
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S'eur,'behavioral To.xirolony and 7'rratoittgy, Vol. J, pp. 673-$%, 1983. ' Ankho Iniernalional Inc. Printed in the U.S.A.
Trimethyltin Induced Pathology
in Sensory Neurons'
LOUIS W. CHANG
Department of Pathology, University of Arkansas for Medical Sciences
4301 West Markltam, Little Rock, AR 72205
AND
ROBERT S. DYER
Division of Neurotoxicology, Health Effects Laboratory
U.S. Environmental Protection Agency, Research Triangle Park, NC 27711
CHANG. L. W. AND R. S. DYER. Trimethyltin induced pathology in sensory neurons. NEUROBEHAV TOXICOL
TERATOL 5(6)673-696. 1983.—Pathologic changes in the retina, inner ear. pyriform cortex, olfactory tubercle, and dorsal
root ganglia as a result of trimethyltin (TMT) intoxication were investigated. Long-Evans rats were orally intubated with
TMT chloride at a dose of 6.0 mg TMT/kg b.w. Swelling of the optic fiber layer and necrotic changes in the ganglion and
inner nuclear layers w ere observed in the retina as early as 72 hours after intoxication. Large segments of the retina were
devoid of ganglion cells at later posttreatment times. The inner ear was also found to be extremely vulnerable to the toxicity
of TMT. Edematous swelling of the hair cells and vacuolar changes of the spiral ganglion cells in the Organ of Corti were
observed 24 hours after TMT exposure. Extensive destruction of these structures was evident 15-30 days after intoxica-
tion. Small neurons in the olfactory cortex (pyriform cortex anil olfactory tubercle) also degenerated rapidly after TMT
exposure. Electron microscopy demonstrated lysosomal accumulation and vacuolar changes in these nerve cells. Exten-
sive destruction of both the pyriform cortex and olfactory cortex was observed 15 days after exposure. Although no
necrotic change was observed in the neurons of the dorsal root ganglia, electron microscopy revealed extensive accumula-
tion of lysosomes and formation of myeloid bodies both in the neuronal bodies and dorsal root fibers. Vacuolar breakdown
and dissolution of the Nissl substance were found in some neurons. Thirty days after treatment, hypertrophy and
hyperplasia of the neuronal mitochondria were observed. Such a change was believed to represent a compensatory
response by these organelles. These findings provide the first morphological evidence of neuronal damage in the sensory
neurons of both the central and peripheral nervous system following acute TMT administration.
TMT Trimethyltin Retina Dorsal root ganglion Hair cells Inner ear Organ of Corti
Spiral ganglion Pyriform cortex Olfactory tubercle
TRIMETHYLTIN compounds (TMT) arc potent
neurotoxicants inducing extensive neuronal changes in the
limbic system, particularly the hippocampal formation.
Acute TMT exposure results in primary degeneration of
Ammon's horn in rats and of fascia dentata in mice [4. 6,8,9,
II]. The rigidly laminated cytoarchitecture of these struc-
tures lends itself to easy recognition of cellular loss. While
careful study has failed to reveal a global neuronopathy, re-
cent studies by Chang and co-workers have revealed patho-
logical "extra-limbic" lesions in certain restricted areas of
the brain stem [7,11] and spinal cord {10,11J following TMT
exposure. Physiological and behavioral studies have
suggested that TMT may produce dysfunctions in the visual,
auditory and somatosensory systems [12. 16, 17, 18, 20, 26],
however, pathological evidence of degenerative changes in
these systems still has not been described. The present re-
port presents histopathological and electron microscopic
evidence of TMT pathology in sensory neurons of the retina,
inner ear, dorsal root ganglia, and olfactory and pyriform
cortices.
method
Forty young adult male Long-Evans hooded rats (60-80
days old) were used in the study. Animals, in groups of five,
were exposed to either trimethyltin chloride (n»20) at a dose
of 6.0 mg TMT/kg b.w. (as base) or saline vehicle (n*20) via
oral intubation. Animals were sacrificed on days I (24
hours), 3, 15 or 30 after TMT exposure.
At sacrifice, animals were anesthetized and perfused
intracardially with saline solution followed by 2.5% buffered
glutaraldehyde. Brains, eyes, base of the skulls, and dorsal
root ganglia (L,-L<) were carefully removed and were further
'This manuscript has been reviewed by the Health tifTccls Research Laboratory, U.S.E.P.A., and approved for publication. Mention of
trade names or commercial product!) does not constitute endorsement or recommendation for use. A preliminary repon on this work was
presented at the International Symposium on Tin Toxicity. Chicago. October. 1982.
673
-------
674
CHANG AND DYER
FIG. I. Retina, control. Seven distinct layers can be recognized in the rat retina: a
thin optic fiber layer (of), ganglion cell layer (g). inner plexiform layer (ip». inner
nuclear layer (in), outer plexiform layer (op), outer nuclear layer (on) and receptor
cell layer of rods and cones (rc). xZ50.
fixed in IOC? buffered glutaraldehyde for electron micros-
copy. All tissue samples were processed and prepared for
light microscopy or electron microscopy by routine
methodologies.
To study the histology of the inner ears, the base of the
skull was decalcified before routine histological preparation.
The specimens were embedded whole and serial sections
were made to avoid sampling and sectioning artifacts.
RESULTS
Chanties in the Retina
Light microscopic examination of the rat retina revealed
the characteristic 7 distinct layers: a thin optic fiber layer,
ganglion cell layer, inner plexiform layer, inner nuclear
layer, outer plexiform layer, outer nuclear layer, and layer of
rods and cones (Figs. I. 2).
Retinal tissue was examined on posttrealment days 3 and
30. Three days after TMT exposure, swelling of the optic
fiber layer was observed in some segments of the retina. This
swelling produced a prominent "thickened" appearance of
the optic fiber layer (Fig. 3). Scattered neuronal pyknotic
changes were also observed in the inner nuclear layer (Figs.
4, 5). as indicated by the clumping of chromatin (pyknosis) in
the cells. Degenerative and pyknotic changes of some of the
cells in the ganglion cell layer could also be found 3 days
after exposure (Figs. 4, 5).
By the 30th day following treatment, swelling of the optic
fiber layer and necrotic activity in various neuronal layers
had subsided and the general morphology of the retina ap-
peared normal. However, careful examination revealed loss
of many ganglion cells (Fig. 6), and ihinning of other retinal
layers, indicating significant additional neuronal loss.
Chances in the Inner Ear
The major sensory organ in the inner ear was the Organ of
Corti consisting of two main types of sensory cells, ihe hair
cells and spiral ganglion neurons (Fig. 7).
The hair cells (outer hair cells) were composed of slender,
finger-like cells innervated by the cochlear nerve and over-
laid by the tectorial membrane (Fig. 8l. As early as 24 hours
posttrealment with TMT. swelling of the hair cells < Figs. 9.
10) became evident. Such changes are believed to be an
edematous condition of these cells. Total loss of the hair
cells was observed in many segments of the Organ of Corti
15-30 days after TMT exposure (Figs. II. 12).
The normal spiral ganglion was composed of large, round
neurons with prominent nuclei and nucleoli (Figs. 7. 13).
Early (24 hours) toxic change was mainly manifested as vacuo-
lation (edematous change) of many of the neurons (Figs 9. 14).
By day 3 of intoxication, while the edematous vacuolation
condition had subsided, increased condensation of the nu-
clear chromatin was observed in many neurons (Fig. 15)
suggesting prenecrotic change in these cells. Extensive
neuronal loss (Figs. II. 16) was evident in the spiral ganglia
15-30 days after intoxication, depicting permanent auditory
damage in these animals.
Chanties in the Olfactory Tubercle and Pyrijorni Curtinil
Ke limns
The olfactory tubercle (olfactory cortex) and pyriform
cortex were examined on posttreatment days 3. 15. and 30.
These areas of the brain normally contain many small
granule neurons (Figs. 17. 18). Scattered neuronal necrosis
in these cortices could be observed as early as 3 days after
TMT exposure (Figs. 19. 20). By the 15-30th day of intoxi-
cation. extensive necrosis and cell loss were observed (Figs.
21. 22) resulting in severe thinning and destruction of these
areas (Fig. 23).
Electron microscopic examination demonstrated vacuo-
lar changes in many of the pyriform and olfactory cortical
neurons three days after TMT exposure (Fig. 24). The mul-
-------
TMT PATHOLOGY OF SENSORY NEURONS
-m
-.-.•v
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FIG. 2. Retina, control. The optic fiber layer (oO was composed of a thin layer of
nerve fibers representing the axons of the large ganglion neurons (g). The inner
nuclear layer (in) was composed of medium size neurons with prominent round nu-
clei. The outer nuclear layer (on) was composed of small, densely stained neurons, ip.
inner plexiform layer: op. outer plexiform layer. x450.
>
Of
<. *!-"V
\
r. i
FIG. 3. Retina. TMT-treated. J days. Swelling of the optic fiber layer (of) was ob-
served in some segments of the retina. x450.
-------
676
CHANG AND DYER
i* v.,
FIG. 4. Retina. TMT-treated, 3 days. Scattered necrotic neurons (-~). bearing very
dense, pyknotic nuclei, could be observed in the inner nuclear layer (in). Vacuolar
degenerative change (-•) of a large neuron in the ganglion layer (g) was also evident.
x450.
I ¦ -
V
- * • * ii t
FIG. 5. Retina. TMT-treatcd. 3 days. Pyknotic neurons (—•) could he found among
the ganglion cells (g) and among the nerve cells in the inner nuclear layer (in) at this
time. x450.
-------
TMT PATHOLOGY OF SENSORY NEURONS
k
FIG. 6. Retina. TMT-treated. 30 days. Large segment of the ganglion cell layer could
be demonstraied to be devoid of neurons. x450.
FIG. 7. Inner ear. control. Organ of Corti. Hair cells (he), tectorial membrane dm),
spiral ganglion (sp). x250.
-------
678
CHANG AND DYER
I
8
FTG. 8. Organ of Corti. control. The outer hair cells (he) have a slender, columnar
appearance, tm. tectorial membrane. x450.
FIG. 9. Inner ear. TMT-treated. 24 hours. Vacuolar changes in the spiral ganglion
-------
TMT PATHOLOGY OF SENSORY NEURONS
67V
f
%
!
0 s?
\ Yjf * -/*5 •
FIG. 10. Organ of Corti. TMT-treated. 24 hours. The outer hair cells (he) of the Organ
of Corti appeared to be swollen and edematous as compared to those of controls (Fig.
8). x450.
f
j
»•
%
% *i
W:' >
.V
JV
1 • \ K
I J*-* i { <
FIG. 11. Inner ear. TMT-treated. JO days. The Organ of Corti displayed an absence of
hair cells (—•) and extensive cell loss in the spiral ganglion (sg). tm. tectorial mem-
br.ine. x250.
-------
680
CHANG AND DYER
k •
9
•s
t m
* * *
FIG. 12. Organ of Corti. TMT-treated 30 days. Total absence of outer hair cells <•)
was observed in the Organ of Corti. tm. tectorial membrane. x450.
K1G. 13. Spiral ganglion, control. The spiral ganglion of the rat inner ear was composed
ol m;iny large, round neurons with prominent nuclei and nucleoli. x4M).
-------
TMT PATHOLOGY OF SENSORY NEURONS
FIG. 14. Spiral ganglion, TMT-treated, 24 hours. Vacuolar changes <-•) were ob-
served in many neurons. x450.
FIG. . Spiral ganglion, TMT-trealed. 3 days. Many neurons displayed dense.
hyperchrui itic nuclei (arrow) and vacuolated cytoplasm (-»). x450.
-------
CHANG AND DYER
ki*' r* \
,-rL , •«' << «L # A. 5- •( >
' * • t. 2 * * 4 «
' /• » ¦*¦-¦-'» * * A Al
t, f ' • %
16 ~ / \ ^ ^ ^ I
a
lSk£& Ci ¦<*
FIG. 16. Spiral ganglion. TMT-treaied. 30 days. Extensive destruction and neuronal
loss in the ganglion was observed. x450.
~~ * *
* fc
• J
«¦ • 4
P.J
Fi' '7 Pyriform cortex, control. The pyriform conical neuronal layer was com-
posed i mtiuII granule neurons, approximately 5-6 cells thick. x250.
-------
TMT PATHOLOGY OF SENSORY NEURONS
%
%» *
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V
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FIG. 18. Olfaciory cortex, control. The olfactory cortex consisted of small neurons lining
the base of the brain. x250.
FIG. 19 Pyriform cortex. TMT-treated. 3 days. Isolated necrotic neurons (-~) could
be found at this time of intoxication. x250.
-------
684
CHANG AND DYER
•>. ' *
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FIG. 20. Olfactory cortex. TMT-treated, 3 days. Many necrotic neurons (-») which
appeared to have pyknotic nuclei and eosinophilic cytoplasm were observed. x450.
* '"mi
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FIG. 21 Pyriform cortex. TMT-treated, 30 days. Extensive destruction of the conical
neuron* v as observed. Few surviving neurons (—~ ) could still be found among the
necrotic ti-'ls. x430.
-------
TMT PATHOLOGY OF SENSORY NEURONS
• * * ~ • • »r s ^
M -M ^ % ' # I k * ¦
* t> & ¦¦ . Y.'>* *
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c *i
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FIG. 22. Olfactory cortex. TMT-treated. 30 days. Many necrotic cells (-») were
observed in this conical area of the brain at this time. x450.
*'«/•« v. V.»« r*:\
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FIG. 23. Olfactory cortex. TMT-treated, 30 days. Extensive neuronal necrosis and
loss occurred resulting in significant thinning i>f the conical neuronal layer. x250.
-------
686
CHANG AND DYER
tiple vacuolation of th? neuronal cytoplasm gave these
neurons a sieved appearance. Close examination revealed
that these vacuoles were membrane-limited (Fig, 25) and
were probably derived from dilatation of the cytoplasmic
membranous structures, such as endoplasmic reticulum and
Golgi complex. Accumulation of electron-dense bodies,
presumably lysosomes, in the neuronal body and processes
(Figs. 24. 26) was also observed.
At later posttreatment tiires. increased neuronal death
was observed in these cortical areas. Extensive lysosomal
accumulation was found in the surviving nerve cells (Fig.
27). Degeneration of axons (Fig. 28) and proliferation of as-
troglial cells were also prominent.
Changes in the Dorsal Root (Spinal) Ganglia
The dorsal root ganglia were examined on posttreatment
days 3 and 30. Although the morphological changes in the
dorsal root ganglion neurons were not remarkable at the light
microscopic level, various changes in the neuronal organel-
les were evident with electron microscopic examination.
Accumulation of lysosomes was the most conspicuous
finding in these neurons 3 days after TMT exposure (Fig. 29).
Close examination revealed dense membranous structures
within some lysosomes (Fig. 30). Loosening or breakdown of
these membranous structures (Fig. 31) to give rise to the
multi-lamellated myelinoid bodies (Fig. 32) was also ob-
served. Accumulation of lysosomal bodies and cellular de-
bris in axons were also evident (Figs. 33. 34). Multiple small
vacuoles similar to those observed in the pyriform cortical
neurons were also found in many dorsal root ganglion
neurons (Fig. 35). Careful examination revealed that clusters
of these small vacuoles corresponded with clusters of Nissl
bodies in the neurons and were believed to be derived from
the dilated endoplasmic reticulum in the Nissl bodies (Fig.
36). Although not a frequent finding, some neurons displayed
extensive dissociation of the ribosomes from the
endoplasmic reticulum membranes leading to cytoplasm
filled with free ribosomes. Severe mitochondrial swelling
and vacuolation were also found in some nerve cells (Fig.
37).
By the 30th day following treatment the general mor-
phological appearance returned to normal. However, careful
survey revealed that areas of many neurons were still devoid
of Nissl bodies (Fig. 38). Conspicuous variations in the size
of mitochondria in both the neurons (Fig. 38) and neuronal
processes (Fig. 39) were also observed. Many mitochondria
displayed budding conformation suggesting mitochondrial
replication. It is believed that the large number of small
mitochondria were resulted from rapid budding or division of
mitochondria. In addition, giant mitochondria measuring
10-25 fim in diameter were also found (Fig. 40).
DISCUSSION
Organotin compounds, particularly the trialkyltins. have
been used for both industrial and agricultural purposes, in-
eluding chemosterilants, fungicides, and plastic stabilizers
[19.21 J. The alkyltin compounds, such as tricthyltin (TETl
and trimcthyltin (TMT). are found to be extremely potent
neurotoxicants with TKT being primarily a myelinotoxicant
[24.25] and TMT being a ncuronotoxicant (4-I1J.
Earlier reports suggested that TMT was a central nervous
system toxicant selectively affecting the limbic system
[14.23] and could account for many of the behavioral effects
of TMT. Recent neuropathologies! investigation by Chang
and co-workers revealed significant lesion production in
many "extra-limbic" neurons including brain stem neurons
and the spinal cord motoneurons in rats and mice [7. 10. 11).
Recent physiological and behavioral studies also indicated
dysfunction in the visual [13,17], auditory [16. 20. 26). and
somatosensory [18] systems. The present study provides the
first morphological evidence of the toxic effects of TMT on
rat visual, auditory, olfactory, and somatosensory systems.
By exposing rats to TMT. Dyer and co-workers [13]
demonstrated an increase in visual evoked response at early
peak latencies with a decreased amplitude recorded in both
the visual cortex and optic tract. These alterations in visual
responses, which were present within 24 hours and persisted
at least 16 days after TMT exposure, suggested retinal
changes. Preliminary demonstration of histological changes
in the retina together with visual dysfunction as a result of
TMT poisoning was first presented by Dyer and Chang
(Neurotoxicity of Heavy Metals Symposium. Chicago.
October, 1982). The present report represents a more de-
tailed histopathological study in the rat retina and revealed
that morphological change could be observed as early as 72
hours after TMT exposure. The earty thickening of the optic
fiber layer was probably a result of edematous change in
these nerve fibers. The transient appearance of TMT-
induced edematous changes has also been reported in other
neurons [6, 7. 9, 10]. Neuronal degeneration and necrosis
was observed in the large ganglion cell layer and in the inner
nuclear layer of the retina. The neuronal loss in these layers
was prominent at later intervals after treatment leading to
thinning of these cell layers and absence of ganglion cells in
large segments of the retina. Because of the extremely dense
staining property of the outer nuclear layer and minute size
of the receptor cells in the retina, necrotic or degenerative
changes in these cells were difficult to be detected at light
microscopic level. However, the overall thinning of the
retina (all cell layers) at later stages of poisoning strongly
suggests cell loss in all layers of the retina. Indeed, recent
electron microscopic investigation not only confirmed our
observations on neuronal changes in and inner nuclear layers
of the retina, but also revealed degenerative changes in the
outer nuclear layer as well as in some receptor cells follow-
ing TMT exposure [3]. These findings suggest that TMT af-
fects all neuronal layers in the retina and the damages are
probably permanent.
Reiter and co-workers [16.20] reported a significant re-
duction in acoustic startle response in rats as early as two
hours following TMT exposure- These investigators indi-
cated that the alterations in the acoustic startle response in
TMT-treated animals did not resemble the effects of limbic
system lesions and suggested that TMT produced damage
within the primary acoustic startle circuit. Similarly. Dyer
and colleagues [12] have observed increased latencies in
Peak 1 of the brain stem auditory evoked response within 24
hours of TMT treatment, suggesting damage to the periph-
eral auditory system. Young and Fechter [26] found that
TMT decreased or eliminated the inhibition of startle re-
sponses by low intensity, high frequency prestimuli. suggest-
ing ototoxicity effects of TMT. The present study also re-
vealed high sensitivity and vulnerability of the inner ear to
TMT toxicity. Edematous swelling of hair cells in the Organ
of Corti and vacuolar changes in spiral ganglion neurons
were observed as early as 24 hours after TMT exposure.
Since this is the earliest time point of our examination, ear-
lier damage to these cells is possible, as suggested by behav-
ioral investigations [20). Progressive destruction and cell loss
was indicated by significant hair cell and neuronal loss in the
-------
TMT PATHOLOGY OF SENSORY NEURONS
^ a ¦ 1 •$..? , • .•
[ V i v
: $
FIG. 24. Pyriform conex. TMT-treaied. 3 days. Multiple vacuolation of the cyto-
plasm gave the neuron a sieved appearance. Lysosomal accumulation (-•) was also
evident, x4.500.
it \ X-r %
fl^N* ¦* ¦» >*ST S* A
rr J\ it w J
r .1 ?V» •*?* 'L.i. Ijfts
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FIG. 25. Pyriform cortex. TMT-treated. 3 days. Closer examination revealed that
these cytoplasmic vacuoles were membrane-limited and probably derived from dila-
tation of the cytoplasmic membranes. X42.000.
-------
CHANG AND DYER
•\v.- ' r*W
FIG. 26. Pyriform cortex. TMT-treated. 3 days. Accumulation of lysosomes in a
neuronal process (—»). X4.500.
vwmm&sg. frsj&
/tfHt ;~!v
L^JKw#w*»a>f si®
'^V» 2'iL# Alk& fc>»s(fj#f: :,'s?V-M~rZ,
p««
mzmm
FIG. 27. Pyriform cortex. TMT-lrealed. 30 days. Extensive accumulation of
electron-dense bodies, presumably lysosomes. in a surviving neuron. N. nucleus.
X5.000.
-------
TMT PATHOLOGY OF SENSORY NEURONS
689
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fJ£ t;
FIG. 28. Pyriform cortex. TMT-treated. 30 days. Degenerative changes in axons (A)
were also observed. X45.000.
FIG. 29. Dorsal root ganglion neuron. TMT-trealed, 3 days. Exiensive accumulation
of clcctron-dcnsc bodies, presumably lysosomes. was observed in many neurons. N.
nucleus. X4.200.
-------
690
CHANG AND DYER
FIG. 30. Dorsal root ganglion neuron. TMT-ireated. 3 days. Some lysosomes were
found to contain membrane-like structures. X45.000.
v?' « >>Xss#
FIG. 31. Dorsal root ganglion neuron. TMT-treated. 3 days. Loosening of the mem-
branous structures in some lysosomes (—~ ) gave rise to myelinoid bodies, x45.000.
-------
TMT PATHOLOGY OF SENSORY NEURONS
• vat. ... v..*
FIG. 32. Dorsal root ganglion neuron. TMT-treated. 3 days. Accumulation of multi-
lamellated myeloid bodies in a neuron, x40,000.
!
FIG. 33. Dorsal root Tiber. TMT-treated. 3 days. Accumulation of lysosomes and
cellular debris within an axon, x27.000.
-------
CHANG AND DYER
FIG 34 Dorsal root fiber. TMT-treaied. 3 days. An axon was found to be filled with
lysosomes and cellular debris, x32.000.
FIG. 35. Dorsal root ganglion neuron. TMT-treated. 3 days. Clusters of small vac-
uoles were found in I he neuron, x 7.500.
-------
TMT PATHOLOGY OF SENSORY NEURONS
FIG. 36. Dorsal root ganglion neuron. TMT-treated, 3 days. Small vacuoles were
found lo be membrane-limited and were believed lo be derived from dilated
endoplasmic reticulum in the Nissl bodies, x 13,000.
SBSB
FIG. 37. Dorsal root ganglion neuron. TMT-lreated. 3 days. Area of the neuronaJ
cytoplasm was filled with free rihosomes. Swelling and vacuolar change in the
mitochondria (-») were also evident, x 10.000.
-------
CHANG AND DYER
FIG. 38. Dorsal root ganglion neuron. TMT-treated. 30 days. Large areas of the
neuronal cytoplasm were totally devoid of Nissl substance. Significant variation of
mitochondrial sizes was also demonstrated, x8.500.
FIG. 39. Dorsal root ganglion. TMT-treated. 30 days. An increase in mitochondrial
number was observed in a neuronal process. Many mitochondria appeared to be
exceptionally small (—»). * 14.500.
-------
TMT PATHOLOGY OF SENSOF.Y NEURONS
695
FIG. 40. Dorsal root ganglion. TMT-treated. 30 days. Significantly enlarged
mitochondria (*) demonstrated in some neuronal processes. Relatively normal sized
mitochondria (—»). x 16.000.
Organ of Coni and in the spiral ganglia 30 days after treat-
ment. Judging from the extent of lesions produced, the dam-
age in these structures is probably permanent, producing
permanent functional deficits.
We have reported damage and neuronal destruction in the
pyriform and olfactory cortices in both rats and mice after
TMT exposure (5. 6. IIJ. This study further affirms that
neurons in these areas, at least in rats, are extremely vulner-
able to TMT toxicity. Total destruction of these conical
areas was usually observed 2 weeks after exposure to TMT.
Since the pyriform cortex and olfactory cortex are important
in olfactory function, the present observations strongly
suggest that olfaction is impaired in rats following exposure
to TMT. Physiological and behavioral studies must docu-
ment the nature and extent of dysfunction.
Dyer and co-workers (18) tested TMT-treated rats for
somatosensory dysfunction and reported an increased pain
threshold (increased hot plate latencies) but normal periph-
eral nerve conduction velocity and action potential
threshold. TMT also produced an increased latency to early
peaks in the somatosensory evoked potential recorded from
the cortex of these rats. Based on their observations, these
investigators suggested that TMT produced no peripheral
nerve effects and the somatosensory dysfunction was be-
lieved to be largely the result of dysfunction somewhere be-
tween the peripheral nerves and the cortex. The present
study demonstrated early submicroscopic (ultrastructural)
changes in the neurons of dorsal root ganglia. Although the
accumulation of lysosomes should not be viewed as specific
to TMT exposure, the formation of multi-lamellated myeloid
bodies in these neurons was intriguing. The significance of
these myeloid bodies is still not clear.
The most interesting and perhaps most important obser-
vations in the dorsal root ganglion neurons following TMT
exposure concern the mitochondrial alterations. Significant
variations in mitochondrial sizes were observed, particularly
at late posttreatment intervals. These changes, hyperplasia
of small mitochondria and hypertrophy of others, may reflect
a functional compensatory response [15]. Aldridge and co-
workers [1.2] reported that trialkyltin compounds are potent
inhibitors of oxidative phosphorylation (mitochondrial func-
tion) and ATP system in cells. The increase in mitochondrial
size and number may represent a biological compensation to
overcome such metabolic suppression. Disturbance in the
plasma membrane ATP system may also be responsible for
the edematous condition of cells and organelles following
TMT exposure. TMT is 30-40 limes less active than triethvl-
tin as an inhibitor of oxidative phosphorylation [2]. a fact
which may prevent the mitochondrial damage as reported in
TET poisoning 122] and allow the surviving mitochondria to
respond and compensate for the functional inhibition.
Disintegration or dissolution of the Nissl substance
(rough endoplasmic reticulum clusters) in some of these
neurons is indicative of a disturbance in protein synthesis
potential. This breakdow n of Nissl substance may be a direct
toxic effect of TMT or may be secondary to toxic injury to
the nerve cells. By the same token, the accumulation of cel-
lular debris and lysosomes in the nerve fibers may only re-
flect a secondary change rather than primary toxic action on
these structures.
In summary, we have demonstrated for the first time
toxic changes in the retina, inner ear. olfactory cortex, and
dorsal root ganglia of rats exposed to TMT. These changes,
which occurred very early after TMT exposure, may under-
lie previously reported behavioral and neurophysiologies!
data indicating dysfunction in the visual, auditory, and
somatosensory systems of TMT-exposed rats. The present
study also reveals for the first time that TMT is not only a
CNS neurotoxicant (e.g.. olfactory cortex) but is also toxic
to certain neurons in the peripheral nervous system (e.g..
dorsal root ganglia) and to special sensory organ neurons in
the inner ear and in the retina.
-------
696
CHANG AND DYER
REFERENCES
1. Aldridge. W. N. The interaction of trialkyltin compounds with
the oxidative phosphorylation system in mitochondria. In: Ef-
fects of Metals on Cells. Subcellular Elements, and Mac-
romolecules. edited by J. Maniloff. }. R. Coleman and M. W.
Miller. Springfield. IL: Charles C. Thomas. 1970. pp. 255-274.
2. Aldridge. W. N. and M. S. Rose. The mechanism of oxidative
phosphorylation, a hypothesis derived from studies of
trimethyltin and triethyltin compounds. FEBS Lett 4: 61-68.
1969. *
3. Bouldin. T. W., N. D. Goines. A. D. Toews and M. R. Krig-
man. Organometal induced retinopathy in the rat. J
Neuropathol Exp Neurol 42: 316 (Abstract #34). 1983.
4. Brown. A. W., W. N. Aldridge. B. W. Street and R. D. Ver-
schoyle. The behavioral and neuropathology sequelae of intox-
ication by trimethyltin compounds in the rat. Am J Pathol 97:
59u8l. 1979.
5. Chang. L. W. and R. S. Dyer. A time-course study of trimethyl-
tin induced neuropathology in rats. Ncurobehav Toxicol Teratol
5; 443-459. 1983.
6. Chang. L. W., T. M. Tiemeyer. G. R. Wengerand D. E. McMil-
lan. Neuropathology of mouse hippocampus in acute trimethyl-
tin intoxication. Ncurobehav Toxicol Teratol 4: 149-156. 1982.
7. Chang. L. W.. T. M. Tiemeyer, G. R. Wengerand D. E. McMil-
lan. Neuropathology of trimethyltin intoxication. III. Changes
in the brain stem neurons. Environ Res 30: 399-411. 1983.
8. Chang. L. W.. T. M. Tiemeyer. G. R. Wenger. D. E. McMillan
and K. R. Reuhl. Neuropathology of trimethyltin intoxication.
I. Light microscopy study. Environ Res 29: 435-444, 1982.
9. Chang. L. W.. T. M. Tiemeyer. G. R. Wenger. D. E. McMillan
and K. R. Reuhl. Neuropathology of trimethyltin intoxication.
II. Electron microscopy study of the hippocampus. Environ Res
29: 445-458. 1982.
10. Chang. L. W., G. R. Wenger and D. E. McMillan.
Neuropathology of trimethyltin intoxication. IV. Changes in the
spinal cord. Environ Res. in press. 1983.
11. Chang. L. W.. G. R. Wenger, D. E. McMillan and R. S. Dyer.
Species and strain comparison of acute neurotoxic effects of
trimethyltin in mice and rats. Ncurobehav Toxicol Teratol Si
337-350. 1983.
12. Dyer. R. S. The use of sensory evoked potentials in toxicology.
Fund Appl Toxicol, in press. 1983.
13. Dyer. R. S.. W. E. Howell and W. F. Wonderlin. Visual system
dysfunction following acute trimethyltin exposure in rats.
Ncurobehav Toxicol Teratol 4: 191-196. 1982.
14. Dyer, R. S.. T. J. Walsh, W. F. Wonderlin and M. Bercegeay.
The trimethyltin syndrome in rats. Ncurobehav Toxit <>! Teratol
4: 127-134. 1982.
15. Ghadially. F. N. Ultrastructural Pathology of tlu- Cell. Boston:
Butterworth. 1975.
16. Howell. W. E.. R. S. Dyer. W. F. Wonderlin. K. Kodd and L.
W. Reiter. Sensory system effects of acute trimethyltin
-------
The Neurobiology of Zinc
Part B: Deficiency, Toxicity, and Pathology, pages 275-290
& 1984 Alan R. Liu, Inc., 150 Fifth Avenue, New York, NY 1001t
TRIMETHYLTIN INDUCED ZINC DEPLETION IN RAT HIPPOCAMPUS
Louis W. Chang and Robert S. Dyer
Univ. oF Arkansas for Medical Sciences, Little Roc
Arkansas, 72205 and Neurotoxicology Div., U.S.
E.P.A., Research Triangle Park, NC 27711
Organotin compounds have been used as stabilizers in
plastic industries, as chemosterilauis and as fungicides.
Alkyltins, including triethyltin (TET) and trimethyltin (TMT)
are found to be extremely neurotoxic. While TET compounds ar
primarily myelinotoxic, inducing.swelling and vacuolar change
in the central myelin (Watanabe 1977; Watanabe 1980), TMT com
pounds are found to be neuronoto:>»ic leading to extensive
neuronal changes and necrosis in*«a number of brain areas, in-
cluding the limbic system and particularly the hippocampus
(Brown et al.. 1979; Bouldin et al. 1981; Chang eit a_l. 1982a;
Chang £t aj_. 1982b; Chang et aj^. 1983).
Various behavioral and sensory changes. Including tremor
aggression, hyper irritability, visual and auditory dysfunc-
tion, have been observed in rats poisoned with TMT. Such
changes were referred to as "the trimethyltin syndrome" (Dyer
et a_l. 1982). Similar behavioral changes and patterns have
been observed in zinc-depleted humans (lleukin ejt a_l. 1975).
Since the normal hippocampal formation contains more zinc tha
most other central nervous system areas, the possibility that
TMT administration interacts with hippocampal zinc to product
its effects becomes an intriguing possibility which deserves
investigation. The present investigation was designed to ex-
plore the effects of TMT on hippocampa1 zinc.
MATERIALS AND METHODS
Young adult male Sprague-Dawley rats with average body
weights of 200 grams were used in this experiment. Animals
-------
276 / Chang and Dyer
were injected (l.p.) with 6.0 mg TMT/kg b.w. Control animals
were injected witli saline solution. In groups of four, TMT-
treated animals were sacrificed at 1, 3, and 15 days after
TMT administration.
At sacrifice all animals were prepared for modified
Timm's method as outlined by Sloviter (1982). In brief,
animals were perfused with 0.4% sodium sulphide solution
followed by 2.5% buffered glutaraldeliyde. After removal from
the skull, brains were further immersion fixed in 102 buffered
formalin and processed for paraffin embedding. Sections
were cut at 10 ym thick, deparaffinized, and developed in a
developer containing silver nitrate, citrate, and hydroquinone
for 60 minutes. Slides were counterstained with cresyl violet.
To ensure consistency in the staining reaction, all tissue
sections were stained and developed together with tissue
sections from control animals. All slides were subjected to
blind examination and evaluated by the same investigator.
Insults
Animals were virtually normal 24 hours after TMT admini-
stration. After 3 days (72 hours) of intoxication, some
hyperirritahility was noted in the rats. Increasing signs of
intoxication, such as behavioral changes and body tremors,
were observed in animals after 10 days of intoxication. By
the end of the second week of intoxication, some of the
animals displayed significant weight loss, coarse coats,
weakness, and seizures.
Ilistopathologlcal examination revealed no significant
cellular damage in the hippocampus until a week after intoxi-
cation. The modified Timm's method demonstrated consistant
depletion of heavy metal staining in the hippocampal formation
of TMT-treated animals.
In the hippocampal formation of control animals, mossy
fibers projecting from the granule cells of the fascia deniata
to fields CA^ and CA of the hippocampus were heavily stained
by Timm's technique (Figs. 1-4) indicating high metal content
in this area of the brain. The molecular layer of llie fascia
dentata appeared to be sharply trilaminar with a clear, un-
stained middle zone, a medium stained inner zone, and a
lightly stained outer zone (Fig. 1). The stratum radiatum of
the hippocampus displayed a medium staining (Fig. 4).
Trlmethyltin Deplete* Hippocampal Zinc / 27
Fig. 1. Hippocampal formation, rat, control. Mossy fibers
(M), were heavily stained by Timm's method. The molecular
layer of fascia dentata (m) had a trilaminar appearance. g
granule cells, SR, stratum radiatum, x 250.
V
Fig. 2. Heavy staining of mossy fibers (M) by Timm's stai
g, fascia dentata granule cells. x 450.
-------
27$ / Chang mod Dyer
f7**f
Fig. 3. Mossy fibers (M) in CA region. Tiram's stain.
450. ja.D
mmrn
Hk- 4. The stratum radiatum (SR) acquired a moderate Timni
staining. |i, pyramidal neurons of Aiiudoh's horn. x 450.
Trlmethyltln Depletes Hippocampal Zinc I 27
Counterstaining with cresyl violet not only left the intens
and pattern of Timm's staining unaltered, hut also provided
clear demonstration for the nerve cells in the hippocampal
f ormat ion.
Fig. 5. Hippocampal format iu^i, 6.0 mg TMT/kg b.w., 24 lioui
Marked reduction of Timm's staining was observed. M, moss;
fibers; m, molecular layer; SK\ stratum radiatum; g, granu
cells, x 250. *
Reduction of Timm's staining in the hippocampal forma
could be visually detected even at 24 hours after TMT admii
stration (Figs. 5-8). Although the mossy fibers were stil
stained, they were not as heavy and dense as those of cont
(Figs. 6 and 7). The trilaminar appearance of fascia dent
molecular layer (Fig. 5) and the staining of the hippocamp
stratum radiatum (Fig. 8), although weaker, was still visi
After 3 days (72 hours) of intoxication, the reductio
of Timm's staining in the hippocampal formation became ver
marked (Figs. 9-12). The staining on the mossy fibers wer
scanLy and weak (Figs. 10 and II). The trilaminar stuinin
pattern of the dentate molecular layer became invisible (I
9) and Lite staining of the hippocampal stratum radiatum wa
also abolished (Fig. 12). However, no significant neurona
necrosis was observed in the granule cells and pyramidal
neurons at this time of Intoxication (Figs. 10-12).
-------
280 / Chang and Dyer
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Fig. 6. 24 hours after TMT treatment, the mossy fibers (M)
became less reactive to Timra's staining. g, fascia dentata
granule cells, x 450.
<*? & *i i .
Fig. 7. Field ^, TMT-treated. Marked reduction of
Timin's staining was'observed in the mossy fibers (M) as early
as 24 hours after TMT exposure. x 450.
Trimelhyllin Depletes Hippocampal Zinc / 28
SIP
mhm
fa':i\A
;s\v>rK:lt
Fig. 8. Some reduction of Timm's staining in the stratum
radiatum (SR) 24 hours after TMT treatment, p, pyramidal
neurons. x 450.
m
9
Fi^. 9. Hippocampal formation, 6.0 mg TMT/kg b.w., 3 days.
Reduction of Timni's staining in the mossy filters (M) uas ol
served and total abolishment of the trilaminar staining
appearance of the fascia dentata molecular layer (m) also
occurred. r, granule cells. x 250.
-------
282 / Chang and Dyer
Fig. 10. 3 days after TMT-exposure, the mossy fibers (M)
became very lightly stained and scanty in appearance. Little
or no remarkable necrotic changes in the granule cells (g)
was observed at this time. x 450.
£&vV:«v-
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H
WV
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ter: '•'
if
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Hg. II. () region of the hippocamp.il Amnion's horn. The
staining of moftsy fibers (M) by Timm's method was drastically
reduced. x 4 50.
I
Trlmethyltln Deplete* Hippocampal Zinc / 28
I •
>.
SR
¦ / • ;K
V • •
12
Fig. 12. Stratum radiatum, 'ITU-treated, 3 days. Total losj
of Tiinm's staining in tlie stratum radiatum (SR) was evident,
p, pyramidal neurons of Ammon's horn, x 450.
V-
t
Fifteen days after intoxiaation, many animals showed
signs of intoxication (aggression and tremor). Examination
of brains from these animals revealed virtually total deple-
tion of Timm's staining in the hippocampal formation (Figs.
13-16). Although neuronal necrosis in both the granule and
pyramidal neurons was evident in this period of intoxicatioi
the extent of cell loss was by no means proportional to the
extent of Timm's (zinc) depletion. Thus, the loss of Timm'i-
staining was not totally a result of granule cell loss.
Discussion. Timm's sulfide-sllver method Is generally
considered as a reliable hlstochemical method for transitioi
metals, group lib meials, and heavy metals (llaug 1973). Sii
1955, both dithizone and Tiinm's su1fide-si1ver methods re-
vealed a high accumulation of metal in the brain, particulai
in the hippocampal formation (Maske 1955; I'l e I schliauer, llor.-
maun 1957; Tinun 1958; Mcl.ardy I9(>0; Mrl.ardy 19(>2; Mcl.ardy
19bJ; Mcl.ardy 19(>4; Eulor 19(>2). These studies demonstratei
lliaL the heavy staining corresponded to the zones receiving
mossy fiber terminals. Later studies with electron microsci
and chemical analyses have confirmed that the metal locallz;
lion w.is confined to the terminal boulon's and was mostly di
-------
284 / Chang and Dyer
to the presence of zinc (Haug 1967; llu, Friede 1968; lbata,
Otsuka 1969).
v '¦ ' ' .1 >«• • ... .f.V
. >% a v *:v
!F^ • • ' «V >1\i.
tr„ ¦¦ • > 'R' . -
f. %. • . . .• • • . 4 V • »•» • *•' .•
' V" .. ' v . a. *1*. f
Fig. 13. Hippocarapal formation, TMT-treated, 15 days. Total
loss of Tinun's staining in the mossy fibers (M), fascia den-
tata molecular layer (m) and stratum radiatum (SK) was ob-
served. g, granule cells; p, pyramidal neurons, x 250.
Fig. 14. 15 days after TMT Intoxication, severe loss of mossy
fiber (M) st.lining was evident. Only scattered neuronal ne-
crosis was lutind among the granule cells (g). x 450.
Trimethyltin Oeplelea Hlppocampal Zinc ,
Fig. 15. CA^ of Ammon's horn, TMT-treated, 15 days. Tota
abolishment of llmm's staining on the mossy fibers (M).
Significant neuronal loss onVthe CA pyramidal cells (p)
occurred at this time; howeve^, the granule cells (g) still
appeared to be relatively intact. x 450.
'* a r
Mg. lb. I MT-t re.ited, 15 days. Total loss of Tirnm's stainl
in stratum radiatum (SK) was also evident. p, pyramidal
eel Is ot Amnion's horn. x 450.
-------
2M / Chang and Dyar
Zinc lias been shown to be an Integral constituent and co-
factor of more than twenty metalloenzymes (Kirchgessner, Roth
1980). Recent evidence suggests that zinc may have a funda-
mental physiological role in nucleic acids and protein metabo-
lism (Underwood 1977; Hsu 1978). There is also evidence
indicating that zinc plays an important role in the stabiliza-
tion of mncromulccules, cellular and lysosomal membranes, as
well as an inhibitor for brain microsomal ATPase and for elec-
tron transport in the mitochondrial respiratory function.
These findings were extensively reviewed and summarized by
Clivapil et al. (1972).
Although the precise function of the exceptionally high
concentration of zinc in the mossy fibers of the hippocampal
formation is still obscure, based on the functional properties
of zinc in the inhibition of brain ATPase activities (Mustafa
et al. 1971; Donaldson et al. 1971; Clivapil et^ a[. 1972) and
mitochondrial respiratory function (Nicliolls, Malviya 1968;
Eliasson 1971; Chvapil et al. 1972), one may reasonably sus-
pect an "inhibitory" or "regulatory" function of zinc on those
nerve fibers and terminals. Indeed, it has been observed in
the nervous system that in certain axons, a marked reduction
in divalent cation, principally calcium, would produce repeti-
tive discharges following a single stimulus. Il has further
been shown that zinc can replace calcium for the same function
and when zinc concentration is varied, the conductance shift
is substantially the same as witli concentration changes of
calcium, i.e., multiple or hyper-conductance (Coldmait 1970).
In view of these observations, we may postulate that reduction
or depletion of zinc in the granule cell mossy fibers may
yield an irregulated, hyperactive, and multiple discharge
phenomenon of these fibers and terminals connected to the CA^
pyramidal neurons.
The present modified Timm's method for zinc localization
in the nervous system provides us with the advantage of visu-
alizing any detectabLe reduction of zinc in the mossy fibers
in relationship with the overall integrity of the granule
cells. Since a reduction of ziuc (Timm's staining) was evi-
dent in very early stages of the IntoxicaL ion (24 and 72
hours) prior to any observable histopathologicaI changes in
the granule cells, the depletion of zinc in the mossy filters
and terminals (axons of the granule cells) was not simply a
secondary consequence of granule cell degeneration. Further-
more, even at a later stage of intoxication uliere some granule
cell damage did occur, the extent of zinc depletion was much
Trimelhyltin Depletes Hippocampal Zinc /
mitre severe than granule cell loss* It is therefore evid
Lliat zinc depletion in the mossy fibers is an early and ui
toxic effect of TMT on the hippocampus. However, nutritii
problems and increasing neuronal damages in the morbidly •
animals at a very late stage of the intoxication may cunt
bute, at least in part, to the extensive zinc depletion
condition of these animals.
Although the Timm's method only provides a qualitati
evaluation of zinc reduction or depletion in the hippocani
formation, a definite TMT-induced change was observed her.
Quantitative assays for zinc changes in the hippocampus a.<
other organs are presently being conducted to provide a m*
precise description of the effect of TMT on zinc metaboli:
The antagonistic action between metal ions in biolog
systems is well known. . In general, there is an inverse r.
tion between the levels of copper and zinc (Whanger, Uesw
1971). Modifications of both cadmium and lead toxicity b>
zinc has also been reported (Flick et. al.. 1971; Hill 1976
Petering et aj[. 1971; Uillou^hby et al. 1972; Sanwall iK .
1978; Prasad 1982). Tin, beHng a divalent metal cation 1
copper, cadmium, or lead, may, also have a potent antagoni-
action on zinc in biological -fissue. This antagonistic
action or interaction on zinc by tin may play an importan
role in tin toxicity. Further investigation is needed to
confirm this hypothesis.
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Although the research described in this article has been
funded wholly or in part by the United States EPA under
assistance agreement number R809452-02 to the University of
Arkansas for Hedical Sciences, it has been subjected to
the Agency's required peer and administrative review. How-
ever, it does not necessarily reflect the view of the
Agency and no official endorsement should be inferred.
The Neurobiology of Zinc
Pari B: Deficiency, Toxicity, and Pathology, page* 291-306
-------
V/iifobehavioral Toxicology and Teratology, Vol. 7, pp. 43-49, 1985. e Ankho International Inc. Printed in the U.S.A.
0275-1380185 $3.00 + .00
Septotemporal Gradients of
Trimethyltin-Induced Hippocampal Lesions
LOUIS W. CHANG'
Department of Pathology. Univ. of Arkansas for Medical Sciences, Little Ruck, AR 72205
AND
ROBERT S. DYER
Neurotoxicology Division, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711
Received 29 February 1984
CHANG. L. W. AND R. S. DYER. Septotemporal gradients of trimethvltin-induced hippocampal lesions.
NEUROBEHAV TOXICOL TERATOL 7(1) 4J-49, 1985.—Rats were administered 6 mg/kg trimethyltin (TMT) PO. and
sacrificed at various time points (up to 60 days) for pathological evaluation. Studies along the fliil extent of the septotem-
poral axis revealed region-specific and cell field-specific damage. Dentate granule cells were most affected at the temporal
pole and were only minimally affected at the septal pole. CAM pyramidal neurons were affected along the full extent of the
septotemporal axis. CA^ and CA1b pyramidal cells were most affected in the septal pole, and virtually unaffected in the
temporal pole. It is suggested that CAU and CA,„ pyramidal ceil damage may be dependent upon functionally intact granule
cells.
Trimethyltin Hippocampal damage Septotemporal axis Dentate granule cells Pyramidal cells
THE toxic effects of trimethyltin (TMT) compounds on the
central nervous system (CNS) are well established. TMT-
induced neuronal destruction occurs in a number of CNS
areas [7], but is most prominent in the hippocampal forma-
tion and other limbic structures of both rats and mice [8].
While published accounts of TMT toxicity in rats describe
high vulnerability of hippocampal pyramidal neurons, the
most sensitive pyramidal cell field has not been convincingly
established. Brown et al. [1] described thinning of CA,., with
sparing of CAm,i, but extensive damage to CAM. Similarly.
Valdes et al. [251 have described sparing of CAM,b in the face
of extensive CAr,c damage. In contrast. Dyer and co-workers
[10,11] demonstrated prominent destruction of CAls, CA,b
and CAk in the hippocampal formation with relative sparing
of CA,.j.
Since identifying the first affected or most sensitive cell
types might provide a due to the mechanism of toxicity it is
important to resolve the issue. We noticed that those studies
showing CAu sensitivity and CAM sparing were based upon
coronal sections, while those showing CA3itb sensitivity
were based upon sagittal sections. Based upon these and
other observations, it was suggested that the vulnerability of
a particular CAj neuron was dependent upon its location
within the hippocampal formation, as well as its subfield
identity [7]. In particular, the anteromedial portion of CA*,
and CAsb in the dorsal hippocampus appeared to be more
vulnerable than more lateral portions. In the present study a
detailed investigation of TMT-induced lesion topography
along the full septotemporal axis of the hippocampal forma-
tion is described for granule cells and pyramidal cells.
METHOD
Young adult (60-80 days old) male Long-Evans hooded
rats (N»40) were used in the experiment. Animals were ex-
posed to trimethyltin chloride via oral intubation in a single
dose of 6.0 mg TMT/kg b.w. (or 7.5 mg TMT-Cl/kg b.w.).
Animals were then sacrificed in groups of 5 at 3 days. 15
days, 30 days, and 60 days following exposure. Control
animals were also sacrificed in groups of 5 at 3,15.30, and 60
days following intubation of the saline vehicle.
At sacrifice, animals were anesthetized with Nembutal
and perfused intracardially with saline solution followed by
2.5% buffered glutaraldehyde. Brains were then removed
surgically and ftuther immersion-fixed in a solution of 10%
buffered formalin for at least 48 hours before tissue process-
ing. After dehydration in graded ethanols and embedding in
P&r&plast, brains were serially sectioned in either coronal or
sagittal planes and stained with hematoxylin-eosin for general
histopathological study [3].
RESULTS
The configuration of dorsal and ventral hippocampal for-
mations of the rat brain is illustrated in a parasagittal section
(Fig. 1). After 3 days of intoxication, TMT produced only
scattered neuronal necrosis in the fascia dentata granule cells
'Requests for reprints should be addressed to Dr. Louis W. Chang, Dept. of Pathology, Univ. of Arkansas for Medical Sciences. 4301 West
Marfcham, Little Rock, AR 72205.
43
-------
44
CHANG AND DYER
FIG. 1. General configuration of the hippocampal formation in the
rat, parasagittal section. DH. dorsal hippocampus; J, dorsal/ventral
hippocampal junction; VH, ventral hippocampus.
in the dorsal hippocampus (Fig. 2a,d), but an increasing per-
centage of necrotic granule neurons as the structure curved
posteriorally and ventrally. This transition was particularly
well demonstrated at the dorsal/ventral hippocampal junc-
tion (Fig. 2b,e). Extensive necrotic involvement of the fascia
dentata was found in the ventral hippocampus (Fig. 2c,f).
By day 15, the pyramidal cells, particularly those in field
CAne, were severely affected in the dorsal hippocampus (Fig.
3a) with only minimal alteration in the corresponding fascia
dentata granule cells (Fig. 3c). On the other hand, the Am-
nion's horn pyramidal neurons were largely spared in the
ventral hippocampus (Fig. 3b,d) with significant neuronal
loss in the corresponding fascia dentata granule cells (Fig.
3b,d).
During the acute intoxicatioo phase, these topographical
differences were most obvious at the extremes of the sep-
totemporal axis (i.e., dorsal vs. ventral). However, with
longer post-treatment intervals (after 2 weeks), it was possi-
ble to detect gradients of damage even within the same dor-
sal hippocampus. At these later periods of intoxication, co-
ronal sections, which illustrate more laterotemporal portions
of the dorsal hippocampus, revealed significant necrotic
changes in the fascia dentata granule cells as well as exten-
sive destruction of CA,., neurons of the Ammon's horn (Fig.
4a,c). While the CAV pyramidal neurons also showed signif-
icant involvement, neurons in CA„ and CA,„ of the Am-
mon's horn were by and large spared (Fig. 4a,b). However, it
is of interest to note that medial parasagittal sections from
the same brain, which illustrate the most septal portion of
the hippocampus, revealed extensive destruction of most
CA3 pyramidal neurons with only little injury in the corre-
sponding fascia dentata and in the CA,.; neurons of the Am-
mon's horn (Fig. 4d,e,f).
DISCUSSION
Assessment of the septotemporal distribution of TMT-
induced neuronal necrosis appears to have resolved the dis-
crepancy between reports claiming that CA:la and CA1h cells
are resistent [1,23] vs. those describing sensitivity (10.11).
Based on serial sections in coronal and sagittal planes, it is
evident that the fascia dentata granule cells were most in-
volved ventrally and dorsolateral^ (temporally) while CA1a
and CA3t) pyramidal cells were most affected dorsomedially
(septally). CAV cells were involved throughout the
entire extent of the dorsal hippocampus with the sep-
tal pole being most severely affected, and CA,., cells
were most affected at mid-septotemporal levels. Since the
population of CA, cells in the ventral hippocampus (extreme
temporal pole) of rats is relatively small and the functional
significance of the ventral hippocampus in rats is still
obscure, the full toxic impact or damage to these cells be-
comes difficult to evaluate accurately. The overall pattern of
these findings is illustrated diagramatically in Fig. 5.
An interesting phenomenon illustrated by these findings is
-------
tmt-induced hippocampal lesions
45
rag
R !«» >\T- V 0? ' * 1
B -~3.«»-^L ^ <*¦ ¦& v •
5 £> •• {CvV •< 1? X
M ^ *9' f*£ /'A> .3< %^*« -->¦ - -vV^. -v.
• yjp:
,, 3«*. * -¦'./£•?<
«Ak* rt-®- -
f -
FIG. 2. Comparison of fascia dentata(fd) granule cell damage in various portions of the hippocampal formation of the same brain section. Rat.
treated with TMT, 3 days. The percent or extent of neuronal necrosis (—•) appeared to be dorsal < junction < ventral, a-d. dorsal
hippocampus; b-e, dorsal/ventral junction; c-f, ventral hippocampus, d-f represent high magnification of similar areas as those represented in
a-c.
-------
46
CHANG AND D
>ir2 4' T-Vlt
| • •• ••/** , r • -V.* TV?
- /•>?
• ¦¦»*** _
• " • • • ' ,
' ¦ w * * • * » • rf'jRVi, 1
•V -V - •. V TSflFT*
r. «f!»» ?fcv -• ¦
** \ »v. •*'
•aV
FIG. 3. Comparison of dorsal and ventral hippocampal damage (same brain) by TMT. Rat, treated with TMT. 15 days, a-c, dorsal
hippocampus (DH). showing extensive damage to the Ammon's horn, particularly the CAV neurons. Granule cells in the fascia deniaia < fd)
only showed moderate involvement; b-d, ventral hippocampus (VH) showing marked damage to the fascia dentata granule neurons with
sparing of (he pyramidal cells of the Ammon's horn (AH). ('Denotes areas of particular damage).
that there seemed to be an inverse relationship between re-
gional lesion severity by the granule cells and for the Am-
mon's horn pyramidal neurons, particularly those in fields
CA3, and CA1b. Portions of the hippocampus which showed
extensive granule cell destruction showed little damage to
the CA5i and CAnb pyramidal neurons, while areas showing
significant damage to these pyramidal neurons had rela-
tively intact panule cells m the corresponding fascia den*
tata.
There are two phenomena which must be explained, the
differential sensitivity of subfield cell types and the gradients
of sensitivity within a subfield at different septotemporal lo-
cations. Two types of hypotheses might explain either phe-
nomenon. First, the inverse relationship between granule
cell and pyramidal cell damage might not have a direct causal
relationship. That is, the existence of a regional relationship
between granule cell damage and pyramidal cell damage
does not imply that the damage is caused by the interaction
of the cells. It is possible that the individual properties of the
cells vary regionally. Furthermore, subtle regional and sub-
field differences in membrane characteristics of cells coupled
with heightened sensitivity of cells with certain membrane
characteristics to TMT might also account for the pattern
of regional damage. It is known, for example, that at a given
septotemporal level there are significant variations in mem-
brane properties across a cell field [2.19]. Perhaps similar
kinds of variation exist along the septotemporal axis within a
given cell field.
An alternative we believe to be more likely is that differ-
ences in synaptic relationships with inputs and outputs
creates a selective vulnerability. It is well known that cells in
the dorsal and ventral hippocampus have different
anatomical connections [20], biochemical properties [16] and
electrophysiological properties [13]. Further, there are dif-
ferent behavioral consequences of lesion in dorsal vs. ventral
hippocampus [18]. One interpretation of the present data is
that the extent of damage to the CA, cells may be a function
of the density of input from intact granule cells. The absence
(cell loss) of granule cells might protect or spare some of
these neurons. Coupled to this hypothesis is the observation
that density of mossy fiber innervations of CAn cells de-
creases as one proceeds more temporally [14], potentially
accounting for the septotemporal gradient of CA1t b damage.
The invariably more extensive damage to CA1r than CAU»
may reflect the heavier input of mossy fibers to these neurons
[15], particularly including the input from the infrapyramidal
bundle [ 17],
It has been demonstrated that heightened synaptic ac-
tivation of granule cells can produce necrosis in the CAi
target neurons of the granule cell axons [23]. Such hyperac-
-------
TMT-INDUCED hippocampal lesions
47
FIG. 4. Comparison of temporal and septal portion of the hippocampus (same brain) in TMT poisoning. Rat. treated with TMT, 30 days, a~c,
coronal section (laterotemporal portion of hippocampus) showing extensive damage of the fascia dentata (fd) granule cells, CA,., and CAW
pyramidal neurons. The neurons in CAj,„ area were spared; 3-f, parasagittal section (medioseptal portion of hippocampus) showing destruc-
tion of all CA, neurons of the Ammon's horn with only mild to moderate damage to the fascia dentate and CA,., pyramidal neurons. ('Denotes
areas of particular damage.)
-------
48
CHANG AND DYER
Mib Ins
Septal Poll
Vantrti
Zk\2
Tnupml Pok
FIG. 5. Diagram illustrating the topographical gradient in cellular
damage of various cell flelds along the septal-temporal axis of the
hippocampal formation. The hippocampal formation is represented
in an "unfolded" form, such that the CA, subfield, which normally
overlies the dentate granule cells, is represented distal to CA, in-
stead. Density of hatching corresponds to extent of damage.
tivation has been postulated as a mechanism of both kainic
acid-induced [24] and TMT-induced [4,9) CA, degeneration.
Indeed, granule cell destruction prevents kainic acid-induced
CA., degeneration [21). There is also some indirect evidence
that TMT induces hyperactivation of granule cells. TMT re-
duces recurrent inhibition in the dentate gyrus [12], which
would increase granule cell activity. Both electrical stimulation
of the entorhinal cortex (perforant path) input to the granule
cells [22J and TMT administration [6] produce a loss of mossy
fiber zinc content, as assessed with the Timm stain. Finally,
Zimmer et ai. [26] have shown that stimulation of the pre-
pyriform cortex produces, via a disynaptic route, a response
in the granule cells which is greatly potentiated following
TMT. Monosynaptic activation of the granule ceils does not
seem to produce this potentiation, thereby suggesting that
the change is not in the granule cells themselves, but rather
in the way in which they are activated.
While the above explanation may account for sparing of
CA, when granule cells are destroyed, and possibly damage
to CA,j when CA, is intact, it does not account for either the
loss of granule cells or the pattern of loss of granule ceils.
Perhaps the answer to this riddle lies in the potent toxic
influence of TMT on the entorhinal and the prepyriform cor-
tices [5,7] which project their fiber connections to the fascia
dentata via the perforant path. Further studies need to be
performed to explore these questions.
ACKNOWLEDGEMENT
This research was supported in part by an EPA cooperative
agreement (CR809360). This manuscript has been reviewed by the
Health Effects Research Laboratory, U.S. Environmental Protec-
tion Agency, and approved for publication. Mention of trade names
or commercial products does not constitute endorsement or recom-
mendation for use.
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5. Chang, L. W. ant R. S. Dyer. Trimethyitin induced pathology
in sensory neurons. Neurobehav Toxicol Teratol 5: 673-696,
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6. Chang, L. W. and R. S. Dyer. Trimethyitin-induced zinc deple-
tion in rat hippocampus. In: Neurobiology of Zinc, edited by C.
J. Frederickson and 0. A. Howell. New York: Alan R. Uss,
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7. Chang, L. W. and R. S. Dyer. A time-course study of trimethyi-
tin induced neuropathology in rats. Neurobehav Toxicol Teratol
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8. Chang, L. W., G. R. Wenger, D. E. McMillan and R. S. Dyer.
Species and strain comparison of acute neurotoxic effects of
trimethyitin in mice and rats. Neurobehav Toxicol Teratol St
337-3JO, 1983.
9. Dyer, R. S. Physiological methods for assessment of trimethyi-
tin toxicity. Neurobehav Toxicol Teratol 4» 659-664, 1982.
10. Dyer, R. S.. T. J. Walsh, W. F. Wonderlin and M. Bercegeay.
The trimethyitin syndrome in rats. Neurobehav Toxicol Teratol
4: 127-133, 1982.
11. Dyer, R. S., W. F. Wonderlin and T. L. Deshields.
Trimethyitin-induced changes in gross morphology of the hip-
pocampus. Neurobehav Toxicol Teratol 4s 141-147. (982.
12. Dyer, R. S., W. F. Wonderlin. T. J. Walsh and W. K. Boyes.
Trimethyitin reduces basket cell inhibition in the dentate gyrus.
Soc Neurosci Abstr 8: 82, 1982.
13. Edinger, H., A. Siegel and R. Trojano. Single unit analysis of
hippocampal projections to the septum in the cat. Exp Neurol
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14. Gaarskjaer, F. B. Organization of the mossy fiber system of the
rat studied in extended hippocampi. I. Terminal area related to
number of granule and pyramidal cells. J Comp Neurol 178:
49-72, 1978.
15. Gaarskjaer, F. B. Organization of the mossy fiber system of the
rat studied in extended hippocampi. II. Experimental analysis of
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16. Gage, F. H. and R. G. Thompson. Differential distribution of
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TMT-INDUCED hippocampal lesions
49
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Damage to hippocampus and hippocampai connections: Effects
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19. Masukawa, L. M., L. S. Bernardo and D. A. Prince. Variations
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20. Meibach. R. C. and A. Siegal. Efferent connections of the septal
area in the rat: An analysis utilizing retrograde and anterograde
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21. Nadler. J. V. and G. J. Cuthbertson. Kainic acid neurotoxicity
toward hippocampal formation: dependence on specific excita-
tory pathways. Brain Res 195: 47-56, 1980.
22. Sloviter. R. S. A selective loss of Timm staining in the hip-
pocampal mossy fiber pathway accompanies hippocampal
granule cell seizure activity produced by sustained electrical
stimulation of the perforant path. In: Neurobiology of Zinc,
edited by C. i. Frederickson and G. A. Howell. New York:
Alan R. Liss. Inc.. in press. 1984.
23. Sloviter. R. S. and B. P. Damiano. Sustained electrical stimula-
tion of the perforant path duplicates kainate-induced elec-
trophysiological effects and hippocampal damage in rats.
Neurosci Lett 24: 279-284, 1981.
24. Sloviter, R. S. and B. P. Damiano. On the relationship between
kainic acid-induced epileptiform activity and hippocampal
neuronal damage. Neuropharmacology 20: 1003-1011, 1981.
25. Valdes, J. J., C. F. Mactutus, R. M. Santos-Anderson. R. Daw-
son, Jr. and Z. Annau. Selective neurochemical and histological
lesions in rat hippocampus following chronic trimethyltin expo-
sure. Neurobthav Toxicol Teratoi 5: 357-361. 1983.
26. Zimmer. L.. Z. Hasan, D. Woolley and L. W. Chang. Evoked
potentials in the limbic system of the rat reveal sites of
trimethyltin toxicity. Neurotn.xicology 3: 135-136, 1982.
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f
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EARLY EFFECTS OF TRIMETHYLTIN
ON THE DENTATE GYRUS BASKET CELLS:
A MORPHOLOGICAL STUDY
Louis W. Chang
Departments of Pathology and Pharmacology/Toxicology,
University of Arkansas for Medical Sciences,
Little Rock, Arkansas
Robert S. Oyer
Division of Neurotoxicology,
U. S. Environmental Protection Agency,
Research Triangle Park, North Carolina
Electrophysiological evidence for reduction of recurrent inhibition in the dentate
gyrus in animals exposed to trimethyltin (TMT) suggested alterations in the inhibitory
neurons (basket cells) by TMT. The present study was designed to investigate the
morphology of basket cells after TMT exposure. Long-Evans hooded rats were injected
with TMT chloride in a dose of 6.0 mg/kg body weight {b. w.). Tissue samples from the
dentate gyri were examined by both fight and electron microscopy at 24 and 72 h alter
TMT exposure. Except for isolated basket cell damage at 72 h. no remarkable patho-
logical changes were observed with light microscopy. Consistent with previous data,
electron microscopy revealed that the basket cells of the dentate gyrus are large
neurons situated just below the granule cell layer with characteristic large, infolded
nuclei and intranuclear filamentous rods. Increased cytoplasmic density and degenera-
tive changes of the Colgi complex were evident in the basket cells as early as 24 h after
TMT exposure. By 72 h, neuronal vacuolation, accumulation of lysosomes, and occa-
sional neuronal necrosis were observed. No significant pathological changes were
found among the granule cells at this time. This report provides the first morphologi-
cal evidence for early damage to the basket cells by TMT, which may account for the
reduction of recurrent inhibition and hyperexcitability among the granule cells
reported previously.
INTRODUCTION
Neuropathological studies with rats have revealed damage to the
pyramidal neurons in Ammon's horn after exposure to trimethyltin
The authors wish to express their thanks to Patty Webb and David Brown for their able technical
assistance and Laurie McDonald for her excellent help in the preparation of the manuscript. The
information in this document has been funded wholly or in part by the U. S. Environmental
Protection Agency under assistance agreement number R 809360-02 to Dr. I. W. Chang. It has been
reviewed by the Health Effects Research Laboratory, U. S. Environmental Protection Agency, and
approved for publication. Mention of trade names or commercial products does not constitute
endorsement or recommendation for use.
Requests for reprints should be sent to Robert S. Dyer, Division of Neurotoxicology, U. S.
Environmental Protection Agency, Research Triangle Park, North Carolina 27711.
Ml
Journal of Toxicology and Environmental Health, 16:641-653,1985
Copyright * 1985 by Hemisphere Publishing Corporation
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642
L W. CHANG AND R. S. OYER
(TMT) (Chang and Dyer, 1983,1984). The pattern of hippocampal dam-
age induced by TMT is reminiscent of damage described by others fol-
lowing excessive stimulation or excitation in the mossy fiber system
(Sloviter and Damiano, 1981a,b; Sloviter, 1984). Among potential mech-
anisms by which excessive stimulation of CA3 pyramidal neurons may
occur is the reduced inhibitory control of the granule cells, whose mossy
fiber axons form excitatory synapses with dendrites of CA3 neurons. Two
lines of evidence support the hypothesis that reduced inhibition is one
of the consequences of exposure to TMT. First, electrophysiological data
suggest a reduction in the recurrent inhibition of granule cells in the
dentate gyrus (Dyer et al., 1982; Dyer and Boyes, 1984). Second, bio-
chemical studies by Doctor et al. (1982) and by Valdes et al. (1983)
revealed a TMT-induced disruption in GABA metabolism, both in vivo
and in vitro.
The disinhibition-induced excessive stimulation hypothesis for TMT-
induced pyramidal cell damage would be further strengthened by
morphological evidence for dysfunction in inhibitory neurons. In the
dentate gyrus, it is generally assumed that basket cells provide a major
source of inhibition onto granule cells. Ramon y Cajal (1911) and
Lorente de No (1934) gave the earliest descriptions of dentate gyrus
basket cells. Most of these cells have a pyramidal-shaped soma that lies
either embedded in the granule-cell layer or directly beneath it. A single
or branching apical dendrite ascends through the granule cell layer, and
basal dendrites run parallel to this layer or flow into the region below it.
The axons of these pyramidal-shaped basket cells usually arise from an
apical dendrite and arborize within the granule-cell layer to form a
basket plexus (Gayoso et al., 1979).
Ribak and Seress (1983) described five different types of basket cells
in the rat dentate gyrus. Although these basket cells differ slightly in
their shapes and dendritic or axonal arborizations, they are believed to
have similar basic functions. Glutamic acid decarboxylase, the enzyme
responsible for synthesis of GABA, has been localized within somata as
well as axon terminals of all types of basket cells (Barber and Saito, 1976;
Ribak et al., 1978; Seress and Ribak, 1983), supporting the contention
that they are GABAergic. Electron microscopic studies of basket cells
have revealed infolded nuclei, intranuclear rods or sheets, and an abun-
dance of organelles in the perikaryal cytoplasm of these cells. Therefore
basket cells are different from granule cells (Seress, 1978; Ribak and
Anderson, 1980; Ribak and Seress, 1983), but similar to many other
GABAergic neurons in the cerebral cortex (Ribak, 1978; Ribak et al.,
1978).
The fact that mossy-fiber collaterals synapse with basket cells, which
in turn synapse onto the somata or proximal dendrites of the granule
cells (Ribak and Seress, 1983), suggests that the basket cells provide
recurrent inhibition of the granule cells (Andersen et al., 1966; Barber
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TRIMETHYLTIN AND BASKET CELLS
643
and Saito, 1976; Ribak et al„ 1978; Seress and Ribak, 1983). Recent
studies have further suggested that basket cells may mediate a feed-
forward inhibition in the dentate gyrus, whereby excitation of commis-
sural axons may cause basket-cell activation that inhibits granule cells
(Buzsaki and Eidelberg, 1981; Douglas et al., 1983; Seress and Ribak,
1984).
The present study was designed to search for evidence of damage to
the basket cells in the dentate gyrus following exposure to TMT. Empha-
sis was placed on time points relatively close to treatment for early
changes in these cells.
METHODS
Young adult male Long-Evans hooded rats (60-80 d old) were used in
the experiment. Animals were exposed to trimethyltin chloride via oral
intubation in a dose of 6.0 mg TMT/kg body weight (b.w.) (or 7.5 mg
TMT-C!/kg b.w.). Animals were then sacrificed in groups of 5 animals at
24 and 72 h following exposure. The saline vehicle was administered to
the control animals, which were sacrificed similarly to the experimental
animals.
At sacrifice, animals were anesthetized and perfused intracardially
with saline solution followed by 2.5% buffered glutaraldehyde. Brains
were then removed surgically and further immersion-fixed in a solution
of 10% buffered formalin for at least 48 h before tissue processing. After
dehydration in graded ethanols and embedding in Paraplast, brains
were sectioned at 6 ^m and stained with hematoxylin-eosin (H&E) for
general histopathological examination.
For electron microscopy, the hippocampal formation was carefully
dissected away from other brain structures and fixed in 2.5% buffered
glutaraldehyde and embedded with Epon. Semithin (1.0 /wm) sections
were made to locate the basket cells before ultrathin sections were
made for electron microscopic examination. Criteria for their identifica-
tion were those specified by others (Seress, 1978; Ribak and Anderson,
1980; Ribak and Seress, 1983), particularly large size, invaginated nuclei,
and intranuclear filamentous rods or sheets.
RESULTS
Light-microscopic survey revealed fusiform or pyramidal-shaped
neurons located just beneath the inner borders of the dentate granule
cell layers (Fig. 1). Except for occasional necrotic changes at 72 h (Fig. 2),
light microscopy did not reveal any remarkable pathology in these puta-
tive basket cells.
Electron microscopy confirmed the presence of large neurons ful-
filling the criteria for basket cells with characteristic infolded nucleus
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644
L W. CHANG AND R. S. DYER
FIGURE 1. Dentate gyrus, rat, control. The basket cell (arrowhead) appeared to be a large pyramidal
or fusiform neuron located just below the granule cell layer (G). *315.
and intranuclear filamentous rods or sheets (Figs. 3 and 4). The basket
cells also displayed well-developed cytoplasmic organelles, including
mitochondria, Golgi complex, and rough endoplasmic reticulum.
At 24 h after TMT exposure, a dense cytoplasmic appearance was
observed in many basket cells of all the animals studied. Careful exam-
ination demonstrated an increase in both rough endoplasmic reticulum
and ribosomal clusters in these neurons (Fig. 5), yielding a dense cyto-
plasmic appearance. A careful survey also revealed degenerative
changes of the Golgi complex giving rise to cytoplasmic membranous
whorls (Figs. 6, 7, and 8) in these neurons. Extensive dilatation and vacuo-
lation of the Golgi complex (Figs. 9 and 10) were also observed, leading
to multiple cytoplasmic vacuolations of many basket cells (Fig. 11). Sim-
ilar vacuolar changes in many of the interneurons within the hilar area
were also found (Fig. 12).
FIGURE 2. Dentate gyrus, TMT-treated, 72 h. Careful survey revealed isolated necrosis of the basket
cells (arrowheads), while the granule cells (G) still appeared intact. *315.
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TRIMETHYLTIN AND BASKET CELLS
645
FIGURE 3. Dentate gyrus, rat, control. A basket cell (B) located just deep to the granule cell (C)
layer, showing the characteristic intranuclear filamentous rod or sheet (arrowhead) and infolded
(arrows) nucleus. *4500.
Vacuolar changes could still be found in the basket cells 72 h follow-
ing TMT intoxication (Fig. 13). At this time of intoxication, many basket
cells also displayed an accumulation of electron-dense bodies, presum-
ably lysosomes, in their cytoplasm (Figs. 14 and 15). Necrotic changes of
the basket cells (Fig. 16), although only an occasional finding, could be
demonstrated. In sum, early degenerative and necrotic changes in the
dentate basket cells were observed following TMT exposure, suggesting
extreme vulnerability of these inhibitory neurons to TMT toxicity.
FIGURE 4. An enlargement of the basket cell from Fig. 3; showing intranuclear filamentous rod or
sheet (arrowhead) and infolded nucleus (arrows). Nu, nucleus. The cytoplasm is also abundant with
organelles. *6300.
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646
L W. CHANG AND R. S. DYER
FIGURE 5. Basket cell. TMT-treated, 24 h. An increase in cytoplasmic density, presumably due to an
increase in ribosomal clusters, was observed. Nu, nucleus; arrow, nuclear infolding; arrowhead,
intranuclear filamentous rod. *5040.
DISCUSSION
The present study provides the first morphological evidence of early
toxic damage to the basket cells of the dentate gyrus. Since these cells
mediate GABAergic inhibition and have reciprocal connections with
granule cells (Ribak and Seress, 1983), this observation is consistent with
the electrophysiological data indicating an early loss of recurrent inhibi-
tion among the dentate granule cells after TMT intoxication (Dyer et al.,
FIGURE 6. Basket cell, TMT-treated, 24 h. Degenerative changes of cytoplasmic membranes to form
myelin-figures (arrowhead) were observed. Nu, nucleus; arrow, intranuclear filamentous rod.
*5040.
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TRIMETHYLTIN AND BASKET CELLS
647
FIGURE 7. Higher magnification of Fig. 6 to show the myelin-figure formation (arrowhead) in a
basket cell cytoplasm after 24 h exposure to TMT. *10,150.
1982; Dyer and Boyes, 1984), It is important to note that significant mor-
phological changes in the basket cells occurred within 24 h following
TMT exposure, a time at which no remarkable behavioral changes and
pathological lesions have been found in either the granule cells or
pyramidal neurons at the dose used here (Chang and Dyer, 1983). While
the nature of the morphological changes (vacuolation, lysosomal
accumulation, necrosis) induced by TMT in the basket cells was nonspe-
cific, the early impact of TMT on basket-cell neurons provides clues to
FIGURE 8. Basket cell, TMT-treated, 24 h. Careful examination revealed degenerative changes of
the Golgi complex (arrowheads), which probably gave rise to the cytoplasmic myelin-figures.
*10,150.
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648
L W. CHANC AND R. S. DYER.
FIGURE 9. Basket cell, TMT-treated, 24 h. Increase in cytoplasmic matrical density with vacuolar
degenerative changes of the Colgi compex (G) typified the early changes of the basket cells. Nu,
nucleus; arrowhead, intranuclear filamentous rod. x5040.
the pathogenic mechanisms underlying the eventual necrosis of CA3
neurons in the hippocampal formation. Although many of the cellular
changes among the basket cells are potentially reversible, these changes
represent cell injuries and would affect the normal functions of these
cells.
Quantitative assessment (Seress and Pokorny, 1981) indicates that the
basket cell/granule cell ratio in the dentate gyrus is approximately 1:200.
FIGURE 10. Enlargement of a basket cell showing vacuolar changes of the Golgi complex (G) after
24 h exposure to TMT. Nu, nucleus. *10,150.
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TRIMETHVLTIN AND BASKET CELLS
649
FIGURE 11. Dentate gyrus, TMT-treated, 24 h. Extensive vacuolation of a basket cell (B) was demon-
strated. x3290.
Although there are many more granule cells than basket cells in the
dentate gyrus, the widely distributed basket-cell axons suggest far reach-
ing and potent inhibitory control. Consequently, loss of a relatively small
number of basket cells would mean a loss of inhibitory influence over a
vast number of granule cells and set the stage for an excess granule cell
output.
Although the precise function of the large polymorphic (pyramidal)
FIGURE 12. Hilus area (field CA4), TMT-treated, 24 h. Two CA4 interneurons (N) also showed similar
vacuolar changes as those observed in the basket cells. The CA4 interneurons (polymorphic neu-
rons) are believed to have physiological functions similar to those of the basket cells. x3290.
t
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650
L W. CHANG AND R. S. DYER
FIGURE 13. Basket cells, TMT-treated, 72 h. Some basket cells still showed an increased cytoplasmic
density and vacuolar changes of the Golgi complex 72 h after TMT exposure. Nu, nucleus; arrow-
head, intranuclear filamentous rod. *5950.
neurons in the hilus (CA4) area is still obscure, they are believed to have
GABAergic inhibitory function similar to that of the basket cells (Amaral,
1978; Seress and Ribak, 1983). Our present study also shows early dam-
age in these interneurons, which further suggests the selective toxic
action of TMT on the GABAergic inhibitory cells in this region of the
nervous system.
FIGURE 14. Basket cell, TMT-treated, 72 h. By this time, many basket cells displayed an increase in
electron-dense bodies, presumably lysosomes, in the cytoplasm. Nu, nucleus; arrowhead, intra-
nuclear filamentous rod. x5040.
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TRIMETHYLTIN AND BASKET CELLS
651
FIGURE 15. Basket cell, TMT-treated, 72 h. Large number of cytoplasmic dense bodies, presumably
lysosomes, was observed within the basket cells. Nu, nucleus; arrowhead, intranuclear filamentous
rod. *5040.
In summary, the present study indicates that alterations occur in
dentate gyrus basket cells shortly after administration of TMT. This find-
ing supports the hypothesis that eventual destruction of CA3 neurons by
TMT may result from overactivation from granule cells that have lost
inhibitory input.
FIGURE 16. Dentate gyrus, TMT-treated, 72 h. A large necrotic cell (asterisk), presumably a basket
cell, was found just below the granule cell layer. The granule cells (G) still showed no remarkable
changes at this time. *5950.
i
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652
L W. CHANG AND R. S. OYER
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TRIMETHYLTIN AND BASKET CELLS
653
Sloviter, R. S., and Damiano, B. P. 1981b. On the relationship between kainic acid-induced epilepti-
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Received lanuary 28, 1985
Accepted May 30,1985
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SeunthrhuvioraI Toxh ohny and Trrutnlogy, Vol. 5. pp. J37-JJ0, 1981. " Ankho InUrmitioiMl. Inc. Primed in lha U.S.A.
Species and Strain Comparison
of Acute Neurotoxic Effects of
Trimethyltin in Mice and Rats'
LOUIS W. CHANG. GALEN R. WENGER. DONALD E. McMILLAN AND ROBERT S. DYER*
Departments of Pathology and Pharmacology. University of Arkansas for Medical Sciences
Little Rock. AR 72205
and *Division of Scuroto.xicology. U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
Received 14 February 1983
CHANG. L. W., G. R. WENGER. D. E. McMILLAN AND R. S. DYER. Species and strain comparison of acute
neurotoxic effects of trimethy'tin in mice and ruts. N'EUROBEHAV TOXICOL TERATOL 3(3) 337-350. 1983.—
Pathological changes in the central nervous system in two strains of mice (BALB/c and C57BU6> and two strains of rats
(Long Evans and Sprague Dawiey > as a result of trimethyltin (TMT) intoxication were compared. Both strains of mice were
administered with trimethyltin chloride at a dosage of 3.0 mg TMT-Cl/kg b.w. while both strains of rats were exposed to 7.5
mg TMT-Cl/kg b.w. Animals were sacrificed at the time of development of observable neurological signs (tremor, aggres-
sion): 2 days for both strains of mice. 3 days for Long Evans (LE) rats, and 5 days for Sprague Daw ley (SO) rats. It was
found that there were both species and strain differences in TMT toxicity. Despite being exposed to a tower dose of TMT
and for a shorter duration of time, mice showed more prominent neurological signs and hippocampal lesions than rats.
Among (hi two strains of rats studied. LE rats were more sensitive than SD rats to TMT toxicity. The regional sensitivity of
the CNS between mice and rats was also different, with mice showing most lesions in the hippocampal fascia dentata and
rats showing more prominent neuronal damages in the olfactory cortices and hippocampal Ammotl't horn. Our present
investigation provides the first species/strain comparison on lesion development as a result of TMT intoxication.
Strain differences Trimethyltin Neurological effects
INDUCTION of overt neurological and behavioral changes
in rodents, including aggression, hyperirritabitity, tremor,
spontaneous seizures, hyperreactivity, and changes in
schedule-controlled behavior by trimethyltin (TMT) com-
pounds are well documented [t—3, tl. 13]. In rats, such be*
havioral changes have been referred to as "the trimethyltin
syndrome" [11).
Neuropathology studies with rats have revealed exten-
sive lesion development in the limbic system, including the
Amnion's horn (pyramidal neurons), fascia dentata.
pyriform cortex, entorhina! cortex, and amygdaloid nucleus
(1. 2, 9, 10], Recent investigations have indicated that mice
are much more sensitive to TMT toxicity, producing rapid
lesions in the limbic system (3,6] and developing significant
pathological changes in "extra-limbic" neurons such as
(hose in the brain stem (4] and spinal cord [7,8).
Since some laboratories have worked with rats and others
with mice, it is not clear if there are species differences in the
neuropathology produced by TMT. If some qualitative and
quantitative differences in neuropathology exist between ro-
dent species and strains, in-depth analysis of those differ-
ences may lead to an understanding of the mechanisms by
which the toxic changes occur. Furthermore, data are be-
ginning to accumulate which suggest different behavioral ef-
fects of TMT on rats and mice [12.131. A comparative
analysis of the morphological consequences of TMT expo-
sure may help to elucidate these species differences. As a
first step toward such analyses, the present investigation was
intended to establish a framework for comparative studies on
TMT-induced lesion development in rats and mice.
METHOD
Two strains of mice. BALB/c and CS7BU6 (CSV. and
two strains of rats. Long Evans (LE) and Sprague Dawiey
(SD), in groups of six were used in this experiment. All
animals used were young day old) adult males.
Trimethyltin chloride (TMT-CU was-dissolved hi a solution
of physiologic saline and administered to mice (both strains)
at a dosage of 3.0 mg TMT-Cl/kg b.w. and to rats (both
strains) at a dosage of 7.5 mg TMT-Cl/kg b.w. Because of the
steep dose-effect function for TMT [I IJ. it is important tt
note that these dosages are here expressed aa the total salt.
'The information in this document has been ftinded wholly or Sn pan by the United Stam Environmental ^wttonAgency^ under
assistance agreement numbers R 809452-01 and R W»#MM to On. L. W. Chang andD. B.McMiltoi». It has few
1-ffects Research Laboratory, U.S. Environmental Protection Agency, and approved for puWicatkwi. Mention or trade names or commercial
products does not constitute endorsement or recommendation for use.
337
-------
338
CHANG ETAL.I
Equivalent dosages of TMT as base would be 2.46 mg/kg for
mice and 6.17 mg/kg for rats. Control animals (six per strain)
were treated with the saline vehicle. Dose response studies
for the SO and LE rats and for the BALB/c mice had been
established in previous studies [2,11, 13]. The doses selected
for our present study represent sub-LD-^ doses which were
found to be effectiv«.in the induction of neurological signs
and lesions in these animals.
Animals were sacrificed at the time when observable
early neurological signs or behavioral changes (tremor and
aggression) developed: 2 days for the mice (both strains), 3
days for LE rats, and 5 days for SO rats. All animals were
deeply anesthetized and sacrificed via intracardial perfusion
with saline solution followed by 2.5% buffered giutaral-
dehyde. After perfusion, brains and spinal cords were re-
moved carefully and further fixed in 10% buffered formalin
for at least 43 hours. Tissue specimens were then dehydrated
with graded ethanols, cleared with xylene, and embedded in
Paraplasm Tissue sections were made (6-8 ^m thick) at both
cross-sectional and parasagittal planes. All sections were
stained with hematoxylin-eoi;n
-------
NEUROTOXIC EFFECTS OF TRIMETHYLTIN
1 "
• —«- m
FIG. I. BALB/c mouse, 3.0 mg/kg TMT-C1, 48 hours. Extensive necrosis and vacuolation of
the granule celJs in the fascia dentata were observed. The extensive vacuolar change gave the
tissue a spongiotic appearance. (x250)
FIG. 2. C57 mouse, 3.0 mg/kg TMT-C1, 48 hours. Large number* of the fascia dentata granule
cells appeared to be necrotic with dense-pyknotic nuclei and eosinophilic cytoplasm. (x250)
-------
FIG 3. BALB/c mouse, 3.0 mg/Vg TMT-CI. 48 hours, Ammon's horn. Despite the extensive
damage in the fascia dentata granule cells, only isolated pyramidal neurons showed vacuolar
changes (—•) in the CA|._. area. (X450)
y
«•
4".
% •
• V
• 1
. r*A
i/
' CA
3*- * *
V '•
• o
-£v'
-7s
i -
FIG. 4. BALB/c mouse, 3.0 mg/kg TMT-CI, 48 hours, hippocampus. While extensive
neuronal necrosis and vacuolation were observed in the fascia dentata (f.d.). only vacuolar
changes (—») were observed in the pyramidal neurons in the CA:lr area. (x450)
-------
NEUROTOXIC EFFECTS OF TRIMETHYLTIN
.5* * . "* * ' : " • , *
FIG. 5. BALB/c mouse, 3.0 trig/kg TMT-CI, 48 hours, pyriform cortex. Isolated neuronal
necrosis (—) also occurred in the pyriform and eniorhina! cortices, (x 100)
»•»?« M
* 1 Z-m
FIG. 6. C57 mouse. 3.0 mg/kg TMT-CI, 48 hours, olfactory tubercle. Neuronal swelling and
necrosis (—») were also evident in this area of the cortex. (x450)
-------
342
1
CHANG ETAL.
AT
. r
• ~
» ft
A * >
§A ¦' V
r , ^ # • k
f . ' ¦¦¦-
• * V -y ft* r }. t
\v >' ¦ v r -> ' ¦ ?q
• ^ • y •
** *) -* ^ .
ffi" V V ,.. \ • 52?-' -
•* ^
FIG. 7. BALB/c mouse, 3,0 mg/kg TMT-C1, 48 hours. Prominent neuronal necrosis (—•) was
found in the medial anterior olfactory nucleus. (x450)
I
r
¦it- •
jm-
'
V
. v
-S» ; f ' .
xWm
v*i.
n-
(r
Del
•>
./
8
FIG. 8. BALB/c mouse, 3.0 mg/kg TMT-C1, 48 hours, anterior olfactory nucleus. Vacuolar
changes (—») were observed in this group of neurons. (x450)
-------
NEUROTOXIC EFFECTS OF TRIMETHYLTIN
V
?v/#
4 > 5 >--V> '.. r^
* / |<* v.- 1,-^^iiViZ^
V- ^ • r i24-1^-1
5r» w
-~ » «,'• . - *
ai'i -¦ J* • - » *
f vf-r #SSlSi
'• •'¦' - •• »'• a* ¦_ :-'v. #•
' ..*'• * • ®F ,•» - „ **^&n
» *
FIG. 9. BALB/c mouse, 3.0 mg/Vg TMT-CI, 48 hours, mesencephalic trigeminal nucleus.
Marked chromatolytic and vacuolar changes (—•) of the large brain stem neurons, especially
those in the mesencephalic trigeminal and raphe nuclei, were observed. (x450)
FIG. 10. BALB/c mouse. 3.0 ring/kg TMT-CI, 48 hours, spinal cord. Extensive chromatolytic
and vacuolar degeneration of many spinal motoneurons (-•) were evident. These changes
were found in both strains of mice studied but not in the rats. (x430)
-------
11
FIG 11. LE rat. 1.5 mg/VgTMT-Cl, 3 days, fasciadentata. Significant neuronal necrosis (-»)
was observed in the granule neurons. (x450)
*
* a
* #
t
I
i
0!
-¦*
%* %
.. 4
# % #
*
i
»*
12
FIG. 12. LE rat. 7.5 mg/kg TMT-CI, 3 days. Ammon's horn. Prominent neuronal changes,
including swelling and necrosis, were observed in the CA„ pyramidal neurons. (x450)
-------
NEUROTOXIC EFFECTS OF TRIMETHYLTIN
4
"V K
FIG. 13. LE rat. 7.5 mg/kg TMT-C1. 3 days. Amnion's horn. Although not markedly exten-
sive. scattered neuronal necrosis (-•) was readily found among the pyramidal neurons in ihe
CAU area. (x 450)
/ ¦
- *4 ' /
• * m ^
'
" • •, V-
- - . V ^
- ' >*
k
>
iT-•
.
*»• , . "j|
• % i • • *
/
•
r
14 '
-jA .
w
>. •* <>:-*• •
•
• 7'
• ¦ ..?¦»*
• • f M
' 7. ,»
Vf
' "A
V *
* •' •
'? - > ; •
•* • ¦%. 2
Si 9 •
CA *• *k
>• " .o _~ % *
- y iic *v _ -
«W • / • *
* ' » r ^ w ! '
* *
. « i • • t '
'~V • / , »
V -T . . • «
ft ' *
FIG. 14. SD rat. 7. J mg/kg TMT-C1, 5 days, hippocampus. Except isolated neuronal death, no
significant damage was observed in the fascia dentata (f.d.). (x 250)
-------
346
CHANG ET AL.
'sJ ; »
J&
» ' t
''i/
*•>
> *
J
- V'J j
t •• j
. L- '
VD
FIG. 15. SD rai. 7.5 mg/kg TMT-C1,5 days, Ammon's horn. While no damage occurred in the
cells in CA:. and CA51.6 areas, isolated neuronal death (-») could be found in the CA:„
neurons. r<430)
,r
Sflk*? A**
\± m'*¦>' #»v.]
^' * -'-"s
f
"¦^r ^-- AS- ^
jte#
16 :
i7— r
•» *
. #
•:v|
FIG. 16. LE rat, 7.5 mg/kg TMT-CI, 3 days, olfactory tubercle. Neuronal swelling and ne-
crosis (—~) were found in this area of the cortex. (x450)
-------
neurotoxic effects oftrimethyltin
i .
<•
r
v. r »
* - ¦ » -»
• .»
T ¦ .
•y « * .
' -
»*
» •
"J" » '*v • N * 0 ** * * ' "**
^ >» • % ' . % ; v
S;
•V ,
T7
FIG. 17. LE rat. 7. J mg/kg TMT-C1, 3 days, pyriform conex. Marked neuronal swelling and
necrosis (—i ".ere observed among the nerve cells of this conex. (x250)
3i
&
i
r'
, I
>
•>v
.i- i
a
. Vv
V'. '
ff
«V-
<
*
¦ >
T+.
%
4
t ~
* &
<5 *
18
&
•1*
• -3
FIG. 18. SD rat, 7.5 mg/kg TMT-C1, 5 days, pyriform cortex. Necrosis of the pyriform conical
neurons (-~). (x450)
-------
I
348
CHANG ET AL.
• V^*" M- "-•¦ rJ» v* - *^iC» 'j
-v
-%
4
M
• -1
>. --1
>t'V .
* ?
i:~ vr " 1.^ -. ~
- T'V*%S> • -- •' ':>• *?£
Vs ^ ^%s42 ^•-./¦•••''.-.-i^
v**'- - .2.4 ~-. T^r'\r ^ -,; 7^-^
FTG. 19 Lc rat. 7.5 mg/kg TMT-C1. 3 days, mesencephalic trigeminal nucleus of brain stem.
Marked .-hromatolytic change (—•) was observed in these neurons. (x450)
FTG. 20. SD rat, 7.5 mg/kg TMT-CI, 5 days, mesencephalic trigeminal nucleus of brain stem.
Although much milder than those observed in LE rats, chromatolytic change of these neurons
(-») was also evident in the SD rats. (x45Q)
raphe nuclei, also occurred in both strains of rats. However,
in rats these lesions were confined to chromatolytic changes
(Figs. 19 and 20) without the vacuolar degeneration as those
observed in mice. No histopathological changes were ob-
served in the spinal motoneurons of rats under the present
experimental conditions.
The extent of lesion development and distribution were
quite consistent among animals in any given strain of rats or
mice. For comparative purposes, the extent of pathological
involvement in each strain of animals studied was estimated
on a scale of - to + + + + and is summarized in Table I.
DISCUSSION
The purpose of this report is to compare the extent of
morphological lesion development in different species and
strains of rodents (mice and rats) under the influence of
TMT. Since details of morphological changes and pathology
related to these species and strains as a result of TMT*
intoxication has been described in previous publications
(1—6. 9. 10], only general and relative pathological chances
are presented in this report. For more detailed pathological
assessment of changes in any specific area of the hi ain of a
-------
NEUROTOXIC EFFECTS OF TRIMETHYLTIN
349
TABLE 1
COMPARATIVE TMT-INDUCED LESIONS IN RATS AND MICE'
CAu
Hippocampus
CAjj.ii CA:(C f.d.
o.t.
p.c./e.c.
S.C.
b.s.
BALB/c
(3.0 mg/kg. 2 d)
-/£
-/£ +T-!-/---r-
*
+ +
+/+ +
C57
(3.0 mg/kg, 2 d)
-1 *
-/£ ++/T + T
£
i
+ +/+ + +
+/+ +
LE
(7.5 mg/kg. 3 d)
£
'-/£ + £/"!-
+
+ /+ +
-
+¦
SD
(7.5 mg/kg, 5 d)
-
£/+ £
£/ +
+
-
* All ratings were based on parasagittal sections of the dorsal hippocampus. All comparisons were made on
approximately the same levels of sectioning (see Diagram I). Spinal cords were examined at levels L,-S,. Extent and
severity of lesions were rated t'rom - to + + + + with - being no observable lesion and + + + + being very extensive
involvement, (f.d.. fascia dentata: o.t., olfactory tubercle; p.c..'e.c., pyriform/entorhinal cortices; s.c.. spinal cord;
b.s.. brain stem.)
particular strain of animal, readers should consult previous
publications [3-9].
Under the present experimental design (single high toxic
dose and short duration), it is evident that there are both
species and strain differences in TMT toxicity. Furthermore,
the species differences appear greater than the strain differ-
ences. Despite being exposed to a relatively lower dose of
TMT for a shorter period of time, both strains of mice used in
our investigation showed more severe symptomatology and
overall lesions than those in rats. Although the difference
between the two strains of mice (BALB/c and C57BL/6) was
not very remarkable, there was a significant difference be-
tween the two strains of rats (LE and SO) studied. It is
apparent that the LE rats were more sensitive than the SD
rats, showing much more damage of the hippocampal
neurons in a shorter period of time (3 days vs. 5 days).
The general lesion distribution or regional sensitivity be-
tween mice and rats was also different. For mice, the order
of neuronal vulnerability to TMT appears to be: granule cells
of fascia dentata > spinal cord motoneurons > brain stem
neurons > olfactory conical neurons (pyriform/entorhinai
cortices and olfactory tubercle) > Ammon's horn pyramidal
neurons. For rats, the equivalent comparison is: olfactory
cortical neurons > Ammon's horn pyramidal neurons (par-
ticularly C A;tc) » granule cells of fascia dentata > brain stem
neurons. No spinal cord motoneuron damage was observed
in rats. Reasons for these species/strain differences are still
not known. It must be emphasized that our present findings
reflect only acute toxic conditions (single high dose, short-
term) in mice and rats. As chronic exposure (multiple low
dose, long-term) may stimulate different toxic responses and
reactions, the distribution and extent of neuronal damage
may also vary.
The present study represents the first investigation, based
on neuropathologies criteria, of species/strain differences in
TMT toxicity. There is no doubt that the experimental design
(single acute exposure and sacrifice at time of symp-
tomatology development) offers a limited scope for compari-
son. However, the study has provided the first morphologi-
cal evidence of species and strain differences in lesion devel-
opment after TMT. This information should serve as an im-
portant cornerstone for future investigations on both struc-
tural and functional (physiological and behavioral) toxicol-
ogy of TMT compounds.
ACKNOWLEDGMENT
The authors wish to express their thanks to Ms. Patty Webb for
her able technical assistance and Mrs. Laurie McDonald for her
excellent help in the preparation of the manuscript.
REFERENCES
1. Bouldin, T. W., N. D. Goines, C. R. Bagnel) and M. R. Krig-
man. Pathogenesis of trimethyltin neuronal activity, ultrastmc-
tural and cytochemical observations. Am J Pathol 104:237-249,
1981.
2. Brown, A, W.. W. N. Aldridge. B. W. Street and R. D. Ver-
schoyle. The behavioral and neuropathologlc sequelae of intox-
ication by trimethyltin compounds in the rot. Am J Puthol 97:
5MI, 1979.
3. Chang. L. W., T. M. Tiemeyer. G. R. Wengerand D. E. McMil-
lan. Neuropathology of mouse hippocampus in acute trimethyl-
tin intoxication. Neurobehav Toxicol Tvratol 4: 149-156, 1982.
4. Chang. L. W..T. M. Tiemeyer. G. R. Wengerand D. E. McMil-
lan. Neuropathology of trimethyltin intoxication. III. Changes
in the brain stem neurons. Environ Res. in press. 1982.
5. Chang. L, W.. T. M. Tiemeyer. G. R. Wenger. D. E. McMillan
and K. R. Reuhl. Neuropathology of trimethyltin intoxication.
I. Light microscopy study. Environ Res 29: 435-444, 1982.
6. Chang. L. W., T. M. Tiemeyer. 0. R. Wenger. D. E. McMillan
and K. R. Reuhl. Neuropathology of Trimethyltin intoxication.
II. Electron microscopy study of the hippocampus. Environ
Fes 29: 445-4J8. 1982.
-------
If
350 CHANG ET AL.
7. Chang, L. W., G. R. Wenger and D. E. McMillan.
Neuropathology of trimethyltin intoxication. IV. Changes in the
spinal cord. Environ Res, in press, 1983.
8. Chang, L. W., G. R. Wenger, D. E. McMillan and R. S. Dyer.
Trimethyltin (TMT) induced lesions in the mouse anterior horn
motoneurons. Taxicologist. in press, 1983.
9. Chang, L. W., £57 Woolley and L. Zimmer. Dose and time-
course effects in neuropathology production in rats following
trimethyltin intoxication. Soc Neuroscience Annual Meeting,
Minneapolis, Minnesota, 1982.
10. Dyer. R. S., T. L. Dcshields and W, F. Womlerlin.
Trimethyltin-induced changes'in gross morphology of the hip-
pocampus. Neurobehuv Toxical Teratol 4: 141-147. 1982.
11. Dyer, R. S., T. J. Walsh. W. F. Wonderlin and M. Bercegeay.
The trimethyltin syndrome in rats. Seurobekciv Toxicol Tenunt
4: 127-133. 1982.
12. Ruppert. P. H., T. J. Walsh. L. W. Reiter and R. S. Dyer.
Trimethyltin induced hyperactivity, time-course; and pattern.
Soc Xcurosci I/th A/iitiial Mvctinii Ah\tr 244.1: 748, 1981.
13. Wenger. G. R., D. E. McMillan and L. W. Chang. Behavioral
toxicology of acute trimethyltin exposure in the mouse.
Ncurobehav Toxicol Tcratol 4: 157-161. 1982.
-------
TOXICOLOGY AND APPLIED PHARMACOLOGY 73, 564-568 (1984)
Distribution of Tin in Brain Subcellular Fractions following the
Administration of Trimethyl Tin and Triethyl Tin to the Rat1
Distribution of Tin in Brain Subcellular Fractions following the Administration of Trimethyl
Tin and Triethyl Tin to the Rat. Cook, L. L. Heath, S. M., and O'Callaghan, J. P. (1984).
Toxicol. Appl. Pharmacol. 73, 564-568. The time course of tin distribution in homogenates and
subcellular fractions of rat brain was determined following the acute administration of trimethyl
tin (TMT) and triethyl tin (TET) to the rat. Exposure to TMT resulted in lower concentrations
but greater persistence of tin in subcellular fractions compared to exposure to TET. A delayed
accumulation of tin in the mitochondrial fraction was observed following the administration of
TMT but not TET. Analysis of total protein and mitochondrial markers did not reveal differences
between the compositions of mitochondrial fractions prepared from control and TMT-treated
subjects.
Trimethyl tin (TMT) and triethyl tin (TET)
are neurotoxic to both the mature and the
developing organism. Neurobehavioral, neu-
rophysiological, neurochemical, and histolog-
ical alterations have been observed following
acute or chronic administration of these com-
pounds [see Dyer et al. (1982b) and Watanabe
(1980) for pertinent reviews]. TET, in vitro,
binds to myelin with high affinity (Lock and
Aldridge, 1975), and exposure to TET in vivo
is primarily associated with morphological and
biochemical changes in central nervous system
(CNS) myelin (Watanabe, 1980). In contrast,
exposure to TMT, while sparing myelin
(Brown et al., 1979), produces lesions in the
neuronal component of the CNS, most no-
tably to neurons of the limbic system (Brown
et al., 1979; Bouldin etal, 1981). The distinct
neurotoxicological profiles of these organotins
make them attractive candidates as neuro-
biological tools for assessing the functional
consequences of damage to different com-
ponents of the mammalian central nervous
system (Gerren et al.. 1976; Brown et al., 1979;
Walsh et al., 1982).
1 This paper has been reviewed by the Health Effects
Research Laboratory, U.S. Environmental Protection
Agency, and approved for publication. Mention of trade
names or commercial products does not constitute en-
dorsement or recommendation for use.
Despite the heightened interest in trialkyl
tin-induced neurotoxicity (Dyer et al., 1982a),
little attention has been devoted to an eval-
uation of the neuroanatomical localization or
subcellular distribution of these compounds
in the CNS. Because TET and TMT not only
affect different components of nervous tissue
but also have dissimilar octanol/water parti-
tion coefficients (Chan and Wong, 1981), some
aspects of the neurotoxic effects of these com-
pounds might be due to differential distri-
bution in nervous tissue. This hypothesis
prompted us to examine the distribution of
these compounds in subcellular fractions of
rat brain.
METHODS
Animals. Male Long-Evans rats (Charles River, Wil-
mington, Mass.) weighing 200-250 g were housed three
per cage in a temperature- and humidity-controlled animal
room maintained on a 12-hr light: 12-hr dark cycle be-
ginning at 0600 hr.
Toxicants. TET bromide was obtained from Alfa Prod-
ucts (Danvers, Mass.) and TMT hydroxide was purchased
from ICN Pharmaceuticals (Plainview, N.Y.). Both tox-
icants were dissolved in 0.9% saline and administered ip
at a dose of 6.0 mg/kg (1.0 ml/kg body wt). Dosages are
expressed in terms of the bromide (TET) or free compound
(TMT) and are equivalent to 2.49 and 4.35 mg tin/kg
body wt for TET and TMT. respectively.
Preparation ofsubcellular fractions. Following death by
decapitation, rat whole brain homogenates were prepared
0041-008X/84 $3.00
Copyright © 1984 by Acadtmic Pna. Inc.
All ri|hu of reproduction in any form raarvad.
564
-------
SHORT COMMUNICATIONS
565
in 10 vol of 0.32 M sucrose, S mm MgSO,, and 5 mM
Hepes, pH 7.4, with a glass-teflon homogenizing vessel
(Arthur Thomas, Type C). Homogenates were fractionated
by a modification of the method of Gurd et al. (1974).
Briefly, a crude nuclear fraction (Pt) was prepared from
the homogenate (H) by centrifugation at lOOOg for 10
min. The resulting supernatant fraction (St) was removed
and centrifuged at 13,000; for 20 min to yield the crude
mitochondrial pellet (P2) and a supernatant fraction (Si)
containing microsomes and soluble material. Microsomes
(P3) were separated from the soluble fraction (S3) by cen-
trifugation at 100,000g for 30 min. The crude mitochon-
drial pellet was subfractionated by centrifugation on a
discontinuous Ficoll gradient (8.0 and 14,0% w/v in 0.32
M sucrose, 5 mM Hepes, pH 7.S). Following centrifugation
for 60 min at 60,000;, the band at the 0.0%/8.0% interface
was taken as the myelin subfraction, the band at the 8.0%/
14.0% interface was taken as the synaptosome-enriched
subfraction, while the pellet at the bottom of the tube was
taken as extrasynaptosomal mitochondria. Each fraction
harvested from the gradients was resuspended in homog-
enizing buffer and then pelleted at 150,000# for subsequent
TABLE 1
Concentration of Tin in Brain Subcellular Fractions following the Administration
of Trimethyl Tin and Triethyl Tin to the Rat
Time H" S, P, P2 P,
TMT
1 hr
1.164 [88]'
0.93 [100]
1.83 [75]
0.38 [78]
0.43 [67]
(0.08)
(0.10)
(0.18)
(0.02)
(0.08)
4 hr
1.26 [95]
0.91 [98]
2.44 [100]
0.38 [78]
0.57 [891
(0.10)
(0.06)
(0.19)
(0.03)
(0.05)
12 hr
1.22 [92 J
0.89 [96]
2.08 [85]
0.44 [90]
0.53 [83]
(0.05)
(0.07)
(0.11)
(0.05)
(0.05)
24 hr
1.32 [100]
0.93 [100]
2.29 [94]
0.41 [84]
0.64 [1001
(0.07)
(0.12)
(0.14)
(0.04)
(0.06)
S days
0.96 [73]
0.70 [75]
1.74 [71]
0.49 [100]
0.57 [89]
(0.11)
(0.07)
(0.13)
(0.06)
(0.06)
10 days
0.74 [56]
0.52 [56]
1.18 [48]
0.46 [94]
0.43 [67]
(0.07)
(0.05)
(0.11)
(0.06)
(0.06)
TET
1 hr
8.40 [72]
7.96 [70]
12.22 [72]
6.10 [59]
5.65 [57]
(1.41)
(1.70)
(2.43)
(0.92)
(1.11)
4 hr
11.72 [100]
11.37 [100]
15.87 [94]
8.51 [82]
9.77 [94]
(0.85)
(1.35)
(1.29)
(1.06)
(0.91)
12 hr
11.00 [94]
11.10 [98]
16.93 [100]
9.37 [90]
9.83 [100]
(0.88)
(112)
(2.27)
(0.50)
(0.33)
24 hr
11.53 [98]
10.85 [95]
13.68 [81]
10.41 [100]
9.84 [100]
(0.71)
(1.17)
(0.49)
(0.08)
(0.92)
S days
4.86 [41]
4.44 [39]
5.07 [30]
4.56 [44]
4.40 [45]
(0.38)
(0.04)
(0.14)
(0.09)
(0.37)
lOdays
2.79 (24]
2.32 [20]
2.34 [14]
2.60 [23]
2.31 [23]
(0.44)
(0.33)
(0.27)
(0.19)
(0.18)
* See Methods for abbreviations.
* Mean ng tin/mg protein (SEM) for six independent observations.
' Values in brackets represent percentage maximum concentration of tin in each subcellular fraction.
-------
566
SHORT COMMUNICATIONS
analysis of tin. Aliquots of each subcellular fraction were
also removed for analysis of total protein (Bradford, 1976).
Evaluation of mitochondrial fraction. (1) Sodium do-
decyl sulfate-polyacrylamide gel electrophoresis (.SDS-
PAGE). Mitochondrial fraction proteins were resolved by
SDS-PAGE according to a modification (O'Callaghan et
al., 1983) of the procedure by Laemmli (1970).
(2) Activity of pyruvate dehydrogenase (PDH). Total
activity of the mitochondrial marker enzyme, pyruvate
dehydrogenase, was assayed according to the method of
Leiter et al. (1978) as modified by Browning et al. (1981).
Prior to assay, mitochondrial PDH was converted to the
dephosphorylated (active) form by incubation in the pres-
ence of calcium and magnesium (Morgan and Routten-
berg, 1981).
(3) Phosphorylation of a submit of PDH. Endoge-
nous protein kinase activity present in the PDH complex
of mitochondrial subfractions was assayed in vitro with
[3iP]ATP as a phosphate donor (Morgan and Routtenberg,
1981). Net incorporation of phosphate was measured by
scintillation spectrometry of gel slices containing the a
subunit of PDH. Prior to assay, mitochondrial PDH was
converted to the dephosphorylated form (Morgan and
Routtenberg, 1981).
Analysis of tin. Aliquots of each subcellular fraction
were added to equal volumes of concentrated nitric acid
and digested overnight at room temperature prior to anal-
ysis of tin by nameless atomic absorption spectrometry.
Standard curves were constructed by analysis of tin in
subcellular fractions (prepared from saline-treated animals)
following the addition of known amounts of TET or TMT.
A linear relationship between the amount of organotin
added and tin absorbance values was obtained for all frac-
tions except the S2 and Sj. Estimates of tin in Si and Si
~ MVUIN
O SVNAPTOKQME
A MTDCH0N0RIA
1,4
TMT
3 0.4
i «
TET
Ihr «»w 12hr M 10d
TIM AFT!* IXMNIMI
Fig. I. Time-effect curves for the distribution of tin in synaptosome, myelin, and mitochondria subfractions
following the acute administration of TMT or TET (6.0 mg/kg). Each point represents the mean ± SEM
for six inuependent determinations.
-------
SHORT COMMUNICATIONS
567
were, therefore, not included in the present study. Tin
levels were quantified in each subcellular fraction prepared
from the oiganotin-treated rats by comparison with values
obtained from the respective standard curves. Endogenous
tin was not detectable in either untreated or saline-treated
rats.
RESULTS AND DISCUSSION
Distribution of tin. The concentrations of
tin in subcellular fractions of rat brain follow-
ing the administration of TMT or TET are
presented in Table 1 and Fig. I. Several gen-
eralizations emerge from these data. (1) In
homogenates or subcellular fractions of whole
brain, substantially higher (4- to 25-fold) levels
of tin were found following the administration
of TET than following the administration of
TMT. This result is likely the reflection of an
almost 4-fold greater octanol/water partition
coefficient for TET (Chan and Wong, 1981)
resulting in greater penetration of the blood-
brain barrier by TET with subsequent parti-
tioning in lipophilic structures. (2) With the
exception of subfractions of P2 (myelin, syn-
aptosome, and mitochondria) (see Fig. 1), tin
accumulated to near maximal levels (90% of
maximum concentration or greater) within 12
hr of exposure to TMT or TET. However, in
comparison to TET, once tin reached maximal
levels, it was eliminated more slowly following
the administration of TMT. (3) For subfrac-
tions of P2 (Fig. 1), the time course of tin
distribution differed for TMT and TET. Tin
distributed more slowly and reached a max-
imum at later time points after exposure to
TMT than after exposure to TET. One striking
finding revealed by subfractionation of P2 was
that tin not only concentrated in the extra-
synaptosomal mitochondrial fraction (and to
a lesser extent in the myelin fraction), but did
not reach maximal levels in this fraction until
5 days after the administration of TMT. Sim-
ilar data were not obtained after the admin-
istration of TET (Table 1, Fig. 1).
Evaluation of mitochondrial fractions. The
onset of neuronal necrosis can occur within
3 days following acute administration of TMT
(6.17 mg/kg) (Chang et al., 1983) to the rat.
Because this effect is manifested in ultrastruc-
tural alterations at the subcellular level (Boul-
din et al., 1981), the accumulation of tin in
the mitochondrial fraction could represent al-
terations in the composition of this fraction
due to neuropathological changes produced
by TMT. To evaluate this possibility, we pre-
pared mitochondrial fractions from rats that
received either saline (0.9%) or TMT (6.0
mg/kg) 5 days earlier. The yield (total protein)
and composition (assessed by SDS-PAGE) of
the mitochondrial fraction did not differ be-
tween the samples obtained from control and
TMT-treated subjects (data not shown). The
concentration of mitochondria in the mito-
chondrial fraction was assessed by two differ-
ent indices of the mitochondrial marker, the
PDH complex. Radiometric measurements of
the activity of PDH or the phosphorylation
of its a subunit by PDH kinase did not reveal
any differences between control and TMT-
treated groups (Table 2). Furthermore, the ad-
dition of TMT, in vitro (1-100 jiM), to assays
for PDH activity was without effect (data not
shown). Taken together, these data indicate
that the concentration of mitochondria in the
extrasynaptosomal mitochondrial fraction was
not altered S days following the administration
of TMT. Therefore, the time-dependent in-
crease in the concentration of tin in this frac-
tion may represent a true accumulation of tin
in mitochondria of extrasynaptosomal origin.
TABLE 2
Pyruvate Dehydrogenase (PDH) activity and
Phosphorylation of the a Subunit of PDH 5 Days
AFTER THE ADMINISTRATION OF SaLINE OR TMT (6.0
mg/kg)
Phosphorylation of a
PDH activity
subunit of PDH
(nmol/mg
(pmol [JJP]phosphate/
protein/min)
mg protein)
Saline
7.90 ± 0.67
32.61 t 2.35
TMT
6.79 ±0.11
28.67 t 1.54
Note. Each value represents the mean ± SEM for six
independent observations.
-------
568 SHORT COMMUNICATIONS
This finding suggests that studies of the func-
tional status of this organelle are warranted
in future investigations of TMT-induced neu-
rotoxicity. These data also indicate that, where
structurally related neurotoxicants have dis-
similar biophysical properties and neurotox-
icological profiles, subcellular distribution
studies may contribute to an understanding
of differences in underlying mechanisms of
neurotoxicity.
REFERENCES
Bouldin, T. W„ Goines, N. D„ Bagnell, C. R., and
Krigman, M. R. (1981). Pathogenesis of trimethyltin
neuronal toxicity: Ultrastructural and cytochemicaJ ob-
servations. Amer, J. Pathol. 104, 237-249.
Bradford M. M. (1976). A rapid and sensitive method
for the quantitation of microgram quantities of protein
utilizing the principles of protein-dye binding. Anal.
Biochem. 72, 248-254.
Brown, A. W., Aldridge. W. N., Street, B. W„ and
Verschoyle, R. D. (1979). The behavioral and neu-
ropathologic sequelae of intoxication by trimethyltin
compounds in the rat Amer J. Pathol. 97, 59-82.
Browning, M., Baudry, M., Bennett, W. F., and
Lynch, C. (1981). Phosphorylation-mediated changes
in pyruvate dehydrogenase activity influence pyruvate-
supported calcium accumulation by brain mitochondria.
J. Neurochem. 36, 1932-1940.
Chan, Y. K., and Wong, P. T. S. (1981). Some envi-
ronmental aspects of organo-arsenic, lead and tin. In
Environmental Speciation and Monitoring Needs for
Trace Metal-Containing Substances from Energy-Re-
lated Processes (F. E. Brinkman and R. H. Fish, eds.),
Proceedings, DOE/NBS, Gaithersburg, Md.
Chang, L. W., Wenger, G. R., McMillan, D. E., and
Dyer, R. S. (1983). Species and strain comparison of
acute neurotoxic effects of trimethyltin in mice and
rats. Neurobehav. Toxicol. Teratol. 5, 337-350.
Dyer, R. S., Deshields, T. L, and Wonderlin, W. F.
(1982a). Trimethyltin-induced changes in gross mor-
phology of the hippocampus. Neurobehav. Toxicol.
Teratol. 4, 141-147.
Dyer, R. S., Walsh, T. J., Swartzwelder, H. S., and
Wayner, M. J., eds. (1982b). Neurotoxicology of the
aikyltins. Neurobehav. Toxicol, and Teratol. 4, 125—
278.
Gerren, R. A., Groswald, D. E„ and Luttges,
M. W. (1976). Triethyltin toxicity as a model for de-
generative disorders. Pharmacol Biochem. Behav. 5,
299-307.
Gurd, J. W.. Jones, L. R., Mahler, H. R„ and Moore,
W. J. (1974). Isolation and partial characterization of
rat brain synaptic plasma membranes. J. Neurochem.
22, 281-290.
Laemmli, U. K. (1970). Cleavage of structural proteins
during the assembly of the head of bacteriophage T4.
Nature (London) 227, 680-685.
Leiter, A. B„ Weinberg, M., Isohashi, F„ Utter,
M. F., and Linn, T. (1978). Relationship between
phosphorylation and activity of pyruvate dehydrogenase
in rat liver mitochondria and the absence of such a
relationship for pyruvate carboxylase. J. Biol. Chem.
253, 2716-2723.
Lock, E. A., and Aldridge, W. n. (1975). The binding
of triethyltin to rat brain myelin. J. Neurochem. 25,
871-876.
Morgan, D. G., and Routtenberg, A. (1981). Brain
pyruvate dehydrogenase: Phosphorylation and enzyme
activity altered by a training experience. Science 214,
470-471.
O'Callaghan, J. P., Miller, D. B., and Reiter, L. w.
(1983). Acute postnatal exposure to triethyltin in the
rat: Effects on specific protein composition of subcellular
fractions from developing and adult brain. J Pharmacol.
Exp. Ther. 224, 466-472.
WatanaBE, I. (1980). Organotins. In Experimental and
Clinical Neurotoxicology (P. S. Spencer and H. H.
Schaumburg, eds.), pp. 545-557. Williams & Wilkins,
Baltimore/London.
Walsh, T. J., Miller, D. B„ and Dyer, R. S. (1982).
Trimethyltin, a selective limbic system neurotoxicant,
impairs radial-arm maze performance. Neurobehav.
Toxicol. Teratol. 4, 177-183.
Larry L. Cook
Stacey M. Heath
James P. O'Callaghan*
Neurotoxicology Division
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, North Carolina 27711
Received August 20, 1983; accepted December 6. 1983
* To whom requests for reprints should be addressed.
-------
TOXICOLOGY AND ArfUED WAHMACOIjOOY 72, 75-81 (1984)
Tin Distribution in Adult and Neonatal Rat Brain
following Exposure to Triethyltin1
Larry L. Cook,* Karen Stine Jacobs,'and Lawrence w. Reiter* !
•Heurotoxicoloty Division. Health Effects Research Laboratory, U.S. Environmental Protection Agency.
Research Triangle Park. North Carolina 27711 and tToxicology Curriculum. Cmversity of
North Carolina School of Medicine. Chapel Hill, North Carolina 27! 14
Received April 4. 1983; accepted August 2. 1983
Tin Distribution in Adult and Neonaul Rat Brain Mowing Exposure to Triethyltin. Cook.
L. L.. Jacobs. K. S.. and Reite*. L W. (1984). Toxicol. Appt. Pharmacol. 71 73-81. The
uptake, distribution, and elimination of tin were determined in adult and neonatal (Postnatal
Day 3) rat brain following ip administration of triethyltin bromide (TET). Groups of five adult
CD rats were killed at 10 mitu 1 hr, 4 hr. 24 hr, 5 days, or 10 days following acute exposure to
6.0 mg/kg TET; an additional group of adult animals was lolled at 24 hr following exposure to
either 3.0. 6.0, or 9.0 mg/kg (Af - i/dosage). The tune course for tin distribution in 5-day-old
rat pups was determined by killing pupa 10 min. JO mia, 1 hr, 4 hr, 8 hr, 12 hr, 24 hr, 5 days,
10 days, or 22 days following exposure to either 3.0 or 6.0 mg/kg TET IN » 4/dosage/tirae).
Tin analyses were performed by flameleas atomic absorption spectrophotometry. The M for
total tin in the adult rat brain following 6.0 mg/kg TET waa determined to be 8.0 days. The
maximum concentration in the adult was reached at 24 hr and corresponded to 4.6, 9,6. and
16.6 ng tin/rag protein for rtniagw of 3.0, 6.0, and 9.0 mg/kg, respectively. Tin was evenly
distributed across all brain ansa studied. For animals exposed to 6.0 mg/kg TET on Postnatal
Day 5. the tVi for total tin in the brain waa 7.3 days, A maximum conceatratioB of 9.9 ng tin/
mg protein was reached at 8 hr postexposure. The ma of elimination of tin from the brain (as
measured by the diminatioa me constant kj did not diflbr significantly between adults and
neonates. However, due to a dilution effect by the rapid brain growth of the neonate, tfae con*
castration of tin in the neonatal brain following TET administration decreased significantly
Outer than that in the adult
The toxic effects of triethyltin (TET) on the
mature vertebrate central nervous system have
been well documented (Watanabe, 1980). Ad>
ministration of TET to the adult rat molts
in interstitial edema in the brain and spinal
cord white matter which is characterized by
vacuolation (spongy defeneration) of the my-
elin sheath at the interperiod line (Magee ft
ai, 1957). The behavioral toxicity of TET ia
1 This paper has beea reviewed by the Health Eftea
Laboratory, UJ. Environmental Protection
Mention of trade
for use.
characterized by impaired motor function re-
sulting in hypoactivity, unpaired grip strength,
and reduced amplitude of the startle response
(Squibb era/., 1980; Reiter era/., 1980). Both
the behavioral and pathological effects are re-
versible upon termination of exposure.
In contrast, neonatal exposure to TET pro-
duces a different profile of neurotoxic effects
(Reiter*ai., 1980; Reiter, 1982). Wender et
ai (1974) showed that TET exposure in the
6-day-old nt resulted in reduced brain weight
and delayed myelinogenesis, but that cerebral
edena does not occur. Acme exposure to TET
on Postnatal Day 3 was reported to produce
IS
004I-008X/8413.00
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. IIM.
-------
76
COOK, JACOBS, AND REfTER
delays in motor development and impaired
startle response, reduced brain weight and hy-
peractivity which persist into adulthood
(Harry and Tilson, 1981; Reiter et al.. 1981).
At least two mechanisms may account for
these age-related differences in the toxicity of
TET. First, during the early postnatal period,
the rat brain undergoes a period of rapid
growth which Dobbing (1968) termed the
"growth spurt." Therefore, differences in tox-
icity may be due to differences in the func-
tional susceptibility of the nervous system re-
lated to these maturational changes. Second,
the neonatal rat has been reported to show a
higher whole body retention of heavy metals
as well as higher levels of accumulation in the
brain (Kostial etal.. 1978: Jugo, 1977). There-
fore, differences in toxicity may be due to dif-
ferences in the amount of chemical reaching
the nervous system.
Previous studies exist describing tin distri-
bution following the administration of TET
in the adult (Rose and Aldridge, 1968: Brody
and Moore, 1962). The object of the present
study was to compare the distribution of tin
in the adult and neonatal rat brain to deter-
mine whether a difference in either peak levels
or persistence of tin could be a contributing
factor to the age-related differences in the
toxicity of TET.
METHODS
Animate. Animals used in these studies wen CD rats
obtained from Charles River Breeding Lata, Wilmington,
Mais. Neonatal exposure utilized offspring of time-preg-
nant ftmales obtained 2 days after mating; adult exposure
utilized mate rats obtained at 60 days of aa* Animals
were boused ia cages measuring 45 x 24 x 20 cm in aa
animal fecility controlled for temperature (22 ± 2'Q and
humidity (50 ± 10*) and maintained on a 12:l2-hr
Kgkcdark cyda beginning at 0600 hr. Adult males were
housed 3/cats whereas dams (with litten) wen housed
individually. Food (Purina Lab Chow) and water were
available ad libitum throughout the experiment.
Adult dotiitf. To determine the time course for tissue
dtaributioa of tin, ran received a single ip injection of
either vehicle (smile isotonic saline) or 6.0 a$/kg TET-
Br (Alpha Products. Danvets. Mass.)-the in.~.
was 1 ml/kg body weight A single group of veh,^^
iN - 5) was lulled 4 hr after injectlo„.
6.0 mg/kg were kilted at 10 min. I hr. 4 hr n hr 5 dl
or 10 days after exposure (tf - 5/groupi. Ap„TOXlmat£
5 mm poor to death, each animal was injected with i j
ml Chloropent (Fort Dodge Laboratories, Fort Dodge
Iowa). The thoracic cavity was opened and a perfusion
tube was placed in the aorta by passing it through the
heart. After perfusion with isotonic saline, the liver, kid-
neys. and brain were removed, sealed in scintillation vials,
and stored at -60°C until the tin assays were performed.
To study total tin levels in whole brain » a (unction
of dosage, rats were exposed to either 3 0. ft 0. or 91) mg/
kg I.V » 5/dosage) and killed 24 hr later. This mter.il
corresponds to the time of peak tin concentrations in the
brain.
Tin distribution in various brain areas ^as also Jeter-
mined 24 hr after administration of ^ 0 mg/kg i.v = >i
The following brain regions were analyzed by the dissection
procedure of Glowinski and Iversen (l%6i' hypothalamus,
stratum, hippocampus, cerebellum, medulla-pons. tore-
brain (minus striatum), and hmdbrain (remaining brain).
Death, perfusion, and storage of tissue were as described
above.
Neonatal dosing. At parturition fDav 0). pups were
randomized and each dam was assigned 4 male and 4
female pups. On Postnatal Day 5. pups received a single
ip injection of either 0. 3.0, or 6.0 mg/kg TET-Br m a
volume of 10 ml/kg body weight. A single group of vehicle
controls {N » 8) was killed 5 days after exposure. Pups
receiving either 3.0 or 6.0 mg/kg TET were killed at 10
min, 30 min, I hr. 4 hr. 8 hr. 12 hr. 24 hr. 5 days. 10
days, or 22 days <
-------
TIN DISTRIBUTION
77
tissue by the method of Lowry el a/. (1951), and tin con-
centration was expressed as ng tm/mg protein in the ho-
mogenized tissue. The percentage of the total TET dose
found in the brain was also calculated. Tin levels in dif-
ferent brain areas were compared by analysis of variance
at the 0.02 level of significance (SAS. 1982).
Linear regression analyses were used to calculate the
elimination rate constant (<:„) and half-life (('/:) of tin in
both adults and neonates. These analyses were performed
for both log (tin concentration) and log (total tin) venus
time, with values from the descending portion of the elim-
ination curves. The was calculated as the slope of the
regression line x 2.303, and half-life defined as 0.693/fc«
(KJaassen. 1980).
Apparatus. A Perkin-Elmer Model 603 atomic ab-
sorption spectrophotometer equipped with a Perkin-Elmer
Model HGA-2100 graphite furnace and Model AS-1 auto
sampling system was used. A Perkin-Elmer tin electrode-
less discharge lamp was used for increased stability and
sensitivity at low tin concentrations. Deuterium back-
ground correction was also employed. Graphite furnace
power supply parameters were set as follows: dry: 90"C,
30-sec ramp, and 30-sec hold: char 1000°C. 30-sec ramp,
and 60-sec hold: and atomize: 2700*0, 5-sec hold. The
analytical wavelength used was 224.6 nm, the slit width
was I mm. and the band pass was 0.7 nm. Untreated
graphite tubes were used, and the How gas was nitrogen,
with interrupt mode used during atomization.
RESULTS
Tin was not detectable in any tissue studied
from vehicle control animals, which were in-
cluded in all studies. The limit of detection
was approximately 10 ng tin/ml of brain ho-
mogenate which was equivalent to 0.3 ng tin/
mg of brain tissue. Data are presented for TET-
exposed animals only.
Adult exposure. The time courses for tin
distribution in brain, liver, and kidneys of
adult rats exposed to 6.0 mg/kg TET are pre-
sented in Fig. 1. The greatest accumulation
of tin was measured in the liver which showed
a peak concentration of 58.2 ng/mg protein
at 4 hr postinfection. For kidneys and brain,
tin levels peaked at 24 hr postinjection and
were 34.S and 12.6 ng/mg protein, respec-
tively. For brain, this peak concentration cor-
responds to 0.33% of the total dose (see bottom
of Fig. I). There was a dose-related increase
in the concentration of tin in whole brain
BRAIN
<
e
H
Z
UJ |
£ 20 f-
3 -
Z 10-
3500
\ 03 w
v ^
\ f
\ =
\ 0 2 S
; u
•0.1
01 ____0
0.1 1.0 10.0 100
TIME AFTER tmtlkg TET. Fir
Fig. I. (Top) Concentration of tin in liver, kidney, and
brain in adult tats following exposure to 6.0 mg/kg TET.
ip. (Bottom) Total tin and percentage of dose in adult
brains after 6.0 mg/kg TET, ip. Percentage of dose: the
mean value for total tin in whole brains of the 24-hr
exposure group is 0.33% of their average dose. Each data
point is the mean ± SE of 5 animals.
measured 24 hr after exposure. Following 3.0.
6.0, and 9.0 mg/kg TET, the resulting whole
brain tin concentrations (±SE) were 4.6
(±0.8), 9.6 (±0.8), and 16,6 (±1.1) ngtin/mg
protein, respectively. However, as shown in
Fig. 2, there was no significant differential dis-
tribution of tin in the brain areas examined
[ANOVA, fi(6,28) - 1.83, p « 0.13].
Neonatal exposure. Neonatal exposure to
either 3.0 or 6.0 mg/kg TET resulted in de-
tectable whole brain levels of tin within 10
min after exposure (Fig. 3). Following 6.0 mg/
kg TET, a maximum concentration of 9.9 ng
tin/mg protein occurred 8 hr after exposure;
the peak amount of total tin corresponded to
1.5% of the total dose (bottom of Fig. 3). The
brain concentration of tin following 3.0 mg/
kg TET was approximately 5 ng/mg protein
1 3000~
5 25001—
?
Z 20001-
2 "001-
o
* 10001-
500).—
-------
78
COOK, JACOBS, AND REITER
20 r
2 181-
16 V-
z
o
121-
t-i
10-
0-
jr j
CER MP HYP HIPP STB FOR HIN
FiC. 2. Concentration of tin in various brain regions
of adult rats measured 24 hr following an ip injection of
9.0 mg/kg TET. Each value is the mean £ SE of 5 animals.
Cerebellum (CER); medulla-pons (MP); hypothalamus
(HYP); hippocampus (HIP); striatum (STR); forebrain
minus striatum (FOR); and hindbrain minus hypothal-
amus and hippocampus (HIN).
between 1 hr and 5 days and no clear peak
was seen.
Elimination rate constants and half-lives.
fcel and i'/i values are presented in Table 1.
When calculated on the basis of total tin, there
was no significant difference in the rate of
elimination of tin from the brain between ne-
onates and adults. However, when the rate of
change of tin concentration was calculated,
tin concentration decreased nearly twice as
fast in the neonate as in the adult brain
(Fig. 4).
DISCUSSION
Rose and Aldridge (1968) studied the dis-
tribution of tin in rats following iv adminis-
tration of triethyltin chloride labeled with
IIJSn. Their data indicate that peak levels of
tin in the brain were reached between 4 and
24 hr. Our data agree with this finding. Tin
concentration in the liver peaked at an earlier
time (4 hr) than in the brain or kidney, as
might be expected following ip administration.
The dose-related increase in tin concentration
indicates that, over the dosage range studied,
entry of tin into the brain is a nonsaturable
phenomenon. The finding that tin is distrib-
uted evenly across brain areas is in agreement
with Rose and Aldridge (1968).
Although peak concentrations of tin are
comparable in the adult and neonate, a greater
percentage of the administered dose is ob-
served in the neonate brain than in the adult
brain (1.5% as compared to 0.33%). One factor
which may contribute to this difference is the
greater relative brain weight of the neonate
(4%) vs that for the adult (0.33O7c). If tin dis-
tribution were determined solely on the basis
of organ weight, however, the neonatal brain
should be receiving 4% of the administered
dose rather than only 1.5%. Therefore the
neonatal brain accumulates a higher percent-
age of the administered dose than does the
adult brain, but less than would be expected
on the basis of brain weight relative to body
weight.
rm
DOSE, mg / kg
Hi
I—1 1 U 1,111
I i 11 Mill 1 i i r 11iii—i i i i rim
I I llll
1.0 10.0 100
TtMi AFTIP TET. hr
1000
Flo. 3. (Top) Time course for tin concentration in neo-
natal rat brain following 3.0 and 6.0 mg/kg TET. ip. (Bot-
tom) Total tin and percentage of dose in neonatal brains
after 3.0 and 6.0 mg/kg TET, ip. Percentage of dose; the
mean value for total tin in whole brains of 24 hr. 6.0-
mg/kg exposure group is 1.5% of their average dose. Each
data point is the mean ± SE of 4 animals.
-------
TIN DISTRIBUTION
79
100
30
SO
40
20
ADULT
TET DOSAGE, mg / kg
•8.0
« 3 0
NEONATE
6 8 10 12 14 IS 18 20 22
TIME AFTER TET day*
Fra. 4. (Top) Log of tin concentration in adult brain following 6.0 mg/kg TET, ip. (Bottom) Log of tin
concentration in neonatal brain following 3.0 and 6.0 mg/kg TET. ip.
TABLE 1
Elimination Rate Constants and Half-Lives or
Tin Based on Total Tin and Tin Concentration in
Adult and Neonatal Rat Brain
Tin
Total tin concentration
**
*-
Adult
0.087
8.0
0.082
8.3
Neonate
0.094
7.3
0.16
4.3
The elimination rate constants and t'h val-
ues for total tin in the brain differ little between
adult and neonates. This finding indicates that
the process for clearance of TET from adult
and neonatal brains may be similar. This con-
clusion differs from results reported by fCostial
et ai (1978) for inorganic metals, which in-
dicate a slower clearance in the neonate than
in the adult. The rate of decrease of concen-
tration, however, is much greater in the ne-
onate. This difference is likely the result of a
-------
80
COOK. JACOBS, AND REITER
dilution effect due to increases in protein and
other constituents in the rapidly growing rat
brain (Agrawal and Himwich. 1970).
These data indicate that following Postnatal
Day 5 administration of TET, a significant
amount of tin persists in the brain for several
days. This time period of exposure encom-
passes the proliferative period of many cell
types, including the maximum proliferation
of oligodendroglial cells close to Day 5
(Schonbach ei al.. 1968) and the beginning of
myelinogenesis at Day 7 (Gottleib et al., 1977),
as well as many other processes {Davison and
Dobbing, 1968). O'Callaghan et al. (1983)
demonstrated that Postnatal Day 5 adminis-
tration of TET causes a decrease in myelin
basic protein (a marker for total myelin con-
tent) and hypothesized that TET interferes
with myelinogenesis. Our data are consistent
with this hypothesis in demonstrating that fol-
lowing Postnatal Day 5 administration of
TET, measurable tin levels persist well into
the period of myelinogenesis.
In considering the toxicity of TET, however,
it may be more appropriate to look at the
concentration of tin (ng tin/mg protein) rather
than total tin. As noted earlier, the rapid
growth of the neonatal brain has the effect of
diluting the tin concentration, and may be as
effective in reducing the toxic potential of TET
as actual elimination of tin from the brain.
This finding would indicate that TET may be
producing its toxic effects in a relatively short
time following Postnatal Day 5 administra-
tion, supporting the findings of Reiter et al.
(1982) that injection of TET on Postnatal Day
10 or IS fails to produce the same spectrum
of behavioral effects as Postnatal Day 5 ad-
ministration. Thus it appears that the sensi-
tivity of the neonate to TET is not due to
higher levels or prolonged exposure, but rather
to the presence of TET during a sensitive crit-
ical period in development.
REFERENCES
Aorawal, H. C., and HtMwtcH, W. H. (1970V Amino
acid*, proteins and monoamines of developing brain.
In Developmental Neurobiology (W. A. Himwich. ed.).
pp. 287-310. Thomas, Springfield. [II.
Brody, T. M., and Moore, K. E. (1962). Biochemical
aspects of triethyltin toxicity. Fed. Proc. 21, 1103-1106.
Davison, a. N„ and Dobbing. J.! (968). The developing
brain. In Applied Seurochemisiiy (a. N. Davison and
J. Dobbing. eds.), pp. 253-286. Blackwell. Oxford.
Dobbing, J. (1968). Vulnerable periods in developing
brain. In Applied Seurochemtsirx (A. V. Davison and
3. Dobbing, eds.). pp. 287-316. Blackwell. Oxford.
Gottleib, a„ keydar. 1.. \nd Epstein. H. T < iy i.
Rodent's brain growth stages: An analytical review Birl
Xeonate 32, 166-176.
GLOW1NSKI, J., and Nersen. L. L. <19661. Regional
studies of catecholamine the rat brain-l. J SeurtKhem.
13, 655-669.
Harry. G. J., and Tilson, H, a. (19811. The effects of
postpartum exposure to tnethyltin on the neurubehav-
ioral functioning in rats. Seurmoxkutw 2, 283-296.
JUGO, S. (1977). Metabolism of toxic heavy metals in
growing organisms: A review. Environ. Res I J, 36-46.
KJ-aassen, C. D. <1980). Absorption, distribution, and
excretion of toxicants. In Toxicology: The Basic Science
of Poisons (J. Doull. C. D. KJaassen. M. O. Amdur.
eds.), pp. 28-55, Macmillan Co., New York.
K0577AL, K.. Kello. D.. luco, S.. Rabar. I.. A.VD Mal-
iJKOVic, T. (1978). Influence of age on metal metab-
olism and toxicity. Environ. Health Persp. 25, 81 -86.
Lowry, O. H.. Rosebrough, N. J.. Farr, a. L., and
Randall, R. J.(1951). Protein measurement with the
Folin phenol reagent. J. Biol. Chem. 193, 265-275.
Magee, P. N, Stoner. H. B.. and Barnes. J M.i 1957).
The experimental production of edema in the central
nervous system of the rat by tnethyltin compounds. J.
Palhoi. Bacterial. 73, 107-124.
Mushak, P., Kjugman, M. R-. and Mailman. R. B.
(1982). Comparative organotin neurotoxicity in the de-
veloping car. somatic and morphological changes and
relationship of accumulation of total tin. Xeurobehav.
Toxicol. Teraiol. 4, 209-215.
O'Cauachan. J. P., Miller. D, B.. and Reiter, L. w
(1983). Acute postnatal exposure to tnethyltin in the
rat; effects on specific protein composition of subcellular
fractions from developing and adult brain. / Pharmacol.
Exp. Ther. 234, 466-472.
REITER, LW.(l 982). Age-related effects of chemicals on
the central nervous system, (n Banbury Report II. pp.
245-267. Cold Springs Harbor Laboratory. Cold Spring
Harbor, N.V.
Retter, l w„ Heavnek, G. B., Dean. K. F.. and R up-
pert, P. H. (1981). Developmental and behavioral ef-
fects of early postnatal exposure to triethyltin in rats.
Seurobthm. Toxicol. Teratoi. 3, 285-293.
Rhteh, L., Kido, K.. Heavnek, G.. and Ruppert. P.
(1980). Behavioral toxicity of acute and subacute ex-
posure to triethyltin in the rat. Neurotoxicology 1,97-
112.
-------
TIN DISTRIBUTION
81
Reiter. L. W.. Ruppert, P. H., and Dean, K. F. (1982).
Developmental and behavioral toxicity of triethyltin in
ran as a function of age at postnatal exposure. Meu-
rotoxicology 3, 134.
Rose, M. S., and Aldrjdge, W. N. (1968). The inter-
action of triettayltin with components of animal tissues.
Biochem. J. 106, 821-828.
SAS Institute Inc. (1982), SAS User's Guide: Statistics,
1982 Edition. Cary, NC: SAS Institute Inc.
Schonbach, J.. Hu, K. H.. andFriede, R. L. (1968).
Cctlular and chemical changes during myelination: his-
tologic. autoradiographic, histochemical, and biochem-
ical data on myelination in the pyrimidal tract and
corpus callosum of rat J. Comp. Neurol. 134, 21 -36.
Squibb, R. E„ Carmichael, N. O., and Tilson, H. a.
(1980). Behavioral and t\eur*>morphotogicai effects of
triethyl tin bromide in adult rats. Toxicol. Appl. Phar-
macol. 55, 188-197.
Watanabe, I. (1980). Organoiins. In Experimental and
Clinical Neurotoxtcology (P. S. Spencer and H. H.
Schaumberg, eds.), pp. 545-557. Williams Sl Wilkins,
Baltimore.
WENDER, M„ MULAREK, O.. AND PtECHOWSKl, A. (19741.
The effects of triethyltin intoxication at the early stage
of extrauterine life on cerebral myelinauon. Seuropathoi.
Pol. 12, 13-17.
-------
13
-------
TOXICOLOGY AND APPLIED PHARMACOLOGY 76, 344-348 (1984)
Tin Distribution in Adult Rat Tissues after Exposure
to Trimethyitin and Triethyltin1
Larry L. Cook,* Karen E. Stine,*-! and Lawrence w. Reiter^
*Neurotoxicology Division, Health Effects Research Laboratory, U.S. Environmental Protection Agency,
Research Triangle Park, North Carolina 2771] and t Toxicology Curriculum, University of
North Carolina School of Medicine, Chapel Hill, North Carolina
Received March 26, 1984; accepted June 8, 1984
Tin Distribution in Adult Rat Tissues after Exposure to Trimethyitin and Triethyltin. Cook,
L. L., Stine, K.. E., and Retter, L W. Toxicol. Appi. Pharmacol. 76, 344-348. The time
course of distribution of tin in the adult rat was determined in brain, liver, kidneys, heart, and
blood following single ip administrations of trimethyitin hydroxide (TMT) and triethyltin
bromide (TET). Adult Long-Evans rats were killed I, 4, 12, and 24 hr, and at S, 10, or 22
days following injection of TMT and TET (N ¦ 6/time), and tissues were analyzed for total
tin by atomic absorbance spectroscopy. TET exposure resulted in higher tin concentrations in
brain, liver, and kidney tissues, while the two trialkyltins resulted in approximately equal tin
concentrations in the heart and blood. Rates of elimination of tin (expressed as elimination
rate constants, were greater in all tissues following TET exposure than following TMT
exposure. The concentration of tin in the brain 12 hr after TMT exposure was 4.4, 8.S, and
12.7 ng tin/mg protein for dosages of 3.0, 6.0, and 9.0 mg/kg, respectively. Tin was evenly
distributed across the cerebellum, medulla-pons, hypothalamus, hippocampus, and striatum
following TMT exposure. These results describe major differences in the disposition and rates
of elimination of tin from body tissues after TMT and TET exposure, and demonstrate that
the regional disposition of tin is not related to the region-specific pathology reported following
TMT exposure, e I9S4 Academic Pre* lac.
Interest in trialkyltin compounds has in-
creased in recent years because of their potent
neurotoxicity and usefulness as model com-
pounds in neurobiological research. The neu-
rotoxicity of trimethyitin (TMT) and tri-
ethyltin (TET) is quite different in the adult
rat both with respect to behavioral effects
(for review see Reiter and Ruppert, 1984)
and neuropathological effects (Brown et ai,
1979; Watanabe, 1980). Neuropathology ob-
served in rats exposed to TMT consists of
1 This paper has been reviewed by the Health Effects
Research Laboratory, U.S. Environmental Protection
Agency, and approved for publication. Mention of trade
names or commercial producn does not constitute en-
dorsement or recommendation for use.
neuronal damage primarily within the hip-
pocampus, amygdala, pyriform cortex, and
neocortex (Brown et al., 1979*, Bouldin et ai,
1981). The characteristic pathological effect
of TET exposure is interstitial edema
throughout the white matter of both brain
and spinal cord resulting from splitting and
vacuolation of the myelin sheath at the in-
terperiod line (Magee et al., 1957; Watanabe,
1980).
Although the differential toxicity of these
two trialkyltins has been documented, their
pharmacokinetic behavior has not been com-
pared using exposure times adequate to cal-
culate half-lives and elimination rate con-
stants. A 5-day distribution profile of TET
in body tissue and brain regions (Rose and
0041-008X/84 $3.00
Copyrisht c I9S4 by Acadtmic Pres. Inc.
All right* of reproduction in *ny ftm itmrrid.
344
-------
TIN DISTRIBUTION
345
Aldridge, 1968) and a 2-day time course of
TMT in the blood, plasma, and brain (Brown
et al., 1979) have been reported. Cook et al.
(1984a) compared the time course of distri-
bution of tin in brains of neonatal and adult
rats, and described the body tissue and the
brain regional distribution of tin in the adult
rat after exposure to TET. A recent study
(Cook et al., 1984b) has compared the distri-
bution of tin in subcellular fractions of adult
rat brain following exposure to TMT and
TET. The present investigation was designed
to compare the time course and rate of
elimination of tin in different body tissues
following exposure to these two trialkyltin
compounds, and to examine a possible rela-
tion between regional pathology and dispo-
sition of tin in the brain after exposure
to TMT.
METHODS
Animals. Male Long-Evans rats were obtained from
Charles River Breeding Labs, Wilmington, Mass. at 60
days of age. Animals were housed in cages measuring 4$
X 24 X 20 cm in an animal facility controlled for
temperature (22 ± 2aQ and humidity (50% ± 10%) and
maintained on a 12:12 hr light-dark cycle beginning at
0600 hr. Animals were housed three/cage; food (Purina
Lab Chow) and water were available ad libitum.
Exposures. To study the time course for tissue distri-
bution of TET and TMT, rats received a single ip
injection of either 6.0 mg/kg TMT hydroxide (ICN
Pharmaceuticals Inc., Plainview, N.Y.) or 6.0 mg/kg
TET bromide (Alfa Products, Dan vers, Mass.). Dosages
of both compounds were calculated as free base and
administered in a vehicle of sterile isotonic saline. The
injection volume was 1 ml/kg body weight. Animals
were killed at I, 4, 12, and 24 hr and at S, 10, and 22
days after injection * 6/group). A single group of
vehicle controls (N ¦ 4) was killed 12 hr after injection.
Approximately 3 min prior to death, animals were
injected with 1j0 ml Chloropent (Fort Dodge Laboratories,
Inc., Fort Dodge, Iowa), the thoracic cavity was opened,
and a blood sample was removed from the heart with a
Vacutainer (Becton-Didrinson) containing EDTA. A
perfbsion tube was placed in the aorta by passing it
through the heart After perfusion with normal saline,
the liver, kidneys, heart, and brain were removed, sealed
in scintillation vials, and stored at -60*C until the tin
assays were performed.
To determine total tin concentrations in whole brain
as a function of TMT dosage, animals were killed 12 hr
after exposure to either 3.0, 6.0, or 9.0 mg/kg (N = 6/
dosage). In a separate group of animals, the tin distribution
in specific brain regions was determined 12 hr after
exposure to 6.0 mg/kg TMT (N = 10). Animals were
perfused as described above and the following brain
regions removed according to Glowinski and Iverson
(1966): cerebellum, medulla-pons, hypothalamus, hip-
pocampus, and striatum. Samples were weighed and
stored in polycarbonate vials at -60°C prior to tin
analysis.
Tin analyses and calculations. Brain, kidney, and
heart tissues were thawed and homogenized in 4 volumes
(w/v) of 2% nitric acid (ACS grade, Fisher Scientific
Company) in glass homogenizers with a Teflon/stainiess-
steel pestle. Liver tissues were homogenized in 2 volumes
(w/v) of 2% nitric acid. Aliquots of the tissue homogenates
were combined with equal volumes of concentrated
nitric acid and digested overnight at room temperature
(Mushak et al., 1982) before analysis by flameless atomic
absorption spectroscopy. Aliquots of blood were similarly
digested in 2 volumes of concentrated nitric acid at
37°C. A Perkin-Elmer Model 603 atomic absorption
spectrophotometer equipped with a Perkin-Elmer Model
HGA-2100 graphite furnace and Model AS-1 auto sam-
pling system was used. A Perkin-Elmer tin electrodeless
discharge lamp and deuterium background correction
were also employed (for more details, see Cook et al..
1984a).
Tin quantification was based on standards which were
prepared from homogenates of appropriate tissues from
control animals and spiked with known amounts of
TMT or TET (free base). These were digested, diluted
according to sample dilutions, and analyzed concurrently
with the experimental tissues. Tin concentrations are
expressed as ng tin/mg protein or n% tin/ml of blood.
Protein concentration in homogenates of brain, liver,
kidney, and heart was analyzed by the method of Lowry
etal. (1951).
Ttn concentrations in different brain regions were
compared by an analysis of variance (SAS, 1982). Linear
regression analyses were used to calculate the first order
elimination rate constants (X^) and half-lives (T,n) of
tin in different body tissues following exposure to TMT
and TET. Hie analysis were performed using log [tin]
versus time, beginning at the time of peak [tin]. The K*
was calculated as the slope of the regression line x 2.303,
and Tta as 0.693/Kt (Klaassen, 1980).
RESULTS
Tissues from control animals were included
in the time course analyses of each tissue to
confirm absence of tin contamination result-
ing from tissue preparation and digestion.
Tin was not detectable in any tissue prepared
from control animals.
-------
346
COOK, STINE, AND REITER
The time courses of tin distribution in
brain, liver, kidneys, heart, and blood follow-
ing exposure to 6.0 mg/kg TMT and TET
are given in Fig. 1. These data indicate that
after ip administration, both TMT and TET
were quickly absorbed into body tissues. Ex-
posure to TET produced higher concentra-
tions of tin in the brain, liver, and kidneys
than TMT for exposure times of 24 hr or
less. The concentration of tin in both the
heart and blood was approximately equal
after exposures to TMT and TET.
The A"e| and 7*t/2 values of tin following
exposure to TMT and TET are presented in
Table 1. TET exposure resulted in shorter
tin half-lives and greater elimination rate
constants than did TMT exposure.
Tin concentration in whole brain, mea-
sured 12 hr after TMT exposure, was dose-
related. Following 3.0, 6.0, and 9.0 mg/kg
TMT, mean tin concentrations (±SE) were
4.4 ± 0.4, 8.5 ± 0.5, and 12.7 ± 0.4 ng tin/
mg protein, respectively. Finally, when tin
concentrations in the cerebellum, medulla-
14
12
10
t
«
4
2
0
70
20
SO
I
41
¦1000
»
21
II
m m ira
M M M II
TIM Af Tf R f XmtUM, kr
Fig. 1. Time coune of tin concentration in brain, liver, kidneys, heart, and blood in adult rats following
6.0 mg/kg TMT and TET. Each data point is i ± SE (S - 6Aime point).
-------
TIN DISTRIBUTION
347
TABLE 1
Tin Half-ufe and Elimination Rate Constants
in adult Rat Tissues after Single Exposures to
6 0 mg/kg TMT and TET
TMT
TET
T *
' 1/2
-A*.
Tw
Brain
0.068
10.2
0.15
4.6
Liver
0.047
14.7
O.ll
6.1
Kidney
0.058
11.9
0.12
5.6
Heart
0.061
11.3
0.20
3.4
Blood
0.071
9.8
0.28
2.5
4 T
tn
slope of semilogarithmic regression X 2.303.
¦ 0.693/A^i, in days.
pons; hypothalamus, and hippocampus were
compared following exposure to 6.0 mg/kg
TMT (Fig. 2), there was no significant differ-
ence in regional tin disposition {ANOVA,
F(4, 45) « 1.74, p < 0.161.
DISCUSSION
The present study compares the pharma-
cokinetic behavior of two trialkyltins, TMT
and TET, in major body tissues, and describes
the regional distribution of tin in the brain
following TMT exposure. Exposure times of
sufficient length were employed for accurate
calculation of elimination kinetics. Following
equal dosages, calculated as the free base, tin
was distributed in and eliminated from several
body tissues differently (Fig. 1 and Table 1).
These data demonstrate that following ad-
ministration of TET, tin reaches higher con-
centrations in the brain, liver, and kidneys,
and is eliminated from all tissues at greater
rates compared to TMT exposure. The con-
centration of tin in the heart, however, is
approximately equal following exposure to
TMT and TET. These results also indicate
that tin concentration in the whole brain
after TMT exposure is linearly related to
dosage and is evenly distributed across specific
brain regions (Fig. 2), which is in agreement
with the even regional disposition of tin in
the rat brain following exposure to TET
(Cook et al., 1984a).
The greater concentration of tin in several
of the tissues following exposure to TET was
likely due to the almost fourfold greater
octanol/water partition coefficient for TET
(Chan and Wong, 1981) permitting greater
penetration of the blood-brain barrier and
enhanced partitioning into lipophilic tissue.
Although the dosages used in this study were
equivalent on the basis of the free base of
the alkyltin, the dosage of elemental tin was
greater by a factor of 1.27 in the. TMT
exposures (1 mg of TMT contains a 1.27-
fold greater amount of tin than I mg of
TET). This difference is noteworthy since the
analysis of tin in this study was based on the
analysis of elemental tin. Therefore, greater
concentrations of tin were observed in the
brain, liver, and kidneys following TET ex-
posure even though this dosage was equiva-
lent to lower dosages of elemental tin; this
observation may be related to the greater
lipophilicity of TET. The directional differ-
ence in the reported LD50 values for the two
compounds (oral TET, 5.7 mg/kg and TMT,
Fio. 2. Concentration of tin in specific brain regions
of adult rats measured 12 hr after ip injections of 6.0
mg/kg TMT. Each data point is x ± SE of 10 animals.
Cerebellum (CER); meduUa-pons (MP); hypothalamus
(HYP); hippocampus (HIP); and striatum (STR).
-------
348 COOK, STINE,
12.6 nig/kg) may also be related to this
difference in lipophilicity.
The T1/2 of tin in the whole brain following
TET exposure in adult Long-Evans rats was
4.6 days in this study, while this value was
previously determined to be 8.5 days in adult
CD rats (Cook et al., 1984a). This shorter
T\a of tin in Long-Evans rats was confirmed
when data presented by Cook et al. (1984b)
were evaluated. The Txn of tin from these
data was 4.2 days. These comparisons indicate
a possible strain difference in the half-life
and rates of elimination of tin in brains of
rats treated with TET.
Since the present study, which was limited
to elemental tin analysis, demonstrates that
tin is evenly distributed in different regions
of the brain after TMT exposure, differential
distribution of tin does not explain the ap-
parently severe pathology described for limbic
structures in animals acutely exposed to TMT
(Brown et al., 1979; Bouldin et al., 1981).
Selective pathology may occur as a result of
vulnerability of certain specific cell types
within the brain to a uniform distribution of
tin or alkyltin. Alternatively, the selective
pathology may be related to differential re-
gional distribution of either the parent com-
pound, TMT, or a specific deacylated metab-
olite, as recently demonstrated in a study
relating region-specific brain pathology and
distribution after methylmercury exposure
(Vandewater et al.. 1983). A more relevant
measure might therefore be the regional dis-
tribution of the parent compound, TMT,
and its metabolites. Employment of proce-
dures now available for the speciation of
alkyltins (Aldridge and Street, 1981; Means
and Hulebak, 1983) might more accurately
relate the distribution of TMT with regional
neuropathy.
REFERENCES
ALDRIDOE, W. N., AND STREET, B. W. (1981). SpectTO-
photometric and fluorimetric determination of tri- and
di-organotin and -organolead compounds using dithi-
zone and 3-hydroxyflavone. Analyst 106, 60-68.
Brown, a. w., aldridge, W. N„ Street, B. W., and
Verschoyle, R. D. (1979), The behavioral and neu-
AND REITER j ^
ropathologic sequelae of intoxication by trimethyltin
compounds in the rat. Amer. J. Pathol. 97, 59-82.
Bouldin, T. W„ Goines, N. D., Bagnell, C. R., and
Krigman, M. R.(198l). Pathogenesis of trimethyltin
neuronal toxicity. Amer. J. Pathol. 104, 237-249.
Chan, Y. K.., and Wong, P. T. S. (1981). Some
environmental aspects of organo-arsenic, lead and tin.
In Environmental Speciation and Monitoring Needs
for Trace Metal-containing Substances from Energy-
related Processes (F. E. Brinkman, and R. H. Fish,
eds.). Proc. DOE/NBS, Gaitsersburg, MD.
Cook, L. L., Jacobs, K. S., and Reiter, L. W. (1984a).
Tin distribution in adult and neonatal rat brain follow,
ing exposure to triethyltin. Toxicol. Appl. Pharmacol.
72, 75-81.
Cook, L. L., Heath, S. M., and O'Callaghan,
J. P. (1984b). Distribution of tin in brain subcellular
fractions following the administration of trimethyl tin
and triethyl tin to the rat. Toxicol Appt. Pharmacol.
73, 564-568.
Glowinski, J., and Iverson, L. L. (1966). Regional
studies of catecholamines in the rat brain-l. J. Neu.ro-
chem. 13, 655-669.
Klaassen, C. D. (1980). Absorption, distribution and
excretion of toxicants. In Toxicology: The Basic Science
of Poisons (L. Doull, C. D. Klaassen, and M. O.
Amdur, eds.). pp. 28-55. Macmiilan, New York.
Lowry, O. H., RosebrouGH, N. J., Farr, A. L., and
Randall, R. (1951). Protein measurement with the
Folin phenol reagent. J. Biol. Chem. 193, 265-275.
Magee, P. N., Stoner, H. B., and Barnes, J. M.
(1957). The experimental production of edema in the
central nervous system of the rat by triethyltin com-
pounds. J. Path. Baa. 73, 107-124.
Means, J. G, and Kulebak, K. L (1983). A method-
ology for speciation of methyltins in mammalian
tissues. Neurotoxicology 4, 37-44.
Mushak, P., Krigman, M. R., and Mailman,
R. B. (1982). Comparative organotin toxicity in the
developing rat: Somatic and morphological changes
and relationship to accumulation of total tin. Neuro-
behav. Toxicol. Teratol. 4, 209-215.
Reiter, L W„ and Ruppert, P. H. (1984). Behavioral
toxicity of trialkyltin compounds: A review. Neuro-
ToxicologyS, 177-186.
Rose, M. S., and Aldridge, W. N. (1968). The inter-
action of triethyltin with components of animal tissues.
Biochem. J. 106, 821-828.
SASINTSTTTUTE Inc. (1982). SAS User's Guide: Statistics,
1982 Edition. SAS Institute Inc., Cary, NC.
Vandewater, L J. S„ Racz, W. j„ Norkis, a. R.,
and Buncel, E. (1983). Methylmercury distribution,
metabolism, and neurotoxicity in the mouse brain.
Canad. J. Physiol. Pharmacol. 61, 1487-1493.
Watanabe, I. (1980). Organotini. In Experimental and
Clinical Neurotoxicity (P. S. Spencer and H. H.
Schaumburg, eds.), pp. 545-557. Williams & Wilkins,
Baltimore and London.
-------
TRIMETHYLTIN EFFECTS ON AUDITORY FUNCTION AND COCHLEAR MORPHOLOGY1
K. M. Crofton^, K. F. Dean^, M. G. Menache^, and R. Janssen^
2Neurotoxicology Division, Health Effects Research Laboratory,
U.S. Environmental Protection Agency, Research Triangle Park, NC
^NSI Technology Services, Inc,
Research Triangle Park, NC
SHORT TITLE: Trimethytin and Auditory Dysfunction
* Portions of these data were presented at the 1989 Annual Meeting of the Society
of Toxicology, Atlanta, GA. This manuscript has been revieved by the Health
Effects Research Laboratory, U.S. Environmental Protection Agency, and approved
for publication. Mention of trade names or commercial products does not constitute
endorsement or recommendation for use.
Correspondencet Kevin M. Crofton,PhD, Neurotoxicology Division, MD-74B, Health
Effects Research Laboratory, U.S. Environmental Protection Agency, Research
Triangle Park, NC 27711 (919)541-2672
Revised 03.'Ofl.'9Q
0}/o3/fo
-------
Page 2
ABSTRACT
Trimethyltin effects on auditory function and cochlear
morphology.Crofton,K.M., Dean,K.F., Menache,M.G. and Janssen,R.
Toxicol. Appl. Pharmacol. 00:000-000. Trimethyltin (TMT) is a
neurotoxicant known to alter auditory function. The present study
was designed to compare TMT-induced auditory dysfunction using
behavioral, electrophysiological, and anatomical techniques. Adult
male Long Evans hooded rats (n»9-12/group) were acutely exposed to
saline, 3, 5, or 7 mg/kg TMT. Auditory thresholds were determined 11
weeks post-dosing for 5 and 40 kHz tones using reflex modification of
the auditory startle response (ASR). Brainstem auditory evoked
response (BAER) thresholds were determined for 5, 40, and 80 kHz
tonal stimuli 9 weeks post-dosing. Cochlear histology was assessed
at 13 weeks post-dosing. Functional endpoints demonstrated a high-
frequency hearing loss. ASR thresholds for 40 kHz tones were
elevated 25-35 dB in all dosage groups. BAER thresholds for 40 and
80 kHz tones were elevated 30-50 dB in the 5 and 7 mg/kg groups.
Organ of Corti surface preparations revealed a a pattern of damage
suggesting classical ototoxicity. That is, outer hair cells died
preferentially in regions associated with high-frequency hearing, in
a dosage-dependent manner from base to apex. These data demonstrate
the utility of the ASR and BAER in detecting functional alterations
in audition and indicate that TMT-induced high-frequency hearing loss
is associated vith cochlear damage.
-------
Page 3
INTRODUCTION
Trimethyltin (TMT) is a neurotoxicant that alters multiple aspects of CNS
structure and function (see Reiter and Ruppert, 1984; Chang, 1987; Norton,
1987). Functional alterations include aggression (Brown et al., 1979),
hyperactivity (Ruppert et al., 1982; Bushnell and Evans, 1985), decreased
startle response (Ruppert et al., 1984), alterations in cognitive function
(Venger et al., 1984), and spontaneous seizures (Brown et al., 1979; Dyer et
al., 1982). Pathological examination of TMT treated rats has revealed extensive
neuronal loss in the hippocampus, and olfactory, pyriform and entorhinal
cortices (Chang and Dyer, 1983a). There is also evidence of pathology in sensory
system organs including the retina and inner ear (Chang and Dyer, 1983b).
TMT-induced depression of the startle response (Ruppert et al., 1984) has
been shown to be partially the result of alterations in auditory thresholds
(Young and Fechter, 1986). These threshold shifts were shown to be permanent,
and restricted to high frequencies (Young and Fechter, 1986; Eastman et al.,
1987). The frequency dependence of the hearing loss, coupled with evidence of
increased thresholds of the cochlear microphonic and auditory nerve compound
action potential (Fechter et al., 1986), are highly suggestive of cochlear
damage. Indeed, Chang and Dyer (1983b) presented evidence for loss of hair
cells.in the organ of Corti. However, these authors did not provide the type of
detailed histological evidence (e.g., surface preparations, cytocochleograms)
that would explain the permanent and selective loss of high frequency hearing
demonstrated by Fechter et al. (1986).
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The specific aims of this research were threefold. The first aim was to
document TMT-induced alterations in auditory thresholds using both a behavioral
technique, reflex modification of the acoustic startle response (ASR), and an
electrophysiological technique, the brainstem auditory evoked response (BAER),
in the same animals. The second was to characterize TMT-induced hair cell loss
in a quantitative and regional manner using the surface preparation of the Organ
of Corti. The third aim was to determine the degree of correlation among the
functional alterations and anatomical damage, in an attempt to more closely
relate the loss of high frequency hearing to specific alterations in cochlear
morphology. Due to evidence of recovery of low frequency hearing loss (Fechter
et al., 1986), animals were tested 9-11 weeks post dosing to insure that
effects seen were lasting.
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METHODS
Animals? Male Long Evans hooded rats (Charles River, Inc.) were obtained at
approximately 60 days of age, and were housed singly in standard plastic hanging
cages (24 x 20 x 45 cm). All animals were given a ten-day acclimation period
and were maintained on a 12:12 hr photoperiod, L:D (0600;1800). Food (Purina Lab
Chow) and water were provided ad libitum. Temperature was maintained at
21.0±2°C and relative humidity at 40±20%.
Exposure and Testing: Animals (n»9-12/group) were acutely exposed to either
saline or 3.0, 5.0, 7.0 mg/kg trimethyltin hydroxide as the base (source). BAER
thresholds were determined 9 weeks post exposure. Startle thresholds were
determined 11 weeks post-exposure. Animals were sacrificed for cochlear
pathology 13 weeks post-exposure.
Reflex Modification: Testing was conducted in 8 sound-attenuated chambers, each
containing a wire mesh plastic-framed test cage, mounted on a load cell/force
transducer assembly designed to measure vertical force (see Ruppert et al., 1984
for details). Three speakers were suspended from the ceiling of each chamber.
One speaker (Motorola 5.0 X 12.5 cm piezoelectric tweeter) presented the
eliciting stimulus (S2) (a 120-dB SPL, 40-msec vhite noise burst, with a 2.5-
msec rise/decay). A second speaker (Creative Acoustics, 16 ohm) delivered
broadrspectrum background noise at 30 dB. The third speaker (Radio Shack leaf
tweeter, #40-1375) delivered the prepulse stimulus (SI), a 5 or 40 kHz, 40-msec
pure tone with a 2.5-msec rise/fall time at variable sound pressure levels
between 6 and 90 dB.
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Page 6
Reflex modification procedures (see Young and Fechter,1983; Ison, 1984)
were used to determine auditory thresholds and the startle reflex amplitude.
Each rat was placed in a test cage and, following a 10-min adaptation period at
a low background noise level (30 db(A)), received a total of 240 trials
consisting of 10 trials at each of 24 different sound pressure levels of SI
(blank, and 6 - 90 dB SPL in 3 or 6 dB increments). Sound pressure levels (dB,
re 20 uN/n^) vere measured vith a BMC 4315 ¦&" microphone and a BMC 2610
Measuring Amplifier. Sound intensities vere estimated by averaging measurements
recorded from 10 locations vithin the animal test cage. The range of
measurements vithin this space vas approximately ±3 dB. For background noise
measurements* a B&K 4165 microphone vas used vith 'A' veighting to eliminate
lov frequency building noise. The blank control trials contained no prepulse
stimulus. The order of presentation of the trials was computer-generated using
randomized blocks, so that within each of ten 24-trial blocks, each Si intensity
vas presented once. The intertrial interval was 15 sec, and the interstiraulus
interval (measured from onset of SI to onset of S2) was 90 msec. Animals were
tested over a tvo day period, one frequency per day.
Data collection began with the presentation of the eliciting stimulus and
continued for 64 msec. The analog signal for each response vas digitized at 1
kHz and converted to grams using a previously determined calibration curve for
each load cell. Response amplitude vas taken from each animal's average
response curve* calculated across all trials at a given stimulus intensity. The
threshold and baseline startle amplitude vere estimated using a nonlinear
regression procedure outlined belov.
The nonlinear regression procedure PR0C NLIN (SAS, 1985) vas used to produce
least-squares estimates of parameters fitting a segmented line model, using the
multivariate secant iterative method. Parameters estimated vere: baseline
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Page 7
startle amplitude (amplitude of the startle response at SI intensities below
threshold); the SI threshold (defined as the join point of the tvo line
segments, or the SI intensity above vhich the response to S2 is inhibited by
Si); and the slope of the descending portion of the fit. These parameters were
estimated for individual animal data and are presented as group means. Animals
vere rejected rrom the data set if their estimates of threshold were less than 5
dB (i.e., 1 dB less than the lowest intensity used) or their slope was not
significantly different from zero (i.e., the slope was flat and not
representative of a true effect of SI on S2). In the case where an animal's data
did not fit the model (less than 2X of the time) or the animal was rejected from
the data set for any other reason, the animal vas retested at that frequency
within 24 hrs. Data on the number of animals either not fitting the model or
rejected were tabulated by treatment for later determination of treatment
effects.
Evoked Response; Rats vere surgically implanted with stainless steel skull
screw electrodes 1 mm posterior to the curve of best fit along the lambdoid
suture (Paxinos and Vatson, 1982) and in the midline (active electrode), and 2
mm anterior to bregma and 2 mm on either side of the midline (ground and
reference). Anesthesia for surgery vas pentobarbital (50 mg/kg) vith
subcutaneous pretreatment vith atropine (0.2 mg/kg). Gold pins attached to
presoldered nichrome vires vere inserted into an Amphenol receptacle vhich vas
cemented to the skull, encasing the rest of the assembly. Vound margins vere
treated vith nitrofurazone and sutured shut. Each rat received an im injection
of 100k units of penicillin 6.
All testing vas conducted in a double-walled anechoie chamber containing a
custom made loudspeaker and animal enclosure (Allotech, Inc., Raleigh,HC) vhich
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Page 8
provides relatively uniform stimulus levels regardless of the animals head
placement. The loudspeaker vas designed specifically to reshape the sharp
vavefront of high-frequency stimuli into a plane wave vhich then propagates past
the animal's ears and is absorbed by acoustical foam several inches below the
cage. The cage is constructed so as to be acoustically transparent to
frequencies as high as 80 kHz. At 80 kHz, sound levels in the cage are least
uniform: there is a 20 dB drop from center to corner. To remedy this condition,
the symmetry of the sound field is used to advantage. Moveable gates at each
end are adjusted to the length of the animal so that it can only assume tvo
positions, i.e., it can face left or right. The levels in these positions are
the same. Artifact rejection at the averager is used to eliminate trials
occurring vhen the rat is changing direction. Sound pressure levels are
measured at the location of the animal's ears. Acoustical measurements vere
made using a Bruel and Kjaer 2636 Measuring Amplifier vith a 1/4-inch 4165
microphone. BAER threshold levels reported are in dB peak sound pressure level
re .0002 ubar. The microphone vas placed at the position of the rat's ears, and
calibration vas performed immediately prior to the experiment. The attenuator
had previously been determined to have a linear response throughout its 110 dB
range at every frequency used, using a combination of acoustical measurements
above, and oscilloscopically read voltage ratios belov, the microphone's
internal noiM level.
Rats vere tested avake and unrestrained a veek following surgery. BAER
thresholds vere determined for filtered clicks at 3 frequencies: 5, 40, and 80
kHz. Responses vere recorded and averaged using a Nicolet Pathfinder II
(bandpass 0.2 Hz to 8 kHz) using a 10 msec recording epoch. Stiauli vere
presented at a rate of 9.7 Hz. Threshold testing vas begun at stimulus levels
high enough to evoke an unambigous BAER, then vere gradually reduced until the
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Page 9
experimenter judged that the averaged response (N-512 averaged sveeps) could no
longer be discriminated from noise. This level was called threshold for a given
frequency, and the procedure was repeated until thresholds were obtained for all
frequencies.
Inner Ear Histology: Rats were deeply anesthetised (pentobarbital) and
immediately sacrificed. Both cochleas were rapdlly dissected and then perfused
(within minutes) with 2.5£ glutaraldehyde in phosphate buffer. The ears were
then later perfused with IX osmium tetroxide and dehydrated by washing with a
graded series of alcohols (35, 50, 70, 80, 90, 95, and 3 x 100Z). The organ of
Corti was microdissected, displaying the reticular surface for hair cell
counting. Surface preparations were analyzed in 0.25 mm sections using light
microscopy. Hair cells were classified as alive or dead (characteristic scars
replace dead cells), and a cytocochleogram constructed for each ear (see
Engstrdm et al., 1966). Hair cell loss was quantified for the three row of
outer and one rov of inner hair cells, as the percentage of damaged hair cells
in each section from base to apex.
Data from the cytocochleograms were used to compute a group average cochlear
damage score. For each animal the length of damaged cochlea, defined as the
length of the cochlea with 0.25 mm sections having more than a 20% loss of
outer hair cells, was determined for the three rovs of outer hair cells. These
scores vere then averaged for each animal. A similar score was calculated for
the one rov of inner hair cells.
Statistical Analysis: Specific statistical variables analyzed weret ASR
thresholds at 5 and 40 kHz, ASR baseline amplitudes, cochlear damage scores for
the inner and 3 outer hair cell rovs, and BAER thresholds at 5, 40, and 80 kHz.
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Page 10
Multivariate analysis of variance (MANOVA) procedures vere used for all main and
simple-effects tests (SAS,1985). In the case of more than one independent
variable, significant interactions were followed by simple effects ANOVA tests
for each independent variable. Repeated-measures ANOVAs (multivariate) were
used when appropriate. Mean contrast comparisons were made using Tukey's
studentized range test (SAS, 1985). To control for main effects multiple
comparisons (a total of 1 ANOVAs and 3 MANOVA's), the a level was set at 0.025
(0.05/^4).
RESULTS
Exposure to TMT resulted in decreases in the amplitude of the ASR and an
increase in the auditory thresholds at 40 kHz (Figure 1). The decrease in the
baseline amplitude of the ASR (F(3,40)ml0.37,p<0.0001) was observed in the
groups receiving 5 and 7 mg/kg TMT (p<0.025). Baseline amplitudes were
decreased 61 and 83X compared to the vehicle control, in the 5 and 7 mg/kg
groups, respectively. There was a significant Treatment*Frequency interaction
(F(3,37)*0.0201), therefore the threshold data vere analyzed separately for each
frequency. There vas no effect of TMT on the ASR threshold at 5 kHz
(F(3,40)*1.06»p<0.3762). There vas a significant effect of TMT on 40 kHz ASR
thresholds (F(3,40)«12.21,p<0.0001). Thresholds vere increased 24, 30, and 39 dB
in thfe 3, 5, and 7 mg/kg groups, respectively (p<0.025).
The BAER threshold data also demonstrated increased auditory thresholds at
40 kHz, as veil as increased thresholds at 80 kHz (Figure 2). There vas a
significant Treatraent*Frequency interaction (F(6,70)«13.95,p<0.0001) and
therefore the data vere analyzed separately for treatment effects at each
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Page 11
frequency. There was no effect of treatment on 5 kHz thresholds
(F(3,36)-l.26,p<0.3041). There were treatment effects for thresholds at both 40
(F(3,36)«65.51,p<0.0001) and 80 kHz (F(3,36)-35.32,p<0.0001). There vas a
significant increase in the 40 kHz thresholds in the groups treated with 5 and 7
mg/kg (p<0.025); thresholds were increased 35 and 50 dB, respectively. For 80
kHz thresholds all TMT treated groups had significantly increased thresholds
(p<0.025). Thresholds at 80 kHz were increased 17, 24, and 31 dB in the 3, 5,
and 7 nig/kg groups, respectively.
The morphological analysis is presented in Figures 3, 4 and 5. The
photomicrographs demonstrate a dosage-dependent loss of hair cells in the
cochlea following TMT treatment (Figure 3). This loss of hair cells was
restricted to the basal region of the cochlea as illustrated in the
cytocochleograms (Figure 4). These effects reflect an increase in the amount of
the cochlea with damaged hair cells in TMT-treated rats and this outcome was
limited to the outer hair cells} there vas no statistically significant
treatment related effect of TMT on inner hair cells (Figure 5). Analysis of the
damage scores revealed a Treatment*Cell type interaction (F(9,54)«3.02,p<0.005),
therefore data were analyzed in two ways. First, ANOVAs were computed to test
for an effect of Treatment vithin each Cell type. Second, ANOVAs were computed
to test for an effect of Cell type vithin each Treatment group. The latter was
used to determine vhether there vas a differential effect of TMT on the three
rows of outer hair cells.
There vas no significant effect of Treatment (F(3,25)-1.94,p<0.148) on inner
hair cell pathology. Although there vas no significance, one (out of 7) aniaals
in the 5 ag/kg group and tvo (out of 9) aniaals in the 7 ag/kg group did have
¦issing inner hair cells in the basal turn and hook regions (see Figure 3c and d
for the vorst case exaaples). In contrast, there vas a significant effect of
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Page 12
Treatment for all 3 rows of outer hair cells (all F's(3,25)>6.72,p's<0.002).
For every row of outer hair cells there was a significant increase in the damage
score in the 5 and 7 mg/kg groups compared to the saline controls (p<0.025).
There was an effect of Cell type in the 7 mg/kg Treatment group
(F(3,32)=ll.10,p<0.0001) that resulted from increased damage in the three outer
rows compared to the inner row of hair cells (p<0.025). There were no instances
in which there were differences between any of the three outer rows of hair
cells (p>0.05) at any of the Treatment levels.
DISCUSSION
TMT administration resulted in a decrease in the baseline amplitude of the
ASR. This is consistent with previous reports (Ruppert et al., 1984$ Young and
Fechter, 1986). As suggested by Ruppert et al. (1984) the depression in the
baseline amplitude of the ASR cannot be explained by the limbic structure damage
induced by TMT (see Brown et al., 1979} Bouldin et al., 1981); lesions of the
hippocampus (Leaton, 1981) or the amygdala (Kemble and Ison, 1971) do not affect
startle responding. Nor can the decreased ASR amplitudes be explained by the
alterations in auditory thresholds seen here and previously (Young and
Fechter,1986} Fechter et al.,1986), since TMT exposed rats have a high frequency
selective hearing impairment and the eliciting stimulus used in the present
study*was a broad band burst o€ white noise, with most power in the 3 to 15 khz
range. It may be possible that TMT damages structures in the primary startle
circuit (Davis et al., 1982). Vhether TMT affects the sensory and/or motor
portions of the circuit remains to be determined.
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Page 13
A second effect of TMT on auditory function was a dosage- and frequency-
dependent elevation in the ASR and BAER auditory thresholds. TMT exposure
resulted in elevations in the high frequency thresholds (40 and 80 kHz), without
any changes in the low frequency threshold (5 kHz). That the increased
thresholds were restricted to high frequencies is consistent with previous
reports for 40 kHz tones (Young and Fechter, 1986; Fechter et al., 1986). The
present results also demonstrate a decreased sensitivity to 80 kHz tonal
stimuli, indicating effects in the ultrahigh frequencies in the hearing range of
the rat.
More importantly, the present results demonstrate TMT-induced damage to the
cochlea. Surface preparations of the organ of Corti demonstrate a loss of hair
cells in the basal turn and hook, regions associated with high frequency
hearing. The effects of TMT were much more prominent on the outer hair cells
than the inner hair cells (Figure 3 and 4). The portion of the cochlea with
damaged outer hair cells exceeded 30X in the 7 mg/kg group, whereas damage in
the inner rov averaged only 9 X in this same group (only 3 animals demonstrated
total loss in the area of the basal turn). These effects are similar to the
classical ototoxicity demonstrated for the aminoglycoside antibiotics in that
there is a progressive increase in damage in a basal to apical direction, with a
greater sensitivity of the outer hair cells compared to the inner hair cells in
some species (Stebbins et al., 1969; Wers&ll,1981). The rows of outer hair
cells are differentially susceptible to aminoglycoside antibiotics in some
ntammais and for some antibiotics (see Harpur, 1986; Moody and Stebbins, 1982;
Ostyn and Tyberghein, 1968| Ylikoski, 1974). TMT shoved no «uch differentially
rov susceptibility in the Long Evans rat.
This demonstration of a direct effect of TMT on cochlear hair cells is
consistent with a previous report by Fechter et al. (1986). These authors
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Page 14
demonstrated TMT-induced alterations in the cochlear microphonic, indicative of
hair cell dysfunction. These authors also reported a functional recovery in the
lower frequencies (<24 kHz). The data presented here were collected at 9 and 11
weeks post-exposure, and the high-frequency selective threshold shifts are quite
consistent. The cytocochleograms taken at 13 weeks post-exposure did not
demonstrate any alterations in hair cells in the areas associated with mid to
low frequencies. The mechanism of this TMT-induced hair cell loss in the
cochlea remains to be determined.
In summary, these data demonstrate TMT-induced alterations in auditory
function that are consistent with previous reports (Young and Fechter, 1986;
Fechter et al., 1986). Both behavioral (ASR) and physiological (BAER)
techniques indicate a high frequency selective hearing loss. Morphological
examination of surface preparations of the Organ of Corti demonstrate a classic
ototoxicity; loss of hair cells preferentially in regions associated with high-
frequency hearing in a dosage-dependent manner from base to apex, and a greater
sensitivity of OHCs compared to IHCs. These data directly correlate the TMT-
induced auditory threshold changes to morphological alterations in cochlear
structure, and demonstrate the utility of the ASR and BAER in detecting
chemically-induced alterations in audition.
Acknowledgements: The authors vould like to thank Rebecca Hamrick and Valt
Faison for technical assistance. Ve also thank C. Sheridan and R. Altschuler
for tissue processing.
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Page 15
FIGURE LEGENDS
FIGURE 1. TMT-induced alterations in auditory thresholds and the ASR amplitude.
(A) TMT-induced depression in the baseline ASR amplitude. Data are presented as
group means (±SE) of the ASR amplitude on blank control trials (S2 alone). (B)
TMT-induced frequency-dependent, increases in auditory thresholds. Data are
presented as group means (+SE) of the auditory thresholds for 5- and 40-kHz pure
tones. Saline = saline control group. [* > significantly different from
respective saline controls, p<0.0251.
Figure 2. TMT-induced elevations in BAER thresholds. Dosage-dependent elevations
in high frequency BAER thresholds in TMT-exposed rats are typical of
ototoxicity. Data are presented as group mean (±SE) BAER thresholds for 5, 40
and 80 kHz filtered clicks. Saline * saline controls [* « significantly
different from respective controls, p<0.025).
Figure 3. TMT-induced loss of hair cells in the basal region of the cochlea;
morphological evidence for classic ototoxicity. (A) Saline-treated control
animal (ASR thresholds ¦ 18 and 36, BAER thresholds - 25, 5, and 38, for 5, 40
and 80 kHz, respectively). Arrovs indicate the three rows of outer hair cells
(OHCs), and the one row of inner hair cells (IHC). (B) A 3 mg/kg TMT-exposed
animajl (worst case) (ASR thresholds * 24 and 57, BAER thresholds * 22, 28, and
62, for 5, 40 and 80 kHz, respectively). Arrovs indicate scattered loss of OHCs.
The IHCs are intact. (C)A 5 mg/kg TNT-treated animal (ASR thresholds - 12 and
63, BAER thresholds • 27,44, and 66, for 5, 40 and 80 kHz, respectively).
Bracket indicates complete loss of outer hair cells. Note intact inner hair
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Page 16
cells (arrow). (D) A 7 mg/kg THT-treated animal (worst case) (ASR thresholds =
36 and 54, BAER thresholds ® 21, 46, and 71, for 5, 40 and 80 kHz,
respectively). Brackets indicate area of total loss of both OHCs and IHCs. All
sections from within the basal turn of the cochlea. Bar is 25 uM.
Figure 4. Cytocochleograms for representative animals treated with either (A)
saline, (B) 3 mg/kg TMT, (C) 5 mg/kg TMT, or (D) 7 mg/kg TMT. Data are
presented as the percentage of remaining hair cells as a function of the total
distance from the apex of the cochlea for the 3 rows of outer hair cells (OHC)
and the one row of inner hair cells (IHC). Data are from individual animals.
Note: the date from the treated animals are the worst case examples.
Figure 5. Effects of TMT on hair cells in the cochlea. TMT treatment caused a
dosage-dependent increase in the amount of damaged (missing) hair cells in the
basal (high frequency) region of the cochlea. Data are presented as group means
(±SE) of the number of 0.25 mm sections of the cochlea with loss of at least 20*
of the hair cells. Saline - saline control group [§- all outer hair cell means
are significantly different from saline controls, p<0.0251.
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Page 17
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TOXICOLOGY AND APPLIED PHARMACOLOGY 97, 113-123 (1989)
Prenatal or Postnatal Exposure to Bis(tri-n-butyltin)oxide in the Rat:
Postnatal Evaluation of Teratology and Behavior1
K.. M. Crofton,* K. F. Dean,* V. M. BoNCEK,t M. B. Rosen,t L.P. Sheets,"
N. Chernoff4 and L. W. Reiter*
*Neurotoxicology Division and $Developmental and Cellular Toxicology Division, Health Effects Research
Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711, and
^Northrop Services. Inc., Environmental Services, Research Triangle Park, North Carolina 27709
Received April 11,1988; accepted August 28, 1988
Prenatal or Postnatal Exposure to Bis(tri-/i-butyltin)oxide in the Rat: Postnatal Evaluation of
Teratology and Behavior. Crofton, K. M., Dean, K. F„ Boncek, V. M.. Rosen, M. B„
Sheets, L. P., Chernoff. N„ and Reiter, L. W. (1989). Toxicol Appl. Pharmacol 97, 113-
123. The results of a series of screening tests to determine the potential teratogenicity and neuro-
toxicity of developmental exposure to TBTO in rats are presented in this paper. For prenatal
exposure, pregnant Long Evans rats were intubated with 0-16 mg/kg/day bis(tri-«-buty]tin)ox-
ide TBTO from Days 6 to 20 of gestation (GD 6-20). For postnatal exposure, rat pups were
intubated with 0-60 mg/kg TBTO on Postnatal Day 5 (PND 5). Following prenatal exposure,
dams were allowed to litter and pups were evaluated using a postnatal teratology screen. Postna-
tal evaluation for both exposures included motor activity (PND 13-64), the acoustic startle
response (PND 22-78), growth, and brain weight. The maximally tolerated dose (MTD) in preg-
nant rats was 5 mg/kg/day, which is one-third the MTD in nonpregnant rats. There were de-
creased numbers of live births, and decreased growth and viability at dosages >10 mg/kg/day.
Cleft palate was found in 3% of the 12 mg/kg/day group. There was mortality following postnatal
exposure to 60 mg/kg and all prenatal dosages >10 mg/kg/day. Preweaning body weight was
significantly decreased for all postnatal dosages, and all prenatal dosages >2.5 mg/kg/day. Body
weight reductions persisted to the postweaning period only in the high dose groups (10 mg/kg/
day and 60 mg/kg). Behavioral evaluation demonstrated transient alterations in motor activity
development (prenatal exposure only) and the acoustic startle response (postnatal exposure
only). Persistent behavioral effects were observed only at dosages that produced overt maternal
toxicity and/or postnatal mortality. The demonstration of the teratogenic and neurotoxic poten-
tial of TBTO in rats is confounded by associated maternal toxicity and/or pup mortality.
<8 1989 Academic Prest, Inc.
INTRODUCTION
Worldwide production of biocidal triorga-
notin compounds in the 1980s has been esti-
1 Although the research described in this article has
been supported by the U.S. Environmental Protection
Agency, it has not been subjected to Agency review and
therefore does not necessarily reflect the views of the
Agency and no official endorsement should be inferred.
Mention of trade names or commercial products does
not constitute endorsement or recommendation for use.
mated to be from 4.5 to 7.2 x 106 kg (Zucker-
man, 1976; Blunden et ai, 1984). Bis(tri-«-
butyltin)oxide (TBTO) is a triorganotin
compound used as a biocide in marine anti-
fouling paint, which is toxic to aquatic species
in both freshwater and marine ecosystems
(for review, see Hall and Pinckney, 1985).
While the effects of TBTO have not been
widely studied in mammalian species, its em-
bryotoxic potential has been shown in both
an in vitro limb bud culture system (Krowke
113
0041-008X/89 $3.00
Copyright © 1989 by Academic Prm* inc.
Ail rights of reproduction in toy form reserved.
-------
114
CROFTON ET AL.
et al., 1986), and an in vivo teratology study
in mice (Davis et al., 1987).
Other organotins have been shown to pro-
duce a variety of neurotoxic effects in rats fol-
lowing prenatal and postnatal exposures. Pre-
natal exposure to triphenyltin produced an
increase in activity in an open field (Lehotzky
et al., 1982), while monomethyltin and tri-
methyltin altered learning (Noland et al.,
1982). Early postnatal exposure (Postnatal
Day 5) to triethyltin or trimethyltin in the rat
reduced adult brain weight and altered the
ontogeny of activity and the acoustic startle
response (trimethyltin only) (Reiter et al.,
1981; Ruppert et al., 1983a. 1985). Only re-
cently has the neurotoxic potential of TBTO
been examined in the developing rat. TBTO
was shown to decrease brain weight of spe-
cific areas and cause alterations in CNS-spe-
cific neurotypic and myelinotypic proteins,
following acute postnatal exposure (O'Cal-
laghan and Miller, 1988a).
Due to the previous findings of neurotoxic-
ity following developmental exposure to
other organotins, it was hypothesized that de-
velopmental exposure to TBTO would also
produce neurotoxicity. Motor activity and
acoustic startle response were selected as be-
havioral endpoints in these studies because
they have been demonstrated to be sensitive
indicators of organotin-induced neurotoxic-
ity (Reiter et al., 1981; Harry and Tilson,
1981; Ruppert et al., 1983a). A postnatal ter-
atology screen (Chernoff and Kavlock, 1982)
was employed due to the previous report of
teratogenic effects of TBTO in mice (Davis et
al., 1987). The purpose of the present studies
was to determine and compare the neuro-
toxic potential of TBTO following both pre-
natal and early postnatal exposures. This pa-
per describes the acute toxicity of TBTO in
nonpregnant and pregnant rats and the re-
sults of a teratology screen following prenatal
exposure, and includes the subsequent post-
natal neurobehavioral evaluation following
both prenatal and postnatal exposures.
METHODS
Animals
Primiparous, Gestational Day 2 (GD 2), and nonpreg-
nant Long-Evans rats were obtained from Charles River
Breeding Laboratories (Wilmington, MA, or Raleigh,
NC) at approximately 60 days of age. Animals were
housed in solid-bottom hanging cages (45 x 24 x 20 cm)
with pine shavings for bedding. Animals were main-
tained on a 12:12-hr, lighrAaik (0600:1800) photo-
period, in a temperature (20 ± 2°C) and humidity (50
± 10%) controlled animal facility. Food and water were
available ad libitum.
Maternal Care
Rats were weighed upon receipt and assigned to treat-
ment groups, balancing groups for body weight. All rats
were weighed daily during exposure and examined for
signs of toxicity. Only females that littered, or were con-
firmed pregnant by necropsy, were used in the determi-
nation of group body weights.
Exposures
In the first two studies nonpregnant females (n = 5/
dosage) were used to determine a maximally tolerated
dosage (MTD) for a 14-day exposure. The MTD was de-
fined as the lowest dosage which resulted in mortality, or
greater than a 10% decrease in body weight. Sprague-
Dawley rats (Charles River), approximately 80 days of
age, were used in the first MTD study. Rats were intu-
bated for 14 consecutive days with either 0, 18.75. 37.5.
75.0, or 150.0 mg/kg/day TBTO [bis(tri-«-buty!tin (ox-
ide] (97%, K. & K Laboratories. Plainview. NY I. TBTO
was administered in corn oil (Eastman Kodak Co.. Roch-
ester, NY) 1.0 ml/kg and vehicle controls were used. Rats
in the second study were nonintubated or intubated for
14 consecutive days with 0, 2, 4, 8, or 16 mg TBTO/kg/
day.
Following the MTD studies in nonpregnant females,
two prenatal dosing studies were conducted. Dams were
intubated for 14 consecutive days during gestation (GD 6
to 20). In the initial study, dams (n = 18/dosage) received
either 0, 12, or 16 mg TBTO/kg/day. Due to evidence of
maternal toxicity (e.g., decreased maternal body weight
gain) at both 12 and 16 mg TBTO/kg/day, a second pre-
natal exposure study was conducted. In this study, fe-
males (n *• 16/group) were intubated with 0, 2.5, 5 .0, or
10.0 mg TBTO/kg/day from GD 6 to 20. Dosages were
adjusted daily based on the dams body weight. The MTD
for pregnant dams was the lowest dosage which produced
a greater than 10% decrease in maternal weight gain, dur-
ing the exposure period, compared to controls (i.e., the
difference in body weight on GD 20 and on GD 6).
In the postnatal exposure study, dams (n « 10) were
allowed to litter and each pup received a single oral dose
of either 0 (vehicle), 40, 50, or 60 mg TBTO/kg on Post-
natal Day 5 (PND 5). TBTO was administered in a vol-
ume of 10 nI/g body weight. A within-litter dosing design
-------
TBTO—TERATOLOGY AND BEHAVIOR
115
was used: 1 male and 1 female from each litter received
each dosage; therefore, each litter contained all treat-
ments.
Teratology
Following prenatal exposure, dams were allowed to
give birth. On PND I and PND 3, litters were counted
and weighed; pups within each litter were counted and
examined for external malformations (Cheraoff and
fCavlock, 1982). Females that had not given birth by GD
24 were killed and examined for uterine implantation
sites.
Postnatal Evaluation
Postnatal growth and behavior were evaluated in ani-
mals from the postnatal and the second prenatal expo-
sure groups. In the prenatal exposure study, pups within
the same treatment group were randomized and four
male and four female pups were redistributed to each
dam on PND 4 (day of birth - PND 0). Because few pups
were born following the 10 mg TBTO/kg/day dosage (N
= 21 by PND 3), pups in this group were randomized
and redistributed to three dams (n » 7/litter). For the
postnatal exposure group, pups were randomized and
each dam was assigned four male and four female pups,
on PND 1. Each pup was tatooed on a paw to provide
unique identification within a litter, using a method
modified from Avery and Spy ker (1977). Toxic signs and
mortality were recorded during daily examination. On
PND 21, pups were weaned and housed with cage mates
by sex. Pups were weighed on PND 5,10,15,20,30,47/
48, and 62/64. Rats in the prenatal exposure group were
also weighed on PND 84 and 110. Rats from the prenatal
and postnatal exposure groups were decapitated on PND
110 and 64, respectively, and their brains removed. Wet
weights of the whole brain, cerebellum, and hippocam-
pus were obtained.
Developmental landmarks. Pups in the prenatal expo-
sure group were examined for sexual maturation. Males
were examined on PND 21-25 for testes descent; females
were examined on PND 30-36 for vaginal opening. The
age of maturation was recorded using the criterion speci-
fied in Adams et al. (1985). Testes descent was evaluated
by placing the forelimbs of each pup on a standard-size
wooden pencil. When the pup grasped the pencil it was
required to independently support its body weight. Dur-
ing this time the scrotal area was visually examined for
complete descent of the testes. Patency of the vagina was
determined by gently separating the vaginal folds with
a pair of blunt-tipped forceps and observing the vaginal
area.
Motor activity. Motor activity of individual rats was
monitored in figure-eight mazes for 30 min daily on
PND 13-21, 43/47, and 61/62. The maze is a series of
interconnected alleys (10 X 10 cm) converging on a cen-
tral arena and covered by an acrylic-plastic top (Reiter et
al.. 1975). Activity is detected by eight phototransistor/
photodiode pairs distributed throughout the maze.
Mazes were housed in a sound-attenuated room main-
tained on the same light:dark cycle as the animal facility.
Testing was conducted during the light portion of the
cycle.
Acoustic startle response. The acoustic startle response
was tested in prenatally exposed rats on PND 30,64, and
78, while postnatally exposed rats were tested on PND
22, 47, and 62. Testing was conducted in eight sound-
attenuated chambers containing a plastic-framed wire
cage mounted on a load/cell force transducer assembly
designed to measure vertical force. Test cages of three
different sizes were used according to the age of the rats:
PND 22 and 30 (9 X 5.1 X 5.1 cm), PND 47 (12.7 x 6.4
X 6.4 cm), and PND 62, 64 and 78 (20 X 7.6 x 7.6 cm).
T wo speakers were mounted on the ceiling of each cham-
ber, 30 cm above the test cage. One speaker presented an
acoustic stimulus, 13 kHz, 120 dB(A), 40-msec tone with
a 2.5-msec rise time; the second delivered background
white noise at each of three intensities (45, 60, and 75
dB). Rats were placed in the test cages and allowed to
acclimate for 10 min at ambient noise levels. Immedi-
ately following acclimation, 30 stimuli were presented at
an interstimulus interval of 20 sec. Ten stimuli were pre-
sented at each of the three background noise levels. The
order of presentation of the three background levels was
balanced across the session. Three components of the
startle response were measured: the number of responses,
latency to onset, and peak amplitude. These components
were measured as previously reported (Ruppert et al..
1983b).
Data Analysis
Data were analyzed using programs on the Statistical
Analysis System (SAS, 1985). Separate general linear
models (GLM) analyses were conducted for body
weights, developmental landmarks, total activity counts,
acoustic startle amplitude, and each brain weight mea-
sure. For the teratology screen, separate GLM analyses
were conducted for litter size and maternal weight gain:
for pup weight, analysis of covariance was performed us-
ing PND 1 litter size as a covariate (ChemofT and Kav-
lock, 1982). Dosage, sex, and the interaction were be-
tween-animal factors for all variables. Age and all inter-
actions were within-animal (repeated-measure) factors
for repeatedly tested variables. Greenhouse-Geisser cor-
rected p values were used when appropriate. Post hoc
comparisons of means were made using Tukey's (a) test.
For all statistical tests, the critical value at p < 0.05 was
accepted as significant. For the four different experi-
ments, statistical significance (p < 0.05) was determined
in the following number of separate GLM analyses: 1/1
-------
116
CROFTON ET AL.
TABLE 1
Results of the in Vi vo Teratology Screen of 0-16 nig/kg/day TBTO"
PND 1 PND 3
Maternal wt
TRT Bred Preg gain(g) Litter size Pupwt(g) Litter size Pupwt(g)
Vehicle 18 15 87.9 + 3.7 11.3±0.8 6.27 ± .22 11.3 + 0.6 7.81+.21
12 mg/kg/day 18 12 33.6*9.7* 3.0 + 0.9c 3.65±.3ic l.3±0.7c 5.03 + .59*
16 mg/kg/day 18 6 -12.8±0.9" 0.4+1.2' 3.45±.59c 0.0C 0.0C
" Group means ± SE.
" Maternal weight gain from GD 6-20 which was greater than the maximally tolerated dose (> 10 % decrease
compared to controls).
L' Significantly different from controls (p < 0.05) least-square means.
in the MTD determination, 6/6 in the first prenatal
study, 11/12 in the second prenatal study, and 3/6 in the
postnatal study.
RESULTS
MTD Studies
The first experiment using nonpregnant fe-
males was terminated after 12 days of dosing,
due to significant weight loss and mortality at
all dosages of TBTO. The 14-day LD50 was
estimated to be between 18 and 37 mg
TBTO/kg/day. Animals receiving 37.5 mg
TBTO/kg/day or greater showed signs of the
irritant properties of TBTO: bloody mouth
and nose, salivation, and inflammation of
oral and anal mucosas (150 mg/kg/day). In
the second study (2 to 16 mg/kg/day), there
was no dosage-dependent mortality. Com-
pared to vehicle controls, the greatest body
weight loss (7.6%) was seen following the sec-
ond dose of 16 mg TBTO/kg/day. Body
weight gain was decreased at the 16 mg/kg/
day dosage only (27 vs 8 g). Based on the body
weight data, the MTD of TBTO for nonpreg-
nant female rats was estimated to be approxi-
mately 16 mg/kg/day.
Prenatal Exposures
In the first prenatal exposure study, all dos-
ages of TBTO reduced gestational weight
gain (Table 1). Weight gain in dams exposed
to 12 mg TBTO/kg/day was reduced 62%,
and there was body weight loss in dams fol-
lowing 16 mg TBTO/kg/day. Vaginal bleed-
ing was observed on GD 14-16 in the TBTO-
treated dams. The incidence of vaginal bleed-
ing was 60% at 12 mg/kg/day and 75% at 16
mg/kg/day. There was no relationship be-
tween the occurrence of vaginal bleeding and
pregnancy. Vaginal bleeding was recorded in
only 2/8 rats not delivering pups but deter-
mined to have been pregnant by necropsy.
No other toxic signs were observed in the
dams during the dosing period. In both the
12 and 16 mg/kg/day groups, there was mor-
tality in 1/18 dams. Based on these effects,
both of these dosages were above the MTD
for pregnant rats.
In the second prenatal exposure study, ma-
ternal weight gain was decreased following 10
mg TBTO/kg/day only (Table 2). Weight
gain in this exposure group was reduced 20%
compared to controls. No toxic signs were
seen at any dosage, although 1/16 dams in the
10 mg/kg/day dosage group died. Based on
maternal weight gain, the MTD of TBTO for
pregnant dams was estimated at 5-10 mg/
kg/day.
Teratology
TBTO produced effects in the offspring of
the treated dams, as reflected in litter size,
pup weight, and pup viability (Table 1). Fol-
-------
TBTO—TERATOLOGY AND BEHAVIOR 117
TABLE 2
Results of the in Vivo Teratology Screen of 0-10 mg/kg/day TBTO "
PND 1 PND 3
Maternal wt
TRT
Bred
Preg
gain (g)
Litter size
Pup wt (g)
Litter size
Pup wt (g)
Vehicle
15
9
95.1 ±4.1
10.1 + 1.1
6.69 ± .23
9.8+1.1
8.91 + .39
2.5 mg/kg
16
12
98.4 + 6.0
8.5 ±0.9
7.00 ± .20
8.5+0.9
9.31 + .35
5.0 mg/kg
16
10
110.2 + 4.5
9.8 ± 1.0
6.41 ±.22
8.5+0.9
8.53 ±.37
10.0 mg/kg
15
7
76.3 ±9.1*
5.0 ± 1.2C
4.56+ .3 lc
3.6 ± 1.2C
5.84 ± ,59c
" Group means + SE.
'Maternal weight gain from GD 6-20 which was greater than the maximally tolerated dose (>10 % decrease
compared to controls).
c Significantly different from controls (p < 0.05), least-square means.
lowing dosing with 12 or 16 mg TBTO/kg/
day, litter size was reduced on PND 1 approx-
imately 73 and 96%, respectively, compared
to controls. Pup viability continued to de-
cline from PND 1 to PND 3. Average pup
weight on PND 1 was decreased approxi-
mately 45% compared to controls, at both
dosages.
A number of pups were born with ob-
servable malformations. Following 12 mg
TBTO/kg/day, cleft palate was seen on PND
1 in 2/71 pups; both these pups were born
dead. In this same group, 6/7 pups were born
dead with placentas attached. No cleft palate
was observed in any control animals in this
study. Only 1/8 rats littered from the 16 mg
TBTO/kg/day group; 5 pups were born alive
with no malformations. Following dosing
with 10 mg TBTO/kg/day, there was a reduc-
tion in litter size (SO and 63%) and pup weight
(68 and 66%) on PND 1 and 3, respectively.
There was also decreased pup survival be-
tween PND 1 and 3 (Table 2). There were no
pups born with malformations at 2.5, 5.0, or
10.0 mg/kg/day of TBTO.
Postnatal Evaluation
There was mortality during the early post-
natal period in pups from both prenatal and
postnatal exposures. By PND 21, prenatal ex-
posure to 10 mg/kg/day TBTO or PND 5 ex-
posure to 50 or 60 mg/kg TBTO resulted in
mortality of 14 and 32%, respectively.
Body weight. Both prenatal and postnatal
exposures produced a decrease in body
weight of animals (Figs. 1 and 2). These de-
creases persisted throughout the course of the
experiments at the highest dosages. Following
prenatal exposure, there was a significant age
by dosage interaction (7^(21,910) = 5.40: p
< 0.0001). On PND 5, body weight was sig-
nificantly reduced at both the 5 and 10 mg/
kg/day dosages. However, by PND 10, only
the 10 mg/kg/day group had decreased body
weight. Body weight remained reduced in
this group throughout the experiment
(PND 110).
Body weight was also reduced following
postnatal exposure to TBTO (F(3,58)
= 10.45; p < 0.0001). By PND 10, there was
a 25% decrease in body weight at all dosages
of TBTO. Body weight remained decreased
at all dosages on PND 30; however, by PND
62, body weight recovered for the 40 and 50
mg/kg dosages but was still decreased for the
60 mg/kg group.
Developmental landmarks. Prenatal expo-
sure to TBTO did not produce a significant
effect on the age of testes descent in male off-
spring (Table 3). The 10 mg/kg/day group
was not used in the analysis due to the small
-------
118
CROFTON ET AL.
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Age (days)
20
Fig. 1. Body weights of rats(n = 40/group, except n * 18 for the high dose) prenatally exposed to TBTO.
Dams were orally dosed on GD 6-20 with 0, 2.5. 5.0, or tO.O mg/kg/day. Postweaning body weights are
shown in insert. Data are presented as group means (±SE). Asterisks indicate that the highest dosage is
significantly different from vehicle control at all ages and that the 5 mg/kg dosage is different only on
PND5.
number of males available; however, the
males in that group were examined and deter-
mined to have descended testes by PND 24.
In female offspring, there was a significant de-
lay in the age of vaginal opening (F(3,69)
= 4.94; p < 0.0036). Females exposed prena-
tally to 10 mg/kg/day TBTO had approxi-
mately a 2-day delay in vaginal opening com-
pared to controls (Table 3).
Motor activity. Following prenatal expo-
sure, there was a significant age by treatment
interaction (/T[24,560) = 2.22; p < 0.0063) for
the 30-min preweaning activity. When indi-
vidual days were examined, there was a sig-
nificant effect only on PND 14. All dosages
of TBTO produced a significant decrease in
activity on that day (Fig. 3). Postweaning ac-
tivity was reduced on PND 47 and 62
(F(3,69) ® 3.84; and 9.15, respectively, p's
< 0.01) in the 10 mg/kg/day dosage only (Fig.
3, insert). In contrast, there was no effect of
postnatal TBTO treatment on either pre-
weaning or postweaning activity at any dos-
age (data not shown).
Acoustic startle response. Neither prenatal
nor postnatal exposure to TBTO produced
persistent effects on the acoustic startle re-
sponse. When postnatally exposed rats were
tested on PND 22, there was a significant
effect of treatment (.F(3,58) = 5.53; p
< 0.0021) on the amplitude of the response.
Post hoc tests revealed a significant decrease
in the amplitude at all dosages of TBTO.
However, on PND 47 and 62 there were no
significant treatment effects. There were no
treatment effects on the amplitude of the re-
sponse of prenatally exposed pups. There
were no effects on the latency to onset or the
-------
TBTO—TERATOLOGY AND BEHAVIOR
119
Dosage of TBTO
O 0 mg/kg
~ 40 mg/kg
A 50 mg/kg
O 60 mg/kg
Age (days)
Fig. 2. Body weights of rats (n =¦= 13-20/group) dosed orally on PND 5 with 0, 40, 50, or 60 mg/kg
TBTO. Postweaning body weights are shown in insert. Data are presented as group means (±SE). Asterisks
indicate that all treated groups are significantly different from vehicle control.
number of responses for either prenatal or
postnatal exposure groups at any test age.
Brain weights. Adult brain weight was sig-
nificantly reduced following both prenatal
and postnatal exposure to TBTO (F(3,69)
= 16.77 and CF(3,58) = 4.22, respectively p's
< 0.007). Brain weights in each of the high
dose TBTO groups were significantly less
than controls (Table 4). Wet weight of the
cerebellum was significantly reduced in the
high dose groups for both prenatal (F(3,69)
= 5.75; p < 0.005) and postnatal exposures
(F(3,58) = 3.16; p < 0.03) (Table 4). Wet
weight of the hippocampus was significantly
TABLE 3
The Age of Sexual Maturity of Pups Exposed Prenatally to TBTO
for 14 Consecutive Days (GD 6-20)°
Dosage of TBTO (mg/kg/day)
0
2.5
5.0
10.0
Male
23.50 ±0.31
23.79 ±0.31
24.15 ±0.25
b
Female
32.00 ±0.28
33.55 ±0.37
33.40 ±0.33
34.00 ±0.41*
' The age in days (X ± SE) of testes descent and vaginal opening is shown for males and females, respectively.
* These animals were excluded from the analyses; however, all had descended testes by PND 24.
* Significantly different from controls (p < 0.0S).
-------
120
CROFTON ET AL.
200
180
1*0
_ 160
HI
CO
IX 140
130
jr
5? 120
uj
= 100
o
o
100
0
o
O
Doasg» of TBTO
O 0 mg/kg/day
~ 2.3 mg/kg/day
A 5.0 mg/kg/day
o to.O mg/kg/day
Age (days)
Fig. 3. Figure-eight maze activity of rats (n * 18-20/group) prenataJly exposed to 0. 2.5, 5.0, or 10.0
mg/kg/day TBTO for 14 days (GD 6-20). Rats were tested for 30 min daily from PND 13-21 and again
on PND 43 and 61 (insert). Data are presented as group means (±SE). Asterisks indicate that all treated
groups are significantly different from vehicle controls on PND 14, but only the highest dosage group was
different from vehicle controls on PND 43 and 61 (insert).
reduced in the high dose prenatal exposure
group only {F(3,69) « 9.62; p < 0.0001) (Ta-
ble 4).
DISCUSSION
Prenatal exposure to TBTO produced sig-
nificant embryo/fetal toxicity in rats, at dos-
age levels which were maternally toxic. Both
prenatal and postnatal exposure to TBTO
produced transient alterations in growth, via-
bility, and postnatal behavior. All measures,
except body weight and brain weight at the
highest dosages, showed recovery by adult-
hood. In addition, the results indicate an ap-
parent differential sensitivity of nonpregnant
Mid pregnant rats to the toxicity of TBTO,
with pregnant rats being more sensitive to the
toxic and lethal effects of TBTO,
The postnatal teratology assay used in
these studies assumes that any change in early
postnatal weight or viability of pups, follow-
ing prenatal exposure, is the result of in utero
fetal damage (Chemoff and Kavlock, 1982).
The findings of this study indicate that prena-
tal TBTO exposure alters fetal development.
Following dosages 5* 10 mg/kg/day, prenatal
TBTO exposure decreased pup viability and
growth. At dosages >12 mg/kg/day direct
signs of in utero damage were found (e.g.,
cleft palate). This concurs with, and extends
to another species, the finding of fetal toxicity
in mice at dosages > 12 mg/kg/day (Davis et
ai, 1987). The fetotoxic effects of TBTO are
found at dosages which also produce mater-
nal toxicity, as measured by maternal weight
gain. This decrease in maternal weight gain
may reflect either maternal toxicity or feto-
toxicity. Therefore, a nonspecific effect of
-------
TBTO—TERATOLOGY AND BEHAVIOR 121
TABLE 4
Brain Weights following TBTO Exposure
Dosage ofTBTO (mg/kg/day)
0
2.5 5.0
10.0
Whole brain (g)
Cerebellum (mg)
Hippocampus (mg)
1. Prenatal exposure (GD 6-20)/sacrifice at PND 110
1.84 ±.02 1.82 ±.02 1.82 ±.02
253 ± 3 251 ±5 249 ± 3
121+4 127 ± 3 131 ± 4
Dosage of TBTO (tng/kg)
1.66 ± .03*
229 ± 4*
106 + 2*
0
40 50
60
II. Postnatal exposure (PND 5)/sacrifice at PND 64
Whole brain (g)
1.74 ±.02
1.67 ±.03 1.67 ±.03
1.64+ .04*
Cerebellum (mg)
234 ±4
220 ±4 222 ± 4
217 + 7*
Hippocampus (mg)
110 ±2
110 ±4 107 ±3
108+ 3
• Significantly different from controls (p < 0.05).
TBTO on in utero development, through ma-
ternal toxicity, cannot be rejected. The report
of teratogenesis in mice (Davis et ai, 1987)
was also demonstrated only at maternally
toxic dosages.
There is a difference in the MTD of TBTO
for nonpregnant and pregnant rats. Follow-
ing 14 consecutive days of dosing in nonpreg-
nant rats, the MTD was determined to be 16
mg/kg/day. However, in pregnant dams, the
MTD was 5 mg/kg/day. This threefold
difference in MTDs indicates greater sensitiv-
ity of the pregnant rat to TBTO toxicity. Fur-
ther work is necessary to assess the mecha-
nism for this difference. The potential for
TBTO to cross the placenta is not known, at
this time. If this compound does not readily
cross the placenta, the difference in toxicity
may be due to the fact that the administered
dosages of TBTO were adjusted for maternal
weight on a daily basis. As maternal weight
increases due to the heavier fetal load, larger
amounts of administered compound may
effectively increase the maternal dose if pla-
cental transfer is limited. A recent report by
Davis et al. (1987) gives preliminary indica-
tion that placental transfer of TBTO may be
limited. The differences in toxicity noted
above, therefore, may not be due to preg-
nancy, but simply reflect an increased dosage
to the maternal compartment.
Both prenatal and postnatal exposure to
TBTO resulted in decreased body and brain
weights. However, TBTO-induced body
weight decreases were persistent only at the
highest dosages; lower dosages showed recov-
ery during the postweaning period. Adult
brain weight reductions were also only sig-
nificant in the highest dosage groups. Follow-
ing prenatal exposure, whole brain and hip-
pocampal weights were decreased. Postnatal
exposure also reduced whole brain weights
and produced a significant decrease in cere-
bellar weights. O'Callaghan and Miller
(1988a) demonstrated decreased whole brain,
cerebellar, and hippocampal weights follow-
ing postnatal exposure to TBTO. It is impor-
tant to note that dosages which produced per-
sistent effects on body and brain weights were
associated with postnatal mortality and/or
overt maternal toxicity. In contrast, PND 5
exposure to TBTO has been shown to yield
-------
122
CROFTON ET AL.
persistent alterations in CNS-specific pro-
teins at dosages that do not produce mortality
or decreases in body weight (O'Callaghan and
Miller, 1988a).
There was also a 2-day delay in sexual mat-
uration of females, as measured by age of vag-
inal opening, in the prenatally exposed ani-
mals. Again, however, it was significant only
in the highest dosage group. The effects of
both prenatal and postnatal exposure to
TBTO on measures of postnatal behavioral
and sexual maturation are seen at dosages
which also produce decreased body weights,
mortality, and/or maternal toxicity. There
was a transient decrease in the development
of preweaning motor activity, on PND 14, for
all prenatal TBTO dosages. At postweaning
ages, hypoactivity was seen again, at the high-
est dosage, and persisted through PND 62.
Following PND 5 exposure to TBTO, there
were no alterations in either preweaning or
postweaning motor activity. Activity counts
for all groups are consistent with control val-
ues for the ontogeny of motor activity, pre-
viously reported (Ruppert et al., 1985). Nei-
ther prenatal nor postnatal exposure to
TBTO produced persistent effects on mea-
sures of the acoustic startle response. Follow-
ing postnatal exposure to TBTO, there was a
transient decrease in startle amplitude on
PND 22, at all dosages. However, when ani-
mals were retested on PND 47 and 62, there
were no significant effects on any measure of
the startle response. The lack of persistent
effects on motor activity and the acoustic
startle response, following PND 5 exposure to
TBTO, are in sharp contrast to the repeated
demonstration of alteration in this behavior
following postnatal exposure to other orga-
notins (Reiter et al., 1981; Harry and Tilson,
1981; Ruppert et al., 1983a,c, 1985).
Neither prenatal nor postnatal exposure to
TBTO produced the magnitude and persis-
tence of neurotoxic effects reported pre-
viously for a number of organotins (Reiter et
al., 1981; Harry and Tilson, 1981; Noland et
al., 1982; Ruppert et al., 1983a,c; and O'Cal-
laghan and Miller, 1988b). Prenatal exposure
to triphenyltin acetate produced only tran-
sient increases in spontaneous locomotor ac-
tivity, an effect that returned to control levels
by 90 days of age (Lehotzky et al., 1982).
PND 5 exposure to TBTO has recently been
shown to transiently depress neurotypic and
gliotypic proteins at dosages that do not pro-
duce alterations in body weight gain or mor-
tality (O'Callaghan and Miller, 1988a). These
alterations in CNS-specific proteins are
thought to result from retarded synaptogene-
sis and myelinogenesis and not cell death or
injury (O'Callaghan and Miller, 1988a).
In summary, the results reported here dem-
onstrate the fetotoxic potential of TBTO in
rats. This agrees with a previous report of TB-
TO-induced teratogenesis in mice (Davis et
al., 1987) and extends these findings to a sec-
ond mammalian species. Due to overt mater-
nal toxicity, a nonspecific effect of TBTO
cannot be ruled out. While both prenatal and
postnatal exposure produced transient alter-
ations in postnatal development, persistent
behavioral effects were demonstrated only at
dosages that produced overt maternal toxic-
ity and/or mortality.
ACKNOWLEDGMENTS
We thank Drs. J. P. O'Callaghan, D. H. Taylor, and
K. F. Jensen for careful review and suggestions on this
manuscript. We also thank F. Poythress and K. Rigsbee
for technical assistance.
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TBTO—TERATOLOGY AND BEHAVIOR
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O'Callaghan, J. P., and Miller. D. B. (1988a). Acute
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-------
OFFTfllNTS FJIOM: Nmnitoxlcnnts and Nciirol>fc>fogfcal FuncUoat
I'll cits of Oi|;amilK.;vv M rials
Edited bjr: liut;li A. TiLon and SKckKm U. Snarlior
Copyright (c) 1967 by Johii Wiley & Sons, Luc.
Macrophyslological Assessment
of Organometal Neurotoxicity
ROBERT & DYER
H»wo»
-------
138 Rofcaitl. Oyar
I. IntroductkMi
Neurophysiologies! methods fall into two broad categories, macrophysiological
and microphysiological. The wit of analysis for microphysiological methods it
cellular or subcellular (e.g., membrane or channel), whereas that for macro-
physiological nelhodi is a population of neurons. Each set of methods may be
used to great advantage or disadvantage, depending upon the experimental
question at hand. This chapter focuses on studies that have used macrophysio-
logical methods to investigate the neurotoxicity produced by organometah. For
each study discussed, the issues considered are( I) appropriateness of the method
to answer (he experimental question. (2) adequacy of experimental design in
using the method, (3) importance of the findings, and (4) identification of
unresolved Imift
Only i lew orgaaometattic compounds have been investigated using macro-
physioiogical techniques, and ll wW become evident that these investigations
have been incomplete. A farther goal of this chapter is to stimulate interest in
pursuing these studies, so that we might achieve a more complete understanding
of the compounds and the methods we use to study them.
II. MacropfiysiotoflicaJ llsthodi
To provide a framework for interpreting the studies involving macrophysiological
methods, it is important to know some general characteristics of the methods. For
example, do we know what the method measures? Can the method be used to
detect or characterize neurotoxicity? Can the method be used to assess
mechanisms of neurotoxicity? Does the method have an analog in human studies
which would allow straightforward extrapolation?
The methods selected for coverage in this review are EEG, evoked potential,
multiple unit, kindling, and aAerdischarge, drug-induced, and maximal-electro-
shock seizures. Unfortunately, no relevant multiple-unit studies were found. The
EEG and evoked-potential studies arc of interest because the methods are easily
applied to both human and nonhuman species. Studies involving various models
of seizures are interesting because they provide some measure of the excitability
of the brain.
Interpretation of EEG studies must always be tempered with an understanding
of the behavioral state of the subject, since in most cases these covary. For
example, in the relaxed wakeful condition, there is a higher incidence of alpha (8-
12 cps) activity than in other normal stales. Depending upon the species and
recording site, a high incidence of theta activity (4-8 cps) might reflect either
sleep or locomotor activity. In recordings from the rat hippocampal formation,
the frequency of theta correlates well with the vigor of activity as well (c/f.
Whishaw and Vanderwotf, 1973). Therefore, a finding that a particular
Mactophinloloalcal AiMtuwnl of Ofaaoomvlal N«u>otoiicMy 139
organometal produced increased theta could only be understood in the context of
knowing whether recordings were made in unrestrained rats, and whether those
rats were more or leu active than controls. High-quality quantitative studies of
EEG are difficult to perform, and few have been done. It is unlikely that this
method will ever be of much use in assessing mechanisms of toxicity, but it may
weH become useful for detecting and characterizing neurotoxicity. It should be
considered largely untested at this point.
Evokedpotential methods assess the responsiveness of a particular system to
a known stimulus. Used prudently, they can detect and help characterize
neurotoxicity. Their ability to determine mechanisms of toxicity is extremely
limited, but they may be useful in determining the direction that mechanism-
oriented studies should take.
Models of seizure activity in the brain differ in the means by which seizures are
elicited and, therefore, in the specific nature of excitability tested. In some cases
there is detailed knowledge of the brain regions or systems activated by the
clicking event, while in others there is not. Unless several models of seizure
activity are utilized in a particular investigation, it is difficult to make accurate
stalrm**"* about the general excitability of the brain.
In most of the work summarized below, the above described methods were
used to aid in characterizing the nature of neurotoxicity, la a few cases, related
measurements have been obtained in humans, and these arc described separately
as clinical studies.
III. Organomarcurlals
A. Msttyhwicwy
1. Qonmral The human methyhnercury poisoning incident at Minimata Bay,
Japan, probably accounts for the most extensive neurotoxicity database on any
toxicant, and is one of the most compelling cases for the heightened vulnerability
of younger subjects. Among the many symptoms of methyhnercury poisoning are
sleep disturbances and changes in motor, cognitive, and sensory function.
Somatosensory and visual systems appear to be particularly affected in humans.
Merigan et ai. (1983) noted that the visual system deficits are relatively unique,
since they appear to result largely from cortical damage, rather than more
peripheral effects. Furthermore, the cortical damage seems to occur primarily in
the depths of the sulci within the visual cortex, thereby raising questions about the
suitability of experimental models of the dysfunction in species that are
lissencephalic. While an extensive scries of behavioral and neuropathological
studies were performed upon animals followingexposure lomethylmcrcury, most
neurophysiologies! studies performed involved either peripheral nerve studies or
studies that arc loo molecular/micro in nature lo fall within the realm of this
-------
140 ItotMrtS. Oyer
review. Relatively Tew studies focus upon macroelectrode recordings from the
CNS.
2. CIMcmI. A number of studies have reported the effects of exposure to
methyimercury on EEGs ia humus. Typical among these is a systematic study
of human EEO alterations by Brtantr and Snyder (1980), who reported a
descriptive longitudinal evaluation of the EEO from a family of 10 ia New
Mexico, 4 of whom suffered from meihyfanercury poisoning. The nature of the
EEG analysis appears to have been visual inspection of the records. The
abnormalities observed included bilateral multifocal spikes, with occasional
generalized spike and polyspike discharges. In addition, background EEO
rhythms showed dMwe slowing and excessive high-amplitude beta waves, a
pattern indicative of severe underlying brain damage. The extent of the EEG
abnormalities correlated well with the age and overt neurological manifestations
of the subjects, such that the mother, who had no EEO abnormalities, also had no
other neurological symptoms. Perhaps the most interesting finding is that no
epileptiform spikes were observed in the recordings from subjects who never
showed clear evidence of behavioral seizures. One might have expected that if
severely affected subjects had seizures, then miktty affected subjects might have
epileptiform abnormalities in the EEG in the absence of seizures, but such was
not the case. Thus the visual scoring of EEO records added nothing to the
neurological exam. Computer analysis of the EEQs might have been awre
sensitive.
Sensory system disturbances are prominent among complaints of individuals
exposed to methyimercury. Mukuno et al. (1981) compared behavioral and
electrophysiological methods for evaluating visual function in 12 humans with
Minimata disease of 15-25 yrs duration. The data were contrasted with those
obtained from seven hcakhy physicians. The behavioral tests included visual
acuity, the Arden grating test (a combination of acuity and contrast sensitivity),
and visual field evaluation. The electrophysiological test was a pattern reversal
evoked potential (PREP) hi which vertical Mack and white bars with 50%
contrast and spatial frequencies fromO.5 to 8.0 cycles/degree were reversed at an
unspecified rate. The authors measured latency of the first positive peak and
peak-to-peak amplitude between the first positive and second negative peaks.
The peak latency tended to increase in the exposed subjects, but the moat useful
dependent variable was based upon the rate of amplitude reduction produced by
increasing spatial frequency. From this variable, the authors calculated the
spatial frequency at which the amplitude was SO% of what it was at 0.5
cycles/degree. There was a very high correlation between this value and the score
obtained by subjects on the Arden grating test (r= .85). While the Arden test is
clearly simpler to administer, the authors point out that it is subject to
Mi-—AmmmmM ol OlfMMWiil M«u> otomtcMy 141
psychogenic factors. The outcome of the PREP test is not easily altered by
motivational or other related factors. Further, this test is readily applied to
nonhuman species.
Sayder and SeeMnger( 1976) evaluated the integrity of the peripheral nerves in
the individuals tested by Brenner and Snyder( 1980) and described above. While
the affected individuals reported somatic sensory disturbances, there were not
alterations in measures of sensory nerve conduction. The authors concluded that
the sensory effects must not be peripheral in nature, supporting a similar
conclusion reached by Von Burg and Rustam (1974), who studied patients
involved in the most recent Iraq epidemic(Bakiret at., 1973). However, Cincaet
al. (1979) showed that in two cases of ethylmercury poisoning there was a clear
and reversible slowing of conduction in sensory nerves.
3. Characterization of Toxicity
A. EFFECTS OF Methylmercury ON THE EEO. Sleep disturbances (both
hypersomnia and insomnia) have been reported in humans exposed to methyl-
mercury, hut no detailed or quantitative assessment of these disturbances has
been reported. Using EEO recordings, Arito et al. (1983) evaluated the
sleep/ wakefulness cycle in rats exposed to three different dosages of methyimer-
cury. The purpose of the study was presumably to determine whether rats also
exhibited sleep disturbances following exposure to methyimercury and, if so, to
characterize the nature of the disturbance.
Arito et al. (1983) presented EEG data obtained from 16 adult Sprague-
Dawley rau with chronic electrodes implanted in the skull over the frontal and
occipital cortex, and in the dorsal neck muscle for recording the EMG. The rats
were divided into three groups, each of which received a different dosage of
methyimercury (1.65 mg/kg/day for 2 days, 5.0 mg/kg/day for 2 days, or IS
mg/kg/day for 2 days). Unfortunately, no animals received only vehicle
treatment. After a 3-day period of habituation to the test chamber. EEGs were
recorded continuously for 4 consecutive lest days prior to administration of the
methyimercury, and on selected test days after administration. On test days, each
minute of EEG was scored visually and classified as either W (wakeful), SWS
(slow wave sleep), or PS (paradoxical sleep), for the full 24 hr. Statistical
treatment of the data consisted oft tests, and therefore must be considered very
nonconservative. Despite this approach, no effects were reported for the low-
dose group.
The authors report data that indicate two types of effects. Only in animals
receiving the highest dosage of methyimercury wu the total amount of lime in
which they were either asleep or awake altered. On the first full day following
treatment, these animals spent 69% of their time in either SWS or PS. whereas
during the baseline (pretreatment) period, animals spent about 54% of their time
-------
142 MMI.DyM
ia sleep. The Mil lest day reported lor this group was 12 days aAer (be end of
treatawat, aad at this poiat total deep Kmc bad reverted to baseliae levds(33%).
la contrast lo the total aaMwat of sleep aad waking time, (he relative distribution
of sleep aad wakiagEEGsaccairiagduifcv the Nght aad dart phases of the day
was altered at lower dosages aad kx laager periods, la general lUi effect was
characterized by a traaiirat lUwpnaiag of the day/night rhythai far W aad S WS
stales, aad samepcrsisteatphass (hill for the PS state. la tbc high-dosage group
the phase shift was stiH e vide at 2 asoalhs after esposure. These resnlts arc
siawiarifcd la Flpw I.
The fiadingr indicate a dear disruption ia the sleep/wake pattern la rats. To
drteimhia the braia levels of aweary related to these effects, the aathors
perfowed regioaal braia dissections aad aaalyied the tissue with AAS. The
high-dosage level produced peak brain levels of 10 jig Hg/g wet whole braia on
A.
o- s.s«a^aaMnmaMncunv
tiBtAfMimn Mtncuav
*M«0|HllimMHCMn
eastuns rmsTD«v Mica ii*oay attin
TMAf%MMT MMMW
Ham I. EfctH aI ¦nfcjlwunn) — tfacy <¦ Sfc«sw-P—tty nn H) Elfccto «fHfomn o«
|p|l| gltcpUMf.
143
-------
144 RoM S. OyM
day 10. and the intermediate dosage produced peak levels of about 2 fig Hg/g. If
the data from the intermediate douse group are Imly statistically significant
(which they may not be. based upon the type of analysis reported), the sleep
disturbance produced by methyhnercury occurs at very low levels compared with
other effects (Arilo el al., 1983).
" EFFECTS OF METOYLMERCURY ON SENSORY SYSTEMS. As mentioned pre-
viously, sensory system effects ate among those most frequently reported
following exposure to methyhnercury. Mattsson et al. (1981) recorded flash-
evoked potentials (FEPs) from three adult beagles, before and following daily0.5
mg/fcg exposure to methyhnercury. To minimize alterations in the FEPs
produced by alterations in attention and arousal, the authors trained the dogs to
perform a cued-avoidance response. The FEPs were obtained during performance
of this task.
This was an entirely within-subjects design, such that each dog's postexposure
waveforms were compared with preexposure waveforms. The analysis was
unconventional in the neurotoxicology literature, since the authors treated the
evoked potential as a complex waveform that should be treated as a whole.
Using this pattern-analysis approach, Mattsson et al. (1981) detected
alterations in the FEPs of the three dogs about I wk after the onset of treatment.
In a parallel scries of tests, it was determined (hat brain levels of mercury in the
occipital cortex were about 1.28 jig/g at this time. The authors detected no
alterations in performance of the cued-avoidance task, nor were there any other
obvious behavioral changes among the dogs. Clinically obvious poisoning did not
occur until nearly 2 months aller the onset of treatment. Therefore, these data
appear to support the argument that FEPs may be useful for screening for early
effects on sensory systems. However, they provide little insight into the nature of
the visual dysfunction produced in dogs at these early exposure points.
Since the damage to the visual system following exposure to methyhnercury
appears to be primarily cortical hi nature and related to the pattern of gyri and
sulci, it is not at aM dear that visual dysfunction should be expected in the
Kssenccphalic rat. Nevertheless, two studies examined the impact of perinatal
exposure to methyimercury upoo visual taction using evoked-potential tedmiques.
Zenick (1976) recorded FEPs from 30-day old male rats which had received
one of live basic exposure conditions, using cross-fostering to evaluate carry-
over between conditions. One group was never exposed to methyimercury. The
other groups received exposure either throughout gestation and nursing, during
nursing only, during gestation only, or during the post weaning period. The
postweaning group received 2.5 mg Hg/kg/day "directly," presumably by
intraperitoneal injection, for postnatal days 21-30. Other exposures were
Mactoyliyatologtcai AuhhmhI ol Organoaialal NaiuotoilcHy 145
indirect, through the mothers, who received approximately 2.5 mg/kg/day in
their drinking water.
Only five rats were evaluated in each treatment condition, and each rat tested
was randomly selected from a different litter. All recordings were made under
chloralose anesthesia. The author did not describe any efforts to control body
temperature, which can be expected to vary under anesthesia, and variations in
which can be assumed to produce large effects on the latency of evoked potentials.
Amplitudes were not evaluated, because the author found them too variable. The
results of this study are difficult to interpret for several reasons. First, while the
representative waveform presented by the author for FEPs recorded from the rat
visual cortex appears similar to those reported by others, the peak designations
are reversed in terms of which peaks are called positive and which are called
negative. Thus the first peak in most FEP recordings is PI, followed by NI, but
by the author's labeling scheme Nl precedes PI. One must either infer that the
peaks were mislabeled or that the waveform was aberrant. For the purposes of
this discussion, it is assumed that the peaks were mislabeled, since the only
technical problem noted was an unduly restricted bandpass (6-600 Hz).
The author indicates that in the control rats the PI (his NI) was 68 msec, and
that the Nl (his PI) latency was about 123 msec. These are extraordinarily long
latencies. For comparison, unanesthetized rats usually have PI and NI latencies
of 20-25 and 28-32 msec, respectively (e.g.. Dyer et al.. 1978). Anesthetized
rats have PI and Nl latencies of 24-26 and 33-35 msec, respectively, and
deeply hypothermic (body temperature about 3I*C) anesthetized rats have PI
and NI latencies of about 35-37 and46-48 msec, respectively (Dyer and Boyes,
1983; Hetzler and Dyer, 1984). The effect reported by the authors was that
exposure to methyimercury shortened PI latencies to about 45 msec and Nl
latencies to about 72 msec. Although these are enormous latency changes, they
still fail to bring the waveforms within normal range. Therefore, despite the care
which the author took to include appropriate controls during the exposures, some
¦¦niitwiiltMl factor in the recording procedure makes the data difficult to interpret.
A different approach to exposure and recording was taken by Dyer et al.
(1978). A single dosage of either corn oil or 5 mg/kg methyimercury was given to
pregnant rate on day 7 of gestation. On postnatal day 65, 18 control and 13
exposed female rate (from 6 and 4 litters, respectively) were implanted with
chronic electrodes for recording FEPs. The FEPs were obtained at four different
flash intensities, and peak latencies as weU as peak-to-peak amplitudes were
measured and analyzed. Increasing flash intensity normally increases peak
but the rats treated with methyimercury exhibited greater increases
in amplitudes of the early peaks( PI NI and NIP2) than did controls. In addition,
latencies of the P2 and N2 peaks were shorter in the exposed group than in the
-------
140 Wofciit». Pyr
control group. indcpcndtal of Duk intensity. The authors interpreted the
amplitude effect im team of a (enenl "bram-damage" model, which U still
ptau«ble( Oyer, 1986b). The latency decreases were attributed to a selective lots
of slowly conducting fibers, since methylmcrcury does appear to produce
selective toxicity to mmM cells. H k «« Hm iK« Ut-«w-y .w.f,w
were about 2 msec for P2 and about 5 msec for N2, and that these changes were
about an order of magnitude leas than (hose reported by Zenick (1976).
While both Zenick (1976) and Dyer et al. (1978) reported altered FEPs in
rata following treat astal with methylmcrcuty.itia not clear whether these findings
reflect specific visual-system deficits or general brain dysfunction. Other studies
have suggested general brain dysfunction (e.g., learning and memory deficits) in
similarly treated rats (Ecdes and Annau, 1982; Zenick, 1974), but no studies
have specifically addressed the possibility that visual deficits exist ia these rats.
The extent to which exposure to methylmcrcury produces auditory dysfunction
has not been determined. Some clinical reports indicated no auditory dysfunction
(e.g., Brenner and Snyder, 1980), white others described sensorineural hearing
Iocs im affected individuals (e.g., Nosaka et al., 1970; Friberg, 1971). The
clinical reports that deacribe auditory dysfunction suggest that it is cortical in
nature, although there it no compelling evidence lo support this contention. From
controlled laboratory studies it ia evident that methylmcrcury can accumulate in
the guinea pig cochlea (Konishi and Hamrick, 1979), and that auditory
dysfunction can be detected in adult rats given 50 mg/kg methylmcrcury and
tested using the acoustic startle test( Wu et al.. 1985). Thcsr findings suggest that
the dysfunction amy be peripheral or subcortical.
Recently Wassick and Yonoviu(1985) used the BACK lo test mice exposed
to methyfanercury as adults. Two exposure groups were utilised, and two sets of
recordings were asade in each groupi in one group(n — 5). mice received 4 mg/kg
subcutaneous methyhncrcury daily for 17 consecutive days, la the other group(w
— 6) the mice received 8 mg/fcg subcutaneous methylmcrcury fori consecutive
days. Although &e first group received more total methylmcrcury (68 mg/kg),
the second group's total exposure of 48 mg/kg was more concentrated.
Recordings were obtained prior to treatment, and on days 7,14, and 28 after the
onset of treatment. There was no control group, and no statistical analysis of the
data was reported.
The first set of recordings performed by Wasaick and Yonovitz (1985) was
intended to determine physiologically the auditory threshold of the rats at dif-
ferent pure-tone frequencies. To do this, BAERs were recorded to pure tones of
descending intensity, until the experimenter could no longer visualize a B AER in
die tracing. The procedure was repeated over a wide range of pure-tone
frequencies (4-78 kHz) to characterize any selective changes. The second set of
recordings consisted of BAERs obtained at 80 dB SPL intensity al each of the
MactopfcyeJotofical AmumimI ol Organomatal NaurotoalcMy 147
frequencies tested previously. In this set the latency of BAER peaks was
measured. This procedure was presumed to allow for localization of dysfunction
produced by methylmcrcury.
Although the data were presented primarily in graphic and tabular form with
no statistical analyses, it appears that exposure to methyfanercury increased the
threshold for producing BAERs. Figure 2 summarizes the effects of methylmer-
cury on BAER thresholds. While the posttreatinent data points arc clearly shifted
with respect to the pretrcatment points, it would have been useful to sec a control
group that showed no such shift with time, particularly in view of the apparent
variability between the two sets of pretrcatment recordings, la addition, a
question of some theoretical interest is whether the shift in threshold was greater
at some frequencies than at others. The authors concluded that there was a
preferentially greater shift at the frequencies of 16 and 32 kHz for the 4
mg/kg/day group; however, it is impossible to determine whether these are valid
conclusions la the absence of statistics.
CMjMi MS TMMMfNT
• <»iA|CN|N|MMn«nn omit
• ¦ «|1| CHjMa MIUMIMM
CM]M« || DAVft A* Tin ONSII
NMOIKMCVtUtol
Figure 1- Efceta of MelhylaKicwy oa pure-tone IkioluUi ia rata. . ¦ determined with the
txafauteaa auditory evoked lopont (BAER). The 4 ms/kfgroup received 1J daily dose»( total dote
69 ms/fcsl, «xi the t «s/ks sfouP received6 daily doses (total dose 41 msAs) The data. icpiuUed
from Wasiict mod Yoaoviu (1915). indicate an apparent losi of teiuitivky at alt frequencies
-------
148 doiMlta. 0»M
The results of the peak-latency uilyiit an even more difficult lo interpret,
since dau are presented for only one mouse in each group. However, (he data
presented suggest that at least in tlie 8 mg/kg group tlie effect of treatment was to
shorten ail peak latencies. These data were considered consistent with those
reported by Zenick (1976) and Dyer el al. (1978) in (lie visual system.
A plot of the interpeak latencies (Peak IV-Peak I), an indicator of central
rather than peripheral dysfunction, la given Cor both treatment groups at the
frequency of greatest hearing sensitivity, 16 kHz. in Figure 3. These data seem lo
indicate a decreased central conduction time, implying increased conduction
velocity through the brainstem. The mechanism by which this might occur It not
clear.
From these studies it is evident that methylmercury produces auditory
dysfunction in mice, and that this dysfunction contains peripheral and brainstem
•< 4 am/be CHjHt «17
0>|n|AtCN)H|a •
1—
mCTIUATMCNT
14
M
DAYS AFTER ONSET Of MtATMINT
Flfart J. ESccti of tmtlhjlmtniny wlAEt lletpoli Irtcuctc* (MIV-hrt 1). w> irfclot ot
ccatialntftef than peripheral dyxftmctioa. Dattareplo*edfa*«li* I6kllt Miaulus. Thcdauwggctf
decreased ccmlrml conduction lint (kaufed ipecd) Data obtained from Wiiiidi and VoaoviU
M act opfcyJotoq teat AiuuhmdI ot Ot|aiwiMl«l Naucoloikctty 140
components. The extent lo which dysfunction in the auditory cortex of mice
occurs remains lo be investigated. Preliminary evidence based upon exposure of
adult squirrel monkeys to methylmercury suggests that there may be significant
species differences in the nature of auditory dysfunction. However, these
findings, presented in abstract form, provide too little detail for evaluation (Zook
and WHpoeski. 1980).
C. EFFECTS OF METHYLMERCURY ON NEURONAL EXCITABILITY. As with
many neurotoxic compounds, poisoning with methylmercury can lead to
seizures. A presumption implicit in the work of a number of investigators, the
validity of which remains to be determined, is that clues to the nature of toxicity
produced by substances that trigger seizures at high dosages may be obtained by
studying neuronal excitability following administration of lower (none on vu Is ant)
dosages. Several such studies have been performed with methylmercury.
Eccles el al. (1981) investigated the influence of prenatal exposure (gestational
day IS) to methylmercury chloride(5 mg/kg by gavage to the mother) or to the
corn-oil vehicle upon hippocampal aAerdischarges recorded during adulthood.
The exposed animals did not differ from controls on variables specific to the
aAerdischargc itself (e.g., threshold and duration); however, two measures of
postictal excitability were affected. Normal hippocampal aAerdischarges end in a
profound EEG depression. The end of the EEG depression is usually signalled
by what has been called the rebound afterdischarge (Dyer et al., 1979). In the
exposed rats, the time from the end of the afterdischarge to the beginning of the
rebound was longer, implying an increased period of hypoexcitability. In addition
to the EEG depression, aAerdischarges are followed by a longer lasting period,
during which it is more difficult lo elicit a subsequent aAerdischargc. This period
was nearly three limes as long in the methybnerctiry-cxposcd rats as it was in the
controls. The functional implications of ibis finding are not clear.
Su and Okita (1976) look a different approach to studying long- term changes
in excitability produced by prenatal exposure to methylmercury. In this study
methylmercury hydroxide was given to pregnant mice subculaneously on
gestational day 10, in dosages of 0.6,8, or 12 mg/kg. The mice were tested on
postnatal day 70 with Aurothyl, inhalation of which is known lo produce
behavioral convulsions. In experiments thai use flurothyl, the dependent variable
is usually the time required for convulsions to appear once the animals are placed
within the exposure chamber. In this study, the highest prenatal dosage of
methylmercury produced an increase in sensitivity to these convulsions, as
measured by decreased latency (threshold) lo exhibit maximal seizures. This lest
produced a different answer than the afterdischarge test described by Eccles et al.
(1981). since it suggests increased excitability, whereas the afterdischarge test
suggested no change in seizure threshold, but decreased postictal excitability.
-------
ISO IMMtt.OfH
Mciuuhi et al. (1982) evaluated audiogenic seizure susceptibility in 28-31
day-old mice exposed prenatally to melhyimercury. la this study 8 rag/kg
methylmcrcury chloride was administered subcutaneously to pregnant mice on
gestational day 12. The audiogenic seizure test involves placing the animal next
to a loud bell or buzzer, taming on the bell, and observing whether or not a seizure
occurs. While the difference between the percentage of control and treated mice
exhibiting seizures was small (11% of the control mice exhibited seizures,
compared with 33% at the treated mice), the large number of mice used (189)
rendered the difference statistically significant. Thus this study too seems to show
a slight but general increase la the excitability of the brains of mice treated
prenataMy with methybnercury.
It is tempting to assume that the differences between the results of Eccleset al.
(1981) and those of Menashi et aL (1982) and Su and Okka (1976) reflect
differences in the tests; however, the difference in species and exposure
paradigms should not be ignored. One must conclude that the issue of persistent
changes in excitability produced by methybnercury it still unresolved.
D. EFFECTS OF METHYLMERCURY ON THE PERIPHERAL NERVOUS SYSTEM.
From the human data it was unclear lo what extent the sensory dysfunction was
produced by peripheral effects, and lo what extent it was produced by central
effects. Mural et al. (1982) reported an experiment designed to resolve whether
sensory deficits produced by methylme reury poisoning were central or peripheral
in nature. Peripheral nerve conduction was studied la rats (sural nerve was
stimulated rerarriingc were nxfa At iritlir) TV ran mrww <
mg/kg of melhylmercuric chloride every other day (or either 32 or 54 days. The
amplitude and latency of the nerve response were recorded. Sensory-evoked
potential data recorded from 2 Macaque monkeys who received 5 mg/kg
melhyhnercury chloride orally once a week for 10 wk were described. Visual-
evoked, somatosensory- evoked, and brainstem- auditory- evoked potentials were
•H recorded, but no details of the recording procedures were described. AN
recordings were made Hnder**mitd anesthesia induced by ketamine hydrochloride."
Sensory and motor conduction properties were studied in the median and sural
nerves of both monkeys.
In rats, exposure for 54 days abolished recordings from the sural nerves.
Recordings from animals treated for 32 days exhibited reduced amplitude of
recordings, but did not exhibit appreciable slowing of conduct ion. In the monkeys
there were no signs of peripheral nerve involvement, although the sural nerve
amplitude measurements were evidently so variable as lo preclude meaningful
comparisons. Both somatosensory and visual- evoked potentials showed progres-
sive slowing, while the brainstem- auditory- evoked potentials were unaffected.
The authors concluded that only very severe poisoning produces peripheral
MactopNirstoloalcal A»M»ww«nt o< OrganoiMlal NaurotoxIcNir 131
Involvement. The work of Cinca et al. (1979) suggests that even in the case of
severe poisoning in humans, the peripheral nerve effects are transient. It may also
be that in rats peripheral nerve involvement is demonstrated more readily than in
other species (Miyami et al., 1983).
B. Other Oraanomurcurlmlt
While other organomercurials arc used as fungicides, few studies have systemati-
cally investigated any of them utilizing electrophysiological approaches. It
therefore remains lo be determined whether the observations of Cinca et al.
(1979), that cthylmcrcury poisoning in humans produces peripheral sensory loss
and demyelination of the ninth and tenth cranial nerves, are true of methylinercury
also, or are peculiar lo ethylmercury.
IV. Organotlna
Of all the organooietaUic compounds, organotins, in particular triethyhin and
trimcthyhiu, have received the most attention from neurophysiology sis using
macrophysiological techniques. In both cases these compounds are viewed as
prototypes for investigating different kinds of neurotoxicity with different
methods.
A. TriethyMn
1. General Triethyhin (TET) is a compound that achieved some attention in
the 1950s, when it was discovered to be the contaminant of Stalinon responsible
for over 100 deaths in France. Triethyhin proved to have some very interesting
properties. Acute administration of TET produces intramyelinic edema, splitting
of the myelin, and vacuolatkm. la addition. It appears lo uncouple oxidative
phosphorylation. A potent action of TET that was not revealed until relatively
recently is that it produces marked changes in thermoregulation and thermoregu-
latory behavior. Rats fayected with 6 mg/kg TET and maintained in normal
ambient conditions (e.g., 22*C) may have a reduction in rectal temperature as
great as 5"C. Many of the acute effects of TET (physiological and other)
described in the literature may in fact be secondary to hypothermia. As discussed
below, some physiological studies have attempted to evaluate the role of
hypothermia in TET neurotoxicity, but with the exception of a study designed to
examine behavioral thermoregulation (Gordon et al., 1984), no behavioral
studies have addressed this confounding variable.
2. Characterization of Neurotoxic Effect
A. EFFECTS OF TET ON THE EEO. Bencdek et al. (1976) evaluated the influence
of a single injection of TET upon the EEG of unaneslhetized cats with chronic
-------
152 KobMtB.OyM
electrodes over the frontal, temporal, and parietal cortex. Two weeks after the
implantation surgery, the cali(aiunbcn unspecified) were treated with either 1.0,
2.5, 3.0, or 10.0 n|/k| TET sulfate. The EEGa were recorded over the first 6
hrs, and then daily lor up to 13 days following injection. Analog filters were used
to divide the signals into frequency bands of I -4 cps (delta), 4-8 cps(lheta), 8-
13 cps (alpha), and 13-30 cps( beta).
The overt behavioral consequences of the injection could be divided into three
phases: aw initial acute toxicity phase Iastiag6-I hr, a phase of apparent recovery
lasting 24-72 hrs, and a return of Ate manifcitatinns of toxicity (e.g., muscle
weakness). A similar patten has been wcH described for rats exposed to acute
TET (e.g., Torack et al., 1970). Cats treated with the 10 mg/kg dosage did not
survive the acute toxicity phase, while those treated with 3 mg/kg died about 30
hr after treatment. Changes In the BEG paralleled the overt behavioral
manifestations of toxicity. The I-mg/kg TET treatment did not alter the CEO.
An injection of 2.5 mg/kg TET produced EEG alterations that were evident
within 15 tin, as evidenced by an increased synchronization and the appearance
of slow waves (3-4 cps). The appearance of slow waves was particularly
apparent In the recordings from the occipital cortex. By the third hour, the
occipital slow waves were newly continuous. At 6 hr the incidence of slow waves
had decreased substantially. However, beginning 24 hr after treatment there was
a return of slow waves over the occipital cortex, which increased over the nest
several days. From the fiAh day on, the occurrence of slow waves again
decreased, but even on the thirteenth day there was still evidence of abnormal
slow-wave activity over the visual cortex.
Despite the absence of statistical evaluation of the data, these findings are
interesting, first because the EEG abnormalities parallel the behavioral ones, and
second because such abnormalities Me to be expected from white- matter lesions
(e.g.,Gloorctal., 1977). It would have been useful to know the status of the white
matter during the different phases of recording. As the authors point out, the
initial slow waves occurred loo early to be considered a result of edema. It is not
dear whether TET-lnduced hypothermia may have contributed to this finding.
Perhaps the most significant finding of this study was the apparent sensitivity of
the visual (occipital) cortex to the effects of TET. The basis for this apparent
sensitivity was not discussed.
B. EFFECTS OF TET ON THE VISUAL SYSTEM. The clinical EEG report of Pniill
and Rompel (1970), the experimental EEO investigation of Benedek et al.
(1976), and reports of visual disturbances in exposed humans (e.g., Barnes and
Sloner, I9S9) have implicated the visual system as an early indicator of TET
neurotoxicity. In addition, neuropalhological studies have suggested special
Macrophyatologlcal AuMsaMnl ot OrgMtoanatal NauiotoxIcUy 153
involvement of the visual system in the neurotoxicity produced by acute
administration of TET (e.g., Scheinberg et al., 1966).
Dyer and HoweN( 1982a) evaluated the integrity of the visual system in TET-
exposed rats using FEPs. The rats were divided into groups for daily treatment
with either saline or 0.188,0.375,0.75, or 1.5 mg/kg TET bromide in saline.
These treatments, administered 2 hr before testing, continued for 6 consecutive
days. The FEPs were recorded on each treatment day and for 2 days following
termination of treatment.
Triethyltin produced evidence of visual dysfunction, as indicated by increased
FEP latencies. While FEP latencies were affected in general, the latency to peak
N3 was most often affected. The N3 latencies were increased at the lowest
dosage of TET (0.188 mg/kg/day), while other peaks were only affected at the
highest dosage used (1.5 mg/kg/day). Effects of TET on amplitudes were
inconsistent and difficult to interpret. There was a considerable lag between the
onset of treatment and the time at which latency alterations began to appear
(several days); however, the increased latencies remained for up to several weeks
after termination of treatment. Figure 4 shows the gradual disintegration of one
waveform over days with treatment.
While these data do not demonstrate the nature of visual system dysfunction
produced by TET, they do indicate that the cortex may be al least as sensitive as
the optic nerve, since N3 latencies were affected al dosages below those required
to affect latencies of peaks PI and Nl. The increased latencies observed are
consistent with what would be expected in the case of disturbances in myelin, but
similar changes could occur as a result of hypothermia. The study also fails to
demonstrate any special sensitivity of the visual system. Concurrent tests (see
below) indicated that hippocampal system function is at least as sensitive as the
visual system function to the neurotoxicity produced by TET.
In a related study. Dyer and Howell (1982b) investigated the impact of
hypothermia upon FEPs recorded from TET-trealed rats. As expected, TET
produced increased latencies. However, contrary to expectation, prevention of
hypothermia appeared to exacerbate the toxic consequences of exposure.
Whereas the rats treated and maintained at normal room temperatures had
normal FEPs 24 hr after treatment, the rats in whom hypothermia was prevented
still had abnormal FEPs 120 hr later. Furthermore, since even the waimed
animals had increased latency, the authors suggested that previous results (Dyer
and Howell, 1982a) could not be accounted for simply on the basis of
hypothermia.
These studies neither substantiate any unique susceptibility of the visual
system nor do they indicate the nature of visual dysfunction produced by TET;
however, they do indicate that monitoring the FEP may provide a marker as to the
magnitude of an acute TET-induced encephalopathy.
-------
CO
AWU
154
Mncropfcyaioioglcal AimimimI ol Otganonieial NauratoiteHy 155
A further step towardcharacterizing the effects of acute exposure toTET upon
the visual system was taken by Boyes and Dyer (1983). In this study, PREPs
were recorded in addition to FEPs, to compare the relative sensitivity and
specificity of the two techniques. While FEP studies are technically simpler to
perform than PREP studies, and for this reason are probably better adapted for
screening purposes, alterations in PREPs are more easily interpreted. Specifically,
under the appropriate circumstances PREPs may be used to describe such
complex visual functions as acuity and contrast sensitivity (e.g., Boyes et si.,
I98S).
Boyes and Dyer(l983) made recordings from chronically implanted Long-
Evans hooded rats immediately before and 24 hr after treatment with either 0,
4.S, or 6.0 m|Ag TET bromide. During the interval between treatment and
testing, the rats were maintained in a warm (30*C) environment to prevent
hypothermia. The results indicated that, as was the case with FEPs (Dyer and
Howell, 1982b), TET produced latency increases in PREPs 24 hr after a 6
mg/fcg treatment. Further, in contrast to FEPs, TET produced a pattern of
amplitude alterations in PREPs that consisted primarily of increased amplitudes
in the early components. While the significance of this finding for visual function
is not clear, it does suggest that TET produced more than a disruption in myelin.
Alterations in evoked- potential amplitude usually reflect the synchrony, magni-
tude, and nature of synaptic events (Dyer, 1986a).
C. EFFECTS OF TET ON NEURONAL EXCITABILITY. As part of their effort to
characterize the efleets of TET, Dyer and Howell (1982a) included an
evaluation of hippocampal afterdischarges (ADs). The results demonstrated a
mixed excitatory/inhibitory effect of TET, depending upon the dose and the
cndpoiM. The low dosage of TET (0.188 mg/kg/day) decreased AD thresholds
relative to control, white higher dosages did nol. All but the lowest dosage ofTET
increased the frequency of spikes within the AD, and the 0.375 mg/kg/day
dosage produced a greater postictal electrical excitability than the other dosages.
All of these effects are arguably a function of increased excitability. However, the
high dosage (1.5 mg/kg/day) produced a decrease in postictal electrical activity
and excitability. Both of these effects are clearly indicative of decreased neuronal
excitability. By virtue of its greater ease of interpretability, this is the more
compelling observation. It is interesting to note that there was not a significant
dose X day interaction described for the analysis, suggesting that the effects were
Figure*. FEPs recorded from (4) one ral in ihc saline control (roup during the firsl 9 days of the
eiperiatcni. ud|l| one (at in the I S ing TET/fcg/day dosage group during the first 9 days of the
operimcnl Figure feprinted from Dyer and llowcN 11982a)
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156 Rokwl I. OfM
of constant magnitude across days. This implies that in some cases a tingle
treatment with • dosage as low as 0.188 mg/kg TET might alter htppocampal
runctioa. Figure 3 illustrates the influence of different dosages of TET upon
postictal electrical activity.
Thf mllH— fa" n~iVmp i« Ih.l lh> «yd«M Aim Bnl
afford unique sensitivity, since TET-Induced effects were observed in the AD at
the same dosages, la addition, TET produced evidence of CNS depression.
Whether there was a biphasic effect of TET (excitation and depression) cannot
be determined until the biological determinants of the different AD variables is
understood mora Ally.
Several studies by Fox and Doctor support the presumption that TET
produces a depressant effect upon the CNS. In theae studies the MES (maximal
eledroshock seizure) model was used to evaluate excitability. These seizures are
produced by application of an ACcuwent pulse cither to the scalp, the ears, or the
corneas, and the timing and sequence of resulting behaviors is recorded. The
140
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120
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Ftgw* S. Efccta of TET oa porticft EEO dcptcuioa. Magnitude of poti AD deptcuioa «m
ralcirt«tc
-------
158 ItabMtS. Oyer
are presented. With this experimental design a repealed-measures analysis of
variance is appropriate so thai dose X time interactions may be assessed. While
(he authors indicate lhal they performed an "overall unweighted means analysis
of variance." they neither specify the nature of the analysis or its outcome. Are
the data ifcey present highly selective or are they representative? Were the
analyses performed upon all data or only those reported? The reader has no way
of knowing.
As part of an effort to compere the neurotoxicity of several trialkykin
compounds. Doctor and Fos (1982b) used the MES test in mice uyccted
intraperitooeally with cither 3.5 X 10~*orl7.5X IO~*moi/kgafTETbromidc.
The low dose produced no significant effects on seizure grade, but the high dose
produced an anticonvulsant effect similar to the one reported in rats. Similarly,
seizure durations were affected by TET treatment. In this case the tow dose
increased the extension/flexion ratios at0.5- and 4-hr time points, but not the 22-
and 96-hr time points. The high dose was effective also at the 22-hr lime point,
but not at 96 hr. Again, the details of the analysis of variance were not made
available.
susacuk in
Ottawa emmmum
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Flpn 4. EBeets of lOdiriy ciposuro to TET ia the dnakiag wHu on pciccaUfc of i*t itowiag •
trade 5 MES The data ifcmmt • depcuwl/aali-coavaltaal cfficct of TET. The data were
obtained feoai Fo> aad Doctor (19S))
Hacfopiiyatoioaical AaHiHMal of Organoimtal NourotoalcHy
159
The sensitivity of the M ES test, and the long duration of the depressant efleet
produced by TET are both impressive. It is not at all clear how this effect might
come about. Possibilities raised by the authors include alterations in the chloride
concentration gradient and a change in the pH equilibrium (Doctor and Fos.
1982a; Fos and Doctor, 1983), or some change in either GABA or alpha
adrenergic neurotransmitter systems (Fox, 1982). While tome evidence exists to
support either view (e.g., Selwyn et al., 1970; Selwyn, 1916; Costa. 1985),
neither argument is persuasive, partly because the organotins most effective al
producing the relevant changes in biochemical parameters are least effective as
anticonvulsants( Selwyn et al., 1970; Costa, 1985; Doctor and Fox, 1982b). and
vice vena. A change in the impede nee of the brain might also produce an
anticonvulsant effect, and such changes have been noted following administration
of TET (Shelton et al., 1982), but the dosages required to produce impedence
changes were probably too high to explain the MES findings. Thus the answer to
this puzzle remains to be discovered.
D. PEIUNATAt- TOXICITY PRODUCED BY TET. Only two studies have assessed
the physiological consequences of perinatal exposure of rats to TET. Dyer et al.
(1981) gave intraperitoneal injections of TET bromide to rat pups on postnatal
day S. A within-litter design was used, such that one male and one female from
each litter received 0, 3, 6, or 9 mg/kg TET, and each litter contained all
treatments ia both genders. Electrodes were implanted when the rals were 60
days old, and testing began 10 days later.
Despite an extensive evaluation of neuronal excitability, there was only trivial
evidence of any long- term alteration produced by TET. Female rats given 3
mg/kg TET on postnatal day 5 had slightly longer cortical ADs during the
hippocampal kindling process than did controls. No other changes in AD and
kindMog parameters or pentylenetetrazol and picrotoxin seizures were significant.
The nondose- related nature of the AD finding, coupled with its relative isolation
in the face of many presumably related endpoints that were unaffected by
exposure, suggest that this may be a type I error.
In contrast to the lack of any long- term effect of perinatal TET on neuronal
excitability. Dyer el al. (1981) found considerable effects on the FEP. In
particular, there was a progressive dose-dependent latency increase in successive
peaks, as indicated in Figure 7. Since the early FEP peaks were unaffected by Ihe
treatment, the enduring effect may be cortical in nature, and could be accounted
for by assuming disturbed myelination in Ihe visual cortex.
More recently. Doctor and Fox (1983) reported a study of Ihe immediate and
long-term effects of perinatal exposure to TET on MESs. In this study rats were
treated intraperiloneally with either 0, 0.25,0.5, 1.0, 2.5, or 5.0 mg/kg TET
bromide on postnatal days 0, 5, 10, 15, and 20. The MES testing occurred on
dayslO, 12.14,16,18,20,23,26; 36,46,56, and 85. The authors reported that
-------
160 Nobart I. Oyar
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fcom wnnlMirf adnh nn. • - Dftrul from laltai mlrel. Reprinted fcum PytmJ. (IMI|.
TET produced a developmental delay in the appearance of (he normal MES
pattern, but IkM when the rata became adults their seizure severity was greater IT
they had been exposed to TET during the early postnatal period. Figure 8a
illustrates the seizure severity scores from this study; these findings appear
nondosc related. However, extrapolation to the age at which each group reaches
60% (the approximate "normal" percentage of stage 5 MESs in rats) indicates a
very clear TET-induced developmental delay. Figure 8b illustrates the duration
I I I 1 1 1 1
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FlftucS. M) Effects ofearty postnatal eipoaure to TET on development of Map SMESsinfcmalc
iMt Rau were given the indicated dosage on postnatal day» 0.5.10. IS, and 20. Tkuefart total
opuuiiu were 0.1.2$. 1.5. J. I I S, and 2S ¦*/** The data obtained bom Doctor and Foa (tttl)
indicaic a TET-induced developmental delay in appearance of il«|c 5 MESs, particularly if the
¦HMmal rale is taken to be about 60%. (A)EflccU of early postnatal cipouire to TET on duration of
hindluab cilciuion (wu t SEMI produced dwW| MESt. Inlcnncdiate dotage (roups appear to
have more severe seizures. Oala from Doctor and Fo« | I9V3).
161
-------
162 IMwtl. Dfr
of blndlimb extension at different ages. Increased duration implies increased
severity. While (be authors suggest that treatment produced a triphasic efled
(retarded development of the response, coupled with an increased severity once it
developed), an alternative explanation may be more parsimonious. Note that
control animals had their longest durations on day 22, and that this peak was
followed by a progressive 20% decline in duration. Treated groups tended to peak
later and decline less, thereby supporting the initial explanation of a development
tal delay.
There are problems with the analyses presented in this study. The seizure
duration data were collected from the same animals, over and over, on the
different lest days. The authors indicated that each rat was tested on each of the
12 different test days, which would asake the design of this study a simple groups
X days repeated measures analysis. However, the authors then indicated that
205 rats were studied between days 10 and 2 6,101 rats, all females, were studied
between days 36 and 56. and 53 females were studied on day 85, including none
from the0.2S mg/kggroup. The authors stated that the analysts performed was an
unweighted means ANOVA, but no further details were given regarding (I) how
the repeated testing nature of the analysis was taken into consideration,
particularly in the face of grossly decreasing numbers with increasing time, or (2)
the outcome of the analyses. Further, since the authors previously indicated that
typically up to 35% of adult rats do not show the normal seizure pattern, it seems
curious that att of the controls did show such a pattern on day 85 (see Figure 8a).
The conUote appear to be the outliers on this lest day.
Annlysis of Figure 8 suggests that very little change in the nature of the elTect
occurs between day 26 and day 85. It is therefore difficult to argue that the effects
observed during adulthood reflect some sort of reorganization or compensation.
The authors point out that a similar pattern of effect (depression initially,
followed by increased activity) is observed when locomotor activity is used as the
endpoint, following a single administration of TET on postnatal day S. However,
in this cue the increased activity observed in adulthood is considerably more
impressive (Reiler et a!., 1981). It is also interesting to note that in the case of the
behavioral studies, the effect was greater in males than females, while only
females were tested by Doctor and Fox(l983).
From these developmental studies one may conclude that perinatal exposure
to TET produces both immediate and persistent effects. The immediate effects
have only been evaluated using the MES lest, and seem to be similar to the
immediate effects observed in adults administered TET. The effects lhat persist
into adulthood have been evaluated with a number of tests. Persistent changes in
excitability appear to reflect delayed development at unspecified loci, while
persistent changes in the visual system are indicative of cortical dysfunction.
Macroplnftologlcal AimimmI of Organom«l»l N«ufo4o«lcMy 163
E. PERIPHERAL nervous SYSTEM TOXICITY. Using the rare but refreshing
combination of electrophysiological, morphological, and biochemical techniques.
Graham et al. (1976) evaluated the consequences of exposure to TET sulfate in
the drinking water (20 mg/l) upon the peripheral nervous system of Osborne
Mendel rats. Assuming a daily intake of about 30 ml/rat, the daily dosage of TET
to these 250-g rats was about 2.4 mg/kg. This dosage was sufficient to produce
paralyais by 20 days, and the authors were required to lake pains to avoid
mortality (they jvere only partially successful).
The sciatic nerve was studied (each nerve was studied only once per rat), and
the endpoinls recorded were (I) electrical threshold for producing a compound
muscle action potential, (2) amplitude of the evoked muscle-act km potential, (3)
and motor conduction velocity. Repeated! tests were performed upon the data,
which was collected on daysO, 10, and 20 of exposure, and at various limes after
exposure was terminated. No alterations were observed in the threshold for
production of the response, or in the amplitude of the muscle-action potential.
The results of the conduction velocity test are illustrated in Figure 9. The figure
shows a decline in conduction velocity, which was reversible over time.
The decline in conduction velocity was correlated with the appearance of
splitting and vacuole formation within the myelin sheath of the sciatic nerve.
There was also an increase in the number of neurofilaments in the axons of both
vacuolated and nonvacuolatcd fibers, but this was not correlated with the
alteration in conduction velocity, since motor-nerve conduction velocity "returned
to normal at a time when florid changes were still present in the sciatic nerves
during the recovery period" (Graham et al, 1976).
While significant alterations were observed in the peripheral nerves of these
rats, the dosage of TET required to produce the alterations was substantial,
compared with the dosages described above as sufficient to produce alterations in
measures of CNS function. This comparison suggests thatCNS is more sensitive
to TET than is the PNS.
3. TET ana Uodel Compound. While few would argue that TET is a compound
lhat poses significant environmental risk, it has nevertheless been used extensively
in the laboratory as a model. Part of the reason for its attractiveness is most surely
that it produces dramatic but reversible effects.
A. DEOENERATIVE/OEMYEUNATING DISORDERS. Gerren et al. (1976) com-
bined neurophysiologies!, biochemical, and behavioral endpoints in an investiga-
tion of the effects of TET on mice. The staled purpose of the study was to assess
the feasibility of using TET as a model of nervous-system demyclinaling
diseases. Among issues the authors addressed were (I) the nature of functional
-------
164 Webert S. Ovw
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Fl$mnt. Motor conduct^ wiocJty <«km ± S£M) rccwdcd faHowfaic Himoltlioa of IkKMc
¦c>v«iaifccratidufia((*alldlia«)arallcr(4oU«dHM)nccMag20a|/ITETtiilItf«iallMirdri«Uag
water. Dal* nbuiwd kom Qrahaai« al. (I'M).
disturbances produced by acute and chronic exposure to TET, (2) the extent to
which the time course of functional disturbances paralleled the know* morpho-
logical disturbances produced by TET, and (3) the extent to which functional
disturbances were central or peripheral in origin. Presumably a good model of a
disease like multiple sclerosis (MS) would show some functional alterations of
CN S origin similar to those produced by MS. These functional alterations should
be correlated with structural alterations, which should be restricted to the CNS.
Ideally the model would ahodemnnsirate llit tpiaidic mmarfUS rf
by dysfunction, remission, and dysfunction. Unfortunately, a variety of proce-
dural shortcomings, including incomplete presentation of methods and analysis
techniques, rendered the results difficult to interpret.
a EDEMA AND DEMYEUNATION. Triethyhin does not produce demyelination
in the classical sense. However, the edema and myelin splitting produced by TET
may amount to a partial functional demyelination. Shah et al. (I978) and
Amochaev et al. (1979) used this property of TET to investigate the influence of
Macrophyaiolofllcal AmhmmM ol Ocgtnomld NaurotoatcHy 165
disordered myelin upon the auditory system. In particular, the authors were
interested in determining the relationship between TET-induced changes in
myelin and latency of peaks in the brainstem auditory-evoked response (BAER)
of Sprague- Da wley rats. Their presumption was that if a strong correlation
existed between BAER latencies and myelin content, BAERs could be used as a
noninvasive measure of myelin delects. In addition, the authors evaluated the
relative effects of TET upon the peripheral and central auditory system by
including a measure of central conduction time. Since the first BAER peak(peak
I) reflects activity in the periphery, while subsequent peaks reflect activity in the
CNS, the difference hi latency between the first and later peaks (known as central
conduction time) provide* an index of alterations which are in addition to any
produced in the periphery and simply passed along.
Amochaev et al. (1979) used an eiposure schedule identical to the one used by
Graham et al. (1976) and described above. Briefly, the rats were exposed to 20
mg/l TET sulfate in their drinking water for 2 wk. The BAERs were recorded
from needle electrodes placed in the scalp while the rats were anesthetized with a-
chloralosc. The stimuli were clicks presented at 60 dBHL above the experi-
menters' threshold. No efforts to control body temperature were described.
Further, the records were highly filtered (100 Hz to 3 kHz), which distorted the
waveform, compared with unfiltcrcd recordings(Janssen el al., 1986). However,
if one can assume that the authors did control body temperature, their findings
indicate that TET produced an increase in those BAER latencies that reflect
activity in both the peripheral and central nervous system. Further, the authors
were able to show that these changes were reversed if the rats were tested 2 wk
after termination of exposure. Myelin content, determined in the same animals,
paralleled the alterations in BAER latencies. Myelin content was reduced by
about 30% at the end of eiposure, but had recovered in the animals tested 2 wk
later. It is important to note that while these findings demonstrate TET-induced
dysfunction in the auditory system, they tell us nothing about what the
consequences of exposure to TET are in terms of hearing. Altered latencies to
click stimuli carry with them only the presumption of disturbed hearing.
0. Trlmmthyltin
1. Genera/. Interest in trimethyltin (TMT) blossomed with the description by
Brown et al. (1979) of the intriguing pathology produced by the compound. A
sequence of neuronal cell loss, which appeared to target certain cell fields within
the hippocampus, made for attractive comparisons between TMT and the
contemporary ncurobiological tool kainic acid. Further, the difference in the
pathology produced by TMT and TET, coupled with the apparent structural
similarity between the two compounds, reinforced our inability to understand
structure/activity relationships. The only known cases of human exposure to
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106 HofcwtS. Oyar
TMT came from otnyiliaiiil accidents |Fortemps el al., 1978; Ross el al.,
1981). In Ibese rqurti, exposed humans reported a variety of symptoms,
including seizHKS thai persisted 6 months after termination of espoaure, EEG
abnormalities (low voltage spikes), lowered threshold to pentylenetetrazol
seizures, aggressive behavior, irritability, insomnia, forgetfulness, and dimming
of vision.
In tbe case of TMT, the neuropatbological and behavioral observations of
exposed sntmsh. "•* In hwrMW*. have guided
the physiological studies. Tbe neuropathology produced by TMT has been most
extensively described in tbe limbic system. In the hippocampal formation,
damage appears to be species- and to some extent strain-dependent, a factor that
must be kept in mind when performing and interpreting physiological (and other)
studies. Tbe nuyor differences in pathology seem to be between rats and mice. In
mice, the primary damage is to the granule cells of the dentate gyrus, while in rata
the primary damage is to the hippocampal pyramidal cells (Chang et al., 1983).
Which pyramidal and granule cells are affected appears to depend upon the
location along the septotemporal axis in Long-Evans rats, although other strains
have not been investigated (Chang and Dyer, 1985a). Gerbila appeared to be
mouse-like, in that damage was more extensive in the granule cells of the dentate
gyrus than in the pyramidal cdla of Anions horn, while hamsters appear to have
even less granule ccH damage than rats. Marmosets showed more difliise damage
than has been reported far most species, including damage in the retina, lateral
geniculate, and visual cortex (Brown et al., 1984).
Aggressive behavior, whether toward conspccifics or toward attempts at
handling by the experimenter, has been described following administration of
TMT in Wistar rats(Porton strain) (Brown et al., 1979), Long-Evans hooded
raU(DycretaL, 1982b), and Marmosets(Brown ct al.. 1984). but not in Fisher
rats (Bushncll, personal communication). Wistar rats (Royalhart strain?)
(LoulhsetaL, l983),hamsters(Brownctal., l984),geihils(Bfowactal.. 1984),
CS7BL/6N or BALB/c asice( Wengcr et al., 1984). grasshopper mice(Hulebak,
personal communication), or primates (Bushncll. personal communication).
Self-mutilation of the tail baa been described in Long-Evans rats and Spraguc
Dawley rats (Dyer ct al., 1982b; Stoviter et al., 1986), but not in other strains
(LouWs et al., 1983). Seizures have been reported by many authors, although
they evidently do not occur in all species or strains tested (e.g.. Oyer et al.,
1982c; Brown ct al.. 1984).
2. Characterization of Tonicity. Most physiological studies of TMT toxicity
have been designed to further characterize the toxicity, rather than to determine
its mechanism. Most of these studies have focused upon either sensory systems or
the issue of CNS excitability. There is very little data on the effects of TMT upon
Macrophyaloloflical Amiimal of Organomatal NvurotoMJcMy 167
the EEQ in animals, which is particularly remarkable since insomnia and EEG
abnormalities have been described in humans (Fortemps el al., 1978).
A. ECO. Ray (1981) recorded the EEG from rats immediately before adminis-
tration of TMT (8-10 mg/kg by gavage), and al varying times over the next 11
days. The dosage of TMT employed was approximately the LDwin the author's
hands. Tbe analysis of the EEG consisted of visual inspection of records, coupled
with peak-to-peak measurements of 30 randomly selected I -sec epochs on each
lest day.
Since the hippocampus is the primary generator of EEG activity in the Ihela
range (4-8 Hz) and is also one of the structures with the most profound TMT-
indticed pathological alterations, it is not surprising thai the only EEG effects
reported by Ray (1981) were derived from the hippocampal recordings.
Hippocampal I beta has been closely tied to movement (e.g., Whishaw and
Vandcrwolf, 1973), and the hyperactivity produced by TMT should therefore
increase the occasions on which one sees Ihela activity in EEG records.
However, since TMT produces loss of hippocampal neurons, it might be
expected that either amplitude or occurrence of Ihela might decline.
Ray( 1981) reported that both the amplitude and occurrence of thcta increased
between days 2 and 4 following treatment. While Ibis period of increased Ihela
corresponded with increased behavioral activity, the author stales that the two
were partly dissociated, since long periods of theta were present, even when the
rat waa immobile. Subsequently, the frequency of the waves within the Iheta
episodes decreased, and after day 8 no Ihela episodes were noted in the
recordings.
These findings are interesting, but do not provide much insight into the nature
of TMT toxicity. With careful recordings from different loci in the hippocampal
formation, more sophisticated analysis of I he EEG, and more systematic
monitoring of behavioral activities during recording sessions. Ibis type of study
might provide some insight into the nature of hippocampal Iheta, since the
generators of Iheta willun tbe hippocampus are still a subject of investigation.
I. SENSORY SYSTEM. Information upon whicb one might predict the presence or
absence of visual dysfunction in TMT toxicity was initially contradictory. While
the clinical reports indicated dimr.tingof vision(Fortemps et al.. 1978). the initial
pathological report in rals sought but failed lo find relinal pathology ( Brown el al..
1979). Based upon the observation lhal TMT- treated rals often ran into walls in
an open field, and lhat some of their other behaviors could be accounted for on the
basis of visual system dysfunction. Dyer el al. (1982a) evaluated the visual
system.
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168 Xobait I. Oy«f
Long Evans rats with chronic electrodes had FEPt recorded from the visual
cortex on days 1,4,8, and 16 following administration (by gavage) of either 0, S,
6, or 7 mg/kg TMT chloride. The findings, illustrated in Figure 10, show that
TMT produced significant alterations in this response. Specifically, latency of
the first positive peak was increased at all dosages, and latency of the second
positive peak was reduced in a dosage-dependent manner. The amplitude of the
first peak was also reduced. There was no dosage X lime interaction, auggesting
that the effect of treatment was immediate and unchanged over the course of Ihe
study. The pattern of amplitude and latency findings was consistent with a
decreased input to the visual cortex, an explanation that was assessed by
recording FEPs from the optic tract in another group of rats (Dyer et a!., 1982a).
As would be expected from a peripheral lesion, optic tract latencies were
increased by exposure to 4-6 mg/kg TMT. Since the magnitude of latency
increase was roughly equivalent to Ihe magnitude of increase observed in the
cortex, Ihe authors concluded lhat the full extent of the lesion occuned distal to
the recording electrodes, in Ihe retina. As with Ihe cortically recorded FEP, the
optic tract response was decreased in amplitude. The most parsimonious
explanation of these findings, thai a decreased output occurred from Ihe retina,
was supported by pathological examinations showing a loss of retinal ganglion
cells (Chang and Dyer, 1983). The TMT-induced retinal lesions have subse-
quently been described by others as well (Bouldin el al„ 1984; Brown el al.,
1984). Figure 10 summarizes the FEP findings following TMT.
The presence of an easily recognizable, albeit different from control, FEP in
TMT-treated Long-Evans rats indicates lhat while they have visual-system
dysfunction, they are not blind. A detailed description of this visual impairment ia
still lacking.
The apparent irritability of some TMT - treated rats suggested that TMT might
produce an increase in the amplitude of the acoustic starlle response. However,
these authors found a profound decrease in Ihe amplitude of acoustic startle
response (Howell el al., 1981). This observation, which has been replicated a
number of times (e.g., Rupperl et al., 1984), suggested that TMT may produce
dysfunction in the auditory system in addition to Ihe visual system. A full
accounting of Ihe effects of TMT on the auditory system has not yet been
produced. However, some evidence indicates lhat TMT increases the stimulus
intensity required to produce a B AER( Janssen and Dyer, 1984), and further that
there is cochlear pathology associated with exposure to TMT (Chang and Dyer,
1983). All of these changes evidently appear quite rapidly, and may be present
within 24 hr of treatment.
Howell el al. (1982) used physiological methods to assess the likelihood that
TMT also produces somatosensory dysfunction. The most compelling suggestions
that this might be Ihe case came from the self-mutilation observed in TMT-
Macrophyatotofltcal to—Iart d Oreanomalal Na wotoxtcHv 18®
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Flfaro It. EITccU of TMT apoa FEPt recorded from Ihe optic tract aad viiutl cortti of rata. (A)
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Both leu of unpiiludc mad laleacy data au|gcal dyifuactioa. Date obtained from Dyer el al.
(IMU
treated raU, and from the effects on the visual and auditory systems, as described
above. To evaluate the somatosensory system, Howell et al. (1982) recorded
somatosensory-evoked potentials from rata with previously implanted chronic
skull electrodes. The authors used electrical stimulation of Ihe tail nerve to elicit
the response, and evaluated the threshold and conduction velocity of this nerve to
assess the role of peripheral factors should cortically recorded alterations be
detected
For a variety of reasons, the somatosensory-evoked potential has not been
well characterized in rats (liyer, 1986b). Il is therefore difficult lo compare the
data recorded by llowell et al. (1982) wilh other studies for purposes of assessing
adequacy of technique. However, Ihe authors describe a waveform following
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I/O Batlrt 1. Pyt
stimulation aflkclii nerve, which was significantly altered in rate treated with
TMT. The primary effect of the treatment appeared lo be an increase in latency,
which could aot be accounted for on the basis of an altered peripheral conduction
velocity. Some latencies wen increased I day after treatment, while others did
not increase until the second posttreatmcat lest point, which was at 4 days. The
authors did not follow die time course of these findings beyond 4 days. As with the
other sensory systems, there may be anatomical correlates of this somatosensory
dysfunction, since Chang and Dyer (1983) reported alterations in dorsal-root
ganglion neurons Mowing exposure lo TMT.
Perhaps the moat significant feature of this collect ion of studies is that they
identified sensory systems as targets far TMT. Neurophysiotogical techniques
have aot bees used effectively lo describe the nature of that toxicity, although in
thecaseofthe visual system the study identified the retina as the probable locus of
dysfunction. Mora sophisticated use of neurophysiological techniques may
provide further fauigM into the real consequences of exposure to TMT for the
sensing organism.
c neuronal EXCITABILITY. There were at least three compelling reasons to
suppose that TMT might produce a change in CNS excitability. First, humans
and rate with sufficiently high exposure levels exhibited seizures. Second,
humans showed a lower threshold Cor elicitation of pentylenetetrazol seizures
(Fcrtemps et al., 1978). FiaaMy, the finding that TMT produces hippocampal
damage and aggressive behavior, coupled with reports that limbic system kindling
produces aggressive behavior (Pincl et al., 1971), suggested that a kindling- like
process might be involved in the reported aggression. The effects of TMT on
neuronal excitability have been approached front a number of physiological
perspectives, and these different approaches have produced occasionally con-
flicting data.
Since TMT produces spontaneous seizures, it would be useful to have EEG
recordings from different brain sites, so that die area of the brain from which
seizures were triggered might be identified. The closest any studies have come to
such recordings is the work of Ray (1981). Ray implanted chronic cortical
electrodes at several loci, and further included depth electrodes In the dentate
gyrus and pyriibna cortex. Of the seven rats treated with 8-10 mg/kgTMT, two
were in the process of having EEOs sampled when spontaneous seizures
occurred. One of these events was on the fourth day following treatment, and the
other was on the eighth posttreatment day. The behavioral seizures described
were similar to those described in rats during limbic system kindling, consisting of
rearing up on the hindlimbs and fordimb clonus. Because of this limbic like
seizure pattern, it is surprising that the only reported EEG changes during the
seizure were from the cortical leads. Ray (1981) indicates that isolated cortical
Macfopfcytlotofiica! timnMl of Organomalsl NiwatoilcKv 171
spike trains lasting 2-5 sec occurred in concert with the behavioral seizure. These
discharges remained localized. Dyer et al. (1982c) attempted to characterize
CNS excitability in TMT-treated rats using a number of different approaches,
including pentylenetetrazol (PTZ) seizure and hippocampal A D characteristics,
an** lappocampal and amygdaloid kindling rate. In the case of hippocampal AD
characteristics, an attempt was made to correlate pathological data with the
physiological findings. This battery of tests was selected to demonstrate whether
any alterations in excitability observed where local or reflected general properties
of the CNS.
To examine the effects of TMT upon PTZ seizures. Dyer et al. (1982c)
treated Long-Evans hooded rats with either0,5.6, or 7 mg/kgTMT chloride.
Five days later, a time when many of the behavioral consequences of TMT
administration in rats are most prevalent, the rats were administered cither 30.
45, or 60 mg/kg PTZ intraperitoneaHy. The authors reported that TMT- treated
rats had more severe seizures with shorter latencies lo clonic seizure activity than
did coat rots. In addition, the 7-mg/kg dosage ofTMT reduced by nearly one-half
the dosage of PTZ required to produce at least a clonic convulsion in 50% of the
group. Browning (1985) suggested that all but the tonic component of PTZ
seizures is probably triggered rostral to the brainstem. These data are therefore
compatible with alterations in excitability occurring in the forebrain, possibly in
the limbic system.
Hippocampal ADs are presumed to reflect the functional integrity of neurons
and pathways within the hippocampus (Dyer et al., 1979). It was presumed that
this measure would be extremely sensitive to TMT, since TMT produces
dramatic changes in the morphology of the hippocampus. To investigate the
influence of TMT upon hippocampal ADs, the authors implanted electrodes in
the dorsal hippocampus of Long-Evans rats. Afterdischarge parameters were
evaluated before treatment and on four day>( 1,4,8. and 16) after treatment with
either 0, 5, 6, or 7 mg/kg TMT. There was a trend toward increased AD
thresholds over time in the TMT-treated groups, but none of the AD parameters
differed significantly from the control. While this finding was quite unexpected,
recent evidence suggests that hippocampal ADs. once triggered, may be
controlled by nonsynaptic events (Traub et al., 1985). Therefore, changes in
synaptic relations among cell groups within the hippocampus may not be well
detected by this technique.
In addition to studying the properties of hippocampal ADs. Dyer et al.
(1982c) evaluated hippocampal excitability by studying the rale al which daily
stimulation of the dorsal hippocampus produced kindling. In this study, rsls were
treated once with either 0,4.5. or 6 mg/kg TMT, and subsequently stimulated
once a day for 30 days. The data indicated that rats treated with 6 mg/kg TMT
kindled slightly (but significantly) more rapidly than did controls.
-------
172 HoMI.D)W
In a manner similar to the hippocampal kindling effect. Dyer el al. (1982c)
also demonstrated thai TMT-treated rats exhibited more rapid kindling of Ike
amygdala An did controls. In both the amygdaloid and hippocampal kindling
studies the threshold for producing an AD was higher at the end of the experiment
in TMT- treated than in control rata: The authors speculated that this may merely
reflect cell loss produced by the treatment, a theory supported by ihe finding of a
correlation between cell Ion and raised hippocampal AD thresholds.
The experiments by Dyer ct ai (1982c) Make a clear case for increased
neuronal excitability following TMT. but fail to pinpoint Ihe locus of the
excitability change. Increased excitability may occur at a number of loci and for a
number of reasons. The AD data seem lo indicate that excitability changes do not
originate within the hippocampus, since TMT increased AD thresholds and did
not change other AD parameters. However, a* indicated above, these findings
may reflect nonsynaptic characteristics of ADs.
An additional study (Dyer and Boyes, 1984) addressed the issue of
hippocampal excitability following TMT somewhat differently. The authors
reasoned thai since the pattern of damage produced in Ihe hippocampal formation
following acute administration of TMT was similar to the pattern of damage
produced by kainic acid, and since kainic acid damage is presumed lo be partially
mediated by increased activity fat the mossy-liber system, secondary to reduced
recurrent inhibition of granule cells ( Sloviler and Damiano, 1981 a. b). TMT may
produce a decrease in the recurrent inhibition of dentate granule cells.
Dyer and Boyes (1984) evaluated the status of recurrent inhibition in the
dentate gyrus using the method described by Anderson el al. (1966). Paired
stimulations arc presented to the perforant path, and Ihe resulting responses are
recorded from the dentate gyms. Dentate gyrus responses lo perforant-path
stimulation are generally characterized by a slow wave, which represents the
EPSP generated by the input. If a sufficiently intense stimulus is presented, a
spike appears on the EPS P. This spike is a reflection of the synchronous action
potentials of a large number of nearby granule cells and is known as the
population spike. The amplitude of the population spike is a reflection of the
number of granule cells responding to the input. Since the first peiforanl-path
stimulus of a pair activates recurrent inhibitory pathways, the second stimulus, if
close enough to the first in time, should produce a smaller population spike. The
magnitude of decrease in Ihe second population spike is a measure of the
magnitude of recurrent inhibition, and the quantity is usually represented as a
ratio (R2/RI).
Using unancsthctized rats, Ihe authors first determined Ihe magnitude of
recurrent inhibition in aN rats at an intentimulus interval of 20 msec. The rats
were subsequently dosed orally with either 0. 5, or 6 mg/kg TMT chloride, and
recurrent inhibition was evaluated at 2, 24. and 120 hr posttreatmenl. The
MacfoptiysioloeJcai Assaawnant of Ors*»°malal Nautoto«ictty 173
findings, which are summarized in Figure 11, indicate thai TMT produced a very
rapid change in recurrent inhibition. Within 2 hr of exposure Ihe effectiveness of
Ihe paired pulse in reducing Ihe amplitude of the second population spike had
declined by nearly 50%. These data support Ihe contention that TMT alters
hippnfimpil excitability by reducing recurrent inhibition in the dentate gynis.
Subsequent morphological studies by Chang and Dyer (1985b) supported this
interpretation by showing that dentate basket cells, the presumed morphological
substrate of recurrent inhibition, exhibit degenerative changes shortly after
treatment with TMT.
In addition lo producing a local excitability change in the hippocampus, TMT
also evidently produces alterationa in the polysynaptic chain which, by way of the
linking process, allows localized ADs in the amygdala and hippocampus lo
become more generalized and ultimately lo trigger behavioral seizures. Further
evidence that TMT produces complex alterations in polysynaptic systems comes
from a study by Hasan et al. (1984).
Hasan et al. (1984) investigated the temporal course of TMT-induced
changes in the limbic system of chronically implanted Sprague- Dawley rats. T wo
3 SO
WC-DO
o a « s a in tz «4 is ta zo az >4 zs sa so
DAV8 AFTCR TMT
Fifarc II. Ellccts of TMT on recui real inhibition in the dentate gyrus of rau. Rl is the amplitude of
Ike populiiMn ipjtc produced in I he dentate jymi by the first of paired stimuli presented to the
pcrfofanl path R2 is the amplitude of the population spike produced by the second of the paiied
stimuli Thesmalkf «lieralioofR2/Rl.the|reaterlliefecurrentinfcib«ion Values plotted arc means
± SI M Rcplutlcd fium Dyer and Boyes (I9R4).
-------
174 ftotefts. Dyar
pathways were examined. Stimulating electrode* placed in the hikis of the
dentate gyrus (DG) were used loelicit monosynaptic EPSPi from CA, neurons.
Stimulating electrodes located in the prepyrifbrni cortex ( PPC) were used to dick
polysynaptic EPSPi from DG neurons. The authors report that 7.5 mg/kg TMT
produced a progressive reduction in the monosynaptic DG-CA, response,
beginning on the second day following treatment and reaching a mini mum at
about 14 days posttreatment. la marked contrast, the same animals exhibited a
transient but profound potentiation of ike PPC-DG response, which became
evident on the third posttreatment day and lasted until about the seventh
posttreatment day. At this point the polysynaptic response began a progressive
decline in amplitude, until the thirtieth day, when it was effectively absent. These
results are summarized in Figure 12.
Based upon this study. It is not dear whether the potentiated PPC-DG
response is an obligatory consequence of the polysynaptic nature of the test or
whether there wast omething unique about either or both of the specific synapses
o SAUNE
A- la*|/knlMI
•• » 14 120
POSf IMCATMCNI (Mi HmumI
FfcanU. Ai»pli>ml«o«y«p>>c(DQ CAH—dputytvatHictPPC DOlfwitcdpoUwlUh
wcordtdkemm—mmWHiidral falh.w«,a.hoMi.nalinaof7.5i|/ttTMT Thepotyyaagic
pathway iNusbMct polcaliatioa kMamtxi by tkprcuioa, while ihc BtoaoayaafHic pathway ihowi uaiy
dcpniUM Rcptotlcd Itoai lluaa d at . (1984).
MaciophysJologlcal Aihimhh) of OfganooMlol MoutoloalcMir 175
involved. These questions may be answered directly with similar studies using
monosynaptic and polysynaptic recordings from the PPC- DG system. The study
provides further evidence that TMT produces an increase in excitability of at
least a segment of the limbic system.
Doctor et al. (1982) addressed the question of TMT-induced changes in
neuronal excitability using a variety of systemic convulsanls. The experiments
described by these authors dilTer from those described above in two important
respects. First, the authors used mice, while the data described above on
excitability were obtained in rats. This is an important difference, since the
neuropathology produced by TMT in the two species is substantially different
(Change! al.. 1983). Second, the authors used dosages of 1.065,2.130.3.195.
and 4.26 mg/kg TMT. While these would be considered relatively low dosages
for a rat. the two high dosages used here are almost certainly lethal dosages for
mice, where the LOM is usually listed as between 2 and 3 mg/kg. The
investigators performed tests of seizure susceptibility at I and 14 hr following
intraperitoneal administration of the TMT. The convulsanls used during the
seizure susceptibility tests were bicuculline. PTZ. strychnine, and isonicotinic
acid hydrazide (INH). and were all administered in lethal dosages. The endpoints
studied were latency to clonic seizure. Ionic seizure, and death.
In general, effects of TMT were restricted to the I -hr posttreatment lime
period and to the two high (lethal) dosages of TMT. Under these conditions.
TMT increased the latency to clonus (bicuculline and PTZ), tonus(INH). and
death (bicuculline, INH. PTZ. and strychnine). It does not seem appropriate to
consider this short-term effect of a lethal concentration of TMT as an
anticonvulsant effect. No convulsions were prevented, and the slight increases in
latency lo different stages of convulsion could be attributed lo metabolic
alterations secondary to a profound acute and general debilitation. These findings
should not, therefore, be considered in serious conflict with the suggestion given
above that TMT increases neuronal excitability.
On the other hand. Doctor and Fox (1982b) investigated TMT- treated mice
using the MES lest described above. The authors provided some evidence of
CNS depression following TMT. Using a dosage of3.5 X 10* mol/kgTMT, the
seizure severity was not affected. However extension/flexion duration ratios
were decreased, in a manner similar to that reported for TET. This attenuated
duration of the most severe aspects of the seizure lasted for about 24 hr after
treatment, which made it more effective than the equimolar dosage of TET. It is
not clear whether this effect reflect! (I) acute debilitation of the mice. (2) a unique
property of alkyltins not evaluated by any of the other procedures for assessing
neuronal excitability, or (3) the fundamental species difference in response lo
TMT.
-------
176 IMwl a. Oy*<
C. Othmr OrgmmoUn*
1. EEQ Studies. Miutv (t al. (1968) reported t study in which rabbits with
chronic electrodes over frontal and occipital cortex were administered either 2 5
mg/kg tetraethyltin or 100 mg/kgdichlotodibutyltia. Both of these dosages were
lethal within 3-7 days. However, the authors reported signs of EEG activation
(low voltage, high frequency) immediately after treatment. No conclusions may
be drawn from this observation.
2. Neuronal ExcilabHHy Studies Using the MES lest described above. Doctor
and Fox (1982b,c) investigated a number of other organotin compounds. Trt-n-
propyltin and tii-a-butyltia both produced a brief (0.5-4 hr) decline in the
duration extension/flexion duration ratios. The effect was not as pronounced as
for TET and TMT, and was only evident at a I7.S X 10 * mol/kg dosage.
A somewhat different pattern of effects was produced by Iricyclohexyltin
(TCT) and triphcnyltin (TPhT). A dosage of I7.S X 110 * mol TCT/kg
produced aa increase in seventy of the MES seizures, as indicated by increased
duration of both Ibrelinib and hmdhmb extension. This effect was evident within
0.S hr of treatment and was slid present al the last test time, 96 hr after treatment.
The TPhT, al a dosage of 17.5 X 10 * mol/kg, produced first an increase in
seizure severity (only evident 0.5 hr after treatment), followed by a decrease in
seizure severity (evident at 4 and 24 hr after treatment), which was followed by
recovery at the last test time of 96 hr after treatment.
V. Othnr Orgsnomslils
Although there have been a number of neurubehavioral studies of other
organometals, virtually none have involved the use of electrophysiological
endpoints. One exception is work reported by Saik>( 1973), who recorded EEGs
from rats exposed to tetraethyllead. In this study 12 adult rats with chronic
cortical electrodes were iqjected intraperitoneally with I ml gasoline without
lead/100 gm body weight, and 9 rats were iigected with leaded gasoline. The
estimated dosage of lead was 16.5 mg/kg. which should have been near the LDW.
The EEGs were recorded I day before and on days 1-5,7, and 10 after injection,
while the animals were unrestrained and unaneslhctized. Unfortunately, no rata
were given an innocuous compound for purposes of control.
The EEG analysis performed consisted of feeding the signal into a 5-channd
frequency analyzer, each channel of which passed only signals within either the
delta (2-4 Hz), thela (4-8 Hz), alpha (8-13 Hz), beta I (13 -20 Hz), or beta 2
(20-30 Hz) bands. Energy within each of these bands was then integrated over
time to provide a quantitative estimate of activity. As noted previously, a
Macroptiyiioloateal ol Ocgwiomalal NauiotonkcMy
177
disadvantage of EEG analyses in unrestrained animals is that meaningful
interpretation can only be made in concert with contemporaneously recorded
behavioral data. While Saito noted behavioral changes over days, no effort was
made to rigorously correlate these changes with the EEG. The most striking
findings in this study were that 7 days after administration of the gasoline
containing lead, there was a great increase in the amount of alpha activity
compared with the group receiving only gasoline, and 10 days after treatment the
lead-exposed group had a slightly higher thela and delta activity than the
gasoline-only group. These changes occurred at a lime when rats were described
as showing "excessive tension and excitement." However, the significance of the
findings is neither clear nor is it discussed.
VI. Summsiy and Conclusions
Macrophysiological methods have been used to evaluate the neurotoxicity of
only a few organomctal compounds. In some cases the methods have failed to
provide any inlerpretablc information. In other cases, the methods have verified
observations made using other approaches. A few studies have used these
methods to identify previously unrecognized characteristics of neurotoxicity
produced by the lest compound.
A. Uathylmmrcury
In the case of methylmercury, EEG studies verified in rats the sleep disturbances
reported in humans (Arilo el al., 1983). Evoked-potential studies have verified
visual dysfunction in dogs exposed as adults (Maltsson el al., 1981) and rats
exposed perinalally (Zenick, 1976; Oyer et al., 1978), without shedding much
light on the nature of dysfunction. The findings in rats were novel, since the rat
brain is lissencephalic and since methylmercury is usually described as producing
damage in the depths of sulci. It would be of interest to determine the extent to
which methylmercury produced visual dysfunction in rats exposed as adults.
However, techniques more sophisticated than recording FEPs from the visual
cortex will be required lo characterize further this dysfunction. It is of interest to
learn whether it is peripheral.
The B AER technique was used productively by Wassick and Yonovitz (1985)
lo demonstrate auditory dysfunction in mice exposed to methylmercury as adults.
Auditory thresholds were raised by exposure. It is still not clear whether this is a
frequency-dependent phenomenon, or whether it applies lo other species, and the
extent lo which peripheral, brainstem, and higher structures are involved. All of
these issues could be resolved using macrophysiological approaches. There have
been few physiological studies of somatosensory function in animals exposed lo
-------
178 IMwK I. Oyt
methylmercury, although a thorough characterization of dyifwiclion should
include such m evaluation.
Neuronal excitability has beea evaluated in rats and mice foNowiagcsposure
to methylmercury. Perinatal exposure produces an increase ia the period of
hypoexdt ability afWr a Mppocampal AD(Eccle». et al.. 1981). However, mice
perinalaiy exposed lo nulhyhatrcary deamastrate increased seasitivity In
flurothyl and audiogenic seizures (Sa and Ottta. 1976; Menashi et al.. 1982)
Thus, while it appear* that perinatal exposure lo methylmercury alien neuronal
excitability when tested later, the tbedioa of (he change may depend upon the
species, age at dosing, and lest used. Future studies might profitably explore the
relationships among different tests of neuronal excitability, because (he case of
methylmercury is not the only one in which the direction of effect may depend
upon the lest used.
0. TrMfcyttte
Attempts to use physiological methods to validate the use of TET as a model for
studying dcgeaerativc/dcmyglinaUng disorders have beea unconvincing. partly
because of the failure to control lor the hypothermic effects o(TET(Gerre*etal .
1976). Similar —certainties cloud At interpretation of studies on the EEG
(Beaedek el al., 1976) and on the auditory system (Amochaev el al., 1979).
Physiological methods have provided some support for the idea that the visual
system is sensitive lo TET, foiowing acute exposure and also following perinatal
exposure. Whether this sensitivity ia unique among sensory systeaas remains lo
be determined. TricthyWn alao produces signs of depressed neuronal excitability
which, when evaluated with the MES lest, arc quite loog lasting, evea foNowing
acute athajniilratioa (Fob and Doctor, 1983). There is as yet no due lo the
mechanism of these effects.
C. THmtUbrHht
Physiological mclhudi have beea particularly useful in characterizing the
neurotoxicity produced by Irimcthylliu. la particular, evoked- potential studies
have identified the rnt visual system as a target, indicating the probable locus as
the retina (Dyer ct al., 1982a). The BAER identified peripheral auditory
dysfunction, and the SEP suggested somatosensory dysfunction. AN of these
findings awe subsequently confirmed using pathological and/or behavioral data.
Evoked-potential techniques have also been useful in suggesting that the
nature of physiological dysfunction ia the hippocampus is in part loss of recuirent
iohibition(Dyer and Boyes, 1984) and ia part polysynaptic potentiation(llasan
et al., 1984). These techniques could be used lo explore further the intriguing
septotemporal distribution of damage produced by TMT in the hippocampus
(Chang and Dyer, 1985a).
MttloylliflMogfcri A»—*umamt al OhmohIW 170
Conflicting evidence exists regarding the influence of TMT on neuronal
excitability. Trimethykin increased rates of amygdaloid and hippocampal
kindling, and increased sensitivity to pentylenetetrazol seizures in rats (Dyer et
al., 1982c). However, Doctor and Foi (1982b) reported thai TMT depressed
excitability ia mice, as measured by the MES test. This effect was short lasting
(about 24 hr), and may reflect either species differences ia TMT toiicity or
differences ia what it measured by the test.
D. Oanaraf
As the summary indicates, evoked potentials have beea particularly useful ia
providing initial characterization of neurotoxicity. However, la all of these cases
the methods were aot used to full advantage. Considerably more could be learned
from evoked-potential tests about the sensory toxicity produced by organooietal
compounds, and this iafbnnatioa could alleviate the task of determining
mechaaisms of toxicity.
Studies of neuronal excitability have oftea provided conflicting information.
While some of the conflict may reflect differences ia species and experimental
desiga. it is also clear that excitability it aot a uailary phenomenon. Considerable
work must be performed lo determiae what relationship (if aay) exists between
the many measures of aeuroaal excitability considered fat this review.
The use of macrophysiologicat techniques has many limitations, most of
which stem from difficulty ia iaterpretatioa. However, the preceding review
should make it evident that there is must lo be learned from these techniques,
whea properly applied. As future studies begia lo unravel the meaning of the
endpoints measured, die techniques should become progressively more valuable.
AcknowriadgmanU
The author thanks Greg Rigdon for reviewing the manuscript, Judy Smith for
preparing the manuscript, and J aha Hoots for assisting with German translation.
This aianuscript has beea reviewed by the Health Effects Research Laboratory.
U. S. Environment al Protection Agency, and approved for publication. Mention
of trade names or commercial products does not constitute endorsement or
recommendation for use.
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Neurobehaviorai Toxicology and Teratology, Vol. 4, pp. 659-664, 1982. ® Ankho Interoationsi. Printed in the U.S.A.
Physiological Methods for Assessment
of Trimethyltin Exposure
ROBERT S. DYER
Neurophysiology Branch, Neurotoxicology Division
U.S. Environmental Protection Agency, Research Triangle Park, NC 27711
DYER, R. S. Physiological methods for assessment of Trimethyltin exposure. NEUROBEHAV TOXICOL TERATOL
446) 659-664, 1982.—Trimethyltin has been reported to produce morphological alterations in the brain which are primarily
restricted to the limbic system. A variety of physiological measures of limbic system integrity are discussed in terms of their
ability to detect TMT-induced dysfunction. In addition, several measures of sensory system dysfunction are discussed. It is
concluded that limbic system dysfunction induced by this compound is detected more efficiently by intrahippocampal
evoked potentials than by more gross measures of dysfunction. It is also concluded that relying upon preliminary descrip-
tions of pathological alterations to direct physiological studies may provide an incomplete description of neurotoxicity.
TMT Trimethyltin Organotin Evoked potentials Hippocampus Neurotoxicity Kindling
Afterdischarge Visual evoked potentials Somatosensory potentials
NEUROPHYSIOLOGICAL methods are most often used to
evaluate mechanisms of brain function. In the context of
toxicology, neurophysiological methods may be chosen
which are useful for assessing dysfunction of a particular
system, or for assessing the functional mechanism of dys-
function. For example, the visual evoked potential recorded
from the cortex may reveal dysfunction of the visual system,
but will not accurately characterize the nature of that dys-
function. Intracellular and extracellular recordings from vis-
ual system neurons can reveal the contributions of mem-
brane properties and synaptic inputs to the general dysfunc-
tion revealed by the gross recordings. The present paper
focuses upon gross measures of brain function for detection
of neurotoxicity to different functional systems (in this case,
sensory and limbic). As a vehicle for discussion, the methods
are considered in terms of their ability to detect dysfunction
induced by trimethyltin (TMT).
TMT has recently become a compound of interest to
neurotoxicologists (e.g. [3, 4, 5]). Two reports have de-
scribed the consequences of human exposure to TMT. In the
first [14] two chemists were exposed to a mixture of dimethyl
and trimethyltin chloride. The major symptoms described
were mental confusion, seizures, and various psychic dis-
turbances (e.g., memory defects, loss of vigilance, insomnia,
anorexia and disorientation). The two chemists also com-
plained of violent pain "sine materia in various organs" [14],
A more recent paper [21] describes chemical workers ex-
posed to either high (n« 12) or low (n= 10) levels of TMT.
The msyor symptoms reported by these investigators were
forgetfulness, fatigue and weakness, headaches, attacks of
rage and temper coupled with bouts of depression, and
dimming of vision.
Although quantitative demonstration are lacking, rats ex-
posed to TMT have been described as aggressive and dif-
ficult to handle [4,11]. Group housed animals often mutilated
and destroyed each other, while individually housed rats
engaged in self-mutilation. (Jnlike the self-mutilation which
occurs in morphine addiction [19], these episodes were
largely restricted to the tail. Furthermore, a single dose of
TMT often produced episodes of seizures beginning 3-5 days
later and lasting for about a week.
At least some of the constellation of symptoms described
above suggest limbic system damage. This suggestion has
been borne out by neuropathological studies in laboratory
animals [3, 4, 5, 8]. Therefore efforts to validate neurophys-
iological methods for detection of neurotoxicity produced by
TMT began with measures of the functional integrity of the
limbic system.
One method for investigating the functional integrity of
the limbic system is the kindling paradigm. Most areas of the
limbic system, when repeatedly stimulated (i.e. once/day for
several weeks), generate behavioral seizures. The process
by which local GEG afterdischarges become behavioral sei-
zures is called kindling [20], and the rate at which animals
kindle has been used to characterize the response of the
brain to a variety of toxicants (e.g. [18]). The kindling proc-
ess in the hippocampus and the amygdala was examined in
rats treated with TMT [12]. In the amygdala kindling experi-
ment, rats were implanted with electrodes in the amygdala
and treated on day 0 with a single dose of either 0, 5 or 7
mg/kg TMT. Electrical stimulation of the amygdala was ad-
ministered at twice the threshold for production of a local
afterdischarge (AO), and occurred once/day for 16 consecu-
tive days. The results of the study, which are shown in Fig. 1
demonstrated that the treated rats kindled more rapidly than
the controls. In the hippocampal kindling experiment the rats
were implanted with electrodes in the dorsal hippocampus,
and treated on day 0 with a single dose of either 0, 4, 5 or 6
mg/kg TMT. Since hippocampal kindling takes longer than
amygdaloid kindling, the rats in this study were stimulated
659
-------
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TMT 00SAGE, mg/kg
1 1
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1 1
1 1
3.5
12
16
TIME POST TREATMENT, days
FIG. I. Effects of TMT upon kindling rate following electrical stimu-
lation of the amygdala in rats [12]. TMT was injected on day 0 only.
Scores on the ordinate are from a scale developed by Racine [20] for
rating seizure intensity, where higher scores indicate more severe
seizures.
TMT DOSAGE, mq/kf
06
~ CONTROL
£ 2.5
u 2.0
15 6-10 1116 1620 21 25
TIME POST TREATMENT, da¥s
26-30
FIG, 2. Effects of TMT upon kindling rate following electrical stimu-
lation of the dorsal hippocampal formation in rats [12], Seizure
scores were averaged into blocks of 5 days each. TMT was injected
on day 0 only.
for 30 days instead of 16. Otherwise the procedures were
identical. Again, the treated animals kindled somewhat more
rapidly than the controls (see Fig. 2).
Studies with other compounds suggested that analysis of
EEG afterdischarge properties following hippocampal stimu-
lation are also useftil for detecting toxicity [7,10]. These
studies may be more rapidly performed than kindling
studies, and provide potentially more information because
more parameters are measured. Consequently rats implanted
with electrodes in the dorsal hippocampus were studied fol-
lowing treatment with either 0, 5, 6 or 7 mg/kg TMT. A
variety of properties of afterdischarges were studied on days
1,4,8 and 16 following treatment, compared to pretreatment
control recordings. The results were surprising since the
only variable which seemed to be affected was the threshold
for production of the afterdischarge. None of the micro-
properties of the AD were changed [12], Based upon this
finding, a retrospective look was taken at the difference be-
tween before- and after-treatment AD threshold values ob-
tained during the kindling experiments. The findings were
similar: treatment with TMT increased the threshold for
elicitation of an AD. These data are summarized on Fig. 3.
These data produced the puzzling conclusion that even
though TMT reduced the number of cells, it did not disrupt
the pattern of cell firing as detected by the AD procedure.
The kindling data seemed to indicate that TMT increased
limbic sensitivity to seizure development once activity was
triggered. The sensitivity of the tests in detecting limbic dys-
function was disappointing. Alterations observed were not at
levels substantially below those at which frank behavioral
disturbances (e.g. self-mutilation) were observed. In an ef-
fort to increase the sensitivity of measures of limbic system
function to damage, a somewhat more mechanistic ap-
proach, the use of intrahippocampal evoked potentials, is
now under exploration.
Kainic acid produces a pattern of hippocampal damage
similar in some respects to that produced by TMT (e.g. [22]).
Since kainic acid-induced damage may result from overac-
tivity of the mossy fiber system [22], an investigation of the
functional integrity of the system which activates the mossy
fibers was begun. In these studies the major input to the
dentate granule cells, the perforant path, was electrically
stimulated, and the resulting evoked potential was recorded
from the granule cells. The output of the granule cells is a
reflection not only of the perforant path input, but also of the
status of other inputs, including recurrent inhibition
produced by basket cells around the axon hillocks of the
mossy fibers. There is a direct way for evaluating the func-
tional integrity of recurrent inhibition in this system. It has
been shown by Andersen et at. [2] that stimulation of the
perforant path with a pair of pulses, each of which is suffi-
cient to excite the granule cells, produces a reduced spike
amplitude of the second response if the second stimulus fol-
lows the first within a particular period of time (20-40 msec).
This time period is considerably greater than the refractory
period of the spikes and the reduction in amplitude is there-
fore taken to directly reflect recurrent inhibition. Figure 4
illustrates this phenomenon.
To study the influence of TMT on recurrent inhibition in
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PHYSIOLOGICAL ASSESSMENT/TRIMETHYLTIN
661
PIG. 3. Effects of TMT upon electrical thresholds for elicitation of an epileptiform afterdischarge from either (A) the hippocampal formation
during an afterdischarge experiment: (B) the amygdala during a kindling experiment: or (C) the hippocampal formation during a kindling
experiment.
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PIG. 4. Paired pulse evoked potentials recorded from the dentate
gyrus of a control rat following electrical stimulation of the perforant
pathway. Each stimulus artifact (indicated by an arrow) is followed
by an CPSP, which is interrupted by a population spike (marked
with *) indicating discharge of the dentate granule cells. Time be-
tween the two stimuli is 20 msec, and the decreased amplitude of the
second spike compared to the first is taken as a reflection of recur-
rent inhibition.
the dentate gyrus, the perforant path stimulating and dentate
gyrus recording electrodes were implanted about two weeks
prior to the onset of testing- These studies are still in prog-
ress, but to date the granule cell responses from treated and
control animals have been recorded before and at several
time points following treatment. Over the course of 3 days
following a single treatment, the threshold for producing
either an EPSP or a spike has not changed, nor do there
appear to be significant differences between the spike ampli-
tudes of treated and control rats. However the magnitude of
recurrent inhibition appears to have been significantly de-
creased by the TMT treatment [13], and this effect appears to
have begun within about 2 hrs of treatment (Fig. 5). This
finding is similar to what has been discovered with kainic
acid, and further reinforces the similarity between these two
compounds [23]. These results and those obtained from
studies of kainic acid raise the distinct possibility that the
morphological damage produced by both agents is at least in
part secondary to physiological dysfunction [13,22].
The rapid nature of the changes observed using the hip-
pocampal recurrent inhibition measurements suggests that
this method may be more useful, than the kindling or AD
methods in detection of TMT toxicity. Although changes are
observed more rapidly with this technique than others, it
remains to be determined whether changes may be observed
at lower exposure levels.
Not all of the symptoms displayed by humans exposed to
TMT can be accounted for by limbic system pathology. The
aches, pains, and dimness of vision in humans, and the
self-mutilation in rats might all reflect concurrent sensory
disturbances. To provide a more complete picture of physi-
ological dysAinction following TMT exposure, several recent
studies have investigated sensory systems. The first of these
-------
662
2.D
_ TMT DOSAGE. m»/kj
_ °6
~ CONTROL
1.0 —
P. 0-2
0.2
TIME POST TREATMENT. d«y>
FIG. 5. Influence of 6 mg/kg TMT administered PO upon recurrent
inhibition in the dentate gyrus. Recurrent inhibition is estimated by
dividing the spike amplitude obtained from the second response of
the pair by the spike amplitude obtained from the first response. The
ordinate reflects Day X-Day 0. Thus negative numbers indicate
more inhibition than on Day 0, and positive numbers indicate less
inhibition than on Day 0. TMT reduced recurrent inhibition begin-
ning within 2 hrs of dosing.
studies investigated the visual system since alterations in the
visual evoked potential often reflect general central nervous
system dysfunction [6]. Based upon the absence of demon-
strated pathology in the retina or other areas of the visual
system [4J, it was initially hypothesized that the visual
evoked potential would not detect TMT-induced damage.
To study the visual system rats were implanted with elec-
trodes in the visual cortex or optic tract. Flash-evoked po-
tentials were recorded immediately before exposure, and at
intervals of 1, 4, 8 and 16 days following exposure. The
findings did not support the preidicted lack of effect. Laten-
cies to the early peaks of the evoked potential were in-
creased, and the fact that this occurred with roughly equal
magnitude in both the cortically recorded and optic tract
recorded potentials indicated that the dysfunction was prob-
ably of retinal origin [9]. Among the more interesting fea-
tures of this finding was its time course. Unlike most of the
behavioral consequences of TMT exposure, the effects on
the visual system were present within 24 hrs and persisted
for the entire test period (Fig. 6). The reports of no retinal
pathology [4] were perhaps premature. A more recent report
from the same laboratory [11] describes damage in both inner
and outer nuclear layers of the marmoset retina following
exposure to TMT. These findings may be related to the dim-
ness of vision reported by the humans exposed to TMT.
A similar although less extensive study has been per-
formed to investigate the effects of TMT upon the
somatosensory system. Based upon observations of self-
mutilation of the tail, it was hypothesized that denervation
might have occurred. To examine this possibility the tail was
DYER
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PHYSIOLOGICAL ASSESSMENT/TRIMETHYLTIN
663
electrically stimulated and the somatosensory evoked po-
tential was recorded from the somatosensory cortex. The
alterations observed were not as extensive as they were in
the visual system, but nevertheless dysfunction could be de-
tected in the TMT-exposed rats [16]. TMT increased laten-
cies and decreased amplitudes, just as it did in the visual
system. Because of the relatively minor changes observed,
the data were interpreted as not supporting a denervation
explanation of the tail mutilation, and this phenomenon
therefore remains unexplained.
Systematic studies of auditory function following TMT
administration have not been published. However the pres-
ence of dysfunction in the visual and somatosensory systems
raises the possibility of a general sensory system defect.
Some data are available to support extension of this gener-
ality to the auditory system. In a pilot study rats were tested
in an auditory startle task immediately before and for several
days following TMT exposure. There was a severe impair-
ment of the response [15], An assessment of the startle re-
sponse in grasshopper mice [17] also demonstrated response
amplitude suppression induced by TMT exposure. These
deficits may, of course, reflect a motor impairment, although
on the grounds of parsimony auditory dysfunction is more
probable. Regardless of the interpretation of the startle data,
it is quite clear that TMT produces sensory deficits in addi-
tion to limbic system dysfunction.
The above data indicate that TMT is not nearly as selec-
tive as a cursory glance at the pathology might suggest.
Based upon the sensory system data and the spontaneous
seizures which occur at high dosages, the possibility that
TMT produces generalized changes in CNS excitability was
investigated with the systemic convulsant pentylenetetrazol
(PTZ). Rats were treated with TMT and 5 days later chal-
lenged with PTZ. The results supported the hypothesis by
indicating that the TMT-treated rats were more likely to
convulse than untreated control rats [12] when both were
administered a dosage of PTZ [12].
CONCLUSIONS
Several important conclusions may be drawn from this
series of experiments. First, while there are many physiolog-
ical methods by which the functional integrity of the limbic
system may be evaluated, the one which appears to give the
most direct information and is perhaps the most cost effec-
tive is the hippocampal evoked potential.
Second, the existence of obvious regional pathology fol-
lowing exposure to a toxicant should not lead to the assump-
tion that there is not functional impairment elsewhere. The
sensory dysfunctions following exposure to TMT would not
have been discovered for some time if the physiology exper-
iments awaited prior demonstration of pathological damage
to these systems.
Finally, it is extremely difficult to generalize between the
functional consequences of closely related organotin com-
pounds. These experiments, when compared to those which
have concentrated on triethyltin, demonstrate how poorly
structure activity relationships are understood in
neurotoxicology.
REFERENCES
1. Aldridge, W. N., A. W. Brown, J. B. Brierley, R. D. Ver-
schoyle and B. W. Street. Brain damage due to trimethyltin
compounds. Lancet 2: 692-693, 1981.
2. Andersen, P., B. Holmquist and P. E. Voorhoeve. Entorhinal
activation of dentate granule cells. Acta physiol. Stand. 66:
448-460, 1966.
3. Bouldin, T. W., N. D. Goines, C. R. Bagnell and M. R. Krig-
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tural and cytochemical observations. Am, J. Path. 104: 237-249.
4. Brown, A. W., W. N. Aldridge, B. W. Street and R. D. Ver-
schoyie. The behavioral and neuropathoiogical sequelae of in-
toxication by trimethyltin compounds in the rat. Am. J. Path.
97: 59-82, 1979.
5. Chang, L. W„ T. M. Tiemeyer, G. R. Wengerand D. E. McMil-
lan. Neuropathology of mouse hippocampus in acute trimethyl-
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1982.
6. Dyer, R. S. Effects of prenatal and postnatal exposure to carbon
monoxide on visually evoked responses in rats. In:
Neurotoxicology of the Visual System, edited by W. H. Merigan
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7. Dyer, R. S., E. Burden, K. Hulebak, N. Schulz, H. S.
Swartzwelder and Z. Annau. Hippocampal afterdischarges and
their post-ictal sequelae in rats: effects of carbon monoxide
hypoxia. Neurobehav. Toxicol. I: 21-25, 1979.
8. Dyer. R. S., T. L. Deshields and W. F. Wonderlin.
Trime thy It in-induced changes in gross morphology of the hip-
pocampus. Neurobehav. Toxicol. Teratol. 4: 141-148, 1982.
9. Dyer, R. S., W. E. Howell and W. F. Wonderlin. Visual system
dysfunction following acute trimethyltin exposure in rats.
Neurobehav. Toxicol. Teratol. 4: 191-196, 1982.
10. Dyer, R. S., H. S. Swartzwelder, C. U. Eccles and Z. Annau.
Hippocampal after-discharges and their post-ictal sequelae in
rats: a potential tool for assessment of CNS neurotoxicity.
Neurobehav. Toxicol. 1: 5—19, 1979.
11. Dyer, R. S., T. J. Walsh, W. F. Wonderlin and M. Bercegeay.
The trimethyltin syndrome in rats. Neurobehav. Toxicol.
Teratol. 4: 127-134, 1982.
12. Dyer, R. S., W. F. Wonderlin and T. J. Walsh. Increased sei-
zure susceptibility following trimethyltin administration in rats.
Neurobehav. Toxicol. Teratol. 4: 203-208, 1982.
13. Dyer, R. S., W. F. Wonderlin, T. J. Walsh and W. K. Boyes.
Trimethyltin reduces basket cell inhibition in the dentate gyrus.
Soc. Neurosci. Abstr. 8: 1982.
14. Fortemps, E., G. Amand, A. Bomboir, R. Lauwerys and E. C.
Laterre. Trimethyltin poisoning. Report of two cases. Int. Arch,
occ up. envir. Hlth. 41: 1-6, 1978.
15. Howell, W. E., R. S. Dyer, W. F. Wonderlin, K. Kidd and L.
W. Reiter. Sensory system effects of acute trimethyltin (TMT)
exposure. Toxicologist 1: 43, 1981.
16. Howell, W. E., T. J. Walsh and R. S. Dyer. Somatosensory
dysfunction following acute trimethyltin exposure.
Neurobehav. Toxicol. Teratol. 4: 197-202, 1982.
17. Hulebak, K. L. and Z. Annau. Behavioral consequences of
trimethyltin exposure in grasshopper mice. Toxicologist 2: 58.
1982.
18. Joy, R. M., L. G. Stark, S. L. Peterson, J. F. Bowyer and T. E.
Albertson. The kindled seizure: production of and modification
by dieldrin in rats. Neurobehav. Toxicol. 2: 117-124, 1980.
19. Leander, J. D., D. E. McMillan and L. S. Harris. Schedule-
induced oral narcotic self-administration: acute and chronic ef-
fects. J. Pharmac. exp. Ther. 195: 279-287, 1975.
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20. Racine, R. Modification of seizure activity by electrical stimu-
lation: II. Motor seizure. Electroenceph. clin. Neurophvsiol. 32:
281-294, 1972.
21. Ross. W. D., E. A. Emmett, J. Steiner and R. Tureen.
Neurotoxic effects of occupational exposure to organotins. Am.
J. Psychiat. 138: 1092-1094, 1981.
DYER
22. Sloviter, R. S. and B. P. Damiano. Sustained electrical stimula-
tion of the perforant path duplicates kainate-induced elec-
trophysiological effects and hippocampal damage in rats.
Neurosci. Lett. 24: 279-284. 1981.
23. Sloviter, R. S. and B. P. Damiano. On the relationship between
kainic acid-induced epileptiform activity and hippocampal
neuronal damage. Neuropharmacology 20: 1003-1011. 1981.
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Neurobehaviorul Toxicology and Teratology, Vol. 6, pp. 369-371, 1984. c Ankho International Inc. Printed in the U.S.A.
0275-1380/84 $3.00 +- .00
Trimethyltin Reduces Recurrent
Inhibition in Rats'
ROBERT S. DYER AND WILLIAM K. BOYES
Neurophysiology Branch, Neurotoxicology Division MD-74B, Health Effects Research Laboratory
U.S. Environmental Protection Agency, Research Triangle Park, NC 27711
Received 16 January 1984
DYER, R. S. AND W. K. BOYES. Trimethyltin reduces recurrent inhibition in rats. NEUROBEHAV TOXICOL
TERATOL 6(5)369-371, 1984.—Rats with electrodes chronically implanted in the perforant path for electrical stimulation,
and dentate gyrus for recording were treated with a single oral administration of either saline. 5 mg/kg trimethyltin (TMT) or
6 mg/kg TMT. Recurrent inhibition was assessed by paired pulse activation of the perforant path input to the dentate gyrus.
The measure of recurrent inhibition employed was the ratio of the population spike amplitudes of the responses to the first
and second of the paired stimuli. Inhibition was assessed immediately before, and at 2, 24 and 120 hr following TMT. The
results indicated a reduction in inhibition as early as 2 hr following treatment, suggesting that TMT-induced destruction of
hippocampal pyramidal cells may be secondary to their over-activation from an uninhibited mossy fiber system.
Trimethyltin TMT Hippocampal evoked potentials Recurrent inhibition Paired pulse paradigm
Dentate gyrus Basket cells
ACUTE oral administration of trimethyltin (TMT) to adult
rats produces widespread morphological, behavioral and
physiological alterations [4], The pattern of morphological
damage is in some respects similar to the pattern of damage
produced by intraventricular kainic acid [8], Cellular nec-
rosis is extensive in the pyramidal cells of the hippocampal
formation, appearing to involve first CA3c, then CA3b and
CA3a [3,5],
The mechanism by which TMT produces this damage is
unknown. However a similar pattern of damage has been
produced by repetitive electrical stimulation of the mossy
fiber system at intensities and durations below those re-
quired to elicit afterdtscharges [9]. Therefore if TMT in-
creased activity in the mossy fiber system, damage to the
hippocampal pyramidal cells could result.
Theoretically, activity in the mossy fibers could be in-
creased by reduction of recurrent inhibition in the basket
cell-granule cell system. The purpose of the present study
was to test the hypothesis that recurrent inhibition in this
pathway is decreased in rats treated with TMT.
METHOD
Adult (60 day old) Long-Evans hooded rats (n»36) were
obtained from Charles River Breeding Co. and housed indi-
vidually in plastic cages on wood chip bedding in a room
maintained at 22°C and with a light:dark cycle of 12:12. The
rats were implanted with a twisted bipolar 0.1 mm nichrome
wire (insulated except at the cut tips) stimulating electrode in
the perforant path (pp) (-6.6 mm from bregma, 3.7 mm lat-
eral and 3.4 below the cortical surface) and a monopolar
nichrome wire recording electrode (insulated except at the
cut tip) in the fascia dentata (fd) (-3.3 from bregma. 2.5
lateral and 3.0 below the cortical surface). Reference and
ground electrodes were 00-90 x '/1»" stainless steel screws
threaded into the skull at 2 mm anterior to bregma and 2 mm
right and left of the midline, respectively. Electrodes were
connected to an amphenol receptacle, which was cemented
to the skull with dental acrylic. Surgery was performed
under 0.35 ml/100 g b.w. Chloropent anesthesia (Fort Dodge
Labs.), with incisor bars elevated 5 mm above the interaural
line.
Following a two week recovery period, recording studies
were begun. At the time of recording the rats were placed in
a small (8x20x 38 cm high) recording chamber, and their
amphenol headplugs were connected to Grass 7P511
amplifiers, with high and low pass filters set at 0.3 Hz and 3
KHz, respectively. Amplified signals (x 500) were lead to an
oscilloscope for monitoring and a PDP 11/70 computer for
averaging and storage. The pp electrode was stimulated at
0.5 Hz, with 0.1 msec biphasic constant current square wave
pulses produced by a Grass S88 stimulator and two PSIU-6
constant current converters. Current level was increased in
steps of 100 nA until the evoked field potential contained a
visually determined maximum amplitude population spike, a
process which usually required about 30 stimuli. The inten-
sity which produced this maximal response was then used to
produce 16 subsequent paired responses, which were aver-
aged by the PDP 11/70 computer. Amplitude of the resulting
population spike was estimated using cursor controls on a
Tektronix 4054 graphics terminal to measure the excursion
from the EPSP to the spike peak and back. These two values
'This manuscript has been reviewed by the Health Effects Research Laboratory, U.S. Environmental Protection Agency, and approved for
publication. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.
369
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370
DYER AND BOYES
CONTROL
8 mg/kg TMT
3MV
STIM 1
SUM 2
FIG. I. Sample Recording from one saline and one 5 mg/kgTMT rat
showing responses elicited by paired pulse stimulation of the perfo-
rant path immediately before and at 2 and 24 hours after treatment.
-- SAUNC
- a TUT
= « mg/kQ TMT
FOIT TMATMINT TIMC {Mural
FIG. 2. Effects of TMT on recurrent inhibition. Rl is the amplitude
of the population spike produced in the dentate gyrus by the first of
the paired stimuli to the perforant path. R2 is the amplitude of the
population spike produced by the second of the paired stimuli. The
smaller the ratio of R2/R1, the greater the recurrent inhibition. Ver-
tical bars are SEM. No bar is present on the 6 mg/kg 120 hr point,
since this is a median value.
were added together and divided by two to obtain spike am-
plitude. This method is similar to that used by others (e.g.,
[11]).
Recurrent inhibition was assessed using the double pulse
method described by Andersen et al. [ 1]. Briefly, the method
entails presenting two identical stimuli at short interstimulus
intervals (ISIs) to the pp electrode. At proper ISIs, the am-
plitude of the population spike recorded from the fd elec-
trode following the second stimulus (R2) was reduced com-
pared to the first (Rl). Recurrent inhibition was expressed as
the ratio of peak amplitudes (R2/R1).
In a pilot study, using different rats, the effect of ISI on
magnitude of recurrent inhibition was assessed in un-
anesthetized and anesthetized rats. Subsequently un-
anesthetized rats were used throughout, and an ISI of 20
msec was selected for all studies.
Once appropriate stimulus parameters were obtained,
each rat was administered either saline (n= 14), 5 (n= 12) or 6
(n=10) mg/kg TMT hydroxide as base by gavage in a volume
of 1 ml/kg. These dosages were chosen because of the ex-
tremely steep dose-response function found with TMT [4,5].
Recording sessions were repeated 2, 24 and 120 hr after dos-
ing. Groups were compared using repeated measures
ANOVA, and comparisons between control and each dosage
were made at each of the time points using Bonferroni-
corrected [7] Mests.
RESULTS
Prior to treatment, all three groups had equivalent recur-
rent inhibition, with mean amplitude of R2 averaging be-
tween 16 and 23% of Rl. Trimethyltin produced an almost
immediate decline in recurrent inhibition, as measured by
the paired pulse technique. At the earliest time point (2 hr),
R2/R1 was nearly twice what it had been in the predose test
in TMT-treated rats. In contrast, the saline-treated rats
maintained a consistent level of inhibition across the test
times. Figure 1 illustrates the recordings obtained from a
TMT-treated animal before and 24 hr following treatment.
Figure 2 illustrates the averaged ratio R2/R1 at different
post-treatment times. Due to death (n=2) and headplug dis-
lodgement (n=4) only 4 of the 6 mg/kg treated rats had re-
cordings made on post-treatment day 5. The median of these
4 scores is plotted in Fig. 2. The ANOVA indicated that the
dose x time interaction was significant, F(6,48) = 28.16,
<0.0001. Saline-treated rats were compared to each of the
two TMT-treated groups at each of the four time points, for a
total of eight comparisons. To maintain an experiment-wise
error rate of 0.05, the required p value for each comparison
was 0.05/8=0.00625. Using this criterion, the 5 mg/kg TMT
group differed significantly from the saline-treated group at
the 2 hr (p=0.0061) and 120 hr (p=0.0011) post-treatment
times, but not at the 24 hr post-treatment time (p=0371). The
6 mg/kg TMT group differed significantly from the saline-
treated group at the 24 hr (p=0.0006) and 120 hr (p=0.0001)
post-treatment times but not at the 2 hr post-treatment time
(p =0.0124).
DISCUSSION
Administration of TMT reduced the size of the population
spike recorded to the second stimulus of a pair, in a manner
similar to what others have shown with bicuculline [111.
These findings suggest that TMT reduces recurrent inhibi-
tion in the dentate gyrus within 2 hr following dosing, and
support the hypothesis that TMT increases activity in the
mossy fiber system by reducing basket cell inhibition of the
dentate granule cells. In this respect, TMT seems to be simi-
lar to kainic acid [10]. Based upon these findings, it is likely
that the loss of CA3 pyramidal neurons in TMT-treated rats
results at least in part from over stimulation.
This study raises several new questions. First, if TMT
-------
TMT REDUCES RECURRENT INHIBITION
371
selectively attacks the basket cells which are presumed to be
responsible for recurrent inhibition, what is the basis for the
selectivity? Possibilities include: (1) selective action against
specific transmitter systems, including those used by basket
cells; (2) a biochemical effect on all neurons with some being
more sensitive than others because of unknown metabolic
factors; (3) altered input to the basket cells (and/or other
cells); (4) altered voltage-dependent conductance relation-
ships; or (5) some combination of the above.
Second, can the loss of basket cell inhibition account for
all of the TMT-induced damage reported in various studies?
The answer to this question is almost certainly no. Neuronal
damage has been described at sites which are either upstream
from the dentate basket cells (e.g., the entorhinal cortex [3]),
or outside the limbic system entirely [2]. Furthermore, there
is evidence that in addition to release of inhibition, TMT may
facilitate activity in certain synaptic systems [12],
Finally, while functional toxicity is not a new concept,
occasions on which changes in neuronal activity are shown
to be responsible for producing changes in brain morphology
are relatively rare. This notion is not new in neuroscience
(e.g., [6,9]) but it is relatively novel in the context of toxicol-
ogy, and suggests new avenues for understanding mech-
anism of neurotoxicity.
ACKNOWLEDGEMENTS
The authors wish to thank Mark Bercegeay for excellent techni-
cal assistance during this study. WKB was supported by a National
Research Council Research Associateship.
REFERENCES
1. Anderson. P., B. Holmquist and P. E. Voorhoeve. Entorhinal
activation of dentate granule cells. Acta Phvsiol Scand 66: 448-
460, 1966.
2. Chang, L. W. and R. S. Dyer. Trimethyltin induced pathology
in sensory neurons. Neurobehav Toxicol Teratol 5: 673-696,
1983.
3. Chang, L. W., G. R. Wenger, D. E. McMillan and R. S. Dyer,
Species and strain comparison of acute neurotoxic effects of
trimethyltin in mice and rats. Neurobehav Toxicol Teratol 5:
337-350, 1983.
4. Dyer, R. S., T. J. Walsh, H. S. Swartzwelder and M. J. Wayner.
Neurotoxicology of the ,4 Iky I tins. New York: Ankho, 1982.
5. Dyer, R. S., T. L. Deshields and W. F. Wonderlin.
Trimethyltin-induced changes in gross morphology of the hip-
pocampus. Neurobehav Toxicol Teratol 4: 141-148, 1982.
6. Greenough, W. T. Enduring brain effects of differential experi-
ence and training. In: Neural Mechanisms of Learning and
Memory, edited by M. R. Rosenzweig and E. L. Bennett. Cam-
bridge,'MA: MIT Press, 1976, pp. 255-278.
7. Muller, K. E., D. A. Otto and V. A. Benignus. Design and
analysis issues and strategies in psychophysiological research.
Psychophysiology 20: 212-218, 1983.
8. Nadler, J. V., 8, W. Perry, C. Gentry and C, W, Cotman.
Degeneration of hippocampal CA3 pyramidal cells induced by
intraventricular Kainic acid. J Comp Neurol 192:333-359, 1980.
9. Sloviter, R. S. and B. P. Damiano. Sustained electrical stimula-
tion of the perforant path duplicates kainate-induced elec-
trophysiological effects and hippocampal damage in rats.
Neurosci Lett 24: 279-284, 1981.
10. Sloviter, R. S. and B. P. Damiano. On the relationship between
kainic acid-induced epileptiform activity and hippocampal
neuronal damage. Neuropharmacology 20: 1003-1011. 1981.
11. Tuff. L. P., R. J. Racine and R. Adamec. The effect of kindling
on GABA-mediated inhibition in the dentate gyrus of the rat. 1.
Paired pulse depression. Brain Res 277: 79-90. 1983.
12. Zimmer, L., Z. Hasan, D, Woolley and L, Chang. Evoked po-
tentials in the limbic systemof the rat reveal sites of trimethyltin
toxicity. Neurotoxicology 3: 135-136 (abstract), 1982.
-------
Neurobehavioral Toxicology and Teratology. Vol. 4, pp. 259-266. 1982. Printed in the U.S.A.
Acute Triethyltin Exposure:
Effects on the Visual Evoked Potential
and Hippocampal Afterdischarge1
ROBERT S. DYER AND WILLIAM E. HOWELL
Neitroto.xicology Division, Health Effects Research Laboratory, U.S. Environmental Protection Agency,
Research Triangle Park, NC 27711
DYER, R. S. AND W. E. HOWELL. Acute triethyltin exposure: Effects on the visual evoked potential and hippucampal
afterdischarge. NEUROBEHAV. TOXICOL. TERATOL. 4(2)259-266, 1982.—Acute administration of triethyltin (TET)
produces a well-described sequence of pathological events characterized by intramyelinic vacuolation, edema, and his-
totoxic hypoxia. Recent behavioral studies have attempted to characterize the functional consequences of TET exposures.
In this study, the effects of exposure on the visual evoked response
-------
260
DYER AND HOWELL
DAILY TESTING PROCEDURE :
VEPTMting , AOI
-------
ACUTE TRIETHYLTIN EFFECTS ON VER
261
START
START
EXPOSURE
EXPOSURE
END
EXPOSURE
EXPOSURE
g XfflllC
JOfttMc
FLASH
FIG. 2. VER's recorded from (A) one animal in the saline control group during the first 9 days of the
experiment; and (B) one animal in the 1,5 mg/TET/kg/day dosage group during the first 9 days of the
experiment. Each tracing is a mean of 64 responses to a flash (setting 16 on the Grass strobe unit). Peak
designations used in this study are indicated on the day 1 waveform.
of variables for purposes of MANOVA, significant MAN-
OVAs were followed by ANOVAs with a set at 0.0038
(0.5/13). Again, significant ANOVAs were followed by Dun-
can's multiple range test (a=0.05).
Histology
Following these studies, all animals were perfused with
potassium ferrocyanide in Formalin, a 1.0 mA 5 sec pulse
was passed through each electrode tip to aid in electrode tip
localization, and the electrodes and brains were removed.
Cresyl violet-stained sections were used to confirm electrode
localization.
RESULTS
The most readily apparent effects of TET were to in-
crease VER latencies and prolong the period of postictal
hippocampal hypoexcitability (increase the number of stim-
uli required to elicit AD3). However, statistical analysis re-
vealed a number of other significant effects, as described
below.
Electrode Localization
The electrode placements were generally accurate.
Forty-three animals had electrodes clearly located in the
hippocampal formation. Of these, 38 were located in the den-
tate gyrus and 5 were in CA1. One electrode was definitely
misplaced (corpus caitosum) and in 3 animals the histological
preparation did not permit electrode localization. Only the
one animal with a clearly misplaced electrode (from the 0.188
mg/kg/day group) was excluded from the AD analysis.
Visual Evoked Response
Alterations in the VER produced by TET are illustrated in
Fig. 2, which contains the VER from one rat in the 1.5
mg/kg/day dosage group and one rat in the 0 mg/kg/day dos-
age group. This figure shows the increased latencies
produced by TET. Supporting statistics for the obviously
increased latencies may be found in Table 1. Significant dos-
age x day interactions (Table !) are accounted for by the
gradually increasing latencies produced by TET. Although
determining the time course of recovery from TET was not
within the design of the study, 4 rats in the 1.5 mg/kg/day
dosage group had VERs recorded on days 10, 15. 20, 25. and
150 following the onset of the experiment. Figure 3 illustrates
the results for the N1 latency measurement. TET continued
to increase N1 latency until day 10. Recovery eventually
occurred, but the time course was prolonged. The pattern of
changes obtained with the NI latency was similar to those
obtained with other latencies. Figure 4 illustrates the main
effect of TET upon latencies of VER peaks PI, Nl, P2, N2,
and N3. Only the N3 latency was affected at a dosage below
1.5 mg/kg/day. This peak appeared most sensitive to TET,
since it was affected at the lowest dosage (0.188 mg/kg/day)
The only VER amplitude which was significantly affected
by TET was N2P3. While N2P3 amplitudes decreased
slightly in normal animals over days, TET increased N2P3
amplitudes. The increase was only statistically significant at
the 0.75 mg/kg/day dosage. Both NIP2 and P2N2 amplitudes
increased significantly over days in a manner which was not
related to TET exposure.
Hippocampal Afterdischarge
Figure 5 illustrates a typical hippocampal AD. An unex-
pected consequence of the 3 AD/day procedure was a blur-
ring of the distinctions between AD types. The 15 min sep-
aration between AD1 and AD2 was clearly not enough to
permit full recovery. Thus, although a 4 x threshold stimulus
-------
262
DYER AND HOWELL
TABLE 1
STATISTICAL SUMMARY OF EFFECTS OF TET ON VERs
Source
MANOVA F (df)
PI
Latency Analyses
ANOVA F's*
N1 P2 N2
P3
N3
Amplitude Analyses
ANOVA F's*
MANOVA F Id/) P1N1 NIP2 P2N2 N2P3 P3N3
Dosage
Day
Squad
Dosage x
Day
9.88<24,402)+«
3.88 (12.230)+*?
4.75 (24.402)+p<0.000l.
should have invariably produced ADs followed by profound
depressions [11], the depressions were often unimpressive.
Rebound ADs, which normally occur during the postictal
depression following a type 1 AD, were seen in the absence
of profound depressions. Due to resulting ambiguity of
classification, AD type was not included in the analyses.
Compared to day 1 values, AD thresholds tended to in-
crease. The increase in AD thresholds was significantly less
for the 0.188 mg/kg/day group than for any other group, in-
cluding control. The threshold increase obtained by the 0.750
mg/kg/day group was significantly greater than the control
increase. Thus, the effects of TET appear to be biphasic.
These results are shown in Fig. 6.
Repeated TET administration appeared to have a biphasic
effect upon magnitude of the PID as well; however, this
appearance was not supported by the statistical analysis.
Figure 7 illustrates the post-AD activity as a percentage of
the day 1 pre-AD activity. The low dosage groups (0.188 and
0.375 mg/kg/day TET) appeared to recover more rapidly than
the control group, but this apparent facilitation by TET was
not statistically significant. Both of the high dosage groups
(0.750 and 1.500 mg/kg/day TET) had depressed post-AD
activity. The 0.750 mg/kg/day group differed significantly
from control only during min 4, but the 1.500 mg/kg/day
group differed significantly from control during all 5 min.
Although TET did not significantly alter duration of the
AD, it did increase the frequency of spikes which occurred
during the AD. Figure 8A shows the effect of TET upon AD
spike frequency during the first 3 sec of the AD. The 0.375,
0.750 and 1.500 mg/kg/day groups had significantly elevated
spike frequencies.
Analysis of postictal hypoexcitability (number of stimuli
to AD3) indicated a biphasic effect of TET. Figure 8B indi-
cates that low dosages decreased while high dosages in-
creased the number of stimuli required to elicit the third AD.
The decrease was significant for the 0.375 mg/kg/day group,
and the increase was significant for the 1.500 mg/kg/day
group. MANOVA and ANOVA summaries are presented in
Table 2.
DISCUSSION
Acute exposure to TET significantly altered both the
VER and the hippocampal AD.
i 1—:
-----0.000 mg/kg N«9
1.500 mg/kg N»9
BE YON 0 DAY 9 N = 4
I L_J
1 3 5 7 10 16 20 25 150
DAY
FIG. 3. Time course of changes in N1 latency following administra-
tion of 1.5 mg/kg TET daily on days 2-7. N1 latency continued to
increase following termination of dosing and did not recover until
after day 25.
Visual Evoked Responses
Latencies observed for all VER peaks except P3 gradu-
ally increased at the 1.5 mg/kg/day dosage. Increased laten-
cies are consistent with abnormalities in myelin since such
abnormalities would be expected to slow conduction veloc-
ity. The absence of significant alterations in the amplitudes
of the PIN 1 and NIP2 components suggests that the popula-
tion of fibers in the optic nerve has not been reduced. Ampli-
tude of these components has been shown to be closely re-
lated to stimulus intensity and, therefore, presumably to
number of activated fibers as well [12].
Although TET did alter the latency of early peaks, the late
VER component appears to have been the most sensitive
detector of toxicity. The N3 latencies were significantly in-
creased over the entire post day 1 test period (i.e., no dosage
x day interaction) at the lowest dosage (0.188 mg/kg/day).
The influence of TET upon N3 latency was not related to
dosage in a linear fashion since the 0.75 mg/kg/day group did
not differ significantly from controls, whereas groups receiv-
-------
ACUTE TRIETHYLTIN EFFECTS ON VER
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FIG. 4. Effects of TET upon peak latencies of the VER, collapsed across days.
TET increased latencies of PI, Nl, P2, N2 and N3.
-------
264
DYER AND HOWELL
HIPPOC AMPAL AFTERDISCHARGE:
STIMULUS AD
12 SEC)
POST ICTAL DEPRESSION
FIG. 5. Typical hippocampal AD.
iu in
20
0.000 mq/kg
0.188 mg/kg
— — — 0.375 mg/kg
— • — • 0.750 mg/kg
1.500 mg/kg
1 2 J 4 5
MINUTES POST-AO
FIG. 7. Effects of TET on postictal (AD2) EEG depression. Mag-
nitude of post-AD depression was calculated using integrated EEG
activity. Each time the integrator reached its maximum value and
reset, 1 count was recorded. During each post-AD minute, the
number of counts/animal was converted to a percentage of the
number of counts that animal had during the min prior to AD2 or day
1. In this figure, these percentages are collapsed across days.
ing both more and less than 0.75 mg/kg/day TET did differ
significantly from control. There may be a relationship be-
tween the observations that (1) only the N3 latency of the
0.75 mg/kg/day group was not different from control and (2)
only the N2P3 amplitude of the 0.75 mg/kg/day group was
different from control. The relationships among the different
VER components are poorly understood, but it is likely that
the morphology of one wave influences the morphology and
thereby the latency of the subsequent wave. Thus, the failure
of N3 to differ from control at 0.75 mg/kg/day TET may have
been secondary to the alterations in N2P3 amplitude at this
dosage. The N2P3 amplitude alterations nevertheless remain
unexplained. This section of the VER is generally considered
to reflect nonspecific thalamic and reticular input [5].
0.000 0.188
0 750 1 500
TET OOSE ifwg/hgj
FIG. 6. AD threshold differences from day 1 as a function of TET
dose. Thresholds generally increase over days. The increase was
significantly smaller than control for the 0.188 mg/kg/day group and
larger than control for the 0.750 mg/kg/day group.
B
o o
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OOSE mg/kg
0 000 188 375 750 1 500
OOSE mg/kg
FIG. 8. A. Effects of TET on frequency of spikes occurring during
the first 3 sec of the AD. Values plotted are means of the differences
obtained between day 1 and all other days. TET significantly in-
creased spike frequency in the 0.375, 0.750 and 1.500 mg/kg/day
groups. B. Postictal hypoexcitability, measured as the number of
stimulus train presentations required to elicit AD3.
The VER data provide physiological correlates (increased
early peak latencies) of TET-induced optic nerve mye-
linopathy. They also suggest that the optic nerve is not
the most sensitive visual system structure, since N3 latency,
which presumably reflects cortical activity, was affected at
lower dosages than more direct measures of optic nerve
function (e.g., PI and N1 latency).
-------
ACUTE TRIETHYLTIN EFFECTS ON VER
265
TABLE 2
STATISTICAL SUMMARY OF EFFECTS OF TET ON ADs
Dose
F(52,1283)=4.93
/><0.0001
MANOVA
Day
F(91.2165)= 1.22
p> 0.08
Day x Dose
F(364,4019)=0.8l
p> 0.90
ANOVA (dose effect only; all df s 4,323)
Threshold
AD Duration
Wet Dog Shakes
Stim to AD3
F= 5.71*
p<0.0002
F= 3.42
p<0.0093
F= 2.95
p<0.02
F= 18.41*
p<0.0001
AD Spike Frequency
First 3 sec
Second 3 sec
Third 3 sec
F=7.43*
p<0.0001
F=7.69*
p<0.0001
F=3.60
p <0.0069
Integrator Resets
Pre AD
Post AD min I
min 2
min 3
min 4
min 5
F= 1.92
p>o.m
F=7.75*
p<0.0001
F=5.41*
p <0.0003
F=6.50*
p<0.000l
F=7.26*
p<0.0001
F=7.30*
p<0.0001
•Significant (Critical Value 0.05/13 = .0038).
Finally, these results are not easily understood as conse-
quences of the histotoxic hypoxia which is produced by
TET. Previous studies have indicated that an early acute
VER response to hypoxia is increased P1N1 amplitude [9],
followed at more severe hypoxia by increased N1 and P2
latency and decreased P3N3 amplitude. Since P1N1 ampli-
tudes were not increased, it is unlikely that the present re-
sults can be explained in terms of hypoxia.
Hippocampal Afterdischarges
TET appeared to have both excitatory and inhibitory ef-
fects upon hippocampal ADs, and the effects of TET upon
some hippocampal AD parameters appeared to be biphasic
m nature. Thus, low dosages of TET produced less than the
n°rmal control increase in AD threshold, decreased the
number of stimuli required to elicit a third AD (decreased
Postictal hypoexcitability), and increased (but not signifi-
cantly) the rate of recovery from postictal depression. These
effects might ail be attributed to increased excitability fol-
lowing low levels of TET. Both low (0.375 mg/kg/day) and
"'gh (1.5 mg/kg/day) dosages of TET increased spike fre-
quency during the first 3 sec of the AD, a finding which is
indicative of increased excitability. On the other hand, expo-
sure to high levels of TET depressed the postictal depression
beyond its normal values (0.75 and 1.5 mg/kg/day), increased
lhe number of stimuli required to elicit a third AD (1.5
j^g/kg/day), and increased the AD threshold (0.75 mg/
kg/day), findings which suggest neuronal depression.
* wo classical CNS depressants, ethanol and sodium pen-
tobarbital [11,20], increase AD thresholds and decrease AD
durations and spike frequency. Only the increased spike fre-
quency at 1.5 mg/kg/day prevents the conclusion that high
dosages of TET produce an anesthetic-like neuronal depres-
sion. Whatever the mechanisms of TET induced depression
in ADs, they are dissimilar to those produced by ethanol and
pentobarbital. These results are also dissimilar to those ob-
tained following acute hypoxia produced by carbon
monoxide which decreased AD spike frequency [10].
In conclusion, both the VER and hippocampal AD tech-
niques appear sensitive to the acute effects of TET. The
VER changes are consistent with myelinopathy and/or CNS
depression. Latency of the N3 peak was the most sensitive
VER measurement, increasing significantly at a dosage of
0.188 mg/kg TET. TET-induced AD changes are indicative
of both excitatory and inhibitory effects. Change in threshold
was the most sensitive AD measurement, since it was signifi-
cantly less than saline control at a dosage of 0.188 mg/kg
TET. Some of these effects, especially those on VER laten-
cies, may be explained by the effects of TET on myelin. It
does not appear likely that cytotoxic hypoxia produced by
uncoupling oxidative phosphorylation can explain the
others. It therefore seems likely that TET produces as yet
undescribed effects upon the CNS.
ACKNOWLEDGEMENTS
The authors thank Mark Bercegeay and Leon Lamm for techni-
cal assistance and the Health Effects Research Laboratory Word
Processing Center for manuscript preparation.
-------
266
DYER AND HOWELL
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1976.
18. Reiter, L., K. Kidd, G. Heavner and P. Ruppert. Behavioral
toxicity of acute and subacute exposure to triethyltin in the rat.
Neurotoxic atony 2: 97-112, 1980.
19. Scheinberg, L. C., J. M. Taylor, I. Herzog and S. Mandell.
Optic and peripheral nerve response to triethyltin intoxication in
the rabbit: biochemical and ultrastructural studies. J.
Neuropath, exp. Neurol. 25: 202-213, 1966.
20. Swartzwelder, H. S. C. T. Johnson, B. C. Cooley. W. E. How-
ell and R. S. Dyer. Alcohol-induced alterations in hippocampal
afterdischarges and afterdischarge thresholds: Dose-response
studies. Neurobehav. Toxicol. 1: 253-258, 1979.
21. Swartzwelder, H. S., S. T. Wegener, C. T. Johnson and R. S.
Dyer. Depressed excitability and integrated EEGs following
hippocampal afterdischarges. Brain Res. Bull. 5: 505-507. 1980.
22. Suzuki. K. Some new observations in triethyltin intoxication of
rats. Expl Neurol. 31: 207-213, 1971.
23. Torack. R., J. Gordon and J. Prokop. Pathology of acute
triethyltin intoxication. Int. Rev. Neurobiol. 12: 45-86, 1970.
-------
Neurobehavioral Toxicology and Teratology, Vol. 4, pp. 267-271, 1982. Printed ill the U.S.A.
Triethyltin: Ambient Temperature Alters
Visual System Toxicity1
ROBERT S. DYER AND WILLIAM E. HOWELL
Neurophysiology Branch, Neurotoxicology Division, Health Effects Research Laboratory,
U. S. Environmental Protection Agency, Research Triangle Park, NC 27711
DYER, R. S. AND W. E. HOWELL. Triethyltin: Ambient temperature alters visual system toxicity. NEUROBEHAV.
TOXICOL. TERATOL. 4(2) 267-271. 1982.—Previous studies have indicated that acute exposure to triethyltin (TET)
increases latencies of the flash evoked response (VER) recorded from the rat cortex. TET also produces hypothermia,
which may be modified by altering environmental (ambient) temperature. In this study, the role of ambient temperature in
determining the effects of acute TET upon the VER was examined. Rats with chronically implanted electrodes were
administered either TET (6 mg/kg) or saline, and maintained in either a warm (30°C) or cool (22°C) environment for the next
7 hrs. VERs were recorded during this 7 hr period, and at regular intervals for the next 2 weeks. TET increased VER peak
latencies. VER peak latencies recorded from animals exposed in a cool room gradually returned to pre-TET values.
Latencies recorded from animals exposed to TET in a warm room remained elevated for a longer period of time, thus
indicating a more severe impact of the TET exposure. This study indicates that toxicant-induced alterations in core
temperature are potential determinants of other toxicant-induced effects.
Tin Organotin Alkyltin Triethyltin Neurotoxicity Core temperature Ambient temperature
Temperature-dependent toxicity Visual system toxicity Visual evoked potential Visual evoked response
TRIETHYLTIN (TET) is a highly neurotoxic compound.
Numerous studies have explored the metabolic, morphologi-
cal, physiological, and behavioral effects of exposure to TET
(e g-, [I, 4. 6, 10, 14]). One of the most extensively studied
consequences of TET exposure is CNS demyelination. Sev-
eral studies have also suggested that TET produces an am-
bient temperature-dependent hypothermia in rats [6,12].
This finding is important, since hypothermia may account for
some of the effects of TET which would otherwise be as-
cribed to myelinopathy. For example. Rose and Aldridge
[15] demonstrated that the depressed incorporation of [32P]
Phosphate into rat brain phospholipids which was found to
occur following exposure to TET [17] was in fact a result of
hypothermia, and could be prevented when hypothermia
was reduced by warm environmental conditions.
Acute exposure to TET has been shown to increase
latencies of the visual evoked response (VER) [9,10]. Since
the optic nerve is sensitive to the demyelinating effects of
TET [16], the most obvious interpretation of the data was
conduction slowing secondary to demyelination. However,
hypothermia also increases evoked potential latencies (e.g.,
[11]), probably due to the relationship between reduced tem-
perature and reduced nerve conduction velocity [7,18].
Therefore, slowed conduction (increased latencies) of the
VER might reflect either hypothermia, alterations in myelin,
or both. The purpose of the present study was to (a) verify
that TET does produce an ambient temperature-dependent
hypothermia; and (b) assess the influence of ambient tem-
perature upon TET-induced alterations in the VER recorded
from the rat visual cortex.
METHOD
Adult male Long-Evans hooded rats were obtained from
Charles River Breeding Laboratories. Except as otherwise
indicated, the animals were maintained in individual plastic
cages with wood chip bedding and ad lib access to food and
water throughout the experiment. Seventy-eight rats were
used in the core temperature study, and an additional 40 rats
were used in the VER study.
Core Temperature
Three experiments were performed to assess the influ-
ence of TET upon core temperature. In all three, core tem-
peratures were recorded using a Yellow Springs Instruments
thermister type multiprobe thermometer. The rats were
placed in a commercial restraining tube and the probe was
inserted 75 mm into the rectum. Digital readings were re-
corded to the nearest 0.1°C. During the 6-hr sessions, the
rats were restrained continuously and the probe remained in
place. All other readings were taken with the rats placed in
the restraining tube for 5 min before recordings. In the first
experiment, 34 rats, kept in a room maintained at 22°C, were
injected IP with either 0 (vehicle, n= 14), 3(n=6), 6(n=8), or
'This paper has been reviewed by the Health Effects Research Laboratory, U. S. Environmental Protection Agency, and approved for
Publication. Mention of trade names or commercial products does not constitute endorsement or recommendation of use.
267
-------
268
DYER AND HOWELL
9(n=6) mg/kg TET bromide in saline vehicle. Core tempera-
tures were recorded immediately before injection and every
0.5 hr thereafter for 6 hrs. The animals injected with 9 mg/kg
TET also had temperatures taken 24, 48, and 72 hrs post-
injection.
In the second experiment, rats were injected with either 0
(n= 10) or 9 (n= 10) mg/kg TET and maintained in a room at
30°C. Again, core temperatures were recorded immediately
before injection and every 0.5 hr afterwards for 6 hr. The
animals exposed to 9 mg/kg TET also had their temperatures
recorded 138 hrs following injection.
In the third experiment, 32 rats in a room maintained at
22°C were injected with either 0 (saline vehicle, n=8), 3
(n=8), 6 (n=8), or 9 (n=8) mg/kg TET. Temperatures were
recorded immediately before injection and 1,2,5, and 6 days
later.
Visual Evoked Responses
Electrodes were implanted in rats for recording the VER,
using surgical procedures described in detail elsewhere
[8], The electrodes consisted of 00-90 stainless steel screws
threaded into the skull. Coordinates for placement were:
visual cortex, 6 mm posterior to bregma, 3 mm lateral
to the midline; reference, 2 mm anterior to bregma, 2 mm
lateral to the midline, and ground, 5 mm anterior, 2 mm
lateral, on the side contralateral to the visual cortex and
reference electrodes. The screws were connected to an Am-
phenol receptacle via nichrome wires, and the receptacle
was cemented to the skull using dental acrylic. Animals were
then administered 100,000 units penicillin G and returned to
their home cage. Recordings were begun no sooner than 1
week following surgery.
VER recordings were made in a box with mirrors on 3
walls and the strobe lamp from a Grass PS-2 photostimulator
on the 4th wail. The intensity setting for the 10 ptsec flash
was 16, which corresponded to a peak power of 4.53 x 10T lux
measured in the center of the chamber. Details of the record-
ing chamber are available elsewhere [8]. During each record-
ing session, the averaged VER to 64 flashes was obtained.
Flashes were presented at 2-sec intervals, and recordings
were obtained through AC coupled amplifiers with low and
high frequency cutoffs set at 1.0 Hz and 10 kHz respec-
tively. Averages were obtained using a Nicolet 1024 signal
averager with 0.4 msec/data point resolution, and copies of
the waveforms were made by a Hewlett-Packard X-Y plot-
ter. Amplitudes and latencies of the first three positive and
negative waves were obtained according to previously de-
termined conventions [9],
The rats were tested on 5 separate days. On the first day,
1 drop of 1% atropine sulfate was placed in each eye 20 min
prior to testing. An averaged VER was obtained as described
above, the rat was injected with either 6 mg/kg TET (n=20)
or saline vehicle (n=20). Following each VER session, the
animal was immediately returned to its home cage. Addi-
tional averaged VERs were obtained 1.5, 4.0, 7.0, 24, 48,
120, and 300 hrs following the injections. Atropine was not
readministered prior to the 1.5 and 4 hr sessions since it was
presumed the initial treatment was still effective.
Half of each injection group was maintained and tested in
a warm environment (30°C) beginning 30 min before the first
test session and continuing until after the 7-hr post-injection
test session. All other tests for these and other animals oc-
curred at normal room temperature (22°C), which was also
the temperature of the animal colony room. No efforts were
c
3
<
X
£
s
ht
FIG. I. Summary of the effects of TET upon core temperature. Data
points are means^SEM. Increasing ambient temperature attenuated
the hypothermia produced by 9 mg/kg TET.
made to record core temperature in these animals. Based
upon the core temperature studies (see Results), it was as-
sumed that the TET-exposed rats became hypothermic for at
least the first 7 hrs, but that the TET-exposed rats main-
tained in a warm environment became considerably less
hypothermic than those maintained at normal room tempera-
ture.
Statistical Analysis
All VER amplitudes and latencies were converted to
difference scores from the preinjection values. A 2 (dose) x
2 (temp) x 6 (time) repeated measures ANOVA was then
performed on each of the 11 variables (PI, Nl, P2. Nl. P3,
and N3 latencies; P1N1, N1P2, P2N2, N2P3, P3N3 ampli-
tudes). In order to approximate an experiment-wise a was
set at 0.05/11 = 0,0045 for each ANOVA. To further reduce
the probability of type I errors, only the following effects
were included in the ANOVAs: Dose, Dose x Temperature,
and Dose x Time x Temperature.
RESULTS
Core Temperature
Acute TET exposure reduced core temperature in a
dose-dependent fashion. The data from all of the core tem-
perature studies are summarized in Fig. 1. All animals, even
controls, had slightly reduced core temperatures over the
course of the experiment. The magnitude of temperature re-
duction was dependent upon dosage of TET, such that the
rats injected with 3 mg/kg TET had temperatures about 1.5°
below controls, while the rats injected with 6 and 9 mg/kg
TET had core temperatures which were about 2.0" and 4.0°
below controls, respectively. The TET-induced alterations
in temperature were largely gone 24 hrs following injection,
although the rats exposed to 9 mg/kg TET were still slightly
hypothermic. Rats injected with 9 mg/kg TET and main-
tained in the warm (30°C) environment did not become as
hypothermic as those maintained in the cool (22°C) environ-
ment. The hypothermia produced by 9 mg/kg TET in the
warm environment was roughly equivalent to that produced
by 3 mg/kg TET in the cool environment.
-------
270
DYER AND HOWELL
120 r i—i i i i mi11 1—r11 11 ii ii 1—i i i 11'ii
*1 LATENCY Nl LATENCY n LATENCY NELATENCY V) LATENCY N3 LATENCY
FIG. 3. Main effects of TET upon VER peak latencies, the values
are means (±SEM) which have been transformed to percentage of
pre-injection values for each rat. and collapsed across both post-
injection hrsand temperature conditions. *=p<0.01, **=p<0.0045.
cates that the daily exposure produced a prolonged time
course of latency alterations. In the present study, VER pa-
rameters of the cool TET-exposed animals had returned to
baseline within 24 hrs after exposure, while in the previous
study the alterations in Nl latency remained for at least 2
weeks following termination of exposure. This difference in
the time course of TET toxicity may have occurred due to
the higher cumulative dosage of TET required to produce
these effects in the previous study (6x 1.5=9 mg/kg vs 6
mg/kg).
Two important results emerge from the present study.
First, it is clear that even though hypothermia produces al-
terations in the VER which are somewhat similar in charac-
ter to those produced by TET [9], the presence of alterations
in Nl latency which are similar in magnitude in both the
warm and cold-exposed rats coupled with the increased dura-
tion of toxicity in warm-exposed rats argues strongly that the
previous results may not be accounted for by hypothermia.
Second, this study points out the temperature-dependent tox-
icity of TET. Even though exposure to warm environmental
temperatures was of relatively short duration (7 hrs), the
effects of this exposure were persistent. N1 latencies of the
warm-exposed TET-injected rats only began to show signs of
returning to normal 12 days following exposure. Cold-
exposed TET-injected rats had normal N\ latencies 24 hrs
following exposure.
The finding that reduction of toxicant-induced
hypothermia increases rather than decreases the toxicity of
the agent in question is not novel. Prevention of carbon
monoxide-induced hypothermia significantly increases le-
thality at a normally non-lethal concentration of CO [3].
o SAUNE
A t mg/kg TET
£ 108 -
?—PERIOD Of--*
. TEMPERATURE
- CONTROL
I L.,1 I I
' ' " ' I I I, I 111
n11 i—i t i i rii| 1—i ) i i nr
O SAUNE
A ( mg/kg TET
?—PERIOD OP-H
TEMPERATURE'
- CONTROL
1
10
102
103
TIME POST INJECTION Ihral
FIG. 4. Time- and temperature-dependent nature of TET effects
upon the latency of VER peak Nl. Top: animals maintained at
"cool" (22°C) room temperature during the period of temperature
control. Differences between TET and control rats were absent 24
hrs following exposure: Bottom: animals maintained in a warm
(30°C) environment during the period of temperature control. Differ-
ences between TET and control rats were still evident 300 hrs fol-
lowing exposure.
These data provide further evidence for the critical nature of
environmental temperature in the assessment of neurotoxic-
ity.
ACKNOWLEDGEMENTS
The authors wish to thank Mark Bercegeay for excellent techni-
cal assistance and Susan Garner for manuscrpit preparation.
REFERENCES
1. Aldridge, W. N. and M. S. Rose. The mechanism of oxidative
phosphorylation. FEBS Lett. 4: 61-68, 1979.
2. Annau, Z. Electrical self-stimulation of the brain: a model for
the behavioral evaluation of toxic agents. Envir. Hlth Perspect.
26: 59-67, 1978.
3. Annau, Z. and R. S. Dyer. Effects of environmental tempera-
ture upon body temperature in the hypoxic rat. Fedn Proc. 36:
579, 1977.
4. Blaker, W. D., M. R. Krigman, D. J. Thomas, P. Mushak and P.
Morell. Effect of triethyltin on myelination in the developing
rat. J. Neurochem. 36; 44-52, 1981.
5. Clark, W. G. Changes in body temperature after administration
of amino acids, peptides, dopamine, neuroleptics, and related
agents. Neurosci. Bio be ha v. Rev. 3: 179-231, 1979.
6. Cremer, J. E. Selective inhibition of glucose oxidation by
triethyltin in rat brain in vivo. Biochem. J. 119: 95-102, 1970.
7. Davis, F. A., C. L. Schauf, B. J. Reed and R. L. Kesler. Exper-
imental studies of the effects of extrinsic factors on conduction
in normal demyelinated nerve. I. Temperature. J. Neurol.
Neurosurg. Psychiat. 39: 442-448, 1975.
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TEMPERATURE-DEPENDENT TET TOXICITY
:69
Ml
NJ
N2
00
40
TO
i 240
FLASH
0.0
7.0
24.0
§ 120.0
MuV
SCAli.
VERs
Figure 2 illustrates the VERs recorded from TET-
exposed rats in the warm and cool environments, and from a
control rat in the warm environment. TET increased laten-
cies of all peaks, although only N1 and P2 latencies differed
statistically from control with a=0.0045. Figure 3 sum-
marizes these data. There were no statistically signficant ef-
fects of TET upon amplitudes. There were no significant
dose x temperature interactions, but TET produced altera-
tions in N1 latency which were dependent upon the temper-
ture condition and the time of measurement (significant dose
x temperature x time interaction). Rats exposed to TET in a
warm room had elevated N1 latencies (compared to saline
controls in the warm room) at all measurement times follow-
ing injection. Rats exposed to TET in the cool room had NI
latencies which were elevated 1.5, 4 and 7 hrs following in-
jection, but returned to control values within 24 hrs. These
data are illustrated in Fig. 4.
DISCUSSION
These studies confirm that TET produces hypothermia
which can be at least partially attenuated by maintenance in a
30°C environment. Even control (saline injected) animals
exhibited a slight drop in temperature over the course of the
experiment. This drop may reflect recovery from an initial
hyperthermia in all rats induced by the stress of being placed
in the restraining tube [13]. The failure to observe this hyper-
thermia in any rats after the first day probably reflects
habituation to the restraining tube.
The failure to completely prevent hypothermia from de-
veloping by maintaining animals exposed to 9 mg/kg TET in
a 30°C environment was surprising, since this environmental
temperature is effective in preventing the more severe
hypothermia which occurs during exposure to either 1000
ppm carbon monoxide or hypoxic hypoxia [3]. It had been
presumed that the hypothermia induced by exposure to TET
reflected the histotoxic hypoxia which this agent is known to
produce by virtue of its uncoupling of oxidative phospho-
rylation nl. These data suggest that other mechanisms may
also be involved.
The VER study produced several significant findings.
First, it replicated the findings of Dyer and Howell [9] in that
TET produced increased VER latencies in the absence of
major effects upon VER amplitudes. Unlike the previous
study, TET was administered in a single injection and the
time course of alterations was studied. Comparing the TET-
exposed animals kept in the cool environment in the present
study with the high dose animals exposed to TET daily in the
previous study (which was done at about the same environ-
mental temperature as the cool exposure of this study), indi-
FIC. 2. Averaged flash-evoked potentials recorded from the visual
cortex of one rat in each of three groups. Top: control rat, record-
ings taken at the times indicated on the ordinate. The peak des-
ignations used in the study are indicated on the 0.0 hr waveform.
This control was injected with saline at 0.0 hrs and maintained in a
30°C environment for 7 hrs; Middle: same as above except the rat
was injected with 6 mg/kg TET at 0.0 hrs; Bottom: 6 mg/kg TET-
treated rat maintained at "normal" (22°C) room temperature
throughout the study. Calibration applies to all 3 sets of waveforms.
Dashed vertical lines are drawn through the pre-injection Nl, N2,
and N3 peaks.
11
i40
I 7#
I
|140
I
? 400
120.0
-------
TEMPERATURE-DEPENDENT TET TOXICITY
271
8. Dyer. R. S. and Z. Annau. Flash evoked potentials from rat
superior colticulus. Phurmuc. Bitn/wm. Be ha v. 6: 453-459,
1977.
9. Dyer, R. S. and W. E. Howell. Acute triethyltin exposure: Ef-
fects on the visual evoked potential and hippocampal afterdis-
charge. Nettrohehav. Toxicol. Tenilol. 4: 259-266, 1982.
10. Gerren. R. A.. D. E. Groswald and M. W. Luttges. Triethyltin
toxicity as a model for degenerative disorders. Pharmac.
Biochem. Behav. 5: 299-307. 1967.
11. Jones. T. A., J. J. Stockard and W. J. Weidner. The effects of
temperature and acute alcohol intoxication on brain stem audi-
tory evoked potentials in the cat. Electroenceph. din. Nettro-
physioi 49: 23-30, 1980.
12. Leow, A. T. C., K. M. Towns and D. D. Leaver. The effects of
triethyltin in the rat following systemic and intercerebroven-
tricular injection. Chem. Biol. Interact. 31: 233-238, 1980.
13. Poole, S. and J. D. Stephenson. Core temperature: Some
shortcomings of rectal temperature measurements, Phwsiol. Be-
hav. 18: 203-205, 1977.
14. Reiter, L. W., K. Kidd. G. Heavnerand P. Ruppert. Behavioral
toxicity of acute and subacute exposure to triethyltin in the rat.
Neuroto.xicolofiy 2: 97-112. 1980.
15. Rose, M. S. and W. N. Aldridge. Triethyltin and the incorpora-
tion of [MP] phosphate into rat brain phospholipids. J. Neiiro-
chem. 13: 103-108, 1966.
16. Scheinberg, L. C.. J. M. Taylor, 1. Herzog and S. Mandell.
Optic and peripheral nerve response to triethyltin intoxication in
the rabbit: Biochemical and ultrastructural studies. J.
Neuropath, ex p. Neurol. 25: 202-213. 1966.
17. Stoner, H. B. and C. J. Threlfall. The biochemistry of organotin
compounds. Effect of triethyltin sulphate on tissue phosphates
in the rat. Biochem. J. 69: 376-385, 1958.
18. Swadlow, H. A., S. G. Waxman and T. G. Weyland. Effects of
variations in temperature on impulse conduction along non-
myelinated axons in the mammalian brain. E.vpl Neurol. 71:
383-389, 1981.
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N euro behavioral Toxicology and Teratology, Vol. 4, pp. 141-147, 1982. Printed in the U.S.A.
Trimethyltin-Induced Changes in Gross
Morphology of the Hippocampus1
ROBERT S. DYER, TERESA L. DESHIELDS* AND WILLIAM F. WONDERLIN3
Neurophysiology Branch, Neurotoxicology Division, U. S. Environmental Protection Agency
Research Triangle Park, NC 27711
DYER, R. S., T. L. DESHIELDS AND W. F. WONDERLIN. Trimethyltin-induced changes in gross morphology of the
hippocampus. NEUROBEHAV. TOXICOL. TERATOl^ 4(2) 141-147, 1982.—Acute exposure to trimethyltin (TMT)
produces alterations in hippocampal morphology. The purpose of this study was to arrive at a simple method for quantita-
tive assessment of the gross changes in morphology which could then be used as a correlate in studies of TMT toxicity.
Adult Long-Evans male hooded rats were treated with a single dose of TMT chloride and sacrificed either (a) within 11
days; (b) following 30 days; or (c) 10S days following treatment. Among a variety of morphological measures explored, the
easiest and most clearly dosage-related was length of the line of pyramidal cells, from CA1 through CA3c. TMT shortened
this line in a dosage- and time-dependent manner. Loss of cells appeared to begin in CA3c and progress through CA3b and
CA3a as dosage and time since treatment increased. It was concluded that this measurement may provide a useful mor-
phological correlate for physiological and behavioral studies of TMT toxicity.
Hippocampus Hippocampal degeneration Trimethytin Organotin Trimethyltin chloride
ACUTE exposure to trimethyltin (TMT) produces altera-
tions in hippocampal morphology [2,4,71, but there has been
no quantitative assessment of hippocampal damage as a
function of TMT dosage. Since a large proportion of current
studies of TMT toxicity have explored behavioral and phys-
iological parameters presumed sensitive to hippocampal dys-
function, a quantitative description of gross changes in hip-
pocampal morphology might be useful to correlate for relating
neuropathological changes to alterations in these endpoints.
Thus, the main intent of the present study was to arrive at a
simple gross measure of TMT-induced hippocampal damage
which could be used as a correlate in other studies. Availa-
bility of such a measure will allow future studies to deter-
mine the relationship between the primary endpoints meas-
ured and a simple measure of hippocampal damage. It is not
presumed that perfect correlations will be obtained between
crude morphological measures and behavioral or physiolog-
ical parameters. On the other hand it is unreasonable to ex-
pect behavioral and physiological studies to routinely pro-
vide detailed morphological assessment of hippocampal
damage. Thus, a simply made measure has merit as a com-
promise between no assessment and detailed assessment for
relating two such data sets.
Brains used for the present study were obtained from three
studies completed for other purposes. In two of these
studies, electrodes were implanted unilaterally in the hip-
pocampus for purposes of either stimulation or recording. In
the other study, only behavioral measures were obtained.
Assessment of brains from these two different types of ex-
periments allowed determination of the role played in hip-
pocampal degeneration by repeated electrical stimulation
and/or electrode placement.
METHOD
Adult male Long-Evans hooded rats, obtained from
Charles River Breeding Company, were housed individually
in plastic cages with wood chip bedding and unless otherwise
indicated given free access to food and water throughout
their stay in the lab. Trimethyltin chloride (TMT) (ICN,
Plainview, NY) was administered by gavage in a volume of 1
ml/kg, and dosages were either 0 (saline control), 5, 6, or 7
mg/kg given as the base.
In the first experiment, 50 rats surgically implanted with
electrodes in the left dorsal hippocampus (see hippocampal
afterdischarge study in Dyer et at., pp. 203-208, this
volume) were used. The animals were divided into groups
accordingly: unstimulated untreated controls (n=10), saline
treated controls (n» 10), 5 mg/kg TMT (n« 10); 6 mg/kg TMT
(n=10) and 7 mg/kg TMT (n»10). Thirty days following
treatment, the rats were perfused through the heart with
saline followed by 10 percent neutral buffered Formalin satu-
rated with potassium ferrocyanide (used for Prussian blue
electrode placement test). Frozen sections (80 mM) were ob-
'This paper has been reviewed by the Health Effects Research Laboratory, United States Environmental Protection Agency, and approved
for publication. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.
Present address: Department of Psychology, University of Georgia, Athens, OA.
'Present address: Department of Environmental Health Sciences, The Johns Hopkins University, 613 North Wolfe Street, Baltimore, MD.
141
-------
142
DYER, DESHIELDS AND WONDERLIN
CA1
CM
c«\v
FIG. 1. Diagrammatic sagittal section through the rat hippocampal
formation illustrating measurements made. (A-B) length of the
pyramidal cell line; (C-D) thickness of the dentate gyrus; (E-F)
thickness of the granule cell layer;
-------
TMT CHANGES IN HIPPOCAMPUS MORPHOLOGY
143
FIG. 2. Sagittal sections through dorsal hippocampus of 4 rats for Experiment 1. Sacrifice time was 30
days following treatment. (A) control; (B) 5 mg-kg TMT; (C) 6 mg/kg TMT; (D) 7 mg/kg TMT.
Calibration bar: 0.5 mm.
RESULTS
In general, the results confirmed previous observations
that the pyramidal cells were most sensitive to TMT, as
measured by cell loss. Figure 2 illustrates typical sagittal
sections obtained from animals in the first experiment.
Measurements obtained from the first experiment indicated
that length of the pyramidal cell line was sensitive to TMT
treatment. Thickness of the dentate gyrus was also de-
creased by TMT, as was density of cells within CA3, (all F's
(4,43)>4.38, all p's<0.005). On the other hand, neither thick-
ness of the granule cell layer, thickness of C A3 or thickness of
CA1 were significantly affected by the TMT treatment, all
F's (4,43)<3.06, allp's>0.03. The TMT treatment x section
interaction tested whether, within blocks of medial (right
side) or lateral (left side) sections, there was an influence of
distance from the midline on presence, magnitude or direc-
tion of treatment effect. The analysis indicated that there
were no significant treatment x section interactions, F's
(4,43)<1.77, p's>0.15.
Figure 3A illustrates that while there were no differences
between the implanted but untreated, unstimulated controls
and the rats receiving saline, there was a dose-dependent
decrease in length of the pyramidal cell line following TMT
administration. The Duncan's multiple range tests indicated
that for the right side the unstimulated and control groups did
not differ from each other. The 6 and 7 mg/kg TMT groups
did not differ from each other, but did differ from all other
groups, and the 5 mg/kg group differed significantly from the
unstimulated group but not from the 0 mg/kg control group.
The same relationships held for the left side, except that the
5 mg/kg TMT group differed from both the saline control and
the unstimulated control. In addition, the 6 and 7 mg/kg
groups differed from each other. Figure 3B illustrates the
effect of TMT upon thickness of the dentate gyrus. As with
length of the pyramidal cell line, TMT reduced the measured
values. In this case, however, the significant paired compari-
son differences were only found on the left side. Here the 7
mg/kg group differed significantly from all groups except the
6 mg/kg group. The 6 mg/kg group differed from both control
groups, but not from the 5 mg/kg group. The 5 mg/kg group
did not differ from either control group.
Figure 3C illustrates the effect of TMT upon density of
cells within CA3. TMT reduced the density of cells within
CA3, but only in the 6 and 7 mg/kg groups, and only on the
right side measurements. The significant overall ANOVA
was not accompanied by any significant individual paired
comparisons on the left side. Figure 3D-F indicates the non-
significant effects of TMT upon the other measurements ob-
tained in this experiment.
Although there were no significant treatment by section
interactions, there were significant effects of section, thus
indicating that within blocks of left and right sections some
of the measurements taken depended upon distance from the
midline. Length of the pyramidal cell line increased with
distance from the midline in both medial (right) and lateral
(left) blocks of sections. In the medial block of sections, the
thickness of the dentate gyrus increased and thickness of the
CA1 cell layer decreased with distance from the midline, all
F's (1,43)>10.46, all p's<0.002. No other measurements
were significantly affected by section number, all F's
(1,43X2.15, all p's>0.15.
From Figure 3, it is evident that in most cases, differences
existed between the medial and lateral blocks of sections
even when no differences existed between sections within
block. The third MANOVA supported the medial versus lat-
eral block differences, F(6,81)»29,64, p<0.0001. Reference
to Fig. 3 indicates the nature of the differences. Those which
were significant were: density of CAS, thickness of the
-------
144
DYER, DESHIELDS AND WONDERLIN
LENGTH OF PYRAMIDAL CELL LINE
THICKNESS Of DENTATE GVMJS
RIGHT SIDE
I I LEFT SlOt
RIGHT SIOE
I | LEFT SIOE
UNTREATED Omg kg Smg/kg 6 mg. kg 7 mg/kg
UNTREATED Omg/kg Smg/kg 6 mg/kg 7mg kg
10
9
•
7
6
S
4
3
2
1
0
CA3 DENSITY
RIGHT SIOE
I I LEFT SIOE
inlifkf
•W
i
150
THICKNESS OF CA3 CELL LATER
UNTREATED Omg/kg Smgkg 6mg kg 7 mg'kg
THICKNESS OF CA1 CELL LAVER
UNTREATED 0 mg'kg Smgkg 6 mg'kg 7 mg kg
THICKNESS OF GRANULE CELL LAVER
RIGHT SIDE
I I LEFT SIOE
RIGHT SIOE
I 1 LEFT SIOE
UNTREATED Omg kg Smgkg Cmgkg 7mgkg
UNTREATEO Omg kg Smgkg 6mgkg 7mg kg
FIG. 3. TMT-induced changes in measurements obtained from sagittal sections during the first experiment (30-day post-treatment sacrifice
time). Left side measurements were about 0.5 mm more lateral than right side measurements. Measurements were averaged across section
number within each side. (A) length of the line of pyramidal cells; (B) thickness of the dentate gyrus; (C) CA3 density (number of cells
intersected by a perpendicular drawn through CA3 at its most sparsely populated point in the elbow of the area); (D) thickness of the CA3
layer at its thickest extent; (E) thickness of CA1 at its thickest extent; (F) thickness of the granule cell layer at its thickest extent (whether
dorsal or ventral blade) in the horizontal plane. *=different from same side unstimulated control (p<0.05, Duncan's multiple range test).
granule cell layer, thickness of the dentate gyrus, and length
of the pyramidal cell line. The main effect of dosage was not
tested using this collapsed set of data, since it was redundant
with the first two MANOVA's. There were no dosage x side
interactions (MANOVA F (24,283)=0.66, p>0.89).
In the second experiment, it was observed that length of
the pyramidal cell line was decreased (by about 20%) in less
than U days, F (1,11)=29.29, p<0.0002. Due to the small
number of subjects, the different post-treatment times were
collapsed to make one control and one exposed group for
purposes of analysis.
In the third experiment, the effects of TMT on the hip-
pocampus were most extensive. Figure 4 illustrates sections
obtained from 1 control and 3 treated animals 105 days fol-
lowing treatment. As shown in Fig. 5A, the length of the
pyramidal cell line was reduced by more than 50 percent. In
some animals, only short segments of CA1 and Ch2 re-
mained. The length of the cell line in these controls is longer
than the controls of the first two experiments because sec-
tions were obtained from a more lateral location. The length
of the line of granule cells was slightly reduced (Fig. 5A) and
the intrahilar thickness was reduced by nearly 30 percent
-------
TMT CHANGES IN HIPPOCAMPUS MORPHOLOGY
145
*- v- ** **? . .. :
P >.11V. *•*»)»» ... ' .
X • •« .... •>. • V
*¦• »-• v.V -•:••••• •
. 4m,
V"
FIG. 4. Sections obtained from 4 animals used for Experiment 3 (105-day post-treatment sacrifice
time). A: section from control rat; B-D: sections from rats treated with 6 mg/kg TMT. Magnification
and section location were identical in all of these pictures. Calibration bar: 0.5 mm.
>.000
I 1 0 mg/kg
• iM/kg
7.200
3.400
4.200
3.600
700
PYRAMIOAL CELLS GRANULE CELLS
INTRAHILAR
EXTRAHILAR
FIG. 5. TMT-induced changes in measurements obtained from sagittal sections during the third experiment (105-day post-treatment sacrifice
time). (A) length of the line of pyramidal cells and granule cells; (B) thickness of the intrahilar and extrahilar region of the dentate gyrus.
(Fig. 5B). These findings were statistically significant, all Fs
(1,13)>9.12, p's<0.01. The measurements of extrahilar
thickness and granule cell layer thickness obtained from the
TMT-treated rats did not differ significantly from those ob-
tained from the control rats, Fs (l,13)<4.23,p's>0.05.
DISCUSSION
As reported by others, systemic administration of TMT
produces a loss of pyramidal cells within the hippocampal
formation, primarily from CA3 [2,7]. This study demon-
strates quantitatively the dose-dependence and time-
dependence of these changes. Following a single administra-
tion of 6 mg/kg TMT, loss of pyramidal cells appears first in
CA3c, then CA3b and a. At long post-treatment intervals
(105 days), damage appears more extensive but cells may
remain in CA1 and the "resistant zone" (see below). The
absence of differences between the two control groups in the
first experiment and the absence of a treatment x side inter-
action in that experiment indicates that changes in hip-
pocampal morphology were not influenced by repeated af-
terdischarge production or presence of an electrode.
Several differences exist between the results reported
here and those reported by Brown et al. [2]. Brown et al. [2]
reported a thinning of CA1, and although there was a slight
thinning of the cell layer in the 30-day post-treatment study
(Experiment 1), it did not approach statistical significance. It
is conceivable that the difference observed by Brown et al.
was secondary to the distance from midline effect reported
here. In other words, if medial control sections were inad-
vertently compared to lateral treated sections, then an ap-
parent treatment-related thinning of the CA1 cell layer might
-------
146
DYER, DESHIELDS AND WONDERLIN
be reported. More probably, some combination of dosage
and/or post-treatment time may produce neuron loss in CA1.
CA1 measurements were not made in the 105-day post-
treatment study (Experiment 3), but inspection of the slides
indicates that it occurred in at least some animals.
Similarly, Brown et al. [21 refer to a sparing of Sommer's
sector by TMT. Sommer's sector is generally described as
either hi [5] or CA1 [15], an area which Brown et al. [2]
report as thinned by TMT. Indeed the present study did find
some sparing of Sommer's sector, especially at low dosages
and short survival times. Brown et al,'s Fig. 13 [2] indicates
sparing of the resistant sector of Spielmeyer [10], which we
observed to be partially spared in some but not all of the
brains measured in this study. The present study indicates
that, at least in terms of gross cellular loss, damage begins in
CA3c and progresses through CA3a and possibly into CA2,
in a dosage dependent manner. Thus at low dosages Spiel-
meyer's zone is entirely spared, while at high dosages it may
not be.
It is important to discriminate between the gross mor-
phological assessment made in the present study and the
more detailed cellular assessments made by others. Bouldin
et al. [1] have reported that in rats the earliest TMT-induced
alterations occur in the dentate granule cells. Similarly,
Chang et al. [4] report that in the hippocampal formation of
mice, the dentate granule cells are most affected. Several
factors might account for the apparent discrepancy between
those studies and the present study. Both studies [1,4] used
electron microscopy, while the present study did not. Boul-
din et al. [1] report that acute exposures to TMT produce
more necrosis in neurons of fascia dentata than cornu am-
monis, and that following chronic intoxication the reverse is
true. However in their study the dosing regimen and the time
course of effects are confounded. Their "acute" experi-
ments involved repeated high dosages and short postdose
sacrifice times. Thus, effects are described as acute if they
follow a dose of TMT by 24 hrs. The dosing regimen is not
strictly acute however since the doses were repeated. The
differences which are ascribed to the differences in dosing (5
mg/kg/day for 3 or 4 days vs 1 mg/kg/2 days for 15 doses) may
in fact be due simply to time since a certain fixed dosage had
accumulated. Thus, if some of the acutely dosed animals had
been sacrificed 30 days after the last dose, the same pattern
of alterations as observed in the 30-dose experiment might
have been obtained. The gross pattern of degeneration re-
ported by Bouldin et al. [1] following "chronic" TMT treat-
ment is similar to the present findings.
The third experiment in the present study does suggest
some loss of dentate granule cells, presumably from the
anterior ends of the blades since the line of cells was
shortened. Scattered loss of cells within the dorsal and ven-
tral blades could well have been missed due to (1) the thick-
ness of the sections examined, (2) the dense packing of the
ceils, and (3) the inappropriateness of the stain and micro-
scopic technique for demonstrating cell death. Loss of
pyramidal cells was so extensive that it overcame these in-
adequacies. The vulnerability of CA3 pyramidal cells is not
surprising. Other evidence suggests that these cells are
especially vulnerable to the effects of a variety of stressors,
including excess stimulation [13, 17, 19], epilepsy [5], is-
chemia [3], carbon monoxide hypoxia [11], di-
piperidinoethane [12], and aging [9]. Indeed, the correlations
between the damage induced by TMT and the damage in-
duced by kainic acid (e.g., [14]), which is presumed to occur
as a result of hyperactivation of the mossy fiber system [13],
may be produced by similar mechanisms. The discrepancy
between the findings of Chang et al. [4] and the present
findings do not contradict this explanation. The differences
may result from a species difference since rats and mice are
known to differ markedly in their response to both kainic
acid [16] and TMT (K. Hulebak, personal communication).
Finally, it should be pointed out that the measurements
made in the present study are quite crude. Thus, the meas-
urement of cell loss in specific regions was thickness of the
cell layer. Although this was the apparent criterion used by
Brown et al. [2], assessing cell loss by this method implies
that either (1) cell loss occurs from one side of the line of
cells or the other, and is not scattered through the layer: or
(2) that loss may occur throughout the layer, but the remain-
ing cells close up the distance. There is no a priori reason to
assume that either of these two possibilities is true. Indeed,
the two methods herein used to measure CA3, which showed
decreased density with no decreased thickness, indicate that
either cell loss is evenly distributed throughout the thickness
of the layer, or at least does not occur preferentially at one
side of the layer or the other.
The thinning of the dentate gyrus in the present study is
demonstrated to result from intrahilar thinning. It is likely
that both cell loss and fiber loss contribute to the change.
CA3c cells are entirely lost from the intrahilar region, and
Bouldin et al. [1] have described loss of small myelinated
fibers within the hilus as well.
In conclusion, from the measurements obtained here it is
possible to single out length of the pyramidal cell line as a
simple dosage-dependent correlate of hippocampal damage
induced by TMT. The data indicate that within a limited
range (between 2 and 3 mm from midline) the effect of TMT
is not lateral distance-specific. However the distance from
midline does affect this parameter, and thus between-
treatment comparisons must be made at a fixed distance
from the midline. The clear dosage-dependence of the meas-
ure suggests that it may be useful as a marker for comparison
with behavioral and physiological studies.
ACKNOWLEDGEMENT
The authors wish to thank Ann Brady for preparation of the
histological material, J. V. Nadler, B. Veronesi and T. J. Walsh for
critical reviews of the manuscript, and Susan Garner for manuscript
preparation.
REFERENCES
1. Bouldin, T. W., N. 0. Goines, C. R. Bagnell and M. R. Krig-
man. Pathogenesis of trimethyltin neuronal toxicity: Ultrastruc-
tural and cytochemical observations. Am. J. Path. 104:237-249,
1981.
2. Brown, A. W., W. N. Aldridge, B. W. Street and R. D. Ver-
schoyle. The behavioral and neuropathological sequelae of in-
toxication by trimethyltin compounds in the rat. Am. J. Path.
97: 59-82, 1979.
3, Bubis, J. J., T. Fugimoto, U. Ito, B. J. Mrsu(ja, M. Spatz and I.
Klatzo. Experimental cerebral ischemia in Mongolian gerbils V.
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neuropath. 36: 285-294, 1976.
4. Chang, L. W., T. Tiemeyer, G. R. Wenger and D. E. McMillan.
Trimethyltin (TMT) induced hippocampal lesions in mice. Sac.
Neurosci. Abst. 7: 153, 1981.
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TMT CHANGES IN HIPPOCAMPUS MORPHOLOGY
147
5. Dam, A. M. Epilepsy and neuron loss in the hippocampus.
Epilepsia 21: 617-629, 1980.
6. Dunn, 0. J. Multiple comparisons among means. J. Am. statist.
Ass. 56: 52-64, 1961.
7. Dyer, R. S., T. J. Walsh, W, F. Wonderlin and M. Bercegeay.
The trimethyltin syndrome in rats. Neurobehav. Toxicol.
Teratol. 4: 127-133, 1982.
8. Geisser, S. and S. W. Greenhouse. An extension of Box's re-
sults on the use of the F distribution in multivariate analysis.
Ann. math. Statist. 29: 885-891, 1958.
9. Lapresle, J. and M. Fardcan. The central nervous system and
carbon monoxide poisoning II. Anatomical study of brain le-
sions following intoxication with carbon monoxide (22 cases).
In: Carbon Monoxide Poisoning. Prog. Brain Res., edited by H.
Bour and I. Leding. 24: 31-74, 1964.
10. Levine, S. and R. Sowinski. Lesions of amygdala, pyriform cortex
and other brain structures due to dipiperidinoethane intox-
ication. J. Neuropath, exp. Neurol. 39: 56-64, 1980.
11. Machado-Salas, J. P. and A. B. Scheibel. Limbic system of the
aged mouse. Expl Neurol. 63: 347-355, 1979.
12. Montgomery, R. L. and E. L. Christian. Pathologic effects of
antimetabolites on the hippocampus of diet controlled mice.
Brain Res. Bull. 1: 255-259, 1976.
13. Nadler, J. V. and G. J. Cuthbertson. Kainic acid neurotoxicity
toward hippocampal formation: dependence on specific excita-
tory pathways. Brain Res. 195: 47-56. 1980.
14. Nadler, J. V., B. W. Perry, C. Gentry and C. W. Cotman.
Degeneration of hippocampal CA3 pyramidal cells induced by
intraventricular kainic acid. J. camp. Neurol. 192: 333-359,
1980.
15. O'Keefe, J. and L. Nadel. The Hippocampus as a Cognitive
Map. Oxford: Oxford Universtiy Press, 1978, p. 108.
16. Olney, J. W. Kainic acid and other excitotoxins: a comparative
analysis. In: Glutamate as a Neurotransmitter, edited by G.
Dichiara and G. L. Gessa. New York: Raven Press, 1981, pp.
375-384.
17. Olney, J. W. and R. S. Sloviter. Ultrastructural analysis of hip-
pocampal damage produced by perforant path stimulation in the
rat. J. Neuropath, exp. Neurol. 40: Abstr. 340, 1981
18. Walsh, T. J., D. B. Miller and R. S. Dyer. Trimethyltin, a selec-
tive hippocampal neurotoxicant, impairs learning and memory
of a radial arm maze task. Soc. Neurosci. Abstr, 7: 647, 1981.
19. Zaczek, R., M. Nelson and J. T. Coyle. Kainic acid neurotoxic-
ity and seizures. Neuropharmacology 20: 183-189, 1981.
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NEONATAL TRIETHYLTIN EXPOSURE ALTERS ADULT
ELECTROPHYSIOLOCY IN RATS'
Robert S. Oyer, William E. Howell, Lawrence W. Reiter
Neurotoxicology Division, Health Effects Research Laboratory,
U.S. Environmental Protection Agency, Research Triangle
Park, NC 27711
In adults, triethyltin (TET) produces degeneration of white mat-
ter, edema, vacuolization of myelin and histotoxic hypoxia. To
determine the functional consequences of perinatal exposure
to TET, albino rats were administered either 0,3,6, or 9 mg/kg
TET on postnatal day 5. Upon reaching adulthood, the rats
were implanted with electrodes for recording visual evoked
potentials (VEPs) and hippocampal afterdischarges (ADs). In
addition to these tests, 17 days of kindling trials were admin-
istered to the rats followed by testing with pentylenetetrazol
and picrotoxin for seizure susceptibility. TET increased laten-
cies of P2, P3, and N3 of the VEP in a dose dependent fashion.
TET also decreased N1P2 amplitudes and produced gender-
specific alterations in both P1,N1, and N2 latencies and N2P3
amplitudes. TET produced alterations in duration of the AD
recorded from cortex during kindling, but did not produce
significant alterations in any of the other variables tested. The
results support previous studies, since they show that the adult
VEP is sensitive to perinatal toxicant exposure.
INTRODUCTION
In adults, triethyltin (TET) produces degeneration of white matter, owing to
edema and vacuolation of myelin (Aleu etai, 1963). In addition, TET produces
a histotoxic hypoxia by inhibiting mitochondrial respiration (Aldridge and
Rose, 1969). In adults, it is difficult to separate the hypoxic and myelinopathic
consequences of exposure to TET. However, if exposure occurs at a develop-
mental stage prior to the onset of myelination, then consequences of the
exposure may be attributed more readily to the hypoxic effects of the sub-
stance, although such exposures nevertheless retard development of full myeli-
nation (Wender eta/., 1974).
Please send requests (or reprints to Or. Robert S. Oyer
'This paper hat been reviewed by the Health Effects Research Laboratory, U.S. Environmental Prelection
Agency, and approved for publication. Mention of trade name* or commercial products does not constitute
endorsement or recommendation for use.
Neurotoxicotofy 2:60*423
Copyright® 1M1
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610
DYER FT AL
Considerable evidence suggests that the developing organism is sensitive
to neurotoxic insult (e.g., Dyer et a/., 1978; Lochry and Riley, 1980; Fechter
and Annau, 1977). In the present experiment, rats were exposed to TET on
postnatal day 5 before the beginning of myelination (Wender et a/., 1974). This
paradigm which has been successfully explored by Reiter et a/. (1980) exposes
the rat while the brain is rapidly developing, yet bypasses many of the problems
attendant to prenatal exposures.
Electrophysiological techniques allow assessment of the integrity of differ-
ent functional units within the CNS. The visual evoked potential (VEP) has
frequently been used as an electrophysiological probe of the functional in-
tegrity of the visual system (e.g., Dyer, 1980). Because the optic nerve was
among the structures damaged following exposure of adults to TET (Scheinberg
et a/., 1966), and because the VEP is under investigation in our laboratory as a
general screen for potential neurotoxicants, animals exposed to TET on post-
natal day 5 were subjected to a VEP test as adults.
Various seizure models have also been proposed as potentially useful in
assessing neurotoxicity (Dyer et a/., 1979b; Fox et a/., 1979). To explore the
value of seizure models in assessment of neurotoxicity induced by perinatal
exposure to TET, one electrical (hippocampal afterdischarge) and two pharma-
cological (picrotoxin and pentylenetetrazol) models \vere used in this study.
The main goals of the project were to determine (1) which of these neurophysi-
ological tests provide the most sensitive index of exposure to TET (2) which
measures making up each test are most useful in assessing the consequences of
die exposure, and (3) some of the functional implications of the exposure.
MATERIALS AND METHODS
Animals
Pregnant CD rats (Charles River, Wilmington, MA) were obtained 2 days
after mating and housed singly in cages measuring 45 x 24 x 20 cm. On the day
of parturition (day 0), pups from 10 litters were pooled and randomly reas-
signed to form litters of 8 pups (4 males, 4 females). TET bromide (ALFA
Products, Danver, MA) was dissolved in sterile saline just prior to treatment;
concentration was adjusted so that each pup received an injection volume of
10 /xl/gm body weight On day 5, pups were injected ip with either 0,3,6, or 9
mg/kg TET. A within-litters design was used for dosing: one female and one
male from each litter received each dose, therefore, each litter contained ail
treatments in both sexes. At 21 days of age, animals were weaned and lit-
termates were housed by sex in groups of 3 or 4 in cages of the same size. The
animal room was mantained on a 12 hr light-dark cycle beginning at 0600 hr,
and food (Purina Lab Chow) and water were available ad libitum throughout
the experiment At 60 days of age, electrodes were surgically implanted and
animals were placed into individual cages. Mortality data obtained from these
animals will be presented elsewhere (Reiter et a/., 1981).
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NEONATAL TRIfTHYlTIN EXPOSURE IN RATS
611
Surgery
Animals were anesthetized with 3.5 ml/kg Chloropent (Fort Dodge Labor-
atories, Inc.). Stainless steel screws (00-90x1/16") were threaded into the skull
5 mm anterior to bregma and 2 mm lateral for ground (left side) and reference
(right side) electrodes. The visual cortex electrode was a screw placed 6 mm
posterior to bregma and 3 mm lateral to the midline. Two sets of bipolar
electrodes were implanted into the hippocampus. Both sets were constructed
of twisted stainless steel teflon-coated .005" wire. Stereotaxic placements were
made with the incisor bar 5 mm above the interaural line. The first set was
aimed at the dentate gyrus and was placed 2.5 mm posterior to bregma, 2.5
mm lateral to the midline, and 3.0 mm below the cortical surface. The second
set of hippocampal electrodes, aimed at the CA1 cell field, was placed im-
mediately anterior and about 0.75 mm dorsal to the dentate gyrus set All
electrodes were connected to an Amphenol receptacle which was subse-
quently cemented to the skull with dental acrylic. Following surgery, each
animal received 100,000 units Penicillin C (im).
Testing Sequence
Following surgery, at least 10 days elapsed before testing was begun. The
following sequence of tests was performed: on the first test day VEPs were
recorded followed immediately by afterdischarge (AO) tests; animals were
then given 17 consecutive days of kindling; 3-6 days later, pentylenetetrazol^
induced seizures were evaluated; and finally, 6-9 days later picrotoxin-in-
duced seizures were studied. At the end of these studies, all animals were
perfused with saline followed by formalin. Brains were removed, sectioned in
the sagittal plane, stained with cresyi violet, and examined for histological
verification of electrode placements.
Visual Evoked Potentials
To ensure pupillary dilation (and thereby eliminate variation in VEP
parameters due to variations in pupil size), a drop of 0.1 % (w/v) atropine sulfate
was placed upon each eye 15 min prior to testing. At the time of testing, each
animal was connected via a recording cable to the amplifier (high and low
frequency cut ofife for the amplifier were 10 khz and 0.1 hz, respectively) and
placed in the recording chamber. Details of the recording chamber have been
provided elsewhere (Dyer and Annau, 1977). Briefly, it was a rectangular box
with mirrors on three sides and the lamp face of a Crass PS-2 photostimulator
mounted against the fourth. Each animal was given a 5 min habituation period
of 0.5 hz flashes before data collection was begun. Following this, a series of 4
averages (64 flashes each) was obtained at each of four flash intensities,
presented in descending order. These intensities were estimated by an EG&G
photometer/integrator to provide peak power of 4.53 x 10', 8.46 x 10*, 4.86 x
10*, and 2.1 x 10* Lux, respectively, and corresponded to settings 16,4,2, and
1 on the photostimulator. Latencies and amplitudes for the major peaks of the
VEP were determined by a Nicolet signal averager. Latencies were recorded to
-------
612
DYIR ET AL
the nearest 0.4 msec, the sampling rate of the averager was 0.4 msec/pt, for
1024 pts. Each averaged evoked potential was also plotted by an X-Y recorder.
Hippocampai Afterdischarges
Three AOs were produced in each animal during each test session. The
first afterdischarge (AD 1) was used to determine the stimulus threshold for AO
production. Each animal was stimulated once/min through the bipolar dentate
gyrus electrodes with an ascending series of stimulus intensities (20,30,40,50,
60, 70, 80, 90, TOO, 125, 150, 175, and 200 uA) until an AD occurred. Each
stimulus period consisted of a 2 sec train of 50 hz pulses. Each pulse was a
biphasic square wave with 0.2 msec/phase. Fifteen min after the end of AD1,
each animal was given a supramaximal stimulus (4 x threshold) to produce
AD2. The purpose of AD2 was to ensure that animals had relatively consistent
types of ADs, so that their properties could be scored (Dyer et a/., 1979b). Ten
min after the end of AD2, stimulation was begun to elicit AD3. The purpose of
AD3 was to determine the time course of postictal hypoexcitability (Swartzwel-
der et a/., 1980). Stimuli occurred once every 2 min at twice threshold until
AD3 was elicited.
For AD 1, the threshold, type and duration (in dentate gyrus) were scored.
Four tyjaes of hippocampai ADs have been described (Dyer et a/., 1979b): type
1 consists of an AD followed by a depression which is interrupted by a brief
rebound AD; type 1a is different from type 1 in that no rebound AD occurs;
type 2 consists of an AD followed by no depression, but with irregular spikes
occurring during the postictal period; type 2a differs from type 2 in that no
postictal spikes occur.
For AD2, the following variables were scored: type; duration (in cortex,
dentate gyrus and CA1); number of wet dog shakes (WDS) (Dyer et al., 1979b);
EEC spike frequency (in three separate 3-sec blocks: beginning, middle, and
end of AD); and postictal depression (P1D) magnitude. PID magnitude was
estimated by connecting the amplified EEC to an integrator. The sensitivity of
the integrator was adjusted so that during a period of normal EEC (i.e., before
stimulation), it reached its maximum and reset about 10 times/min. The
number of resets occurring for 5 consecutive min following the AD was then
determined. In order to adjust for individual differences in prestimulation EEC
resets, each min of post AD activity resets was divided by the prestimulation
value.
The variables recorded for AD3 were: number of stimuli until AD3 was
elicited (a minimum duration of 5 sec was required for a given AD to be
counted); AD type; and AD duration.
Kindling
The kindling experiment required daily stimulation for 17 consecutive
days at a stimulus intensity of 4 times AD threshold. During each test, ADs were
recorded from dentate gyrus, CA1, and cortex and the rafs behavior was
observed. Behavioral seizures were rated on a 5 point scale (Racine, 1972): (1)
facial movements; (2) head nodding; (3) forelimb clonus; (4) rearing; and (5)
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NEONATAL TRIETHYITIN EXPOSURE i
613
rearing and falling. The dependent variables were seizure score and AO
duration. In order to reduce the number of dependent variables, only the scores
for days 1,2,5,8,11,14, and 17 were used.
Pentylenetetrazol
During pentylenetetrazol (metrazol) testing, the EEC was recorded from
the cortex. Pentylenetetrazol was administered sc at 70 mg/kg and the follow-
ing dependent variables were scored: latency to first twitch; latency to clonus,
latency to end of first seizure episode; latency to first burst of EEC spikes;
number of EEC bursts in the 60 sec after the first burst; and number of EEC
bursts 60 sec prior to the behavioral seizure.
Picrotoxin
Picrotoxin was given in a dosage of 2 mg/kg ip and the following measure-
ments were made; latency to first twitch, latency to clonus, latency to end of the
first episode, seizure score (using the 5 point rating scale used in the kindling
experiment); and time from clonus to the end of the first episode.
Number of Subjects/Experiment
All of the experiments reported here were performed on the same popula-
tion of animals. Although all animals were implanted with the same electrode
arrays, not ail of the recordings were equally satisfactory (i.e., some were
electrically noisy). Thus, some animals with satisfactory visual cortex record-
ings did not have satisfactory hippocampa! recordings, etc, In addition, as the
experiment proceeded, some animals either pulled their head plugs or died
during a seizure. These animals obviously were excluded from the rest of the
studies, but their data were included for the studies they had completed. Table
1 shows the distribution of subjects by dosage and gender for the different
studies reported here.
TABU 1. Distribution of Subjects by Doug*. Gander, and Experiment
TET
Visual
Dosage
Total
Evoked
After*
(mg/kg) Gender
Implanted
Potential
discharge
Kindling
Metrazol
Picrotoxin
0
malt
8
7
7
7
7
8
female
8
6
6
6
3
3
3
male
8
8
7
7
6
S
female
9
9
7
7
S
5
6
male
S
4
S
5
S
3
female
7
&
7
7
6
6
9
male
3
3
2
2
1
1
female
S
S
S
S
4
4
-------
614
DYKETAL.
Statistical Analysis
The visual evoked potential data were analyzed using multivariate statisti-
cal techniques (MANOVA). The six latencies (P1, NT, P2, N2, P3, and N3) and
five peak-to-peak amplitudes (P1N1, N1P2, P2N2, N2P3, and P3N3) obtained
under each of the 4 stimulus intensities were the dependent variables. Separate
MANOVAs were performed using the general linear models procedure (GIM)
of SA579 (Helwig and Council, 1979) to determine overall effects of treatment,
gender, intensity of flash, gender x treatment interactions, intensity x dosage
interactions, and gender x intensity x dosage interactions. A Bonferroni correc-
tion was used to adjust the required significance level for each MANOVA to
compensate for the fact that 6 MANOVAs were done (.05/6 - .0083). If overal I
MANOVAs were significant, univariate ANOVAs were performed, which, if
significant, were followed by Duncan's Multiple Range Test. This same anal-
ysis strategy was adopted for all of the other studies. To ensure an overall a level
of .05, the Bonferroni correction was also applied to ANOVAs. Since 11
dependent variables were measured in the VEP study (6 latencies, 5 amp-
litudes) any given univariate ANOVA was required to have a ps.05/11 (.0045)
in order to be considered statistically significant.
RESULTS
VEP
Of the different tests performed upon the TET-treated animals, the one
which clearly differentiated among the different exposure groups was the VEP.
The results of the MANOVA and ANOVA (when appropriate) are indicated in
Table 2.
TABLE 2. Statistical Summary for Day S TCT Exposure Visual Evoked Potential Study
Source
Latency ANOVAs
Amplitude ANOVAs
MANOVA' PI Nt P2 N2 P3 N3 P1N1 N1P2 P2N2 N2P3 P3N3
Dosage
Gender
Intensity
Oosage
x
Gender
Oosag*
x
Intensity
Dosage
x
Gender
x
Intensity
p<.0001
p<.0001
p<.0001
pc.0001
p<1.000
P< 1.000
.0651 .1800 .0001'.0001'.0001
.0016* .0112 .70 .35 .205
.0001'.0001' .99 .96 .117
'.0001*.209 .003S" .157
.0013* .0001' .0001' .0065
.501 .0001'.0001'J 12
.00011.0609
.0001*.0001'
.906 .0079
.0043'.00091.063 .0001'.54 .227 .0501 .0163 .0839 .001V .0391
1 Using exact multivariate F (see Rao. 1965).
'Considered significant after Bonferroni correction (critical value * .0045).
-------
NEONATAL TRIETHYLTIN EXPOSURE IN RATS
615
Treatment In general, TET increased latencies in a dosage-dependent
fashion which was most pronounced in the later peaks (N2, P3, N3). TET
decreased N1P2 amplitude, but increased N2P3 amplitude. TheMANOVA for
dosage was significant [F(33,589) = 8.70, p < .0001], ANOVA indicated that
the dependent variables which were significantly influenced by TET (after the
Bonferroni correction) were: P2; N2; P3; and N3 latencies; and N1P2 and
N2P3 amplitudes [all F's(3,210) > 4.70, ail p's < .0035]. P2 and N2 (see Figs. 1
and 3) latencies increased at 3 and 6 mg/kg. Although latencies of these two
peaks were also above controls at 9 mg/kg, they were below those at 6 mg/kg.
Duncan's Multiple'Range Test (a * .05), used to make comparisons between
dosages, indicated that for P2 latency the 6 mg/kg group was significantly
different from all other groups. No other dosages significantly altered P2
latency. The presence of an increased P2 latency in the absence of alterations
in P1 and N1 latencies implies a broadening of the P1N1P2 wave. To test this
hypothesis, an ANOVA was performed upon the P2-P1 latency differences.
The results supported the hypothesis [F(3,246) = 16.56, p < .0001], and the
Duncan's Multiple Range Test indicated that the results were accounted for by
the 6 mg/kg group which was significantly different from all others. For N2
latency, the 0 mg/kg group was significantly different from all other groups, and
the 6 mg/kg group was significantly different from the 3 and 9 mg/kg groups.
Increasing dosage of TET also increased peak latency of P3 and N3. In the case
of P3, the 0 mg/kg group was significantly different from all others, as was the 3
mg/kg group. The function appears to asymptote at 6 mg/kg. In the case of N3,
all groups were significantly different from each other. Figure 1 shows the
influence of dosage upon the latencies of P2, P3 and N3.
With respect to amplitudes, the Duncan Multiple Range Test indicated
that only the 9 mg/kg group had N1P2 amplitudes which significantly differed
from any other group, and these were different from all other groups. Similarly,
the N2P3 amplitudes were only significantly affected by the highest dosages of
TET. The N2P3 amplitudes of the 9 mg/kg group were nearly twice as large as
those of any other group (see Fig. 3). Figure 2 illustrates the influence of TET
dosage upon N1P2 amplitudes.
To further characterize the effects of the day 5 TET exposure upon adult
VEPs, a trend analysis was performed upon those variables which were signi-
ficantly affected by TET. This analysis is summarized in Table 3. Both the P3
and N3 latency functions are best described by a linear trend although the P3
latency function has a significant quadratic trend as well. The P2 latency had
significant linear, quadratic, and cubic trends.
Gender In general, females had higher amplitudes than males. Females
also had shorter P1 latencies and longer N3 latencies. The MANOVA for
gender was significant [FO1,200)» 7.34, p < .0001 ]. ANOVAs indicated that
the dependent variables which were significantly influenced by gender (after
the Bonferroni correction) were: PI and N3 latencies; and P1N1, N1P2, N2P3
and P3N3 amplitudes [all F's (1,210) a 10.27, ail p's < .0016]. Table 4
summarizes these results. A separate MANOVA indicated that there was a
significant treatment x gender interaction [F(33,589) « 2.76, p < .0001].
-------
616
OYER HAL.
215
210
200
190
N3LAT
P3LAT
P2LAT
180
170
i!
6
> 'M
1 120
<
mi
110
100
o-~
9
0
3
DOSAGE TST
FICURC 1. Influence of day 5 postnatal exposures to TET upon mean
-------
NEONATAL TRIETHYLT1N EXPOSURE IN RATS
617
UJ
a
D
H
Zj
a.
S
<
CN
a.
DOSAGE TET
FIGURE 2. Influence of day 5 postnatal exposures to TET upon mean (s SEM) VEP amplitude o/NiP2 peak
recorded from adult unanesthefized rats. N t P2 was the only amplitude for which the main effect of dosage
was significant and no dosage x gender interaction was significant The 9 mg/ltg group differed significantly
from ail others using Duncan's Multiple Range Test (p < .091.
TABU 3. Trend Analysis of VHP Components Influenced by TFT'
n
P3
N3
N1P2
Linear:
F-
to.3a«
77.57*
79.20*
S.SO
Quadratic:
F »
19.80*
12.02*
2.09
1.48
Cubic:
f-
18.46*
1.68
1.23
4.66
•All dfs - 1,246.
'p<.01.
»p<.001.
~p<.0001.
-------
618
DYER ET AL
FCMAUC w
OOSAOITTT
a s
OOSASC TCT
3 •
DOSAGI TIT
3 S
OOMCi TIT
FIGURE 3. Differential effects of day 5 postnatal exposures to TFT upon mean (x SEMI latency and amplitude
components of the VEP recorded from adult unanesthetized male and female rats. A: Pi latency, B: N1
latency; C: N2 latency, and O: N2P3 amplitude. * ¦ Oiffefent from same gender 0 mg/kg TET control using
Duncan's Multiple Range Test (p < .05).
TABLE 4. Mean Amplitudes and Latencies (xSEM) for Peaks with Significant Cender Differences'
Latencies (mSec)
Amplitudes
Cender
PI
N3
PINt
N1P2
N2P3
P3N3
Female
Male
25.1 ±0.3
26.9x0.5
186.6=2.4
176.3 £2.3
57.2x3.3
38.1x2.0
91.8x5.0
60.8x2.7
40.4X2.4
23.4X1.4
78.3x5.1
49.1x2.2
'All tabled gender comparisons differed significantly fall p's < .0016). Values based on all males and all
females.
Intensity In general, increasing the flash intensity produced decreased
latencies and increased amplitudes. The MANOVA for intensity was significant
[F{44,767 • 2.81), p < .0001 ]. Following Bonferroni correction, the following
dependent variables were indicated to have been significantly altered by the
intensity variable: PI, and N1 latencies; and P1N1 and N1P2 amplitudes.
However, there was not a significant treatment x intensity interaction
[F{ 132,165 3) * 0.53, p < 1.00] nor was there a significant treatment x gender x
intensity interaction [F{132,1653) » 0.55, p < 1.00].
-------
NCONATAl THICTHYLTJN EXPOSURE IN RATS
619
HippocainpaJ AOs
MANOVAs indicated that there was no significant effect of gender
[F(13,25) » 0.32, p < .99], treatment [F<39,81) - 0.75, p < 0.84], or gender x
treatment [F(39,81)31 0.81, p < 0.76],
Hippocampai Kindling
MANOVAs indicated that there were overall significant effects of treat-
ment, gender, stimulation number (i.e., day), and treatment x gender. These
MANOVAs and the ANOVAs which followed are summarized in Table 5.
TABLE 5. Statistical Summary for Day 5 TET Exposure Hippocampai Kindling Study
ANOVA'l
Seizure
Cortex AO
CA1 AO
DC AO
Source
MANOVA'
score
duration
duration
duration
Dosage
p<.QQ0l
0.0346
0.0003'
0.0591
0.9865
Gender
p<.0004
Q.0S23
0.7825
0.1469
0.1483
Day
p<.0285
—
—
—
—
Dosage
X
p<.0001
0.1289
0.001C
0.3865
0.0084'
Gender
Dosage
X
p<.9991
—
—
—
— x
Oay
1 Using Exact multivariate f (see Rao, i 965).
'Considered significant after Bonferroni correction (critical value ¦ .0 T 25).
Dosage of TET significantly altered only cortical AD duration. There were no
significant treatment x day interactions, but the treatment x gender interaction
was significant for cortex AD duration and dentate gyrus AD duration. Day of
testing did not significantly alter any seizure variables. These results are sum-
marized in Fig. 4.
Pentylenetetrazol
MANOVAs indicated that there was no overall effect of treatment
[F(27,444) « 0.93, p < .571, nor was there a significant treatment x gender
interaction (F(27,44) - 1.38, p > .168].
Picrotoxin
MANOVAs indicated that there was no overall effect of treatment [F(9,53)
* 1.05, p > .4141 "or was there a significant treatment x gender interaction
CF(9,53)- 1.38, p>.221].
Histology
Examination of electrode placements revealed that all depth electrodes
were appropriately placed.
-------
620
OYER ET AL
28 r-
— 26
a
UJ
v»
Z
o
H
<
S
3
a
a
<
x
UJ
»-
ce
8
24
22
20
18
16
• - MALE
v - FEMALE
0 3 6 9
DOSAGE TET
FIGURE 4. Summary of kindling experiment. Females exposed to the 3 mg/kg dosage of TET had signifi-
cantly longer cortical AOs than all other groups.
DISCUSSION
Only the VEP technique detected any clear influence of the perinatal
exposure to TET. Considering how insensitive the other techniques were, it was
surprising to find that the VEP technique demonstrated treatment-related differ-
ences on so many of the dependent variables. The pattern of alterations in the
VEP allows some inferences to be made about the nature of TET-induced
alterations in CNS function. The characteristics of PI and N1 are generally
taken to reflect input to the cortex via the retinogeniculate pathway, whereas
later peaks are recognized as reflecting nonspecific or recurrent activity (Creel
et a/., 1974). Therefore, increased latencies of peaks P2, P3, and N3 at 6 mg/kg
TET, in the absence of significant increases in latency of peaks PI and N1,
suggest that TET exposure did not affect the retina, optic tract, or lateral
geniculate body. The failure to find alterations in P1N1 amplitude at any
dosage and in N1P2 amplitude below the 9 mg/kg dosage provides support for
ruling out these noncortical structures as target loci for the lower dosage level
effects. It thus seems likely that in the present experiment, the conduction or
synaptic properties of either the visual cortex or nonspecific inputs to the visual
cortex have been altered. These results are very different from those obtained
when adult rats are given 7 daily exposures to TET. In that case, the optic nerve
is presumed to be involved, and the main effect observed is increased latency
of all VEP peaks (Howell and Oyer, 1980).
-------
NEONATAL TRJETHYITJN KPOSUtt IN HATS
621
Some of the effects of TET upon the VEP were gender-dependent includ-
ing the PI, NT, and N2 latencies and N2P3 amplitudes. In most cases, the
effects of TET on the two genders were similar at dosages of 0,3, and 6 mg/kg.
At 9 mg/kg, the males had significantly increased PI and N1 latencies. Thus,
males appear more sensitive to whatever process increases P1 andNI latencies
at high dosages of TET than do females.
In contrast, the gender x dosage interactions for N2 latency and N2P3
amplitude appear to result from uncharacteristically low and high readings,
respectively, for the females at the 9 mg/kg dosage. N2 latencies increased for
both males and females at 3 and 6 mg/kg TET, and although they remained
elevated at 9 mg/kg TET in males, the females' N2 latencies returned to control
levels at the 9 mg/kg dosage. Effects of TET on N2P3 amplitudes were some-
what more complex, but the major difference is the substantial increase
observed in the females given 9 mg/kg TET. The fact that most of the gender x
dosage interactions are easily accounted for by values at the 9 mg/kg dosage,
coupled with the small sample size in the 9 mg/kg dosage (3 male, 5 female),
could indicate that a few aberrant animals contributed to the findings. These
findings should be replicated before they are extensively discussed.
These results point out the very different consequences for adult electro-
physiology of hypoxia produced by prenatal low level carbon monoxide (CO)
exposure and hypoxia produced by early postnatal TET exposure (via uncoupl-
ing of oxidative phosphorylation). Continuous gestational exposure to 150
ppm CO (maternal HbCO * 15%) has been shown to increase the amplitude of
PI N1 and N1P2 amplitudes, but only in females (Dyer et a/., 1979a). No other
amplitudes were affected by the exposure nor were any latencies affected. The
qualitative differences between the CO experiment and the presentTET experi-
ment may result from either the time at which the exposures were made, the
cellular mechanism by which hypoxia was produced, or an effect of TET
unrelated to hypoxia. For the following reasons, it is unlikely that exposure
level (i.e., extent of hypoxia) accounts for the difference. The only variable
which was affected by both procedures, N1P2 amplitudes, was affected only at
high dosages of TET (9 mg/kg), and at this point the amplitudes were decreased,
whereas CO increased the amplitude of this peak. It cannot be argued that the 9
mg/kg TET exposure was below the equivalent level of CO exposure in severity
since none of the other variables affected by TET were also affected by CO
exposure. If the CO exposure was more severe than the TET exposure, then
every variable affected by TET should have been affected to an even greater
extent by exposure to CO.
These data suggest that visual function is impaired by the day 5 exposure
to TET. The nature of this impairment is not directly discernible from these data,
but the pattern of increased latencies in the later peaks is suggestive of slowed
processing of visual information at the cortical level.
The failure of any of the seizure tests to detect TET-induced alterations in
brain function was surprising. At the time of treatment (day 5 postnatal), the
cells of the hippocampus are still undergoing rapid growth (Bayer, 1980).
Consequently, if toxicity depends solely upon rapid growth occurring at the
time of treatment, the hippocampal AD and/or kindling tests should have
-------
622
DYfRETAL.
produced some separation of groups. If the alterations occurred generally in
cortical regions, either of the pharmacological models of seizures should have
detected toxicity since these are presumed to generate seizures by acting
primarily upon cortical areas (Stone, 1972).
The findings thus indicate that although TET is supposed to be a non-
specific metabolic inhibitor (Aldridge and Rose, 1969), there is a regional
specificity of effect produced by day 5 exposure. This regional specificity
cannot be explained entirely by knowing which cells are actively undergoing
development at the time of treatment since visual cortex cells are also rapidly
differentiating on postnatal day 5 (Juraska and Fifkova, 1979). The dimensions
of the visual system damage remain to be explored with behavioral techniques.
Finally, these results demonstrate that (1) the evoked potential technique
provides the most sensitive neurophysiological index of TET exposure yet
tested: (2) altered latencies are most useful in assessing the consequences of
day 5 TET exposure; and (3) visual function is permanently impaired following
day 5 TET exposure. The extent to which brain regions other than those
examined here are damaged by day 5 TET exposure remains to be determined.
ACKNOWLEDGMENT
The authors wish to thank Mark Bercegeay and Leon Lamm for assistance
with surgery and data collection, William Wonderlin for statistical assistance,
and the U.S.E.P.A. Health Effects Research Laboratory Word ProcessingCenter
for manuscript preparation.
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intoxication.) Neuropath Exper Neurol 1963; 22:403-13
Bayer SA. Development of the hippocampal region of the rat II. Morphogenesis during embryonic and early
postnatal life.) Comp Neurol 1980; 190:115-34
Creel D, Dustman Rt, Beck EC Intensity of flash illumination and the visual evoked potential of rats, guinea
pigs and cats. Vis Res 1974; 14:725-9
Dyer RS. Effects of prenatal and postnatal exposure to carbon monoxide on visually evoked responses in
rats. In: Neurotoxicity of the Visual System. Merigan WH, Weiss B. eds. New York: Raven Press. 1960.
pp. 17-33
Dyer RS, Aiwimi Z. Flash evoked potentials from rat superior colliculus. Pharmacol Biochem Behav 1977;
6:453-9
Dyer RS, Ecclei CU, Aimau Z. Evoked potential alterations following prenatal methyl mercury exposure.
Pharmacol Biochem Behav 1978; 8:137-41
Dyer RS, Ecdes CU, SwaiUwekJer HS, Fechter ID, Aimaai 2. Prenatal carbon monoxide and adult evoked
potentials in rats. J Environ Sci Health 1979a; C13:107-20
Dyer RS, SwartiweMer HS, Eccle* CU, Annan Z. Hippocampal afterdischarges and their post-ictal sequelae
in rats: A potential tool for assessment of CNS neurotoxicity. Neurabehav Toxicol 1979b; 1:5-19
Fechter LO, Arwiau Z Toxicity of mild prenatal carbon monoxide exposure. Science 1977; 197 .680-2
Fox DA, Overmami SR, Wool ley DC. Neurobehavioral ontogeny of neonatally lead-exposed rats. II.
Maximal eiectroshock seizures in developing and adult rats. Neurotoxkol 1979; 1:149-70
Hefcvig |T, Council KA. eds. SAS Users Guide, 1979 Edition. Raleigh: SAS Institute, Inc. 1979
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NEONATAL TRIETHYLTIN EXPOSURE IN RATS
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Howell WE, Oyer RS. Acuta triethyltin alters visual evoked potentials and hippocampai afterdischarges.
Neuros Abst 1980; 6:727
Jurasica JM, Fifkova E. A Co'gi study of the early postnatal development of the visual cortex of the hooded rat
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Loduy EA, Riley EP. Retention of passive avoidance and T-maze escape in rats exposed to alcohol
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Racine R. Modification of seizure activity by electrical stimulation: II. Motor seizure. Electroenceph Clin
Neurophysiol 1972; 32:281-94
Rao CI Linear Statistical Inference and Its Applications. New York: John Wiley and Sons. Inc. 1965
Reiter LW, Heavner C, KM K, Ruppert P. Developmental and behavioral effects of postnatal exposure to
triethyltin in rats. Neurobehav Toxicol 1981; in press
Reiter LW, Heavner C, Ruppert P, KSdd K. Short-term vs. long-term neurotoxicity: The comparative
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25-26,1980
Sdwinberg LC, Taylor |M, Herxog I, Mandeil S. Optic and peripheral nerve response to triethyltin intoxica-
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Stone WE. Systemic chemical convuisants and metabolic derangements. In: Experimental Models of
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Swartzweider HS, Wegener ST, Johnson CT, Dyer RS. Depressed excitability and integrated EECs following
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S£UROTOXICOLOCY MS N-14 J
Submitted March JO. 1981
Accepted May 5. 1981
-------
Neurobehaviural Toxicology and Teratology, Vol. 4, pp. 191-193, 1982. Printed in the U.S.A.
Visual System Dysfunction Following Acute
Trimethyltin Exposure in Rats1
ROBERT S. DYER,2 WILLIAM E. HOWELL AND WILLIAM F. WONDERLIN3
Neurophysiology Branch, Neurotoxicology Division, Health Effects Research Laboratory
U, S. Environmental Protection Agency, Research Triangle Park, NC 27711
DYER, R. S., W. E. HOWELL AND W. F. WONDERLIN. Visual system dysfunction following acute trimethyltin
exposure in rats. NEUROBEHAV. TOXICOL. TERATOL. 4(2) 191—195, 1982.—Trimethyltin (TMT) has been shown to
produce damage in the limbic system and several other brain areas. To date, damage to sensory systems has not been
reported. The present study investigated the integrity of the visual system following acute exposure to TMT. Rats were
chronically implanted with electrodes for recording the evoked response from either the visual cortex or optic tract
following photic stimulation. Following recovery, the animals were exposed to either 0 (saline), 4, 5, 6, or 7 mg/kg
trimethyltin chloride (TMT). Evoked potentials were averaged and peak-to-peak amplitudes and latencies were deter-
mined. The results indicated that exposure to TMT produced alterations in the visual evoked response. The pattern of
changes suggested two effects, an alteration in retinal processing and an alteration in arousal. The manifestation of these
changes was an increase in early peak latencies recorded from the visual cortex and the optic tract, a decreased amplitude
recorded from the visual cortex and optic tract early peaks (all suggestive of retinal changes) and a decreased P3N3
amplitude and N3 latency recorded from the visual cortex (suggestive of increased arousal). The results demonstrate that
TMT does produce alterations in sensory systems as well as in the limbic system.
Visual evoked response Optic tract Trimethyltin Visual cortex Visual toxicity
TRIMETHYLTIN (TMT) has been reported to produce a
pattern of neuronal degeneration which is largely restricted
to the limbic system [2]. Intraventricular application of
kainic acid produces a pattern of damage similar to that
produced by systemic TMT administration [15], Thus, local
administration of kainic acid mimics systemic TMT adminis-
tration. In addition to the hippocampal pathology produced
by kainic acid following intraventricular administration,
retinal damage has been described following injection of
kainic acid into the vitreous humour [12,13]. Therefore, if the
parallel between local kainic acid and systemic TMT is main-
tained, then systemically administered TMT may produce
retinal damage which has not yet been detected.
Neuropathological findings do not lead one to suspect
that the visual system would be affected by TMT. However,
TMT has been shown to produce both aggressive behavior
[2,9] and hyperactivity [16]. Impaired vision is one of many
factors which may account for these two types of behavior
[10,11]. One method by which visual system dysfunction
may be ruled out as contributing to these abnormal behaviors
is to demonstrate the functional integrity of the system by
using the visual evoked response (VER).
A variety of studies have suggested that the VER tech-
nique may be used as a means to detect general CNS dys-
function as well as specific damage to the visual system (e.g..
[4]). Evaluation of the VER in TMT-exposed animals would
allow a partial test of that hypothesis. Finally, the late peak
of the VER (i.e., P3N3) has been .assumed to correlate in-
versely with arousal [1,4]. If this is true, it would be expected
that the hyperactive and hyperreactive TMT-treated rats
would have low amplitude P3N3 amplitudes.
Thus, there are a variety of reasons why TMT might be
expected to produce alterations in the VER. The present
study was designed to determine whether any such altera-
tions occurred.
METHOD
Adult male Long-Evans hooded rats (n= 108) were ob-
tained from Charles River Breeding Company and housed
singly in plastic cages on woodchip bedding. Access to food
and water was ad lib throughout the experiment. At 60 days
of age, under 3.7 ml/kg Chloropent (Fort Dodge Laborato-
ries, Inc.) anesthesia, the rats were surgically implanted with
electrodes for recording from the optic tract and/or the visual
cortex. The electrodes for cortical recordings were stainless
steel screws (00-90x1/16") threaded into the skull 5 mm
anterior to bregma and 2 mm lateral for ground (left side) and
reference (right side). The visual cortex electrode was a
screw placed 6 mm posterior to bregma and 3 mm lateral to
¦This manuscript has been reviewed by the Health Effects Research Laboratory, U. S. Environmental Protection Agency, and approved for
publication. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.
'Address reprint requests to Dr. Robert S. Dyer, Neurophysiology Branch, MD-74B, Neurotoxicology Division, HERL, USEPA, Research
Triangle Park, NC 27711.
'Present address: Department of Environmental Health Sciences, The Johns Hopkins University, 615 North Wolfe Street, Baltimore, MD
21205.
191
-------
192
DYER, HOWELL AND WONDERLIN
SCHEMATIC VER PROM VISUAL CORTEX
150 /-
-100 r
-250
30 120
PEAK LATENCY (mMcl
SCHEMATIC VER FROM OPTIC TRACT
200
S
<
SO 100 150 200 250
PEAK LATENCY (imkI
FIG. 1. Schematic evoked potentials indicating the peaks measured
in this study. A: Schematic VER recorded from the visual cortex of a
rat. B. Schematic VER recorded from the optic tract of a rat. Ampli-
tudes are in arbitrary units. Dashed lines represent the changes in-
duced by TMT.
the midline. The optic tract electrode consisted of a twisted
pair of 0.25 mm nichrome wires insulated except at the tip.
Coordinates for these depth placements were (with the in-
cisors 5.0 mm above the interaural line) O.S mm posterior to
bregma, 3.0 mm lateral and 8.1-8.8 mm below the cortical
surface. If the electrode struck the base of the skull during
surgery, it was raised 0.15 mm. Following placement, all
electrodes were connected to an Amphenol receptacle which
was subsequently cemented to the skull with dental acrylic.
Following surgery, each animal received 100,000 units
Penicillin G (IM).
At the beginning of each test session, a drop of \% at-
ropine was placed in each animal's eyes 30 minutes prior to
VER testing in order to dilate the pupils. At the time of
testing, the animal's head plugs were connected to the re-
cording amplifier (low and high filters set at 0.8 Hz and 1
kHz) by a cable and placed in a small chamber with mirrors
on three walls and a Grass strobe lamp on the fourth wall [5].
During each test session, the evoked response to 64 con-
secutive flashes of the 10 jtsec strobe was averaged. Flashes
were presented at 0.5 Hz, and the flash intensity was esti-
mated by an EG&G photometer to have a peak power of
4.53x 107 lux above background in the center of the record-
ing chamber (intensity 16 on the Grass strobe).
The epoch duration was 409.6 msec, and samples were
taken every 0.4 msec. The evoked potential averages were
obtained using a Nicolet computer from which X-Y plots
were obtained along with digital readouts of peak amplitudes
and latencies. Latencies of VER peaks PI, Nl, P2, N2. P3.
and N3 were obtained. Peak-to-peak amplitudes of PIN1,
N1P2, P2N2, N2P3, and P3N3 were also obtained.
Peak designations from optic tract recordings have been
less standardized than VER's from the cortex. In this study,
the first three positive and negative peaks were analyzed,
using the same procedures described above. Figure 1 illus-
trates schematic averaged evoked potentials obtained from
the visual cortex and optic tract.
In the first study, animals were treated with either 0
(saline vehicle) (n= 10), 5(n=10), 6(n=9), or 7 (n = 13) mg/kg
trimethyltin chloride (ICN Pharmaceuticals. Plainview. New
York) by gavage in a volume of 1 ml/kg, and recordings were
obtained from their visual cortex, as described above, on
days 1,4, 8, and 16 following treatment. In the second study,
animals were treated with either 0 (saline vehicle, n= 16), 4
(n=l6), 5 (n=16), or 6 (n=16) mg/kg TMT by gavage in a
volume of 1 ml/kg, and recordings were obtained from both
the visual cortex and the optic tract on day 0 (immediately
prior to treatment), day I, and day 4. Thus, the main differ-
ences between the first and second studies were that in the
second study, a low dosage (4 mg/kg) was substituted for the
high dosage (7 mg/kg); days 8 and 16 were eliminated; day 0
was included; and recordings were obtained from both cor-
tex and optic tract.
Data from these studies were analyzed by multivariate
analysis of variance (MANOVA) techniques. For each ex-
periment, MANOVAs were performed for amplitude and la-
tency measurements and, in the second experiment, separate
MANOVAs were performed upon the optic tract and cortical
recordings. MANOVAs which were significant after Bonfer-
roni corrections were followed by ANOVAs which in turn
were followed by Duncan's multiple range test for individual
comparisons. The overall alpha for the experiment was thus
maintained at 0.05. In addition to the overall analysis per-
formed upon the data obtained from the visual cortex in the
second experiment, planned comparisons (using Duncan's
multiple range test) were made between the different dosage
groups for the variables which were statistically significant in
the first study.
Following these studies, all animals with optic tract elec-
trodes were perfused with potassium ferrocyanide in For-
malin, a 1.0 mA 5 sec pulse was passed through each elec-
trode tip to aid in electrode tip localization, and the elec-
trodes and brains were removed. Cresyl violet-stained sec-
tions were used to confirm electrode localization.
RESULTS
TMT produced increases in early peak latencies of the
evoked potentials recorded from both the optic tract and the
visual cortex. In addition, early peak amplitudes were de-
pressed in both locations, and the P3N3 amplitude was de-
pressed in the visual cortex.
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TRIMETHYLTIN AND VISUAL DYSFUNCTION
193
VISUAL CORTEX PEAK LATENCIES
VISUAL CORTEX
PEAK-TO-PEAK AMPLITUDES luVI
0 5 8 7
DOSAGE TMT m,/V,,
0 5 6
DOSAGE TMT. mg/kg
5 S
DOSAGE TMT, mg/kg
0 5 6
DOSAGE TMT.mg/kg
FIG. 2. EfFects of TMT on peak latencies recorded from the visual
cortex. A: PI latency; B: N1 latency; C: P2 latency; and D: N3
latency. In this and other figures, the vertical lines represent +SEM.
The main effects of TMT on VER latencies in the first
study are illustrated in Fig. 2. TMT increased PI and N3
latencies, but shortened P2 latencies, all F's (3.152)>3.74,
p's<0.01. There were no other significant TMT-induced
changes in VER latency. There were also no significant ef-
fects of day of testing, nor were there any significant treat-
ment x day interactions. Thus, the statistics indicate that the
effects of TMT upon PI, N3, and P2 latencies were present
at the first post-treatment test and remained for the entire
16-day period of the experiment.
The main effects of TMT on VER amplitudes in the first
study are illustrated in Fig. 3. TMT significantly decreased
the amplitude of the PIN1, N2P3, and P3N3 peaks, and sig-
nificantly increased the amplitude of the P2N2 peak, all F's
(3,152)>4.59, p's<0,0044. Differences between the 0 (control)
and low dose (5 mg/kg) groups were significant for the PIN1,
N2P3, and P3N3 amplitudes and for the PI latencies. The
dose-dependent effects described above were supported by
an overall significant MANOVA, F(33,149)=4.73,p<0.0001.
In the second study, the results obtained from recordings
of the optic tract indicated that alterations in amplitude and
latency of response could be obtained at this early level of
visual processing. All amplitudes were decreased in TMT-
treated rats, F's (3,179)>4.87, p's<0.003, and latencies of the
Nl, N2, and P2 peaks were all significantly increased, F's
140 -
> 120
8
= 100
I 80
60
¦ 0 mg/kg TMT ~ 6 mg/kg TMT
Q 5 mg/kg TMT ~ 7 mg/kg TMT
1
Jiff,
nn
Li
LL
P1N1 N1P2 P2N2 M2P3 P3N3
FIG. 3. Effects of TMT on peak-to-peak amplitudes recorded from
the visual cortex.
OPTIC TRACT PEAK LATENCIES
17 -
\
1t -
>
X
15 -
<
14 -
z
13
JL
A
X
1
56
2.
£l
4 S
TMT, mg/kf
0 4 5
TMT. mq/kq
30
29
£ 28
6 27
> "
2 25
ui
5 24
-» 23
Z 22
21
20
JL
120
IIS
| 110
>106
Z 100
Ui
< 9S
a
rx 90
a.
85
- 80
_L
jEl
TMT mg/kg
TMT. mg/kg
FIG. 4. Effects of TMT on peak latencies recorded from the optic
tract. A: Nl latencies; B: PI latencies; C; N2 latencies; and D: P2
latencies.
(3,179)>7.68, p<0.0001. Duncan's multiple range test indi-
cated that differences between the 0 (control) and low dose
(4 mg/kg) groups were significant for the N1P1, P1N2. and
N2P2 amplitudes and for the NI, N2, and P2 latencies. These
results (Figs. 4 and 5) were supported by an overall signifi-
cant MANOVA for dose, F(27,500) = 3.64,p<0.0001. In con-
trast to the first study, the MANOVA for the cortical record-
ings was not statistically significant. The planned compari-
sons indicated that only the P2 latency differed significantly
among the different dosage groups. As with the first study,
P2 latencies were shorter in treated animals than controls,
-------
194
DYER, HOWELL AND WONDERLIN
OPTIC TRACT
PEAK-TO-PEAK AMPLITUDES (*V)
125
115
105
95
\ 85
UJ
§ 75
Z 65
I 55
46
35
25
15
I 0 mg/Vq TMT ~ 5 mq/kq TMT
£1 4 mq/kg TMT Q 6 mq/Vq TMT
FIG. 5. Effects of TMT on peak-to-peak amplitudes recorded from
the optic tract.
although the only significant difference was between the 0
and 6 mg/kg groups.
Analysis of the histological data indicated that all elec-
trodes were accurately placed in the optic tract.
DISCUSSION
The results indicate that TMT produces visual system
dysfunction. This dysfunction is characterized by a reduc-
tion in amplitude of the retinal input to the rest of the brain
and by a delayed onset (i.e., increased latencies) of the re-
sponse. While the changes observed were significant, the
presence of an easily recognizable VER indicates that the
rats were definitely not blind. The pattern of change in the
cortical VER was different from changes observed following
exposure to other toxicants and drugs. For purposes of
analysis, the effects may be divided into two parts: the effects
on early components and the effects upon the late (P3N3
amplitude, N3 latency) component.
Early VER Peak Alterations
Alterations in early peaks of the cortical VER are usually
assumed to reflect abnormal input to the cortex [3,7], The
present study supports such an interpretation, because simi-
lar alterations were obtained from the optic tract. Increased
cortical latencies may reflect either slowed conduction in the
optic tract and optic radiations or slowed synaptic activity in
the retina or lateral geniculate nucleus. If decreased conduc-
tion velocity has occurred, then the magnitude of the in-
creased latencies should be greater in the cortex than in the
optic tract, by virtue of the greater distance from the retina
to the visual cortex. Similarly, if the decrement has occurred
in either the lateral geniculate or the optic radiations, then
the increased latencies should be greater in the cortex than in
the optic tract. In the present study, the optic tract record-
ings were most sensitive to the disrupting effects of TMT
(i.e., changes were observed at a lower dosage level). Fur-
thermore, the magnitude of change (in msec) was at least as
great, and in some cases greater in the high dose optic tract
recordings than in the corresponding cortical recordings.
Thus, the alterations in the early peaks of the cortical VER
are probably due to changes in retinal function.
The decreased P2 latencies observed in the cortical VER
do not contradict the above interpretation. If the first wave
of the cortical VER (P1N1P2) is viewed as a Gaussian distri-
bution (over time) of inputs to the cortex, then a nonspecific
reduction (i.e., randomly distributed) of input would (I)
lower the amplitude of the wave; and (2) move the tails of the
distribution towards the mean. Thus, PIN I and N 1P2 ampli-
tudes should be reduced. PI latencies should be increased.
P2 latencies should be decreased, and Nl latencies should
stay about the same. Therefore, a general reduction in corti-
cal input may explain the changes observed in the first part
of the VER. It is not clear why the increased latencies ob-
served at the optic tract are not reflected at the cortical level.
Perhaps the variability of cortical responses masks the
changes.
A variety of other procedures, including acute exposures
to triethyltin [6|, both acute adult and prenatal exposure to
carbon monoxide [4], depletion of dopamine [7], and reduced
flash intensity [8] have been found to produce alterations in
the first wave of the cortical VER. None of these procedures
duplicate the pattern of changes observed in the first wave
following TMT exposure. Although the present study cannot
indicate by what mechanism the changes have occurred, it
must be assumed that the mechanism is different from the
mechanism by which the other alterations mentioned above
have occurred.
Late VER Peak Alterations
The TMT-induced alterations in the late VER peak may
be explained in terms of the presumed relationship between
this wave and arousal. Several studies have indicated that
the P3N3 wave is largest in a relaxed, wakeful subject, and
smallest in an aroused subject [1,7, 14], TMT-treated rats
have been described as hyperactive, hypenrreactive. and ag-
gressive [2, 9, 16], Therefore, the P3N3 amplitudes should
be smaller than controls in these rats and, in fact, the data
indicate that the amplitudes are smaller in treated rats. These
data support the hypothesis that P3N3 amplitudes are in-
versely correlated with arousal, and that TMT-treated rats
have high arousal states.
In summary, the present experiment has described alter-
ations in the visual system following acute exposure to TMT.
The alterations are best characterized as reduced retinal out-
put and increased cortical arousal, a pattern of changes not
observed following exposure to other environmental condi-
tions. The apparent "all-or-nothing" character of the
changes in PI latency and P1N1 amplitude suggests that ex-
posure to lower concentrations of TMT than those used here
may also produce changes in these measures.
ACKNOWLEDGEMENTS
The authors wish to thank Mark Bercegeay, Teresa Deshields.
and Mike Isley for excellent technical assistance during these exper-
iments, and Susan Garner for manuscript preparation.
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TRIMETHYLTIN AND VISUAL DYSFUNCTION
195
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The trimethyltin syndrome in rats. Neurobehav. Toxicol.
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in several strains of mice. Physiol. Behav. 15: 43&-441, 1975.
11. Fordyce, D. J. and J. F. Knutson. Influence of blinding and
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Changes of electroretinogram and neurochemical aspects of
GABAergic neurons of retina after intraocular injections of
kainic acid in rats. Brain Res. 211: 305-314, 198!.
13. Hampton, C. K., C. Garcia and D. A. Redburn. Localization of
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14. Joseph, R., N. M. Forrest, D. Fiducia, P. Como and J. Siegel.
Electrophysiological and behavioral correlates of arousal.
Physiol. Psychol. 9: 90-95, 1981.
15. Nadler, J. V., B. W. Perry, C. Gentry and C. W. Cotman.
Degeneration of hippocampal CA3 pyramidal cells induced by
intraventricular kainic acid. J. comp. Neurol. 192: 333-359.
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Neurobehavioral Toxicology and Teratology, Vol. 4, pp. 203*208, 1982. Printed in the U.S.A.
Increased Seizure Susceptibility Following
Trimethyltin Administration in Rats1
ROBERT S. DYER, WILLIAM F. WONDERLIN1 AND THOMAS J. WALSH
Neurophysiology Branch, Neurotoxicology Division, Health Effects Research Laboratory
U. S. Environmental Protection Agency, Research Triangle Park, NC 27711
DYER, R. S., W. F. WONDERLIN AND T. J. WALSH. Increased seizure susceptibility following trimethyltin adminis-
tration in rats. NEUROBEHAV. TOXICOL. TERATOL. 4(2) 203-208, 1982.—Acute treatment with trimethyltin (TMT)
produces a multitude of behavioral effects including spontaneous convulsions in some animals. The present study used
several different experimental seizure models to investigate seizure susceptibility in TMT-treated rats. Rats surgically
implanted with electrodes in the amygdala and treated with TMT kindled more rapidly than saline-treated controls.
Similarly, rats implanted with electrodes in the dorsal hippocampus kindled more rapidly than controls. Although TMT did
not alter the properties of hippocampal afterdischarges, the threshold for production of afterdischarges was increased in
both hippocampal and amygdidoid kindled rats, probably due to cell loss. TMT also increased the sensitivity of rats to
pentylenetetrazol, thus suggesting that the increased seizure susceptibility was not limited to the limbic system.
Trimethyltin Seizures Kindling Amygdala Hippocampus Afterdischarge Pentylenetetrazol
TRIMETHYLTIN (TMT) administration produces a variety
of neurobehavioral sequelae which include spontaneous sei-
zures as a prominent feature [2,11]. The mechanism underly-
ing this effect has not been elucidated; however, the
neuropathological alterations produced by TMT (i.e., nec-
rosis of pyramidal neurons in hippocampal cell field CA 3) do
resemble those observed in other experimental models of
epilepsy [3,16]. For example, systemic, intraventricular and
intrahippocampal injections of the excitotoxic glutamate
analogue, kainic acid, each produce a neurological disorder
consisting of spontaneous seizures and wet-dog shakes in
conjunction with degeneration of pyramidal cells in area CA3
[7, 15, 20, 22, 24], Further, it is interesting to note that a
similar pattern of neurodegeneration has been frequently ob-
served in human epileptic brains at autopsy [6]. While it is
uncertain whether hippocampal damage is a causative factor
in the production of epileptogenesis or the result of sustained
abnormal neuronal activity within certain neuronal pathways
[1, 13, 18, 25], it is clear that a relationship exists between
hippocampal dysfunction and seizure susceptibility. Fur-
thermore, since human epileptics and kainic acid-treated
animals are more vulnerable to the experimental production
of seizures [4,5], it is possible that TMT-treated rats would
exhibit an enhanced sensitivity to the electrical or chemical
induction of seizures. Based upon the incidence of spon-
taneous seizures and hippocampal degeneration in TMT-
treated animals and the report of behavioral convulsions fol-
lowing accidental human exposure to TMT [12], the present
experiments examined the susceptibility of TMT-treated rats
to seizures.
Due to the documented effects of TMT on the hippocam-
pus, we first utilized three seizure models which are particu-
larly useful for assessing functional changes in the limbic
system: hippocampal kindling, hippocampal afterdischarges,
and amygdaloid kindling. Secondly, we examined the sus-
ceptibility of TMT-treated rats to the generalized CNS con-
vulsant pentylenetetrazol.
The results of these experiments should provide a more
complete profile of TMT-induced neurotoxicity and also
assess the utility of using this compound as a tool for inves-
tigating the neural substrates of epilepsy.
METHOD
Adult male Long-Evans hooded rats obtained from
Charles River Breeding Company were used in the present
series of experiments. They were housed individually in
plastic cages with wood chip bedding.
Hippocampal Afterdischarge
In the first experiment, 76 rats (220-230 g) were implanted
with electrodes for stimulating and recording the hippocam-
pal afterdischarge. The surgical procedures have been previ-
ously described [10], Briefly, animals were anesthetized with
Chloropent (Fort Dodge), 0.35, ml/100 g body weight. Rats
were placed in the stereotaxic instrument with incisors 5.0
mm above the interaural line and twisted bipolar nichrome
wire (0.25 mm) electrodes were implanted at 2.5 mm
posterior to bregma, 2.5 mm lateral to the midline, and 3.0
mm below the brain surface. Tip separation was approx-
'This manuscript has been reviewed by the Health Effects Research Laboratory, U. S. Environmental Protection Agency, and approved for
publication. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.
'Present address: Department of Environmental Health Sciences, The Johns Hopkins University, 615 North Wolfe Street, Baltimore, MD.
203
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204
DYER, WONDERLIN AND WALSH
imately 0.5 mm in the vertical plane. Ground and anchor
screws (00-90x'/t«") were threaded into the skull, and all
electrodes were connected to an Amphenol receptacle. The
entire assembly was then covered with dental acrylic. Fol-
lowing surgery, the rats were administered 100,000 units
procaine penicillin G IM and returned to their home cages.
Animals were allowed at least 1 week to recover from the
surgery before any testing was begun. The recording situa-
tion proceeded as follows: The animals were connected to a
Grass model 7B polygraph with model 7P511J EEG
amplifiers with high and low frequency filters set at 10 kHz
and 1 Hz, respectively. A low noise Microdot cable was used
to minimize movement artifact. The hippocampal electrode
leads were connected to a relay which could be thrown either
to the recording amplifiers or to the stimulator. The
stimulator used was either a Nuclear Chicago Model 750
constant current stimulator or a Grass S88 stimulator with
two PSIU6 constant current converters. Stimuli were
biphasic constant current square waves of 0.4 msec duration
in a 2 sec train of 50Hz pulses. The output of the EEG
preamplifiers was interfaced with 1 7P10B polygraph integ-
rator. The integrator was adjusted for each individual animal
to ensure that on day 1 of the experiment, before any stimu-
lation, at least 10 and less than 100 integrator resets occurred
during 1 minute (resets occur when the integrator reaches it
maximum amplitude and resets to baseline).
The first test session consisted of determining afterdis-
charge (AD) thresholds by using an ascending series of
stimulus trains, beginning at 20 piA and incrementing by 10
fxA once each minute until an AD of at least 6 sec duration
occurred. The stimulus intensity which produced an AD was
defined as the threshold. Based upon earlier data [10],
animals with thresholds greater than 100 fiA were assumed
to have misplaced electrodes and were not included in the
study (n»12). The dosages of trimethyltin chloride (TMT)
(ICN, Plainview, NY) administered in this and the other
studies described below were calculated as the base and
were always administered by gavage in a volume of 1 ml/kg.
The remaining animals were divided into four groups coun-
terbalanced for threshold and weight. The groups, with their
thresholds (±SEM) and weights (±SEM) were: (1) 0 mg/kg
TMT (saline vehicle, 38.1 /aA ±5.7, 277 g ±7.9 g), n>=16; (2)
5 mg/kg TMT (37.5 jtA ±5.4, 278 g ±6.4), n= 16; (3) 6 mg/kg
TMT (37.5 fiA ±5.1, 284g ±5.2), n= 16; and (4)7 mg/kg(37.5
fiA ±5.1, 279 g ±6.4), n=16. Due to attrition (plug pulls,
recording problems, etc.), the final group sizes were smaller
(n=» 11,12,11, and 11 for groups 0,5,6, and 7 mg/kg, respec-
tively), and the average threshold values on day 1 were no
longer the same across groups (see Fig. 4A).
At least 1 week following the threshold determination, the
animals were treated with TMT. AD tests were performed I,
4, 8, and 16 days following treatment. Each AD test con-
sisted of three parts: (1) (ADl) the AD threshold was
redetermined. Redetermination runs began 40 /±A below the
previously determined threshold. (2) Fourteen minutes fol-
lowing ADl, the polygraph was engaged for recording pre-
stimulation hippocampal EEG integrator resets, a quantita-
tive measure of the maximal energy content of the EEG.
After 1 minute of pre-stimulation EEG was recorded, the
animal was stimulated at 4 times the most recently deter-
mined threshold. During the ensuing AD (AD2), wet-dog
shakes were counted by an observer and marked on the EEG
paper with a push-button marker. Five minutes of post-AD
EEG were recorded to assess the magnitude of the postictal
EEG depression [10]. (3) Five minutes after the end of the
5-minute post-AD period, stimulations were begun at 2 times
the threshold. These stimuli occurred once every 2 minutes
in order to assess the duration of postictal hippocampal
hypoexcitability. The number of stimuli required to elicit the
AD (AD3) was the measure of postictal hypoexcitability.
Other measures taken were duration of AD2, spike fre-
quency within AD2 (a 1-second sample from the middle of
the AD), and a ratio of prestimulation integrator resets to
post-stimulation integrator resets for AD2. These measures
and those described in the above paragraph were analyzed
by multivariate analysis of variance (MANOVA), followed
by ANOVA and Duncan's multiple range test when appro-
priate.
Amygdala Kindling
In the second (amygdaloid kindling) study, 54 rats were
implanted with electrodes for amygdala stimulation and re-
cording. Surgical procedures were as described above, but
the coordinates for electrode placement were 1.0 mm
posterior to bregma, 5.0 mm lateral to the midline, and 8.7
mm below the brain surface.
Animals were gentled by daily handling for at least 1 week
after a 1-week post-surgery recovery period, and AD
thresholds were determined as described above. Based upon
pilot data, animals with thresholds above 400 fxA were as-
sumed to have misplaced electrodes and were discarded.
The 45 remaining rats were divided into 3 groups. The groups
were randomly assigned to receive either 0 mg/kg (saline), 5
mg/kg, or 7 mg/kg TMT. Treatment occurred at least 3 days
after the AD determination. Twenty-four hours later, kin-
dling stimulation was begun. The kindling stimulus had the
same parameters as the AD eliciting stimulus described for
Experiment 1, except that it was applied at 2 times threshold,
once/day for 16 consecutive days. On the 17th day.
thresholds were redetermined.
EEG's were monitored immediately following stimulation
to (1) ensure that an AD occurred, and (2) allow subsequent
determination of AD duration. The behavioral seizures were
scored according to the criteria established by Pinel and
Rovner [21]: l=facial movements; 2=facial movements and
head nodding; 3=facial movements, head nodding and
foretimb clonus; 4=facial movements, head nodding,
forelimb clonus and rearing; 5=all of the above plus falling;
6=all of the above with multiple rearing and falling episodes;
7=a running fit. Animals were considered to have been kin-
dled when the seizures reached stage 5. Scoring was done in
a blind fashion.
For purposes of data analysis, the daily seizure scores
were collapsed into average scores for blocks of 5 days (in
order to keep block size constant, day 1 was not used in the
analysis). MANOVA was performed upon seizure score. AD
duration and threshold difference (day 17 minus day 0).
Hippocampal Kindling
In the third experiment, 75 rats were implanted with elec-
trodes in the hippocampus as described above. At least 1
week following surgery, their AD thresholds were deter-
mined as described above, except that pulse duration was
shortened to 0.3 msec. The Nuclear Chicago stimulator had a
lower limit current of 20 pi A. With a 0.4 msec pulse, a
number of animals in other studies had thresholds deter-
mined to be 20 (iA. Reducing pulse duration should slightly
increase the threshold and ensure that no thresholds were
below the lower limit of the stimulator.
-------
TRIMETHYLTIN AND SEIZURE SUSCEPTIBILITY
205
% + CONTROL
O—O 5 m«/k| TMT
6 mf/kg TMT
7mg/k«TMT
70
60
50
40
30
20
10
16
8
4
1
DAYS POST TMT INJECTION
FIG. I. Percentage of animals showing signs of behavioral seizures
during the course of hippocampal afterdischarge testing. Any sign of
a seizure was counted, including kindling-type grade 1. Seizures
were common in the high dosage TMT group, particularly on the last
day of testing.
Following threshold determinations, animals with
thresholds above 200 /xA or with poor quality EEG record-
ings were discarded. When rats were randomly assigned to
groups in the amygdaloid kindling study, the starting
thresholds turned out to be different for the different groups.
Therefore, in this study, the animals were divided into four
groups matched with respect to thresholds before receiving
their treatment. Attrition during the experiment left the fined
group sizes at: saline (n= 14), 4 mg/kg TMT (n= 15), 5 mg/kg
TMT (n= 15), or 6 mg/kg TMT (n= 15).
Hippocampal kindling takes longer than amygdala kin-
dling [23]. Therefore, in this study, animals were stimu-
lated 6 days/week for 30 days. As with the amygdala kindling
experiment above, stimulation was accomplished at two
times the threshold determined before dosing, and seizures
were scored according to the same criteria in a blind manner.
For purposes of data analysis, the seizure scores were aver-
aged across 5 days to form 6 blocks of scores. A MANOVA
was performed on seizure score and day 0 - day 30 threshold
difference. Subsequent ANOVAs, when significant, were
followed by Duncan's multiple range test for group mean
comparisons.
Following termination of these studies, all animals with
electrodes were subjected to intracardiac perfusion with
saline followed by 10% neutral buffered Formalin. Frozen
brain sections were taken (80 fi) and stained with cresyl vio-
let for localization of electrodes.
Pentylenetetrazol
To examine whether TMT treatment altered sensitivity to
CNS activation in general, rats were administered TMT and
then challenged with various doses of PTZ, a generalized
CNS analeptic. The rats were intubated with either saline
(n=21), 5 mg/kg (n-21), 6 mg/kg (n=21), or 7 mg/kg (n«21)
TMT. Five days later, a time when spontaneous seizure
TABLE 1
CORRELATIONS BETWEEN AD PARAMETER AND HIPPOCAMPAL
MORPHOLOGY (LENGTH OF THE PYRAMIDAL CELL LINE)
Spike
Post-ictal
Threshold
Frequency
Depression
AD Duration
JO
ii
i
S
-.50
-.19
-.47
p= 0.0002
0.008
0.34
0.014
activity is observed in rats receiving high dosages [11], each
treatment group was divided into 3 subgroups (n=7 each)
and injected IP with either 30, 45, or 60 mg/kg PTZ (Sigma).
Animals were subsequently observed for 30 minutes during
which their reactions to the convulsant were systematically
rated. The following parameters of seizure activity were
examined: latency and incidence of first myoclonic jerk,
foretimb clonus, and clonic episodes. The incidence of mor-
tality was also recorded. Scoring was done in a blind manner
and the resulting data were analyzed with a x2 test.
RESULTS
A single acute administration of TMT produced dose-
dependent alterations in all seizure models tested. TMT in-
creased thresholds for production of ADs in both kindling
experiments. TMT also increased rate of kindling from both
sites, and increased seizure susceptibility to PTZ.
Afterdischarge Study
The multivariate analysis of variance indicated that there
were no overall significant effects of dosage on any of the
variables (using Wilks' criterion, F(36,349)=0.78; p<0.10).
During AD testing, behavioral seizures were observed. This
completely unexpected occurrence (in view of the typically
slow rate of hippocampal kindling) was analyzed by chi-
squared, and the results are indicated in Fig. 1.
Parametric analyses also indicated that although there
were significant day effects with several of the measured
variables, there were not significant day x dosage interac-
tions.
Measurements were made of a number of dimensions of
the dorsal hippocampus from the animals in the afterdis-
charge study and, from these, the most clearly dosage-
related was length of the line of pyramidal cells extending
from CA1 to CA3c (see [8] for details). To assess the rela-
tionship between hippocampal damage and AD parameters,
a Pearson product moment correlation coefficient was calcu-
lated to relate this parameter with (1) AD threshold on day
16; (2) AD2 spike frequency on day 16; (3) average number of
integrator resets following AD2/average number of integ-
rator resets before AD2 on day 16; and (4) AD2 duration on
day 16. These physiological endpoints were selected to
sample the range of activities which occur within an AD.
Day 16 was chosen because it was the date closest to the
sacrifice time for the animals (day 30). The results are shown
in Table 1. Threshold, spike frequency, and AD duration
increased with increasing damage to the pyramidal cells.
Figure 4A indicates that rats treated with 6 or 7 mg/kg TMT
exhibited a tendency towards increased AD thresholds.
-------
206
DYER, WONDERLIN AND WALSH
5.0
4.0 -
ill
3.0 -
ui
c
2.0
1.0 -
t 1 r
O——O 0 mg/kg TMT
O — —-O S mg/kg TMT
O O 7 mg/kg TMT
a ft 0 mg/kg TMT
4- — —«s 4 mg/kg TMT
A— -4 S mg/kg TMT
* A • mg/kg TMT
5
a
<9
>
I
(A
a
6
3
j.
_L
4.0
3.S
s
§
S 3.0
cc
a
2.5
§ 1.60
S 1.50
1.40
1.30
TMT mg/kg
0 5 7
~OSAGE TMT. m«/kg
FIG. 3. Mean seizure scores collapsed across all days of testing for
TMT and control rats (illustrates the main effect of dosage). A.
amygdaloid kindling experiment; B. hippocampal kindling experi-
ment.
S DAY BLOCKS POST-TMT
FIG. 2. Development of kindling over blocks of 5 days in both the
amygdaloid and hippocampal kindling experiments. Amygdaloid
kindling is more rapid than hippocampal kindling, which was not
fully developed even at the 30th day of stimulation.
no
100
•o
70
3 w
i n
CONTROL
>*o 8 mf/kf TMT
• m«fr| TOT
<*-< 7 mf/k| TMT
KPOfU
TMT
19 DAYS
AFTtR TMT
CONTROL
5 ma/M
7
SALINE
4 m«/hf TMT
& m«/V« TMT
• mt/k| TMT
17 DAYS
AFTER TMT
APTCR TMT
FIG. 4. Effects of TMT upon afterdischarge thresholds. A. afterdischarge thresholds before adminis-
tration of TMT and 16 days after TMT in animals used for the afterdischarge study; B. afterdischarge
thresholds before administration of TMT and 17 days after TMT in the animals used for the amygdaloid
kindling experiment; C. afterdischarge thresholds before administration of TMT and 30 days after
.TMT in animals used for the hippocampal kindling experiment. In all cases, high dosages of TMT
raised afterdischarge thresholds.
-------
TRIMETHYLTIN AND SEIZURE SUSCEPTIBILITY
207
TABLE 2
PERCENTAGE INCIDENCE OF PTZ SYMPTOMS IN TMT-TREATED RATS
Symptom Dosage PTZ/Dosage TMT: 0 mg/kg 5 mg/kg 6 mg/kg 7 mg/kg
Myoclonic
30 mg/kg
14
71
57
71
jerks
45
43
100
86
86
60
43
86
86
100
Forelimb
30
0
28
55
80
clonus
45
55
100
80
80
60
55
85
80
80
Clonic
30
0
0
30
50
seizure
45
0
70
55
85
60
55
45
45
85
Death
30
0
0
0
0
45
0
0
55
75
60
0
0
55
45
Kindling Studies
In the amygdala experiment, kindling occurred more
rapidly in TMT-exposed rats than in controls. Analysis of
seizure scores indicated no significant dosage x day interac-
tion, but the main effect of dosage was significant,
F(2,505)=5.52; /><0.001. Figure 2 illustrates the rate of kin-
dling in TMT-treated and control rats. Figure 3 indicates the
main effect of TMT. TMT-treated animals had a greater
difference between their AD thresholds on day 0 and day 17
than controls. These results are summarized in Figure 4B.
In the hippocampus, kindling occurred more rapidly in
the TMT-treated animals than in controls (Fig. 2, Fig. 3B),
but there was no significant dosage x day interaction. Again,
the high dosage TMT-treated animals had a greater differ-
ence between their starting and ending AD thresholds than
controls, F(3,156)= 12.76; p<0.Q00l. These results are sum-
marized in Fig. 4C.
Pentylenetetrazol-Induced Seizures
TMT administration markedly affected certain indices of
PTZ-induced seizure activity. When the data were collapsed
across PTZ dosage, the results indicated that TMT-treated
rats had a higher incidence of myoclonic jerks (x*(2)«15.3,
pcO.OOl), forelimb clonus (x3(2)=21.5, /?<0.001), and clonic
seizures (x2(2)=8.6, p<0.05) than did controls. Furthermore,
between 55% and 75% of the rats treated with 6 or 7 mg/kg
TMT died in ftill tonic extension following challenge with 45
or 60 mg/kg PTZ. These data are summarized in Table 2. The
PTZ dose required to produce clonic convulsions in 50% of
the animals in any given treatment group (CDM) was calcu-
lated according to Litchfield and Wilcoxon [14] to be 58.5
mg/kg for the controls and 44.5 mg/kg for both the 5 and 6
mg/kg TMT groups and 30 mg/kg for the 7 mg/kg TMT treat-
ment group. These data demonstrate a dramatic shift to the
left in the PTZ dose-response curve and could indicate that
TMT produces a generalized enhanced sensitivity to the ex-
perimental production of seizures.
While TMT did significantly affect seizure incidence, it
did not alter other parameters of seizure activity such as
latency to onset of first myoclonic jerk, forelimb clonus, or
clonus, TTiese data were difficult to analyze statistically,
2SO -
I II
- 1W- ¦
!»,- | I
s '*" I I
Mil
0 S • 7
OOSAQE TMT
FIG. 5. Latency to clonus in animals treated with 60 mg/kg pen-
tylenetetrazol. The apparent latency shortening effects of high dos-
ages of TMT were not statistically significant.
however, since not all animals had seizures. Figure 5 illus-
trates a trend towards shorter latencies to clonic seizures in
rats receiving 6 or 7 mg/kg TMT and challenged with 60
mg/kg PTZ. Future experiments will be addressed to
whether TMT alters the characteristics (i.e., latency, dura-
tion, topography, etc.) as well as the incidence of PTZ-
induced seizures.
DISCUSSION
The results of the present series of experiments indicate
that TMT increases the susceptibility of rats to the electrical
and pharaiacological production of seizures. This enhanced
sensitivity is manifested as an augmented response to the
chemical convulsant PTZ. These data indicate that TMT not
only increases the vulnerability of the limbic system to the
production of seizures, but also promotes seizure sensitivity
in general. Since kainic acid is also reported to increase sen-
-------
208
DYER, WONDERLIN AND WALSH
sitivity to PTZ in conjunction with a pattern of hippocampal
degeneration similar to that observed following TMT [5], it
might be argued that disruption of selected hippocampal cell
fields and their associated pathways produces an epilepsy-
prone nervous system.
The increased AD thresholds found following treatment
with TMT seem to contradict the increased rate of kindling.
However. AD threshold may be assumed to reflect the cur-
rent spread required to recruit enough neurons to produce an
AD. Under normal circumstances, a required increase in
current spread to produce an effect might be assumed to
indicate that more neurons must be stimulated. Thus, an
increased AD threshold would indicate that more neurons
must be stimulated to produce an AD, and the inference that
the cells were less excitable than normal would be a logical
one. The case of TMT is not so simple. Since TMT is known
to produce cell loss in the kindled areas [2,11], a more plaus-
ible explanation for the increased AD thresholds is that more
current spread is required to recruit the same number of
neurons as required to produce an AD before TMT treat-
ment.
Failure to find alterations in hippocampal AD parameters
following TMT treatment was surprising. An underlying as-
sumption of this particular study was that anything which
altered hippocampal morphology should certainly be ex-
1. Bernard. P. S., R. E. Sobiski and K. M. Dawson. Antagonism
of a kainic acid syndrome by baclofen and other putative
GABAmimetics. Brain Res. Bull, 5: Suppl. 2, 519-523, 1980.
2. Brown. A. W.. W. N. Aldridge, B. W. Street and R. D. Ver-
schoyle. The behavioral and neuropathological sequelae of in-
toxication by trimethyltin compounds in the rat. Am. J. Path.
97: 59-82, 1979.
3. Corsellis, J. A. N. and B. S. Meldrum. Epilepsy. In:
Greenfield's' Neuropathology. 3rd edition, edited by W.
Blackwood and J. A. N. Corsellis. London: Edward Arnold,
1976, pp. 771-795.
4. Czuczwar, S. J., L. Turski, W. Turski and Z. Kleinrok. Effects
of some antiepileptic drugs in pentetrazol-induced convulsions
in mice lesioned with kainic acid. Epilepsia 22: 407-414, 1981.
5. Czuczwar, S. J.. L. Turski and Z. Kleinrok. Atropine reversal
of kainic acid-induced decrease in the leptazol convulsive
threshold. J. Pharm. Pharmac. 33: 44-45,1981.
6. Dam. A. M. Epilepsy and neuron loss in the hippocampus.
Epilepsia 21: 617-629, 1980.
7. Dyer, R. S., E. Burden, K. Hulebak, N. Schultz, H. S.
Swartzwelder and Z. Annau. Hippocampal afterdischarges and
their postictal sequelae in the rat: Effects of carbon monoxide
hypoxia. Neurobehav. Toxicol. 1: 21-25, 1979.
8. Dyer, R. S., T. L. Deshields and W. F. Wonderlin. Trimethyltin-
induced changes in gross morphology of the hippocampus.
Neurobehav. Toxicol. Teratol. 4: 141-147, 1982.
9. Dyer, R. S., C. U. Eccles, H. S. Swartzwelder, L. D. Fechter
and Z. Annau. Hippocampal afterdischarges and prenatal car-
bon monoxide. Soc. Neurosci. Abstr. 3: 104, 1977.
10. Dyer, R. S., H. S. Swartzwelder, C. U. Eccles and Z. Annau.
Hippocampal afterdischarges and their post-ictal sequelae in
rats: a potential tool for assessment of CNS neurotoxicity.
Neurobehav. Toxicol. 1: 5-19, 1979.
11. Dyer, R. S., T. J. Walsh, W. F. Wonderlin and M. Bercegeay.
The trimethyltin syndrome in rats. Neurobehav. Toxicol.
Teratol. 4: 127-133. 1982.
12. Fortemps, E., C. Amand, A. Bomboir, R. Lauwerys and E. C.
Laterre. Trimethyltin poisoning. Report of two cases. Int. Archs
occup. envir. Hlth 41: 1-6, 1978.
13. Fuller, T. A. and J. W. Olney. Kainate neurotoxicity is suppres-
sed by some anticonvulsants but not by others. Soc. Neurosci.
Abstr. 6: 400, 1980.
pected to alter the properties of hippocampal ADs. The find-
ings reported here indicate that the hippocampal AD is a
robust measure of hippocampal function: drastic rearrange-
ment of hippocampal morphology (demonstrated by loss of
pyramidal cells) had no effect. There seems to be a qualita-
tive difference between the effects of certain other
neurotoxic agents and TMT since the AD procedure detects
exposure to a variety of other agents [7,9, 10], It may be that
the AD procedure detects only generalized CNS effects
(e.g., depression) and is insensitive to specific alterations in
the hippocampal system.
These data only address the acute effects of TMT. It
would be of interest to determine whether altered seizure
susceptibility, as reported here, is a persistent feature of
TMT treatment or whether it is coincident with an acute
phase of TMT toxicity. Further studies examining the time
course and potential neural substrates of altered seizure sus-
ceptibility should substantially contribute to understanding
the neurotoxicology of trimethyltin and also perhaps provide
certain insights into the biology of epilepsy.
ACKNOWLEDGEMENTS
The authors wish to thank Mark Bercegeay and Teresa Deshields
for excellent technical assistance during these studies, and Susan
Garner for manuscript preparation.
14. Litchfield. J. T., Jr. and F. Wilcoxon. A simplified method of
evaluating dose-effect experiments. J. Pharmac. c.xp. Ther. 96:
99-113, 1949.
15. Lothman, E. W. and R. C. Collins. Kainic acid induced limbic
seizures: metabolic, behavioral, electroencephalographie. and
neuropathological correlates. Brain Res. 218: 299-318. 1981.
16. Meldrum, B. S. and J. R. Brierley. Neuronal loss and gliosis in
the hippocampus following repetitive epileptic seizures induced
in adolescent baboons by allylglycine. Brain Res. 48: 361-365.
1972.
17. Nadler, J. V.. B. W. Perry and C. W. Cotman. Preferential
vulnerability of hippocampus to intraventricular kainic acid. In:
Kainic Acid as a Tool in Neurobiology, edited by E. G. McGeer,
J. M. Olney and P. L. McGeer. New York: Raven Press. 1978.
pp. 219-237.
18. Nadler, J. V. and G. J. Cuthbertson. Kainic acid neurotoxicity
toward hippocampal formation: dependence on specific excita-
tory pathways. Brain Res. 195: 47-56. 1980.
19. Nadler, J. V., B. W. Perry, C. Gentry and C. W. Cotman.
Degeneration of hippocampal CA3 pyramidal cells induced by
intraventricular kainic acid. J. comp. Neurol. 192: 333-359.
1980.
20. Olney, J. W., V. Rhee and O. L. Ho. Kainic acid: a powerful
neurotoxic analogue of glutamic acid. Brain Res. 176: 91-100.
1979.
21. Pinel, J. P. J. and L. I. Rovner. Electrode placement and
kindling-induced experimental epilepsy. Expl Neurol. 58: 335—
346, 1978.
22. Pisa. M., P. R. Sanberg. M. E. Corcoran and H. C. Fibiger.
Spontaneous recurrent seizures after intracerebral injections of
kainic acid in rat: a possible model of human temporal lobe
epilepsy. Brain Res. 200: 481-487, 1980.
23. Racine, R. Modification of seizure activity by electrical stimu-
lation: II. Motor seizure. Electroenceph. din. Neurophxsiot. 32:
281-294,1972.
24. Schwarz, R., R. Zacekand J. T. Coyle. Microinjection of kainic
acid into the hippocampus. Eur. J. Pharmac. 50: 209-220. 1978.
25. Zaczek, R., M. Nelson and J. T. Coyle. Kainic acid neurotoxic-
ity and seizures. Neuropharmacology 20: 183-189, 1981.
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NeurobehavioraI Toxicology and Teratolony. Vol. 4, pp. 127-133. 1982. Printed in the U.S.A.
The Trimethyltin Syndrome in Rats1
ROBERT S. DYER, THOMAS J. WALSH, WILLIAM F. WONDERLIN
AND MARK BERCEGEAY
Neurophysiology Branch, Division of Neurotoxicology, U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
DYER. R. S., T. J. WALSH, W. F. WONDERLIN AND M. BERCEGEAY. The trimethyltin syndrome in ruts.
NEUROBEHAV. TOXICOL. TERATOL. 4(2) 127-133, 1982.—Trimethyltin (TMT) chloride, administered to adult male
Long-Evans hooded rats, produced a unique and distinctive behavioral syndrome consisting of spontaneous seizures, tail
mutilation, vocalization and hyperreactivity. The LDW for TMT was weight dependent; in large rats (e.g., 450 g), 7 mg/kg
TMT produced significant weight loss and lethality, whereas in small rats (e.g., 250 g), 7 mg/kg produced neither weight loss
nor lethality. TMT produced mild hypothermia and tremors, Results are discussed in comparison with kainic acid-induced
morphological alterations and septal lesion-induced behavioral alterations. Histopathological evaluations of hippocampal
tissue revealed cell loss that was largely confined to regio inferior pyramidal cells. TMT offers potential as a tool for
investigations of limbic system structure and function.
Seizures Trimethyltin Hypothermia Tremor Trimethyltin syndrome Hippocampal degeneration
Tail mutilation Weight-dependent toxicity Irritability Hyperreactivity
ORGANOTIN compounds have been used for a variety of
industrial and agricultural purposes. The trialkyltins, for
example, have been used as stabilizers of plastic, as chemos-
terilants, and as biocides for the control of fungus, bacteria,
and insects [21], A number of alkyltins are also reported to
induce neurobehavioral toxicity [14]. Recently, Brown and
his colleagues [71 reported that trimethyltin (TMT) produces
brain damage which is primarily restricted to limbic system
structures. These investigators observed neuropathological
changes in the hippocampus two days following administra-
tion of 10 mg/kg TMT. Neuronal degeneration was subse-
quently evident in the pyriform cortex, neocortex, and the
amygdala, attaining maximal severity 21 days following ad-
ministration. The hippocampal damage involved most re-
gions of the hippocampus although the fascia dentata was
less affected than hi—5. The regional damage was bilaterally
symmetrical and was characterized by the following sequence
of cellular changes: clumping of nuclear chromatin, naked
nuclei, cell "ghosts" and eventual death.
The neuropathological changes induced by TMT are
markedly different from the effects of its structural analogue,
triethyltin. TET, for example, has been commonly used as a
model compound for the production of cerebral edema,
myelinopathies and spongy degeneration of the brain [2,12].
While TET produces a marked histotoxic hypoxia via the
uncoupling of oxidative phosphorylation, TMT has minor
effects on energy metabolism in the nervous system [1]. It is
evident therefore that these trialkyltins induce differential
patterns of neurological toxicity. Furthermore, these com-
pounds produce dissimilar behavioral effects following acci-
dental human exposure. For instance, TET produces muscu-
lar weakness, nausea, vomiting and sensory disturbances
[14], while TMT exposure results in memory loss, episodes
of visceral pain, mental confusion, anorexia, and generalized
epileptic seizures [10].
Because of the widespread use of trialkyltins and the lim-
ited literature on TMT exposure, we present here a general
description of the behavioral sequelae which result from
acute TMT administration. These sequelae are so unique and
distinctive we have called them collectively the TMT Syn-
drome. Due to the apparent selective toxicity of TMT, we
believe this compound to be a potentially important
neurobiological tool for elucidating the behavioral and phys-
iological function^ of the limbic system.
METHOD
Adult male 50 day old Long-Evans hooded rats (n-428)
were obtained from Charles River Breeding Co., and housed
in individual plastic cages upon wood chip bedding. Unless
otherwise noted, the animals were maintained with free ac-
cess to food and water on a 12:12 light:dark cycle. Some
animals were used immediately (175 g) but most remained
undisturbed for periods ranging from 2 weeks to 7 months
(685 g). All rats were naive until treated.
Solutions of trimethyltin chloride (TMT, ICN Phar-
maceuticals, Plainview, NY) were prepared by mixing the
compound with normal saline. Dosages are presented as mg
of the base/kg body weight. Injection volume was always 1
ml/kg, and dosages ranged from 1.25 to 10 mg/kg. Lethality was
assessed in 258 rats. During the 10 days following treatment,
108 of these animals were used in either the weight loss,
behavioral reactivity or temperature studies.
Behavioral observations were made during daily weighing
of all animals as well as during formal testing sessions for
subsets of the population. Behavioral reactivity was assessed
both subjectively and with a rating scale [20], During the formal
behavioral reactivity testing sessions, rats were administered
either 0 (n-15), 5 (n—13) or 7 (n-16) mg/kg TMT, and the
reactivity was rated for 16 consecutive days using the scale
'This paper has been reviewed by the Health Effects Research Laboratory, U.S. Environmental Protection Agency, and approved for
publication. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.
127
-------
128
DYER ETAL.
A. B.
H2)
100
80
(46)
>
I-
£ $o -
1 so-
2 40 —
U
c
it M-
(91
1601
W;
6
10
S
8
9
100
90 -
80
40
200
300
400
500
600
DOSAGE TMT Img/kg) MEAN STARTING WEIGHT Igml
FIG. I. TMT-induced mortality. (A) Mortality as a function of TMT dosage, independent of weight of
animal. Numbers in parentheses are n's for the indicated dosage. (B) Mortality of animals administered
7 mg/kg TMT as a function of their weight (starting weight±SEM). Weight groups were chosen to
represent roughly equal intervals.
described by Pinel et al. [20]. This scale consists of two
subtests, each of which is rated on a scale from 0-4. Scoring
on the first subtest, resistance to capture, was as follows:
0=remains calm when approached and grasped; 1=shies
from hand when grasped; 2=avoids hand by running, strug-
gles when captured, or both; 3=leaps to avoid capture and
struggles vigorously when captured; and 4= leaps, struggles
and bites when captured. Scoring the second subtest, re-
sponse to pencil tap on base of tail, was as follows: 0=no
response; l=flinches or twists; 2=flinches and jumps;
3=moves away; and 4=jumps at least 6 inches. Data were
analyzed by ANOVA with repeated measures. Multiple
comparisons (when appropriate) were performed using Dun-
can's multiple range test.
Since triethyltin has pronounced effects on body tempera-
ture, we also recorded TMT-induced alterations in core tem-
perature using a modified multistation Yellow Springs ther-
mometer [8]. Rats were treated with either 0 (saline), 1.25,
2.5, 5 or 10 mg/kg TMT (n=6/group) and restrained in com-
mercial restraining tubes. The thermometer probe was inserted
about 75 mm into the rectum, and the digital reading was
recorded to the nearest 0.1°C at ambient temperatures of
22°C (±1°C). The probe was left in place during the 6 hr
session. To determine long-term changes, animals were
treated with either saline (n= 10) or 7.0 mg/kg TMT (n= 10),
placed in the tubes once each day and the probe was inserted
2 min prior to recording. Data were anlayzed by ANOVA,
followed by Duncan's multiple range test when appropriate.
Histopathological evaluation of the brain was performed
upon rats perfused with saline followed by 10% neutral buf-
fered Formalin. Frozen sections (40 n) were taken in the
sagittal plane, mounted and stained with cresyl violet.
RESULTS
Lethality
The slope of the lethality x TMT dosage curve is steep.
With IP dosages as high as 10 mg/kg, some mortality occurs
within 48 hr. At lower dosages, maximum mortality is
achieved by 10 days post-treatment. Only about 17% mortal-
ity is observed with dosages of 7 mg/kg, but 7.5 mg/kg
produces about 55% mortality. Based upon these data, the
LDjo determined according to the method described by
Litchfield and Wilcoxon {16] was 7.45 mg/kg. These data are
illustrated graphically in Fig. 1. The data presented in Fig. la
are derived from 258 rats. In the course of collecting these
data, it appeared that mortality produced by intermediate
dosages was dependent upon the weight of the animal at the
time of dosing. To determine the influence of body weight
upon mortality, the weights of 71 rats receiving 7 mghcgTMT
were divided into four groups: > 500 g (mean wt=590 g; n= 14);
400-499 g (mean wt=433 g; n= 12); 300-399 g (mean wt = 339
g; n=32); and <300 g (mean wt=279 g; n=13). Figure lb
depicts the percentage mortality produced by TMT in each
of these weight groups. Heavy animals were clearly at
greater risk than light animals.
Weight Loss
Immediately following IP injection of 6 mg/kg or more of
TMT, signs of gastrointestinal distress (characterized by
barrel rolling) were evident. Subsequently the animals be-
came quiescent for a period lasting at least one hr. At high
dosage levels (7 mg/kg and above) weight loss was often
evident. The period of weight loss lasts for about one week
and is followed by recovery over the next two weeks. TMT-
induced alterations in body weight are illustrated in Fig. 2. In
some studies, whole groups treated with 7 mg/kg TMT lost
weight, while on other occasions they did not. As with mor-
tality, a m^jor determinant of weight loss appears to be start-
ing weight of the animals. For example, weights were re-
corded on day 0 (the day of treatment) and on day 5 (chosen
arbitrarily) for 124 rats receiving 7 mg/kg TMT. Day 0
weights ranged from 175 g to 685 g. A regression analysis of
day 5 weights as a function of day 0 weights was performed
and the results are plotted in Fig. 3. The slope of the regres-
sion line for TMT animals was 0.738 and the R' was 0.78.
The Y intercept was 71.73. The graph indicates that animals
weighing more than 276 g lose weight from day 0 to day 5 if
administered 7 mg/kg TMT on day 0. A comparable regres-
sion analysis was performed on 58 saline-ityected control
rats for the day 0 and day 5 weights. This line, also indicated
in Fig. 3, demonstrates that even animals injected with saline
on day 0 will have lost weight on day 5 if their day 0 weights
-------
TMT SYNDROME
129
«0Q
100
0
0
J
i
i
i
T
9
10
I
FIG. 2. TMT-induced alterations in body weight. Effects of different
dosages of TMT on body weight (±SEM). Note that there were two
groups administered 7 trig/kg TMT. The heavy group (n= 10) lost
weight, the light group
-------
130
DYER ET AL.
—— ¦ 7.0 mg/kg TMT
AMBIENT TEMP - 21
FIG. 6. Severe tail mutilation 6 days following 7 mg/kg TMT.
DAYS (poll inaction)
FIG. 5. Time course of apparent 7 mg/kg TMT-induced hypother-
mia. Core temperatures are presented as change from pre-treatment
values (±SEM) obtained on day 0. Saline n=I0, TMT n=IO.
§
—o
surface. One animal, however, chewed off the distal 75 mm
of its tail. Figure 6 illustrates one of the more severe cases of
tail mutilation.
Following TMT exposure, many rats engaged in bouts of
vocalization. Although the vocalization often occurred with-
out apparent provocation, it could also be triggered in some
animals by either auditory or visual stimuli (shaking of keys
or movement of a hand). In contrast to the vocalizations of
normal rats, TMT-induced vocalization could be elicited
without handling.
Seizures
Seizures, which were usually noticed at weighing time
(-10:00 a.m.), took several forms: episodes of rearing and
forelimb clonus, generalized tonic-clonic episodes, and run-
ning fits. Tonic-clonic episodes were the least common and
were only rarely observed. Of the more than 200 rats likely
to have seizures (i.e., that received 6 mg/kg of TMT or
more), less than 10 displayed a generalized tonic-clonic
episode. Running fits and episodes of rearing and forelimb
clonus occurred with about equal frequency and were ob-
served in about 50% of animals receiving 7 mg/kg TMT. The
episodes of rearing and forelimb clonus were best charac-
terized by their short duration (usually 5-10 sec) and benign
character. The movements were not as sharply defined as
those which occur in kindled seizures (i.e., movements ap-
peared smooth rather than choreiform). The episodes were
not followed by any appreciable postictal behavioral de-
pression. Indeed, on some occasions, two or three succeed-
ing episodes rapidly followed each other. Running fits were
charcterized by vigorous running about the cage, during
which the animal frequently collided with the walls and ceil-
ing of its cage. Cages with wire tops not weighted with water
bottles occasionally had their tops knocked off by rats during
these running fits. Systematic evaluation of the frequency of
these different types of seizures was not made. However, it
appeared that in many animals they were triggered by han-
dling.
Following treatment, the frequency with which these dif-
ferent characteristics of the TMT syndrome occurred in-
creased for a period of about 1 week and then gradually
declined through the next week. In one subset of 10 control
(saline treated) and 10 TMT (7 mg/kg) treated rats (see Fig. 2,
heavy groups), none of the controls and 6 of the TMT-treated
animals had seizures within 8 days following treatment
(X* = 5.95, df= 1, p<0.02). Seizures were observed in 2 of
these rats within 72 hr of treatment. On the 8th posttreatment
day only I animal had a seizure.
Hyperreactivity
With a time course closely paralleling that of the seizures,
self mutilation and vocalization, animals demonstrating the
TMT syndrome became gradually intractable. The intract-
able behavior was not simply non-specific aggression. A
hand or glove placed in the cage did not elicit attack in these
animals. Indeed, an otherwise undisturbed 7 mg/kg TMT
animal's cage may be quietly opened and its back stroked
without eliciting abnormal behavior. However, animals picked
up by placing the hand under the thorax and lifting were
likely to react violently. Following one such handling, the
animals appeared to be sensitized and were thereafter more
difficult to pick up.
Analysis of the data obtained using the behavioral reac-
tivity scale indicated that TMT exposure altered reactivity
on both the first, F(2,615) = 22.64. p<0.000l, and the second
F(2,615)=4.76, p<0.0089, subtest. Animals exposed to 7
mg/kg TMT were more reactive to handling (subtest 1) than
the other two groups.
The animals exposed to 5 mg/kg TMT were less reactive
than either control or 7 mg/kg groups on both reactivity tests.
Figure 7 indicates that reactivity as subjectively appreciated
by the investigators was not well detected by this scale.
Even the 7 mg/kg group did not have an average score of 1 on
the resistance to capture test.
Tremor
Animals receiving high dosages of TMT exhibited what
appeared to be a fine tremor-at-rest. Tremor first appeared 2
days following injections of 8.75 mg/kg TMT and 3 days
following injections of 7.5 mg/kg TMT. Tremors were rarely
observed in animals receiving TMT in dosages of 7 mg/kg or
-------
TMTSYNDROME
131
0.8
0.7
06
0.5
u
< 0.4
0.3
0.2 -
0.1
00
REACT 1
REACT 2
rh
TABLE 1
PERCENTAGE OF SURVIVING ANIMALS EXHIBITING TREMORS
DOSAGE TMT. mg/kg
FIG. 7. Reactivity in TMT-treated and control rats (uSEM). The
scale, derived from Pinel et at. (201 has two parts. React I measures
resistance to capture. React 2 measured reactivity to a pencil tap on
the tail.
less. Table I indicates the distribution of tremors across days
in animals receiving different dosages of TMT.
Pathology
Necropsies were performed upon a sample of surviving
animals from several studies for histopathological examina-
tion. Five animals received TMT treatment only. Two of
these rats received TMT by gavage and showed no evidence
of pathology in the peritoneal cavity. The administration of
TMT by IP injection to the other three animals was observed
to lead to the development of an acute inflammatory reaction
in the peritoneal cavity, consisting of fibrin proliferation and
the accumulation of free bloody fluid. The extent of fibrin
proliferation ranged from small, isolated patches to more
generalized adhesions of the abdominal viscera, particularly
the liver, stomach, spleen, and small intestine. In one ex-
treme case, extensive adhesions led to blockage and disten-
sion of the small intestine.
Cresyl violet-stained sections of the brain confirmed the
findings of Brown et al. [7] that neuronal cell loss occurred in
the hippocampal formation. Damage was consistently evi-
dent in the intrahilar and extraventricular or lower end and
middle portion of regio inferior [5,15]. Occasionally damage
extended to the ventricular or upper end of regio inferior.
There was no obvious cell loss in regio superior. Figure 8
illustrates extensive cell loss in an animal administered 7
Dose (mg/kg)
N
(Day 0)
0
2
4
Days
6
8
10
12
7.0
47
0
0
0
0
0
0
0
7.5
9
0
0
38
14
40
25
0
8.75
39
0
39
66
50
40
50
38
10
6
0
100
FIG. 8. Cresyl violet-stained saggital sections through the hip-
pocampus. (A) Control (saline treated). (B) 7 mg/kg TMT-treated
rat. Animals were sacrificed 30 days following treatment.
mg/kg TMT and sacrificed 30 days later. Damage to other
brain areas was not systematically assessed.
DISCUSSION
The present report describes a unique complex of behav-
ioral effects which result from acute exposure to TMT.
These effects, which we have chosen to call the TMT syn-
-------
132
DYER ET AL.
drome include tail mutilation, hyperreactivity, vocalization
and episodes of spontaneous seizures. The LD,0 for acute
TMT exposure, regardless of route of administration, was
determined to be approximately 7.45 mg/kg. This value is
somewhat less than the values reported by other inves-
tigators, however, differences in dose expression (mg of salt
vs mg of base), vehicle (saline vs arachis oil), age or gender
might account for the discrepancies. A potentially more im-
portant determinant of TMT toxicity is that it appears to
depend upon body weight and/or age at time of TMT treat-
ment (Fig, 1). Heavier animals are more susceptible to TMT
toxicity than leaner animals. This weight dependence could
indicate that there is either a critical quantity of TMT which
must reach the brain for weight loss and lethality to occur or
that TMT is excreted or stored more efficiently in leaner
animals. It has been reported [22] that in albino male rats, a
196% difference in body weight corresponds to only a 9%
difference in whole brain weight. Therefore, over the weight
range of the animals used in the present series of experi-
ments (175-685 g), there could be approximately 391%
difference in total dose received by animals in any given
dosage group. The relationship between body weight and
lethality (Fig. I) and weight loss (Fig. 3) is clear. However,
the relationship between body weight and other charac-
teristics of the TMT syndrome is less clear. For example, 7
of the 15 animals in the low weight 7 mg/kg group depicted in
Fig. 2 had at least one seizure by day 8, but did not lose
weight. Of the 10 heavy animals receiving 7 mg/kg depicted
in Fig, 2, all lost weight and 6 had seizures. The difference in
seizure frequency between these two groups was not signifi-
cant, x4(l)=0 06, p<0.l0. Therefore the neurotoxicity of
TMT does not depend upon the debilitating effects of weight
loss.
While TMT-treated rats are hyperreactive, several factors
suggest that the TMT-syndrome does not result from in-
creased sensitivity to sensory stimuli. Neither stroking the
back of a TMT-treated rat nor tapping the tail vigorously
with a pencil elicits a major response. Furthermore, prelimi-
nary data indicate that TMT-treated rats are hyporesponsive
to auditory startle stimuli [13]. Hyperreactivity is usually
only evident following attempts to lift the animal by placing
hands around its thorax. Conceivably, the TMT-treated
animals experience visceral pain which is exacerbated by
handling. This concept might have validity since organ pain
is one of the presenting symptoms in humans exposed to
TMT.
Sensory impairment might also account for the TMT-
induced tail mutilation. Preliminary data suggest an impaired
somatosensory system in TMT-treated rats [9], but in the
absence of specific anatomical data on the integrity of
somatosensory pathways from the tail, it is difficult to ac-
count for the specificity of the tail mutilation. At any rate,
TMT-induced self-mutilation is unique by comparison to
self-mutilation induced by pharmacological manipulations
[17], since the latter is characterized by biting and mutilating
the digits and thorax in stereotyped grooming sequences.
While TMT induced a unique complex of behavioral ef-
fects, certain components of the TMT syndrome are also
induced by lesions of the septum or its associated fiber
tracts. For example, electrolytic lesions which include the
lateral septum induce a state of hyperreactivity (i.e., septal
rage) which is characterized by difficulty in handling, vocali-
zation, increased auditory startle response, and aggressive
reaction to tactile stimuli [6,11]. To date, septal damage has
not been a reported consequence of TMT exposure although
a more complete anatomical analysis is necessary to deter-
mine whether TMT-induced hyperreactivity is dissociated
from septal damage.
TMT is one of a growing class of compounds which ap-
pears to produce relatively selective neuronal damage. The
compound which produces neuropathological effects most
similar to those of TMT is kainic acid, an excitotoxic analog
of glutamate. Systemically administered kainic acid
produces wet dog shakes, behavioral convulsions, and
amygdaloid epileptiform electrical activity [4. 18. 19]. While
a single systemic administration of TMT (present study) se-
lectively damages CA3, systemic administration of kainic
acid damages CA1 and CA3. Browner al. [7], using repeated
dosing, found that TMT may also damage CAI. Differences
between the pattern of hippocampal damage reported here
and that reported by Brown et al. [7] may be due to the use of
repeated versus simple doses. It will be important for future
studies to correlate the specific neural damage produced by
these two compounds with their respective mechanisms of
toxicity and behavioral sequelae.
In summary, the present report demonstrates that acute
administration of TMT produces an intriguing behavioral
syndrome. Clearly, TMT offers potential as a tool for the
investigation of limbic system structure and function.
ACKNOWLEDGEMENT
The authors thank Teresa Deshields for technical assistance. Dr.
James Wright for performing pathological evaluations, ihe animal
care technicians of Program Resources Inc. for handling "danger-
ous" rats, Ann Brady for tissue preparation and histology, and the
U.S.E.P.A. Health Effects Research Laboratory Word Processing
Center for manuscript preparation.
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TMTSYNDROME
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W. Reiter. Sensory System Effects of Acute Trimethyltin (TMT)
Exposure. Paper presented to, 20th A. Meet., Society of To-
xicology, March, 1981.
14. Kimbrough, R. Toxicity and health effects of selected organotin
compounds: a review. Envir. Hlth Perspect. 14: 51-56, 1976.
15. Laurberg, S. Commissural and intrinsic connections of the rat
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19. Nadler, J. V., B. W. Perry. C. Gentry and C. W. Cotman.
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-------
TOXICOLOGY AND APPLIED PHARMACOLOGY 73, 543-550 (1984)
Effect of TriethyItin on Autonomic and Behavioral
Thermoregulation of Mice1
Christopher J. Gordon, Merritt D. Long, and Robert S. Dyer*
Experimental Biology Division and *Neurotoxicology Division, Health Effects Research Laboratory,
U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711
Received September 2, 1983; accepted December 22. 1983
Effect of Triethyltin on Autonomic and Behavioral Thermoregulation of Mice. Gordon,
C. J., Long, M. D., and Dyer, R. S. (1984). Toxicol. Appl. Pharmacol. 73, 543-550. The
organotin compound, triethyltin (TET), produces toxic effects in a variety of physiological systems.
Thermoregulatory control appears to be especially susceptible to TET toxicity, since TET ad-
ministration has been shown to cause a pronounced hypothermia in rats. To further elucidate
effects of TET on thermoregulation, we measured metabolic rate, evaporative water loss (EWL),
body temperature, and preferred ambient temperature (?,) of mice treated intraperitoneal^ with
TET (bromide salt). At a T, of 23 to 24aC, TET (6 and 8 mg/kg) inhibited metabolic rate by
23 and 66%, respectively. TET resulted in hypothermia at 7",'s of 20 and 30°C but not 35°C.
TET had little effect on EWL. Mice given TET at doses of 4, 6, and 8 mg/kg selected a cooler
T, (ca. 25°C) compared to controls (ca. 29°C). Thus, the mice selected a T, associated with a
hypothermic body temperature. At a relatively cool T„ mice treated with TET had a reduced
rate of heat production and, consequently, were hypothermic. At a relatively warm T„ TET
had no effect on heat production and did not increase active heat dissipation (i.e., EWL), thus
the mice remained normothermic. The behavioral data indicate that TET evokes a type of
regulated hypothermia in mice.
Triethyltin (TET), an extremely toxic organ-
otin compound (Kimbrough, 1976), has been
shown to be a potent inhibitor of cellular me-
tabolism (Brody and Moore, 1962; Aldridge
and Rose, 1969; Cramer, 1970). A variety of
anatomical and functional neurotoxic effects,
such as demyelination (Blaker et al1981),
ataxia (Gerren et al., 1976), and increased la-
tency for a visual-evoked response (Dyer and
Howell, 1982), have been demonstrated in
animals injected with TET.
Intraperitoneal TET administration to the
rat at an ambient temperature of 22°C results
1 This manuscript has been reviewed by the Health Ef-
fects Research laboratory, U.S. Environmental Protection
Agency, and approved for publication. Mention of trade
names or commercial products does not constitute en-
dorsement or recommendation for use.
in a prolonged hypothermic state than can
last up to 24 hr (Dyer and Howell, 1982).
Intravenous administration of TET to the rat
also causes hypothermia, whereas, intracere-
broventricular injection causes a biphasic
temperature response, hypothermia followed
by hyperthermia (Leow et al., 1980). TET-
induced hypothermia can be partially atten-
uated by raising ambient temperature from
22 to 30°C (Dyer and Howell, 1982). Inter-
estingly, one neurotoxic effect of TET, delay
in the visual evoked response, is more pro-
nounced at a higher ambient temperature
(Dyer and Howell, 1982). Thus, the observed
hypothermia following TET may be indepen-
dent of the neurotoxicity. In a related study,
Cretner (1970) found that TET effects on glu-
cose metabolism in the brain were not directly
attributable to hypothermia.
543
0041-008X/84 $3.00
Copyright © 1984 by Academic Prtsk Inc.
AU ri|hti of reproduction in any form reterved.
-------
544
GORDON, LONG, AND DYER
Toxic as well as other agents which affect
thermoregulation may induce either a forced
or regulated change in body temperature
(Gordon, 1983). For example, TET-induced
reduction in body temperature could be a
forced hypothermia meaning that heat gain
and/or heat conservation is inhibited with no
change in set-point. That is, body temperature
is below its regulated or preferred level and
the animal's response is to select a warm en-
vironment and/or increase heat production (if
possible) in an attempt to raise body temper-
ature back to normal. If the hypothermia is
regulated, then there is a decrease in set-point
whereby the animal lowers heat production,
increases heat loss, and behaviorally selects a
cooler environment which leads to a con-
trolled decrease in body temperature.
Discerning forced from regulated changes
in body temperature is important to toxic
studies. The ambient conditions may be cru-
cial depending on whether a toxic-induced
temperature change is forced or regulated. For
example, during forced hypothermia, a warm
ambient temperature is preferable to the an-
imal since there is a physiological and behav-
ioral drive to elevate body temperature back
to normal. However, a warm ambient tem-
perature becomes stressful in regulated hy-
pothermia since the response of the animal is
to dissipate heat and maintain body temper-
ature at a lower than normal level. The data
of Dyer and Howell (1982) suggest that such
mechanisms may occur since it was shown
that preventing the TET-induced hypothermia
by raising ambient temperature accentuated
its neurotoxic effects.
This study was designed to measure body
temperature, metabolic rate, evaporative heat
loss, and behavioral temperature preference
of mice treated intraperitoneally with TET.
METHODS
Animals used in this study were young adult, male mice
of the BALB/c strain obtained from Jackson Laboratories
(Bar Harbor, Maine). The animals were maintained in
groups of five in cages lined with wood chip bedding at
22#C under a 12:12-hr light;dark photoperiod with ad
libitum access to feed (Lab-Blox, Wayne Pet Food) and
water.
Metabolic rate, evaporative heat loss, and body tem-
perature. Metabolic rate was measured in single mice
maintained in a temperature controlled, stainless-steel en-
vironmental chamber which had a volume of 1950 ml.
Single mice were placed in a wire cage which was inserted
into the environmental chamber. Dry air was pulled
through the chamber at a flow rate of approximately 2500
ml/min. A small fraction of the effluent air from the en-
vironmental chamber was pulled through a dew point
hygrometer (General Eastern, Model 1200 APS) to mea-
sure the dew point temperature of the air. The same air
sample was dried over calcium anhydrite and then passed
through an oxygen analyzer (Applied Electrochemistry)
to measure the percentage oxygen. Evaporative water loss
was calculated from the dew point temperature by pre-
viously published methods (Wood, 1970; Gordon. 1982).
Consumed oxygen was calculated from the change in per-
centage oxygen of the air passing through the chamber
multiplied by the flow rate. Metabolic rate (watts) was
calculated assuming that 1.0 ml of consumed oxygen was
equivalent to 20.1 joules (Kleiber, 1975). A layer of mineral
oil placed beneath the wire-mesh cage prevented the urine
and fecal moisture from interfering in the measurement
of dew point temperature.
An initial experiment (N = 19) was run to assess the
effects of acute TET administration on metabolic rate.
The average body weight of the mice in this experiment
was 25.4 ± 1.8 (SD) g. The experiments were run at an
ambient temperature (7.) of 23 to 24°C. Single mice were
injected ip with the saline vehicle or TET (bromide salt)
at doses of 2, 4. 6, and 8 mg/kg in a volume of 0.3 ml/
100 g body weight. Naive mice were injected and placed
in the environmental chamber. Four to five mice were
used in each dose group. Oxygen consumption data be-
tween 30 to 60 min post-TET injection were averaged to
one value and converted to metabolic rate (watts per ki-
logram). Sixty minutes after injection, the animal was
removed from the environmental chamber, marked, and
returned to the animal quarters.
In a second experiment (W =» 46) metabolic rate, evap-
orative water loss (EWL), and body temperature were
measured at T,'s of20, 30, and 35°C. In these experiments
TET doses of 0, 6, and 8 mg/kg were administered ip.
The average body weight was 24.8 ± 1.5 g. The animals
were treated as described above. Four to five mice were
used in each treatment group. Immediately after TET
injection, the mice were placed in the environmental
chamber maintained at either 20, 30, or 35°C. Sixty min-
utes after being placed in the chamber the mouse was
removed and a thermistor temperature probe (Yellow
Spring Instruments) was inserted 2.5 cm past the anal
sphincter. The probe was maintained for approximately
10 sec while the measurement of colonic temperature
stabilized. Similar to the earlier experiment, metabolic
rate and evaporative water loss were calculated from av-
-------
THERMOREGULATION IN MICE
545
erages of percentage oxygen and dew point temperature,
respectively, taken over 30 to 60 min post-TET injection.
Behavioral temperature regulation. Preferred ambient
temperature of mice (N = 45) was measured by using a
longitudinal temperature gradient-shuttlebox system
which has been described (Gordon et al.. 1983). Briefly,
the system consists of a rectangularly shaped metal tube
positioned between hot and cold water baths which gen-
erate a temperature gradient along the length of the metal
tube. A Plexiglas shuttlebox is placed inside the metal
tube and the position of the mouse inside the shuttlebox
is recorded continuously by a phototransitor-based de-
tection system.
Temperatures in the shuttlebox ranged from 19°C at
the cool end to 36°C at the warm end. The shuttlebox
had a length of 76 cm. Single mice were injected ip with
TET at doses of 0, 2, 4, 6, and 8 mg/kg and placed in
the gradient on the coolest end. The average body weight
of the mice was 22.3 ± 1.5 g. Nine naive mice were used
for each treatment group. The position of the mouse in
the shuttlebox was monitored for 60 min. Average pre-
ferred T, in the temperature gradient during 0 to 20, 20
to 40, and 40 to 60 min post-TET injection was calculated.
Statistical analysis. For each of the three experiments,
a was maintained at 0.05 by the following procedures.
The data collected on dose of TET and metabolic rate
were analyzed with a univariate ANOVA, followed by
Bonferroni-corrected T tests for individual comparisons.
The experiment testing the effect of TET on metabolism,
body temperature, and evaporative water loss at different
ambient temperatures was statistically analyzed by a Mul-
tivariate two factor (dose • ambient temp) analysis of vari-
ance (MANOVA), Hotelling-Lawley procedure, with body
weight of the animal as a covariate. The MANOVA was
followed by univariate Bonferroni-corrected ANOVAs,
which were followed in turn by Bonferroni-corrected T
tests. The behavioral temperature selection experiment
was analyzed with a univariate two factor repeated mea-
sures design (dose • time). Between group comparisons were
made with Tukey's tests (Muller et at., 1983).
RESULTS
Examples of the time course of metabolic
rate of mice injected ip with saline or TET at
8 mg/kg are shown in Fig. 1. Immediately
after TET injection there was an inhibition of
metabolic rate lasting for the 60-min experi-
mental period. Overall, metabolic rate was re-
duced significantly by TET (,F(4,18) = 22.41,
p < 0.0001). The hypometabolic effect of TET
was due entirely to the reduction by 23 and
66% (p < 0.001) at doses of 6 and 8 mg/kg,
respectively (Fig. 2). TET doses of 2 and 4
mg/kg had no effect on metabolic rate.
We used two dose levels of TET that inhibit
metabolism in the BALB/c mouse (6 and 8
mg/kg) to test the interaction between dose
and ambient temperature on metabolism.
S
3
• (CONTROU
Fig. 1. Mean ± standard error of metabolic rate of mice treated with 0 and 8 mg/kg triethyltin (TET)
at an ambient temperature (TJ of 23 to 24°C. N - 4 to 5 for each treatment W/kg * watts/kg.
-------
546
GORDON, LONG, AND DYER
a
¦M
%
i
14*
K
<
CC
u
3
o
a
<
DOSE, ma/ka
Fig. 2. Overall effect of dose of TET on metabolic rate
of mice treated at a T, of 23 to 24°C. N = 4 to 5 for each
dosage. Vertical bars represent one standard error from
mean. *Differer from control, p < 0.05.
body temperature, and evaporative water loss.
The MANOVAs indicated significant overall
dose (F( 10,54) = 10.9, p < 0.0001) and dose-
ambient temperature interactions on these
dependent variables (F(20,106) = 7.70, p
<0.0001). ANOVAs indicated that meta-
bolic rate and colonic temperature contrib-
uted to these interactions (/"s(4,31) > 6.4, p's
< 0.0007). Figure 3A illustrates that at a 7a
of 20°C metabolic rate was inhibited by TET
in a similar manner as shown in the initial
experiments run at 23 to 24°C (Fig. 2). Raising
ra to 30°C reduced the inhibitory effect of
TET on metabolism, and at 7*a of 35°C there
was no significant inhibition of metabolism
by TET. In the control groups, metabolic rate
was significantly higher at a of 20°C com-
pared with 30 (p < 0.0012) and 35°C (p
< 0.0002). There was no significant difference
in metabolic rate of control animals exposed
to 77s of 30 and 35°C.
Body (colonic) temperature at a 7*a of 20°C
decreased an average of 6.1 and 11.2°C at
TET doses of 6 and 8 mg/kg, respectively (p
< 0.0001) (Fig. 3B). At 30°C TET induced
relatively small drops in body temperature,
while at 35°C there was no change in tem-
perature. While EWL did not show a signif-
icant ra-dose interaction, there was a signif-
icant overall dose effect (/r(2,31) = 5.69, p
< 0.0078). This significant effect was due
largely to the inhibition by TET at a dose level
of 8 mg/kg at a 7"a of 20°C (p < 0.0019)
(Fig. 3C).
In the temperature gradient, control mice
selected a Ta of 28 to 29.5°C (Fig. 4). ANOVA
indicated that TET significantly altered this
preferred temperature (F(4,40) = 3.26, p
<0.021). There was no apparent difference
in thermoregulatory behavior between the 0-
and 2-mg/kg treatment groups. Doses of 4, 6,
30
<
K
O •
a*
<
20
10
5 9
u
2
o
u
®
P E
<
cc
o
K
<
>
40
30
20
0 2
0.1
0 mg
I I 6 mg
r .
20° C 30° C 3S°C
AMBIENT TEMPERATURE. »C
Fig. 3. Interaction of T, and dose of TET on metabolic
rate (A), colonic temperature
-------
THERMOREGULATION IN MICE
547
c
3
5
tr
UJ
a.
2
30
20
A
L
J,
lid
0-20 min
~ 20-40 min
40-60 min
A
fU
&
DOSE, mg/kg
Fig. 4. Effect of TET on the preferred 7", of mice during 0 to 20, 20 to 40, and 40 to 60 min post-TET
injection. N = 9 for each treatment group. Vertical bars represent one standard error from mean.
and 8 mg/kg all caused a reduction in preferred
Ta, and at 8 mg/kg the preferred Ta was 25.2
to 26.5°C. The analysis of preferred Ta data
was split into three 20-min slots with the no-
tion that there might be a change in behavioral
temperature selection with time. However,
ANOVAs for time (^(2,80) = 0.42, p > 0.65)
and dose • time (/^.SO) = 1.15, p > 0.34) both
failed to reach significance. TET-treated an-
imals were notably sluggish in the temperature
gradient compared to controls; however, most
of the animals exhibited movement to and
from the warmest and coolest areas of the
temperature gradient.
DISCUSSION
Intraperitoneal injection of TET inhibited
metabolic rate (heat production) and induced
hypothermia at Ta's of 20 and 30°C. At a
relatively high of 35 °C, TET had little effect
on metabolism and no effect on body tem-
perature. When mice were provided with an
option to select their preferred T,, they selected
a cooler Ta at TET doses of 4, 6, and 8 mg/
kg. By selecting a cooler T3 the mice were
apparently maintaining their body tempera-
ture at a hypothermic level. Although body
temperature was not measured during the be-
havioral thermoregulatory experiments, it was
observed earlier that TET at 6 and 8 mg/kg
caused significant hypothermia at 77s of 20
and 30°C (Fig. 3B). The observation that the
mice selected a of approximately 25°C
when given TET at 6 and 8 mg/kg suggests
that their core temperature was below normal.
Since high doses of TET resulted in de-
pressed locomotor activity, one might question
if the behavioral experiments were biased by
introducing the animals on the cold side of
the temperature gradient. However, when the
TET-injected animals entered the temperature
gradient they usually moved from the cold to
the warm end repeatedly before settling down
in the cool side of the gradient.
There is little direct evidence on the mech-
anism by which TET induces hypothermia.
However, results from studies not concerned
specifically with thermoregulation indicate
that TET may inhibit muscular thermogenesis.
The release of acetylcholine in the rat phrenic
-------
548
GORDON, LONG, AND DYER
nerve-hemidiaphragm preparation during
phrenic nerve stimulation is attenuated fol-
lowing TET treatment (Bierkamper and
Valdes, 1982). The resting membrane poten-
tial of the rat soleus muscle is significantly
reduced following chronic TET administration
(Millington and Bierkamper, 1982). Tonus
control of skeletal muscle in mice (e.g., shiv-
ering) is critical to the normal mechanisms of
heat production (Hart, 1971). This reaction
may explain why, at low ambient temperatures
where mice must shiver to maintain an ele-
vated heat production, TET was most effective
in inhibiting metabolic rate and inducing hy-
pothermia.
Is the reduction in body temperature fol-
lowing TET administration forced or regu-
lated? Distinctions between forced and reg-
ulated temperature changes were discussed
earlier (Introduction). In the present study
TET either directly or indirectly inhibited
metabolic rate which apparently led to the
reduction in body temperature. If the TET-
induced reduction in temperature were forced,
one would expect the mice to select a very
warm Tx. The observation that the animals
selected a cold in the temperature gradient
suggests a volitional reduction or regulated-
like decrease in body temperature. Yehuda
and Wurtman (1974) found that rats given d-
amphetamine and placed in a cold environ-
ment avoided a heat lamp and allowed body
temperature to decrease. Rats given ^-am-
phetamine and placed in a warm environment
remained close to a heat lamp and allowed
body temperature to increase. This "para-
doxical" effect of ^-amphetamine is somewhat
similar to our results except that mice given
TET tended to always avoid the warm side of
the temperature gradient.
At a r, of 35*C there was no reduction in
metabolic rate or body temperature. If TET
induced a regulated hypothermia at this T%,
one would expect an increase in heat dissi-
pating effectors (i.e., EWL). However, EWL
was not elevated significantly at either 6 or 8
mg/kg. Mice are not well adapted to dissipate
heat by EWL (Hart, 1971; Gordon, 1982).
Thus, when given TET at a high T„, a regu-
lated hypothermia may be invoked but not
accomplished because of poor autonomic heat
dissipatory reflexes. The behavioral test
showed that TET-treated mice avoided warm
in favor of cool 7Vs. Thus, following TET
exposure at a high ra, mice apparently can
employ behavioral but not autonomic mech-
anisms to reduce body temperature.
In view of the effects of TET on behavioral
thermoregulation, it is apparent that the neural
control of body temperature is affected by
TET. The effect of TET on temperature reg-
ulation may be due to an alteration in the
levels and/or binding of neurotransmitters in
the central nervous system. A lethal dose (10
mg/kg, ip) of TET to the rat causes significant
decreases in brain levels of norepinephrine
(NE) and 5-hydroxytryptamine (5-HT) (Rob-
inson, 1969). Cook (1983) found decreases in
NE and dopamine in the rat brain within 30
min after TET administration (6 or 9 mg/kg,
ip). On the other hand. Mailman et al. (1983)
found little change in the levels of 7-amino-
butyric acid or acetylcholine of various brain
sites in rats treated daily with TET (0.3 mg/
kg/day) postnatally from Days 2 to 29. The
concentration and turnover of NE and 5-HT
in the brainstem are crucial to thermoregu-
latory control (Myers, 1980). Thus, the ther-
moregulatory effects of TET may be partially
attributable to the influence of the organotin
compound on neurotransmitter levels in the
central nervous system.
Dyer and Howell (1982) found that the im-
pact of TET exposure on the latency of visually
evoked responses was greater with increasing
r,. That is, the animals recovered sooner from
TET injection if they were allowed to remain
hypothermic at a cooler 7\. If TET induces
a regulated hypothermia in the rat as it does
in the mouse, then maintaining the animal at
a relatively warm T, and attenuating the TET-
induced hypothermia may accentuate the
neurotoxic effect of TET. It is well known that
changes in ambient temperature above or be-
-------
THERMOREGULATION IN MICE
549
low thermoneutral levels can magnify the tox-
icity of various drugs and toxic agents (Gold-
berg and Salama, 1969; Klauenberg and Spar-
ber, 1983a,b). As shown in the present study,
TET-induced hypothermia was associated
with a lower preferred Tt. If, following TET
administration, the preferred lower ra and
body temperature cannot be selected, then the
same dose of TET may be more toxic, and
elevated temperatures may therefore be more
stressful.
In summary, ip TET administration results
in hypothermia which increases in magnitude
with decreasing 7*a. The hypothermia is as-
sociated with the inhibitory effects of TET on
metabolic heat production. However, when
given the option of selecting their thermal en-
vironment, TET-treated mice select a cooler
and consequently, lower body temperature.
The fact that TET induces regulated hypo-
thermia may be related to the effect of on
the neurotoxicity of this compound.
Since TET-induced neurotoxicity is more
pronounced in rats prevented from becoming
hypothermic (Dyer and Howell, 1982), one
might view the regulated-hypothermia re-
sponse of mice treated with TET as a survival-
motivated behavioral and physiological re-
sponse. Presently, these observations from the
present study and Dyer and Howell (1982)
are coincidental. It remains to be shown con-
clusively if TET-induced regulated hypother-
mia is a true toxicity-mediated survival re-
sponse.
ACKNOWLEDGMENTS
We thank Drs. R. Luebke, L. Gray, and P. Watkinson
for their critique of the manuscript and M. Bercegeay for
his technical support. We also thank P. KiUogh for the
design and construction of the metabolic chamber.
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GORDON, LONG, AND DYER
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Mailman, R. B„ Krigman, M. R., Frye, G. D„ and
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Slturobehavioral Toxicology and Teratology. Vol. 4, pp. 197-201, 1982. Printed in the U.S.A.
Somatosensory Dysfunction
Following Acute Trimethyltin Exposure1
WILLIAM E. HOWELL, THOMAS J. WALSH AND ROBERT S. DYER
Neurotoxicology Division MD-74B, Health Effects Research Laboratory
U. S. Environmental Protection Agency, Research Triangle Park, NC 27711
HOWELL. W. E., T. J. WALSH AND R. S. DYER. Somatosensory dysfunction following acute trimethyltin exposure.
NEUROBEHAV. TOXICOL. TERATOL. 4(2) 197-201. 1982.—A variety of trimethyltin (TMT)-produced sensory and
behavioral dysfunctions have been reported. In this study the functional integrity of the somatosensory system was
evaluated. Animals were tested using three different measures prior to (day 0) and 1,4, and 16 days following dosing with
either 0 (saline) or 7 mg/kg TMT. The tests employed were: (1) hot-plate pain threshold; (2) dorsal caudal nerve response
threshold and conduction velocity; and (J) the somatosensory evoked response (SER). In the hot-plate test, TMT treated
animals took longer to lick the hind paws in response to the heat. No effects were seen in the nerve function evaluation
(conduction velocity and threshold), although increased N1. PI, and P2 latencies and decreased N1 PI amplitude were seen
in SER recordings. The fact that no peripheral nerve function effects were observed suggests that the hot-plate and SER
findings were the result of central nervous system dysfunction.
Trimethyltin neurotoxicity Somatosensory evoked response Pain threshold Peripheral nerve function
THE neuropathpology associated with trimethyltin (TMT)
exposure primarily involves the limbic system, and in par-
ticular the hippocampal CA3-CA4 pyramidal cell line [3, 9,
22], In spite of this restrictive damage, a variety of
neurobehavioral effects have been reported to follow TMT
exposure. For example, acute administration of TMT
produces hyperreactivity, spontaneous seizures, sensory
system disturbances, and learning and memory impairments
[5,8,10,16,18,20,23,24). Furthermore, we have observed
that a characteristic behavior of TMT treated rats is self-
mutilation restricted to the tail [8]. Several agents such as
morphine [13], caffeine [4], and pemoline (17], have also been
shown to produce self-mutilation. The self-mutilation follow-
ing these treatments however, is not typically restricted to
any particular anatomical region. The regional specificity of
TMT-induced self-imrtitioa suggests that deafferentatioo of
the tail might be a caaaative factor [14,15], In the following
series of experiment* we examined the integrity of the
somatosensory system at three levels of neural organization
following TMT exposure: peripheral nerve (taction; CNS
processing of sensory information; and behavioral reactivity
to a painAil thermal stimulus.
MRBOO
Forty-two male Long-Evans hooded rats (320-340 g)
anesthetized with 3.7 ml/kg Chloropent (Fort Dodge Labora-
tories, Inc.) were implanted with skull electrodes for record-
¦This manuscript has been reviewed by the Health Effects Reseat
publication. Mention of trade names or commercial products does
ing the SER. Stainless steel jeweler screws (00-90 x '/is in.)
were threaded into the skull at 2.3 mm posterior and 2.5 mm
left of bregma (primary somatosensory cortex), and 2.0 mm
anterior and 2.0 mm left and right of bregma (reference and
ground, respectively). Electrodes were connected to an Am-
phenol receptacle and encased in dental acrylic. Following
surgery each animal received 100,000 units Penicillin G (IM).
Animals were given a seven day postoperative recovery
period before testing was begun and were individually
housed fat plastic cages with wood chip bedding. A 12:12
light:dark schedule with ad lib food and water was followed.
The sequence of events on each test day was: (I) hot-plate
test; (2) Chloropent injection; (3) nerve conduction evalua-
tion; and (4) SER recording. Immediately following testing
on day 0, animals were treated with either 0 (n» 17) or 7
(n-19) mg/kg trimethyltin chloride as base by gavage and
re-tested 1,4, and 16 days after dosing.
Hat-Plate
Behavioral reactivity to thermal pain was assessed with a
hot-plate method modified from Dennis et at. [7]. During
testing, each rat was placed on a Plexiglas-surrounded slide
wanner which was preheated to 51±0.5*C. The dependent
measure of pain sensitivity was the latency to lick either of
the hind paws. Two trials (LICK 1 ami LICK 2) separated by
a 180 sec intertrial interval were ran, each with a 60 sec
maximum duration to avoid damaging tissue.
sboratory, U. S. Environmental Protection Agency sod approved for
constitute endorsement or recommendation for use,
197
-------
198
HOWELL, WALSH AND D'
Dorsal Caudal Nerve Conduction
Immediately following hot-plate testing animals were
anesthetized with Chloropent (3.7 ml/kg) and placed into a
stereotaxic frame mounted above a temperature controlled
paraffin oil bath (37.5°C); pivoting the frame allowed immer-
sion of the tail into the oil. Before the tail was immersed,
three pairs of platinum needle electrodes (Grass E2B) were
inserted into the tail: two stimulating and one recording. The
proximal electrode of each stimulating pair was the anode.
The recording pair was positioned at a distal location within
the right dorsal caudal muscle. The two stimulating pairs
were separated by 100±0.5 mm (cathode to cathode) and
positioned proximal to the recording electrodes (cathode of
distal stimulating pair 10±0.5 mm from proximal electrode of
recording pair). Electrodes of each stimulating and recording
pair were separated by 1.0 mm. The tail was grounded be-
tween the recording and most distal stimulating pair (5 mm
distal from the cathode). The stimulating electrodes were
inserted alongside the right dorsal caudal nerve, penetrating
the skin surface by approximately 1 nun. Following elec-
trode insertion, the tail was lowered into the warm oil and
allowed to equilibrate for 5 tnin before testing was begun.
Leads from the recording electrodes were connected to a
high impedance amplifier with high* and low-pass filter set-
tings at 0.3 and 10 kHz, respectively. A cable leading to an
identically configured second amplifier was connected to the
head socket for recording the SER. A Grass S-88 stimulator
with PSIU6 constant current adapters was used for generat-
ing all stimulus pulses. Each stimulus consisted of a square
wave pulse (5 Hz, 0.1 msec duration). Threshold stimulus
intensity for eliciting a compound nerve action potential was
determined by gradually increasing the current level until the
compound nerve action potential was visually detected on an
oscilloscope. The proximal stimulating pair was used to de-
termine this threshold. This pair was then supramaximally
stimulated at an intensity level 50% above that level at which
any further action potential amplitude increase could no
longer be detected. Thirty-two compound action potential
responses were averaged with a Nicolet 1072 averager (20
{jmg sampling rate). The latency of the take-off point for the
first positive deflection was determined. The same proce-
dure was then followed for the distal stimulating pair. The
latency difference between these two measures and the in-
terelectrode pair distance (100 mm) were then used to com-
pute the mixed nerve conduction velocity (m/sec).
Somatosensory Evoked Response
The proximal stimulating electrode pair used during nerve
conduction testing was used to elicit the SER (proximal elec-
trode as cathode). The caudal nerve was stimulated at as
intensity sufficient to produce a maximal compound nerve
action potential, with a square pulse (1 Hz, 0.1 msec dura-
tion). Sixty-four responses were used to derive an average
waveform. M^or peak latencies (to the nearest 0.002 msec)
and peak-to-peak amplitudes were determined.
RESULTS
Data from all three experiments were analyzed within a
single repeated measures multivariate analysis of variance
(MANOVA) [21]. Data from day 16 were not included in the
analysis because of head plug pulls and TMT related mortal-
ity. Difference scores (Day X-Day 0) derived from collected
data were used in all analyses. Pillai's Trace indicated a sig-
CONTROL
FIG. 1. Difference scores for latency to hind-paw lick XlDays 1
4)-Day 0 (MeanssSE). "Significantly different from con
p<0.005.
and
urol
'd ^
10.
ied
kit
5
nificant main effect of dose, F(13,21)»3.64.p<0.0043. art*
significant dose by day interaction, F< 13,17)=3.
p <0.0156. Individual variables were subsequently assess
using univariate ANOVAs.
Hot-Plate
TMT significantly increased latencies to hind-paw li
during the first (LICK 1), F(l,65)»7.J4, p<0.0097, but n
the second (LICK 2) test session (Fig. 1). No dose by d;
interactions were observed for either variable.
Dorsal Caudal Nerve Conduction
TMT exposure produced no significant changes in eitht
conduction velocity or stimulus intensity threshold for elici
ing the compound nerve action potential (Table 1).
Somatosensory Evoked Response
The SER elicited by caudal nerve stimulation consisted c
three negative peaks at approximately 11, 18, and 42 msec
and two positive peaks at 14 and 23 msec. Schematic SER
using the mean latencies and amplitudes for the control an'
TMT groups on Day 4 are plotted in Fig. 2.
In general, TMT increased SER latencies and decrease'
SER amplitudes. ANOVAs demonstrated significant dose b)
day interactions for N1 and PI latencies (Fig. 3A) and N1P1
amplitude (Fig. 4), Fs(t,29)>12.40, p's<0.0014; while a
significant main effect of dose was observed for P2 latency
(Fig. 3B). F(1,29)-5.88, p <0.0200. Within day analysis re-
vealed that Nl and Pt latencies were significantly increased.
Fs(l,29)>6.41, p's<0.0170, and N1P1 amplitude was de-
creased, F(l,29)«8.74,p<0.0061, on day 4.
DISCUSSION
The results demonstrate that exposure to TMT produces
somatosensory dysftmction. The absence of effects on
measures of peripheral mixed nerve function suggests that
the observed hot-plate and SER changes were the result of
central nervous system impairment. This hypothesis might
receive Anther indirect support from the observation that
while TMT increases hot-plat* latencies it does not alter
-------
tmt and somatosensory dysfunction
199
TABLE 1
MIXED NERVE CONDUCTION VELOCITY (m/sec) AND STIMULUS INTENSITY LEVEL
THRESHOLD (mA) FOR ELICITING A COMPOUND NERVE ACTION POTENTIAL
FOLLOWING 7 mg/l£g TRIMETHYLTIN (TMT) EXPOSURE'
Dose
X Days 0, 1 and 4
X Difference Score
X (Days I and 4)-Day 0
Conduction
Control
129.28
£ 1.23
0.90
s 0.61
Velocity (m/sec)
N-33
F( 1.65)-1,72
p-0.1985
7 mg/kg
130.53
s 0.32
-0.32
= 0.50
N-33
Threshold (mA)
Control
2.01
* 0.01
0.13
£ 0.08
F(l,65)«2.21
N-33
p =0.1463
7 mg/kg
2.05
£ 0.01
-0.13
~ 0.10
N-33
'Means ± SE; no significant treatment effects.
Conduction velocity values (m/sec) were determined using the compound nerve action
potential elicited by supramaximal electrical stimulation of the dorsal caudal nerve. The
minimum stimulus intensity level (mA) necessary to visibly detect the nerve action
potential on an oscilloscope was used as the threshold value.
UkTOKTana
FIG. 2. Schematic somatosensory evoked potentials (SEJt) with
mean (Day 4) peak Isasncias and amplitudes plotted for control and
TMT treated]
1:: ***\
FIG. 3. Effects of 7 mg/kg TMT on peak latencies of the SER elicited
by electrical stimulation of the dorsal caudal nerve. (A) Nf and PI
latency dttbmce scorn for days t and 4 (Day X-Dey 0); a dose by
day interaction with significance on day 4 for each was observed
(solid line-coe«roi; dashed line-TMT mated). (B) Difference
scores for runaming SEJt peak latencies, X(Deys 1 and 4)-Day 0),
with a significant oaia effect of dose for PZ latency. Positive differ-
enee scone it Figs. 3A and 3B represent increased latencies
(MeansxSE) (open bars>*control; crose-hatched ban-TMT -
treated). *Hgnrtlcantly different from control p <0.03.
flinch-jump responses to electric shock [23]. It haa been
suggested by a number of investigators that these two meas-
ures of pain sensitivity are organized at different levels of the
neuraxis, with flinch-jump responses mediated at a spinal
reflex level and hot-piate responses at a mors central inte-
grative level [2,6]. Therefore, the data faun the present ex-
periment do support a central (at least supraspinal), and not
peripheral somatosensory system locos of action for TMT.
The early components of the SER (N1-P1-N2) am gener-
ally thought to represent the arrival of input to primary
somatosensory cortex via specific somatosensory pathways
of the lemniscal system. This system is primarily concerned
with the mediation of touch and kinesthesia. The later com-
ponents at the SER (P2-N3) an thought to be produced by
inputs to somatosensory cortex from non-specific, extra-
IttniHafal brain legions [1,19]. Specific somatosensory
pathway input is transmitted via long, large diameter nerve
fiber*, whereas shorter, small diameter fibers are found in
tiie nonspecific pathways [11]. Since both earty (N1 and PI)
and lata (R) component peak latencies were increased, it is
likely that TMT influences both specific and nonspecific
somatosensory systems. These lately increases represent
either (1) decreased axonal conduction velocity along
sontatoseneory pathways within the CNS; (2) increased
-------
200
HOWELL, WALSH AND DYER
«•
I
S
t
0
s
t
N.'l '("J Vl I-JWJ
OA* 1
"I'l 't«l «J»! 'j«S
o»v«
FIG. 4. Effects of 7 mg/kg TMT on SER peak-to-peak amplitudes
recorded I and 4 days following exposure. Positive difference scores
(Day X-Day 0} represent increased amplitudes t Means rSE). 'Sig-
nificantly different from control?<0.01.
synaptic delay; or (3) non-synchronous timing of somatosen-
sory information within the CNS.
A CNS axonal conduction velocity decrease could be
produced by axonal demyelination. Such a mechanism is not
supported by the fact that no velocity change was evident in
the caudal nerve, nor have there been any reports of de-
myeiination following TMT exposure. Loss of cells with fast
conducting axons i.e., large neurons with large diameter
axons, or the degeneration of these axons within somatosen-
sory pathways, would also increase SER latencies. Although
there have been no reports of TMT related pathology within
somatosensory systems, TMT does produce damage within
the limbic system 15, 9, 22]. Of particular interest is the ob-
servation that in the rat hippocampus, the largest neurons
are among the most affected [9]. A loss of neurons might also
account for the decreased NIP1 amplitude.
Potential causes for an increased synaptic delay include
disruption of transmitter availability, release, or postsynap-
tic receptor binding. A decrease in synaptosomal uptake of
gamma-aminobutyric acid (GABA) following TMT exposui*
has been reported [22). Non-synchronous timing
somatosensory information within the CNS could be a by
product of such synaptic impairment. Any influence on the
dispersion of nerve impulses elicited by somatosensory input
might serve to temporally broaden components of the SER;
this in turn might increase the temporal separation of these
components and decrease their corresponding peak ampli-
tudes (such as the observed N1P1 amplitude decrease). To
test this we compared the mean time (r SE) between N t and
N2 for the control and TMT treated groups: there was no
significant difference for this measure (71.82:3.0 msec vs
70.4±2.* msec, respectively). Thus, if observed latency in-
creases are due to synaptic delay, this delay does not appear
to influence the timing of somatosensory events within the
CNS.
The P2 peak latency increase suggests that TMT influ-
ences non-specific somatosensory systems. The increased
pain threshold or decrease in reactivity to pain demonstrated
by the hot-plate experiment may be the behavioral counter-
part of this electrophysiological finding. The reticular for-
mation has been associated with processes of pain percep-
tion, as well as the generation of the late SER components
(such as P2) (1, 3, 19]. However, in tight of the fact that
barbiturate anesthesia suppresses the reticular activating
system (12], the contribution of this system to the SER
should be questioned. It is more likely that TMT damage
occurred in other non-specific somatosensory regions, such
as the lateral spinothalamic tract which also mediates pain.
In conclusion, somatic sensation from the tail is clearly
altered but not eliminated by TMT exposure. The question
therefore remains whether the self-mutilation produced by
TMT is related to this sensory dysfunction. SER findings
suggest that the origin of the dysfunction is probably central
and that cell loss within specific and nonspecific somatosen-
sory pathways may be involved.
1. Beck, E. C. Electrophysiology and behavior. A. A«v. pxychoi.
26: 233-262* 1975-. .... .
2. Bennan, D. W., S. Caidecott-Hazard, Y. Shavit and I. C.
Liebeskiad. Analgesia produced by kindled seizure* in rats.
Soc. Newmci. Abstr. 7: 31, 1911.
3. Bowsher* 9. Role of the reticular formation in responses to
noxious liaUai. Pain 2s 361-378, 1976.
4. Boyd, B. M., M. Dolman, L. M. Knight and E. P. Sbeppard.
The chronfc oral toxicity of caffeine. Can. J. Physioi. 43s 955-
1005,1963.
5. Brown, A. W., W. N. AJdridge. B. W. Street and R. D. Vee
scboyle. The behavioral and oeuropathoiogical sequelae of in-
toxication by trimethyltin compounds. Am. J. Path. 97:39-42*
1979.
6. CaJdecott-Hazard, Y. Shavit and J. C. I.ieheskmd. Kindled sei-
zures appear to cause analgesia preferentially on the affective
component at pain. Soc. Nturosci. Abstr. It Ml, 1981.
7. Dennis, S. 0„ R. Melzack, S. GuUnaa end P. Boucher. Pain
modulation by adrenergic agents and morphine as measured by
three pain tens. Lift Sci. 3tt 1247-1299, 1910.
8. Dyer. R. S..T. J, Walsh, W. P. Wooderfa and M. Bereegaay.
The trimethyltin syndrome in rats, Seurobthav. Toxicol.
Ttratoi. 4t 127-133, 1982.
9. Dyer. R. S., T. L, Deshields and W. F. Wonderlin.
Trimethyltin-induced changes m gross morphology of the hip-
pocampus. Neurobfkav. Toxicol. Teratol. 4t 141-147, 1982.
10. Dyer. R- S. W, E. Howell and W. P. Wonderlin. Visual system
dysfunction following acute trimethyltin exposure in rats.
Nturobrhav. Toxicol. Ttratoi. 4t 191-195, 1982.
11. Katz, S., Martin, H. P.. and J. G. Blackburn. The effects of
interaction between large and small diameter fiber systems on
the somatosensory evoked potential. Elearoenceph. din. Stu-
ropkysioi. 4St 43-32,1971.
12. KiDaaa, E. K. Drag action on the bain-stem reticular formation.
Pharmmc. ttn. 1* 173-223, 1962.
13. Leander, J. D., D. E. McMillan sad L. S. Harris. Schedule-
jmtiKni oral narcotic self-administration: acute ahd chronic ef-
fects. J. Pharmac. txp. Thtr. 19St 279-287. 1973.
14 Levitt. M. and J. P. Heybach. The deafferentation syndrome in
"y Mind tats: a model of the painflil phantom limb. Pain
I* 67-73, 1981.
13. Lomberd, M. C., B. S. NasboM. Jr., D. Albe-Fessard. N. Sal-
0MB and C. Sakr. Deaffmnuoon hypersensitivity in the rat
after dorsal rMaotoay: a possible animal model of chronic pain.
Paim to 163-174, 1979.
-------
TN(T and somatosensory dysfunction
201
<6 Mactutus. C. F., J. J. Vaides, J. S. Young and Z. Annau.
Trimethyltin neurotoxicity: a potential neurobiologicai tool for
teaming and memory. Soc. Seurosci. Abstr. 7: 647, 1981.
17 Mueller. K. and S. Hsaio. Pemoline-induced self-biting in rats
and self-mutilation in the deLange syndrome. Pharmac.
Bioihem. Behav. 13: 627-631, 1980.
18. Myers. R. D.. H. S. Swartzwelder and R. S. Dyer. Acute treat-
ment with trimethyltin alters alcohol self-selection. Psycho-
phurmm'otouy. in press. 1982.
19. O'Brien. J• H. and S. H. Rosenblum. Contribution of non-
specific thalamus to sensory evoked activity in cat post-arcuate
conex. J- Seurophysiol. 37: 430-442. 1974.
20. Ruppert. P- H., T. J. Walsh. L. W. Reiter and R. S. Dyer.
Trimethyltin-induced hyperactivity: Time course and pattern.
Seurobehav. Toxicol. Teratol. 4: 135-139, 1982.
21. Statistical Analysis System (SAS). Raleigh, NC: SAS Institute,
1979.
22. Vaides. J. J., C. F. Mactutus, R. M. Santos-Anderson, R. Daw-
son and Z. Annau. Selective chemical and neuronal lesions in
hippocampus of trimethyltin treated rats. Soc. Seurosci. Abstr.
7: 713, 1981.
23. Walsh, T, J„ M. Gallagher, E. Bostock and R. S. Dyer,
Trimethyltin impairs retention of a passive avoidance task.
Seurobehav. Toxicol. Teratol. 4: 163-167. 1982.
24. Walsh, T. J., D. B. Miller and R. S. Dyer. Trimethyltin, a selec-
tive limbic system neuroioxicant, impairs radiaJ-arm maze per-
formance. Seurobehav. Toxicol. Teratol. 4: 177-183, 1982.
-------
^[
-------
/«WJ fWSctrmr rmhKtktrt 0 ¥
/VtW'fMVMi m tkr Snemft W Frmctut of Tonfoiogy
4 W Hmrtt. * (' SrWJi aWTS Mtrm rJt
A*>KYLTlNs
ill
INHIBITION OF ATPASE ACTIVITIES OF BRAIN AND LIVER HOMOGENATES AY
TRIE THYL TIN
ATPase activity in liver was below detectable limits. Tissue tin levels were deter-
mined by atomic absorption spectroscopy (10).
RESULTS AND DISCUSSION
The relative sensitivities to Inhibition by TET for homogenate ATPases were:
liver mitochondrial ATPase » brain mitochondrial ATPase, brain Na*/K*-ATPase
» brain and liver nonspecific ATPase (Fig. I and 2| Table 0. The Kj for brain
Na*/K*-ATPase was comparable with literature values for brain microsomal frac-
tions, while the K. lor the brain mitochondrial ATPase was much higher than for
isolated brain mitochondria (I). The higher apparent K- value for brain homoge-
nate mitochondrial ATPase may be due to protective partitioning of TET into
myelin and other lipid-rich structures, or to a basic difference in the sensitivity of
-------
5lt
newal and hepatic mitochondrial ATPases to TET. The latter, however, seems un-
likely since Moore and Brody (10 report that TCT has comparable effects on nttf*
al and hepatic Mitochondria.
<*
K
Snot
u
* <
M
<
SO
»-«
i*
D
TET (Ml
F%. I. Mibitioa oI tram ho«0|enalr ATPases by TET.
|100
* '
•»
>-
g SO
2
5
>ui*.
„-4
TET (Ml
Fig. 2. Mllilion of liver homogenate ATPases by TET.
519
TABLE I
APPARENT K. VALUES FOR TET INHIBITION OF ATPases IN HOMOGENATES
Tissue
Na*/K*-
Mitochondrial
Nonspecific
ATPase
ATPase
ATPase
Brain
2.J x 10'* M
2.* x 10"* M
I.I x 10*' M
Liver
2.) x 10"* M
4.2 x 10'3 M
In order to determine if tissue concentrations of TET become sufficient to
inhibit ATPases following a neurotoxic dosage, tin levels were determined in brains
and livers from rats given 4.0 mg/kg TET-Br (10). Peak brain tin concentration was
17 pM at 2% hr, a concentration which was insufficient to inhibit ATPases in brain
homogenates. In liver, however, peak concentration was I)) pM, which should be
sufficient to inhibit hepatic mitochondrial ATPaie.
To test whether inhibition of netral or hepatic ATPases occurs following a
neurotoxic dose of TET, animals were treated with 4.0 mg/kg TET-Br, and ATPase
activities of brain and liver homogenates measured (Fig. 3). Animals were tested at
I hr (to evaluate early effects) and at 24 hoirs (time of peak tin accomodation in
brain). Neither brain nor liver ATPase activities differed from controls at either I
or 24 hr post-exposure. Although these results were predicted for brain* the
S
| too
r
l40
~s.
I
_ 90
S
I"
Fig. J. ATPase activities m tiuue homo,twit— Inm rats injected with TET.
m
jm
|i
•m
~ c«NI
E9 TET
nsf!
m
m-
241
<
vs.
r*tt.
rf
rh
g
M
L*CA
«
-Mm
rf
1
M
i
ft
j
£
NaK M
_ LtVCR-MIn
; fi n
N
1
s
-------
520
expected inhibition in liver was not observed, even taking info account the dilution
o/ the samples inherent m the ATPase assay. The lack of inhibition of liver mito-
chondrial ATPase after in vivo exposure to TET si^gests that measured tin, even
at one hr post-injection, was no longer predominately m the form of TET. TET and
other aftflims Have teen shown to be dealkyiated m the liver by a cytochrome
P-*JO dependent process (12), and the metabolites may be less potent inhibitors.
CONCLUSIONS
Higher concentrations of TET are necessary to inhibit mitochondrial ATPase
in brain homofenates than an isolated brain mitochondria or in liver homoge nates.
The greater apparent K. lor mitochondrial ATPase in bram homogenate may be due
to binding of TET to other tissue constituents such as myelin. Tin concentrations
•n brain following a neurotoxic nposwe to TET are insufficient to inhibit any
brain ATPase activity measured. In liver, by contrast, tin concentrations ought to
be sufficient to produce inhibition, but liver homogenates from TET-treated ani-
mals showed no difference in activity. The lac* of apparent inhibition in the liver
indicates that TET m liver may be bound, metabolized or otherwise rendered
Incapable of ATPase Htutanian.
ACKNOWLEDGEMENTS
This research was Hfported by EPA CRMMM-02 aid MH GM-28999.
REFERENCES
1. Wassenaar. 3.$^ and Kroon, AM. 1973. Eur. Netvol. 10: *9-370.
2. Li*m*y, f., and Abfridge, ®.N. 1973. Biochem. Pharm. 2*: l*53~I*3S.
3. Akfcidge, V.fL, and Street, B.W. J971. Biochem. 3. 124: 221-23*.
~. Aldridge, #.N„ and Street, B.». 1970. Biochem. 3. Ills 171-179.
3. Farrow, R.C, and Dawson, A.P. 197*. Etr. 3. Biochem. 177: *61-470.
4. ENsot, BJkl., Akbidge, *.«L, and Bridges, 3.#. 1979. Biochem. 3. 177-. *41*70.
7. Lock, E.Am and Attidge, W.N. 1973. 3. Neurochem. 23: 171476.
t. Pullman, M.E^ Penelsky, H.S^ Dalta, A. and Racket, E. I960. 3. Biol. Chem.
133s 3322-3329.
9. Sorenson, R.O, and Mahler. H.R. I9f I. 3. Nwodien. 37: 1*07-1*18.
10. Cook, LLn Jacobi, K.S*, and fteiter, L.V. 1911. Ton. Appf. Pharm. (in press).
11. Moore, KX, and Brady. T.M. 1961. Biochem. Pharm. 6: 123-1)3.
12. VieMdn, P„ ProMgh, R.A., and Bridges, 3.*. I9S2. Tor. Appl. Pharm. 62:
*09-*20.
-------
-------
Neurobehavioral Toxicology and Teratology. Vol. 4, pp. 225-230. 1982. Printed in the U.S.A.
Studies on the Flavor Aversions
Induced by Trialkyltin Compounds1,2'3
R. C. MacPHAIL
Neurotoxicology Division, U. S. Environmental Protection Agency
Research Triangle Park, NC 27711
MacPHAIL, R. C. Studies on the flavor aversions induced by trialkyltin compounds. NEUROBEHAV. TOXICOL.
TERATOL. 4(2) 225-230, 1982.—These experiments were undertaken to determine the suitability of a flavor-aversion-
conditioning paradigm for detecting the effects of trimethyltin and triethyltin. Both organotins produced flavor aversions
whose magnitude depended jointly on the dosage administered and the number of flavor-organotin pairings. Estimated ED
50s (for triethyltin. 1.8 mg/kg; for trimethyltin, 3.1 mg/kg) were smaller than the dosages reported to affect other classes of
behavior, but nevertheless represented a substantial percentage (25-45%) of the respective published LD-50 values.
Flavor-aversion conditioning may represent a valuable tool for studying the effects of organic and inorganic heavy metals.
Trimethyltin Triethyltin Flavor aversion
ORGANOTINS form a broad category of compounds that
are widely used commercially and which produce a mul-
titude of biological effects [42]. Mono- and di-substituted
organotin compounds are primarily used industrially in the
manufacture of polyvinyl chloride and polyurethane foams.
Tetrasubstituted organotins are used mainly in the prepara-
tion of other organotin compounds. Many trisubstituted or-
ganotins have been widely used as agricultural, marine and
general biocides, most notably tributyl-, triphenyl- and
tricyclohexyl-tin. Although trimethyl- and triethyl-tin have
been used in fewer agricultural and industrial contexts, they
are being increasingly used in research because of their
prominent effects on behavior and the nervous system.
Triethyltin produces cerebral edema in adult rats that is as-
sociated with intramyelinic vascuolation [26], Repeated
treatment of adult rats produces ataxia and hindlimb
paralysis, which are generally reversible if dosing is stopped
before the animals become severely debilitated [26]. Acute
and short-term-repeated treatment of neonate rats produces
persistent hyperactivity in adults and a different profile of
central nervous system effects that are thought to be due to
the inhibitory effect of triethyltin on cellular respiration.
Trimethyltin produces destruction of portions of the hip-
pocampus and surrounding area by an unknown mechanism
[1.8]. Acute treatment of adult rats produces hyperactivity
under many test conditions [35]; acute treatment of adult rats
with targe dosages induces a multifaceted syndrome of be-
havioral change somewhat similar to that produced by le-
sions of the limbic system [14].
There is now great concern over the development of test
methods for detecting and characterizing the behavioral and
neurological consequences of exposure to toxic substances
[12, 19, 29]. One very promising method involves flavor-
aversion conditioning. Garcia and his colleagues, for exam-
ple, showed that rats' preferences for distinctly flavored so-
lutions could be simply yet substantially reduced by pairing
flavor intake with exposure to x-irradiation ([15]; for early
review see [32]). Numerous subsequent experiments have
both replicated these findings and extended them to include a
wide array of drugs and other biomedically relevant chemi-
cals. The ease and rapidity with which flavor-aversion tests
can be carried out make them choice candidates for the
routine preliminary assessment of neurotoxicants. The pres-
ent series of experiments was therefore undertaken to de-
termine the suitability of a flavor-aversion-conditioning
paradigm for detecting the effects of trimethyltin and
triethyltin. These experiments were part of a research pro-
gram on flavor-aversion paradigms and their use in assessing
the effects of agricultural, industrial and environmentally
relevant chemicals. Flavor aversions have already been
produced by arsenic [36], cadmium [24], copper sulfate
[10,27], lead [11,20], methylmercury [6,22], pesticides such
as chlordimeform [25] and 2, 4, 5-T [37,38], and acrylamide
[3]. In addition, Leander and Gau [20] have recently shown
'A preliminary report of a portion of these data was presented at the annual conference of the Society of Toxicology. 1981 (The Toxicofoitist
1; 4*45. 1981). ,
'I thank J, D, Leander for specific advice and general support, R. S. Dyer and T. J. Walsh for patience, and P. Keeter for secretarial
assistance.
This manuscript has been reviewed by the Health Effects Research Laboratory, U. S. Environmental Protection Agency, and approved for
publication. Mention of trade names or commercial products does not constitute endorsement of recommendation for use.
22S
-------
226
MacPHAIL
that flavor aversions can be conditioned by triethyltin. The
present series of experiments involved a systematic replica-
tion of the Leander and Gau [20] findings for triethyltin, and
an extension to collect data on the flavor-aversions con-
ditioned by trimethyltin.
METHOD
Animals
Adult male Long-Evans-derived (hooded) rats (Charles
River Breeding Laboratories, Wilmington, MD) were used
throughout except in one experiment (Experiment IV) which
also used adult females. The rats were housed in groups of
two or three in clear plastic cages (45 x 24 x 20 cm) with direct
bedding and with food (Purina Laboratory Chow No. 5001,
St. Louis. MO) provided ad lib. Cages were located in a
temperature-controlled (21 ±0.6"C) colony room in which
artificial lighting was provided from 0600 to 1800 hr daily.
Apparatus
The test apparatus was the same as described previously
[25] and briefly consisted of a rack of small metal ceiling-
suspended cages (Wahmann Mfg. Co., Timmonium MD,
24.5x 18x18 cm) to which either one or two fluid-filled glass
bottles with metal drinking spouts could be attached.
Procedure
In general, the procedures were similar to those described
previously [25] although unique details are given below for
each of the experiments. All rats were first acclimated to the
colony room for several days and then placed on a 23-'/j hr
water-deprivation schedule. Tap water was provided for 30
min daily to individual rats in the test cages. The position of
the water bottle was counterbalanced for each rat across test
sessions and across rats for any one test session. Once in-
takes had stabilized, each rat was given access to one bottle
containing tap water and one containing a 0.1% (w/v) solu-
tion of sodium saccharin, or to saccharin alone, for one
30-min session. Experiment II differed in that the distinctive
flavor was 0.9% saline solution. Approximately 20 min after
the end of the session, the rats received either a dosage of the
toxicant, the toxicant vehicle, or no treatment. Six rats were
generally included in each treatment group. All rats contin-
ued to receive limited acess to tap water alone for the next
two sessions. On the third session after dosing, each rat
received concurrent access to tap water and the flavored
solution. In some experiments, the same treatment was ad-
ministered again to each rat and followed by concurrent
availability of tap water and flavored solution three sessions
later; in different experiments, this cycle was in effect for a
total of two to six pairings of flavor availability and toxicant
administration (see below).
Experiment I: Triethyltin. This experiment provided nor-
mative data on the flavor aversions produced by triethyltin,
and preliminary data on the effect of repeated pairing of
flavor intake and triethyltin administration. After six days of
adaptation to limited water availability, rats were given con-
current access to water and saccharin solution for 30 min and
then approximately 20 min later, either triethyltin or the
normal saline vehicle. Saccharin preference was determined
three days later, as described above, and followed by a sec-
ond administration of triethyltin or the saline vehicle, and a
final determination of saccharin preference three days later.
Triethyltin-Br (Alfa Products, Danvers, MA) was dissolved in
the saline vehicle and administered IP in a volume of 1 ml/kg
body weight, in dosages of 0.375, 0.75, 1.5 or 3.0 mg of the
base/kg body weight. Each treatment group, including a
vehicle-control group, was comprised of 6 rats. An addi-
tional 6 rats received access to saccharin and water in the
test cages, and were treated with 1.5 mg/kg triethyltin, but
were not otherwise adapted to the test cages and received
access to water in their home cages. This group was included
to determine the importance of direct experience with the
test environment during stabilization of restricted water in-
takes.
Experiment II: Triethyltin. This experiment extended the
above findings to include more pairings of flavor availability
with triethyltin administration, and a different flavor
stimulus. The design of this experiment was very similar to
that of Experiment I except that the distinctively flavored
solution was 0.9% saline, and flavor intake was determined
after each of five or six pairings of the flavor with triethyltin.
Groups of six rats received 0.188, 0.375, 0.75 or 1.5 mg of
triethyltin base/kg body weight, or the normal saline vehicle,
as described above for Experiment I. Previously all rats had
been studied in a different test environment and had received
a single pairing of saccharin availability and triethyltm ad-
ministration, after which choice of saccharin was determined
on five occasions. The test arrangement proved to be inap-
propriate for our purposes and therefore the rats were reas-
signed to groups and adapted to restricted water availability
in the cages described above for an additional II days.
Triethyltin in this experiment was administered more than
five weeks after the initial treatments.
Experiment III: Trimethyltin. This experiment provided
normative data on the flavor aversions induced by
trimethyltin and on the efficacy of repeated pairings of flavor
intake and trimethyltin administration. After 11 days of ad-
aptation to limited water availability, all rats received 30 min
access to saccharin solution and approximately 20 min later
an injection of trimethyltin, normal saline, or no treatment.
Trimethyltin-OH (K and K Laboratories, Plain view NY) was
dissolved in normal saline solution and injected IP, in a vol-
ume of I ml/kg body weight, in dosages of 0.625, 1.25, 2.5
and 5.0 mg of base/kg body weight. Six rats comprised each
treatment group. Relative saccharin intake was determined
at three-day intervals after each of three pairings of saccha-
rin availability and trimethyltin administration.
Experiment IV: Trimethyltin. This experiment determined
whether there were sex differences in the flavor aversions
induced by a large dosage of trimethyltin. After 7 days of
adaptation to limited water availability, male and female rats
received 30 min access to saccharin solution and, approx-
imately 20 min later, an IP injection of trimethyltin-C 1 (5 mg
of base/kg body weight) or the normal saline vehicle (1 ml/kg
body weight). Three days later saccharin preferences were
determined. Six rats comprised each treatment group.
Trimethyltin-C 1 was obtained from the same source as in
Experiment III.
Data Analysis
In all experiments, treatment groups were compared on
the third day following toxicant administration for effects on
total fluid intake (expressed in ml) and relative saccharin (or
saline) intake (expressed as a proportion of total intake). In
addition, in some experiments estimates of ED-50 values
were made graphically: Average relative saccharin intake of
-------
TRIALKYLTIN COMPOUNDS AND FLAVOR AVERSIONS
227
0,i—i—i 1 1 r
5 o.« -
o
H
0
1
h-
Ui
s
" " 1
£ K20r&
2 p
5 " !
<,°LX
L-L I I I L_l
0 V 0.378 0.79 15 3.0
TRIETHVITIN Img/kgl
FIG. I. Flavor aversions induced by triethyltin. Each symbol repre-
sents the average effect ~ SEM of the saline vehicle (V) or a dosage
of triethyltin on the saccharin intake of rats (N=6 per group), ex-
pressed as a proportion of the total intake. Effects of each treatment
on total intake are shown in the insert. Solid symbols represent the
effects of a single pairing of flavor and toxicant and open symbols
represent the effects of two flavor-toxicant pairings.
the vehicle-control rats was first determined and divided by
two. A horizontal line was drawn from that value on the
y-axis to its point of intersection with the dose-effect func-
tion, from which a perpendicular was dropped to the x-axis.
The resulting ED-50 value was estimated by interpolation.
RESULTS
Experiment I: Effects of Triethyltin
Saccharin intake (expressed as a proportion of total in-
take) did not differ between rats receiving the two smallest
dosages of TET and those receiving the saline vehicle, but was
decreased in an orderly fashion in rats receiving the two largest
dosages (Fig. 1). The ED 50 was estimated to be 1.8 mg/kg.
The flavor aversion produced by 1.5 mg/kg was augmented
after a second pairing of saccharin and TET, whereas the
effect of smaller and larger dosages remained unchanged.
Figure 1 also shows there was no systematic effect of TET
on total fluid intake.
Table 1 compares the effect of 1.5 mg/kg of TET on sac-
charin intake of rats that were adapted to limited fluid avail-
ability in the test cages and of those placed in the test cages
on only the days when saccharin or saccharin and water
were available. Data for the former group (A) were taken
from Fig. 1. Although the average effect of TET is compara-
ble for both groups after each saccharin-TET pairing, indi-
vidual variability was substantially greater on both occasions
for the group (B) not previously adapted to the test cages.
TABLE 1
COMPARISON OF THE EFFECTS OF TRIETHYLTIN (1.5 mg/kg)
ON SACCHARIN INTAKE (AS PROPORTION OF TOTAL INTAKE)
GROUP
TEST 1
TEST 2
A
0.37
0.11
±0.08
±0.05
B
0.42
0.22
± 0.14
±0.10
SEM - B
SEM-A
1.75
2.00
Group A had extensive exposure to the test chambers whereas
Group B did not. Values are means ± SEM for groups of six rats.
0.7
_ a* -
oj •
0.4
0J
< 28
0J
0.1
X
I
T
I
0
1
4
1
I
TKIITHVITIN (TOTAL mt/kll
FIG. 2. Flavor aversions induced by triethyltin. Each symbol repre-
sents the average effect ± SEM of triethyltin (•, 0.188 mg/kg: ¦.
0.375 mg/kg; ~, 0.75 mg/kg; and ~, 1.5 mg/l
-------
228
MacPHAIL
NIC
v OMn iJi
TRMNTHVLTIN (mAtf
L
-L.
NIC
OAS 1.2t
TRIMCTHYLTIN
1.8
OJ
"g
S
" 8
0,3
s
OJ 2
02
0.1
FIG. 3. Flavor aversions induced by trimethyltin. Each symbol represents the average effect r SEM of no treatment
(NIC), the saline vehicle (V) or dosages of trimethyltin on the saccharin intake of rats (n=6 per group), expressed as a
proportion of total intake, after one (A) or three (B) flavor-toxicant pairings. Inserts show the effects of each treatment on
total intake.
dosage administered showed considerable overlap in the ef-
fects of all but the largest dosage of TET. Total fluid intake
was inversely related to TET dosage; the insert in Fig. 2
shows the effect of TET on total intake after the fifth pairing
of flavor intake with toxicant administration.
Experiment HI: Effects of Trimethyltin
The smallest dosage of TMT (0.625 mg/kg) had no effect
on relative saccharin intake whereas the largest dosage (5.0
mg/kg) produced an almost maximal aversion to saccharin
(Fig. 3). Intermediate dosages of TMT produced inter-
mediate effects. The ED 50 was estimated to be 3.1 mg/kg.
None of the dosages produced a change in total fluid intake.
The flavor aversion produced by 2.5 mg/kg was more
pronounced after three flavor-toxicant pairings. A second
administration of 5.0 mg/kg proved to be lethal to all treated
rats. Figure 3 also shows that the flavor aversions induced by
TMT were non-systematically related to its effects on total
intake.
Experiment IV: Effects of Trimethyltin
Figure 4 shows that regardless of sex, prominent flavor
(saccharin) preferences were obtained in vehicle-treated rats
whereas equally prominent flavor aversions were obtained in
TMT-treated (5 mg/kg) rats. Sex differences were evident in
total fluid intake, but for either sex the total intake did not
differ between treatment groups (data not shown).
DISCUSSION
Administration of triethyltin or trimethyltin to rats shortly
after consuming a distinctively flavored solution produced
aversions to that flavor that depended on both the dosage
and the number of flavor-toxicant pairings. Such conditioned
flavor aversions have been demonstrated after exposure to
x-irradiation (e.g., [15]) and a wide variety of psychotropic
drugs (see [33]). It is only recently, however, that flavor
aversions produced by agricultural, industrial and environ-
mental chemicals have received experimental attention. To-
gether with other results, the present finding of flavor aver-
sions produced by trimethyltin and triethyltin, suggests that
conditioned-flavor-aversion paradigms may represent a val-
uable tool for studying toxic heavy metals.
A single pairing of flavor availability and triethyltin (IP)
produced flavor aversions that had an ED 50 of 1.8 mg/kg,
and which were maximal at 3.0 mg/kg (Experiment I). After a
second flavor-triethyltin pairing, effects were also produced
by 1.5 mg/kg. In Experiment II, in which flavor and toxicant
were paired several times, flavor aversions were noticeable
in rats receiving 0.75 mg/kg. These results are in good
agreement with those obtained by Lewder and Gau [20) for
rats receiving a different salt form of triethyltin, by a differ-
ent route of administration, and tested under somewhat dif-
ferent conditions. By comparison, the locomotor activity of
rats in a figure-eight maze was decreased by only about 30%
after 3.0 mg/kg of SC triethyltin [31]. In addition Squibb et
al. [39] reported behavioral effects of triethyltin (PO) only
during the second week of twice-weekly dosing of rats with 2
-------
TRIALKYLTIN COMPOUNDS AND FLAVOR AVERSIONS
229
MALII FEMALES
FIG. 4. Flavor aversions induced by trimethyltin. Each histogram
represents the average effect + SEM of the saline vehicle (SAL) or
5.0 mg/kg of trimethyltin (TMT) on saccharin intake, expressed as a
proportion of total intake, of male and female rats.
mg/kg. The present data together with those of Leander and
Gau [20], indicate that substantial flavor aversions can be
produced by triethyltin at dosages that have much less effect
on other classes of behavior.
The LD50 for acute triethyltin has been determined to be
between 4.0 and 5.7 mg/kg, and is somewhat independent of
the route of administration [4,40]. Therefore, the ED 50 for
triethyltin obtained after one flavor-toxicant pairing is be-
tween 32 and 45% of the reported LD50 values. Although
flavor aversions were obtained at smaller dosages when they
were repeatedly paired with flavor intake, the total amount
of triethyltin actually delivered represents an even greater
percentage of the reported LD50. Thus, triethyltin produces
many prominent behavioral effects, and in the absence of
grossly observable signs of intoxication, but the effective
dosages are nevertheless a substantial fraction of those
producing lethality. Some caution is appropriate, however,
in interpreting these data since the LD50 values may be un-
usually low. In Experiment II. for example, no deaths were
obtained in rats treated repeatedly with 1.5 mg/kg. Similarly,
Squibb el al. [39] noted lethal effects in rats after the third
administration of twice-weekly 3.0 mg/kg but not after the
second administration.
A single pairing of flavor availability and trimethyltin (IP)
produced flavor aversions that had an ED 50 of 3.1 mg/kg
and that were maximal at 5.0 mg/kg. Flavor aversions were
also produced by 2.5 mg/kg after repeated pairing of flavor
and toxicant. At the same time, however, a second injection
of 5.0 mg/kg proved lethal. Moreover, after three injections
of 2.5 mg/kg rats displayed prominent signs of intoxication
similar to those previously described by Dyerer ul. [14] after
a single administration of 6 mg/kg of trimethyltin. Acute ef-
fects of trimethyltin in dosages of 5-7 mg/kg have also been
reported for locomotor activity [35] and radial-arm-maze
performance [41]. The LD50 for trimethyltin has been re-
ported to be between 7.4 mg/kg [14] and 12.6 mg/kg [8].
Therefore the ED50 for trimethyltin obtained after one
flavor-toxicant pairing is between 25 and 42% of the LD50:
these values are comparable to those for triethyltin.
Despite the fact that flavor aversions can be produced by
trialkyltin compounds at dosages that are generally below
those reported to appreciably affect other classes of behav-
ior, very little is known about their mechanism(s) of action.
Triethyltin's neurotoxic effects are in part related to the
compound's inhibition of cellular respiration that results in
cytotoxic hypoxia [2,34]. It would be interesting, therefore, to
determine whether flavor aversions can be conditioned with
other toxicants having similar effects, for example,
hexachlorophene [9] and 2'-chloro-2,4-dinito-5',6-di(trifluoro-
methyD-diphenylamine [23], Although cyanide also inhibits
cellular respiration [7] this compound was reported in two
experiments to be ineffective in inducing flavor aversions
[18,28], These negative results may be due. however, to the
particular experimental design and therefore not necessarily
representative of the effects of compounds that inhibit cellu-
lar respiration.
Trimethyltin is thought to exert many of its effects via
destruction of certain regions of the hippocampal formation
and surrounding areas [1,8]. Although similar central effects
have been produced by systemic administration of di-
piperidinoethane [21,30] and kainic acid (e.g., [5]) whether or
not these toxicants produce conditioned flavor aversions has
yet to be determined. The impoartance of hippocampal in-
volvement in the flavor aversions induced by trimethyltin
could also be assessed by determining the effects of both
prior lesioning of the hippocampus and neuropharmacologi-
cal manipulations on the conditioning of flavor aversions.
Alternatively, the flavor aversions induced by trimethyltin
may be related to changes in gustatory sensitivity. This type
of an effect would be consistent with alterations produced by
trimethyltin in the visual, auditory and somatosensory sys-
tems [13, 16, 17], In this regard further experiments are re-
quired that compare the flavor aversions and preferences
produced by prototype flavored solutions (e.g., saccharin,
quinine) in normal animals and in those pretreated with
trimethyltin.
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TOXICOLOGY AND APPLIED PHARMACOLOGY 72, 557-565 (1984)
Pre- and Postweaning Indices of Neurotoxicity in Rats:
Effects of Triethyitin (TET)1
Diane B. Miller2
Biological Sciences Research Center. University of North Carolina at ChapeI Hill, Chapel Hill, North Carolina
27514 and Neurotoxicology Division, USEPA, Health Effects Research Laboratory (MD 748),
Research Triangle Park, North Carolina 27711
Received August 17, 1983; accepted October 10. 1983
Pre- and Postweaning Indices of Neurotoxicity in Rats: Effects of Triethyitin (TET). Miller,
D. B. (1984). Toxicol. Appl. Pharmacol. 72,557-565. Pre- and postweaning measures of learning
and locomotor activity were used as indices of CNS function after early perinatal neurotoxic
insult. Triethyitin (0.0,4.0, or 6.0 mg/kg, ip) administered on Postnatal Day 5 (PND5) was used
as the neurotoxicant. Learning deficits and alterations in locomotor activity were observed during
both the pre- and postweaning periods. Preweamng learning ability was evaluated with an appetitive
alleyway paradigm, while an automated radial-arm maze (RAM) was used to assess juvenile
learning. Preweamng open-field locomotion was evaluated in the presence and absence of home-
cage litter while postweaning activity was measured in an automated device, the Motron, or as
a component of performance in the RAM. The 6.0-mg/kg TET-exposed animals required more
trials to acquire the alleyway task. RAM subjects receiving 6.0 mg/kg of TET were less accurate
than those given 3.0 mg/kg or vehicle control. TET did not affect open-field activity on PNDI0;
low levels of spontaneous locomotion occurred regardless of treatment. On PND13, there was
a dose-related decrease in locomotion over home-cage litter while all groups exhibited equivalent
low rates of locomotion in the absence of home-cage cues. All treatment groups were more
active when tested over litter than over no litter. In contrast, on PND21 and throughout the
RAM testing period, TET-exposed subjects exhibited dose-related increases in activity. TET
produced treatment-related decreases in wet weight of whole brain, hippocampus, and cerebellum
in both developing (PND23) and adult (PND200) animals with the hippocampus being most
affected on a percentage basis. In contrast, no treatment-related body weight differences were
observed at these times. The neurotoxicity of TET can be demonstrated during the preweaning
period whether a commonly used endpoint in assessing CNS function (locomotor activity) is
monitored or a more complex endpoint (learning) is evaluated provided (I) the method used to
monitor locomotor activity incorporates both the normally low levels of preweaning locomotion
and the more elevated levels induced by home-cage cues and (2) the learning paradigms utilized
are appropriate for the capabilities of the animal.
1 A preliminary report of this investigation appeared in
Toxicologist, 2, 28, 1982. This paper has been reviewed
by the Health Effects Research Laboratory, U.S. Envi-
ronmental Protection Agency, and approved for publi-
cation. Mention of trade names or commercial products
does not constitute endorsement or recommendation for
use.
] Address correspondence to the author at: Neurotox-
icology Division (MD-74B), Health Effect Research Lab-
oratory, U.S. Environmental Protection Agency, Research
Triangle Park, N.C. 27711.
Triethyitin (TET) is neurotoxic and its ad-
ministration during the early postnatal period
markedly interferes with central nervous sys-
tem (CNS) development. This disruption is
manifested in behavioral, neurochemical, and
electrophysiological alterations observed dur-
ing the entire lifespan of the organism (Dyer
et ai, 1981; Harry and Tilson, 1981; Reiter
etal., 1981; Ruppert et ai., 1983; O'Callaghan
557
004I-008X/84 $3.00
Copyright €> 1984 by Academic Pro. Inc.
AU rithti at reproduction in any form rewrved.
-------
558
DIANE B. MILLER
et al., 1983). In the rat a single exposure to
TET on Postnatal Day 5 (PND5) results in
neurobehavioral effects which include changes
in activity (Reiter et al., 1981; Ruppert et al.,
1983), deficits in sensorimotor capabilities as
well as altered learning ability (Harry and Til-
son, 1981), and electrophysiological changes
indicative of slowed sensory processing (Dyer
et al., 1981). This broad spectrum of neuro-
toxic effects makes TET an ideal compound
with which to evaluate the utility of tests de-
signed to assess the functional integrity of the
neonate or juvenile animal after early perinatal
insult. Tests designed to detect neurotoxicity
during any period of the lifespan should in-
clude evaluations of a range of CNS functions
(Adams and Buelke-Sam, 1981). However,
most evaluations of neurotoxicity during early
developmental periods have utilized the end-
points of physical maturation or reflex and
motor development (e.g., Maker et al., 1975;
Vorhees et al., 1979). In general, learning or
memory paradigms are used to assess the im-
pact of early neurotoxic insult on the mature
adult rather than on the neonate or juvenile
animal (e.g., Harry and Tilson, 1981).
The limited sensory and response capabil-
ities of the neonate make it difficult to evaluate
learning and other complex CNS functions
during the preweaning developmental period.
However, tests structured to the capabilities
of the young animal used in conjunction with
home-cage cues make such evaluations pos-
sible. Faster learning and retention of response
contingencies, as well as increases in spon-
taneous alternation and activity, are obtained
if appropriate paradigms are used to evaluate
the functional capabilities of the neonate
(Smith and Spear, 1978). The present research
demonstrates that with the use of suitable tests
the developmental perturbation produced by
early postnatal exposure to TET is evident
during both the preweaning and juvenile de-
velopmental periods whether a commonly
utilized functional endpoint (e.g., locomotor
activity) or a more complex CNS function
(e.g., learning) is evaluated.
METHODS
Animals. Gravid Long-Evans rats (Blue Spruce Farms.
Altamont, N.Y.) were obtained on Day 2 of gestation and
housed individually in plastic tub cages (43 x 25 x 20
cm) with wood shavings as bedding. Pups bom between
1700 and 0500 hr were considered to be born on the same
day (Day 0). On PND1 pups were reassigned to litters
such that each dam received four male and four female
pups and no more than one male and one female of her
own offspring. Litter was not changed from PND5 to
PND16. At 21 days of age littermates were weaned, sep-
arated by sex. and housed four per cage. Animals were
maintained in a temperature- (22 ± 2°C) and humidity-
(50 ± 10%) controlled colony room on a 12:12-hr light:dark
cycle beginning at 0600 hr.
Dosing. Intraperitoneal injections of 0.0. 3.0. or 6.0
mg/kg of triethyltin bromide (Alfa Products. Danvers,
Mass.) with saline (0.9%) as the vehicle control (10 Ail/g
body wt) were given on PND5; dose levels are expressed
as the salt. The doses used were based on previously pub-
lished work with this compound (see Ruppert et al.. 1983).
All animals in a litter received the same dose and were
foot tatooed with India ink for later identification. To
control for possible test-retest interactions, animals were
evaluated in only one test paradigm. A limited number
of randomly chosen animals (no more than one or two
of each sex) from a litter was used; the remaining animals
were removed for biochemical evaluation (see O'Callaghan
etaL 1983).
Preweaning Indices
Open field. Separate groups (n = 24) comprised of two
males and two females from six litters at each dose and
age were tested at 10 and 13 days of age for open-field
locomotion in the presence or absence of their own home-
cage litter. The apparatus was constructed of clear Plexiglas
(25-cm high. 21-cm wide, and 48-cm long) with a steel
mesh floor placed over a 2-cm deep Plexiglas pan. The
open field was demarcated into a 3 x 4 matrix of 7 em
(i.e., the length of a 10- to 13-day-old pup) squares. The
bedding pan was either empty or filled to immediately
below the mesh floor with litter (900 ml). Separate open
fields were used in the two test conditions and the entire
apparatus was placed in a covered 10-gal aquarium to
minimize ambient noise and odor. Each animal was placed
in a 1000-ml plastic beaker for 10 sec prior to the start
of testing and between conditions. Order of testing was
counterbalanced across groups and sex. Animals were
weighed immediately prior to testing which began with
the placement of the pup in the center of the open field
with its body straddling a line. Squares crossed were ob-
served for 3 min in each condition, and a count was
recorded each time the front paws crossed a line. The
-------
NEUROTOXICITY OF EARLY POSTNATAL TET
559
wire mesh floor allowed substantial forward locomotion
especially on PND13 and pivoting was rarely observed.
Interobserver reliability ratings of .90 were obtained for
the two observers prior to the start of the study.
Alleyway. On PND13 pups were tested in an appetitive
alleyway task (Amsel el al.. 1979). However, the procedure
was modified to insure treated animals were not debilitated
by the testing conditions. The reinforcer was access to
home-cage litter and littermates rather than nonnutritive
suckling on an anesthetized dam. Animals were separated
from the litter for 1 rather than 8 hr. One male and one
female per litter were evaluated (n = 10 and 12 for 0.0-
and 6.0-mg/kg TET-exposed groups, respectively).
Test procedure. During testing the home cage was
maintained at approximately 33°C by a commercial heat-
ing pad. A pup was removed from the home cage ap-
proximately I hr before testing and maintained in an
incubator (33°C); the dam was kept in a different room
during separation and testing. The Plexiglas apparatus
consisted of a 38 x 7.5 x 10-cm alleyway and a 17 x 25
x 10-cm goalbox (GB); the rear of the GB was wire mesh.
Testing included aquisition, performance, and extinction
phases. The first trial was initiated with two GB placements:
first, a 10-sec confinement in an intertrial interval (ITI)
chamber and second, placement into the GB immediately
followed by 30-sec access to litter and littermates. During
acquisition, litter odors were provided in the alleyway by
tilting the home cage against the wire end of the GB so
"homing" could facilitate the acquisition of the response.
A pup was placed in the start area facing away from the
GB and given 120 sec to traverse the distance. If the
response did not occur within the required time, the pup
was pushed to the GB. A GB entrance was followed by
30 sec access to the homecage. Acquisition trials continued
until a criterion of five consecutive trials in less than 120
sec was reached. The alleyway was wiped between trials.
The performance phase consisted of 15 consecutive trials
with a 10-sec ITI in which the reinforcement conditions
were identical to those of the acquisition phase but the
odor cues in the alleyway were eliminated. During ex-
tinction the pup was confined to the goalbox for 30 sec
and not given access to the home cage. Extinction trials
continued until two consecutive trials occurred in which
the GB was not reached within 60 sec.
Postweaning Indices
Motron. Equal numbers of male and female pups (n
~ 12/group, 2 males and 2 females/litter) were tested in
an automated activity monitoring device early in the ju-
venile period on PND21. Dams were removed in the early
morning, and pups were tested between 1300 and 1600
hr. Motor activity was recorded in six commercial photocell
devices (Motron Electronic Motility Meters, Motron
Products, Stockholm, Sweden). Each consisted of a plat-
form with a matrix of 40 photocells that were illuminated
by a single overhead incandescent lamp (GE 30R20, 30
W). Any horizontally directed movement that interrupted
a photocell was recorded and will be referred to as lo-
comotion. A Plexigias chamber (33 x 21 X 26 cm), placed
on top of the platform, was used to contain the rats and
had a removable lid. Each entire chamber and photocell
apparatus was placed in a wooden enclosure that was
lined with acoustically absorbent ruber (FM11. Southern
Kinetics. Raleigh N.C.) and equipped with an exhaust
(Rotron WR2A1, Woodstock. N.Y.). The six chambers
and enclosures were housed permanently in a small test
room (5 X 11 X 10 ft) that was lined with acoustic tile.
Data collection was carried out in an adjacent room with
an Intel 80/80 microprocessor. Rats were tested individ-
ually for a 25-min session with photocell interruptions
recorded in 5-min blocks.
Radial-arm maze (RAM). Animals (3 males and 3 fe-
males/group, randomly selected from different litters) were
tested for learning and memory in an automated RAM
during the juvenile period (from PND37 through PND53).
The automated RAM allows a simultaneous determination
of performance accuracy and activity and is described in
detail elsewhere (see Miller?/ al.. 1982). Briefly. 24 LED-
photodiode pairs, located directly in front of the entrance
to each arm, 5 cm into the arm, and in the food cup at
the end of each arm, monitored movement. A Gerbrands
feeder dispensed 45-mg Noyes pellets directly into the
food cup. The operation of the doors and pellet dispensers
as well as the recording of the time and location of pho-
todiode interruptions was controlled by a Nonhstar "Ho-
rizon" computer. The criterion for an arm selection was
the following sequence of photodiode interruptions: center,
arm, and food cup. The RAM. elevated 70 cm above the
floor, was located in a well-lighted 110 ft: room with
distinguishing characteristics on several of the walls.
Animals were allowed access to Purina rat chow in their
home cage for approximately 2 hr following daily testing:
this schedule allowed adequate deprivation but also en-
sured growth. Tap water was available ad libitum in the
home cage. During the first test phase, each cup was baited
with a 45-mg Noyes pellet. An interruption of the food
cup photocell resulted in the immediate presentation of
an additional pellet for a possible total of 16 pellets per
session. Phase 2 started the day after an animal had entered
all the arms and eaten all pellets. During the 14 days of
the second phase, pellets were available only when the
photocell in the feeder cup was interrupted, reducing the
possible pellets per session to eight. On the final test day.
no pellets were available in any alley (i.e.. extinction). A
daily session (Mon-Fri) was 10-min long: arm selections
could be made the entire time, but only the first eight
correct selections were reinforced.
Brain and body weight. Wet weight of whole brain and
areas known to have significant postnatal development,
the hippocampus and cerebellum (Altman and Das. 1965;
-------
560
DIANE B. MILLER
Pelligrino and Altman, 1979), were obtained on PND23
from animals not used for behavioral testing and from
the RAM animals following retesting at 190 to 200 days
of age (RAM retest data not presented here). Body weights
were also measured at the time of testing.
Statistical analyses. For tests with several measurements
on the same subject (e.g.. RAM) data were analyzed by
a multivariate general linear model (Morrison, 1967); all
MANOVAs indicated no significant effect of sex of the
subject so data for males and females were combined in
all subsequent analyses. When significant differences
(Wilk's criterion used throughout) were found, individual
variables were analyzed by ANOVA. When significant
interactions with the within-subjects factor were found
for repeated measures ANOVA (Keppel. 1973), simple
main effects were examined at each level of the repeated
factor. For tests with a single measurement, only ANOVAs
were performed. F values greater than those associated
with the critical value at p > 0.05 were accepted as sig-
nificant. Post hoc individual mean comparisons were made
with Duncan's multiple range test. Details of the statistical
evaluation for a particular measure appear in the figure
legend.
DAY 13
nHOMJ-CAGf
unss
I 1 NO UTTER
DAY 10
0 3 6 0 3 6
TRIETHYLTIN DOSE. m9/l
-------
NEUROTOXICITY OF EARLY POSTNATAL TET
561
u 60
1/1 40 -
0.0
6.0
-------
562
DIANE B. MILLER
2- 4
i- «/t
z =
$S 5
t *
t* I
.,820
•—•00m(/kQ
a—A ] 0nig/kg rcT
r460«|/k|
1-5 6-10 11-15 E*t
SLOCKS OF 5 DAYS
Ss<
II
u
ut
5
•—tOOiM/k«
4-4jQ*«A« r«T
•-« *0<*«/k«
ng
1-S 6-» >1-15 S«
BLOCKS Of 5 DAYS
Fig. 4. Accuracy in the RAM expressed as mean correct
choices in the first eight choices and total choices needed
to select eight different arms for Phase 2 and the day of
extinction (EXT) as a function of dose (0.0, 3.0, or 6.0
mg/kg, ip) on PND5. A repeated measure ANOVA showed
an effect of treatment (F\2,13) = 27.45, p < .0001) and
blocks (F(2,26) = 15.76, p < .0001) for the correct choices
in the first 8 choices and an effect of treatment (fU,13)
= 11.78, p < .0012) and blocks (F12.26) = 5.95, p < .0075)
for the total choice measure. The 6.0-mg/kg TET-exposed
group required more choices than either the 3.0- or 0.0-
mg/kg TET-exposed groups for either measure of accuracy.
A repeated measure ANOVA comparing the last block
of Phase 2 and the day of EXT did not show any dose-
dependent differences.
3.0-, and 6.0-mg/kg TET-exposed groups, re-
spectively). Cerebellar weight decreases were
also found on PND23 (0.220,0.201, and 0.185
g for the 0.0-, 3.0-, and 6.0-mg/kg TET-ex-
posed groups, respectively) as well as in the
adult (0.315, 0.296, and 0.265 g for 0.0-,
3.0-, and 6.0-mg/kg TET-exposed groups, re-
spectively). There were no body weight dif-
ferences at either age. Similar changes in brain
weights as a function of early postnatal ex-
posure to TET have been reported with a dif-
ferent strain (albino) and a slightly different
dosing regimen (see Reiter et at., 1981; Rup-
pert et al., 1983).
DISCUSSION
The neurotoxic effects of acute postnatal
administration of TET were manifest early in
development by learning deficits and altered
motor behavior. That pronounced neurobe-
accuracy; all groups were at least as accurate
during extinction as during rewarded testing
(Fig. 4). In fact, the 6.0-mg/kg TET-exposed
group appeared to improve when correct
choices were not rewarded, but this result may
be a function of their pronounced decrease in
activity during extinction. The effects of
nonreward on activity were dependent on
treatment (Fig. 5). The control group showed
an increase in activity while both TET-exposed
groups showed decreases. However, the drop
was more pronounced for the 6.0-mg/kg TET-
treated group.
Brain weight. Postnatal TET produced
treatment-related decreases (n = 6 for all mea-
sures) in wet weight of whole brain on PND23
(1.58, 1.514, and 1.275 g for the 0.0-, 3.0-,
and 6.0-mg/kg TET-exposed groups, respec-
tively) and in adults (2,157, 2.017, and 1.639
g for 0.0-, 3.0-, and 6.0-mg/kg TET-exposed
groups, respectively). Weight of the hippo-
campus was affected at PND23 (0.100,0.087,
and 0.064 g for the 0.0-, 3.0-, and 6.0-mg/kg
TET-exposed groups, respectively) and in the
adult (0.108, 0.096, and 0.067 g for 0.0-,
1-5 6-10 IHS Ext.
BIOCKS Of 3 DAYS
1-5 6-10 11-15 Ex.
blocks of 5 days
Fig. 5. Activity in the RAM expressed as mean pho-
todiode interruptions/min and total alley choices in a 10-
min session for Phase 2 and the single day of extinction
(EXT) as a function of dose (0.0, 3.0, or 6.0 mg/kg, ip)
on PND5. A repeated measure ANOVA showed an effect
of treatment for photocell interruptions (F(2,13) = 6.95,
p < .0089) and total alley choices (/i(2,13) = 5.82. p
< .0157). Dose-related increases in activity were manifest
throughout testing. A repeated measure ANOVA com-
paring the last block of Phase 2 and the single day of EXT
showed no dose-related changes in total photocell inter-
ruptions but separate ANOVAs indicate there was a sig-
nificant difference in activity during the last block (F{2.13)
= 6.35, p < .0110), but not during EXT. A comparison
of the last block of Phase 2 and EXT for total choices
indicated an interaction between treatment and test con-
dition (fl[2,13) = 11.97, p < .00II). Separate ANOVA's
indicate the treatment effect observed on the last block
of Phase 2 (H2.I3) = 5.58, p < .0178) was reduced by
the removal of the reinforcer (F\2,13) = 3.16, p < .0760).
-------
NEUROTOXICITY OF EARLY POSTNATAL TET
563
havioral changes were still observed during
the juvenile period suggest that the CNS func-
tional alterations induced by TET are persis-
tent and may be permanent. The present study
demonstrates that paradigms tailored to the
limited capabilities of the neonate can be used
to determine functional competence in im-
mature rats and thus provide the means for
assessing the neurotoxicity of other com-
pounds during early development in this spe-
cies. Whether neonatal tasks tailored to the
response capabilities of other species, including
humans, will be useful in the detection and
characterization of neurotoxicity should be
determined. In this regard, human neonates
display altered physical development and later
reduced intellectual capacity after exposure to
certain neurotoxicants (e.g., alcohol, methyl
mercury) (Clarren and Smith, 1978; Darby et
al., 1981; Reuhl and Chang, 1979). Human
infants exposed to alcohol in utero are reported
to be irritable and tremulous and to display
weak sucking ability during the early devel-
opmental period (Clarren and Smith, 1978).
These reported alterations in behavior suggest
it may be possible to use neurobehavioral tasks
as well as physical indices to assess and char-
acterize toxicity in human neonates. In ad-
dition to the detection and characterization
of neurotoxicity, appropriate tests of function
in the neonate may be useful in determining
whether early deficits are related to or pre-
dictive of later disturbances. However, the
limited neonatal capabilities of most species
with the attendant assessment difficulties have
precluded any extensive experimental vali-
dation of this concept (see Adams and Buelke-
Sam, 1981).
Postnatal TET exposure caused perturba-
tions in the motor activity of both the neonate
and juvenile rat and these findings again rein-
force the idea that alterations in the activity
of an organism may be a sensitive index of
altered CNS function (Grant, 1976; Reiter and
MacPhail, 1982).
Although locomotor activity is clearly af-
fected after early postnatal exposure to TET,
it is difficult to draw firm conclusions regarding
TET's specific effects on locomotor activity
during the preweaning period. For example,
Reiter and colleagues (1981) reported that
TET did not affect the development of open-
field activity, but behavior was not monitored
in the presence of home-cage cues and activity
levels were low. In contrast, they found a re-
tarded motor development when some aspect
of the home environment was included in
testing. Delays in locomotor development fol-
lowing early TET exposure are also suggested
by the present data. Thus, it appears that a
preweaning activity task in which both low
and moderate levels of locomotion can be ob-
tained is best suited for the detection of per-
turbations during this early developmental
stage. That a compound's effects on a specific
behavior can be determined in part by the
baseline rate of that behavior has been re-
peatedly demonstrated (e.g., Clark and Steele,
1966; Leander, 1975).
Motor ability of untreated rats gradually
improves during the period of most active
myelination (Davison and Dobbing, 1966; Ja-
cobson, 1963). TET is known to produce vac-
uolization and splitting of myelin in adult rats
(Suzuki, 1971) and exposure to TET during
the early postnatal period induces biochemical
alterations suggestive of perturbation in the
myelination process (O'Callaghan et al, 198 3).
However, the notion that the activity changes
observed in this study, which include both
preweaning motor deficits and persistent in-
creases in adult activity, are solely the result
of a TET-induced myelin deficit is unlikely.
TET's extensive effects on pre- and postwean-
ing activity are more likely a reflection of insult
to a number of different brain structures or
systems through either an interference with
processes occurring during the postnatal de-
velopmental period, such as synaptogenesis
and myelinogenesis, or direct cell toxicity.
The treatment-related increases in activity
exhibited in both the Motron and RAM during
the postweaning period replicate and extend
previous work characterizing the develop-
mental neurotoxicity of TET (Harry and Til-
son, 1981; Reiter et al., 1981; Ruppert et al..
-------
564
DIANE B. MILLER
1983). These data indicate that early postnatal
TET exposure produces persistent changes in
activity which include a pervasive hyperac-
tivity, evident at weaning and demonstrable
in a variety of monitoring devices.
The RAM has proven sensitive to hippo-
campal dysfunction (Jarrard, 1978; Olton et
al., 1979) and effective in identifying learning
deficits following preweaning treatments
known to affect both hippocampal morphol-
ogy and function (Petit et al, 1983; Taylor et
al, 1982). Despite the persistent, treatment-
related decreases in hippocampal weight re-
ported here, it is doubtful that the learning
deficits obtained after postnatal TET exposure
are mediated solely through a selective inter-
ference with development of the limbic system
in general, or the hippocampus specifically.
First, the present study as well as other work
(Ruppert et al, 1983) shows that TET pro-
duces persistent weight decreases in other brain
areas, not just the hippocampus. Second,
functions in other brain systems or areas con-
tribute to preweaning learning capacity (Sahley
and Nonneman, 1979). Third, TET has been
reported to alter electrophysiological end-
points in a manner indicative of slowed sen-
sory processing (Dyer et al, 1981). Damage
to brain areas involved in processing and uti-
lization of visual or sensory information is
known to disrupt RAM performance (Dean
and Key, 1981). Thus, the alterations in learn-
ing induced by acute postnatal TET exposure
may reflect a generalized neurotoxicity rather
than relatively specific hippocampal damage.
ACKNOWLEDGMENTS
P. Patrick, IC. R. Lavin, and J. Garner are acknowledged
for skilled technical help and their aid in data preparation.
Dr. R. C. MacPhail is thanked for the use of his Motions.
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N euro behavioral Toxicology and Teratology. Vol. 4, pp. 185-190. 1982. Printed in the U.S.A.
Chronic Neonatal Organotin Exposure
Alters Radial-Arm Maze
Performance in Adult Rats1'2
DIANE B. MILLER, DAVID A. ECKERMAN, MARTIN R. KRIGMAN
AND LESTER D. GRANT
Neurobehavioral Toxicology Branch, Neurotoxicology Division,
U. S. Environmental Protection Agency, Research Triangle Park, NC 27711
and
The Departments of Psychology, Pathology and Psychiatry, University of
North Carolina at Chapel Hill, Chapel Hill, NC 27514
MILLER. D. B.. 0. A. ECKERMAN, M. R. KRIGMAN AND L. D. GRANT. Chronic neonatal organotin exposure
alters radial-arm maze performance in adult rats. NEUROBEHAV. TOXICOL. TERATOL. 4(2) 185-190, 1982.—Long-
Evans rats were intubated with 0.3 or 1.0 mg/kg of triethyltin sulfate (TET) or 0.3 mg/kg of trimethyltin hydroxide (TMT)
from postnatal day 3-29.1.0 mg/kg of TMT was given on alternate days beginning on postnatal day 3. Learning and memory
were assessed in an automated radial-arm maze when the rats were 180-200 days old. With this maze accuracy and activity
data can be collected simultaneously. TET or TMT treatment resulted in an increase in the number of days required to
adequately perform the radial-arm maze task, and a transient deficit in accuracy. However, the most pronounced effect in
both TET and TMT-treated animals was hyperactivity which became manifest on the second day of testing and persisted
throughout the remainder of testing.
Triethyltin sulfate Trimethyltin hydroxide Automated radial-arm maze Neonatal exposure
Learning and memory Behavioral toxicology
THE toxicity of tin compounds has been of interest since the
early 19th century and Orfila is credited with first describing
the toxicity of inorganic tin [II]. Recent interest in the tox-
icity of tin has centered on the neuropathological and
neurobehavioral effects of the organotins, especially the
structurally related trialkyltins, triethyltin (TET) and
trimethyltin (TMT). Bouldin et al. [3] have described in de-
tail the ultrastructural cytopathology found in both neonates
and adults after chronic exposure to TET and TMT. One
mg/kg of TET produced pathological changes in myelin in
both adults and neonates characterized by widespread
intramyelinic vacuolization. These data provide corrobora-
tive evidence for previous research indicating TET primarily
damages central nervous system myelin [1]. In contrast
chronic expsosure to 1.0 mg/kg of TMT resulted in selective
neuronal necrosis in both adults and neonates. Tissue dam-
age was particularly evident in the CAB and CA4 fields of the
hippocampal formation and in the pyriform cortex. Although
Bouldin et al. [3] have described the neuropathological con-
sequences of chronic exposure to TET and TMT during the
neonatal period, data on the possible functional conse-
quences of the exposure regime was not presented. The pur-
pose of the present experiment was to characterize the
learning-memory functioning of adult animals after chronic
exposure to TET and TMT during the neonatal period.
The effects of chronic neonatal organotin exposure on
learning and memory were assessed with a radial-arm maze
(RAM). The RAM has received considerable attention as a
paradigm for assessing learning and memory in the rodent
[17]. RAM performance has also been shown to be sensitive
to disruption by drugs and lesions [3, 10, 21] as well as
neurotoxic insult [6,12]. In this spatial learning task the
animal is required to obtain food located at the end of arm a
which radiate from a center platform. Optimal performance
consists of selecting all still-baited aims before repeating
previous choices. Rats rather quickly acquire efficient maze
performance, demonstrating that the animal remembers
which arms have been visited. Our version of the maze (14]
is automated and easily allows accuracy and activity to be
measured concurrently. An index of activity is important
'This paper has been reviewed by the Health Effects Research Laboratory, U. S. Environmental Protection Agency, and approved f"r
publication. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.
'Presented at the Society of Toxicology Annual Meeting, San Diego, CA, 1981.
'Send reprint requests to Dr. Diane B. Miller, Neurotoxicology Division (MD-74B), Health Effects Research Laboratory, U. S. Environ-
mental Protection Agency, Research Triangle Park, NC 27711.
18}
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186
MILLER ET AL.
both because many neurotoxic compounds have been found
to affect activity [18] and because changes in activity might
confound RAM performance. In particular TET and TMT
exposed animals have been shown to be hyperactive in a
variety of test situations [17, 19, 20], Therefore, accuracy,
activity, time to complete the task, and time per correct
selection (efficiency) were all used as measures of RAM per-
formance.
METHOD
Animals and Treatement
Neonatal Long-Evans rats were obtained from our lab-
oratory colony and were bred from stock originally obtained
from Charles River Breeding Laboratory (Somerville, MA).
During pregnancy the dams had free access to tap water and
a pelleted rat chow (Wayne Lab-Blox®). The vivarium was
maintained at 26°C with 12 hr:12hr light-dark cycle. Pups
were cross-fostered on day 2 of life (birth=»0) and reassigned
such that each litter contained 4 males and 4 females. A dam
never received more than 1 male and I female from her own
litter. Rats were weaned at 21 days of age. On day 30 rats
were separated by sex and housed 4/cage for the remainder
of the study.
The organotin compounds were prepared in milk plus
Tween-80® as vehicle. Mixtures of one drop Tween-80®/10
mg of agent were diluted with homogenized milk to a pre-
defined concentration. The volume of milk used was ad-
justed so that 10 ml of solution contained the appropriate
dose. Trimethyltin hydroxide (TMT) was obtained from
Alfa-Ventron (Danvers, MA) and triethyltin sulfate (TET)
was a gift from Organische Chem. Inst., Utrecht, the Nether-
lands. Tween-80* was obtained from Nutritional Biochemi-
cals, Inc. (Cleveland, OH).
The litters were assigned in a random manner to a given
treatment group. Rat pups received vehicle or vehicle plus
0.3 or 1.0 mg/kg of TET or 1.0 mg/kg of TMT in a volume of
0.01 ml/g of body weight from postnatal day 3-29. A dose of
1.0 mg/kg of TMT was given every other day due to the
greater toxicity of this dose when administered on a daily
basis (see Bouldin et al. [3] for details).
Rats were tested as adults for learning and memory per-
formance. Each treatment group contained rats from at least
3 litters and an approximately equal distribution of males and
females. The numbers of subjects per group were as follows:
vehicle control (n**19); 0.3 mg/kg TET (n» 16); 1.0 mg/kg of
TET (n»16); 0.3 mg/kg TMT (n«8); and 1.0 mg/kg of TMT
(n=8).
Apparatus and Testing
All animals were tested for learning-memory function in
an automated RAM beginning at 180-200 days of age. The
automated RAM (Fig. 1) has a center octagonal area (7.5 cm
to a side and 32 cm in diameter) with 8 arms (26.0 cm long x
10.5 cm wide x 9.5 cm high) radiating from it like the spokes
of a wheel. Solenoid-operated guillotine doors controlled ac-
cess from the center of the maze to the arms, and the entire
maze was covered with clear Plexiglas. Twenty-four LED-pho-
todiode pairs monitored movement in the maze. An LED-
photodiode pair was directly in front of the entrance to each
ann, 5 cm into the arm. and in the food cup located at the end of
each arm. A Gebrands feeder was also located at the end of
each arm and dispensed 45 mg Noyes pellets directly into the
food cup. The operation of the doors and pellet dispensers as
well as the recording of the time and location of photodiede
interruptions was controlled by a Northstar "Horizon" com-
puter. The criterion for an arm selection was the following
sequence of photodiode interruptions: center, arm and food
cup. The RAM was located in a well-lighted 110 square foot
room with distinguishing characteristics on several of the
walls (e.g., window, electrical outlet). The entire maze was
elevated 70 cm above the floor.
One week before testing rats were reduced to 85 Jc of their
free-feeding weight by restricting access to Purina rat chow
to 1 hr daily; tap water was available ad lib in the home cage
throughout the study. Testing was conducted in two phases.
During the first phase each cup was initially baited with a 45
mg Noyes pellet. If the rat interrupted the photocell in the
feeder cup, an additional pellet was presented for a possible
total of 16 pellets per session. Once an animal was entering
all the arms and consuming all the available pellets within
one 10-fflin trial, the second phase was started. During the 8
days of the second phase, pellets were available only when
the photocell in the feeder cup was interrupted, reducing the
possible pellets per session to 8. Subjects were tested daily
Monday through Friday. A daily trial ended when 8 correct
selections were made or 10 min had elapsed.
Statistical Analyses
For behavioral measures with repeated indices data were
analyzed by a multivariate general linear model [14]; when
significant differences (Wilk's Criterion used throughout)
were found, individual variables were analyzed by ANOVA.
For tests with a single measurement such as weight and days
to criterion, only ANOVAs were performed. F-values
greater than those associated with the critical value at
/><0.05 were accepted as significant. Post hoc individual
comparisons were made using Duncan's Multiple Range
Test.
RESULTS
The effect of neonatal organotin treatment on adult RAM
performance was assessed with the following dependent var-
iables; days to enter and eat in all 8 arms, unrepealed arm
selections in the first 8 choices (accuracy), total photocell
interruptions (activity), time to finish a session dime), and
time per correct arm selection (efficiency).
The mean number of days to enter and eat in all arms
(days to criterion) varied as a function of treatment.
F(4,51)>"2.67, p<0.0422. No significant sex effect or sex-
by-treatment interaction was found, so data were combined
for males and females (Fig. 2). The 6.0 mg/kg TET and TMT
groups required significantly more days to reach criterion
than the vehicle control or the 3.0 mg/kg TET and TMT
groups (p<0.05). Thus, chronic treatment with TET or TMT
during the neonatal period resulted in a significant increase
in the time required to adequately perform the RAM task In
addition, a number of animals in the treated groups failed to
satisfy the criterion to enter the second phase; 1 in the 0 ?
TET group, 3 in the 1.0 TET group, and 2 in the 1.0 TMT
group.
Phase I
The MANOVA indicated significant effects for treatment.
F( 16,162)—2.15, p<0.0086; day of testing, F(4,53) = 2h \<.
p<0.0001; and treatment x day of testing, F(16.162)= l *>.
p<0.0233. Data from males and females were pooled in thi>
and the remaining analyses since a MANOVA indicated no
significant sex effect. Only data from the first and last ti.
-------
188
MILLER ET AL.
rn
OQSAGI. m«/kf
• VI MICH
• J TIT
A I I TIT
# • 3 TUT
* ItTHT
MMT IUT IIMT LAST HUT I AIT
PIUT ANO LAST OAVI Of MA|( t
] I 1 1 ) 1 1 J
IIOCU OP OAVI IN PNAII 2
FIG. 4. Activity (photodiode interruptions) in RAM expressed as
mean responses/minute for the first and last days of Phase I and the
3 blocks of Phase 2 as a function of dose of organotin. Data are
pooled for males and females and are presented as means £ SE.
'Different from vehicle control at p<0.0J.
HUT I All
OA VI Of
• LOCKS 0»
OAVSOf
PMASi I
810CKS 0»
PHASI I
PHAJI J
PNAII I
FIG. 5. Time to complete a session expressed as mean minutes and
efficiency expressed as mean minutes/choice for the first and last
day of Phase 1 and the 3 blocks of Phase 2 as a function of organotin
dose. Data are collapsed across groups, pooled for males and
females, and expressed as means ± SE. All animals required less
time to complete a session and became more efficient as training
progressed. 'Different from the first day of Phase 1 or the 1st block
of Phase 2 at p<0.05.
Phase I were included in the analyses because animals var-
ied in the number of days they remained in Phase 1.
ANOVAs indicated the measures affected by organotin
treatment were accuracy and activity.
Accuracy. For the number of selections correct in the first
8 choices (Fig. 3) on the first and last day of Phase 1 there
was a significant treatment, F(4,56)-3.17, p<0.0091; day of
testing, F( 1,56)-17.27, p<0.0001; and treatment x day of
testing interaction, F(4,56)-2.90, p<0.0298. Further
analysis indicated significant treatment effects for both the
first, F(4,56)-3.68, p<0.0099 and final day, F(4,56)-0.0437
of Phase 1. On the first day of training, treated animals were
significantly impaired in their ability to remember which al-
leys they had visited (p<0.05). The high dose groups for both
TET and TMT made significantly less correct selections than
the control or low dose groups (p<0.05). All groups im-
proved as a function of training but both TET groups were
still making significantly (p<0.05) fewer correct selections in
the first 8 choices than the other groups on the final day of
Phase 1. The first day differences are not due to a lower
overall number of selections since there was no significant
difference in the total correct choices in the session; this
implies that the treated groups made more repeat selections.
Activity. Photocell interruptions per minute (Fig. 4) were
used as a general index of activity in RAM. The repeated
measure ANOVA on responses/minute for the first and last
day of Phase 1 indicated significant treatment, F(4,56)-3.96,
p<0.0067; day of testing, F( 1,56)-34.47, p<0.0001; and
treatment x day of testing interaction, F(4,56)»3.10,
p<0.0226 effects. The groups were not significantly different
in activity on the first day of Phase 1. However, by the final
day of Phase 1 the groups differed significantly in activity,
F(4,56)-3.68, p<0.0099. Treatment with 1.0 mg/kg of either
TET or TMT produced animals which were more active than
the controls (p<0.05) or animals treated with 0.3 mg/kg of
either compound (p<0.05).
Time and efficiency. The repeated measures ANOVAs
on time to finish the session and number of minutes/correct
response indicated all groups improved with training (Fig. 5)
requiring less time per session and less time per correct re-
sponse. These results are reflected in the significant day of
testing effect, F( 1,56)= 111.3. p<0.000l for time and
F(l,56)=45.2,pcO.OOOl for efficiency and the nonsignificant
treatment x day of testing interaction.
Phase 2
The data analysis of Phase 1 was limited to the first and
last day of training because the subjects differed in the
number of days required to acquire the task. However, all
subjects received 8 days of Phase 2 training and rather than
including only the first and last day in the analyses, the data
was collapsed into blocks and all blocks included in the
analyses to provide concise and complete information about
performance during Phase 2. Blocks I and 2 are the mean of
3 days each while block 3 is the mean of the final 2 days
MANOVA indicated organotin treatment affected Phase 2
RAM performance. The MANOVA for treatment. Fi Ih.lh2)
-2.50, p<0.0020, and day of testing, Ft8.2IH> =
2.62, p<0.0094 was significant; but there was no M^ni-
ficant treatment x day of testing interaction. Repeated
measure ANOVAs indicated the only measure affected by
treatment in this phase was activity (Fig. 4). The ANOV A on
the 3 blocks of Phase 2 activity resulted in a significant
treatment, F(94,56)=4.2, p<0.6048, and day of testing.
F(2,l l2)-4.59, p<0.0121, effect but a nonsignificant treat-
ment x day of testing interaction. All groups became more
active as training progressed (p<0.05). More importantly,
the high dose groups for both TET and TMT were signifi-
cantly more active than the control or the low dose group
(p<0.05). In addition, the low dose TET group was signifi-
cantly more active than the vehicle controls (p<0.05 > ! hese
results indicate chronic neonatal treatment with either of
-------
NEONATAL ORGANOTINS AND RADIAL-ARM MAZE 187
FIG. 1. Automated radial-arm maze.
VlHICll I) l|
'IT mT
QftGAMaimOOSACi
FIG. 2. Time to criterion expressed as the mean days required to
enter and eat in all 8 arms of the RAM for control and organotin
treated rats. Data are pooled for males and females and are pre-
sented as means ±SE. Both TET and TMT produced a dose-related
increase in days required to reach criterion. 'Different from vehicle
control at p<0.05.
VIHICLi • J II II II
III I«T
OOSAGC. Mykf
s—iAir OAV-—
1.3 II 01 II
TIT TMT
OOSACi.
¦flMT QAV
J V ,l
~ V-J
'MAS!I —
OAV or PHASC I
FIG. 3. Accuracy expressed as the mean number of correct selec
tions in the first 8 arm choices for the first and last day of Phase I
and the 3 blocks of Phase 2 as a function of dose of organotin. Dai.i
are pooled for males and females and are presented as means . SK
The high doses of TET and TMT produced a deficit in accuracy onK
on the first day of Phase 1. ^Different from vehicle control ui
p<0.05.
-------
NEONATAL ORGANOTINS AND RADIAL-ARM MAZE
189
these organotins produced a profound hyperactivity which
was manifest throughout most of the testing.
Accuracy, time and efficiency. The repeated measure
ANOVA for number of correct selections in the first 8
choices for the blocks of Phase 2 resulted in no significant
main, effects or interactions. These results indicated all
groups were at their asymptotic level of performance by the
end of Phase I and made no significant improvement during
Phase 2 (Fig. 3). These data support the contention that the
RAM is a rapidly acquired task.
The repeated measure ANOVAs for time and efficiency
indicated, as does Fig. 5, that all groups became faster and
required less time/correct selection as Phase 2 continued;
F(2,112)-7.8, p<0.0007 for time and F(2,l 12J-3.98,
p <0.0214 for efficiency for the day of testing variable. In
neither analysis was there a significant treatment or treat-
ment x day of testing interaction.
DISCUSSION
Chronic treatment with both triaikyltins, TET and TMT,
during the neonatal period resulted in altered RAM perform-
ance in the adult. The treated animals required more days to
adequately perform the task, showed a treatment decrease in
accuracy, and displayed a consistent hyperactivity from the
second day of testing until the end of the study.
Both TET and TMT treatment animals required more
days to enter alt arms and eat the available pellets. In addi-
tion, all the control animals reached criterion while there
were a number of TET and TMT-treated animals that failed
to ever satisfy the criterion despite several weeks of training.
These time to criterion increases reflect differences in the
number of pellets eaten since the groups did not differ in the
total number of correct selections made even on the first day
of testing. While these treatment effects may reflect motiva-
tional differences between the treated and untreated groups
it is more likely a difference in the initial habituation to the
RAM. The finding that neonatal organotin treatment results
in an increase in days to criterion and the failure of some
animals to reach criterion suggest that this measurement of
habituation to RAM may serve as a useful index of
neurotoxic effects. Information about other measures of
habituation to the RAM is scarce since lesions or other
treatments are usually administered after training (e.g.. Ec-
kermen et at. [5]). Consistent with the present Phase 1 con-
trol findings. Jarrard (101 reported gradually increased explo-
ration of RAM during the initial training phase. Jarrard also
reported that rats with hippocampal lesions show higher
exploration levels than controls, a finding which parallels the
results obtained with the organotin groups in the present
study. „ . ,
Reiter et ul. [18! and Harry and Tilson [71 have deter-
mined that a single exposure to TET early in the neonatal
period results in increased motor activity in the adult. The
increases in activity found after TET or TMT exposure m the
present study provide evidence that chronic neonatal treat-
ment with either of these organotins will also result in in-
creased activity in the adult. Interestingly, activity increased
for all groups across days of testing although the treated
groups were always more active than the control groups
other than on the first day of testing. A number of inves-
tigators [2,10] have reported that limited but regular feeding
resulted in increased activity in the time immediately pre-
ceeding feeding and that this effect became more
pronounced with time. Also lesions of the hippocampus re-
sults in more dramatic increases as a function of limited feed-
ing. The subjects in the present study were fed immediately
following the daily session; it is likely that the activity in-
creases across testing were related to limited food access.
TET and TMT clearly produce different neuropathologi-
cal effects (see Bouldin et al. [3]). Yet in the present study
treatment with either of the compounds on a chronic basis
during the neonatal period produced the same behavioral
effects, a clear hyperactivity and marginal learning-memory
deficits. Treatment with either compound resulted in an in-
crease in the number of repeated arm choices only during the
early portions of testing. Although there is no previous data
on the functional consequences of chronic exposure to TET
or TMT during the neonatal period several studies have
demonstrated that a single exposure to TET on postnatal day
5 produced a variety of neurobehavioral effects in the
juvenile and adult rat. These effects included deficits in
learning memory [13], changes in sensorimotor capabilities
[7], increases in locomotor activity [7,18], decreases in
myelin-basic proteins [15], and alteration in elec-
trophysiological parameters such as the visual-evoked re-
sponse [4,8]. Only . limited data is available on the
neurobehavioral effects of neonatal exposure to TMT. Miller
and Dyer [12] exposed rats to TMT on postnatal day 5 and
found that when tested as juveniles in the RAM these rats
were not hyperactive but were less efficient than controls.
Thus, the apparent absence of qualitative differences be-
tween the effects of TET and TMT observed in the present
study may be related to the chronic dosing.Certainly future
studies of the neurotoxicity of TET and TMT should provide
a further characterization of the neurobehavioral. elec-
trophysiological, and neurochemical effects of TET and
TMT. Clearly these studies must provide a comparison of
TET and TMT toxicity at different ages in chronic versus
acute dosing models to allow for a truly integrated interpre-
tation of organotin neurotoxicity.
ACKNOWLEDGEMENTS
We would like to thank JoAnne Gamer for testing the animal*
and suggesting the days to criterion measure as well as George
Anderson, Walt Kozel, and Bill Parrott for the construction and
programming of the automated radial-arm maze and Mrs. Ph>llt>
Keeter for typing this manuscript.
REFERENCES
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2. Bolles. R. C. Theory of Motivation. New York: Harper-Row,
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3. Bouldin, T. W„ N. D. Gaines, C. R. Bagneli and M. R. Krig-
man. Pathogenesis of trimethyltin neuronal toxicity; Ultmtruc-
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ijlr
190
6. Gordon, W. A., D. A. Eckerman, S. L. Elliott, J. A. Garner and
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11. Kimbrough, R. Toxicity and health effects of selected organotin
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13. Miller, D. B., J. A. Gamer, J. D. Edwards, G. E. Anderson, W.
M. Kozel, W. A. Parrott, D. A. Eckerman, M. R. Krigman and
L. D. Grant. Postnatally administered triethyltin and trimethyl-
tin affects spatially controlled behavior. Toxicologic 1: 45,
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MILLER ETAL.
14. Morrison, D. Multivariate Statistical Methods. New York:
McGraw-Hill, 1967.
15. O'Callaghan, J. P., D. B. Miller and L. W. Reiter. The effect of
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16. Olton, D. S. and R. J. Samuelson. Remembrance of places
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19. Ruppert, P. H., T. J. Walsh, L. W. Reiter and R. S. Dyer.
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0O22-3&65/M/23L3-074U0O.00/0
The Journal or Pharmacology and Expcrimintal Therapeutics
Copyright © 1984 by Th® Amarican Socitty for Pharmacology and Ezpsrimantal Tharapautica
Vol. 231, No. 3
Printed in U.S.A.
Biochemical, Functional and Morphological Indicators of
Neurotoxicity: Effects of Acute Administration of Trimethyltin to
the Developing Rat1
DIANE B. MILLER and JAMES P. O'CALLAGHAN
Neurotoxlcology Division, Health Effect Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina
Accepted for publication August 27,1984
ABSTRACT
The neurotoxic organometal, trimethyltin (TMT), was adminis-
tered to rats on postnatal day (PND)5. Neurotoxicity was as-
sessed throughout subsequent development using morphologi-
cal, biochemical and functional endpoints. These consisted of
brain weight measures and histology (morphology), assays of
nervous system-specific proteins (biochemistry) and neurobe-
havioral indices of activity and learning (function). All three indices
were affected. TMT caused dose-related decreases in brain
weights at all ages examined, PND13, 22 and 66, with the
hippocampus being the most severely reduced. Histological ex-
amination of the hippocampus revealed loss of pyramidal neu-
rons in CA3 to CA4. Exposure to TMT was followed by dose-
and age-dependent reductions in synapsin I, a neuron-specific
phosphoprotein associated with synaptic vesicles; these effects
of TMT were greater in hippocampus than in forebrain. TMT did
not alter the concentration or protein composition of isolated
myelin. The ontogeny of locomotion was altered in a dose- and
time-dependent manner; TMT induced hypoactivity early in de-
velopment (PND13) and hyperactivity by weaning (PND21); hy-
peractivity was also observed in the adult. Finally, TMT also
affected learning ability throughout development Deficits were
observed in: 1) acquisition and retention of an instrumental
alleyway response; 2) acquisition of a step-through passive
avoidance response; and 3) radial-arm maze performance. These
results demonstrate the use of a multi-endpoint, multi-timepoint
strategy for the detection and characterization of neurotoxicity.
In the preceding paper (O'Callaghan and Miller, 1984) we
reported that acute administration of TMT to the adult rat
decreases neuron-specific phosphoproteins in the hippocampus,
findings consistent with the effects of this compound on limbic
system morphology. Acute administration of TMT to the de-
veloping rat also results in morphological alterations in the
CNS (Ruppert et a 1983). We reasoned that these neuropath-
ological effects of TMT, like those in the adult, might be
accompanied by alterations in neuron or other cell-type specific
proteins. Because the postnatal development of the rat CNS is
characterized by neuronal maturation, glial proliferation and
myelinogenesis (Dobbing, 1968), we chose to examine the ef-
fects of TMT on nervous system-specific proteins associated
with these processes.
Severe functional deficits mult from exposure of adult rats
to TMT as indicated by neurobehavioral assessments of activity
Received for publication Much 12,1984.
1 This paper hu been reviewed by the Health Effect* Research Laboratory,
U.S. Environmental Protection Agency, and approved for publication. Mention
of trade ntmea or commercial product* doe* not conatitute endowment or
recommendation for u*e. A preliminary report of thi* inveitifation appeared in
Soc. Neurosci. Abetr. 8: 268,1983.
and learning (Dyer et al,, 1982) and it appears likely that similar
alterations are associated with exposure to this compound early
in development (see Ruppert et al., 1983). Thus, in addition to
morphological and biochemical measurements, we employed
learning and activity paradigms tailored appropriately to the
age and capabilities of the rat (see Miller, 1984) as indices of
functional changes induced by early postnatal exposure to
TMT. Our results indicate that the effects of TMT on CNS
development are reflected in age-related alterations in mor-
phology, nervous system-specific proteins and behavior.
Materials and Methods
Materials. Materials wen obtained as described in the preceding
paper (O'Callaghan and Miller, 1984).
Subjects. Gravid Long-Evans rats (Blue Spruce Farms, Altamont,
NY) ware obtained on day 2 of gestation and housed individually in
plastic tub cages (46 x 28 x 20 cm) with wood shavings as bedding.
Pup* bom between 5:00 P.M. and 5:00 a.m. were considered to be bom
on the same day (PND0). On PNDl pups were reassigned to litters
such that each dam received four male and four female pups and no
more than one male and one female of her own offspring. Bedding was
not changed from PND5 to PND13; from PND14 to 21 half of the
soiled bedding was removed every 3rd day and replaced with clean
ABBREVIATIONS: TMT, trimethyltin; CNS, central nervous system; PND, postnatal day; SDS-PAGE, sodium dodecyl sutlate-polyacrylamide gel
electrophoresis; RAM, radial-arm maze; TET, trtethyMn. i
744
-------
1984
Brain Ocvtiopmant and TMT 745
bedding. At 21 days of age littermatea were weaned, separated by sex
and housed four pet cage. Animals were maintained in a temperature
(22°C ± 2)- and humidity (50% ± 10)-controlled colony room on a 12-
hr light/12-hr dark cycle beginning at 6:00 A.M.
Dosing. Pups were injected (i.p.) on PND5 with 0.9% saline (0.0
mg/kg) or TMT hydroxide (5.0 or 6.0 mg/kg) dissolved in saline; this
regimen was chosen because it results in minimal weight loss and
mortality (see Buppert et at1983). Doses of TMT are expressed as
the free base and were administered in a volume of 10 nl/g b.wt. AH
animals in a litter received the same dose and were foot tatooed with
india ink for later identification. To control for the possibility of
interactions between testing procedures animals were evaluated in only
one behavioral paradigm. In addition, no more than two animals of
each sex from a litter were used in a particular behavioral experiment;
the remaining animals were removed for biochemical evaluations. Ex-
cept where brains were prepared for histology, all animals were killed
by decapitation.
Brain and optic nerve dissection. Immediately after decapitation
whole brains were excised from the skull; the hippocampus, forebrain
and cerebellum were removed by freehand dissection and wet weights
were obtained. For synapsin I assays the forebrain was defined as the
region remaining after removal of hippocampus and cerebellum from
whole brain. For myelin analysis, the forebrain also included the
hippocampus. Optic nerves (intraorbital foramen to optic chiasm) were
removed from one group of animals on PND22 and were immediately
disrupted by Bonification in 0.05% SOS. An aliquot of the optic nerve
homogenate was taken for determination of total protein and samples
were stored at -70* C before electrophoresis (see below). All dissections
were performed on a refrigerated plate maintained at 0-4"C (Thermoe-
lectrics Unlimited, Wilmington, DL). Samples from control and treated
animals were always obtained at the same time.
Histology. On PND22, one group of animals that received TMT
(6,0 mg/kg only) or saline was anesthetized (Chloropent, Fort Dodge
Laboratories, Fort Dodge, IA) and perfused intracardially with 0.9%
saline followed by buffered 10% Formalin. Whole brains were then
carefully removed and stored in 10% Formalin for subsequent process-
ing. Beginning at midline, sequential sagittal sections 10-ft thick were
cut at 100-ji intervals from the left side of each brain. Sections were
stained with cresyi violet.
Radioimmunoassay of synapsin I. On PND13, 22 and 66, the
hippocampus and forebrain from one group of control and treated
animals were dissected and homogenized immediately in 10 volumes of
1% SDS at 90* C. The relative specific activity of the neuron-specific
phosphoprotein, synapsin I (De Camilli et al., 1983a,b), was determined
by detergent-based radioimmunoassay (Goeiz et al., 1981) as described
in the previous paper (O'Callaghan and Miller, 1984). The data are
presented on both a total and concentration basis as described previ-
ously (O'Callaghan and Miller, 1984).
Myelin isolation. On PND22, forebrains from one group of treated
and control animals were dissected and homogenized immediately in
0.32 M sucrose. After an aliquot was removed for determination of
total protein, myelin was isolated from the homogenate by the method
of Norton and Poduslo (1973) using an SW-28 rotor in a Beckman L3-
50 Ultracentrifuge maintained at 4*C. The isolated myelin was assayed
for total protein before electrophoresis (see below).
SD8-PAGE sad microdensitometry. Forebrain myelin and optic
nerves were dissolved in an SDS-PAGE sample buffer (O'Callaghan et
al., 1983) and heated to 90-9&*C for 2 min. The specific protein
composition of these samples was then analyzed by SDS-PAGE. A
detailed description of the SDS-PAGE procedures was given previously
(O'Callaghan et al., 1983; O'Callaghan and Miller, 1984) with the
exception that the resolving gel contained 12% acrylamide. The con-
centration of small myelin basic protein, large myelin basic protein and
proteolipid protein in samples of forebrain and optic nerve was deter-
mined by microdensitometry (LKB Ultrascan) of the dried gels. The
areas under the peaks corresponding to each myelin protein are ex-
pressed as a percentage of total protein (see Toews et al., 1980).
Protein assays. Protein was determined as described in the preced-
ing paper (O'Callaghan and Miller, 1984).
Assessment of locomotor activity. On PND10,13, 15, 17 and 21
the development of locomotor activity was determined in a Plexiglas
open field (21 cm wide x 28 cm long x 25 cm high) with the wire-mesh
floor demarcated into a 3 x 4 matrix of 7-cm squares. Individual
subjects were observed for 3 min over home-cage litter with a count
recorded each time the front paws crossed a line (see Miller, 1984).
In a separate set of animals, the generality of the effects of TMT on
locomotor activity at weaning was measured in a photocell device
(Motion Electronic Motility Meters, Motron Products, Stockholm,
Sweden). Individual activity was determined only on PND21 in a 25-
min session as described previously (see Miller, 1984).
Assessment of learning ability. An appetitive instrumental alley-
way task in which home-cage litter and littermates serve as the rein-
forcer was used to determine the effect of TMT on learning ability
during the preweaning period (PND14). In this task the animal is
placed facing away from the goaibox, must learn to tum around and
locomote down the alleyway to the goaibox in 120 sec or less. The
procedure was as described by Miller (1984) with the following excep-
tions: 1) the floor of the alleyway was changed from Plexiglas to steel
mesh to allow for the placement of home-cage litter beneath the entire
apparatus during the acquisition period; 2) the acquisition criterion
was changed from 5 to 2 consecutive trials and testing continued for
only 5, rather than 15, trials during the performance phase; and 3) a
0.5-hr isolation period was interposed between the acquisition and
performance phases so that memory or retention of the alleyway
response could be determined.
The effect of TMT on learning ability during the postweaning, early
juvenile period was determined in a second experiment by a passive-
avoidance task. The passive-avoidance apparatus consisted of a V-
shaped trough divided by a guillotine door into a small illuminated
start box and a larger darkened box (Jarvik and Kopp, 1967). A trial
was initiated by raising the guillotine door and a Coulbourn Instru-
ments Constant Current Solid State Shocker (model E13-16) delivered
a 0.5-mA shock each time the darkened chamber was entered. The AC
shock was delivered to the feet of the animal through the stainless-
steel panels forming the walls and floor of the darkened chamber.
Testing began on PND22 and continued with one trial per day until
two consecutive trials occurred in which the animal remained in the
startbox for 5 min.
In a third experiment an automated RAM (see Miller et al., 1982)
was used to assess the effect of TMT on learning in the young adult.
The maze consists of eight equidistantly spaced arms radiating from a
central octagonal confinement area. A limited amount of food is avail-
able at the end of each arm and efficient performance requires the
animal not to re-enter arms from which food has already been obtained.
Photodiode pairs located in the center, arms and feeder cups allow the
simultaneous determination of both accuracy and activity. Five-minute
sessions (Monday-Friday) began on PND42 and continued for a total
of 15 sessions past the session in which all arms were entered and all
sucrose pellets (BioServe, Frenchtown, NJ) were eaten (see Miller,
1984 for details).
Statistical analyses. The Statistical Analysis System (SAS, 1982)
was used for data analyses. When several measures (e.g., body and
brain weights) were collected on the same subject data were analyzed
by a multivariate analysis of variance procedure (Morrison, 1967).
Where multivariate analyses of variance were significant individual
variables were analyzed by an analysis of variance or a repeated
measure analysis of variance (Keppel, 1973). Sex of the subject was
included as a variable in all analyses but was not found to interact with
treatment; therefore, data from males and females were combined. In
some instances dose-response relationships in the data were explored
through the use of trend analyses (Winer, 1971). Duncan's Multiple
Range Test was used to make mean comparisons. Details of the
statistical evaluation for a particular measure appear in the figure or
table legend.
-------
746 Millar and O'Callaghan
Vol. 231
TABLE 1
Effect of acuta poatnatai administration of TMT on brain and body weights
Each value represents the mean (±S.E.M.).
Day
N Dom
Body Wt.
Bram Wts.
Hippocampus
Wts.
CsraMlumWts.
Foratirain Wts.
mg/kg
9
13
13
13
8 0.0
8 5.0
8 6.0
30.2 (±1.0)
27.9 (±0.4)
24.9 (±1.5)*
1.22 (±0.02)
1.06 (±0.03)*
0.88 (±0.09)*t
0.071 (±0.002)
0.051 (±0.003)*
0.033 (±0.007)*t
0.141 (±0.003)
0.127 (±0.004)*
0.118 (±0.006)*t
1.009 (±0.014)
0.880 (±0.240)*
0.671 (±0.080)*t
CM CM CM
CM CM CM
8 0.0
8 5.0
8 6.0
49.2 (±2.8)
45.3 (±1:1)
43.8 (±2.3)
1.48 (±0.01)
1.23 (±0.02)*
1.20 (±0.05)*t
0.082 (±0.003)
0.048 (±0.003)*
0.041 (±0.003)*
0.199 (±0.004)
0.181 (±0.004)*
0.181 (±0.004)*
1.188 (±0.011)
0.995 (±0.023)*
0.905 (±0.032)*t
66
66
66
8 0.0
8 5.0
8 6.0
268.9 (±19.3)
255.8 (±21.5)
247.3 (±17.5)
1.90 (±0.03)
1.62 (±0.03)*
1.47 (±0.03)*t
0.113 (±0.004)
0.071 (±0.004)*
0.053 (±0.004)*t
0.281 (±0.004)
0.265 (±0.004)*
0.252 (±0.004)*
1.508 (±0.020)
1.284 (±0.030)*
1.131 (±0.030)*f
* Significantly different from the control, P < .05; f significantly different from 5.0 mg/kg. P < .05.
Results
Body and brain weights. Administration of TMT on
PND5 resulted in marked reductions in wet weight of whole
brain, hippocampus, cerebellum and forebrain at PND13, 22
and 66 (table 1). Of the three brain regions examined, the
hippocampal formation was the most affected; at a dose of 6.0
mg/kg, approximately a 50% reduction in the weight of this
structure was observed at all three time points. In comparison
to hippocampus, wet weights of cerebellum and forebrain were
affected to a lesser degree at all time points and the greatest
change was observed at PND13. In contrast to the effects of
TMT on brain weight, body weights were minimally affected.
Significant decrements were observed only in the high dose
group during the preweaning period (PND13); no body weight
changes were observed in the juvenile (PND22) or adult
(PND66).
Histology. The effects of TMT (6.0 mg/kg, PND5) on the
gross morphology of the hippocampus at PND22 are shown in
figure 1. Representative sections obtained from dorsal hippo-
campus of a saline-treated subject (fig. 1A) and subjects mod-
erately (fig. IB) and severely (fig. 1C) affected by exposure to
TMT are presented. The decreased hippocampal weights re-
sulting from exposure to TMT (table 1) were reflected in a
decreased size of this structure in all sections prepared from
TMT-treated subjects; the range of effects of TMT on the size
of the hippocampal formation are typified by the examples in
figure 1. TMT also affected the cytoarchitecture of the hippo-
campus as evidenced by a loss of pyramidal cells in CA3 to CA4
and a diminished density of the remainder of the cell line. In
contrast, no changes wen observed in the gross morphology of
cerebellum or forebrain after administration of TMT (data not
shown).
Radioimmunoassay of synapsis I. The effects of TMT
on the neuron-specific phosphoprotein, synapsin I, are pre-
sented in table 2. Administration of TMT on PND5 resulted
in large dose-related decreases in total hippocampal synapsin I
on PND13, 22 and 66. The decreases in total hippocampal
synapsin I on PND13 and 22 were greater than the correspond-
ing reductions in hippocampal wet weight aa indicated by the
24 and 36% decrease in concentration of hippocampal synapsin
I on PND13 and 22, respectively. By PND06 the concentration
of hippocampal synapsin I did not differ significantly between
saline and TMT-treated groups.
In comparison to the values obtained for hippocampus, ra-
dioimmunoassay values (total and concentration) for synapsin
I in forebrain were less affected as a result of administration of
TMT on PND5. Total synapsin I was reduced by as much as
45% on PND13 but by PND66 only a 21% reduction was
observed at the 6.0-mg/kg dose. Dose-related decreases in the
concentration of synapsin I were observed on PND13 and 22;
however, these TMT-induced effects were never as large in
magnitude as those observed for synapsin I concentration in
hippocampus.
CNS myelin. The effects of TMT on indices of CNS mye-
lination at PND22 are presented in table 3. Total protein and
total myelin protein in forebrain as well as total protein in
optic nerve were reduced as a function of the dose of TMT
administered on PND5. However, because the decrease in iso-
lated forebrain myelin was parallel to the decrease in forebrain
weight, myelin concentration (milligrams of myelin protein per
gram of forebrain) was not affected by TMT. In contrast, the
decrease in total forebrain protein was greater than the decrease
in forebrain weight as indicated by the slight reduction (ap-
proximately 5%) in forebrain protein concentration at the 6.0-
mg/kg dose; a finding that indicates the possibility that non-
myelin protein components of forebrain were affected by TMT.
Representative electrophoretic profiles of isolated myelin and
optic nerve protein are presented in figure 2. SDS-PAGE
resolved the proteins characteristic of rat forebrain myelin:
proteolipid protein (M, ~ 24 K), large basic protein (M, «¦ 18
K) and the small myelin basic protein (M," 14 K). These same
proteins constitute a large percentage of total optic nerve
protein (fig. 2., right side). The data obtained from densitome-
try scans (table 4) indicate that the protein composition of
forebrain myelin and optic nerve was not affected by TMT.
Locomotor activity. Dose-dependent alterations in the
development of locomotor activity were observed in the open
field (fig. 3, left panel). The developmental curve for locomotion
generally follows an inverted U-shaped Auction (see Bronstein
et al., 1974) similar to that observed in the saline-treated
animals. TMT altered the shape of this curve; both TMT-
treated groups ware hypoactive relative to controls early in
development (PND13) but became extremely hyperactive by
weaning (PND21). The ontogeny of these toxicant-induced
effects differed with respect to the dose of TMT administered
on PND6. Thus, the time point chosen for activity assessment
is a critical factor in determining both direction and magnitude
of the effects of early postnatal exposure to TMT.
Figure 3 (right panel) illustrates that the hyperactivity found
-------
1984
Brain Development and TMT 747
J ' ^ . :¦-. '• '
B
^\»A
4'^ivV*C
TABLE 2
Effect of acute postnatal administration of TMT on synapsin I
Each value represents the mean (±S.E.M.) of at least seven independent obser-
vations.
Oay Dose
Hippocampus
Forewarn
Total
Cone.
Total
Cone.
13
13
13
22
22
22
66
66
66
mg/
kg
0.0
5.0
6.0
0.0
5.0
6.0
0.0
5.0
6.0
1.00 (±0.13)
0.57 (±0.06)*
0.38 (±0.11)*
1.00 (±0.10)
0.51 (±0.07)*
0.33 (±0.05)*
1.00 (±0.06)
0.60 (±0.07)*
0.41 (±0.03)*t
1.00 (±0.10) 1.00 (±0.10)
0.81 (±0.08) 0.75 (±0.02)'
0.76 (±0.05)' 0.55 (±0.09)*
1.00 (±0.07)
0.90 (±0.09)
0.65 (±0.09)*
1.00 (±0.07)*
0.94 (±0.06)
0.89 (±0.04)
1.00 (±0.09)
0.78 (±0.08)
0.62 (±0.06)*
1.00 (±0.08)"
0.91 (±0.05)
0.79 (±0.06)
1.00 (±0.10)*
0.86 (±0.02)
0.81 (±0.06)
1.00 (±0.08)'
0.94 (±0.09)
0.82 (±0.08)
1.00 (±0.08)*
1.08 (±0.08)
1.03 (±0.06)
c -
Fig. 1. Morphology of dorsal hippocampus on PND22 after acute admin-
istration of 6.0 mg/kg of TMT on PND5. Sections of brains from two
TMT-treated animals are included to show moderate damage (B), severe
damage (C) and control (A). The magnification is the same for all
photomicrographs.
at weaning in the open field can be generalized to a photocell-
based measure of activity, using a Motron device. The group
treated with 6.0 mg/kg of TMT was hyperactive, as indicated
by an increase in photocell interruptions relative to controls.
TMT did not interfere with habituation to the apparatus, fewer
responses were recorded during the final 5-min of testing rela-
tive to the first 5-min for all groups. A group exposed to 7.0
mg/kg of TMT on PND5 was included and showed almost a
400% increase in photocell interruptions relative to controls.
However, this dose does cause some deaths before weaning.
Learning ability. Dose-related deficits in learning ability
were apparent at all ages tested. In the preweaning alleyway
task (table 5) animals treated with 6.0 mg/kg of TMT required
*F(1,20) and P values (linear trend analysis) were 1 92, P < .1806 for concen-
tration of synapsin I in hippocampus at PND66: 3.50, P < .0753 for total synapsin
I in foreOrain and 3.77. P < .0665,2.30, P< 1440; 0.10, P< .7538 for concentration
of synapsin I in forebrain at PND13. 22 and 66, respectively.
* Significantly different from 0.0 mg/kg at P < .05; f significantly different from
5.0 mg/kg at P < .05.
slightly more trials to exhibit a "homing" response. More
importantly, these subjects were unable to retain the response
over a 30-min delay. Only 40% of the animals receiving 6.0 mg/
kg of TMT retained the response compared to 100% of the
controls. Thirty percent of the group exposed to 5.0 mg/kg of
TMT did not retain the homing response even though they
acquired the response at the same rate as the saline-treated
controls. TMT also increased the time required to reach the
goalbox, a finding that may reflect the hypoactivity observed
at this time (see fig. 3).
TMT also interfered with the acquisition of the passive
avoidance response in a dose-related fashion (table 6); the group
exposed to 6.0 mg/kg of TMT required almost twice as many
days of training before they avoided as well as the controls.
The group exposed to 5.0 mg/kg of TMT also required more
days to avoid but this increase was not significant. Inasmuch
as there were no differences in time to emerge from the startbox
on the first day of training (table 6), the learning deficits
probably cannot be attributed to treatment-induced increases
in activity. Although it is possible that the deficiencies in
passive avoidance are the result of treatment-related changes
in shock sensitivity, this explanation also is not likely. All
animals were observed to flinch or vocalize when shocked and
in later testing with a nociceptive stimulus (hot-plate method,
see O'Callaghan and Holtzman, 1975) the treated animals were
found to be hyperalgesic (5.1, 4.9 and 3.0 sec to respond on a
51.5"C hot plate for the control and the groups exposed to 5.0
and 6.0 mg/kg of TMT, respectively [F(2,29) = 5.48, P <
.0096].
Deficient RAM performance was also observed after TMT
(fig. 4). Although no acquisition differences were found (i.e.,
the number of days required to enter and eat in all alleys),
differences were found in the accuracy with which the task was
performed. The group exposed to 6.0 mg/kg of TMT always
made more errors than controls (fig. 4, left panel), as well as
selecting fewer correct arms in the first eight choices (6.3, 6.5
and 7.0; 6.2, 6.5 and 6.6; 5.6, 5.9 and 5.9 choices in the three
consecutive 5-day blocks of testing for the control and the
groups exposed to 5.0 and 6.0 mg/kg of TMT, respectively
[ir'(2,31) = 6.94, P < .0003; 2.79, P < .0769; 16.36, P < .0001)
-------
748 Miller and O'Callaghan
Vol. 231
TABLE 3
Effect of acute postnatal administration of TMT on indices of myelinatlon at PND22
Each value represents the mean (± S.E.M.) of at least six independent observations.
Forewarn
Optic Nerve
Dose
Total
Cone.
Total
Wt.
Protein
Myelin protem
Protein
Myelin protein
Protein
mg/kg
0.0
5.0
6.0
1.300 (±0.010)
1.037 (±0.034)*
0.832 (±0.028)'
mg
199.22 (±5.88)
175.03 (±9.96)*
127.79 (±5.75)'t
mg
1.566 (±0.086)
1.023 (±0.262)*
1.034 (±0.084)'
m9/9
151.29 (±2.32)
150.42 (±1.77)
144.84 (±1.94)*
">9/9
2.40 (±0.14)
2.29 (±0.13)
2.46 (±0.17)
mg
0.431 (±0.008)
0.400 (±0.010)'
0.359 (±0.006)*t
* Significantly different from control. P < .05; t significantly different from 5.0 mg/kg, P < .05.
FOREBRAIN
MYELIN
OPTIC
NERVE
I
O
X
O
<
Z>
u
LU
O
50
24
18
17
14
Discussion
0 5 6 0 5 6
DOSE OF TMT
(mg /kg)
Fig. 2. Staining pattern of PND22 forebrain myelin and optic nerve
proteins after resolution by SDS-PAGE. A total of 10 /ig of protein were
loaded on each lane.
for the treatment effect in the three respective blocks). No
learning deficits were observed in the group exposed to 5.0 mg/
kg of TMT but this and the group exposed to 6.0 mg/kg of
TMT were hyperactive as indicated by an increase in photocell
interruptions (fig. 4, right panel) and total arm selections (23.0,
22.6 and 23.5; 23.9, 28.3 and 29.9; 32.3, 35.1 and 35.2 arms in
the three consecutive 5-day blocks of testing for the control
and the groups exposed to 5.0 and 6.0 mg/kg of TMT, respec-
tively [.F(2,31) = 7.64, P < .002] for the overall treatment
effect).
The neurotoxic organometal, TMT, interferes with brain
development based on the following observations: 1) permanent
weight deficits in forebrain, hippocampus and cerebellum; 2)
loss of pyramidal cells in hippocampus; 3) age-related decreases
in the neuron-specific phosphoprotein, synapsin I; and 4) al-
terations in activity and learning throughout the earlier por-
tions of the lifespan of the animal. Data are discussed by
category according to the three indices used to characterize the
developmental neurotoxicity of TMT.
Morphology. Normal postnatal maturation of the rat brain
requires the balanced interaction and timing of several critical
processes, including cell division, migration and differentiation.
Thus, it is not surprising that chemical d physical interven-
tion during the early postnatal period auers many aspects of
brain morphology, including brain weight (e.g., Bayer et al.,
1973; Bohn, 1980; DeKosky et al., 1982; Seress, 1978). In this
study, TMT caused a large dose-related decrease in the weight
of the hippocampus and less pronounced weight deficits in
forebrain and cerebellum, findings that replicate the work of
Ruppert et al. (1983) and extend it to several earlier postnatal
ages. Whereas it is obvious that overall brain development was
affected by the administration of TMT on PND5, our morpho-
logical findings are also indicative of an adult-like effect, i.e.,
preferential involvement of the hippocampus. This notion is
supported by three lines of evidence: 1) weight deficits in
forebrain and cerebellum (expressed as a percentage of control)
diminished with age whereas deficits in hippocampal weight
remained constant; 2) damage to the hippocampus was char-
acterized by loss of pyramidal cells [cells formed prenatally
(Bayer, 1980)]; and 3) the pattern of pyramidal cell loss (CA3-
CA4) resembled that seen in the adult (Brown et al., 1979;
O'Callaghan and Miller, 1984). Our morphological assessment
of the developmental neurotoxicity of TMT was at best prelim-
inary. Future studies would benefit from a detailed morpho-
metric analysis of the neuronal damage as well as immunocy-
tochemical characterization of the affected nervous system-
specific proteins (see below).
Nervous system-specific proteins. We have proposed
that toxicant-induced interference with nervous system devel-
opment should be accompanied by alterations in nervous sys-
tem-specific proteins (O'Callaghan and Miller, 1983). One such
protein is synapsin I (formerly protein I), a neuron-specific
phosphoprotein associated with synaptic vesicles (De Camilli
et al., 1983a,b). As synapsin I is apparently present in all nerve
terminals (De Camilli et al., 1983a) and its ontogeny coincides
with synapse formation (Lohmann et al., 1978), measurement
of this protein may provide a procedure for determining the
-------
1984
Brain Development and TMT 749
TABLE 4
Effect of acute postnatal administration of TMT on protein composition of forebrain and optic myelin at PN022
Each value represents the mean (±S.E.M.) of at least six independent observations.
% Total Prowin
Dosa
Forebrain
Optic netve
Small basic
Large basic
Protsotptf
Small basic
Largs basic
Proieotpid
prolan
protein
protein
proton
protein
protein
mgjkg
0.0
17.85 (±0.85)
15.22 (±0.81)
31.89 (±1.47)
14.29 (±1.25)
4.64 (±0.41)
22.21 (±1.95)
5.0
18.58 (±1.11)
15.89 (±0.88)
30.65 (±1.52)
12.00 (±1.10)
4.41 (±0.47)
19.31 (±2.72)
6.0
18.60 (±1.31)
16.21 (±0.86)
30.36 (±1.49)
14.56 (±1.90)
4.99 (±0.51)
15.82 (±2.41)
Fig. 3. Open-field activity over home-cage litter for
the same animals at 10, 13, 15, 17 and 21 days
of age as a function of dose (0.0, 5.0 or 6.0 mg/
kg of TMT i.p.) on PN05. Repeated measure
analysis of variance indicated an interaction be-
tween treatment and age [^(8,124) - 9.63, P <
.0001]. Treatment effects were observed at
PND10 [F(2,31) - 4.10, P < 0264]; PND13
[F(2,31) - 12.44, P < .0001); PND17 {f(2,31> =
8.22, P < .0014]; PND21 [F(2,31) - 13.76, P <
.0001]; but not PND15 [f(2,31) = 0.78, P <
.4685]. Activity (photocell interruptions) at PN021
in the Motron (right panel) expressed as mean
interruptions in the consecutive 5-rnin intervals of
the 25-min session and mean total interruptions
as a function of dose. A repeated measure analysis
of variance showed an effect of treatment (F(3,20)
- 17.06, P < ,0001 ] and interval (F(4,80) =» 12.49,
P < .0001] but no interaction between these vari-
ables. "Significantly different from control at P <
.05; +significantly different from 7.0 mg/kg of
TMT-exposed group at P < .05.
TABLE 5
Effect of acute postnatal administration of TMT on prewesning instrumental alleyway conditioning at PND14
Each value represents the mean (±S.E.M.).
No. of Trials to
Time on final
No. Retaining on
Mean Time on Final
Dosa
N
Body wt.
Homing
Honing
first Retention
Five Performance
Criterion
Trial
Trial
Trials
ty)ls
3
MC
MC
0.0
10
32.4 (±0.8)
2.4 (±0.4)
11.8 (±2.5)
10/10
15.2 (±3.0)
5.0
10
30.9 (±1.0)
2.4 (±0.3)
18.2 (±5.6)
7/10*
24.4 (±6.4)
6.0
10
31.2 (±1.0)
3.9 (±0.5)*
37.1 (±6.0)*
4/10~
41.5 (±11.3)*
* Significantly different from control, P < .05; * significantly different from control, P < .05; ** significantly different from control, P < .002 using test for significance of
difference between two proportions (Bruning and Kirrtz, 1906),
synapain I was reduced, findings suggestive of a reduction in
synaptic denaity within the hippocampus. Several interpreta-
tions for these data can be advanced, among which are the
following: 1) decrements in synapsin I concentrations may
indicate that TMT interferes with events related to synapto-
genesis and thereby delays synapse formation and 2) decre-
ments in synapain I concentration (PND13 and 22) may reflect,
as they appear to in the adult (O'Callaghan and Miller, 1984),
the extensive loss of pyramidal cells.
The effects of TMT on the concentration of hippocampal
synapsin I during the preweaning (PND13) and juvenile
(PND22) periods could not be extended to the adult (PND66).
This apparent "recovery" in synapsin I concentration makes it
tempting to speculate that the developing hippocampus may
respond to TMT-induced injury with homotypic reinnervation
and subsequent synapse formation, a phenomenon referred to
as reactive synaptogenesis (Cotman and Nadler, 1978; Cotman
et al„ 1981). Several recent observations support this hypoth-
esis. For example, reactive synaptogenesis occurs more readily
- O—O OOm^/kaTMT
A ¦ 6 5 0 mg/kj TMT
O—O d0«g/kg TMT
O—O0 0
50
O—TMT
O-Q70
-2700
2 500
- 1900
13 15 17
DAYS OF AGE
1 2 3 4 5 Total
SUCCESSIVE 5MIN INTERVALS
TABLE 6
Effect of acute postnatal administration of TMT on passive
avoidance conditioning
Each value represents the mean (±S.E.M.). Tasting began at PN022.
NaofOtytto
Time to emerge from
Doat
N
Avoidance
the Start Box on
Criterion
Oay 1 of Training
rng/dg
m
0.0
11
6.9 (±0.4)
5.9 (±2.6)
5.0
9
8.2 (±0.5)
2.4 (±0.9)
6.0
12
10.8 (±0.7)*
11.1 (±3.2)
* Significantly different from control, P < .09.
density of nerve terminals in neural tissue (Goelz et aL, 1981).
In the present study we found that administration of TMT on
PND5 resulted in large dose-related decreases in hippocampal
synapsin I without causing comparable changes in forebrain
synapsin I. These data indicate that the hippocampus was
preferentially affected by exposure to TMT on PND5. Of
particular significance is the fact that the concentration of
-------
750 Miller and O'Callaghan
Vol. 231
ACCURACY
ACTIVITY
Doit of TMT ( mg/kg)
0.0
*-65 0
6.0
OC
Q£
o
u 16
6-10 11-15 1-5 .6-10 11-15
BLOCKS OF 5 DAYS
Fig. 4. Accuracy in the RAM expressed as mean total selections required
to get eight correct arms for as a function of dose (0.0, 5.0 and 6.0 mg/
kg i.p.) on PND5. Testing began at PN037. A repeated measure analysis
of variance showed an effect of treatment [F(2,31 » 9.74, P < .0005]
for total choices. The 6.0 mg/kg of TMT-exposed group consistently
required more total choices to obtain all eight pellets compared to either
the 5.0 or 0.0 mg/kg of TMT-exposed groups. Activity in the RAM is
expressed as mean photodtode interruptions per minute. A repeated
measure analysis of variance showed an effect of treatment [f(2,31) -
4.10, P < .0263] and block of testing [F(2,62) -10.31, P < .0001], TMT-
treated groups were always more active than the saline-treated control
but the activity of all groups increased as testing continued. 'Significantly
different from saline at P < .05.
in the immature animals than the adult (Gall and Lynch, 1980;
Tsukahara, 1981; McWilliams and Lynch, 1983) and is espe-
cially prevalent in the hippocampal formation (Cotman and
Nadler, 1978; Cotman et al., 1981; Tsukahara, 1981). Although
reactive synaptogenesis has been documented more extensively
by reinnervation of dentate granule cells after lesions of the
entorhinai cortex (Cotman et al., 1981; Finger and Stein, 1982;
Oswald and Vinsant, 1983), recent studies have shown that
kainate-induced pyramidal cell loss in CA3 to CA4 is followed
by gradual reinnervation, apparently by surviving homotypic
fibers (Nadler et al., 1980a,b). Thus, it is possible that reactive
synaptogenesis in the hippocampus, like that observed with
kainic acid, may result from exposure to TMT on PND5 and
thus account for the apparent recovery in synapsin I concen-
tration that occurred by PND66. Regardless of the changes
associated with the recovery in hippocampal synapsin I, these
events were not accompanied by a return to control values with
respect to total synapsin I, hippocampal weight or neurobehav-
Loral function.
Other nervous system-specific proteins may be used to mon-
itor postnatal events associated with nervous system develop-
ment. For example, oligodendroglia proliferation occurs just
before the onset of myelination. Proteins specifically associated
with myelin, e.g. myelin basic protein and proteolipid protein,
become detectable at this time and increase in concentration
as myelination proceeds (Norton, 1981). Thus, proteins specific
to myelin may serve as a biochemical markers for events
associated with the process of myelinogenesis. We have shown
previously that TET, an organotin that is structurally related
to TMT and known to damage rat myelin sheath (Jacobs et al.,
1977), appears to affect myelin after administration of TET on
PND5 (O'Callaghan et al., 1983). Although TMT and TET
cause almost equivalent whole and regional brain weight de-
creases (O'Callaghan et al., 1983; Miller, 1984), our present
findings indicate that TMT does not affect myelin concentra-
tion or protein composition. The impact of TET on neuronal
elements of either the developing or adult hippocampal for-
mation has not been determined.
Function. Persistent alterations in activity often accompany
toxic insult or injury to both developing and mature CNS
(Bayer et al., 1973; DeKosky et al., 1982; Miller, 1984; Ruppert
et al., 1983; Walsh et al., 1982). Here changes were observed in
both the developing and mature animal and in a variety of
monitoring devices. Our findings again reinforce the notion
that the activity of an animal can serve as an index of CNS
function (Grant, 1976; Reiter and MacPhail, 1982) and that
the incorporation of home-cage cues into the test situation
allows for the functional assessment of the preweaning rodent
(see Miller, 1984; Smith and Spear, 1978). The effect of TMT
on the development of locomotor activity presents an interest-
ing paradox because the effect, hypoactivity, hyperactivity or
no change in activity, is dependent on when in the develop-
mental sequence the behavior is measured. The cerebellum and
hippocampus both figure prominently in the expression of
activity and interfering with the normal ontogeny of either
structure by direct cytotoxicity or by preventing cell division
and growth alters the characteristic pattern of locomotor de-
velopment (Bayer et al., 1973; Pelligrino and Altman, 1979).
Thus, the ontogeny of TMT effects on preweaning locomotion
may be due to damage in either of these brain areas. Whatever
the cause, the presence of these differential effects does suggest
the importance of including more than one time point when
the development of a behavior is in question.
The weight deficits, loss in neurons and lowered synapsin I
levels found in hippocampus provide support for the contention
that TMT interferes with the development of this structure
and it is reasonable to assume that hippocampal function
should also be altered. In general, the behavioral deficits ob-
tained, hyperactivity, poor retention of the preweaning learning
task, slower passive avoidance acquisition and, in particular,
the deficient RAM performance, are consistent with the notion
that hippocampal Auction is altered (see Jarrard, 1976,1980).
However, to attribute the observed behavioral deficits solely to
a dysfunction of the hippocampus, in particular, or the limbic
system, in general, is unwarranted. Other brain areas are ob-
viously affected by TMT. The forebrain is reduced in size and
the possible alterations in the processing and integration of
sensory information caused by the dysfunction of cortical areas
cannot be dismissed as a potential contributing factor (see
Dean and Key, 1981). No obvious functional deficits that can
be attributed to compromised cerebellar function (e.g., faUs in
the open-field or ataxia) accompanied the reduction in cerebel-
lar size. However, the putative role of this structure in learned
motor responses (see Thompson, 1983) as well as the ability of
TMT to interfere with tasks reflecting cerebellar development
(Ruppert et al., 1983) suggests that the possible contribution of
cerebellar dysfunction to the observed deficits cannot be over-
looked.
AckaowladgBMBU
The author* with to thank Dr. Hany A. Makkawy and Drawry M. Vincent for
•xealhot technical aaaietance, Chriatine Booth tor expert preparation of the brain
(action* and Dr*. Paul Greengard, Michael D. Browning and Linda J. Burdetta
for uaeftil diacuaaiona.
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Btff*, S. A., Bxuknsr, R. L,, Hun, R. AMD ALTMAN, J.: Behavioural effects
-------
1984
Brain Development and TMT 751
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-------
Psychopharmacology (1982) 78:19 - 22
alkyltins
Psychopharmacology
'("> Springer-Verlag 1982
Acute Treatment with Trimethyltin Alters Alcohol Self-Selection
R. D. Myers1, H. S. Swartzwelder1, and R. S. Dyer2
1 Center for Alcohol Studies, Department of Psychiatry and Pharmacolog}. University of North Carolina School of Medicine.
Chapel Hill, NC 27514. USA
Neurotoxicology Division, US Environmental Protection Agency. Research Triangle Park. NC 27711. USA
Abstract. Male rats of the Long-Evans strain were divided
into two equal groups of nine each and given either 7,0 mg. .kg
trimethyltin (TMT) or 0.9% saline by intragastric gavage.
The pattern of self-selection of alcohol in concentrations of
3% — 30% was examined in both groups at 21 and 150 days
following the gavage. The TMT-treated rat consistently
drank less alcohol than did the controls at every concen-
tration of alcohol. This difference in alcohol intake was
equally significant when the rats were tested in a food-
contingent, schedule-induced polydypsia situation. Further,
although the TMT-lesioned animal consumed fewer calories
per day in the form of alcohol, their overall daily caloric
intakes were slightly higher than those of the controls. These
results are interpreted as a consequence of damage to
structures of the forebrain and as part of a syndrome of
behavioral and neurological pathology.
Key. words: Alcohol — Trimethyltin — Quinine — Oral
consumption of alcohol — Polydipsia - Hippocampus
The hippocampal formation has been shown to be highly
sensitive to the effects of both acute (Grupp and Perlanski
1979; Swartzwelder et al. 1979) and chronic (Riley and
Walker 1978; Walker et al. 1980) ETOH administration.
In this connection, the hyperactivity, convulsions, and
hippocampal histopathology that characterize acute TMT
toxicosis suggest that the TMT-treated rat may be differen-
tially sensitive to ETOH.
The present study was designed to examine the effect of
acute treatment with TMT upon volitional ETOH drinking.
In these experiments, we monitored ETOH intake both in
terms of self-selection in the home cage and drinking during
responding for food under a fixed-ratio (FR) schedule of
reinforcement. Both the untreated control and TMT-treated
rats were offered a wide range of ETOH concentrations. In
addition, an identical profile of caloric intake of these animals
was analyzed in conjunction with this ETOH-preforcnce
sequence. Finally, the gustatory sensitivity of each of the rats
was also quantified by a preference test for quinine solutions
of varying concentration.
Chemical lesions of neurotransmitter pathways in the brain of
the rat are known to alter voluntary drinking of ethyl alcohol
(ETOH) (Kiianmau et al. 1975; Myers and Melchior 1975).
An injection of 5,6-dihydroxytryptamine (5,6-DHT) into the
cerebral ventricle of the rat can augment its consumption of
ETOH offered in different concentrations, whereas 6-hy-
droxydopamine (6-OHDA) may exert the opposite effect on
ETOH self-selection (Melchior and Myers 1976). Kainic acid,
which lesions glutamine-containing neurons iit the forebrain
(McGeer et al. 1978), also may shift the preference for ETOH
(Myers et al. 1981). Though reports differ on the effect on
drinking of a neurotoxic lesion (Kiianmaa et a). 197S; Brown
and Amit 1977), it is clear that a functional imbalance in
central neurotransmitter activity may serve to alter ETOH
preference (Myers and Veale 1968).
Trimethyltin (TMT, [CHj]3Sn) is an alkyl tin derivative
that has marked neurobehavioral effects when administered
systemically (Barnes and Stoner 1958; Dyer et al. 1982). For
example, conspecific aggression (Barnes and Stoner 1958;
Brown et al. 1979), hyperactivity (Swartzwelder et al. 1981).
and convulsions (Oyer et al. 1982) have been observed within
1 week following TMT treatment. In addition. Brown et at.
(1979) noted marked cytological damage to the hippocampal
formation following systemic TMT treatment.
Offprint requests to: R. D. My en
Materials and Method*
Male Long-Evans rats, obtained from Charles River and
weighing 348-460 g, were housed in individual case* and
maintained throughout on a 12-h light-dark cycle. Atu-r tiie
animals were divided into two groups of nine each, they were
given either 7.0tng/kg {CH,]3SnCl (TMT: ICN) or ivr(>
saline by intragastric gavage. The tin compound w.i» Ji>^oi-
ved in the saline vehicle and administered in a volume of
1.0 ml. kg body weight. The single dose was selected because
preliminary observations showed that this concentration
produced a well-defined syndrome ol"acute toxicity (Dyer et
al. 1982).
ETOH Prefitrvm i". At 21 days after TMT treatment, the rats
weighed 384- 477 #. The case of each animal was fitted w ith
three 100-ml Kimax drinking tubes: one contained water. .t
second contained a V, V solution of ETOH prepared with tap
water and 95ETOH: and a third dummy tube wa* empty.
By random rotation ol* the three bottles on each day of the
8-day testing sequences, the development of a position habit
was prevented (Myers and Holman l%6l. The ETOH
solution was increased in concentration on each of eight
successive days as follows: 3%. 'V ?"«• l,"»- '-V
20%. and 30 V Fluid intakes were recorded and the tubes
containing water and the freshly prepared ETOH solution
were refilled on each day at the same time. A second test
0033-3158.82,0078,0019 $01.00
-------
20
sequence, identical to the first, was begun 150 days after the
TMT treatment with the rats weighing 456-587 g. Records
of fluid intakes were taken at the same time in the morning of
each day.
Preference Test for Quinine Solutions. To test for possible
differences in gustatory sensitivity between the TMT-treated
and control groups, an analysis of the preference-aversion
profile for quinine compared with water was undertaken for
each rat using the three bottle-two choice method already
described for alcohol preference. Quinine selection was tested
across concentrations of 0.5 —16.0 mg/1 according to meth-
ods described previously (Myers and Oblinger 1977). This
procedure was initiated immediately following the termi-
nation of the second alcohol preference test, i.e., 159 days
after TMT had been administered.
Food Intake and Caloric Measurement. The intake of food by
each rat was monitored during the preference sequences. The
total number of calories consumed either in the form of
ETOH or food biscuits (Purina Rat Chow) was calculated
according to the tables of Veale and Myers (1968).
Adjunctive ETOH Drinking. At 5 days after the first home
cage ETOH preference test, each of eight TMT-treated and
eight control rats was reduced to 85 % of its free-feeding body
weight by limiting food intake to one-half the normaJ daily
amount over a period of 5 — 7 days. Then, each rat was trained
to depress a lever in a Plexiglas one-lever operant chamber
during a 3-h session on each day. Each chamber was fitted
with a food dispenser that, when activated, delivered a 45-mg
Noyes food pellet.
Two calibrated drinking tubes were mounted on the wall
opposite to the lever: one contained tap water and the other a
9% solution of ETOH (V/V). Each tube was fitted with a
lickometer device so that the rate and periods of drinking
during the operant sessions could be measured, as well as the
volume of each fluid consumed. A microprocessor-controlled
system permitted reinforcement schedules to be varied daily
in an ascending scries from FR 2 to FR 99. On the final test
day, each animal was placed in the operant chamber with the
pellet dispenser inactivated so that the pattern of responding
during extinction could be determined. At the end of every
test session, each rat was weighed and given a portion of food
that was sufficient to maintain the animal at 85 % - m.M.-r MenscuiiMNtontlv observed
*',v M.wt of these Mj-m declined
4C'
JO
I
<5 2C'
*
\
3 to-
-J I Of
<
0 8-
1 6'
s
4.
b 2 ¦
i o
. ccktbolQo O
1 tm-tinQ«-«
11 1 i
J
.14141
m
Y\
3 5 7 91215 20 JO
* ETOH ON EACH OAT
Fig. I. Mean ± SE grams of aclohol per kg body weight consumed imp)
and mean±SE proportion (PROP.) of alcohol to total fluid intake
(bottom). Concentrations of alcohol (ETOH) are plotted for each
successive day on the abscissa. The test sequence was initiated -1 days
after treatment with trimethyltin (TM-T1N) or a saline-control vehicle
control ~ 0-0
TM-TlNQ»-»
4.0
3.0-
2.0 ¦
I. Of
5 7 9
6 20 30
* ETOH ON CACH OA*
Fi». 2. Mean £ SE aranu of alcohol per kg body weight coiiMimed < V'ii
and mean ± SE proportion (PROP.) of alcohol to total fluid ir.r.ike
(bottom). Concentration! of alcohol (ETOH) are plotted for each
successive day on the abscissa. The test sequence was initiated 150 days
after treatment with trimethyltin (TM-TIN) or a saline-control vehicle
within 21 days after treatment, although some of the animals
remained difficult to handle.
Throughout each ETOH-preference sequence, the TMT-
treated rats consumed markedly less ETOH than controls.
Figure 1 presents the noteworthy differences across all
concentrations observed between the two groups in terms of
both g/kg alcohol consumed per day (r(14)« 5.41, P< 0.011
as well as the proportion of ETOH to total fluid consumed
Mt4)» 3.86, P< 0.01). During the first sequence. 21 days
post-treatment, the intake of ETOH by the controls was
essentially 1.5-3.3 g/kg/day (Fig. 1>. With the exception of
the 3% and 5% concentrations, the intake of the TMT-
treated rats fell below 1.0 g/kg and. at the five higher
concentrations, were virtually negligible. Similarly, as shown
in Fig. I. the proportion of ETOH to total fluid reflected this
progressively marked suppression of intake with no overlap
of the SE values below 15 ETOH concentration.
-------
FB SC-EOULE
H;;. 3. Mean + SE proportion (PROP.) of licks on the drinking tube
¦-•''iitaining 9°^ alcohol (ETOH) to the total number of licks per 3-h lest
•c>Mon. The increasing fixed ratio (FR) schedules on which the TM-TIN-
trcated and control rats were tested are plotted for each day on the
¦ib>cissa
fable I. Mean + SE daily intake of kilocalories as alcohol (ETOH)
and lab chow by TMT-treated and control rats during the first alcohol
>olf-sclection test
Saline-treated
TMT-treated
Calories as ETOH
5.95+1.73
1.89 + 0.48
Calories as lab chow
85.22 + 3.16
110.81 ±5.07
Temporal Persistence of Alcohol Aversion. At 150 days after
TMT treatment, the separation between control and treated
animals was just as clear, both in terms of the g/kg/day
alcohol consumed [r( 14) — 12.78, /><0.0l] and the pro-
portion of ETOH to total fluid intake [/(14) = 7.15, P < 0.01],
This is illustrated in Fig. 2, which shows also that the ETOH
intakes of both groups had increased during this test
sequence.
Schedule-Induced Drinking. During the 12-day period of
'¦.Mint} under the condition of FR-induced drinking, the
isoiureated control rat generally licked the ETOH tube at a
ijlio of 0.4:0.65, in contrast to the water-containing tube. As
Portrayed in Fig. 3, this proportionality was maintained at
^ach of the FR schedules of reinforcement during the operant
^.'Ssion. However, the TMT-treated animals maintained their
Version to ETOH in this adjunctive drinking situation. This
K reflected in the low proportion values (Fig. 3).
Quinine Drinking and Other Measures. In examining the
£u*tatory sensitivity of the two groups, the selection-rejection
ratio (proportion) between water and the inert quinine
impound was essentially identical for both control and tin-
seated rats.
There were no significant differences between the groups
>n the drinking of quinine solution across the concentrations
"•5 —16.0 mg/l [/(16) = 0.43. P> 1.0]. During the test
sequence the mean consumption of quinine failed to exceed a
ratio of 0-50.
An analysis of the caloric intake of each animal revealed
•hat the TMT-treated rat consumed significantly more total
Glories per day than controls [/(16) = 2.95, P < 0.01 J. As
Presented in Table I, however, the untreated rats consumed
significantly more of their daily calorics as ETOH during the
preference sequence and less in the form of food than the
Fig. 4. Sagittal section stained with cresyl violet of a representative rat
given saline gavage (top) and 7.0 mg kg TM-T1N ((CH,],SnC1. bottom).
Histological material was obtained 200 days after the respective treat-
ments. .4rrmt depicts region of hippocampal damage
TMT-treated rats. That is, the control rats averaged
5.95 kcal, day as ETOH in comparison to only 1.89 kcal day
ETOH for the TMT animals.
Detailed histological analysis of the sagittal sections
revealed that the hippocampal formation of each of the TMT-
treated rats exhibited severe cytological damage. A typical
magnified section taken from the brain of the non-TMT-
treated control rat is shown at the top of Fig. 4. The bottom of
Fie. 4 depicts a distinct loss of cells in the CA4 field
accompanied by cellular thinning of the CA 1 - CA 3 regions,
clearly visualized in the representative photomicrograph.
Discussion
A single systemic dose of TMT given to the rat remarkably
reduced its volitional drinking of ETOH at all concentrations
offered. This powerful effect was observed during both of the
experimental test situations, i.e., home cage and operant
chamber. Equally significant is the apparent permanence of
the aversive effect of the compound on ETOH intake.
Moreover, TMT-treated animals consumed fewer of their
daily calories as ETOH than did controls, though their daily
caloric intakes were higher. This suggests a distinct neuro-
toxic effect on the mechanisms, possibly located in the brain,
involved in the consumption of alcohol and perhaps other
drugs. Furthermore, this alteration in drinking was accom-
panied by cell loss in the hippocampus particularly evident in
the CA 4 field as described previously by Brown et al. (1979).
The effect of TMT is clearly not due to a nonspecific
change in gustatory sensitivity. That is, quinine intakes of
both groups coalesced throughout all concentrations offered
in a nearly identical self-selection test paradigm. In this
-------
22
connection, the avoidance of alcohol by the TMT-treated rat
cannot be attributed to the development of a conditioned
taste aversion. The typical mode of conditioned taste aversion
induction involves the temporal pairing of a novel taste with
an acute drug-induced toxicosis (Garcia et al. 1966). In the
present study, the rats had not tasted ETOH prior to the TMT
poisoning, nor did they have their first exposure to ETOH
until fully 3 weeks had elapsed from the time of the treatment.
Whether secondary toxicological effects of TMT on the rat's
liver could have altered the metabolism of ETOH is presently
unknown.
Although the central mechanism of TMT action is not
clear, the toxic compound is known to produce neurological
and behavioral pathology. Behaviorally, TMT-treated ani-
mals exhibit a transient and complex syndrome consisting of
anorexia, aggression, self-mutilation, and convulsions which
persist for several weeks following treatment (Dyer et al.
1982). A somewhat more permanent effect of TMT on
activity levels has been reported (Swartzwelder et al. 1981). A
marked loss of neurons in the hippocampal formation occurs
following the systemic administration of TMT (Brown et al.
1979; Dyer et al. 1982).
Since ETOH but not quinine intake is altered by TMT, it
is possible that the toxicant may selectively after the animal's
physiological response to many centrally acting drugs includ-
ing morphine. Thus, the hippocampal damage that is engen-
dered by TMT could result in a disruption of the normal
central effects of alcohol. As a result, alcohol could be more
aversive\o the rat and, as a consequence, self-selection of the
fluid is reduced. Evidence for this view is based on obser-
vations thaL^la^ifiLfiaa^fiiiLmaduji^ndjcjkctivedTec^on^
tht-hippocampus (Grupp and Pgrlaaski—*979; Pj|jj nnil
Walker 1978; Walker et al. 19S§^Siiailzwelder et al. 1929)^
Acknowledgements. Supported in part by NIAAA grants AA 05160-0IA
to H. Swartzwelder and AA 040200-01A to R. Myers. The authors are
indebted to W. Holahan, H. Leverenz. S. King, P. Miller, and J. Hepler
for their expert technical assistance throughout these experiments.
References
Barnes JM, Stoner H8 (1958) Toxic properties of some dialkyl and
triaikyi tin salts. Br J Indust Med 15:15-22
Brown AW. Aldridge WN, Street BW, Verschoyle RD (1979) The
behavioral and Muropathotogk sequelae of intoxication by tri-
methyltin compound* in the ret. Am J Pathol 97:59-82
Brown ZW, Amit Z (1977) The effects of selective catecholamine
depletions by 6-hydroxydopaminc on ethanol preference in ruts.
Neurosci Lett 5:333-336
D>er RS, Walsh TJ. Wonderlin W.F, Bercegeay M (1982) The tri-
methyltin syndrome in rats. Neurobehav Toxicol 4:127— 134
Garcia J. Ervin F. Koelling R (1966) Learning with prolonged delay of
reinforcement. Psychonom Sci 5:121 — 122
Grupp LA. Perlanski E (1979) Ethanol-induced changes in the spon-
taneous activity of single units in the hippocampus of the awake rat:
A dose-response study. Neuropharmacology 18:63-70
Kiianmaa K. Fuxe K. Jonsson G. Ahtee L (1975) Evidence for
involvement of central NA neurones in alcohol intake: Increased
alcohol consumption after degeneration of the NA p-.iihw.iy to the
cortex cerebri. Neurosci Lett 1:41-45
McGeer EG, Olney JW, McGeer PL (1978) Kainic acid as a tool in
neurobiology. Raven. New York
Melchior CL, Myers RD (1976) Genetic differences in eihanol drinking
of the rat following injection of6-GHDA. 5,6-DHT or 5.7-DHT into
the cerebral ventricles. Pharmacol Biochem Bchav 5:63-72
Myers RD. Holman RB (1966) A procedure for eliminating position
habit in preference-aversion tests for eihanol and other fluids.
Psychonom Sci 6:235-236
Myers RD, Melchior CL (1975) Alcohol drinking in the rat after
destruction of serotonergic and catecholaminergic neurons in the
brain. Res Commun Chem Pathol Pharmacol 10:363-378
Myers RD, Oblinger MM (1977) Alcohol drinking in the rat induced
by acute intracerebral infusion of two tetrahydroisoquinolincs and a
0-carboline. Drug Alcohol Depend 2:469 - 483
Myers RD. Veale WL (1968) Alcohol preference in the rat: Reduction
following depletion of brain serotonin. Science 160:1469- 1471
Myers RD, Swartzwelder HS. Leverenz H (l98l)Glutaminergic neuron
lesions in the brain of the rat: Effects on alcohol drinking.
Alcoholism Clin Exp Res 5:162
Riley JN, Walker DW (1978) Morphological alternations in hippo-
campus after long-term alcohol consumption in mice. Science
20t :646 — 648
Swartzwelder HS. Dyer RS, Holahan W, Myers RD (1981) Activity
changes in rats following acute trimethyltin exposure. Neuro-
toxicology 2:589 - 594
Swartzwelder HS. Johnson CT. Cooley BC. Howell WF. Dyer RS (I97«#»
Alcohol-induced alternations in hippocampal afterdischarges and
afterdischarge thresholds: Dose-response studies. Neurobehav
Toxicol 1:253-258
Swartzwelder HS, Hepler J. Holahan W. KineS. I.ewrenz H. P.
Myers RD (1982) Impaired maze performance hi ihc rut c.iuiw'.l
by trimethyltin treatment: Problem-solving detkits and persevera-
tion. Neurobehav Toxicol Teratol 4-. 169-176
Veale WL, Myers RD (1968) Table for determining gram and caloric-
values of ethanol solutions. Purdue Neuropsychol Ser 1:1-4
Walker DW, Barnes DE. Zornetzer SF. Hunter BE. Kuban is p (1980)
Neuronal loss in hippocampus induced by prolonged ethanol
consumption in rats. Science 209:711 - 713
Received September 22, 1981; Final version April I. 1982
-------
NeuroToxlcology® 10:393-406,1989
Copyright © 1989 by Intox Press, Inc.
Assessment of Chemically-Induced Alterations in Brain Development
Using Assays of Neuron- and Glia-Localized Proteins
James P. O'Callaghan and Diane B. Miller
Health Effects Research Laboratory U.S. Environmental Protection Agency
Research Triangle Park, North Carolina 27711, USA
ABSTRACT: Chemical-induced injury of the developing central
nervous system (CNS) is often manifested by alterations in the
cellular ontogeny of specific neuroanatomical regions. Within the
affected area, critical developmental processes encompassing a
variety of neuronal and glial cell types may be transiently or per-
manently altered. Because the cellular heterogeneity of the
developing CNS is expressed by unique neuronal and glial pro-
teins, we proposed that radioimmunoassays of these proteins can
be used to define normal and chemically- altered patterns of CNS
development. We are testing this hypothesis by administering
prototype neurotoxicants to the developing rat and then assessing
the effects of these agents on previously characterized neuronal
and glial proteins. Using this approach, we have characterized
several features associated with perinatal chemical exposure: (1)
region-dependent patterns of altered brain development are
revealed by changes in the amounts of specific neuronal and glial
proteins; (2) chemical-induced changes in neuroQal and glial pro-
teins depend on the time of exposure and nature of the insult; and
(3) significant changes in neuron• and glial-localized proteins can
be observed in the absence of cytopathology or decreases in
brain weight. Data obtained from studies of toxicant-induced
injury of the CNS will be presented as models for the use of neu-
ron- and glial-localized proteins as biochemical indicators of
altered brain development e 1989 intox Press, inc.
Key Words: Development, Brain, Neurotoxicity, Neurons, Glia, Astrocytes, Synapsin I, p38,
Myelin Basic Protein, Glial Fibrillary Acidic Protein, Alkyltins
INTRODUCTION
Chemical intervention during prenatal or
postnatal ontogeny can result in complex bio-
chemical, morphological and behavioral alter-
ations in brain development (Suzuki, 1980;
Miller and O'Callaghan, 1984; Rodier, 1986;
Ruppert, 1986). As has been shown at this con-
ference (e.g., by Hammer and coworkers), the
knowledge of specific actions of a given
drug/chemical in the adult can serve as a guide
for choosing processes affected by the same
compound administered during development.
Commonly, however, the neuronal or glial sub-
strates for chemical action in the developing
brain are not known and cannot be easily pre-
dicted on the basis of effects observed in the
mature animal. Indeed, there are a multitude of
structurally dissimilar compounds that affect
diverse and unpredictable targets in the devel-
oping brain (Spencer and Schaumburg, 1980;
Craruner and Tilson, 1986). The focus of this
review is to present an approach for detecting
Please send requests for reprints to Or. James P. O'Callaghan, Neuro toxicology Division (MD-74B) Health
Effects Research Laboratory U.S. Environmental Protection Agency Research Triangle Park, NC 27711 USA.
Presented at the Sixth International Neurotoxicology Conference, Little Rock, Arkansas USA, October 10-14,
1988.
-------
394
O'CALLAGHAN AND MILLER
and characterizing chemically-altered brain
development that can be used regardless of the
information available on the chemical in ques-
tion
Selective Vulnerability
Because specific brain regions arc selective-
ly vulnerable to the effects of a given chemical,
a common dilemma in the detection of develop-
mental neurotoxicity is deciding where to look.
To illustrate this problem, we have shown the
selective effects of three chemicals on the
developing rat brain following exposures dur-
ing the early postnatal period (Fig. 1). The first
example shows the effects of neonatal exposure
to the organometallic compound, trimethyltin
(TMT). As in the adult, extensive loss of hip-
pocampal pyramidal cells is observed after a
single injection, in this case, on postnatal day
five. Further, this frank pathological change is
accompanied in both neonate and adult by a
permanent reduction in the size of this structure
(O'Callaghan and Miller, 1984; Miller and
O'Callaghan, 1984; Brock and O'Callaghan,
1987). The second example shows that acute
administration of cadmium to the neonatal rat
causes a large cystic cavitation of the neostria-
tum (O'Callaghan and Miller, 1986) whereas in
the third example neonatal hyperbilirubinemia
results in marked cerebellar hypoplasia
(O'Callaghan and Miller, 1985). In contrast to
TMT, systemic exposure of the adult to cadmi-
um or bilirubin does not result in effects predic-
tive of sites of neurotoxicity in the neonate. In
fact, both compounds are not considered to
adversely affect the adult CNS because they do
not readily pass the blood-brain barrier
(Diamond and Schmid, 1966; Cantilena and
Klaassen, 1981; Cherian and Rodgers, 1982).
Thus, for cadmium and bilirubin, not only are
the targets not known in the neonate, but on the
basis of the adult data they would not even be
suspected to alter brain development. These
data illustrate two features of the effects of
chemicals on brain development: 1) chemical-
induced alterations of the developing nervous
system often are manifested by changes in the
cytoarchitecture of different neuroantomical
regions even when the exposures occur during
the same stage in brain development; and 2) the
sites of chemical action on the developing brain
are not necessarily predicted by sites known to
be affected in the adult.
A feature of developmental neurotoxicity
that is not revealed by the above examples is
that brain regions affected by chemical expo-
sures often are not revealed by Nissl-based his-
tological procedures (e.g. see O'Callaghan and
Miller, 1985; 1988a; 1988b) or surveys of clas-
sical neurotransmitter systems (Mailman et al.,
1983; Singer et al., 1983). Moreover, within
the affected areas, the response to chemical
administration may encompass several types of
glia as well as neurons (e.g. see Mikoshiba et
al., 1980 and O'Callaghan and Miller, 1985; for
a review see Spencer and Schaumburg, 1980).
Neuron- and Glia-localized Proteins
Given the extreme cellular and molecular
heterogeneity of the brain it is not unexpected
that different brain regions and cell types are
differentially sensitive to chemical alteration.
Determining the effects of chemicals on the
developing brain is further complicated by the
fact that different brain regions may be under-
going critical ontogenetic processes (e.g. cell
division, migration, differentiation, synaptogen-
esis, myelinogenesis) at the time of exposure.
Thus, patterns of chemically-altered brain
development result from the vulnerability of
different brain regions, exposure parameters
(Rodier, 1986) and effects specific to the com-
pound in question. The complexity of the
developing brain, however, need not serve as an
insurmountable obstacle to the detection and
characterization of chemical-induced alter-
ations. This is due to the fact that differences
between nervous system cell types and, there-
fore, among cellular processes associated with
brain development, can be distinguished on the
basis of specific neuronal and glial proteins
(Nestler and Greengard, 1984; DeBlas et al.,
1984; Hockfield and McKay, 1985;
O'Callaghan and Miller, 1988a). We have pro-
posed that assays of these proteins, in turn, may
be used to assess chemical-induced alteration in
the mature and developing nervous system
(O'Callaghan and Miller, 1983). Some of the
proteins we have examined, their associated
developmental processes and their cellular and
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CHEMICALLY-INDUCED ALTERATIONS IN BRAIN DEVELOPMENT
395
TABLE 1. Neuron- and Glia-localized Proteins Used to Characterize
Developmental Neurotoxicity.
Protein CNS Call Cellular Developmental
Type Distribution Process
NF-200
Neurons
Intermediate Filaments
Ontogeny of Axonal
Cytoskeleton
Synapsin 1
Neurons
Synaptic Vesicles
Synaptogenesis
p38
Neurons
Synaptic Vesicles
Synaptogenesis
MAP-2
Neurons
Dendrites
Dendritic
Maturation
G-Substrale
Purkinje Cells
Cylosol
Ontogeny of
Purkinje Cells
PCPP-260
Purkinje Cells
Membrane
Ontogeny of
Purkinje Cells
MBP
Oligodendroglia
Myelin
Myelinogenesis
GFAP
Astrocytes
Intermediate Filaments
Astrogliogenesis
Astroglial Response
to Injury
^•Tubulin
Neurons and Gila
Microtubules
Cell Division
Axon Dendrite
Formation
Abbreviations: NF-200 » neurofilament Mr. 200,000; PCPP-260 » E,urkinje s,ell-specific
fthosphoflrotein Mr, 260,000; MAP-2 » microtubule associated prot«in-2; MBP • myelin basic
protein; GFAP • glial fibrillary acidic protein. For extensive references documenting the regional
and cellular distribution of these proteins see O'Callaghan and Miller, 1988a and O'Callaghan,
1988.
subcellular distribution are shown in Table 1.
We have assayed proteins associated with
the ontogeny of both neuronal and glial pro-
cesses (Table 1). For example, the synaptic
vesicle-localized protein, synapsin I, has been
used to monitor synaptogenesis; myelin basic
protein was chosen to assess myelinogenesis
and glial fibrillary acidic protein was used to
follow the ontogeny of astrocytes. In the case
of neurons an effort was made to examine a
variety of subcellular components, from soma
to synapse, that are known to be affected by
chemical exposures in the adult. Thus, we have
evaluated 6-tubulin and MAP-2 for effects on
dendrites, neurofilament proteins for effects on
the axonal cyloskeleton and p38 and synapsin I
for effects at the synapse. Finally, we have also
focused attention on proteins that are restricted
to only a specific cell type localized to a specif-
ic brain region known to be vulnerable to
developmental insult. Examples of such pro-
teins include the cerebellar Purkinje cell pro-
teins, PCPP-260 and G-substrate.
Given the paucity of data on the effects of
chemicals on nervous system development, any
brain region must be viewed as a potential tar-
get By using assays of a large "battery" of
neuron- and glia-localized proteins combined
with an analysis of specific brain regions, we
felt it would be possible to detect areas affected
by chemical exposure and the cell types/pro-
cesses involved. Moreover, by examining sam-
ples obtained throughout ontogeny it should be
possible to detect transient as well as perma-
nent effects of chemicals on various stages of
brain development. The only limits to this
-------
1
t
A"'
i
Saline
, ^
i
i
*v
A3
Saline
CdCi2
-------
FIG. 1. Examples of selective vulnerability to toxic insult. Top panels: Morphology ot dorsal hippocampus on postnatal day 22 alter administration of saline or
TMT (6.0 mg/kg) on postnatal day 5 Middle panels: Morphology of whole brain on postnatal day 22 after administration of saline or cadmium chloride (CdCI2)(3 0
mg/kg) on postnatal day 5. Lower panels: Morphology of adult cerebellum in heterozygous control (Jj) or homozygous reoessive (hyperbilirubinemia (jj) Gunn rats.
All sections were cut in the sagittal plane and stained with cresyl violet. (Adapted from Miller and O'Callaghan, 1984; O'Callaghan and Miller, 1965; 19B6).
to
CD
-J
-------
398
O'CALLAGHAN AND MILLER
approach are the availability of enough proteins
with which to distinguish the altered cells or
processes. Current advances continue to reveal
the extensive heterogeneity of the nervous sys-
tem based on the discovery of unique glial and
neuronal proteins (HocWield, 1987; Nestler
and Greengard, 1984; Hockfield and McKay,
1985). Thus, in the near future, the availability
of known cell-type-specific proteins may not be
the limiting factor for detecting the effects of
chemicals on brain development
Experimental Approach to Validating the
Use of Neuron* and GIia-localized Proteins
in the Assessment of Chemically-Altered
Brain Development
Our initial studies of chemical-induced
effects on both the mature and developing ner-
vous system have followed a straightforward
approach. Prototype chemicals known to affect
brain cytoarchitecture have been used as "posi-
tive controls" for altering the levels of a variety
of neuron and glia proteins (for a review see
O'Callaghan, 1988). The results of such stud-
ies have shown the predicted changes in pro-
teins associated with the affected cell types (see
O'Callaghan, 1988). Recently, in our studies of
the developing nervous system we have
expanded this approach by studying a group of
structurally similar organotins, trimethy-, tri-
ethyl- and tributyltin (TMT, TET & TBT)
(O'Callaghan and Miller, 1988a; 1988b and
O'Callaghan, 1988). The use of these com-
pounds as models for altering brain develop-
ment will be the focus of the remainder of this
review.
TMT causes neuronal degeneration through-
out the CNS in both the adult (Brown and
Aldridge, 1979; Balaban et al., 1988) and
neonate (Miller and O'Callaghan, 1984; Reuhl
and Cranmer, 1984) rat A striking pattern of
damage occurs in the hippocampus character-
ized by loss of pyramidal cells (see Fig. 1).
TET, in contrast, is a known myelin toxicant
(Stoner et al., 1955; Jacobs et al., 1977; Blaker
et al., 1981; Toews et al., 1983). It causes vac-
uolization, edema and splitting of the myelin
sheath in the adult and in neonates after the
onset of myelination. TBT differs from both
TMT and TET in that it has no prominent
actions on the nervous system; its effects are
predominantly immunotoxic (Snoeij et al.,
1985). For our purposes we group these com-
pounds into 3 categories; (I) a prototype "posi-
tive control" that causes overt cytopathology
(TMT) (see Fig. 1); (2) a known toxicant in the
adult lacking prominent cytopathic effects in
the neonate (TET); and (3) a compound with no
known effects on the nervous system of the
adult or neonate (TBT). As a prototype neuro-
toxicant, TMT could be used as a denervation
tool to validate specific neuronal and glial pro-
teins as indicators of altered brain development.
TET and TBT, on the other hand, would be
used as test compounds to assess whether
effects of these agents could be demonstrated in
the absence of cytopathology. Because a large
percentage of brain development in the rat
occurs postnatally (for a review see Ruppert,
1986), we used early postnatal exposures of the
developing rat to assess the effects of these
alkyltins on brain development
METHODOLOGY
Dosing and Tissue Preparation
Long-Evans rats bom in our animal colony
were cross-fostered and reared in litters culled
to 8 pups (4 males and 4 females). Pups were
dosed with TMT, TET, TBT or vehicle on post-
natal day 5, a period of maximal brain growth
(spurt) in the rat (Dobbings and Sands 1979).
The highest dosages of each compound were
just below those known to cause body weight
deficits. Thus, effects of each organotin com-
pound on brain development would not reflect
an overall reduction in growth. Animals were
killed by decapitation on postnatal days 13,22,
40 or as adults (day 60 or 66). Hippocampus
and cerebellum were dissected, free-hand, from
the rest of the brain (forebrain). Tissue was
homogenized in 1% SDS heated to 90°C and
stored frozen before radioimmunoassay.
Radioimmunoassays. Aliquots of the tis-
sue homogenates were diluted in assay buffer
and spotted on nitrocellulose sheets with the aid
of a slotted template. Proteins were fixed to the
-------
CHEMICALLY-INDUCED ALTERATIONS IN BRAIN DEVELOPMENT
399
110 ~ TET
100 o TBT -r- . . •
¦5 90 . wmm ~ r i
80 m + I
o 70 ¦ _Z_ ¦ |gj
O 60
l I ll
Whole Brain Hippocampus Cerebellum
FIG. 2. Acute exposure to TMT, TET or TBT on postnatal day five
results in persistent decrements in brain weight without affecting body
weight. Subjects were killed at weaning (postnatal day 22). Dosages
were as follows: TMT, 6.0 mg/kg; TET, 6.0 mg/kg; TBT, 3.0 mg/kg.
Each value represents the mean ± SEM for eight independent observa-
tions. 'Significantly different from control, P < 0.05. TET and TBT data
adapted from O'Callaghan and Miller, 1988a and 1988b, respectively.
paper in acidic propanol and assayed by our
modification (e.g., see O'Callaghan and Miller,
1985; 1988a) of the immunoassay procedure of
Jahn et al. (1984). In addition to the sensitivity
and specificity inherent to radioimmunoassays,
there are two major advantages to this assay
system: (1) antigens do not need to be purified
(data can be presented simply as a percent of
adult controls) and (2) whole homogenates can
be assayed directly, therefore, the data obtained
are indicative of effects that occurred in the tis-
sue sample not an extract or an operationally
defined membrane ("particulate") fraction.
Effects of TMT, TET and TBT on Brain
Development
Brain Weight. In much of our work on
neurotoxicity, we have observed that many
chemicals alter the size and weight of the brain
without affecting body weight. In general,
these permanent reductions in brain weight are
found to be accompanied by changes in neuron-
and glia-localized proteins. Consequently, we
routinely survey the weight of whole brain and
certain brain areas as a first step in detecting
alterations in brain development. As was the
case in our studies of organotins (highlighted
below), we often limit our dissections of
defined neuroanatomical structures to hip-
pocampus and cerebellum. These brain areas
undergo extensive postnatal maturation in the
rat (see Ruppert, 1986), a factor which may
predispose these structures to chcmical-induced
changes. In a practical sense, these regions
have the added advantage of being amenable to
rapid, accurate and reproducible dissection.
At dosages just below those resulting in
decrements in body weight, all three organotins
caused permanent reductions in either whole
brain or brain area weights (Fig. 2). TMT and
TET, however, proved to be much more potent
than TBT. Both TMT and TET reduced the wet
weights of whole brain, hippocampus and cere-
bellum, with the most substantial deficits
occurring in hippocampus (decreases of 60%
and 30% for TMT and TET, respectively).
TBT, in contrast, affected only the cerebellum
where it caused a decrease (6%) equivalent to
that produced by TMT and TET. Available
kinetic data do not indicate that the effects of
these compounds are due to differences in their
distribution to the brain (Mushak et al., 1982;
Cook etal., 1984a; 1984b). While brain weight
data do not reveal ontogenetic processes or cell
types altered by chemical exposure, our find-
-------
400
O'CALLAGHAN AND MILLER
Hippocampus
120
100
30
60
40
20
0
120
o
Js 100
c
o
O 80
Synapsin I
MBP
jGFAP
s s / *
/ i |
/ ' 4
'« .¦ !
i
//
I
. !
I
Ooaag* <" TWTI
¦' • 5.0
—a— «.o I
3
¦a
<
60
_ 40
C
-------
CHEMICALLY-INDUCED ALTERATIONS IN BRAIN DEVELOPMENT
401
ings suggest that the hippocampus is a major
target of TMT and TET and that the effects of
TBT, if any, may be greatest in cerebellum.
Moreover, in the case of TMT, our "positive
control", these observations serve to confirm
that brain weight decreases are reliable indica-
tors of areas known to be affected by chemical
exposure.
Relative (Concentration) vs Absolute (Total)
Expression of Protein Alterations. Changes
in the relative amounts (concentration) of a spe-
cific protein reflect the abundance of that pro-
tein in relation to all others in the test sample.
Changes in the absolute (total) amount of a spe-
cific protein reflect the total amount of that pro-
tein in the sample. Because permanent brain
weight reductions occurred as a consequence of
exposure to all the organotins examined, the
absolute amount of most proteins was perma-
nently reduced. Below we have highlighted
organotin-induced changes in the concentration
of specific proteins. Thus, we only present rep-
resentative data for protein alterations that
reflect organotin-induced changes over and
above the observed changes in tissue weight
Neuron-Localized Proteins. Ail three
organotins decreased specific neuron-localized
proteins in hippocampus (Fig. 3) and cerebel-
lum (Fig. 4). These effects differed with
respect to protein, region, and compound. For
example, in hippocampus (Fig. 3), TMT and
TET caused permanent decreases in the con-
centration of synapsin I or p38, the synaptic
vesicle localized proteins. TBT, in contrast,
was without effect on p38. These data suggest
that TMT and TET, but not TBT, caused either
a permanent retardation in synaptogenesis or a
loss of synapses after their formation. Thus,
neurons in hippocampus can be considered sites
of damage for a compound known to affect
neurons, TMT, and a compound generally
regarded as a myelin selective toxicant, TET.
Analysis of p38 in cerebellum (Fig. 4) also
revealed differences in the effects of the three
organotins. TMT was without effect on p38,
TET caused slight (10%) but permanent
decreases and TBT caused large (25%) but
transient decrements in this protein. Because
p38 is predominantly localized to granule cells
in cerebellum (Navone et al., 1986), the
decreases caused by TET and TBT suggest that
both compounds preferentially affect this cell
type.
Glia-Localized Proteins: All three organ-
otins altered the concentration of glia-localizcd
proteins in hippocampus (Fig. 3) and cerebel-
lum (Fig 4), In hippocampus, MBP, a protein
specific to myelin, was permanently decreased
by TMT and TET, but was largely unaltered by
TBT. In cerebellum, TMT and TET again pro-
duced permanent deficits in MBP. In contrast
to hippocampus, however, TBT also affected
this protein, although its concentration returned
to control levels by adulthood. The decreases
in MBP indicate that both TMT and TET may
have killed or injured oligodendrocytes in both
the hippocampus and cerebellum with subse-
quent alterations in myelinogenesis in these
areas. The transient nature of the effect of TBT
on MBP in cerebellum was suggestive of a
retardation in the process of myelinogenesis.
A characteristic feature of damage to the
adult brain is an enhanced expression of GFAP
(Eng, 1987; Brock and O'Callaghan, 1987), the
major intermediate filament protein of astro-
cytes. Thus, increases in GFAP in the neonate
may also reflect an astroglial response to neu-
ronal or glial damage. Indeed, exposure to
TMT and, to a lessor extent, TET caused an
increase in this protein in hippocampus (Fig. 3).
Increases were as great as 400 and 160% of
control for TMT and TET, respectively. TMT
also caused a small but permanent increase in
cerebellar GFAP (Fig. 4) but TET did not.
TBT did not affect this protein in either brain
region. The pattern of GFAP responses
observed in hippocampus after TMT and TET
suggests that these compounds kill or injure
cells in this area, findings consistent with the
known effects of TMT on morphology, but that
would not have been predicted for the effects of
TET. Because TBT did not affect the concen-
tration of GFAP suggests that its effects on
other cell type proteins was not due to injury or
death of neurons or glia. Indeed, the transient
nature of most of the effects of TBT would be
consistent with a developmental delay induced
by this compound.
-------
402
Cerebellum
O'CALLAGHAN AND MILLER
150 "
125 "
10Q "
75 •
50 -
25 ¦
0
150
MBP
p 38
GFAP
o
125 "
C
o
o
100
3
T3
<
O
Doug* ol TET
img/xgj
—• 0.0
• *¦' 3.0
—m— «.o
0)
a
150
125
100
75
50
25
0
0 10 20 30 40 50 60 0 10 20 30 40 50 60 0 10 20 30 40 50 SO
Postnatal Age (days)
FIG. 4. Effects of acute neonatal administration of TMfT, TET or TBT on the concentration of neuron-localized and
glia-locaJized proteins in cerebellum. Each value represents the mean ±SEM for eight independent observations.
Where S.E. bars are not shown they are within the data point 'Significantly different from 0.0 mg/kg, P < 0.05.
TET and TBT data adapted from O'Callaghan and Miller, 1988a and 1988b, respectively.
GFAP
Oougt at TMT
imj.xs)
•— 0.0
—•— s.o
—¦— «.o
GFAP
Dong* ot TBT
tmg/ka)
* J.O
¦ 3.0
-------
CHEMICALLY-INDUCED ALTERATIONS IN BRAIN DEVELOPMENT
403
DISCUSSION
Chemical exposure during brain develop-
ment can result in transient or permanent alter-
ations in specific cellular elements or processes.
In most cases, the absence of prior knowledge
of the targets of a given agent make it difficult
to detect the vulnerable brain regions and cell
types. Our data suggest that assays of neuron-
and glia-localized proteins represent a viable
first approach for detecting the affected brain
regions and for identifying and quantifying
responses of affected cell types. By combining
RIA data with immunohistochemical evalua-
tions of specific proteins, it should be possible
to achieve an integrated biochemical and mor-
phological characterization of the effects of
broad classes of chemicals on brain develop-
ment.
In this review we summarized some of our
data on structurally similar triakyltins to illus-
trate the range of protein changes that occur
after exposure of the developing brain to com-
pounds resulting in cytopathology, to com-
pounds with no known effects on the brain. All
organotins (TMT, TET and TBT) caused alter-
ations in neuronal and glial proteins over and
above the decrements they caused in tissue
weight Moreover, protein changes proved to
be sensitive indicators of developmental neuro-
toxicity. Alterations were observed at com-
pound dosages that did not reduce tissue weight
or cause histological changes. Although com-
plex, the protein profiles for each compound
revealed distinct patterns of neurotoxicity from
which structure activity relationships can be
established. The prototype neurotoxic com-
pound, TMT, caused the expected decrease in
hippocampal neuronal proteins. This effect was
shared by TET, an unexpected finding because
it is known as a myelin toxicant in adult rats.
In addition, both compounds reduced myelin
basic protein in hippocampus and cerebellum.
Finally, both TMT and TET appeared to dam-
age or kill cells in the hippocampus because
they caused an increase in GFAP. Thus, com-
pounds with strikingly dissimilar ncurotoxico-
logical patterns in the adult rat show markedly
similar patterns when administered early in
postnatal ontogeny, findings which suggest tint
these chemicals act at similar sites in the devel-
oping brain. In contrast, the effects of TBT dif-
fered markedly from those of TET and TMT:
the cerebellum, not hippocampus, was affected;
the effects (p38 and MBP decreases) were tran-
sient; and there was no indication of cell dam-
age or death because GFAP concentration did
not change. From these data, it is apparent that
the effects of TBT on the developing brain are
qualitatively different from those of TET and
TMT. Moreover, one can infer that increasing
trialkyltin side-chain length from methyl or
ethyl to butyl results in a compound with
reduced potential for causing developmental
neurotoxicity.
Some features of developmental neurotoxic-
ity that we mentioned earlier (see under
Selective Vulnerability) also emerge from the
brain weight and specific protein changes
caused by the trialkyltins. For example, our
data indicate that targets of neurotoxicity in the
adult do not necessarily predict targets in the
developing brain as illustrated by the effects of
TET (an adult myelin toxicant) on hippocampal
weight and neuron-localized proteins. Our
findings also suggest that specific stages of
postnatal brain development do not necessarily
confer vulnerability to chemical insult. For
example, in the cerebellum a major postnatal
growth spurt occurs which peaks on the day
that we administered the organotins, day 5
(Dobbing and Sands, 1979). This growth phase
coincides with a period of rapid division of
cerebellar granule cells (Rodier, 1980). Such
periods of cell proliferation are widely regarded
as being especially vulnerable to chemical dis-
ruption (Suzuki, 1980; Rodier, 1986; Wiggins,
1986; Oster-Granite, 1988). If vulnerability
associated with cell division was a major deter-
minant of chemical effects on brain develop-
ment, then all three organotins would have had
the greatest effects on cerebellum. This
appeared to be the case for TBT but was cer-
tainly not observed for TMT and TET where
hippocampus was the major target. Thus, other
factors unique to the affected cell type(s) must
account for the selective vulnerability of hip-
pocampus to these chemicals.
A final point that can be made on the basis
of our oiganotin data is that increases in GFAP
can be used to detect areas of cellular damage
-------
CHEMICALLY-INDUCED ALTERATIONS IN BRAIN DEVELOPMENT
405
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Mailman RB, Krigman MR, Frye GD,
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GABA, and acetylcholine systems in the rat
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Mikoshiba K, Kobsaka S, Takamatsu K,
Tsukada Y. Cerebellar hypoplasia in the
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1318
Miller DB, O'Callaghan JP. Biochemical,
functional and morphological indicators of
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tion of trimethyltin to the developing rat. J
Pharmacol Exp Ther 1984; 232:744-751
Mushak P, Krigman MR, Mailman RB.
Comparative organotin toxicity in the devel-
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Navone F, Jabn R, Di Gioia G, Stukenbrok
H, Greengard P, De Camilli P. Protein p
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2527
Nestler EJ, Greengard P. Protein
Phosphorylation in the Nervous System,
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O'Callaghan JP. Neurotypic and gliotypic
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O'Callaghan JP, Miller DB. Nervous system-
specific proteins as biochemical indicators
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O'Callaghan JP, Miller DB. Diethyl-
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O'Callaghan, JP,. Miller, DB. Acute expo-
sure of the neonatal rat to triethyltin results
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1988a; 244:368-378
O'Callaghan JP, Miller DB. Acute exposure
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Oster-Granite ML. The development of the
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1988, pp. 203-226
Reinhard JF, Jr, Miller DB, O'Callaghan JP.
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Reuhl KR, Cranmer JM. Developmental
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22:525-545
Rodier PM. Time of exposure and time of
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Singer HS, Weaver D, Tiemeyer M, Coyle
JT. Synaptic chemistry associated with
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Snoeij NJK, Van Iersel AAJ, Penninks AH,
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-------
0022-3585/88/246l-0394$00,00/0
The Journal op Pharmacology and Experimental Therapeutics
Copyright £> 1988 by The American Society for Pharmacology and Experimental Th«r»p«uttcs
Vol. 246. No. I
Printed in U.S.A.
Acute Exposure of the Neonatal Rat to Tributyltin Results in
Decreases in Biochemical Indicators of Synaptogenesis and
Myelinogenesis1
JAMES P. O'CALLAGHAN and DIANE B. MILLER
Neurotoxicoiogy (J.P.O'C.) and Developmental and Cell Toxicology (D.B.M.) Divisions, Health Effects Research Laboratory, U.S. Environmental
Protection Agency, Research Triangle Park, North Carolina
Accepted for publication April 4,1988
ABSTRACT
Assays of neuron-localized (neurotypic) and glia-localized (gli-
otypic) proteins were used to detect and characterize the toxic
effects of tributyltin (TBT) on the developing rat central nervous
system. Four proteins associated with specific aspects of neu-
ronal and glial development were evaluated: 1) p38, a synaptic
vesicle-associated protein; 2) neurofilament 200, an intermediate
filament protein of the neuronal cytoskeleton; 3) myelin basic
protein, an oligodendroglia and myelin-sheath associated protein;
and 4) glial fibrillary acidic protein, an intermediate filament pro-
tein of astrocytes. On postnatal days 13, 22 and 60, the amount
of each protein in homogenates of cerebellum, forebrain and
hippocampus was determined by radioimmunoassay. A single
administration of TBT (2, 3 or 4 mg/kg i.p.) on postnatal day 5
caused dose- and region-dependent decreases in brain weight
with the cerebellum being most affected. These decrements
were not associated with light microscopic evidence of altered
brain development (on postnatal day 61) but were accompanied
by large dose- and region-dependent decreases in p38 and
myelin basic protein. Decrements in both the per tissue (total)
and per milligram of tissue protein (concentration) values for
these proteins were observed in cerebellum and forebrain; hip-
pocampus was largely unaffected. TBT-induced reductions in
p38 and myelin basic protein were seen at dosages that did not
affect brain, thymus or body weight. At dosages of TBT that did
not affect body weight, reductions in brain weight, p 38 and
myelin basic protein did not persist into adulthood. The data
indicate that exposure to TBT on postnatal day 5 is toxic to the
developing nervous system. The data also suggest that neuro-
typic and gliotypic proteins can serve as sensitive indices for
detecting and characterizing the effects of toxic exposures on
the developing central nervous system.
The short-chain trialkyltins, TMT and TET are toxic to
neuronal and glial elements of the adult and developing CNS
as evidenced by persistent alterations in brain morphology (for
reviews see Reuhl and Cranmer, 1984; Krigman and Silberman,
1984; Reuhl, 1987) and biochemistry (for reviews see Morell
and Mailman, 1987; O'Callaghan, 1988). Previously, we dem-
onstrated that assays of neuron-localized (neurotypic) and glia-
localized (gliotypic) proteins can be used to quantify neuronal
and glial responses to toxic exposures of the CNS (for a review
see O'Callaghan, 1988), including those caused by TET (O'Cal-
laghan et of., 1983;) and TMT (Miller and O'Callaghan, 1984;
O'Callaghan and Miller, 1984; Brock and O'Callaghan, 1987;
Balaban et aL, 1988). More recently, we found that neonatal
exposure to TET results in permanent alterations in a variety
Received for publication December 8,1987.
1 Thi* pap«r ha* been reviewed by the Health Effect! RmmkIi Laboratory,
U.S. Environmental Protection Agency, and approved for publication.
of neurotypic and gliotypic proteins (O'Callaghan and Miller,
1988) without affecting brain morphology at the light micro-
scopic level. Such findings raise the possibility that compounds
not found to be toxic to the nervous system based on morpho-
logical evaluations may be considered toxic based on alterations
in neurotypic and gliotypic proteins.
Although CNS damage constitutes a dominant feature of the
toxicity profile of lower trialkyltins, intermediate chain length
compounds such as tri-n-propyltin and TBT, are characterized
primarily by their immunotoxic properties (Voa et aL, 1984;
Snoeij et ad., 1985). Morphological assessments have not re-
vealed CNS involvement in the toxic effects of tri-n-propyltin
or TBT (Snoey et al., 1985; Mushak et al„ 1982). As was the
case for TET, however, effects of intermediate alkyltins on the
CNS may be manifested by changes in specific brain proteins
in the absence of overt changes in morphology. Therefore, the
purpose of the present investigation was to assess the effects
ABBREVIATIONS: TMT, trimethyWn; TET, trlethyltln; CNS, central nervous system; TBT, tributyltin; RIA, radioimmunoassay; NF-200, neurofilament
200 (M, - 200 kilodaltona); MBP, myelin baste protein; QFAP, glial fibrillary acidic protein; IgQ, Immunoglobulin G; SDS, sodium dodecyl sulfate;
PNO, postnatal day; BSA. relative specific activity.
394
-------
Neonatal T8T and Brain Protaina 395
! compounds, TBT, on proteins that serve as
Micators of neuronal and glial development.
i'svious studies (see O'Callaghan and Miller, 1985;
Callaghan, 1987; O'Callaghan and Miller, 1988),
iple neurotypic and gliotypic proteins were used
Potential effects of TBT on the developing CNS.
assayed were p38, an integral synaptic vesicle
protein (Jahn et ai, 1985; Navone et ai, 1986;
11987); NF-200, a cytoskeletal protein of neurons
P Simon, 1981; Debus et al„ 1982); MBP, a protein
flodendroglia and myelin (Sternberger et ai, 1978;
¦ a/., 1982); and GFAP, the intermediate filament
Ktrocytes (Bignami et a/., 1972; Eng, 1985). Addi-
f* concerning the cell-type-specificity of these pro-
jwe developmental processes with which they are
We been summarized in tabular form (O'Callaghan
1988). Hippocampus, cerebellum and forebrain,
Pns with different developmental profiles, were ex-
| our previous studies of TMT and TET (Miller and
1984; O'Callaghan and Miller, 1988). Homoge-
P«se same regions were evaluated in the present study
^ the effects of TMT and TET with those of TBT.
Materials and Methods
j, - t13SI]Protein A (2-10 nCi/ng; 1 Ci - 37 GBq) was
Jfrom New England Nuclear (Boston, MA). Monoclonal anti-
Monoclonal antineurofllament (p200) were purchased from
F'r Mannheim Biochemicals (Indianapolis, IN). Antiserum to
^®ry acidic protein and rabbit anti-mouse IgG were obtained
11 Corp. (Santa Barbara, CA). Antisera to p38 was the generous
, • Reinhard Jahn (Max-Plank-lnstitut fur Psychiatrie, Mar-
RRG). Nitrocellulose paper (0.2 am pore size, German manu-
form) was purchased from Schleicher and Schuell (Keene,
PS was obtained from Bio-Rad Laboratories (Richmond, CA);
Pon X-100 was from New England Nuclear. Other materials
t'he RIAs were obtained from the sources described by Jahn et
r>- Bovine serum albumin was obtained from Sigma Chemical
^ Louis, MO). Bis (tri-n-butyltin) oxide was obtained from ICN
^suticals, K & K Laboratories (Plainview, NY); corn oil was
*d from Sigma. All other chemicals were of at least analytical
''grade and were purchased from a variety of commercial sources,
wcu. Primigravida Long-Evans rats (Blue Spruce Farms, AI-
^Y) were received 2 days after conception and housed individ-
Plastic tub cages with pine shavings as bedding. The colony
! *** maintained under filtered positive pressure ventilation at a
constant temperature (22*C ± 2), humidity (50% ± 10) and on a 12-hr
light/12-hr dark lighting cycle beginning at 6:00 a.m. EDT. Pups bom
between 5:00 p.m. and 5:00 a.m. were considered to be born on the
same day (PND 0). On PND 1 pups were assigned to litters such that
each dam received four male atv.i tour female pups with no more than
one male and one female of her own offspring. Pups were tatooed on
the foot with India ink as a means of identification (Avery and Spyker,
1977). At 21 days of age littermates were weaned, separated by sex and
housed four per cage.
Dosing. Pups were injected on PND 5 with TBT oxide (2.0,3.0, 4.0
or 5.0 mg/kg i.p.) or with its vehicle control (corn oil). Doses of TBT
are expressed as the base and were administered in a volume of 10 y\/
g b.wt. All animals in a litter received the same dose.
Tissue preparation. Animals were weighed and then sacrificed by
decapitation on PND 13, 22 and 60. Brain, thymus and spleen were
removed immediately and weighed. Brains subsequently were dissected
on a cold plate (model TCP-2, Thermoelectrics Unlimited, Wilmington,
DE) maintained at Q-4°C. Cerebellum and hippocampus were removed
by free-hand dissection; the remaining portion of the brain was desig-
nated as forebrain. Each brain region was weighed, homogenized with
a sonic probe (Kontes Cell Disrupter, Vineland, NJ) in 10 volumes of
hot (90-95'C) SDS and stored frozen (-70"C) before RIA.
Histology. On PND 61, one group of animals (n ¦ 8) that received
TBT (5.0 mg/kg only) and one group that received vehicle (n » 8) were
sacrificed by decapitation. The brains were removed, fixed by immer-
sion in 10% neutral-buffered Formalin, embedded in paraffin, sectioned
at 6 Mm in the coronal plane at lOO-pm intervals and stained with
cresyl violet for subsequent histological examination.
Protein assay. Total protein was assayed by the method of Lowry
etal. (1951) or the method of Smith et at. (1985). Bovine serum albumin
was used as the standard.
RIA of neurotypic and gliotypic proteins. All neurotypic and
gliotypic proteins were measured by solid-phase RIA according to
modifications (O'Callaghan and Miller, 1985; Brock and O'Callaghan,
1987; O'Callaghan and Miller, 1988) of the dot-immunobinding proce-
dure of Jahn et ai (1984). Briefly, samples were assayed for total
protein, adjusted to equal protein concentration in dot-immunobinding
buffer and applied to nitrocellulose sheets (0.1 to 0.2 nm pore size)
using a slot-blot manifold (Minifold II, Schleicher and Schuell, Keene,
NH). Typically, samples containing 10 tig of total protein in 20 m1 of
sample buffer were loaded into each slot. The spotted sheets then were
fixed, blocked with gelatin, washed, incubated with primary antibody
and then with {''"I] protein A as described previously (Jahn et aI.,
1984). Where monoclonal antibodies were used, additional incubations
in rabbit antimouse IgG (1:500), blocking and washing solutions were
required (O'Callaghan and Miller, 1985; Brock and O'Callaghan, 1987).
The primary antibody stocks were used at the following dilutions:
MBP, 1:500; GFAP, 1:500; p38,1:300; and NF-200; 1:1000. The speci-
_ i of TBT on PND 5 raaulta in doaa-dapandant dacraaaa* in body and brain weight
represents the mean ± (S.E.M.).
J* n
cfiyj
13
22
60
Dosage
Body
Bran
Forebrain
Hippocampus
mtlkg
9
9
9
9
9
0
33.4
(±0.48)
1.25 (±0.02)
0.145(0.002)
1.011 (±0.012)
0.072 (±0.003)
2
31.1
(±1.04)
1.18 (±0.02)*
0.131 (±0.002)*
0.962 (±0.014)*
0.071 (±0.001)
3
32.5
(±1.13)
1.18 (±0.01)*
0.130 (±0.003)*
0.982 (±0.015)*
0.068 (±0.002)*
4
29.2
(±0.53)*
1.12 (±0.01)*
0.123 (±0.003)*
0.919 (±0.018)*
0.067 (±0.002)*
0
57.5
(±0.91)
1.52 (±0.01)
0.207 (±0.002)
1,210 (±0.008)
0.087 (±0.001)
2
54.3
(±1.77)
1.51 (±0.02)
0.206 (±0.002)
1,198 (±0.015)
0.087 (±0,001)
3
54.1
(±1.50)
1.46 (±0.02)
0.196 (±0.005)*
1.180 (±0.014)
0.084 (±0.002)
4
45.8
(±2.22)'
1.39 (±0.02)*
0.187 (±0.003)*
1.107 (±0.010)*
0.080 (±0,002)*
0
262.0
(±26.0)
1.86 (±0.04)
0.279 (±0.006)
1.441 (±0.033)
0.108 (±0.002)
2
287.0
(±17.0)
1.85 (±0.03)
0.277 (±0.005)
1.448 (±0.024)
0.103 (±0.002)
3
281.0
(±20.0)
1.82 (±0.02)
0.275 (±0.003)
1.422 (±0.014)
0.103 (±0.002)
4
238.0
(±18.0)*
1,88 (±0.03)*
0.243 (±0.007)*
1.304 (±0.020)*
0.100 (±0.002)
Significantly different from 0 mg/kg, P < .05,
-------
396
O'Callaghan and Miller
Vol. 246
TABLE 2
Administration of TBT on PND 5 causes dose-dependent decreases
in thymus but not spleen weight
Each value represents the mean ± (S.E.M.).
Age
n
Dosage
Thymus
Spleen
days
mg/kg
9
9
13
8
0
0.142 ±0.005
0.199 ±0.008
2
0.112 ± 0.008
0.151 ± 0.010*
3
0.108 ± 0.009
0.203 ±0.010
4
0.095 ± 0.009*
0.227 ± 0.011
22
8
0
0.222 ± 0.006
0.388 ± 0.020
2
0.239 ± 0.012
0.360 ± 0.025
3
0.215 ± 0.013
0.346 ± 0.022
4
0.184 ± 0.012*
0.275 ±0.019*
60
8
0
0.632 ± 0.045
0.752 ± 0.057
2
0.578 ± 0.047
0.721 ± 0.076
3
0.590 ± 0.041
0.809 ± 0.059
4
0.585 ± 0.036
0.842 ± 0.076*
• Significantly different from 0 mg/kg, P < .05.
ficity and linearity of the assays for all proteins have been verified
according to the criteria described previously (Jahn et al., 1984; O'Cal-
laghan and Miller, 1985).
Expression of RIA data. For all RIAs, standard curves were
constructed from dilutions of a single control sample. By comparing
the immunoreactivity of individual samples (both control and TBT
gToups) with that of the sample used to construct the standard curve,
the RSA of each sample was obtained (e.g. see O'Callaghan and Miller,
1984, 1985). The RSA of each sample (from control and TBT groups)
then was multiplied by a constant that resulted in a mean RSA of 1.0
(100%) for the adult (PND 60) control groups. Data are expressed on
a total (per tissue) and concentration (per milligram total protein)
basis. Changes in total values are indicative of alterations in the amount
of a specific protein per brain region; whereas changes in concentration
values are indicative of alterations in the amount of a specific protein
per milligram of total protein in a given brain region.
Statistical analysis. The Statistical Analysis System (SAS, 1982)
was used for data analyses. Individual variables were evaluated by
analysis of variance followed by Duncan's Multiple Range Test for
mean comparisons. Because sex-related differences were not observed
for any measure, data for males and females were combined.
Results
Body and brain weight. Acute administration of TBT on
PND 5 resulted in dose-dependent decreases in body weight
and wet weights of whole brain, hippocampus, cerebellum and
forebrain (table 1). The lower dosages (2.0 and 3.0 mg/kg)
decreased brain weight without affecting body weight whereas
the high dosage (4.0 mg/kg) decreased both brain and body
weight. A dosage of 5.0 mg/kg resulted in approximately 40%
mortality by PND 13 and a dosage of 6.0 mg/kg was fatal to
100% of the pups by PND 13 (data not shown). TBT-induced
decreases in whole brain weight and in brain region weights
were most apparent at PND 13. Deficits in brain weight per-
sisted into adulthood (PND 60) only at the high dosage of
TBT. At all time points the most affected region was the
cerebellum and the least affected region was the hippocampus;
deficits in cerebellum weight were as great as 15% (PND 13)
whereas reductions in hippocampus weight did not exceed 7%
(PND 13).
TBT
*
CONTROL.
MOL.
•
« •
i •
.-V
MED
Fig. 1. Photomicrographs of cresyl violet-stained coronal sections of cerebellum on PND 61 after acute administration of corn oil vehicle (control) or
5.0 mg/kg of TBT on PND 5. Bar ¦ 50 Mm. No apparent differences were observed between sections prepared from oil- or TBT-treated animals.
MOL, molecular layer; PCL, Purkinje cell layer; GCL, granule cell layer; MED, medullary layer. *
-------
Neonatal TBT and Brain Protein*
397
TBTU
*
CONTROL ^ ^ >&£*•< & Sv
-a&jjSe;_
Fig. 2. Photomicrographs of cresyl violet-
stained coronal sections of dorsal hippocam-
pus on PND 61 after acute administration of
com oil vehicle (control) or 5.0 mg/kg of TBT
on PND 5. Bar » 100 nm. No apparent
differences were observed between sections
prepared from oil- or TBT-treated animals.
Thymus and spleen weights. Acute (Funahasi et al., 1980)
or chronic (Vos et al., 1984; Snoeij et al., 1985) exposure of rats
to TBT is known to cause persistent immunosuppression.
Consistent with these data we found that acute administration
of 4.0 mg/kg of TBT on PND-5 was sufficient to cause pro-
tracted (PND 13 and 22) decrements in wet weight of thymus
(table 2). Lower dosages of TBT, however, did not affect thymus
weights. In contrast to its effects on thymus weight, spleen
weight was not consistently affected by TBT.
Histology. Representative micrographs obtained from cer-
ebellum and hippocampus of 61-day-old animals are shown in
figures 1 and 2, respectively. The cytoarchitecture of these
postnatally developing structures did not appear to be affected
by TBT (5.0 mg/kg). Nissl-stained sections of forebrain also
did not reveal alterations as a consequence of the administra-
tion of TBT (data not shown).
RIA of Neurotypic Proteins
p38. TBT caused dose-, time- and region-dependent de-
creases in the synaptic vesicle protein, p38 (fig. 3). On an
absolute (total) basis, p38 was decreased in cerebellum and
forebrain throughout ontogeny; values were decreased by as
much as 33% (PND 22 cerebellum) in comparison to controls.
On PND-22 in cerebellum and PND 13 and 22 in forebrain,
the magnitude of the decreases in total p38 exceeded the
magnitude of TBT-induced decreases in tissue weight, findings
which are reflected in the large (as great as 26%) decreases in
the concentration of p38 at these postnatal ages. When ex-
pressed on either a total or concentration basis, values for p38
were not affected in hippocampus.
NF-200. On both a total and concentration basis, values for
the neuronal cytoskeletal protein, NF-200, were decreased as a
result of exposure to TBT (fig. 4). Decreases in total NF-200
were observed in all brain regions. In comparison to its effects
on p38, however, TBT caused decreases in NF-200 that were
smaller in magnitude and that occurred at fewer time points.
In general, reductions in total NF-200 followed the TBT-
induced reductions in tissue weight. Thus, reductions in the
-------
398
O'Callaghan and Miller
Vol. 246
O
a:
o
o
o
100
80
60
40
20
>- ao
O a
0£
100
80
60
40
20
CEREBELLUM
CEREBELLUM
ITOTALl
FOREBRAIN
ICONCENTRATIONI
FORE BRAIN
Doiagt of TBT (mg/^al
HIPPOCAMPUS
HIPPOCAMPUS
"0 10 20 30 40 50 60
10 20 30 40 50 60
AGE (days)
10 20 30 40 50 60
Fig. 3. Effects of acute neonatal administration of TBT on p38 in cerebellum, forebrain and hippocampus. Each value represents the mean ± S.E.M.
for eight independent observations. Where S.E. bars are not shown they are within the data point. Oata are expressed on a per tissue (total) and a
per milligram of tissue protein (concentration) basis. Additional details appear under "Materials and Methods." * Significantly different from 0.0 mg/
kg, P < .05.
concentration of NF-200 were small in magnitude and were
limited to PND 13 (10% decrease) and 60 (12% decrease) in
forebrain and occurred only on PHD 60 (11% decrease) in
hippocampus. Moreover, the high dosage of TBT accounted for
all but the PND 13 effects.
RIA of Gliotypic Proteins
MBP. TBT caused large dose-, time- and region-dependent
reductions in the myelin-associated protein, MBP (fig. 5).
Although total MBP was reduced at all time points in cerebel-
lum and forebrain, the greatest decrements were observed on
PND 22, the only time point in which a decrease in total MBP
also was observed in hippocampus. On PND 22, total MBP was
reduced by as much as 32% in cerebellum and by as much as
34% in forebrain. Because the magnitude of the decreases in
total MBP on PND-22 greatly exceeded the magnitude of
corresponding decreases in wet weight, the concentration of
MBP waa reduced on PND-22 in all regions; decreases of as
much as 28% (forebrain) were seen at the high dosage of TBT.
Small decreases (10%) in the concentration of MBP also were
seen on PND 13 (forebrain), but no alterations in the concen-
tration of this protein were seen in any region by PND 60.
GFAP. TBT caused a decrease in the ontogeny of the
astrocyte-associated protein, GFAP, that generally coincided
with the degree of TBT-induced reductions in tissue weight
(fig. 6). Thus, total GFAP was reduced by TBT at several time
points in all three regions but, with the exception of the
cerebellum, these effects were restricted to the high dosage (4.0
mg/kg). The concentration of GFAP in all regions was largely
unaffected by postnatal exposure to TBT.
Discussion
Our data demonstrate that acute administration of TBT to
the neonatal rat is toxic to the developing nervous system as
evidenced by 1) decrements in brain weight; 2) decrements in
-------
1988
Neonatal TBT and Brain Proteins 399
O
cm
O
O
O
- 8
i
Q
100
80
60
40
20
o
QC
0
100
80,
60
40
20
CERE8CLLUM
CEREBELLUM
I TOTAL I
FOR£8«AfN
ICQNCENTRATIONI
Dotag* « TBT (g)
HIPPOCAMPUS
HIPPOCAMPUS
0 10 20 30 40 50 60
10 20 X 40 50 60
AGE (days)
10 20 30 40 50 60
Pig. 4. Effects of acute neonatal administration of TBT on NF-200 In cerebellum, forebrain and hippocampus. Details appear in the legend to figure
3 and under "Materials and Methods." * Significantly different from 0.0 mg/kg, P < .06.
p38, a biochemical indicator of synaptogenesis (Knaua et ai.,
1986; O'Callaghan and Miller, 1988); and 3) decrements in
MBP, a biochemical indicator of myelinogenesis (Sternberger
et ai, 1978; Hartman et ai., 1982; O'Callaghan and Miller,
1988). With respect to each of these endpoints, the data also
revealed that the effects of TBT are less persistent and quali-
tatively dissimilar to the actions of the neurotoxic trialkyltins,
TET and TMT.
Permanent decrements in brain weight without changes in
body weight are characteristic effects of exposure to TET and
TMT on PND-5 (O'Callaghan et al., 1983; Miller, 1984; Miller
and O'Callaghan, 1984; O'Callaghan and Miller, 1988), At the
lower dosages used in this study, administration of TBT on
PND-5 also resulted in decreases in brain weight in the absence
of decreases in body weight. These findings indicate that TBT
can affect brain growth without causing a general decrease in
overall growth and, accordingly, it can be considered a neuro-
toxicant. Unlike the effects of TET and TMT, however, TBT-
induced brain weight deficits persisted into adulthood only at
a dosage that also resulted in reductions in body weight. The
rank order of regional brain weight decreases induced by TET
and TMT also distinguish the effects of these compounds from
those of TBT. TET and TMT cause the greatest deficits in wet
weight of hippocampus followed in order by forebrain and
cerebellum (Miller and O'Callaghan, 1984; O'Callaghan and
Miller, 1988). In contrast, the rank order for wet weight de-
creases caused by TBT was cerebellum > forebrain > hippo-
campus. Thus, the actions of TBT on the developing CNS
differ from those of TET and TMT with respect to the persist-
ence of the observed effects and with respect to the brain
regions affected.
Assays of neurotypic and idiotypic proteins provided farther
verification of the neurotoxic potential of TBT by revealing
the regions and cell-type-specific processes affected by exposure
to this compound. Of the proteins assayed, the neurotypic
protein, p38, and the gliotypic protein, MBP, were the most
affected by 1ST. On both a total (per structure) and concen-
tration (per milligram of protein) basis, large dose-dependent
-------
in and Miliar
Vol. 246
I TOTAL I
FOREBRAIN
CEREBELLUM
HIPPOCAMPUS
[CONCENTRATION!
FOREMAIN
CEREBELLUM
HIPPOCAMPUS
Dosog* of TBT1 mg/WqJ
O—O 00
20
~—a 30
O—Q 40
10 20 30 40 50 60
10 20 30 40 50 60
AGE (days)
10 20 30 40 50 60
of acute neonatal administration of TBT on MBP in cerebellum, forebrain and hippocampus. Details appear in the legend to figure 3
tertals and Methods." * Significantly different from 0.0 mg/kg, P < .05.
p38 and MBP were seen in cerebellum and fore-
is the hippocampus was largely unaffected. The
?38 and MBP coincide with synaptogenesis (Knaus
3'Callaghan and Miller, 1988) and myelinogenesis
f et al., 1978; Hartman et aL, 1982; O'Callaghan and
8), respectively, and both processes continue
postnatal development in all regions examined (e.g.
dan and Bloom, 1967; Bunge, 1968). Our findings,
ire consistent with a TBT-induced retardation of
esis and myelinogenesis with the cerebellum and
eing affected preferentially. Although hippocampal
BP were relatively unaffected by TBT, neurotypic
tic proteins in this structure are altered severely by
TMT (Miller and O'Callaghan, 1984; O'Callaghan
, 1988), again suggesting that different regions of the
; CNS are affected preferentially by these two classes
s.
of p38 and MBP were sensitive indicators of the
TBT on the developing nervous system. Decreases
in both proteins were observed at dosages of TBT that did not
affect body or brain weight. Surprisingly, decreased thymus
weight, an index of immunotoxicity (e.g. see Seinen and Wil-
lems, 1976; Hioe and Jones, 1984; Snoeij et al., 1986) and,
therefore, an expected effect of exposure to TBT, was not
observed at dosages of TBT that altered neurotypic or gliotypic
proteins. Moreover, as in our previous investigations, changes
in neurotypic and gliotypic proteins could be demonstrated in
the absence of overt morphological alterations at the light
microscopic level (as determined in adult subjects). Thus, on
the basis of assays of p38 and MBP, early postnatal exposure
to TBT can be considered neurotoxic at doses that are not
immunotoxic and that do not affect other indices of nervous
system toxicity.
Values for total p3S and MBP in cerebellum and forebrain
and values for total NF-200 in all brain regions were reduced
by TBT on PND-60. These effects represent an absolute re-
duction of each protein and may, therefore, reflect a permanent
reduction in the cell type or maturational process with which
-------
Neonatal TBT and Brain Proteins
401
O
100
80
60
40
Q.
< 2
o o
UL.
O
20
Of
"O
ad
100
80
60
40
20
CEREBELLUM
CEREBELLUM
of-
ITQTALI
FOWMAtN
[CONCENTRATION I
FORE BRAIN
Doioq« of TIT (mgAw
HIPPOCAMPUS
HIPPOCAMPUS
0 10 20 30 40 50 60
10 20 30 40 50 60
10 20 30 40 50 60
AGE (days)
Fig. 6. Effects of acute neonatal administration of TBT on GFAP in cerebellum, forebrain and hippocampus. Details appear in the legend to figure 3
and under "Materials and Methods.' * Significantly different from 0.0 mg/kg, P < .05.
each protein is associated Because these effects occurred only
at a TBT dosage that also resulted in decreased body weight, a
more plausible interpretation is that they reflect an effect of
TBT coincident with an overall reduction in growth. In general,
TBT-induced changes in the concentration of neurotypic and
gliotypic proteins, Le., changes in the relative abundance of
specific proteins, did not persist into adulthood; data which
indicate that the specific developmental processes represented
by each protein were not permanently affected by TBT. These
findings stand in marked contrast to the permanent changes
in neurotypic and gliotypic proteins caused by TET and TMT
(Miller and O'Callaghan, 1984; O'Callaghan and Miller, 1988).
A characteristic feature of damage to the CNS is an enhanced
expression of GFAP (Bignami and Dahl, 1976; Lagenaur et ai.,
1982; Eng, 1987; O'Callaghan and Miller, 1985; Brock and
O'Callaghan, 1987), the major intermediate filament protein of
astrocytes (Eng, 1986). Previously, we demonstrated that ex-
posure of the developing CNS to known neurotoxicante, in-
cluding TET or TMT, results in large and persistent increases
in GFAP (Billingaley et ai,, 1987; Brock and O'Callaghan, 1987;
O'Callaghan and Miller, 1988). Light microscopic evidence for
overt cell loss or injury was not a concomitant of these effects
even in the presence of large increases in this protein (Brock
and O'Callaghan, 1987; O'Callaghan and Miller, 1988). Thus,
the absence of an effect of TBT on brain morphology does not
preclude an effect on GFAP. Our evidence that TBT did not
increase GFAP, therefore, indicates that this compound affects
the developing CNS in a manner that is qualitatively different
from TET and TMT. Moreover, these findings suggest that the
alterations in indices of nervous system toxicity that did result
from exposure to TBT occurred in the absence of conditions
normally associated with an astrocytic (GFAP) response, such
as cell death or injury. The transient effects of TBT on p38
and MBP, therefore, are likely a reflection of retarded synap-
togenesis and myelinogenesis rather than a loss of synapses
and myelin from which there is eventual recovery.
In summary, our data suggest that early postnatal exposure
to TBT causes transient deficits in brain development involving
-------
102
O'Callaghan and Millar
Vol. 246
i
nultiple cell types and regions. Together with our previous
>bservations (Miller and O'Callaghan, 1984; O'Callaghan and
^filler, 1984, 1985, 1988; Brock and O'Callaghan, 1987), the
ireaent data also suggest that assays of neurotypic and gliotypic
iroteins can be used to detect and characterize the temporal
ind regional patterns of neuronal and glial response to other
>;oxic exposures of the developing CNS.
Acknowledgments
The authors are grateful to Ms. Donna Jenkins for preparation of the brain
Sections and Ms. Julia A. DaviB and Dr. Karl F. Jensen for photographic
issistance, We appreciate the critical evaluations of this work provided by Drs.
Vtichael D. Browning, Linda J. Burdette, Karl P. Jensen, Diane M. Niedzwiecki
ind Larry P. Sheets.
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0022.3566/86/2441-0368100.00/0
The Journal or Pharmacology and Experimental Therapeutics
Copyright 1988 by The American Society for Pharmacology and EipwrimentaJ Therapeutics
Voi. 244. No. i
Printed in USA.
Acute Exposure of the Neonatal Rat to Triethyltin Results in
Persistent Changes in Neurotypic and Gliotypic Proteins1
JAMES P. O'CALLAGHAN and DIANE B. MILLER
with the technical assistance of Michael E. Viana
Aleurotox/cology (J.P.O'C.) and Developmental and Cell Toxicology (D.B.M.) Divisions, Health Effects Research Laboratory, U.S. Environmental
Protection Agency, Research Triangle Park, North Carolina
Accepted for publication September 22.1987
abstract
Measurements of neuron-specific (neurotypic) and glia-specific
(gliotypic) proteins were used to characterize the toxic effects of
triethyltin (TET) on the developing central nervous system. Six
proteins, each of which is associated with specific aspects of
neuronal and glial development, were evaluated as follows: 1)
neurofilament-200, an intermediate filament protein of the neu-
ronal cytoskeleton; 2) synapsin I, a synapse specific, synaptic
vesicle localized protein; 3) p38, another synaptic-vesicle local-
ized protein; 4) myelin basic protein, a protein unique to myelin-
forming oligodendroglia; 5) glial fibrillary acidic protein, the inter-
mediate filament protein of astrocytes; and 6) tubulin, a con-
stituent primarily of neuronaJ microtubules. The amount of each
protein in homogenates of hippocampus, forebrain and cerebel-
lum, brain regions with different developmental profiles, was
determined by radioimmunoassay. After a single administration
on postnatal day 5, TET (3 or 6 mg/kg i.p.) caused permanent
dose- and region-dependent decrements in brain weight, with
the hippocampus being the most affected. These effects were
not associated with light microscopic evidence of cytopathology
but were accompanied by large dose-, time- and region-depend-
ent alterations in all neurotypic and gliotypic proteins evaluated.
On a per structure (total) basis, TET caused permanent de-
creases in most neurotypic and gliotypic proteins in all areas. On
a per milligram of tissue protein (concentration) basis, changes
in specific proteins also were observed in all regions but were
most prevalent in hippocampus and cerebellum, in hippocampus
and cerebellum, decrements in the concentration of neurotypic
and gliotypic proteins were observed in the absence of TET-
induced decreases in the weights of these structures. The data
indicate that 1) neonatal exposure to TET causes permanent
deficits in neuronal as well as giiai development, 2) the effects of
TET are region-dependent but do not appear to be related to
region-dependent stages in development and 3) assays of neu-
rotypic and gliotypic proteins may be used to characterize the
temporal and regional patterns of neuronal and glial responses
to toxic exposures of the developing central nervous system.
TET is toxic to the CNS of the developing rat (for reviews
see Reuhl and Cranmer, 1984; Ruppert, 1986). The effects of
acute or chronic exposure to TET during postnatal develop-
ment are characterized by persistent deficits in the morphology
and biochemistry of CNS myelin (Wender et oL, 1974; Blaker
et al, 1981; Toews et oL, 1983; O'Callaghan et al, 1983). These
findings are not entirely unexpected. TET is known to damage
the myelin sheath after administration to the adult rat (Stoner
et al., 1955; Barnes and Stoner, 1958; Magee et al, 1957; Jacobs
et al, 1977). Moreover, oligodendroglia proliferation (Altman,
1970) and subsequent myelinogenesis (Norton, 1981) are post-
natal events known to be susceptible to toxic insult (e.g. see
Suzuki, 1980; Wiggins, 1986).
Received fot publication June S, 1987.
1 Thii paper has been reviewed by the H**lth Effect* Rmarch Laboratory,
U.S. Environmental Protection Agency, and approved for publication.
As has been suggested previously (O'Callaghan et al., 1983;
Mailman et al, 1983; Reuhl and Cranmer, 1984), there are
reasons to suspect that neurons, as well as glia, can be affected
as a consequence of exposure to TET during postnatal devel-
opment. First, proliferation, migration, dendritic maturation
and synaptogenesis are critical processes in the postnatal de-
velopment of neurons (for reviews see Jacobson, 1978; Rodier,
1980; Bayer, 1985; Suzuki, 1980) and all are sensitive to chem-
ical intervention (Suzuki, 1980; Rodier, 1980; Ruppert, 1986).
Second, postnatal administration of TET permanently de-
creases wet weight of whole brain and specific brain regions
without altering body weight (O'Callaghan et al., 1983; Ruppert
et al, 1983; Toews et al, 1983; Miller, 1984). Because two of
the areas decreased in weight by TET, hippocampus and cere-
bellum (Ruppert et al, 1983; Miller, 1984), are known to
undergo extensive postnatal proliferation and maturation of
neurons (Jacobson, 1978; Rodier, 1980; Bayer, 1985), develop-
A68REVIAT10N8: TET, triethyltin; CNS, central nervous system; PNO, postnatal day; NF-200, neurofflament 200 (M, - 200 kHoda/tons); GFAP,
glial fibrillary acidic protein; RIA, radioimmunoassay; IgG, immunoglobulin; SDS-PAGE, sodium dodecyi suifate-polyacrytamide gel electrophoresis;
MBP, myelin basic protein; RSA, relative specific activity.
3M
-------
1988
Neonatal TET and Brain Proteins
369
ment of neuronal elements with these and possibly additional
structures may be affected. Finally, postnatal administration
of TET is accompanied by permanent alterations in behavioral
measures of sensation, activity, learning and memory (for a
review see Ruppert, 1986), findings suggestive of an involve-
ment of several aspects of neuronal development.
In view of the potential for postnatal exposure to TET to
disrupt neuronal as well as glial development, the purpose of
the present investigation was to assess the effects of postnatal
administration of TET on proteins that serve as biochemical
indicators of neuronal and glial development. Previously, we
proposed (O'Callaghan and Miller, 1983) and subsequently
demonstrated that assays of proteins that distinguish among
several neuronal and glial cell types can be used to quantify
neuronal and glial response to chemical-induced injury of the
central nervous system (Miller and O'Callaghan, 1984; O'Cal-
laghan and Miller, 1984, 1985, 1986; Brock and O'Callaghan,
1987). In the present investigation, six proteins, each associated
with the ontogeny of a specific developmental process, were
used to characterize the neurotoxicity of TET after its admin-
istration on PND 5. The proteins measured, their cell-type
specificity and the developmental processes with which they
are associated are summarized in table 1. Recent observations
indicate that damage to the CNS or peripheral nervous system
is accompanied by degradation of two of the proteins described
in table 1; NF-200 (Schlaepfer et al., 1984; 1985) and GFAP
(Schlaepfer and Zimmermann, 1981; DeArmond et al., 1983).
Consequently, we also included an analysis of NF-200 and
GFAP degradation products to determine the extent of TET-
induced in situ proteolysis.
As a first step in localizing the brain sites affected by TET,
we subjected three regions (hippocampus, forebrain and cere-
bellum) to morphological (brain weight and histology) as well
as biochemical (RIA of neurotypic and gliotypic proteins) eval-
uations. These brain areas were selected for three reasons.
First, they are amenable to rapid, accurate and reproducible
dissection. Second, they are known to be permanently reduced
in weight by TET (Ruppert et al., 1983; Miller, 1984) an effect
that frequently is accompanied by changes in neurotypic and
gliotypic proteins {e.g. see Miller and O'Callaghan, 1984; O'Cal-
laghan and Miller, 1985). Finally, specific stages in postnatal
ontogeny (e.g. cell proliferation) often have been proposed to
coincide with periods of vulnerability to chemical insult (Ro-
dier, 1976,1980*, Suzuki, 1980). Because hippocampus, forebrain
and cerebellum are at different stages in development on PND
5 (e.g. see Jacobson, 1978), we reasoned that examination of
these areas might reveal regional differences in vulnerability to
the neurotoxic effects of TET.
Materials and Methods
Materials. ("®I]Protein A (2-10 iiCi/itf, 1 Ci — 37 GBq) was
purchased from New England Nuclear (Boston, MA). Monoclonal
antimyelin basic protein and monoclonal antineurofilament (p200)
were purchased from Boehringer Mannheim Biochemicals (Indianap-
olis, IN). Antisera to GFAP and rabbit antimouse IgG were obtained
from Dako Corp. (Santa Barbara, CA). Monoclonal antisynapsin I
(cl0.22 and cl0.31) and monoclonal anti-p38 (c7.1, c7.2 and c7.3) were
the generous gifts of Dr. Reinhard Jahn (Rockefeller University).
Monoclonal anti-fl-tubulin was purchased from Ametshatn Corporation
(Arlington Heights, IL), Nitrocellulose paper (0.2 and 0.06 tm pore
size, German manufacture, roll form) was purchased from Schleicher
and Schuell (Keens, NH). Additional materials used in the RIAs were
from the reported sources (Jahn et al., 1984). Reagents used for SDS-
PAGE were obtained from Bio-Rad Laboratories (Richmond, CA) with
the exception of Tris which was from Calbiochem (San Diego, CA).
Bovine serum albumin and molecular weight standards were purchased
from Sigma Chemical Co. (St. Louis, MO). X-ray film (XRP) was from
Kodak (Rochester, NY). TET bromide was obtained from Alfa Prod-
ucts (Danvers, MA). All other chemicals were of at least analytical
reagent grade and were obtained from a variety of commercial sources.
Subjects. Pregnant Long-Evans rats (Blue Spruce Farms, Alta-
mont, NY) were received 2 days after conception and were housed
individually in plastic tub cages (45 x 25 x 20 cm) with pine shavings
as bedding. The colony room was maintained at a constant temperature
(22°C ± 2) and humidity (50% ± 10) with a 12-hr light/12-hr dark
lighting cycle beginning at 6:00 A.M. Pups born between 5:00 P.M. and
5:00 A.M. were considered to be born on the same day (PND 0), On
PND 1 pups were reassigned to litters such that each dam received
four male and four female pups with no more than one male and one
female of her own offspring. Pups were tatooed on the foot with India
ink as a means of identification. At 21 days of age littermates were
weaned, separated by sex and housed four per cage.
Dosing. Pups were injected on PND 5 with TET bromide (•'?.() or
6.0 mg/kg i.p.) or with its vehicle control (0.9% saline). These dosages
of TET were used to effect slight (3.0 mg/kg) to moderate 16.0 mg/kg)
decrements in brain weight without altering body weight (see O'Cal-
laghan et aL, 1983; Ruppert et al., 1983; Miller, 1984). Doses of TET
are expressed as the bromide and were administered in a volume of 10
ttl/g b.wt. All animals in a litter received the same dose.
Brain dissection. Animals were sacrificed by decapitation on PN'D
13, 22, 40 or 66. Whole brains then were excised from the skull and
placed on a cold plate (Thermoelectrics Unlimited, Wilmington. DE)
maintained at 0-4*C. Cerebellum and hippocampus were removed by
free-hand dissection; the remaining portion of the brain was designated
as forebrain. The dissected regions then were weighed, homogenized bv
Bonification (Kontes Cell Disrupter, Vineland, NJ) in 10 volumes of
hot (90-95°C) SDS and frozen at —70°C before RIA.
Histology. On PND 22, one group of animals that received TET
(6.0 mg/kg only) and one that received saline were anesthetized,
perfused and the brains were processed for histology as described
previously (Miller and O'Callaghan, 1984).
Protein assay. Total protein was determined by the method of
Lowty et al. (1961) using bovine serum albumin as the standard.
RIA of neurotypic and gliotypic proteins. All proteins were
assayed by modifications (O'Callaghan and Miller, 1985; Brock and
O'Callaghan, 1987) of the dot-immunobinding procedure of -lahn et al.
(1984). Briefly, samples were assayed for total protein, diluted in dot-
immunobinding buffer and applied to nitrocellulose sheets (0.2 )im pore
size) using a slot-blot apparatus (Minifold II, Schleicher and Schuell.
Keene, NH) as a template. Samples containing 5.0 u$ (GFAP assays)
or 8.75 jig (ail other assays) of protein in 20 pi of sample buffer were
loaded into each slot. The spotted sheets then were fixed, blocked,
washed and incubated with primary antibody and [rJSI]Protem A de-
tection solution as described previously (Jahn et al., 1984). Where
monoclonal antibodies were used, additional incubations in rahbit
antimouse IgG (1:500), blocking and washing solutions were required
(O'Callaghan and Miller, 1985; Brock and O'Callaghan, 1987). The
primary antibody stocks were used at the following dilutions: MBP,
1:500; GFAP, 1:500; synapsin I, 1:2500 (cl0.22) and 1:3000
-------
370
O'Callaghan and Millar
Vol. 244
TABLE 1
Neurotypic and gliotypic protein* used to characterize the developmental neurotoxicity of TET
Designation
CNS Cad-Type Specificity
CeHulaf Distribution
Associated Developmental
Process or Cell-Type Response
Reference
NF-200
Neurons*
Intermediate filaments*
Synapsin I
p38
MBP
GFAP
Neurons'
Neurons*
Synaptic vesicles"
Synaptic vesicles*
Myelin forming oilgo-
denroglia"
Astrocytes'
Myelin®
Intermediate filaments'
^-Tubulin
Neurons and giia'
Microtubules (primarily in
neurons)'
Ontogeny of axonal cy-
tosketeton (in specific
neurons)"
Synaptogenesis"
Synaptogenesis'
Myelinogenesis'1
Aatrogliogenesis'
Astroglial response to
injury* -
Cell division, axon and
dendrite formation""
* Willard and Simon, 1981;
"Shawef al., 1981;
'Debus atal., 1982;
'Lazarides, 1982;
" Shaw and Weber,
1982; ° Willard and Si-
mon, 1983;0 Pachter
and Liem, 1984;
° Cambray-Oeakin and
Burgoyne, 1986
0 De Camilli at a I., 1983a,
c 1983b;c Huttner at a/.,
1983;c Navone at a/.,
1984;" Lohmann et al.,
1978
•Jahnefa/., 1985;
* Wiedenmann and
Franks, 1985; 'Navone
at at., 1986; * Obata et
al., 1986,'Knausef al.,
1986
3 " Stemberger at at., 1978;
'Norton, 1981;
Colman et al., 1982;
" Hartman ef al., 1982
' Bignami ef a/., 1972,
1980;' Eng, 1980,
' 1985;1 Eng and De Ar-
mond, 1982; 'Sapirstein,
1983;' Weir eta/., 1984
' Bignami and Dahl, 1976;
* Eng, 1987; * Brock and
O'Callaghan, 1987
' Caceres et al.. 1984;
' Brady and Black, 1986;
'¦m Morrison etal., 1981;
m Morrison and Griffin,
1986
obtained by comparing the immunoreactivity of individual samples
(from saline and TET groups) with that of the sample used to construct
the standard curve (e.g. see O'Callaghan and Miller, 1984, 1985). The
RSA of each sample (from saline and TET groups) then was multiplied
by a constant that resulted in a mean RSA of 1.0 (100%) for the adult
(PND 66) saline groups. Data are expressed on both a total (per tissue)
and a concentration (per milligram of tissue protein) basis. Total values
reflect the absolute amount of a given protein; whereas concentration
values reflect the abundance of a specific protein in comparison to all
other proteins. Thus, changes in total values are indicative of altera-
tions in the amount of a specific protein per brain region; whereas
changes in concentration values are indicative of alterations in the
amount of a specific protein per milligram of total protein in a given
brain region.
Immunoblota of NF-200 and GFAP. Samples obtained from
PND 22 hippocampus and forebrain (saline and 6.0 mg/kg of TET
only) were subjected to SDS-PAGE and then were transferred electro-
phoretically (Towbin et aL, 1979; Burnette, 1981) to sheets of nitrocel-
lulose (0.05 mM pore size). Using the procedures described above, one
sheet was assayed for GFAP and another duplicate transfer was assayed
for NF-200. The resulting GFAP- and NF-200-immunoreactive bands
were revealed by autoradiography.
Statistical analysis. The Statistical Analysis System (SAS, 1982)
was used for data analyses. Individual variables wen evaluated by
analysis of variance followed by Duncan's Multiple Range Test for
mean comparisons. Because sex-related differences were not observed
for any of the measures, data for males and females were combined.
Results
Body and brain weight. In agreement with previous ob-
servations (O'Callaghan et aL, 1983; Ruppert et al., 1983; Miller,
1984), acute administration of TET on PND 5 resulted in dose-
dependent decreases in wet weight of whole brain, hippocam-
pus, cerebellum and forebrain (table 2). Decrements in wet
weight were observed at all time points after exposure to 6.0
mg/kg of TET; only slight recoveries were observed by adult-
hood (PND 66). At all time points the most affected region was
the hippocampus and the least affected region was the cerebel-
lum; deficits in hippocampus weight were as great as 35% (PND
40) whereas reductions in cerebellum weight did not exceed
12% (PND 13). With the exception of PND 13, decreases in
brain weight were not accompanied by decreases in body weight.
TET did not change the concentration of protein (Lowry value,
1951) in any brain region at any dose or time point (data not
shown).
Histology. Representative micrographs obtained from dor-
sal hippocampus and cerebellum of 22-day-old saline- and TET
(6.0 mg/kg)-treated subjects are shown in figure 1. The cytoar-
chitecture of these poatnatally developing structures was not
affected by TET. Gross morphology of forebrain also was not
altered as a consequence of the administration of TET (data
not shown). In agreement with the brain weight data, the
-------
7988
Neonatal TET and Brain Proteins 371
TABLE 2
Effects of acute postnatal administration of TET on body and brain weights
Each value represents the mean (±S.E.M ).
Oay H Dose Body wt. 8rain wt Hippocampus wt. Forebrain wt. Cerebellum wt.
mg/kg
9
13
8
0
33.6 ± (0.6)
1.224 ± (0.012)
0.073 ±(0.001)
1.015 ±(0.012)
0.145 ±(0.003)
8
3
33.4 ± (0.6)
1.226 ± (0.010)
0.071 ±(0.001)
1.003 ±(0.010)
0.142 ±(0.002)
8
6
29.3 ± (0.8)*
0.992 ±(0.015)*
0.049 ± (0.002)*
0.802 ± (0.014)*
0.128 ±(0.003)*
22
8
0
55.5 ± (2.6)
1.475 ± (0.019)
0.088 ± (0.002)
1.165 ±(0.015)
0.200 ± (0.003)
8
3
57.1 ±(1.6)
1.425 ± (0.009)
0.087 ± (0.002)
1.125 ±(0.008)
0.190 ± (0.004)'
8
6
56.0 ± (2.1)
1.226 ± (0.041)*
0.060 ± (0.004)*
0.955 ± (0.035)*
0.185 ± (0.003)*
40
8
0
167.9 ± (7.5)
1.736 ± (0.022)
0.106 ± (0.002)
1.368 ±(0.017)
0.253 ± (0.006)
8
3
177.1 ± (7.5)
1.696 ± (0.026)
0.102 ± (0.003)
1.326 ±(0.019)
0 248 ± (0.006)
8
6
170.1 ± (9.8)
1.398 ± (0.029)*
0.069 ± (0.004)*
1.087 ±(0.024)*
0.224 ± (0.005)*
66
8
0
296.8 ± (22.8)
1.862 ± (0.023)
0.113 ±(0.003)
1.441 ± (0.018)
0.283 ± (0.006)
8
3
303.9 ± (26.6)
1.830 ± (0.044)
0.108 ± (0.003)
1.411 ±(0.033)
0,279 ± (0 009)
8
6
289.0 ± (26.3)
1.612 ± (0.038)*
0.084 ± (0.007)*
1.238 ± (0.029)*
0.267 ± (0.005)
' Significantly different from 0 mg/kg, P < .05.
overall size of the hippocampal formation was consistently
smaller in the TET-treated group (fig. 1).
RIA of Neurotypic and Gliotypic Proteins
Administration of TET on PND 5 caused dose- and time-
dependent changes in neurotypic and gliotypic proteins in all
brain areas examined. These data are presented by area starting
with the region with the greatest weight deficit (hippocampus*
and proceed in order to the area with the least weight deficit
(cerebellum). For a given brain region, values for neurotypic
proteins (NF-200, synapsin I and p38) are presented first,
followed by values for gliotypic proteins (MBP and GFAP ) and
ending with values for j3-tubulin.
Fig. 1. Morphology of dorsal hippocampus (top) and cerebellum (bottom) on PND 22 after acute administration of saline or 6.0 mg/kg of TET on
PND 5. The micrographs shown were of sagittal sections obtained approximately 500 ym from midline. Bars - 200 »m. No apparent differences
were observed between sections prepared from saline- or TET-treated animals.
-------
372 O'Callaghan and Millar
Vol. 244
Hippocampus. TET caused dose- and time-dependent de-
creases in all neurotypic proteins in hippocampus (fig. 2). At
the high dosage (6.0 mg/kg), values for total NF-200, synapsin
I and p38 were reduced throughout ontogeny (fig. 2, left panels);
at some time points values were decreased by as much as 60%
(i.e., synapsin I and p38 values on PND 13). With the exception
of PND 13, the magnitude of the decreases in total NF-200
(fig. 2, left panel) coincided with the magnitude of the decreases
in hippocampal weight; therefore, the concentration of this
protein in hippocampus was largely unaffected by TET (fig 2,
right panel). In contrast, the TET-induced decreases in total
synapsin I (3.0 and 6.0 mg/kg) and p38 (6,0 mg/kg) (fig. 2, left
panels) exceeded those in tissue weight throughout postnatal
development, findings which are reflected in the large (20-40%)
100'
80'
§ 60-
2
o—o
00
A—A
10
CONCBttUTION
100
80 ¦
decreases in the concentration of body synaptic vesicle proteins
(fig. 2, right panels).
On a per hippocampus (total) basis, TET caused dose- and
time-dependent decreases in the ontogenetic profile of both the
myelin protein, MBP and the astrocyte protein, GFAP (fig. 3,
left panels). At the high dosage (6.0 mg/kg), these TET-induced
decreases did not parallel decreases in hippocampal wet weight.
The magnitude of the reductions in MBP were greater and the
magnitude of the reductions in GFAP were less than the extent
of the reduction in hippocampal wet weight. Because the effects
of TET on MBP and GFAP did not parallel TET-induced
decreases in hippocampus weight, the concentration of these
proteins was altered accordingly (fig. 3, right panels). The
concentration of MBP in hippocampus was reduced by as much
as 15% between PND 40 and PND 66. In contrast, the high
dosage of TET caused a marked (1.6-fold) increase in the
concentration of GFAP on PND 13 with elevations persisting
through PND 40,
The effects of TET on hippocampal ^-tubulin are presented
in figure 4. Consistent with the known ontogenetic profile of
this protein (Morrison et ai„ 1981; Nunez, 1986), values for
total ^-tubulin remained relatively stable despite the increase
in tissue growth. Consequently, values for concentration of 0-
tubulin declined as postnatal development progressed. TET
reduced total 0-tubulin (fig. 4, left panel) to a degree that, in
general, coincided with the degree of TET-induced reductions
in tissue weight. The concentration of 0-tubulin in hippocam-
pus, therefore, was largely unaffected by postnatal exposure to
TET (fig. 4, right panel).
Forebrain. In comparison to hippocampus, the effects of
AGE (days)
Pig. 2. Effects of acuta neonatal administration of TET on neurotypic
proteins in hippocampus. Each value represents the mean ± S.E.M. tor
eight independent observations. Where S.E. bars are not shown they
are within the data point. Data are expressed on a per tissue (total) and
a per milligram of tissue protein (concentration) basis. Values for synapsin
I on PND 40 ware not determined. Additional details appear under
"Materials and Methods." 'Significantly different from 0.0 mg/kg, P <
.OS.
(Of At
CONCINflUfKJN
50 ~ 10 ' 30
AGE (days)
Pig. 3. Effects of acute neonatal administration of TET on gliotypic
proteins in hippocampus. Details appear in the legend to figure 2 and
under "Materials and Methods." 'Significantly different from 0.0 mg/kg.
P < .05.
-------
1988
Neonatal TET and Brain Proteins 373
s
120 •
hippocampus!
2
O
o too^
5
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<
Q
1 80-
60-
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40-
OeMte •<
ii
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10
30
COfCcNntATION
WO Q
50 10
AGE (days)
Fig. 4. Effects of acute neonatal administration of TET on /^tubulin in
hippocampus. Details appear in the legend to figure 2 and under "Mate-
rials and Methods." 'Significantly different from 0.0 mg/kg, P < .05.
TET on neurotypic and gliotypic proteins in forebrain were
smaller in magnitude and were observed at fewer time points
(figs. 5 and 6). For example, although total NF-200, synapsin I
and p38 were reduced at all time points after the administration
of 6.0 mg/kg of TET (fig. 5, left panel), the magnitude of these
decreases, unlike those in hippocampus, was similar to the
magnitude of the decreases in forebrain weight (see table 2).
Consequently, corresponding values for the concentration of
these proteins in forebrain were generally not affected by TET
throughout most of postnatal development (fig. 5, right panels).
Like the neurotypic proteins in forebrain, total MBP was
reduced at all time points after the administration of 6.0 mg/
kg of TET (fig. 6, left panel), with the magnitude of these
decreases corresponding to the magnitude of the decreases in
forebrain weight (see table 2). Total GFAP in forebrain was
reduced by TET (6.0 mg/kg) only on PND 66 (fig. 6, left panel).
Because the weight of forebrain was reduced at all time points,
the concentration of GFAP in forebrain, as in hippocampus,
increased from PND 13-40 after exposure to 6.0 mg/kg of TET
(fig. 6, right panel). The magnitude of these increases in fore-
brain GFAP, however, were not as great as those observed in
hippocampus (e.g. a 36% increase on PND 22 in hippocampus
vs. a 20% increase on PND 22 in forebrain).
As in hippocampus, the ontogenetic profile of 0-tubulin in
forebrain, in general, was altered by TET (fig. 7) in accordance
with its effects on forebrain weight (see table 2).
Cerebellum. All neurotypic proteins in cerebellum were
reduced as a consequence of the administration of TET (fig. 8)
and the magnitude of these decreases generally exceeded the
magnitude of the reductions in cerebellar weight (see table 2).
NF-200 was reduced at both dosages of TET, effects which
could be observed by PND 22 (fig. 8, left panel) and which
increased in magnitude with increasing postnatal age (fig. 8,
left and right panels). In contrast to the effects of TET on
neurotypic proteins in hippocampus, cerebellar NF-200 was
affected to a greater degree than cerebellar synapsin I and p38
(fig. 8, left and right panels).
TET caused large reductions in MBP in cerebellum and,
unlike its effects on this protein in hippocampus and forebrain,
a dosage of 3.0 mg/kg caused significant reductions at all but
the earliest time point (fig. 9, left and right panels). In contrast
to hippocampus and forebrain, values (total and concentration)
•I minffN
o—O 00
30
~—o 40
60 5 >
50 10 30
AGE (days)
no-s. Effects of acute neonatal administration of TET on neurotypic
proteins In forebrain. Details appear in the legend to figure 2 and under
"Materials and Methods." 'Significantly different from 0.0 mg/kg, P <
.05.
for GFAP in cerebellum were largely unaffected by TET (fig.
9, left and right panels).
As in hippocampus and forebrain, values for 0-tubulin in
cerebellum varied largely as a function of decreases in tissue
weight (fig. 10, left panel). The concentration of this protein,
therefore, was only slightly affected as a consequence of the
administration of TET on PND 5 (fig. 10, right panel).
Immunoblota of GFAP and NF-200. Immunoblots ob-
tained from samples of hippocampus revealed evidence sugges-
tive of proteolysis (fig. 11). In comparison to samples obtained
from hippocampus of control subjects, samples from hippocam-
pus of subjects exposed to TET (6.0 mg/kg) showed increased
GFAP immunoreactivity and the presence of GFAP immuno-
reactive bands below the 50 kilodalton band characteristic of
the native protein. Similarly, in samples from TET-exposed
hippocampus, NF-200 immunoreactivity was associated not
only with a prominent 200 kilodalton band but also with several
-------
374
O'Callaghan and Millar
Vol. 244
I FOREBRAIN I
100-
80-
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Fig. 6. Effects of acute neonatal administration of TET on gliotypic
proteins in forebrain. Details appear in the legend to figure 2 and under
"Materials and Methods." "Significantly different from 0.0 mg/kg, P <
.05.
[FOREBRAIN
'OTAt
CQNCINTUTIOM
•< TIT
MURIUM]
CCNCINtHWION
TfT tn«/Va)
a 60
60 ®
50 10
AGE (days)
Fig. 7, Effects of acute neonatal administration of TET on /3-tubuUn in
forebrain. Details appear in the legend to figure 2 and under "Materials
and Methods." 'Significantly different from 0.0 mg/kg, P < .05.
bands of lower molecular weight. In general, immunoblots of
forebrain samples from TET-exposed subjects exhibited
GFAP- and NF-200-immunoreactivity that was limited to
bands corresponding to the electrophoretic mobilities of the
native proteins (data not shown).
Discussion
A single administration of TET on PND 5 is toxic to the
CNS of the developing rat based on evidence of alterations in
10 30 50 10
AGE {days)
Fig. 8. Effects of acute neonatal administration of TET on neurotypic
proteins in cerebellum. Details appear in the legend to figure 2 and under
"Materials and Methods." 'Significantly different from 0.0 mg/kg. P <
.05.
functional (Ruppert et aL, 1983; Miller, 1984), morphological
(O'Callaghan et aL, 1983; Ruppert et at., 1983; Miller, 1984)
and biochemical endpoints (O'Callaghan et aL, 1983). Our
results support the views that: 1) the effects of acute postnatal
exposure to TET on the developing CNS are manifested by
permanent changes in neurons as well as glia; 2) the toxic
effects of TET on the CNS are region-dependent but are not
linked to region-dependent stages in brain development ongo-
ing at the time of exposure; and 3) TET-induced changes in
neurotypic and gliotypic proteins can be demonstrated in the
absence of overt histopathology and at dosages below those
necessary to cause reductions in brain weight.
The permanent decreases in hippocampus, forebrain and
cerebellum weight observed after exposure to TET (6.0 mg/kg)
are consistent with the results of previous investigations where
TET was administered acutely on PND 5 (O'Callaghan et aL,
1983; Ruppert etaL, 1983; Miller, 1984) or throughout postnatal
ontogeny (Blaker et aL, 1981; Mailman et al„ 1983; Toews et
-------
1988
Neonatal TET and Brain Proteins 375
rorii
£ m 60
ssnrmszi
o—o oo
10
Bsfl 40
60
GFAP NF 200
D«ap at TIT (wm
200
CO
9 1001-
50-
40
50 0 30 50
AGE (days |
F19. 9. Effects of acute neonatal administration of TET on gliotypic
proteins in cerebellum. Details appear in the legend to figure 2 and under
"Materials and Methods." 'Significantly different from 0.0 mg/kg, P <
.05.
Splint TET
50 10
AGE (days)
Fig. 10. Effects of acute neonatal administration of TET on 0-tubulin in
cerebellum. Details appear in the legend to figure 2 and under "Materials
and Methods.' "Significantly different from 0.0 mg/kg, P < .05.
aL, 1983). Previously, we observed that such toxicant-induced
reductions in brain weight often are associated with changes in
the amounts of neurotypic and gliotypic proteins in the affected
structures (Miller and O'Callaghan, 1904; O'Callaghan and
Miller, 1984, 1986, 1986; for a review see O'Callaghan, 1988).
Thus, we expected to find TET-induced changes in specific
neurotypic or gliotypic proteins in all areas. The data obtained
were consistent with these expectations; exposure to TET
caused dose- and region-dependent changes in both the total
amount and the concentration of all neurotypic and gliotypic
proteins evaluated.
30
Saline TET
Fig. 11. GFAP and NF-200 immunoblots obtained from homogenates of
hippocampus. Samples were prepared from saline- and TET (6.0 mg/
kg>-treated animals on PNO 22. Note presence of additional immunore-
active bands in blots obtained from the TET-treated animal.
The decreased total values for specific neurotypic and gli-
otypic proteins were suggestive of an absolute reduction in the
number or maturational state of the cell type with which the
protein is associated (see table 1). Because total values for most
neurotypic as well as gliotypic proteins were permanently re-
duced in all regions, we conclude that TET caused permanent
decrements in neuronal as well as glial development throughout
the brain. In many instances these decreases, like reductions
in tissue weight, may be a reflection of a general reduction in
brain growth caused by TET. The decreased total values could
not, however, be attributed to weight loss associated with a
general toxic response, because reductions in brain weight were
observed without corresponding changes in body weight.
The alterations in concentration values for specific neuro-
typic and gliotypic proteins, in contrast to changes in total
values, were indicative of TET-induced effects that did not
coincide with the overall reductions in brain growth. Therefore,
where such effects occurred they may reflect a preferential
action of TET on a given brain region and on a specific cell
type within that region. Although TET affected the concentra-
tion of all neurotypic and gliotypic proteins, the specific protein
affected and the degree to which it was affected varied consid-
erably from region to region.
With respect to neurotypic proteins, TET caused permanent
effects on concentration values in two brain regions, hippocam-
pus and cerebellum. These data suggest that neuronal celL types
within these structures were preferentially and irreversibly
affected by TET. In hippocampus, TET caused a permanent
decrease in the concentration of synapsin I and p38. These data
are consistent with a preferential decrease in synaptic density,
-------
376 O'Callaghan and Millar
Vol. 244
an effect which may have resulted from a TET-induced de-
crease in synaptogenesis or loss of synapses after their forma-
tion. In cerebellum, a permanent decrease in NF-200 with lesser
decreases in synapsin I and p38 resulted from exposure to TET.
Because NF-200 immunoreactivity in cerebellum is associated
predominantly with basket cells (Cambray-Deakin and Bur-
goyne, 1986), whereas synapsin I and p38 are predominantly
localized to parallel fibers of granule cells (Dolphin and Green-
gard, 1981; De Camilli et al, 1983a; Navone et al, 1986), our
data are suggestive of preferential decrements in basket and
granule cells, respectively. Immunohistochemical localization
of individual proteins using the same antisera used in the RIAs
would aid in determining the precise regions and cell types
affected by TET (see Brock and O'Callaghan, 1987).
Of the two gliotypic proteins examined, only MBP was
reduced in concentration by TET and, as was true for the
neurotypic proteins, this effect was limited to hippocampus and
cerebellum. These data are compatible with the reported defi-
cits in myelination resulting from acute (Wender et al, 1974;
O'Callaghan et al, 1983) or chronic (Blaker et al., 1981; Toews
et al., 1983) exposure to TET during postnatal development.
The effects of TET on MBP in cerebellum were observed at a
dosage of 3.0 mg/kg, findings which indicate that changes in
this protein, as well as in NF-200, can be demonstrated in the
absence of alterations in tissue weight.
The fact that the concentration of MBP in forebrain was not
altered by TET does not indicate that myelin was unaffected
in this region because the absolute amount (total) of MBP, like
all neurotypic proteins, was reduced. These data do suggest,
however, that administration of TET on PND 5 does not
preferentially affect forebrain myelin, as is the case when dosing
is continued throughout postnatal ontogeny (Blaker et ai, 1981;
Toews et al, 1983). Previously, we suggested that the admin-
istration of TET on PND 5 may cause a selective reduction in
myelin, based on an analysis of subcellular fractions of whole
brain (O'Callaghan et al., 1983). Our current findings, which
are based on regional analyses combined with more sensitive
methodology, now make it seem likely that TET affects several
cell types in multiple regions of the developing CNS.
GF AP, the major intermediate filament protein of astrocytes,
increases in response to trauma (Bignami and Dahl, 1976; Latov
et al., 1979; Lagenaur et ai, 1982; Eng, 1987)- or toxicant
(O'Callaghan and Miller, 1985, 1986; Brock and O'Callaghan,
1987)-induced injury of the adult CNS. Our data indicate that
this astroglial response can be extended to the neonate as early
as PND 13, based on the marked and protracted increases in
GFAP concentration (hippocampus and forebrain) that re-
sulted from exposure to TET. We interpret these findings to
mean that TET damaged or killed cells in hippocampus and,
to a lesser extent, in forebrain. Our reasoning is supported by
the following lines of evidence: 1) an increase in GFAP concen-
tration is associated with damage or loss of neurons in the adult
(Brock and O'Callaghan, 1987) or developing (O'Callaghan and
Miller, 1986) CNS, observations consistent with the increase
in GFAP and the decrease in synapsin I and p38 (hippocampus)
due to TET; 2) greater toxicant-induced increases in GFAP
concentration are associated with proportionately greater de-
creases in tissue weight and greater cell loss or damage (O'Cal-
laghan and Miller, 1985), findings in agreement with the pres-
ent data; and 3) the data suggestive of in situ proteolysis of
GFAP and NF-200 in hippocampus are consistent with cell loss
or damage due to TET. Although the cerebellum can exhibit
tiauma (Lagenaur et al, 1982) or toxicant (O'Callaghan and
Miller, 1985)-provoked increases in GFAP as early as PND 13
(J. P. O'Callaghan and D. B. Miller, unpublished observation),
we did not observe such an increase in response to TET. These
data suggest that TET did not cause cell damage or cell death
in the cerebellum. Consequently, the marked effects of TET on
MBP, NF-200, synapsin I and p38 in cerebellum probably were
not the result of damage or loss of the cell types with which
these proteins are associated.
TET caused an increase in the concentration of GFAP in
forebrain without altering the concentration of other neuro-
typic and gliotypic proteins. Possibly, the damaged neuronal or
glial cell types in forebrain were not those associated with the
panel of neurotypic and gliotypic proteins measured in this
study. Alternatively, the increases in GFAP may have been
provoked by damage to cell types that indeed were associated
with the neurotypic or gliotypic proteins chosen for examina-
tion. If, however, the cell types affected by TET comprised only
a small fraction of the forebrain, then corresponding changes
in the concentration of neurotypic and gliotypic proteins may
have been so small in magnitude as to escape detection. This
line of reasoning is supported by our recent demonstration of
a marked (300% of control) increase in the concentration of
GFAP in striatum of mice exposed to l-methyl-4-phenyl-
1,2,3,6-tetrahydropyridine (MPTP) (Reinhard and O'Cal-
laghan, 1986; Miller et al., 1986), an effect elicited by loss of no
more than 5% of nerve terminals in the affected area (Arluison
et al, 1984) and, as a consequence, an effect which was not
characterized by significant loss of nerve-terminal proteins.
Evaluation of additional neurotypic proteins that delineate
subsets of neurons (for reviews see Nestler and Greengard,
1984; Nestler et al, 1984) combined with an examination of
more discrete areas of the developing brain, may aid in the
identification of the forebrain cell types affected by TET.
During postnatal development of the rat, periods of cell
proliferation are widely regarded as being especially vulnerable
to disruption by environmental insults (Dobbing and Sands,
1971; Rodier, 1976, 1980; Suzuki, 1980; Wiggins, 1986). In the
cerebellum a major postnatal growth spurt occurs which peaks
on PND 5 (Dobbing et al., 1970). This growth phase coincides
with a period of rapid proliferation of granule cells (Altman,
1972; Clark et al., 1978; Rodier, 1980). Proliferation of dentate
granule cells, although not a dominant feature of the postnatal
ontogeny of hippocampus (Dobbing et al, 1970, 1971), is also
ongoing during the 1st week after birth (Altman, 1970; Rodier,
1980). If vulnerability associated with cellular division is a
major determinant of the neurotoxic effects of TET adminis-
tered on PND 5, then the cerebellum and, to a lesser degree,
the hippocampus should be preferentially affected. Several lines
of evidence obtained in the present study argue against this
hypothesis. First, growth retardation was pronounced in hip-
pocampus, less so in forebrain and, by comparison, only minor
in cerebellum, findings which do not support the concept that
rapid proliferation confers sensitivity to TET. Second, synthe-
sis and accumulation of ^-tubulin during the 1st week of
postnatal development is correlated with cellular proliferation
(Morrison et aL, 1981). Because cell division is a dominant
feature of the postnatal development of the cerebellum (Alt-
man, 1972), disruption of this process by TET might be ex-
pected to result in permanent decrements in the concentration
of (3-tubulin. In cerebellum, as in other brain regions, no such
reductions were observed. Finally, a TET-induced increase in
-------
1988
Neonatal TET and Brain Proteins
377
the concentration of GFAP, an effect strongly linked to toxic
responses of CNS cell types (see above and Eng, 1987; O'Cal-
laghan and Miller, 1985, 1986; Brock and O'Callaghan, 1987),
was limited to brain regions in which the majority of cells on
PND 5 are postmitotic, i.e., forebrain and hippocampus. Thus,
taken together, the evidence suggests that innate vulnerability
of specific cell types to TET, rather than vulnerability associ-
ated with cellular division, dictates the pattern of neurotoxicity.
We cannot rule out the possibility, however, that the loss or
disruption of a few proliferating cells early in postnatal devel-
opment could account for subsequent changes in proteins as-
sociated with the ontogeny of other specific maturational proc-
esses (e.g. synaptogenesis and myelinogenesis). Indeed, this
possibility offers an attractive explanation for the effects of
TET on the concentration of NF-200, synapsin I, p38 and MBP
in the cerebellum.
In summary, using assays of neurotypic and gliotypic pro-
teins, we have obtained evidence suggesting that early postnatal
exposure to TET causes permanent, multicell-type and mul-
tiregion deficits in brain development. Together with our pre-
vious observations (Miller and O'Callaghan, 1984; O'Callaghan
and Miller, 1984, 1985, 1986; Brock and O'Callaghan, 1987),
the present data also suggest that assays of neurotypic and
gliotypic proteins can be used to detect and characterize the
temporal and regional patterns of neuronal and glial response
to other toxic exposures of the developing CNS.
Acknowledgments
The author* are grateful to Mi. Chriitine Booth for preparation of the brain
sections and Ma. Julia A. Davia for photographic assistance. We appreciate the
critical evaluations of this work provided by Drs. Michael D. Browning, Linda J.
Burdette, Karl Jensen, Christopher Lau and Diane Niedzwiecki.
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0022-3&66/&4/23l3-0?36tO<}.00/0
Thi Journal or Pharmacology and Exfuimintal Th*ju«ltics
Copyright C 1984 by Tht AoMncaa Socitty for PhanMcoiofy ud Erptnoxnui Thtraptutica
Vol, 231, No. 3
Pruutd in U S A
Neuron-Specific Phosphoproteins as Biochemical Indicators of
Neurotoxicity: Effects of Acute Administration of Trimethyltin to
the Adult Rat1
JAMES P. O'CALLAGHAN and DIANE B. MILLER
Naurotoxicology Division, Health Effacts ftaaaareh Laboratory, U.S. Enviromrmtal Protection Agancy, Raaaarch Triangla Park, North Carolina
Acotpted for publication August 27,1984
ABSTRACT
The cytoarchitecture of the adult central nervous system is
expressed by proteins specific to individual ceil types. In this
investigation, a subclass of these proteins, the neuron-specific
phosphoproteins. was examined after the administration of tri-
methyitin (TMT), a neurotoxicant which preferentially damages
neurons in limbic structures. After acute administration of TMT
(0.0-9.0 mg/kg i.v.), effects on neuronal phosphoproteins were
examined by three separate techniques: 1) endogenous phos-
phorylation of total synaptic membrane proteins; 2) radiometric
assay of synapsin I, a neuron-specific phosphoprotein associated
with synaptic vesicles; and 3) radioimmunoassay of synapsin I
and protein III, another synapse specific, synaptic vestcie-locaJ-
ized phosphoprotein. All three procedures gave similar results.
TMT caused dose- and time-dependent decreases in hippocam-
pal phosphoproteins. These effects were large in magnitude and
were still evident 14 weeks after exposure to TMT. Microdissec-
tion of slices of dorsal hippocampus did not reveal significant
regional differences in the extent to which TMT affected synapsin
I. Phosphoproteins in frontal cortex, unlike those in hippocampus,
were not affected by TMT. Our findings are consistent with the
neuropatftofogicat effects of this compound and suggest that
neuron-specific phosphoproteins may be useful biochemical in-
dicators of neurotoxicity.
The cellular heterogeneity of the developing and mature
nervous system is expressed by proteins specific to individual
cell types (for reviews see: Zomzely-Neurath and Keller, 1977;
Bock, 1978; Raff at oL, 1979; Kennedy, 1982; Schachner, 1982;
Nestler and Greengard, 1983). The availability of techniques
for identifying and, in tome cases, measuring these proteins
makes it feasible to characterize nervous tissue cell types on
the basis of biochemical and immunochemical as well as, mor-
phological criteria. Our interest in nervous system-specific pro-
teins is based on extensive neuropathologies! data which indi-
cate that neurotoxic effects of environmental pollutants are
often highly selective (Spencer and Schaumberg, 1980), For
example, toxicants are known preferentially to affect specific
cell types neurons and oligodendroglia) or subsets of a
given cell type (e.g., specific types of neurons), often within
only a discrete region of the nervous system. The possibility
that the response of an organism to neurotoxic insult could be
Received for publication March 12,19S4.
'TWi paper ha* been reviewed by the Health Bitot* Reeeenfa Laboratory,
U.S. Environmamal Protection Asiney, and approved fat publication. Mention
of trade name* or commerciaJ product* doe* not constitute endowment or
recommendation tor A preliminary report of this iaveetifatiea appealed in
Soc. Neurowi. Abetr. 9t 387,1983.
characterized and quantified by changes in nervous system-
specific proteins led us to propose the use of these proteins as
broadly applicable biochemical indicators of neurotoxicity (0'-
Callaghan and Miller, 1983). In order to evaluate this hypoth-
esis, we are assessing the effects of known neurotoxicant* on
proteins specific to cellular and subcellular elements of the
mature and developing nervous system.
The orgsnometallic compound, TMT, is neurotoxic to the
adult rat Acute or chronic exposure to TMT, while sparing
myelin (Brown at aL, 1979), damages the neuronal component
of the CNS (Brown at aL, 1979; Bouldin et aL, 1981; Chang and
Dyer, 1983). Evidence from both light microscopic (Brown et
oL, 1979; Chang and Dyer, 1983) and ultrastructural (Bouldin
at at, 1981) studies indicate that the cytopathologic effects of
TMT are most pronounced in limbic system neurons and are
accompanied by neurobehavioral alterations indicative of lim-
bic system dysfunction (Walsh at aL, 1982). This regional and
cell-type profile of TMT-induced neurotoxicity moke this com-
pound a use Ail tool for validating the use of nervous system-
specific proteins as biochemical indicators of neurotoxicity.
In the present study we examined the effects of TMT on a
subclass of nervous system-specific proteins, the neuronal phos-
phoproteins (for reviews see Nestler and Greengard, 1983,
ABMKVIATIONS! TMT, nimethyWn; CNS, central nervous system; SOS-PAQE, sodium ctodecyt sulfats-potyacrylamide qei electrophoresis; cAMP.
cycle AMP. ¦ ¦ .
7M
-------
1984
Bnrtn Ptwaphoproteina and TMT 737
1984), including the nerve terminal specific phosphoproteins,
synapsin 1 (De Camilli et aL, 1983a,b; Huttner et aL, 1983) and
protein III (Huang et aL, 1982; Tsou and Greengard, 1982;
Browning et aL, 1982). Of the neuron-specific phosphoproteins,
synapsin I is the most well characterized (for reviews see
Greengard, 1981; Nestler and Greengard, 1983, 1984) and it
was, therefore, the focus of the present investigation. Measure-
ments were limited to two regions of the CNS, hippocampus
and frontal cortex. By examining neuronal phosphoproteins in
an extralimbic region (frontal cortex) as well as a limbic struc-
ture known to be severely damaged by TMT (hippocampus),
we expected to find changes consistent with the effects of this
compound on the morphology of these brain regions. On the
basis of evidence obtained from both biochemical and immu-
nochemical assays, we report that acute administration of TMT
results in dose- and time-related decreases in hippocampal
phosphoproteins without causing similar changes in phospho-
proteins from frontal cortex. In the following paper, we dem-
onstrate that the neurotoxic effects of TMT in the developing
rat are characterized by alterations in nervous system-specific
proteins, neurobehavioral function and morphology (Miller and
O'Callaghan, 1984).
Materials and Methods
Materials. [*rMPlATP (10-40 Ci/umol was purchased from New
England Nuclear (Boston, MA). Piperszine-N.N'-bis(2-ethanesulfonic
acid) and Tris were obtained from Calbiochem (San Diego, CA). Re-
agents used for SDS-PAGE were of electropboretic grade and were
purchased from Bio-Rad Laboratories (Richmond, CA). Sucrose (grade
II) and catalytic subunit (beef heart) were from Sigma Chemical Co.
(St. Louis, MO). Synapsin 1, protein III and corresponding antisera
(rabbit) were the generous gifts of Drs. M Browning, E. Perdahl and
Jesse Chan, Rockefeller University. The specificity of the antisera
against rat antigen has been demonstrated by 1) the double agar
immunodiffusion test, 2) immunoprecipitation from brain extracts and
3) radioimmunolabeling of SDS-poJyacrylamida gels (see D« Camilli et
aL, 1979; Goelz et aL, 1981 for additional details). Other materials used
in the radioimmunoassays were from (he sources described (Goelz et
aL, 1981). TMT hydroxide was obtained from ICN Pharmaceuticals,
Inc. (Plainview, NY).
Subjects. Male, Long-Evans rats (Charles River Breeding Labora-
tories, Inc., Wilmington, MA) weighing 175 to 225 g were housed
individually in a temperature (22'C ± 2)- and humidity (60% ± 10)-
controlled colony room maintained on a 12-hr light/12-hr dark cycle
beginning at 8:00 A.M.
Dosing. Rats were injected i.v. via tail vein with saline (0.9%) ot
TMT hydroxide (3.0, 6.0 or 9.0 mg/kg) dissolved in saline. Doses of
TMT are expressed as the free basis and were administered in a volume
of 1.0 ml/kg b.wt. Except where brains were prepared for histology (see
below), all subjects were killed by decapitation.
Brain dissection. Immediately after decapitation whole brains were
excised from the skull and weighed. The hippocampus and frontal
cortex were then obtained by free-hand dissection and weighed. Care
was taken to remove overlying white matter from hippocampal samples.
The frontal cortex waa defined as the region between the anterior pole
of the brain and the anterior commissure. Brain regions obtained from
control subjects were slways prepared in tandem with those obtained
from subjects receiving TMT. In one experiment, 700-m thick trans-
verse sections of the dorsal hippocampus (unilateral) were prepared
with a Mcllwain Tissue Chopper (Brinkman Instruments, Westbury,
NY). Each slice waa subdivided into ams GA1, CA3 and fascia dentals
as described by Danacher et aL (1976) with the exception that the
regiona designated 11 and III wen combined. When dissected in this
manner areas CA1 and CAS would each contain a portion of CA2; areas
CAS and CA4 as well as the molecular and granular layers of the
dentate gyrus would comprise the area designated fascia dentata (for a
discussion of nomenclature, see Shepherd, 1979). Ail dissections were
performed on a refrigerated plate maintained at 0-4*C (Thermoelec-
tric* Unlimited, Wilmington, DL). Areas obtained from five slices were
pooled before acid extraction of membrane proteins (see below).
Histology. Three weeks after the administration of saline or TMT
(9.0 mg/kg), rata were anesthetized (3.5 ml/kg of Chloropent; Fort
Dodge Laboratories, Fort Dodge, IA) and then were killed by transcar-
dial perfusion with isotonic saline, followed by 10% neutral-buffered
Formalin. Brains were then removed and sliced in saggital section at
midline. Beginning at midline, sequential saggital sections 10-«i thick
were cut at 100-m intervals from the left side of each brain. Beginning
at the anterior pole of the brain, sequential coronal sections 10-n thick
were cut at 50-p intervals from the right side of each brain for a total
anterior-posterior distance of approximately 5 mm. Sections were
stained with cresyl violet.
Endogenous phoephorylation of synaptic proteins. Individual
(bilateral) samples of hippocampus and frontal cortex were homoge-
nized in 50 volumea of ice-cold 0.32 M sucrose, 5 mM Tris HC1 (pH
7.2), 5 tiM MgSO* using s glass-Teflon homogenizer (0.1&-0.22 mm
rated clearance, Arthur H. Thomas, Philadelphia, PA) and a crude
synaptic membrane fraction (Mt) waa prepared aa described (De Rob-
ertia et aL, 1967). These samples ware then pTeincubated at 0*C for l
hr for the purpose of converting endogenoua phosphoproteins entirely
to the dephosphorylated form by the action of endogenoua phospho-
protain phosphatases (Fom and Greengard, 1978; Sieghart et at, 1979;
Lohmann et al, 1978). Endogenous phosphorylation of synaptic mem-
brane proteins prepared from hippocampus and frontal cortex was
assayed aa described previously (O'Callaghan et aL, 1983). The standard
aaaay contained: 100 Mg of membrane proteins, 10 mM piperazine-
N,N'-bis(2-ethane-sulfonic acid) (pH 6.8), 10 mM MgCl-j and 5 pM
ATP containing [7-MPJ ATP (1 X 10T dpm/nmol) in the absence or
presence of 10 pM cAMP (to detect endogenous synapsin I present in
the Mi fraction). The final volume was 200 til and assays were con-
ducted st 30*C. The phosphorylation reaction was initiated by addition
of a 25-«l aliquot of the Mi fraction and was terminated IS sec later by
the addition of 100 pi of an SDS-PAGE sample buffer (O'Callaghan et
aL, 1983). All samples were heated to 90-95*C before SDS-PAGE.
Acid extraction and phosphorylation of synapsin I. Synapsin
I was extracted from total membrane proteins using a modification of
the procedure described by Goelz et aL (1981). Using microfuge tubes
in combination with a custom-fitted Teflon pestle, samples of hippo-
campua (unilateral and dissected contralateral) and frontal cortex
(unilateral) were homogenized in 10 volumes of ice-cold 0.32 M sucrose,
5 mM Tris HC1 (pH 7.2) and 5 jiM MgSO*. The homogenates were
allowed to stand on ice for 60 min for the purpose of converting
endogenous synapsin I entirely to the dephosphorylated form (see
above). Ice-cold piperazine-N,N,-bis(2-ethanasulfonic acid), pH 7.4,
containing 5 mM 2n (OAc)i waa then added to each homogenate at a
volume equal to 10 volumes of the original sample. Following this
addition, the aamples were centrifuged at 3000 x g for 10 min and the
supernatanta were discarded. In the original studies by Greengard and
co-workers (Forn and Greengard, 1978; Sieghart et aL, 1979) Zn (OAc)j
was included in the initial homogenizing buffer to inhibit phosphopro-
tain phosphatases and protein kinases. In the present study, however,
we employed Zn (OAc)j to aggregate membranes (Forn and Greengard,
1978) in order to effect a more uniform recovery of the pelleted samples.
After centrifugation, each pellet waa re homogenized in 11 mM ice-cold
citric acid (600 pi for unilateral samples of hippocampus and frontal
cortex and 200 i>l for each region dissected from slices of hippocampus)
and centrifuged at 8000 x g for 6 min. Aiiquota of the synapsin I-
containing supematants were removed and neutralized with 0.2 M
Na,HP04. The relative amounts of synapsin I in the extracta obtained
from control or TMT-treated subjects was assayed by back phosphor-
ylation (Forn and Greengard, 1978) in the presence of (t-mP]ATP and
added protein kinase. The standard assay contained 20 vl of neutralized
extract, 50 mM piperaxiae-N,N'bia(2-ethaneeuifonic acid), pH 7.4,10
mM MgClf, 0.4 mM ethylene glycol bisd-aminoethyl ether)-N,N'-
-------
738 O'Callagtan and Miller
Vol. 231
tetraacetic acid, 1.0 oM dithiothreitol, 100 nM catalytic nibunit and 5
mM ATP (I x 107 dpm/nmol) in a final volume of 200 pi The
phosphorylation reaction waa conducted at 30'C for 30 min and then
terminated with an SDS-PAGE aampla buffer (O'Callaghan et aL,
1983). All samples were heated to 90-96'C before SDS-PAGE.
SDS-PAGE, autoradiography and microdensitometry. The
endogenoualy phosphorylated synaptic proteins and the exogenously
phosphorylated acid extracts were subjected to SDS-PAGE as described
previously (O'Callaghan et aL, 1983). The acrylamide concentration
was 6 and 10% in the stacking and resolving gels, respectively. After
electrophoresis, the gels were stained for protein with Coomaasie bril-
liant blue R-250 and destained by diffusion in a solution of 30%
methanol and 10% acetic acid. Gels were dried between sheets of
dialysis membrane using heat (80*C) and reduced pressure. Auto radi-
ographs of the phosphorylated protein banda were obtained using
Kodak RP X-ray film in combination with intensifying screens (Du-
pont Cronex Lightening-Plus). Molecular weight standards (U.S. Bio-
chemical Corporation, Cleveland. OH) were used for estimations of the
relative molecular mass of resolved proteins. Autoradiographs of en-
dogenously phosphorylated proteina were scanned with a microdensi-
tometer (LKB Ultrascan Laser Densitometer, LKB Instruments,
Gaithersburg, MD). Proteins below Mr • 30 K were not scanned The
background darkness of the autoradiographs waa taken aa the base line
for densitometry determinations. Microdensitometry values obtained
were linear with respect to the full range of autoradiographic exposures.
Comparisons between control and experimental groups were only made
between samples that had been subjected to electrophoresis on the
same gel. Autoradiographs of the extracted exogenously phosphorylated
proteins were used to localize the M, « 80 to 86 K synapsin I doublet.
These bands were then cut from the dried gels and the absolute amount
of«P incorporation was determined in duplicates by liquid scintillation
spectrometry. The amount of phosphorylated synapsin I in extracts of
control subjects was linear with respect to the total amount of protein
subjected to extraction (data not shown).
Redioimnraaoaaaay of synapsin I and protein 111. Three weeks
after the administration of saline or TMT (9.0 mg/kg), rata were killed,
the hippocampus and frontal cortex (bilateral) were dissected and each
tissue sample was immediately homogenised in 10 volumes of 1% SDS
heated to 90*C. Synapsin I and a similar neuron-specific phosphopro-
tein, protein III, were measured by detergent-baaed radioimmunoassay
(Goelz et aL, 1981). All samples were analyzed in quadruplicate and
assays wen performed in 96-well microtiter plates (Becton Dickinson
Labware, Oxnard, CA) instead of microfuge tubes as described previ-
ously (Goelz et aL, 1981). Dilutions of a single control sample were used
as a standard for quantification of synapsin I and protein III in samples
obtained from saline- and TMT-treated subjects. By comparing the
amount of [lMl]synapstn! and [>M!]protein 111 immunoprecipitated in
the tissue samples of interest to that immunoprecipitated from the
sample used as a standard, the specific activity of synapsin ! and
protein III in a sample relative to that in the standard was obtained.
This expression of immunoreactivity (in arbitrary units) is hereafter
referred to aa relative specific activity. The relative specific activity of
synapain I or protein III for a given sample was multiplied by a factor
in order to set the mean relative specific activity of each control group
equal to 1.0. The data are presented on both a total and concentration
basis, (¦«., relative specific activity per hippocampus or frontal cortex
and relative specific activity per milligram of hippocampus or frontal
cortex protein, respectively. Separate standard curves were generated
for each microtiter plate. The length of time for incubation step iii (see
Goelz et aL, 1981) was 4 hr for synapsin I assays and 12 hr for protein
III assays.
Protein aaeaya. Protein waa determined according to the method
of Bradford (1976) with the exception of the radioimmunoassays, where
the method of Lowry et aL (1951) waa employed. Bovine gamma
globulin was used aa a standard in the former, whereas bovine serum
albumin was used in the latter.
Statistical analyse*. Programs on the Statistical Analysis System
(SAS, 1982) were used to analyze the data. Data sets in which multiple
measures were collected from the same animal were analyzed by mul-
tivariate analysis of variance procedures (Morrison, 1967). When treat-
ment effects were found for the multivariate tests individual variables
were analyzed by analysis of variance or Student's t test for group
observations if the comparisons involved only two groups. Significant
analyaea of variance were followed by appropriate group comparisons
using Duncan's multiple range teat In some instances relationships in
the data were explored by trend analysis (Winer, 1971). Details of the
statistical evaluation for a particular measure appear in the figure or
table legend.
Results
IndicM of body and brain weight. Acute administration
of TMT to the adult rat resulted in a dose-dependent decrease
in a number of brain weight parameters without affecting body
weight (table 1). Three weeks after administration of TMT (9.0
mg/kg), hippocaxnpal wet weight and total protein were reduced
significantly. Although whole brain weights were reduced, this
effect cannot be attributed solely to the decrease in hippocam-
pal wet weight because the values for brain weights minus
hippocampus wen also depressed significantly. None of these
effects wen transient; 98 days after exposure to TMT (9.0 mg/
kg), the wet weights of hippocampus, whole brain and whole
brain minus hippocampus wen reduced by 30, 8 and 6%,
respectively (data not shown). A direct relationship between
body weight at dosing and TMT-induced lethality has been
reported (Dyer et aL, 1982). In agreement with these findings,
we observed that the administration of 9.0 mg/kg of TMT did
not result in lethality if body weights at dosing wen not greater
than approximately 225 g.
Histology. The effects of TMT (9.0 mg/kg at 3 weeks) on
gross morphology of the hippocampus and frontal cortex an
presented in figure 1. In general, our results an consistent with
the effects of TMT administered per os (Dyer eta/., 1982). The
TABLE 1
effects of TMT on body and brain weight Indices
Rets were kited three weeks after treatment eeoh value i sprsienis the mean ± &.E-M.
N
Oose
CfTMT
MyWt«
Oosng
Body Win
OssD
A(i Body Wl
Brain MM,
Bran wa, ma
Hppocr**
Hppocsnipui Wis.
Hippocampus
Prow
«¥/*»
0
5
0.0
21413
328 ±6
114 ±6*
1.93 ±0.03'
±0,03-
0.122 ±0.004*
5.52 ±0.25*
9
3.0
222*3
381 ±11
126 ±5
1.96 ±0.04
1.84 ±0.03
0.123 ±0.006
8.40 ± 0.17
S
6.0
186 ±18
308 ±12
119 ±14
1.88 ±0.03
1.77 ±0.03
0.113 ±0.003
8.38 ± 0.26
S
9.0
193 ±11
306 ±9
112 ±11
1.78 ±0.08*
1.89 ±0.03*
0.092 ± 0.002*
3.96 ± 0.03*
*f(3,l5) and P values were 1.3$, P > 1.000; 8.19, P < .0117; 4.28, P < 0226; 1S.76. P < .0001; 9.33, P < .001 far body wt, brain wis., brain wis. minus
hippocampus, hippocampus wts. snd ttppocsmpus protein vsMs, rsspectvsty,
• Sgnwcandy dWareni >wi 0.0.3.0 and 8.0 mg/fcfl ot TMT-expoaed grcupa. P < .06.
-------
1984
gross appearance of the hippocampus was characterized by a
loss of pyramidal cell neurons, most notably in CA3-CA4 re-
gions, with sparing of the dentate granule cells. Pyramidal cell
loss, manifested in diminished density and length of the cell
line, was observed in each of the 30 sections prepared from the
dorsal hippocampus of each of four TMT-treated rats. Similar
changes were not observed in sections obtained from an equal
number of control subjects. Two other morphological charac-
teristics distinguished hippocampal sections from TMT-treated
subjects: 1) the overall size of the hippocampal formation was
reduced, a finding consistent with the effects of TMT on wet
weight of this structure and 2) the stratum radiatum and
stratum oriens layers were populated with cells of unknown
origin, suggesting the possibility of glial proliferation (e.g., see
Nadler et al., 1978). In contrast to these observations, the gross
morphology of frontal cortex was qualitatively similar for
TMT- and saline-treated animals (fig. 1).
Endogenous phosphorylation of synaptic proteins. In
initial experiments, the staining pattern and phosphorylation
profile of specific synaptic membrane proteins were employed
as indices of TMT-induced neurotoxicity. Representative stain-
ing patterns and corresponding autoradiographs of synaptic
proteins from hippocampus and frontal cortex are shown in
figure 2. The pattern of protein staining in samples of hippo-
campus and frontal cortex did not differ between control and
TMT-treated groups. Autoradiographs obtained from the dried
gels revealed the substrates of protein kinases present in syn-
aptic membrane fractions. As reported previously (Ueda et aL,
Brain Phosphoproteins and TMT 739
1977), the addition of cAMP to the standard phosphorylation
assay stimulated the phosphorylation of two bands (M, = 80
and 85 K) that correspond to the neuron-specific phosphopro-
tein doublet, synapsin I. In membranes prepared from hippo-
campus both the basal and cAMP-dependent phosphorylation
of synapsin I were reduced as a consequence of the administra-
tion of TMT (9.0 mg/kg, 3 weeks). Treatment with TMT also
caused a decrease in the endogenous phosphorylation of other
synaptic proteins from hippocampus, both in the presence and
absence of cAMP. A small but consistent reduction in cAMP-
dependent and independent phosphorylation of proteins from
frontal cortex was also observed 3 weeks after the administra-
tion of TMT. The effects of TMT on endogenous phosphory-
lation of synaptic membrane proteins were not the result of a
direct inhibition of endogenous kinases because addition of
TMT (1.0-100 mM) to the assay did not affect phosphorylation
(data not shown). Representative time course data for the
effects of TMT (9.0 mg/kg) on endogenous phosphorylation
are shown in figure 3. These data are presented as microden-
sitometric scans of autoradiographs obtained from individual
subjects. The maximal decrease in phosphorylation of all hip-
pocampal proteins occurred 3 weeks after the administration
of TMT. At this time, microdensitometry values for peaks
corresponding to synapsin I were reduced in samples obtained
from TMT-treated subjects to 35% of the values obtained from
saline-treated subjects (data not shown). Endogenous phos-
phorylation was still depressed 98 days after treatment, sug-
gesting that the effects of TMT on hippocampal phosphopro-
Rg. 1. Morphology of dorsal hippocampus (A,B) and frontal cortex (C,D) three weeks after the acute administration of saline (A,C) or TMT (9.0 mg/
kg). (B,D). The magnification is the same for al photomicrographs.
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740
O'Callagtian and Miller
Vol. 231
HIPPOCAMPUS
protein
autorodiography itoining
FRONTAL CORTEX
150
IOO
85
II
- 2
50
40
li
• •
I5
CAMP -
r r
h i
*
TMT
F
i i
is
to to
li
autoradiography
protein
staining
E
6
I
It if e e
*
¦
r-
i
C TMT
~ ~
C TMT C TMT
Fig. 2. Effects of TMT on
staining and endogenous
phosphorylation of synaptic
membrane proteins. Crude
synaptic membrane fractions
were prepared from rats
treated with 0.9% saiine (C) or
(TMT) (9.0 mg/kg) 3 weeks
earlier. Proteins were phos-
phoryiated in the absence (-)
or presence (+) of 10 mM
CAMP.
teins were permanent. The effects of TMT on synaptic mem-
brane phosphoproteins in the hippocampus could not, in gen-
eral, be extended to the corresponding phosphoproteins in the
frontal cortex.
Extraction and phosphorylation of synapsin I. The
persistent reduction in phosphorylation of hippocampal syn-
aptic membrane proteins, especially synapsin I, was suggestive
of a TMT-induced loss of neuron-specific phosphoproteins. To
pursue this lead we employed the back-phosphorylation method
to quantify synapsin I in control and TMT-treated subjects.
Representative autoradiographs of extracts prepared from hip-
pocampus and frontal cortex are shown in figure 4. Dose-
response curves for the effects of TMT on synapsin I in
hippocampus and frontal cortex are presented in figure 5. Back-
phosphorylation of extracts of hippocampus and frontal cortex
showed that TMT caused a decrease in synapsin I in hippocam-
pus but not in frontal cortex (fig. 4). Other hippocampal phos-
phoproteins, including those with electrophoretic mobilities
corresponding to the neuron-specific phosphoproteins collec-
tively referred to as protein III (Mr ™ 74 and 55 K) (Browning
et aL, 1982), also appeared to be affected by TMT (fig. 4). The
effects of TMT on synapsin I concentration were dose-related
and resulted in a loss of as much as 50% of this protein in
hippocampus without significantly altering its concentration in
frontal cortex (fig. 5). Microdissection of slices of dorsal hip-
pocampus from saline-treated controls did not reveal differ-
ences in the concentration of synapsin I among the three
regions examined (table 2). TMT caused a significant and
nearly comparable decrease in the concentration of synapsin I
in all three areas of the hippocampus (table 2).
Radioimmunoassay of synapsin I and protein III. In
addition to the back-phosphorylation technique, a detergent-
based radioimmunoassay was used to quantify the effects of
TMT on synapsin I (table 3). Because the data obtained from
the phosphorylated extracts (fig. 4) indicated that protein III
as well as synapsin I might be affected by TMT. protein III
was also measured by radioimmunoassay (table 3). Administra-
tion of TMT (9.0 mg/kg at 3 weeks) resulted in a 61 and 59%
decrease in total synapsin I and protein III, respectively, in the
hippocampus. This reduction in total synapsin I and protein
III did not simply parallel the loss in hippocampal wet weight
and total protein that occurred at the high dose of TMT (see
table 1) because the values for the concentration of these
phosphoproteins in the hippocampus were also reduced signifi-
cantly (48 and 45%, for synapsin I and protein III, respectively);
findings suggestive of a loss of neurons in the hippocampal
formation. Radioimmunoassay values for concentration of hip-
pocampal synapsin I were in agreement with those obtained by
back-phosphorylation (see table 2; fig. 5). Radioimmunoassay
values (total and concentration) for synapsin I and protein III
in frontal cortex were not affected as a consequence of the
administration of TMT.
Discussion
The neurotoxic effects of acute administration of TMT to
the adult rat are characterized morphologically by a loss of
pyramidal cells in the hippocampal formation (Brown et al.,
1979; Bouldin et aL, 1981; Dyer et aL, 1982; fig. 1, this paper).
Our results demonstrate that these neuropathological effects of
TMT are accompanied by dose- and time-dependent decreases
in neuronal phosphoproteins, including the 9ynapse-specific
-------
1984
Brain Phosphoproteina and TMT
741
HIPPOCAMPUS
FRONTAL CORTEX
a 4
E
O
9=
X
UJ
S U
S 21
v>
i
32
9S
AAA
••••-'l*'.'A—
HIPPOCAMPUS
FRONTAL
CORTEX
fO
1
O
O
UJ
$
a:
<
3
U
UJ
o
5
125
85
60
25
cr
125 85 50 30 125 85 50 30
MOLECULAR WEIGHT * 10*®
Fig. 3. Time course of the affects of TMT on endogenous phosphorylation
of synaptic protains. Tha pattern of synaptic membrane phosphorylation
from control (0.9% salina) ( ) and TMT (9.0 mg/kg)-treatad ( )
rats is raprasantad by dansitomstrtc scans of tha autoradiographa. AM
scans wara prepared from samples phosphorylatad in tha prasanca of
10 (iM cAMP. For each time-point and brain region, scans wara prepared
from each of at least four control and four TMT-treated subjects. A
representative scan was than chosen from each group. Relative differ-
ences between scans obtained from salina and TMT-treated subjects
are valid across days or brain regions. Comparisons of scana across
days and brain regions are not valid with respect to absolute values
(peak heights or areas).
phosphoproteins, synapain I and protain III. Phosphoproteins
in tha frontal cortex wara largely unaffected, findings that are
also consistent with tha cytopathologic affects of TMT. Thus,
by employing a known neurotozicant to effect a chemically-
induced denervation, wa have shown that neuron-specific phos-
phoprotaina can serve as biochemical indicators of damage to
the neuronal component of tha mature CNS. Whereas it ia
tempting to speculate that the decrease in synaptic phospho-
protaina reflects a loss in synapses accompanying neuronal
damage, a decrease in either the number of vesicles par synapse,
or in the amount of protain par vasida could also account for
our findings (see Goelz et aL, 1981).
Whera toxicant or lesion-induced cell loss ia examined in
brain regions composed of few call types arranged in a uniform
pattern (*.{., hippocampua, cerebellum and cerebral cortex), we
reasoned that microdissection of tissue slices combined with
TMT
TMT
Pig. 4. Effects of TMT on the phosphorylation of extracts of hippocampus
and frontal cortex. After preincubation to convert endogenous phospho-
proteins to the dephospho form, total particulate fractions were extracted
and the proteins in each extract ware hosphorytated by the addition of
protein kinase and (7-"P]ATP. Rats were killed three weeks after the
administration of 0.9% saline (C) or TMT (9.0 mg/kg).
TABLE 2
Effects of TMT on synapain I in regions of dorsal hippocampua
Assays of synapain I ware based on phosphorylation of extracts of total membrane
protains. Incorporation of "P into synapain I was determined by santiNation
spectrometry of slioas cut from the dried gels. Rats were kWed 3 weeks attar me
edmWatration of saline (0.9%) or TMT (9.0 mg/kg).
^ taorpontm mo Syrepsn I
at
CAS
^Hoedimsta
pmof/fflgpramn
Control
19.0 * 2.0*
21.9 ±2.1
18.1 ± 0.9
TMT
8.8*1.3t
9.8±1.7t
10.1 ±1.4t
' Each value represents the mean * S.E.M. for Owe independent observations,
t Significantly different from oorrtroi, p < .004.
assays of nervous system-specific proteins would aid in defining
the biochemical basis of neurotoxicity. Therefore, we subdi-
vided slices of hippocampus into areas roughly approximating
the major internal structures to determine the intrahippocam-
pal distribution of synapain I in both control and treated
animals. Our results indicated that synapsin I concentration in
slices prepared from saline-treated subjects did not vary sig-
nificantly with respect to region. These results are compatible
-------
742
O'Callaghan and Millar
Vol. 231
il 20
HIPPOCAMPUS
FPONTAL CORTEX
DOSE OF TRIMETHYITIN (mg/kg)
Fig. 5. Effects of TMT on synapsin i in hippocampus and frontal oortax.
Assays of synapsin I ware based on phosphorylation of extracts of total
membrane proteins obtained three weeks after treatment. Each value
represents the mean ± S.E.M. for five independent observations. Incor-
poration of *P into synapsin I was determined by scintillation spectrom-
etry of sAcas cut from dried gels. Separate analyses of variance showed
an effect of treatment on incorporation of *P in extract of hippocampus
[£(3,15) - 23.14, P < .0001) but not frontal cortex [£(3,15) - 0.24]. In
addition, a significant linear trend was found for hippocampus [£(1,15) ¦
47.89, P < .0001]. The extract from the hippocampus of me 9.0 mg/kg
of group treated with TMT incorporated significantly less *P than at
other groups (asterisk denotes P < .01).
with those of immunohistofluorescence studies showing that
synapsin I is distributed in a relatively uniform fashion
throughout the stratum radiatum and stratum oriens layers of
CA1 and CA3 (De Camilli et aL, 1983a). Our histological
findings indicated that TMT affected pyramidal cells of CA3
to CA4 to the greatest extent; however, cell loss waa observed
throughout the length of the pyramidal cell line. Because py-
ramidal cells in regions CA3 to CA4 interconnect all areas of
the hippocampal formation (Cotman et aL, 1981; Nadler et aL,
1980a), their destruction by the neuron-specific toxicant, kainic
acid, denervates stratum radiatum and stratum oriens of CA1
and the inner molecular layer of the dentate gyrus (Nadler et
aL, 1980a, b). Thus, it waa not surprising that even mors
widespread destruction of pyramidal cell* by TMT resulted in
a reduction in synapsin I concentration that was large in
magnitude (50% of control) and that waa not confined to a
•pacific region of tha hippocampal formation.
Endogenous phosphorylation of synaptic membrane proteins,
back-phosphorylation of extracts of total membrane proteins
and radioimmunoassays wen employed to evaluate the effects
of TMT on neuronal phosphoproteins. All three procedures
gave similar results; however, only the latter two provided
quantitative data with respect to the amount of specific phos-
phoproteins (synapsin I and protein III). This is due to the fact
that changes in endogenous phosphorylation may reflect alter-
ations not only in the amount of specific phosphoproteins, but
also in the amount or activity of protein kinases or other factors
that affect the net incorporation of phosphate into protein (for
a discussion, see Sieghart et aL, 1978). Although inconsistencies
between the amount and the state of phosphorylation of a given
protein were observed in a few instances, it is likely that the
effects of TMT on endogenous phosphorylation were largely
an expression of changes in the concentration of neuron-spe-
cific phoaphoproteins. This contention is based on the following
lines of evidence: 1) because TMT is known to destroy neurons
in limbic structures, its effects on endogenous phosphorylation
as well aa on values for synapsin I and protein III are consistent
with this profile of neurotoxicity; 2) the effects of TMT were
not the result of a direct inhibition of the phosphorylation
reaction; 3) the time course for the neuropathological effects of
TMT coincides with effects on endogenous phosphorylation
(Brown et aL, 1979; Chang and Dyer, 1983); and 4) the admin-
istration of the neuron-specific toxicant, kainic acid, like TMT,
results in a decrease in the endogenous phosphorylation of
moat aynaptic membrane proteins (Sieghart et aL, 1978). Thus,
the TMT-induced decrease in the phosphorylation of several
hippocampal proteins in addition to synapsin I (see fig. 2) can
be taken as an indication that at least some of these proteins
are neuron-specific. Indeed, several neuron-specific phospho-
proteins in addition to synapsin I and protein III have been
described (Nestler and Greengard, 1983, 1984) and identifica-
tion of those affected by TMT would seem warranted. If any
of these additional proteins prove to be affected by other
neuron-specific toxicants, they may be suitable candidates as
biochemical indicators of neurotoxicity (for proposed criteria,
see O'Callaghan and Miller, 1983).
In agreement with the findings of previous investigators
(Bouldin et aL. 1981; Chang and Dyer, 1983), our histological
examination of hippocampal samples from TMT-treated sub-
jects revealed tha presence of cells suggestive of an astrocytic
response to neuronal damage. Toxicant- or trauma-induced
injury to the CNS often elicits a response characterized by
astrocyte proliferation at the site of injury (Nadler et al., 1978;
Finger ami Stein, 1982; Billingsley and Mandel, 1982). Glial
fibrillary acidic protein, a CNS protein specific to fibrous
astrocytes (Eng and DeArznond, 1983), has been shown to
reflect astrocyte proliferation in response to injury (Latov et
aL, 1979) and may therefore be an excellent marker for assess-
ing toxicant-induced damage to the CNS (see O'Callaghan and
TABLE3
Radioimmunoassay of synapsin I and protein HI after aetil* administration of TMT to 9m rat
Ratswsre kMed three weeks sfter the admWwsMon of sstne (0.9%) or TMT (9.0 mg/kg). M - 6 tor determinations of synapsin i in frontal cortex; N - 7 tor a* other
ftMMuvii,
rfmw nravonv
Tot* Cm ToM Cane.
*sWBa t>w>sfti mm* Syrepawt Pnswi tmmi awm
Control 1.00 ± 0.08* 1.00 ±0.05 1.00*0.08 1.00 ±0.03 1.00 ±0.09 1.00 ±0.04 1.00 ±0.10 1.00 ±0.03
TMT 0.39 ± 0.Q3'" Q.41 ± 0.04"' 0.82 ± 0.03'" 0,58 ± 0.04— 0.98 ± 0.05 0.99 ± 0.10 1.02 ± 0.08 1.03 ±0.09
. * fee* rapmants the mean rsnov spease aettvtty * 3.B.M. enpraaasd on * total (per structural or oonuantraUun (psr mWgrani al protwn) Basis; saa 'Materia*
•na Methods lor addWcnai define.
—SignMeanay different from comm. P < .0001.
-------
1984
Miller, 1983). Indeed, we have found recently that glial fibrillary
acidic protein increases dramatically after the administration
of TMT to adult rats (Brock and O'Callaghan, 1984). Because
the neuropathological sequela* of exposure to TMT may en-
compass several CNS cell types, alterations in additional ner-
vous system-specific proteins, both neuronal and glial in origin,
may result from the administration of this toxic organometal.
We are currently exploring this possibility. Whether the effects
of TMT on nervous system-specific proteins can be generalized
to broad categories of neurotoxicants must await the outcome
of future investigations.
AcknowladcBMata
The authors wish to thank Or. Hany A. Makkawy and Ml. Christina Booth
for excellent technical aasiaunce. Jackie D. Farmer for doaing tha rata and On.
Paul Greangard, Michael D. Browning and Linda J. BurdatU for uaaftil discus-
•ioni.
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-------
^V22-:)SHS/83/'>242-iH66$00.00/0
The Journal op Pharmacology and Experimental Therapeutics
Copyright $ 1983 by The American Society for Pharmacology and Experimental Therapeutics
Vol. 224, No. 2
Printed tn U.S.A.
Acute Postnatal Exposure to Triethyltin in the Rat: Effects on
Specific Protein Composition of Subcellular Fractions from
Developing and Adult Brain1
JAMES P. O'CALLAGHAN, DIANE B. MILLER and LAWRENCE W. REITER
Neurotoxicology Division, Health Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina
Accepted for publication November 8, 1982
ABSTRACT
The morphological maturation of the central nervous system is
characterized by ontogenetic changes in proteins associated
with specific developmental processes. In this investigation we
examined the effects of acute postnatal administration of tri-
ethyltin (TED on the ontogeny of proteins associated with
myelinogenesis, synaptogenesis and synaptic junction forma-
tion. Myelin, synaptic plasma membrane and synaptosomal
cytosolic fractions were prepared from 13-, 22- and 66-day-
old Long-Evans rats that had received either saline or TET (3.0
or 6.0 mg/kg i.p.) at 5 days of age. The specific protein
composition of each fraction was evaluated by resolution of the
fraction proteins on sodium dodecyl suifate-polyacrylamide
slab gels. The presence of marker phosphoproteins associated
with each fraction was determined by three criteria: electro-
phoretic mobility, developmental profile and autoradiography
of incorporated phosphate. The specific protein and phospho-
protein content of each fraction was quantified by microdensi-
tometry. The postnatal day 5 administration of TET produced
a dose-related decrease in the concentration of myelin basic
protein, a myelin-specific marker. This effect was large in
magnitude (40-60% of control) and was observed in myelin
fractions prepared from 13-, 22- and 66-day-old subjects. No
changes in the protein or phosphoprotein content of other
subcellular fractions were observed after the day 5 administra-
tion of TET. These data indicate that acute postnatal adminis-
tration of TET may interfere selectively with myelinogenesis.
Recently, both behavioral and neurochemical effects of TET
have been assessed in the developing rat. A single exposure to
TGT on postnatal day 5 results in disruption of developmental
behaviors and causes permanent changes in brain weights,
behavior and sensorimotor functions (Harry and Tilson, 1981;
Reiter et al., 1981). Few neurochemical correlates of this toxi-
cant-induced change in brain and behavior have been examined.
Before the onset of myelination at postnatal day 10 (Norton,
1981), maturation of the rat brain is characterized by rapid
oligodendroglia proliferation (Altman, 1970). This process of
cellular proliferation has been shown to be particularly vulner-
able to toxic insult (Suzuki, 1980). Although cell division during
the early postnatal period is primarily glial in nature, there is
a considerable degree of postnatal neurogenesis in certain brain
areas, e.g., hippocampus and cerebellum (Altman, 1970). Fur-
thermore, the formation of synapses in various areas of the rat
brain markedly increases between 1 and 4 weeks postnatally
Received for publication June 17, 1882.
1 Thit paper haa been reviewed by the Health Effect* Reaearch Laboratory,
U S. Environmental Protection Agency, and approved for publication. Mention
of trade name* or commercial producti doe* not conatitute endonement or
recommendation for uae. A preliminary report of thia investigation appeared in
The Toxicologin 2: 58,1982.
(Aghajanian and Bloom, 1967,- Armstrong-James and Johnson,
1970; Crain et al., 1973). Thus, the administration of TET on
postnatal day 5 may result in changes in neuronal as well as
myelin elements of the CNS. In order to delineate which cellular
elements are affected by acute postnatal exposure to TET, we
examined the protein composition of three subcellular fractions:
M, SPM and SC.
In the nervous system, specific functions are thought to be
associated with various cellular and subcellular structures
(Bock, 1978). These functions presumably are mediated to a
large extent by soluble or membrane-bound proteins that are
unique or characteristic to the nervous system. Indeed, a num-
ber of nervous system-specific proteins have been identified in
the mammalian CNS (for reviews see Zomzely-Neurath and
Keller, 1977; Bock, 1978). Thus, nervous system-specific pro-
teins may serve as specific markers of the unique structure and
function of the CNS and as such would provide useful indicators
of selective neurotoxicity. Therefore, in the present investiga-
tion we examined not only the total protein composition of
specific subcellular fractions, but extended our observations to
include measurements of M basic protein, protein 1 and neu-
rotubulin. These proteins serve as biochemical markers of mye-
linogenesis, synaptogenesis and synaptic junction formation,
ABBREVIATIONS: TET, triethyltin; CNS, central nervous system; M, myelin; SPM, synaptic plasma membrane; SC. synaptosomal cytosol; MBP,
myelin basic protein; SOS-PAGE, sodium dodecyl aulfate-polyacrylamlde gels; M„ molecular weight.
4M
-------
1983
Postnatal TET and Brain Proteins
467
respectively. Because all three marker proteins are phospho-
rylated by endogenous protein kinases (Steck and Appel, 1974;
Lohmann et al., 1978; Burke and DeLorenzo, 1981), we were
able to detect and quantify each in SDS-PAGG using protein
staining combined with autoradiography of the 32P-labeled pro-
tein bands.
Methods
Subjects. Gravid Long-Evans rats (Blue Spruce Farms, Inc., Alta-
mont, NY) were obtained on day 2 of gestation and housed individually
in plastic tub cages (45 X 25 x 20 cm) with direct bedding. Pups born
between 5:00 p.m. and 5:00 a.m. were considered to be bom on the same
day (day 0). On day 1, pups were reassigned to litters such that each
dam received four male and four female pups and no more than one
male and female of her own offspring. At 21 days of age, littermates
were weaned and separated by sex and housed four per cage. Animals
were maintained in a temperature (22°C ± 2)- and humidity (60% ±
Uncontrolled colony room on a 12:12 hr light/dark cycle beginning at
6:00 a.m.
Dosing. Pups were injected (i.p.) on postnatal day 5 with 0.0, 3.0 or
6.0 mg/kg of TET bromide with saline (0.9%) as the vehicle control (10
fd/g b.wt.). All pups in a litter received the same dose and 20 separate
litters were used in this study. Subjects were randomly selected from
separate litters and sacrificed at 13, 22 or 66 days of age. At each time
point no more than one female and one male were selected from each
litter.
Preparation of subcellular fractions. After sacrifice by decapi-
tation, brains were removed within 30 sec and immersed in ice-cold 0.32
M sucrose. Brains obtained from control subjects were always prepared
in tandem with those obtained from subjects receiving TET. SC, M
and SPM subtractions were prepared as described below. Whole brains
were homogenized in 10 volumes of 0.32 M sucrose and a crude
mitochondrial fraction (Pi) was prepared by differential centrifugation.
The P2 fraction from each treatment group was then subjected to
hypoosmotic shock in 500 jil of 5.0 mM Tris HC1 (pH 8.0), essentially
as described (Jones and Matus, 1974, without Bonification, except where
noted). The resulting lysate was centrifuged at 140,000 X g for 30 min
and the supernatant fraction obtained was used as a source of SC (see
O'Callaghan et al, 1980, 1982). The membrane fractions recovered
during the preparation of the SC fraction were subjected to isopycnic
sedimentation on discontinuous sucrose gradients (10, 28.5 and 34%)
(w/w) as described (Jones and Matus, 1974). The M-enriched subtrac-
tion was harvested from the 10:28.5% interface and an SPM-enriched
subtraction was harvested from the 28.5:34% interface. The protein
content of each subcellular fraction was assayed according to the
method of Bradford (1976) and expressed as a function of brain weight.
Bovine plasma yglobulin was used as a standard. In a given experiment,
all protein values for different subcellular fractions (regardless of treat-
ment group) were equalized before electrophoresis.
Resolution of subcellular fraction proteins by SDS-PAGE. The
SC, M and SPM proteins were resolved on SDS-polyaaylamide vertical
slab gels (10 x 16 x 0.16 cm) (Hoefer Scientific Instruments, Inc., San
Francisco, CA). Routinely, a 50-|d aliquot containing 17 pg of fraction
protein was applied to each of the 20 wells in a slab. Except where
noted, the acrylamide concentration was 6 and 10% in the stacking and
resolving gels, respectively (36.5:1, acrylamide:bisacrylamide). The
stacking, resolving and running gel buffers were those described by
Laemmli (1970). The samples were subjected to electrophoresis under
conditions of constant power (10 w/gel) at 18-20*C until the tracking
dye reached the bottom of the gel (3.5-4.0 hr). After electrophoresis,
the gels were fixed and stained for protein overnight with 0.1% Coo-
massie brilliant blue R-250 in 50% methanol and 10% acetic acid. Oris
wen destained by diffusion in 30% methanol and 10% acetic add and
dried under heat and partial vacuum. Autoradlographa of the MP-
iabeled proteins (see below) were obtained by placing the dried gels in
close contact with Kodak RP X-ray film for a period of up to 24 hr.
Short exposure times were achieved by using intensifying screens
(Dupont Cronex Lightning-Plus, CGR Corp., Raleigh, NC). The appar-
ent Mis of the resolved proteins were determined from M, standards
that had been subjected to electrophoresis under conditions identical
to the tissue samples.
Detection of marker proteins. Specific marker proteins were
located in the dried gels and corresponding autoradiographs on the
bssis of electrophoretic mobility, pattern of incorporated phosphate
and developmental profile. MBP, a metabolically stable protein (Sabri
et al., 1974; Fischer and Morell, 1974; Shapira et al., 1981) unique to M
and glial cell lines that produce M (Shapira et al., 1978), was chosen as
a marker of the M fraction. The synapse-associated, neuron-specific
protein, protein 1 (for a review, see Greengard, 1979), was utilized as a
marker of the SPM fraction. Tubulin, a major cytoplasmic protein in
brain that plays a role in axoplasmic transport, axonal elongation and
synapse formation (see Shelanski and Feit, 1972). was employed as a
marker of SC. The M, [Norton, 1981 (MBP); Ueda and Greengard,
1977 (protein I); Blitz and Fine, 1974 (tubulin)], endogenous phosphor-
ylation characteristics [Steck and Appel, 1974 (MBP); Huttner and
Greengard, 1979 (protein 1); Burke and DeLorenzo, 1981 (tubulinl ] and
ontogenetic profile [Banik and Smith, 1977 (MBP); Lohmann et al..
1978 (protein 1); Schmitt et al., 1977 (tubulin)] have been determined
by SDS-PAGE for all three marker proteins. The specific marker
proteins were detected in the. stained gels and corresponding autoradi-
ographs. Both MBP and tubulin were identified in the stained gels on
the basis of electrophoretic mobility (Mr) and ontogenetic profile.
Protein I could not be readily identified in stained gels. Autoradi-
ographic detection of all three markers was achieved by phosphoryl-
ating the subcellular fractions, in vitro, before electrophoresis. The
standard phosphorylation assay contained: 100 fig of the SC. M or SPM
proteins; 50 mM piperazine-N,N'-bis(2-ethanesulfonic acid) (pH 6.5);
10 mM MgCU; and 5 nM ATP containing [y-'-P]ATP (I x 10: dpm/
nmol). The SC proteins were incubated in the presence of 50 jiM CaCL.
The SPM subfraction was incubated in the presence of 10 /iM cyclic
AMP. Assays were incubated at 30°C in a final volume of 200 pi. The
phosphorylation reaction was initiated by the addition of a 25-nl aliquot
of the SC, M or SPM subfraction (suspended in 5.0 mM Tris HC1, pH
8.0). The reaction was terminated after 1 min (SC) or 15 sec (M and
SPM) by the addition of 100 jd of an SDS-PAGE sample buffer
containing 0.125 M Tris HC1 (pH 6.8), 4% SDS, 20% glycerol and 10^
2-mercaptoethanol. All sample* were then heated in a water bath for
2 min at 90"C before electrophoresis.
Quantification of marker proteins. The amount of each marker
protein resolved by SDS-PAGE was measured by microdensitometry
of the stained gels and corresponding autoradiographs using a Quick-
Scan (Helena Laboratories, Beaumont, TX) transmission microdensi-
tometer. The amount of protein (stained bands) or phosphoprotein
(autoradiographic bands) was quantified from the densitometry scans
by determining the areas under the peaks corresponding to specific
protein or phosphoprotein bands. A linear relationship was observed
between microdensitometry values and the full range of observed
staining intensities or autoradiographic exposures, The values obtained
for the two peaka corresponding to the large MBP were combined as
were those corresponding to «• and /9-tubulin. Values for the small
basic protein were not determined. Densitometric values for protein 1
were limited to those obtained from the autoradiographs. The back-
ground color of the dried gets or the background darkness of the
corresponding autoradiographs was taken as the base-line reading for
densitometric determinations. The data are expressed as a percentage
of total protein or as a percentage of control and values obtained from
males and females were combined. Comparisons between control and
experimental groups were only made between samples that had been
subjected to electrophoresis on the same gel. Statistical comparisons
were based on Student's t test for group observations.
Materials. [y:1,P]ATP (10-40 Ci/mmol) was purchased from New
England Nuclear (Boston, MA). Piperarine-N,N'-bu(2-ethanesulfonic
acid) and Tris were obtained from Calbiochem (San Diego, CA).
Sucrose (grade II) was from Sigma Chemical Co. (St. Louis, MO). All
other materials uaed in the preparation of the polyacrylamide gels and
associated buffers were of electrophoresis grade and were purchased
-------
468
O'Callaghan et al.
Vol. 224
from Bio-Rad Laboratories (Richmond, CA). TET bromide was pur-
chased from Alfa Products (Danvers, MA).
Results
Brain weights and subcellular fraction protein yields.
Administration of TET on postnatal day 5 resulted in a signifi-
cant decrease in brain weights at 13, 22 and 66 days of age
(table 1). Total protein yields for each subcellular fraction were
also obtained at these ages (table 1). M, but not SC or SPM,
proteins were significantly reduced at 22 days of age as a
consequence of TET administration (6.0 mg/kg) on postnatal
day 5. At 13 or 66 days of age, the protein yields for all three
subcellular fractions did not differ between the control and
TET groups. The body weights of the TET-treated rats did not
differ significantly from controls at either 22 or 66 days of age;
however, at 13 days of age the body weights of rats that had
received 6.0 mg/kg of TET were reduced by 13% (P < .01) (data
not shown). Sex-related differences were not observed for any
of the measurements employed in this investigation.
Electrophoretic profiles and autoradiographs of sub-
cellular fraction proteins. The electrophoretic profiles and
corresponding autoradiographs of SC, M and SPM proteins
were obtained from control and TET-treated subjects at 13, 22
and 66 days of age. A representative series of protein staining
patterns and corresponding autoradiographs obtained from 22-
day-old animals is presented in figure 1. SDS-PAGE of SC, M
or SPM proteins resulted in the resolution of at least 50 Coo-
massie blue stained bands (Mr, 10,000-200,000) in each fraction.
With the exception of protein 1, protein bands corresponding
to the specific marker proteins associated with each subcellular
fraction could be identified in both the staining and autoradi-
ographic profiles. A band corresponding to the neuron specific
protein, protein 1, could only be detected in the SPM fraction
with the aid of autoradiography. Qualitative and quantitative
analysis of the protein composition of the SC or SPM fractions
did not reveal any differences between the control or TET
treatment groups. In contrast, a reduction in specific M fraction
proteins was observed in both the staining and autoradiographic
profiles obtained from subjects that had received 6.0 mg/kg of
TET on day 5. This effect was not limited to the M marker
protein, the phosphorylated MBP doublet (M„ 15,000-17,000),
TABLE 1
Effect of acute postnatal administration of TET on protein yields obtained from brain subcellular fractions
Each value represents the mean ± S.E.M of six independent observations.
Age
N
Dose of TET
Brain Wis.
SC
M
3PM
days
mg/kg
9
mg protein / g Oram
13
6
0.0
1.36 (±0.02)
1.33 (±0.18)
1 40 (±0.15)
2 45 (±0 22)
6
3.0
1.25 (±0.02)* •
1 36 (±0.16)
1 36 (±0 10)
2.17 (±0.20)
6
6.0
1.06 (±0.05)* '
1 78(±0.15)
1.25 (±0.10)
2 40 (±0.25)
22
6
0.0
1.59 (±0.02)
3.50 (±0.1 4)
2.21 (±0.25)
3.17 (±0.41)
6
3.0
1 49 (±0.02)' •
3.72 (±0.16)
2.18 (±0.28)
3.19 (±0.42)
6
6.0
1.33 (±0.02)'*
3.55 (±0.11)
1 65(±0.19)*
2.96 (±0.39)
66
6
0.0
1.98 (±0.04)
2.44 (±0.06)
3.36 (±0.25)
2.60 (±0.18)
6
3.0
1.90 (±0.03)
2.47 (±0.12)
3.36 (±0.23)
2.55 (±0.19)
6
6.0
1.55 (±0.02)* *
2.41 (±0.11)
2.90 (±0.18)
2.82 (±0.25)
• Significantly different from control, P < 05; * "significantly different from control, P < .01.
O
X
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—I
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U
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130
100
80
60
50
40
30
20
15
MYELIN
PS **
* n
SPM
» ft
ii
0 3 6 *0 *3 6
ni
0 3 6 0 3 6
DOSE OF TET (mg/kg)
CYTOSOL
PS All
-3 f f t
di
Hi*
•••
0 3 6 0 3 6
Fig. 1. Protein staining (PS) pattern and corresponding autoradiographs (AR) of M, SPM and synaptosomal cytosolic (cytosol) fractions from 22-
day-old male rats. Fractions were obtained from rats treated on day 5 with saline or TET. Proteins were stained with Coomassie Blue R-250.
Proteins or phosphoproteins with electrophoretic mobilities corresponding lo proteolipid protein (PLP), large MBP, protein 1 (P,} and a- and /i-
tublin (a and /3T) are indicated by arrows. See Methods ' for criteria used to identify specific proteins. *
-------
1983
Postnatal TET and Brain Proteins 469
but was also observed for a protein of apparent M, of 22,000.
The ontogenetic profile (day 13, 22 and 66) and electrophoretic
mobility of this protein (Banik and Smith, 1977; Jones and
Matus, 1975; Burgoyne et al., 1981) as well as its failure to
incorporate phosphate in vitro (Steck and Appel, 1974) identify
it as the M-associated proteolipid protein. Although MBP and
proteolipid protein were both consistently affected by postnatal
administration of TET, only MBP is significantly phosphoryl-
ated; therefore, quantitative data were obtained only for this
protein. Furthermore, as the small MBP was not highly phos-
phorylated on postnatal day 13, MBP determinations at all
time points were restricted to the large basic protein compo-
nent.
M fractions prepared as described under "Methods," without
sonification, are known to be contaminated with SPM-derived
fragments (Jones and Matus, 1974). It is apparent (fig. 1) that
several protein staining bands in the M fraction exhibit electro-
phoretic mobilities that are similar to major bands found in the
SPM fraction. Because contamination of the M fraction with
SPM-derived proteins may have contributed to the results
observed in figure 1, we subjected the homogenates from several
experiments to sonification before density gradient centrifuga-
tion. The resulting M fraction proteins were then resolved in
15% gels. Typical electrophoretic profiles of M proteins pre-
pared in this fashion (from 22-day-old subjects) are presented
in figure 2. The proteolipid protein, large basic protein doublet
and small basic protein compose the major M proteins in the
sonified preparation (fig. 2). In agreement with the results
presented in figure 1, TET produced a dose-related decrease in
the proteolipid and large MBP. Additionally, the small MBP
was also decreased by TET administration on postnatal day 5.
The densely staining protein of Mr 55,000 may be the M specific
Wolfgram protein (fig. 2). It was not affected by TET admin-
istration.
Quantification of marker proteins. The administration of
TET on postnatal day 5 did not affect the microdensitometry
values for neurotubulin or protein 1 obtained from 13-, 22- or
66-day-old subjects (data not shown). Microdensitometry val-
ues obtained from the staining profiles of MBP are shown in
figure 3. As has been reported (Jones and Matus, 1975; Bur-
goyne et al., 1981), the MBP content of M subfractions could
not be detected at 13 days of age. Therefore, quantitative data
based on protein staining was limited to values obtained from
the 22- and 66-day-old subjects. Postnatal day 5 administration
of TET resulted in a dose-related decrease in J5 at both 22 and
66 days of age. This decrease was greatest (50% of control) at
22 days but was still significantly reduced at 66 days. After
phosphorylation, in vitro, MBP was detected by autoradiogra-
phy and quantified by microdensitometry. These data are
shown in figure 4. In agreement with previous work (Steck and
Appel, 1974), we found that endogenously phosphorylated MBP
could be detected and quantified during the early postnatal
period. Postnatal TET administration caused a dose-related
decrease in the phosphorylated MBP at 13, as well as 22 and 66
days of age. These data extend our observations based on
protein staining to 13-day-old animals, a point in postnatal
development that coincides with the earliest stages of myeli-
nogenesis (Norton, 1981).
Discussion
We have demonstrated that exposure to TET on postnatal
day 5 in the rat results in a persistent decrease in total M
protein and in the M marker, MBP. The total and specific
U
LU
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50
24
18
17
14
3X3
r. s
•— PLP
> LBP
SBP
0 3 6
DOSE OF TET
(mg/kg)
Fig. 2. Protein staining pattern ot sonified M fractions obtained from
22-day-old female rats Fractions were obtained from rats treated on
day 5 with saline or TET. The acrylamide concentration was 6 and 1 5a6
in the stacking and resolving gels, respectively (36.5:1, acrylamide-
bisacrylamide). Proteins were stained with Coomassie Blue R-250.
Abbreviations are as follows: proteolipid protein. PLP; large myelin
basic protein, LBP; small myelin basic protein, SBP
protein content of other CNS subcellular fractions were not
affected, suggesting that the effects of TET were specific to the
M. Previous investigators have also found deficits in mvelina-
tion after acute (Wender et al., 1974) or chronic (Blaker et al..
1981) administration of TET to the rat during the early post-
natal period.
MBP, protein 1 and neurotubulin were chosen as specific
markers of the M, SPM and SC fractions, as their ontogeny
coincides with three critical developmental processes in the
CNS; myelinogenesis (MBP) (Banik and Smith, 1977; Jones
and Matus, 1975; Burgoyne etal., 1981), synaptogenesis (protein
1) (Lohmann et al., 1978) and the formation of synaptic junc-
tional complexes (tubulin) (Shelanski and Feit, 1972; Jones and
Matus, 1975). The use of SDS-PAGE to examine polypeptide
components of each subcellular fraction and to detect and
quantify nervous system-specific marker proteins has been used
previously [e.g., see Barbarese et al., 1978; Reigner et al.. 1981
(MBP); Lohmann et al., 1978 (protein 1); Schmitt et al., 1977
(tubulin)] and proved to be the most sensitive indicator of the
effects of acute postnatal exposure to TET. Several proteins
may comigrate in SDS gels with the proteins employed as
markers for the specific subcellular fractions. The us^e of phos-
-------
470
O'Callaghan et al.
Vol. 224
phorylated markers with known ontogenetic profiles makes the
positive identification of these proteins more likely. However,
identification of the specific marker proteins will require reso-
lution by two-dimensional gel techniques or the use of specific
radioimmunoassays (Cohen et al., 1980; Goelz et al., 1981).
Our results indicated that TET only affected the M fraction
and, within this fraction, only the M-specific proteins were
altered. These included both the proteolipid protein (see figs.
1 and 2) as well as the large and small MBPs. Quantification of
the large MBP by microdensitometry of the autoradiographs
or of the stained gels revealed a persistent 40 to 60% reduction
in this marker of myelinogenesis. The content of MBP in most
areas of the CNS of the rat reaches a peak by postnatal day 40
(Zgorzalewicz et al., 1974; Banik and Smith, 1977; Day, 1981)
and stabilizes at that level well into adulthood (Zgorzalewicz et
al., 1974; Banik and Smith, 1977). MBP that accumulates
during development remains essentially stable (Shapira et al.,
1981), a finding that supports the hypothesis that MBP may
play a role in stabilizing the compaction of M lamellae (Smith,
1977). In light of these reports, the deficits in MBP
as a
1,
o
*
i4
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GE J
a.
vi
<
CO
m 1
>
3
DAY 22
DAY 66
: X
•
t
1 1,.. . L_ _
,1
1 . .
0 3 S 0 3 6
DOSE OF TET (mg/kgl
Fig. 3. The effects of TET on the concentration of MBP. Values were
obtained from M fractions prepared from 22- and 66-day-old rats that
had received TET on day 5. Each point represents the mean + S.E.M.
of at least six independent observations. fSignificantly different from
the control, P < .05; * significantly different from control, P < .01.
consequence of TET administration on postnatal day 5 are
indicative of a permanent deficit in myelination.
We suggest that the reduction in MBP on days 13, 22 and 66
is a consequence of a TET-induced alteration of the prolifer-
ating oligodendroglia. Such an effect could be due to a cytotoxic
action of TET or the result of a depression of early M synthesis.
A general cytotoxic action of TET on day 5 is unlikely since the
SPM and SC fractions were not affected. However, a general
metabolic insult on day 5 would be expected to affect the
rapidly proliferating oligodendrogliocyte population and may
account for the results obtained in the present investigation.
That TET is a general metabolic inhibitor has been well doc-
umented by studies demonstrating its effects on mitochondrial
function, in vitro (Stockdale et al., 1970). Additionally, it is
possible that the effect of TET on day 5 is mediated through a
direct action on MBP. On postnatal day 5, MBP is present in
high concentration in M-forming oligodendroglia (Sternberger
et al., 1978) and may play an important role in the initial
development of the M sheath (Sternberger et al., 1978).
Although TET did not affect the protein yield, composition
or specific marker content of the SPM or SC fractions, an
action of TET on these non-M components of the CNS cannot
be ruled out. Whereas MBP is metabolically stable (Fisher and
Morell, 1974; Sabri et al., 1974; Shapira et al., 1981), most
proteins in the developing brain are subject to turnover (Lajtha
and Dunlop, 1981). As only steady-state levels of specific pro-
teins were examined, the possibility exists that TET may influ-
ence the dynamic state of proteins present in cell types and
subcellular organelles that are both M and non-M in origin. It
is also possible that the effects of TET may be restricted to an
action on proliferating cell populations. Because extensive post-
natal neurogenesis is largely restricted to the hippocampus and
cerebellum (Altman, 1970), an effect of TET on these structures
may have been masked by an examination of the whole brain.
We have employed endogenous phosphorylation reactions to
quantify as well as identify three marker proteins in heteroge-
neous mixtures of proteins resolved from subcellular fractions
of whole brain. Because protein 1 could not be identified in
protein staining profiles of SPM fractions, it was routinely
detected by endogenous phosphorylation (see Greengard, 1979).
Quantification of protein 1 in particulate fractions of developing
100
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o 0
2#
°Z 50
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§£
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5®
O- A
DAY 13
' N,
i
DAY 22
(
DAY 66
-- i- —
N
J- 1
3 6 3 6 3 6
DOSE OF TET (mg/kg)
Fig. 4. The effects of TET on the amount of phosphomyelin basic protein. The net incorporation of phosphate into protein was quantified from the
densitometric scans of the autoradiographs and is expressed as a percentage of control phosphorylation. Each point represents the mean +
S.E.M. of at least six independent observations.
-------
1983
Postnatal TET and Brain Proteins
471
brain was also achieved by endogenous phosphorylation, as the
amount of protein 1 assayed in this fashion is indistinguishable
from that obtained for the extracted and purified protein (Loh-
mann et al., 1978). Although MBP and tubulin were detected
and quantified by dye-binding, they were also analyzed by
protein phosphorylation. Whereas endogenous phosphorylation
assays can indicate the presence of a specific protein, the net
incorporation of phosphate into a given protein may be influ-
enced by a number of factors other than the amount of endog-
enous substrate present (see Bar et al., 1981, for a discussion).
Our data indicated that TET caused a dose-related decrease in
phosphomyelin basic protein without affecting protein 1 or
phosphoproteins that comigrate with a- and /8-tubulin. The
relative decrease in MBP assessed by endogenous phosphory-
lation was roughly equivalent to that observed by dye-binding
(see figs. 3 and 4). Thus, it appears that although several factors
may affect the net incorporation of phosphate into the marker
proteins employed in this study, these and possibly other phos-
phoproteins may be detected and measured after phosphory-
lation by endogenous protein kinases.
Several authors have suggested that the observed toxicity of
the heavy metals may be in part due to heavy metal-induced
deficiencies in essential trace elements such as copper and zinc
(Mahaffey, 1980). For example, Klauder and Petering (1977)
have noted the similarities between lead and copper deficiency-
induced anemias. Interestingly, anomalies in M formation have
been linked to deficiencies in copper during both the prenatal
and neonatal periods (Everson et al, 1968; DiPaolo et al, 1974;
Zimmerman et al., 1976). Copper deficiency is also known to
produce depigmentation as it is an essential component of
tyrosinase which participates in the conversion of tyrosine to
melanin (Malmstrom and Ryden, 1968). In the present study,
pigmented coat areas of the TET-treated rats showed a dose-
related decrease in pigmentation and these areas were "grayish"
in coloration. These effects gradually dissipated and were not
present after approximately 50 days of age. We are currently
investigating the possibility that some of the effects of TET
observed in the present study may be related to a copper
deficiency.
References
Achajanian, G. K. and Bloom, P. E.: The formation of synaptic junctions in
developing rat brain: A quantitative electron microscopic study. Brain Res. 8s
716-727, 1967.
Altman, J.: Postnatal neurogenesis and the problem of neural plasticity. In
. Developmental Neurobiology, ed. by W. A. Himwich, pp. 197-237. Charles C
Thomas, Springfield, IL, 1070,
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-------
Brain Restarch Bulletin, Vol. 22, pp. 637-642. Pergamon Press pic. 1989. Printed in the U.S.A.
0361-9230/89 $3.00 + .00
Variations in the Neurotoxic
Potency of Trimethyltin
J. P. O'CALLAGHAN,* D. M. NIEDZWIECKI* AND J. C. MEANSt
*Health Effects Research Laboratory, U.S. Environmental Protection Agency
Research Triangle Park, NC 27711 and tInstitute for Environmental Studies
Louisiana State University, Baton Rouge, LA 70803
Received 20 October 1988
O'CALLAGHAN. 3. P., D. M. NIEDZWIECKI AND J. C. MEANS. Variations in the neurotoxic potency of trimethyltin. BRAIN
RES BULL 22(4) 637-642, 1989.—The organometallic compound, trimethyltin (TMT). is used as a selective denervation tool to
validate moiphological, biochemical and functional approaches to the detection and characterization of neurotoxicity. Variations in
nervous system response TMT have been repotted and may complicate the use of this compound as a research tool. We examined the
contribution of sample-to-sample differences to variations in TMT-induced neurotoxicity. Seven samples of TMT obtained from three
commercial sources were evaluated for neurotoxic potency in the rat. Hippocampus weight, histology and assays of the astrocyte
protein, glial fibrillary acidic protein (GFAP), were used as indices of neurotoxicity. A single administration (8.0 mg/kg, IV) of
different samples of TMT resulted in markedly different degrees of neurotoxicity as assessed by hippocampus weight and GFAP
assays. Subsequent analysis of each sample for trace metal and speciated organotin content revealed that sample-to-sample differences
in neurotoxic potency could be attributed to the presence of several impurities. Indeed, in several samples, sodium was present at levels
high enough to affect neurotoxic potency simply by diluting the TMT content. A number of samples also showed contamination with
the nonneurotoxic organotin. dimethyltin. The data indicate that different sources of TMT produce quantitatively different degrees of
neurotoxicity, differences that may be attributed to sample-to-sample variations in TMT content.
Trimethyltin Astrocytes Glial fibrillary acidic protein Neurotoxicity GC/mass spectrometry
THE pathological effects of trimethyltin (TMT) on the central
nervous system are widely documented [for a review see (22)].
Destruction of neurons, especially those localized to limbic
structures, is the hallmark of damage caused by TMT [e.g.. see
(1)]. Because these effects of TMT can be obtained following a
single systemic administration, this compound is gaining wide-
spread use as a denervation tool for producing defined damage to
specific neural structures. As such, TMT has been used to validate
a diverse variety of biochemical (2, 13, 25) and behavioral (3,11,
16,26) approaches to neurotoxicity detection and characterization.
The reported potency of TMT for damaging specific neuronal
cell types (e.g., hippocampal pyramidal cells) is subject to a
number of variables. These include species (4,3), strain (3), age
(6), histological orientation (7), body weight (9) dosing vehicle
(17), the route of administration [e.g., compare (9) with (18)] and
the salt form of the compound (J. P. O'Callaghan, unpublished
observation). Other factors also may influence the apparent
Potency of TMT. We recently encountered large experiment-
to-experiment differences in the effects of a given dosage of TMT
on a number of endpoints including lethality, neuropathology,
brain weights and the concentration of neurotypic and gliotypic
Proteins (J. P. O'Callaghan, unpublished observations). In these
studies, we attempted to limit apparent sources of variability by
Using a single salt form of TMT (the hydroxide) administered to
Long-Evans rats of equal body weight. Moreover, the intravenous
route of administration was chosen to eliminate rate of compound
absorption as a contributing variable. Different samples of TMT,
however, were used in these experiments. It was possible,
therefore, that variations in the composition of TMT samples may
have been a source of variability in our results.
In order for a chemical toxicant to gain acceptance as a research
tool, a standard response to its administration should be obtained
across laboratories. Unfortunately, establishing a definitive basis
for nervous system response to TMT has been hampered by at least
two factors: 1) the lack of methodology for quantitative assessment
of TMT-induced damage to the CNS and 2) the lack of method-
ology for analyzing samples of organotins. Recent advances now
make it possible to quantify the neurotoxic effects of TMT (2) and
we report here a procedure to quantitatively analyze samples of
TMT. Using these methods, we demonstrate that quantitative
differences in die toxic effects of TMT on the CNS may be due to
sample-to-sample variations in chemical composition.
METHOD
Materials
Six different bottles of TMT hydroxide and one bottle of TMT
chloride were purchased from three commercial sources. All
samples were treated as though they were 100% pure (verbal
quotes from the manufacturers indicated purities of greater than
95%). For convenience, each sample of TMT has been assigned a
numerical designation (Table 1). Four different bottles of TMT
hydroxide were obtained from K & K Laboratories (a division of
ICN Pharmaceuticals) (Plainview, NY) on die following dates:
637
-------
638
O'CALLAGHAN, NIEDZWIECKI AND MEANS
TABLE 1
EFFECTS OF DIFFERENT SAMPLES OF TMT ON HIPPOCAMPUS WEIGHT
AND CONCENTRATION OF GFAP
Salt
GFAP
Form
Hemihippocampus
Concentration
Sample
and
Wt. (grams
(% of control
Designation
(pH)
±SEM)
±SEM)
0.9% saline
(7.00)
0.051 ±
0.002
100 ± 3
1
hydroxide
0.040 x
0.002*
373 a: 28*+
(7.00)
2
hydroxide
0.047 £
0.001*
217 X U*
(7.25)
3
hydroxide
0.043 x
0.001*
403 x 67*+
(7.50)
4
hydroxide
0.042 x
0.002*
405 x 46*+
(7.70)
5
hydroxide
0.049 *
0.002
166 ± 19*
(7.30)
6
hydroxide
0.049 ±
0.002
204 * 30*
(6.00)
7
chloride
0.044 x
0.001*
272 x 38*
(3.20)
N = 5.
•Significantly different from saline, p<0.05.
tSignificantly different from saline and samples 2. 5, 6 and 7, p<0.05.
Hemihippocampus weights obtained for sample 1 in our previous study
(2) were: saline = 0.056 ± 0.004; TMT = 0.044 x 0.001.
The concentration of GFAP obtained for sample 1 in our previous study
(2) was 329 x 34% of control.
January 1981 (No. 1), July 1984 (No. 2), January 1985 (No. 3),
and August 1987 (No. 4). We do not know whether these different
shipments were from different lots of TMT hydroxide. Another
bottle of TMT hydroxide (No. 5) was purchased from Organome-
tallics. Inc. (East Hampstead, NH) in December 1983. Alfa
Products (Danvers, MA) was the source of the final bottle of TMT
hydroxide (No. 6) purchased in November of 1982. The bottle of
TMT chloride (No. 7) was bought from K & K Laboratories in
November of 1982. All bottles of TMT were stored at room
temperature in a desiccator.
The materials used in the radioimmunoassay of GFAP and the
assay for total protein were those described previously (2,20) and
were obtained from the reported sources.
Animals
Male Long-Evans rats, 40 days of age, were obtained in a
single shipment from Charles River Breeding Laboratories (Wil-
mington, MA). The subjects were housed individually in a
temperature- (22°C ± 2) and humidity- (50 ± 10%) controlled col-
ony room maintained on a 12 hr light: 12 hr dark cycle beginning
at 0600 EDT. Food (Purina Rat Chow) and water were continu-
ously available.
Dosing
With the exception of the sample obtained from Alfa Products,
all samples of TMT were dissolved in 0.9% saline. Sample
numbers 3 and 5, however, dissolved only after Bonification
(Kontes Cell Disrupter. Vineland, NJ) and vigorous mixing. The
TMT from Alfa (sample 6) was not soluble in 0.9% saline or
distilled, deionized water. An aqueous solution of this sample was
achieved by first dissolving the compound in 1 N NaOH followed
by near neutralization of the pH by addition of HC1. Rats weighing
300 grams were administered TMT (8.0 mg/kg) or its vehicle
(0.9% saline) by injection into a lateral tail vein. The dosage of
TMT is expressed as the free base and was administered in a
volume of 1.0 ml/kg body weight. The final pH of each injection
solution is presented in Table 1.
Tissue Preparation
Previously we demonstrated that the effects of TMT (8.0
mg/kg) on brain weight, hippocampai pyramidal cell number and
concentration of GFAP were maximal by three weeks postdosing
(2). Therefore, in the present study, rats were weighed and then
killed by decapitation three weeks following administration of
TMT or saline. Brains were immediately removed, weighed and
bisected in the midsagittal plane. The right hemibrain was pre-
pared for histology (see below). Within 30 sec. the hippocampai
formation was dissected free-hand from the left hemibrain. weighed
and homogenized in 10 volumes of hot (90-95°C) sodium dodecyl
sulfate with the aid of a sonic probe (Kontes Cell Disrupter.
Vineland. NJ). The hippocampai homogenates were stored at
-70°C until radioimmunoassay.
Histology
The right hemibrains were immersion-fixed in Bouin's fixative
for 1-2 days and then cleared with neutral-buffered formalin over
at least a 2 week period. The tissue was embedded in paraffin and
sectioned (8 jtM) in the sagittal plane through the medial-lateral
extent of the brain. Photomicrographs of representative cresyl-
violet stained sections were prepared using an Olympus Vanox
microscope. The nomenclature of Lorente de No' (14) was used to
describe subdivisions of the hippocampai formation [see (2)].
Protein Assay
Total protein was assayed by the method of Smith et al. (24).
Bovine serum albumin was used as the standard.
Radioimmunoassay of GFAP
The homogenates of hemihippocampus were assayed for GFAP.
the intermediate filament protein of astrocytes (10) by a modifi-
cation of the procedure of Jahn et al. (12) as described previously
(20,21). Briefly, aliquots of homogenate were diluted in assay
buffer and applied to nitrocellulose sheets using a slot-blot
apparatus (Minifold II; Schleicher and Schuell, Keene, NH). The
sheets were then sequentially incubated in blocking solution.
GFAP antiserum and USI Protein A detection solution. The
concentration of GFAP in hippocampus is expressed as a percent
of saline control values. Other details concerning the expression of
the RIA data have been published (2,21).
Statistical Analysis
Data were analyzed using the Statistical Analysis System (26).
Individual variables were analyzed by analysis of variance fol-
lowed by Tukey's Studentized range test for mean comparisons.
Preparation of TMT Samples
Each sample of TMT was weighed and dissolved in 10 ml of
0.01 N HC1; two samples (No. 5 and No. 6) required the addition
of 0.1 ml of 4 N HC1 to dissolve fully. Dilutions of these stock
-------
VARIABILITY IN TRIMETHYLTIN NEUROTOXICITY
639
TABLE 2
TRACE METAL ANALYSIS OF DIFFERENT SAMPLES OF TRIMETHYLTIN
Sample Na Zn Cd Pb Cr Ni As Fe Mn Ca Mg Mo Si P A1 K Cu
(ppm)
1
1000
159
14
97
3
48
183
41
2
234
23
12
152
237
60
0
1188
2
419803
75
29
257
26
162
292
11
2
376
35
37
3648
689
431
0
70
3
58406
112
43
481
25
254
464
64
4
754
80
62
3616
1203
170
2362
126
4
23378
12
3
48
6
37
77
8
0
76
9
6
838
138
95
0
13
5
109419
10
5
55
3
32
84
9
0
77
6
8
57
173
5
0
50
6
7557
53
4
98
5
57
179
64
1
129
14
14
1736
224
18
0
201
7
2099
49
6
78
4
44
85
30
0
151
18
10
15
227
87
181
250
solutions were subjected to trace metal analysis and speciated tin
analysis.
Organotin and Metal Analysis
Multielemental analyses of the amounts of twenty elements
were performed on the stock solutions of each organotin sample
using a Jerrel-Ash Model 800 inductively coupled plasma atomic
absorption spectrometer. Each element was determined as the
average of six measurements.
Speciated organotin analyses were performed using a modifi-
cation of the procedure of Means and Hulebak (IS). Aliquots of
each stock solution were transferred to a 25 ml purging tube
containing 23 ml of saturated borate buffer with 4% (w/v) KOH.
Methvltins were converted to their corresponding volatile methyl
stannanes by the addition of 100 p.1 of a 2% (w/v) solution of
NaBH4. The methylstannanes were cryogenically trapped at the
head of a 50 m x 0.32 mm DB-5 capillary column held at - 30°C
for 4 min. The column was then temperature programmed from
-30 to 100"C at 10°C/min. A Hewlett-Packard 5970B mass
selective detector was scanned from tn/e 100-300 at I scan/sec.
RESULTS
Histology
All sections obtained from rats dosed with the various samples
of TMT showed a loss of hippocampal pyramidal cells, especially
in regions CA3-CA4 (Fig. 1). The qualitative variability in the
histological effects of a given sample of TMT made it difficult to
reliably distinguish among the effects caused by different samples.
In general, however, the degree of cell loss in regions CA3-CA4
was greatest in sections prepared from rats dosed with samples 1,
3 and 4.
Body and Brain Weights
None of the samples of TMT caused a reduction in body weight
or whole brain weight (data not shown). In contrast, all but
samples 5 and 6 caused reductions in weights of hemihippocampus
(Table 1). These effects, however, varied considerably from
sample to sample with deficits ranging from a high of 22%
(sample 1) to a low of 8% (sample 2).
Radioimmunoassay of GFAP
The concentration of GFAP in hippocampus was increased by
all samples of TMT (Table 1). Again, however, the effects were
highly sample-dependent, with increases ranging from a low of
66% (sample 5) to a high of 305% (sample 4). Consistent with the
hippocampal weight and histology data, the effects of samples I.
3, and 4 on GFAP were greater than the effects of the other TMT
samples. The effect of sample 1 an GFAP observed in our
previous study (2) did not differ from the effect observed in the
present study (Table I).
Chemistry
Multielemental analyses indicated wide variations in both the
elements and the amounts of those elements found as contaminants
in the seven different samples of trimethyltin. These results,
presented as the concentration of each element in the indicated
sample, are presented in Table 2. For each metal analyzed, widely
different concentrations were observed among the different sam-
ples of TMT. In two samples, numbers 2 and 5, sodium salts made
up such a large percentage of the sample mass so as to significantly
dilute the concentration of TMT.
Speciated organotin analyses revealed the presence of signifi-
cant quantities of dimethyltin in some samples of TMT (Table 3).
Although exact quantification could not be achieved without a
confirmed pure reference standard, relative peak areas obtained
suggest that dimethyltin was present at approximately 13%, 5%,
20%, and 10% in samples 2, 3, 6, and 7, respectively. The
variations in dimethyltin content of samples are illustrated in Fig.
2, which compares a GC/mass spectrogram from a high dimeth-
yltin sample (No. 6) with a low dimethyltin sample (No. 4).
DISCUSSION
We have demonstrated that different samples of TMT obtained
from the same or different commercial sources produce widely
varying degrees of CNS toxicity as revealed by changes in
histology, hippocampal weight and concentration of the astrocyte
protein, GFAP. Speciated organotin analysis and trace metal
FOLLOWING PAGE
HO. 1. Cresyl-violet stained sagittal sections of dorsal hippocampus obtained from rats treated with saline and the various samples of TMT identified in
Table 1. Bar-200 ilM.
-------
640
O'CALLAGHAN, NIEDZWIECKI AND MEANS
Saline
mam
-------
VARIABILITY IN TRIMETHYLTIN NEUROTOXICITY
641
TABLE 3
ESTIMATION OF DIMETHYLTIN CONTENT IN DIFFERENT
SAMPLES OF TRIMETHYLTIN
Sample % Dimethyltin
<2
13
5
<2
<2
20
10
analysis of the TMT samples indicated the presence of several
impurities that may account for the variable neurotoxic effects of
different samples of TMT.
Previously we demonstrated that brain regions affected by
known neurotoxicants show dose-dependent decreases in wet
weight and dose-dependent increases in GFAP [e.g.. (2,21)]. The
present data are consistent with these observations. Samples of
TMT that caused the greatest reductions in hemihippocampus
weights caused the greatest increases in GFAP and were associated
with the most apparent pyramidal cell loss in area CA4 of
hippocampus. Of the seven samples evaluated, numbers 1. 3 and
4 were most neurotoxic. Sample 1 was the source of TMT used in
the original studies [see (2,14)]. The percentage decrease in
hemihippocampus weight and percentage increase in GFAP ob-
tained with sample 1 in the present study replicated the values
obtained previously. The samples that led us to undertake this
study, i.e., those that failed to produce effects consistent with our
original findings, were numbers 2 and S, two of the least potent
samples. Taken together the data support our hypothesis that
potency differences among samples of TMT are major sources of
experiment-to-experiment variability. Because the oldest sample
tested (No. 1) was one of the most neurotoxic, sample age did not
seem to contribute to loss of potency.
Trace metal and organotin analysis of the samples revealed
impurities that may account for some of the variability in the
neurotoxic effects caused by different samples of TMT. For
example, wide variations in both the number and amount of
specific elements were found across different samples of TMT,
even among samples obtained from the same vendor. In several
cases these included toxic trace metals such as arsenic, copper,
lead, nickel or cadmium. The aggregate concentration of these
contaminants in any sample, however, never exceeded 0.1% of the
total. It is doubtful, therefore, that these elements contributed to
the spectrum of neurotoxic effects caused by different samples of
TMT. Sodium, in contrast, may have contributed significantly to
potency differences among the samples of TMT by serving as a
diluent. For example, samples 2 and 5 contained levels that would
constitute a large percentage of the mass of the organotin sample.
This "dilution" effect of sodium could explain the relatively low
potencies of samples 2 and 5. Speciated organotin analysis also
revealed the presence of significant quantities of another impurity,
dimethyltin. Although exact quantification could not be achieved
without a confirmed pure reference standard, relative peak areas
obtained suggest that diinethyltin constituted up to 20% of total
organotin content. Dimethyltin has low neurotoxic potency rela-
tive to the trialkyltins (17); therefore, it would be expected to
S
1
0 2 4 6 • 10 13 14 1ft
Retention Tlm«, mln.
s
0 2 4 « 8 10 12 14 l«
Retention Tim*, min.
FIG. 2. Representative scans obtained for speciated organotin analysis of
samples 4 and 6. The small peak in sample 6 that precedes the large peak
common to both samples 4 and 6 represents dimethyltin contamination.
dilute the neurotoxic potency of TMT if present in sufficient
quantities. Indeed, the samples with the highest concentrations of
dimethyltin, numbers 2, 6 and 7 showed the lowest potency in the
neurotoxicity evaluations. Thus, the low neurotoxic potencies of
samples 2, 5, 6, 7, relative to samples 1, 3 and 4 may have been
due in large measure to contamination with either sodium (samples
2 and 3) or dimethyltin (samples 2, 6 and 7).
In summary, our findings suggest that care must be exercised in
the interpretation of data obtained from samples of TMT with
unknown purity. One implication of our data is that experiment-
to-experiment and laboratory'to-laboratory comparisons with re-
spect to the effects of a specific dosage of TMT on a particular
morphological, biochemical or functional endpoint may not be
valid unless all data were obtained from the same source of TMT.
Moreover, in the absence of data to the contrary, we see no reason
why the same situation would not apply to investigations of the
effects of other organotins. We realize that valuable contributions
to the fields of alkyltin toxicity, cognitive neuroscience and
cellular neuroscience have been made by researchers using tri-
methyltin of unconfirmed purity. Where resources are not avail-
able to conduct appropriate sample analyses, we recommend that
simple bioassays, such as hippocampal wet weight, be employed
to establish a quantitative basis of trimethyltin-induced neurotox-
icity. Such an approach would serve to "standardize" the effects
of trimethyltin from experiment*to-experiment and minimize labo-
ratory-to-laboratory variability. The reproducibility of future stud-
ies of organotin toxicity, however, would be enhanced by the
availability of compounds with confirmed purity.
ACKNOWLEDGEMENTS
The authors are grateful to Dr. Karl F. Jensen for assistance in
photographing the histological sections and to Dr. Melvin L. Billingsley
for a critical review of the manuscript.
1
3
4
5
6
7
3«mpJ«6
A
-U
" •
—,—i—
Stmpki4
B
1 '
¦U—
i i I i
-------
642
O'CALLAGHAN, NIEDZWIECKI AND MEANS
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Psychopharmacology (1989) 97:521-528
Psychopharmacology
© Springer-Verlag 1989
Time-dependent deficits in delay conditioning produced by trimethyltin*
David B. Peele1, Jackie D. Farmer2, and Janice E. Coleman2
' NSI - Environmental Sciences, P.O. Box 12313, Research Triangle Park, NC 27709, USA
2 Neurotoxicology Division, Health Effects Research Laboratory, US Environmental Protection Agency,
Research Triangle Park, NC 27711, USA
Abstract. Trimethyltin (TMT) produces behavioral and
cognitive deficits resulting, in part, from limbic system toxi-
city. To determine whether these effects result from learning
deficits or accelerated memory loss, the present experiment
examined two delay conditioning paradigms in rats pre-
viously treated with either saline or TMT. Saline-treated
Long-Evans rats receiving injections of lithium after con-
suming saccharin-flavored water later avoided saccharin in-
gestion: the degree of avoidance varied inversely with the
time (0.5, 3 or 6 h) separating initial saccharin availability
and lithium injection. Rats treated with TMT (8 mg/kg
IV, 30 days prior) showed impaired conditioning at the
long but not the short or intermediate delay conditions,
suggesting that the deficits were mnemonic and not associa-
tive. Similar delay-dependent deficits in rats treated with
TMT were observed in a passive avoidance task that ar-
ranged one of two delays between response emission and
shock delivery during training. The effects of TMT on delay
conditioning were accompanied by reduced bodyweight and
hippocampal pathology. In summary, TMT appears to alter
the temporally dependent association of events (entering
darkened compartment versus saccharin consumption) and
consequences (foot shock versus lithium administration)
during acquisition. Furthermore, the observed deficits in
delay conditioning produced by TMT did not appear to
be task specific, with similar effects determined with tests
of both somatosensory and gustatory avoidance learning
designed to distinguish between functional alterations due
to deficits in memorial processes from those due to altered
sensory, motor, or associative processes.
Key words: Trimethyltin - Delay conditioning - Condi-
tioned flavor aversion - Passive avoidance - Rat
* Although the research described in this article has been supported
by the United States Environmental Protection Agency (through
contract 68-02-4450 to NSI - Environmental Sciences), it has not
been subjected to Agency review and therefore does not necessarily
reflect the views of the Agency and no official endorsement should
be inferred. Mention of trade names or commercial products does
not constitute endorsement or recommendation for use, A prelimi-
nary report of these data was given at the 1987 annual meeting
of the Society of Toxicology, Washington, DC
Offprint requests to: D.B. Peele
Trimethyltin (TMT) is a potent neurotoxicant capable of
inducing a range of neurobehavioral symptoms in acutely
exposed organisms (Dyer et al. 1982; McMillan and
Wenger 1985). Acute administration of TMT to rats pro-
duces weight loss, convulsions, and a delayed behavioral
sequelae characterized by whole-body tremor, tail mutila-
tion, vocalization, and aggressive sparring (Brown et al.
1979). Other symptoms of TMT intoxication include altered
performance on several behavioral assays of memory in
laboratory animals (e.g., delayed matching-to-sample,
Bushnell and Evans 1984; passive avoidance, e.g., Walsh
et al. 1982a; serial pattern learning, Fountain etal. 1985).
These neurobehavioral effects are accompanied by CNS pa-
thology including selective destruction of neurons (Area
CA3/4) in the pyramidal cell layer of the hippocampus
(Chang and Dyer 1983).
In a novel application of the conditioned flavor-aversion
paradigm, Riley and colleagues have reported deficits in
long-delay flavor-aversion conditioning (LD-FAC) induced
by TMT (Riley etal. 1984). In that experiment LD-FAC
was used as a learning and memory assay; rats learned
not to consume saccharin-flavored water that had pre-
viously been paired with lithium chloride administration.
Memorial capacity was determined by imposing delays (0,
3 or 6 h) between flavor intake and lithium administration
during conditioning. The 0-h delay condition was included
to determine if TMT also altered associative conditioning.
Both controls and rats treated with TMT showed robust
flavor aversions after two saccharin-lithium pairings with
a 0-h delay. However, when the delay separating saccharin
and lithium was increased to 3 h, the rats treated with TMT
showed deficits (relative to controls) in conditioning. These
time-dependent learning deficits, observed after long but
not short delays, were interpreted by the authors as an
acceleration of short-term memory loss due to TMT admin-
istration. These results provided additional evidence that
TMT produces an acceleration of short-term memory loss.
The present experiment was designed to address two
specific issues. The first issue was whether the deficits in
delay conditioning induced by acute administration of
TMT were task specific, i.e., whether similar deficits would
occur in other delay conditioning paradigms. Therefore,
a passive avoidance conditioning paradigm utilizing a delay
conditioning protocol was conducted with the same animals
to determine the generality of effects obtained in the LD-FAC
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522
experiment. Second, the present experiment addressed the
issue of the specificity and utility of the LD-FAC paradigm
by partially replicating, refining, and validating the results
of the Riley et al. (1984) experiment. The latter issue was
of concern primarily because of the need in toxicology for
rapid screens for learning and memory deficits.
Conditioning was assessed using a two-bottle choice test
(relative flavor intake) rather than the single-bottle test (ab-
solute flavor intake) that was used in the Riley et al. (1984)
experiment. This decision was made for several reasons.
First, two-bottle tests have been demonstrated to be a more
sensitive measure of flavor-aversion conditioning (see Grote
and Brown 1971). Second, two-bottle tests are capable of
determining the effects of conditioning in a manner that
is not dependent on total fluid intake. This precaution was
particularly relevant in the present experiment given the
fact that TMT has been reported to produce alteration in
fluid intake in rats (Bushnell and Evans 1985) which, in
turn, could bias the results from a single-bottle test. Finally,
in assessing the specificity of effects, a taste (saccharin) reac-
tivity test was conducted with rats treated with either TMT
or saline to determine whether alteration in gustatory sensi-
tivity might have contributed to the obtained results.
Method
Animals
One-hundred forty-four naive male, Long-Evans hooded
rats (Charles River Breeding Company, Raleigh, NC) were
obtained at 40 days of age. After arrival at the laboratory,
rats were individually housed in ceiling-suspended, metal
cages and were given unlimited access to food (Purina Lab
Chow) and limited access to water, as described below. Ex-
perimental procedures were initiated 10 days after arrival.
Body weights were determined for all animals on each day
of the experiment and ranged from approximately 230 to
300 g at the beginning of the experiment. Group assign-
ments, as described below, were made to equate group mean
body weight.
Procedure
A summary of group assignments and experimental condi-
tions is provided in Table 1.
TMT administration. Rats were randomly assigned to one
of two groups and were administered a single IV tail-vein
injection of either isotonic saline (SAL) or 8 mg base/kg
body wt trimethyltin hydroxide (TMT: ICN Pharmaceuti-
cals, Inc., Plainview, NY). Over the following 14 days, rats
received no additional treatments, and were allowed ad lib
access to both food and water.
Flavor aversion conditioning. Beginning 15 days following
SAL or TMT injections, two squads composed of 36 rats
treated with TMT and 36 rats treated with SAL were main-
tained under a 23.5-h schedule of water deprivation that
was preceded by 30 min access to tap water each day from
50-ml plastic centrifuge tubes (Nalgene Co., Rochester,
NY) equipped with rubber stoppers and curved stainless-
steel drinking spouts. The tubes were attached to the front
of the cages by coil springs and centered 3.8 cm from the
cage floor. Total daily intakes were calculated (to the near-
Table 1, Summary of conditions for rats in squads 1 arid 2.
Numbers refer to the number of rats exposed to each condition,
while day numbers refer to number of days following either saline
(SAL: 0.9%, vot=l ml/kg, IV) or trimethyltin (TMT: 8 mg.kg
as base, vol= 1 ml/kg, IV) administration
Day
Procedure
Squad one
Squad two
SAL
TMT
SAL
TMT
1
Intravenous
36
36
36
36
injections
15-26
Acclimation to
36
36
36
36
restricted water
27
Flavor-aversion
36
36
36
36
conditioning
28
Passive-avoidance
—
—
27
27
conditioning
29
Passive-avoidance
-
—
27
27
testing
JO
Flavor-aversion
36
36
36
36
testing
31
Necropsy
10
10
10
10
31-37
Restricted water
—
—
9
9
(1 h)
38-55
Taste-reactivity
-
—
9
9
testing
est 0.1 g) from drinking tube and spout weights determined
before and after the water-access period.
Rats from squad 1 were tested using procedures for
determining TMT effects on both body weight and LD-
FAC. On day 27 post-treatment, 27 rats treated with TMT
and 27 rats treated with SAL received IP injections of lithi-
um chloride (0.9 mEq/kg body wt; volume: 1 ml/kg body
wt) at either 0.5, 3.0 or 6.0 h after access to a novel sac-
charin solution (0.1% w/v, sodium saccharin dissolved in
tap water; Fisher Scientific Products, Pittsburgh, PA). Ad-
ditional groups («=9) of rats treated with either TMT or
SAL received injections of isotonic saline at the 0.5-h post-
saccharin delay. After 2 intervening days of water only (30
min/day), all rats received concurrent access to water and
saccharin for 30 min (two-stimulus choice test) and their
intakes of each fluid were determined.
Rats from squad 2 were maintained under conditions
identical to those for rats in squad 1 with the following
exceptions. In addition to receiving saccharin-lithium pair-
ings at one of three delays, equal numbers of rats treated
with SAL and TMT were used in a passive avoidance test
and a test of taste reactivity, as described below. Rats in
squad 2 were given concurrent access to water and sac-
charin for 30 min on the 3rd day following delay condition-
ing and their preference for the saccharin solution was de-
termined.
Passive avoidance conditioning. Rats from squad 2 were ex-
posed to single-trial, passive avoidance conditioning in a
V-shaped step-through apparatus similar to that described
by Jarvik and Kopp (1967). The device was a two-compart-
ment trough, with an opaque guillotine door separating
the two compartments. One compartment (light compart-
ment) was lighted from above by a 30-W high-intensity
lamp with galvanized steel side-walls and clear plexiglas
-------
523
end-walls and top. The other compartment (dark compart-
ment) consisted of galvanized steel side walls and opaque
plexiglas top and end wall. A constant-current AC shock
was delivered through the walls and floor of the darkened
compartment only, with the current adjusted to deliver a
2-s 0.7-mA shock from a line- and load-regulated shock
generator (for complete description of the shock generator,
see Ali and Reiter 1977).
Passive avoidance training and testing occurred during
the 2 water-only days separating flavor aversion condition-
ing and testing (days 28, 29 post-treatment for training and
testing, respectively) according to a procedure adapted from
Kapp et al. (1978). Rats from both the SAL and TMT
groups were randomly assigned to one of three groups (N,
D-0, D-30; /i = 9/group), and placed individually into the
lighted compartment of the apparatus. After 30 s, the door
separating the compartments was raised and the step-
through latency (s) determined. A step-through was defined
as a rat placing all four paws into the darkened compart-
ment. Rats in group N received no shock upon stepping
through in order to determine the baseline latency of a
retest step-through response. Rats in groups D-0 and D-30
were trapped in the darkened compartment after stepping
through and the shock was delivered either 0 or 30 s later,
respectively. All rats were removed from the apparatus at
the termination of shock delivery and returned to their
home cages. Twenty-four hours after training, all rats were
again placed in the light compartment with the door sepa-
rating the two compartments closed. After 30 s, the door
was raised and the retest latency was determined. Rats were
removed when they either (a) stepped-through into the dark
compartment or (b) when 300 s had elapsed, which ever
came first. Training and testing occurred approximately 1 h
after the rats received their daily access to water.
Taste reactivity testing. Eighteen rats treated with either
TMT or SAL from squad 2 that received no lithium chlo-
ride injection were selected to determine if treatment with
TMT altered proportional intakes of varying concentra-
tions of sodium saccharin (see Oakley and Pfaffmann 1962).
Rats were given 60 min access to water only for 1 week
following LD-FAC (days 31-37 post-treatment). Beginning
on day 38, and on alternate days for 18 days, rats were
given 60-min access to both water and saccharin solution.
Saccharin solutions varied in concentration (0.001, 0.01,
0.03,0.1,0.3,0.56,1.0,3.0%, w/v), with the order of presen-
tation randomly determined. Acceptance-rejection scores
were expressed as proportion of total fluid consumed from
the saccharin bottle (relative saccharin intake).
Histology, Randomly-selected rats (n»40) from squads 1
and 2, treated with TMT or SAL were decapitated immedi-
ately after behavioral testing (day 31) and their brains re-
moved and sliced in saggital sections, beginning at the mid-
line, 10 microns thick at 100-micron intervals. Sections were
subsequently stained (cresyl violet), mounted, and photo-
graphed in order to verify treatment effects on the hippo-
campus.
Data analysis. The statistical significance of the data from
the flavor-aversion conditioning and taste-reactivity experi-
ments was determined using SAS General Linear Model
procedures (SAS 1985). The LD-FAC data were analyzed
by a 3-way ANOVA (treatment x delay x block); simple ef-
fects were subsequently assessed following the determina-
tion of a significant interaction term. The data from the
taste-reactivity determinations were subjected to a 2-way
MANOVA with repeated measures (treatment x concentra-
tion, with concentration as a repeated factor), with subse-
quent simple effects tests performed if significant interac-
tions were determined. The data from the passive avoidance
conditioning experiment were analyzed using the non-para-
metric, JCruskal-Wallis test (Marascuilo and McSweeney
1977). In order to make within-group comparisons, the data
from rats treated with either TMT or SAL were analyzed
separately, with subsequent orthogonal contrasts arranged
for those instances of a significant main effect. An alpha
level of 0.05 was used throughout in determining statistical
significance.
Results
The administration of TMT resulted in reduced body
weight relative to SAL-treated rats (see Fig. 1). During the
first 14 days following TMT or SAL administration, rats
from squad 1 showed moderate weight gain, with little if
any difference in the mean body weight for the two treat-
ment groups.
Following water restriction on day 14, rats treated with
either TMT or SAL both showed a weight loss of approxi-
mately 10% of their free-water weights. However, as the
water-restriction schedule continued, the body weights for
the two groups began to diverge. Apart from minor fluctua-
tions, rats treated with TMT showed a 10% reduction in
body weight compared to SAL-treated rats. This reduction
in body weight persisted for the remainder of the experi-
ment. The body weights for rats from squad 2, treated with
either TMT or SAL, showed a similar trend with an approx-
imate 10% reduction in weight for rats treated with TMT
(compared to controls) at the time of delay conditioning
(data not shown). A single rat from squad 2 died 24 days
after receiving TMT.
Following 10 days of restricted water availability, rats
from squad 1 treated with TMT or SAL consumed'a mean
of 20.8(±1.2) and 18.5(±1.1) ml water, respectively, on
day 26. When saccharin was substituted for water on day
27, rats treated with TMT or SAL consumed a mean of
17.7(±1.1) and 15.8(±0.7) ml, a reduction of 15% and
S 10 IS 20
Dayi
Fig. I. Body weights for raw treated with either saline (open sym-
bols) or TMT (filled symbols) IV, over the course of 30 days. Each
point represent* the mean (±SEM) of 36 rats from squad 1. The
reduction of weight at day 14 corresponds to the 1 st day of restrict-
ing fluid access during flavor-aversion conditioning
-------
524
3J 0.8
Jt
«
5 0.7
Delay (hours)
Fig. 2. Relative saccharin intake (saccharin intake expressed as a
proportion of total fluid intake) for rats receiving lithium chloride
following saccharin consumption from squad 1 (connected symbols)
and squad 2 (unconnected symbols) during the flavor preference
test. Rats treated with TMT or SAL are represented by triangles
and circles. respectively. Additional unconnected points, located
over and offset from the 0.5-h abscissa tick-mark, represent rats
receiving saline following saccharin consumption from squads 1
and 2, respectively. Each point represents the mean (±SEM) pref-
erence score of nine rats as a function of the delay separating
saccharin availability and lithium chloride administration
15% for rats treated with TMT and SAL, respectively. Sim-
ilarly, rats from squad 2 treated with either TMT or SAL
consumed a mean of 15.1(±0.8) and 15.8(±0.4) ml water,
respectively, on day 26 post-treatment. Consumption of
saccharin on day 27 by rats treated with either TMT or
SAL fell by 12 and 17%, respectively (consumption of
13.3 ±0.8 and 13.0 ±0.5 ml saccharin, respectively, for rats
treated with TMT and SAL).
The effect of TMT administration on LD-FAC was de-
pendent on the time separating saccharin intake and lithium
administration during training, as shown in Fig. 2. Rats
from squad t (connected symbols, Fig. 2) pretreated with
either TMT or SAL that received saline injections after
consuming saccharin showed a high preference for sac-
charin. Saccharin intake accounted for approximately
85-93% of their total fluid intake on the test day. Rats
pretreated with SAL and that received saccharin-lithium
pairings exhibited saccharin intakes (approximately 5-35%)
that varied as a function of the delay interval. While the
saccharin intake scores for the rats receiving TMT showed
a similar dependence on the delay value, differences
emerged between the treatment groups at the 6.0-h delay.
Rats treated with TMT had saccharin intakes of approxi-
mately 10% of total fluid intake at the 0.5-h delay, 37%
at the 3-h delay and just less than 60% at the 6-h delay
value. During the test trial, rats treated with TMT con-
sumed a mean of 20.5(±t.l) ml total fluid intake, while
the SAL-treated rats consumed a mean of 18.4(±0,8) ml.
Rats from squad 2 (unconnected symbols, Fig. 2)
showed similar effects of TMT pretreatment on LD-FAC.
Rats treated with TMT or SAL, and receiving saccharin-
saline pairing, showed relative saccharin intakes of approxi-
mately 85% of their total fluid intake. Rats treated with
SAL and receiving lithium at one of three delays after their
« 0.8
0.001 0.003 0.01 0.03 0.1 0 3 0.56
Saccharin Concentration
Fig. 3. Relative saccharin intake determined during taste-reactivity
testing for rats from squad 2. Each point represents \.he mean
(±SEM) preference score of nine rats as a function of saccharin
concentration. The scores for rats treated with SAL or TMT arc
represented by open and Jilted symbols, respectively
initial access to saccharin showed saccharin intakes that
were proportional to the delay. As the delay value increased
from 0.5 to 6 h, saccharin intakes for SAL-treated rats in-
creased from approximately 10 to 28% of their total fluid
intakes, respectively. Rats treated with TMT, while showing
similar relative saccharin intake scores at the nominal 0.5-h
delay value, consumed proportionately more saccharin than
SAL-treated rats at both the 3- and 6-h delay interval. This
effect was most evident at the 6-h delay, where rats treated
with TMT consumed approximately 60% of their total fluid
intake from the saccharin bottle on the test day. The mean
total fluid intake during the test trial for rats treated with
TMT and SAL was 18.1(± 1.2) and 18.9(±0.8) ml, respec-
tively.
The statistical analysis of the saccharin preference scores
from the LD-FAC experiment revealed a significant effect
of TMT treatment [F\l,128) = 7.88, P<0.000t] and delay
[F(S, 128)= 113.68, ?<0.0001], and a significant treatment-
by-delay interaction [/(3,128) = 6.41, P< 0.0004]; the dos-
age x block, block x delay, and the dosage x block x delay
interactions were all non-significant. Simple effects analysis
showed that the relative intakes of the rats treated with
either TMT or SAL were significantly different at the 6.0-h
delay only (P< 0.0002).
The effect of TMT on passive avoidance conditioning
was to reduce the retest latencies for rats receiving TMT
compared to that for SAL-treated rats. Rats treated with
SAL in both the 0-s and 30-s delay groups showed median
retest latencies of 300 s (arbitrary limit). In contrast, rats
treated with TMT showed reduced latencies that were de-
pendent (in magnitude) on the delay value: 290 s and 200 s,
respectively, for the 0-s and 30-s delay groups. Rats treated
with either TMT or SAL and receiving no shock during
training showed latencies that were far lower than their
shocked counterparts, taking a median of 6.0 and 8.0 s.
respectively, to enter the darkened compartment upon re-
test. In order to make within-group comparisons, the data
from rats treated with either TMT or SAL were analyzed
separately with the non-parametric ICruskal-Wallis tfst. The
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525
Fig. 4a, b. Photomicrographs of representative saggital. cresyl vio-
let stained sections of the hippocampus from rats treated with
saline (a top panel) or TMT (b bottom panel) from squad 1. The
magnification was held constant for a and b
results indicate a significant treatment effect for both
groups (TMT: H= 10.838, /><0.004; SAL: W = 22.674,
PcO.OOl). Orthogonal contrasts revealed a significant dif-
ference (PcO.Ol) between the no-shock and the 0-s and
30-s delay groups for rats treated with SAL, while only
the 0-s delay group was significantly (/><0.01) different
from the respective no-shock group of rats treated with
TMT.
The results from the taste-reactivity experiment are
shown in Fig. 3. Rats treated with SAL demonstrated no
preference for saccharin over water at the lowest saccharin
concentration (i.e., saccharin preference scores of approxi-
mately 0.5), with preference scores showing a peak at con-
centrations of 0.1 and 0.56%. The saccharin preference
scores for rats treated with TMT paralleled those for the
SAL-treated rats, with the exception of a lowered preference
for saccharin at concentrations of 0.01 and 0.03%. Total
intake during the taste-reactivity tests (data not shown) was
not different between groups, and remained at comparable
levels at all concentrations (approximately 20 ml/trial). The
statistical analysis of these data revealed a significant treat-
ment x concentration interaction [/"(7,10) = 3.16, < 0.04];
the effects of treatment and concentration were both non-
significant. Assessment of simple effects determined that
rats treated with either TMT or SAL showed significantly
different saccharin intakes at the 0.01% concentration only.
Hippocampal damage produced by TMT administra-
tion can be seen in the photomicrographs presented in
Fig. 4, showing saggital, cresyl violet stained sections of
representative control and treated rats from squad 1. In
rats from both squads, TMT produced a destruction of
pyramidal cells that was most prominent in area CA3. The
photomicrograph in Fig. 4b shows the typical, dramatic
reduction in the length of the pyramidal cell line, with a
notable thinning in areas CA3a-c (as compared to control.
Fig. 4a). There was little if any thinning observed in rats
treated with TMT in areas CA1 or CA2 of the pyramidal
cell layer or in the granule cell layer.
Discussion
The results from the present experiment indicate that LD-
FAC was impaired by TMT in a time-dependent manner
and that the deficits produced by TMT administration were
accompanied by hippocampal pathology and body weight
loss. The hippocampal pathology and weight loss are con-
sistent with previous reports in Long-Evans rats treated
30 days prior to sacrifice (Dyer et al. 1982). The acceleration
of memory loss induced by TMT in the LD-FAC experi-
ments was consistent with the time-dependent behavioral
deficits determined using a passive avoidance delay condi-
tioning paradigm. The weight loss, occurring only after
water access was restricted, suggests that rats treated with
TMT may have difficulty maintaining body weight once
homeostasis is challenged. By comparing the aversions to
saccharin in the 0.5-h delay groups, is is clear that rats
treated with TMT or SAL were identical in their ability
to associate saccharin consumption and the consequences
of lithium administration. This was not the case, however,
when a 6-h delay was imposed between saccharin and lithi-
um administration; at this delay, aversions to saccharin
were attenuated in the rats treated with TMT, compared
to their SAL-treated counterparts. The use of a two-stimu-
lus test allowed an assessment of conditioning independent
of the rate or total amount of fluid consumption. Also,
by using a single training session, the results avoid problems
of determining whether the conditioning deficit resulted
from deficits of memory versus acquisition. Collectively,
the results replicate and extend the findings of deficits in
LD-FAC originally reported by Riley et al. (1984).
The selective attenuation of flavor-aversion condition-
ing with long but not short delays embedded in the condi-
tioning protocol was accompanied by a similar time-depen-
dent deficit in passive avoidance conditioning. Previous re-
ports on the effects of TMT on passive avoidance condi-
tioning (without delays) have been equivocal. For instance
both Loullis et al. (1985) and Walsh et al. (1982a) have
reported a decrease in retest latencies (compared to con-
trols) in rats treated with TMT either 2 or 3 weeks, respec-
tively, prior to training. These findings are in contrast to
those of Hagan et al. (1988), who found no deficits in pas-
sive avoidance conditioning 2 weeks following TMT admin-
istration. While further research is necessary to determine
the factors contributing to these conflicting findings, the
results from the present experiment indicate that the reten-
tion deficit in passive avoidance conditioning produced by
TMT administration can be exacerbated by imposing delays
in training. By examining step-through latencies during
training and the results from the no-shock condition it is
clear that rats treated with TMT had median step-through
h
-------
526
latencies that were shorter than the SAL-treated rats. De-
spite this general tendency shown by rats receiving TMT,
only the 0-s delay condition resulted in retest latencies that
were significantly different from the no-shock condition.
On the other hand, the latencies for both the 0- and 30-s
delay conditions were significantly different from that from
the no-shock condition in SAL-treated rats. Rats treated
with SAL. but not TMT, were able to acquire the avoidance
response despite temporal delays between behavior and the
aversive consequences of footshock. This observation, cou-
pled with the results from the flavor-aversion conditioning
experiments, appear to have, as a common mechanism, a
deficit in the ability of rats treated with TMT to overcome
temporal delays in conditioning that separate responses
(i.e., consumming saccharin, entering a darkened compart-
ment) and consequent events (i.e., lithium administration,
shock delivery).
The effects of TMT on long-delay flavor-aversion condi-
tioning were originally reported for rats exposed to 6 mg/kg
trimethyltin chloride via the oral route of administration
(Riley etal. 1984). The outcome of that experiment was
important for two reasons: first, it confirmed the observa-
tion of memory deficits induced by TMT in tests using
other sensory modalities (e.g., spatial memory in the radial-
arm maze: Walsh et al. 1982b), and second, it demonstrated
the feasibility of using long-delay flavor-aversion condition-
ing as a test for memory deficits induced by chemicals.
Even though other tests exist for assessing toxicant-induced
memory deficits in laboratory animals, they typically in-
volve extensive training and expensive equipment which
preclude rapid screening of a large number of suspected
chemicals. Alternatively, while home cage behaviors have
the potential for being useful in screening for toxicant ef-
fects on locomotor activity and consummatory behavior
(Bushnell and Evans 1985), there was and remains a paucity
of simple home cage test procedures for assessing potential
learning and memory deficits.
While the results of Riley etal. (1984) were important
in demonstrating the feasibility of studying memory pro-
cesses with a relatively simple homecage test, there were
a number of alternative explanations of the observed effects
other than memory loss produced by TMT administration.
First, the effect of TMT on flavor-aversion conditioning
at the longest (6-h) delay was equivocal. Thus, the conclu-
sions relied on a lack of effect at 0-h delay and an atten-
uated effect after a 3-h delay. It is likely that the equivocal
effects at the 6-h delay were due to the lack of effective
conditioning in rats treated with either TMT or SAL. Other
features of the experiment also weakened the conclusion
that the toxicity produced by TMT administration included
a selective memorial component. For instance, TMT ad-
ministration might have affected the aversiveness of the
lithium injections. This possibility was subsequently ad-
dressed by Mastropaolo et al. (1986), showing that similar
TMT treatment did not alter the lithium dosage-effect func-
tions in single-stimulus flavor-aversion conditioning.
While TMT administration did not affect sensitivity to
lithium chloride-induced flavor aversion conditioning (0-h
delay), other questions remain concerning the mechanism(s)
involved in producing the effects reported in the Riley et al.
(1984) study. For instance, neither the potential for pathol-
ogy induced by TMT nor the sensory integrity of the rats
treated with TMT was assessed. Both of these issues were
addressed in the present experiment, showing that similar
time-dependent behavioral alterations occurred in similarly
treated rats with demonstrated destruction of the pyramidal
cell layer in region CA3 of the hippocampus. A dosage
of 8 mg/kg IV was selected based in part on reports of
the biochemical and morphological damage observed fol-
lowing IV-administered TMT in dosages of 6-9 mg/kg
(Brock and O'Callaghan 1987). Also, previous experiments
in our laboratory indicated that a dosage of 7 mg/kg IV
was without effect on LD-FAC. body weight or hippocam-
pal morphology. In terms of pathology, it appears that the
potency of TMT is lower when administered intravenously
than when it is administered orally or intraperitoneally
(Dyer et al. 1982, versus Brock and O'Callaghan 1987), al-
though a firm conclusion awaits a systematic comparison
of the pathology resulting from TMT administered via var-
ious routes.
Because of the nature and specificity of the pathology
produced by TMT administration, namely neuronal de-
struction in the pyramidal cell layer of the hippocampus,
several investigators have drawn parallels between the cog-
nitive deficits induced by TMT and those due to electrolytic
or chemical lesions of the hippocampus (e.g., Brown et al.
1979; Walsh et al. 1982a, b). Similarly, a number of studies
have reported that hippocampai lesions attenuate flavor-
aversion conditioning (Miller et al. 1971, 1986; Nonneman
and Curtis 1978; Best and Orr 1973), and that the attenua-
tion of conditioning was enhanced by utilizing two-stimulus
tests of conditioning or delay-conditioning techniques
(Krane etal. 1976). Others, however, have reported that
hippocampai lesions produce either transient effects (Mur-
phy and Brown 1974) or no effect on flavor-aversion condi-
tioning (McGowen et al. 1972; Miller et al. 1975; Thomka
and Brown 1975). One possibility for such equivocal find-
ings concerning the effects of limbic system pathology on
flavor-aversion conditioning may be that the proper tech-
niques for assessing mnemonic deficits in rats with hippo-
campai lesions have not been employed (Gaston 1978; Non-
neman and Curtis 1978). The only study to manipulate the
delay separating flavor intake and toxicant administration,
Krane et al. (1976), varied the delay from 0 to 15 min. As
shown in the present experiment with rats treated with SAL
(control), more appropriate delay values for demonstrating
altered, time-dependent conditioning would be on the order
of several hours rather than 15 min. This observation has
been reported by others investigating LD-FAC (Bures and
Buresova 1977).
A number of investigators have attributed attenuated
flavor-aversion conditioning in rats with lesions of the hip-
pocampus to attenuated neophobia, that is a decrease in
the tendency of rats to avoid novel flavors (see Krane et al.
1976; Miller et al. 1986). The data from the present experi-
ment do not resemble those from experiments with hippo-
campally lesioned animals. In the present experiment, rats
treated 'with TMT or SAL showed nearly identical suppres-
sions of intake on their first exposure (training trial, day
11) to the novel saccharin flavor. One possibility is that
the mechanism responsible for attenuated LD-FAC in the
present experiment is the combination of damage to the
hippocampus and the amygdala produced by TMT admin-
istration. While the effects of TMT on the structural integri-
ty of the amygdala were not determined in the present ex-
periment, damage to this limbic structure has been pre-
viously reported for rats exposed to TMT (Brown et al.
1979). Damage to the amygdala haw been previously shown
-------
527
to greatly attenuate lithium-induced flavor-aversion condi-
tioning in rats (McGowan et al. 1972; Nachman and Ashe
1974; Kolb et al. 1977; Ashe and Nachman 1980; Simbayi
etal. 1986; but see Freeman etal. 1978). Further research
on the respective contributions of limbic structures to LD-
FAC, including the effects of joint lesions, would add to
our knowledge of not only the mechanisms responsible for
the memory deficits induced by TMT but also the neural
substrates of memorial processes underlying delay condi-
tioning.
A final issue in assessing the effects of neurotoxic insult
(e.g., TMT administration) on learning and memory con-
cerns the specificity of effects when a multitude of variables
within the experimental protocol are capable of altering
the results. For instance, altered gustatory thresholds for
detecting saccharin in the present experiment could have
radically altered the behavior of rats treated with TMT.
In this regard, it is conceivable that instances of attenuated
neophobia seen in hippocampally lesioned rats could reflect
a shift in taste-reactivity to a novel flavored solution. The
use of the taste-reactivity test in the present experiment
was designed to assess the ability of rats treated with TMT
to discriminate saccharin solution from water. At concen-
trations of 0.1-3.0%, both groups of rats show nearly ident-
ical acceptance/rejection functions. Since a concentration
of 0.1% was used in the LD-FAC experiments, it appears
that both groups were equally prepared to at least discrimi-
nate the novel flavor paired with lithium administration.
On the other hand, the results from concentrations of
0.001-0.03% suggest that the rats treated with TMT may
have higher thresholds for detecting saccharin. While taste-
reactivity to saccharin has not been reported, similar tests
of gustatory toxicity have been made with solutions
(0.5-16.0 mg/l) of quinine in rats treated with TMT or
SAL showing no difference in their sensitivity to this flavor
(Myers etal. 1982). These observations deserve further in-
vestigation with other flavors (e.g., acetic acid and salt solu-
tions) in order to determine if and to what extent TMT
alters gustation.
Acknowledgement. We thank Donna Jenkins and Julia Davis for
preparing the photomicrographs, and Drs. K.M. Crofton, M.I.
Gage, and A.L. Riley for helpful comments on the manuscript.
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Received November 20, 1987 / Final version September 21, 1988
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SturoToxicoloty 5 (2); 177-186 (1984)
Copyright © 1984 by Intox Press, Inc.
BEHAVIORAL TOXICITY OF TRIALKYLTIN COMPOUNDS:
A REVIEW
Lawrence W. Reiter and Patricia H. Ruppert
Neurotoxicology Division, US Environmental Protection Agency, Re-
search Triangle Park, North Carolina 27711
ABSTRACT: Triethyltin (TET) and trimethyttin (TMT)
are neurotoxic organotin compounds which produce dif-
ferent patterns of toxicity in adult animals. Exposure to
TET produces behavioral toxicity (decreased motor activ-
ity, grip strength, operant response rate and startle re-
sponse amplitude) which reflects impaired neuromotor
function. These deficits are consistent with the reported
myelin vacuolation and cerebral edema produced by TET,
and with its direct effects on muscle. Exposure to TMT
produces both hyperactivity and impaired learning and
performance. These impairments are consistent, with re-
ported neuronal cell death produced by TMT, particularly
in limbic system structures. White the behavioral deficits
produced by repeated exposure to TET are reversible
when dosing is terminated, the behavioral impairments
produced by a single exposure to TMT appears to be ir-
reversible.
Keywords: Triethyltin, Trimethyltin, Behavioral Toxicity.
INTRODUCTION
To a limited extent, the neurotoxicity of both inorganic and
organic tin compounds has been recognized for many years. In the
early 19th century, Orfila described muscular weakness, loss of
pain sensation and immobility in dogs exposed to tin chloride (Kim-
brough, 1976). In 1880, White reported that vapors of triethyltin
acetate produced headaches, general weakness, nausea and diarrhea.
The following quote from Sollmann's (1906) textbook on pharmacology
refers to the report of White and recognizes that the CNS is a prin-
cipal target organ for tin compounds:
Please send requests for reprints to Or. Lawrence W. Reiter.
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17»
REITER AND RUPPert
"This metal is absorbed in part even from non-corrosive
preparations...The symptoms on injection devolve to some
extent on the central nervous system, as stimulation and
•subsequent paralysis.. .With more chronic poisoning the
gastroenteritis is most marked, but there is also an a-
taxia and motor paralysis, resembling chronic lead-poi-
soning (White, 1880)".
The present review will focus on the behavioral toxicity of the
alkyltin compounds. Since the major research emphasis has been on
the trialkyltins, and more specifically on triethyltin (TET) and
trimethyltin (TMT), we will review and contrast the behavioral ef-
fects of these two compounds.
SIGNS AND SYMPTOMS OF POISONING IN HUMANS
The signs and symptoms associated with human exposure to TET
and TMT are summarized in Table 1.
TABLE 1. Signs and Symptoms of Trialkyltin Poisoning in Humans
TRIETHYLTIN
TRIMETHYLTIN
Headaches
Abdominal Pain
Visual Disturbances
Vert igo
weight Loss
Hypothermia
Paralysi s
Papi 11oedema
Headaches
Pa in (genera Iized)
Visual Disturbances
Disorientation
Loss of Appetite
Memory Deficits
Sleep Disturbances
Loss of Libido
Bouts of Depression
Attacks of Rage
Source: Barnes end
Stoner, 1959
Sources:
(1) Fortemps et
(2) Ross et al.,
a I., 1978
1981
An extensive episode of TET poisoning occurred in France in the
1950's resulting from the contamination of Stalinon, a proprietary
preparation used in the treatment of skin infections (for review see
Barnes and Stoner, 1959). Stalinon capsules, which contained di-
ethyl tin and linoleic acid, were contaminated with monoethyltin and
TET. Of the 217 people reportedly poisoned by this preparation, 100
died.
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BEHAVIORAL TOXICITY OF TRIALKYLTINS
179
Occupational exposures have accounted for two reported episodes of
TMT poisoning. The first episode involved two chemists who were
synthesizing dimethyltin and were inadvertently exposed to vapors
of dimethyl- and trimethyltin chloride for approximately 3 months
(Fortemps et al., 1978). The second episode involved twenty-two
chemical workers who were exposed to both dimethyl- and trimethyl-
tin dichloride and methyl bromide due to a malfunctioning
ventilation system (Ross et al., 1981). Since some of the
non-specific signs and symptoms of toxicity in humans overlap be-
tween TET and TMT, the toxicity of these two compounds was generally
thought to be qualitatively similar, differing only in relative po-
tency. In retrospect, however, while certain symptoms are common to
both compounds (e.g., headaches, pain, loss of weight/appetite),
there are important differences in the overall clinical syndrome.
TET toxicity reflects primarily a sensorimotor deficit while TMT
reflects specific behavioral impairment. As we know now, these dif-
ferences in symptoms reflect marked differences in the underlying
neurotoxicity associated with exposure to these two compounds.
SIGNS OF POISONING IN ANIMALS
Stoner et al. (1955) described the acute toxicity resulting
from organotin exposure in animals. TET was the most potent, al-
though other alkyltins produced similar signs of poisoning. Muscle
weakness was observed within hours after dosing; following a short
period of recovery, tremors developed, leading to convulsions and
death 2-5 days after dosing. Tremors were more pronounced after ex-
posure to TMT; aggressive sparring with cage mates was also observed
(Stoner et al., 1955; Barnes and Stoner, 1958). Even in recent re-
ports, the acute toxicity of TET and TMT has been described as being
qualitatively similar: trembling, irritation, twitching, loss in
body weight and progressive oaralysis (Tan and Ng, 1977). The ap-
parent similarity in signs of poisoning between TET and TMT during
the acute phase of toxicity led these compounds to be classified as
general examples alkyltin intoxication.
Although increased water content of the brain and spinal cord
warn observed with TET and not TMT (Barnes and Stoner, 1958), the
neuronal damage produced by TMT within the limbic system was not de-
scribed until recently (Brown et al., 1979). Behavioral studies
have clearly differentiated between the neurotoxicity of TET and
TMT. Tables 2 and 3 summarize behavioral studies of TET and TMT
toxicity, respectively. Although toxicity produced by TET becomes
¦ore pronounced with continued exposure, reversal of behavioral de-
ficits occurs within weeks after termination of dosing. In
contrast, following acute exposure to TMT, toxicity progresses from
acute effects, through a period of hyperreactivity characterized by
tail mutilation, vocalization and seizures (Oyer et al., 1982), to a
stage where overt signs of poisoning are no longer evident but be-
havioral deficits persist.
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180
TABLE 2. Behavioral Effects of Triethyltin Exposure in Adult Rodents
BEH4 'ORAL
ME'- .RE
(SPECIES)
EXPOSURE LEVELS
(DURATION)
EFFECT
REFERENCE
Moto r Act i v i ty
(Mouse)
2 mg/kg; ip
(27 days)
Hypoactivity Gerren et a I.. 197$
(Rat)
1.5 or 3.0 mg/kg;
sc (1 dose)
Hypoactivity Reiter et ai., 1930
5 or 10 ppm; water Hypoactivity
(3 weeks)
Rei ter et a 1., 1980
Startle Response
(Rat)
5 or 10 ppm; water Reduced
(3 weeks) Amplitude
Rei ter at a 1., 1980
1 or 2 mg/kg; po
(3 doses)
Reduced
Amp I itude
Squibb et a I., 1980
Landing Foot-Spread
(Rat)
5 or 10 ppm; water Reduced
(3 weeks)
Rei ter et a i., 1980
Grip Strength
(Rat)
1 or 2 mg/kg; po
(3 doses)
Reduced
Squibb et a I., 1980
Response to Pain
(Rat)
0.25 or 0.5 mg/kg; Impel red
sc (14 doses)
Til son and Burne, 1981
Active Avoidance
(House)
2 mg/kg; ip
(27 days)
NO Effect
Cerren et al., 1976
Schedule-Control led
Performance (Rat)
0.5, 1.0 or 1.5 Reduced Rates
mg/kg; ip (4 doses)
Do Haven et a I., 1982
Flavor Aversion
(*»t)
1 or 3 mg/kg; po
(4-5 doses)
PosItIve
Leender and Gau, 1990
0.375, 0.75, 1.5 or Positive
3.0 mg/kg; Ip
(2 doses)
MacPhall, 1982
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BEHAVIORAL TOXICITY OF TRIALKYLTINS 181
TABLE 3. Behavioral Effects of Trimethyltin Exposure in Adult Rodents
BEHAVIORAL
measure
(SPECIES)
EXPOSURE LEVELS
(DURATION)
EFFECT(S)
REFERENCE
Motor Activi ty
(Rat)
7 mg/kg; po
(1 dose)
Hyperactivi ty
Swa rtzweIde r
et a I ., 1981
5, 6 or 7 mg/kg;
po (1 dose)
Hyperactivity Ruppert et a I,,
Altered Pattern 1982
of Act ivi ty
(Mouse)
3 mg/kg; ip
(1 dose)
Hypoactivity Wenger et a I.,
Altered 1982
Ci read ian Rhythm
RadiaI-Arm Maze
(Rat)
6 mg/kg; po
(1 dose)
Decreased
Accuracy
Wa I sh et a I.,
1982b
Schedule-
ControI led
Performance
(Rat)
7 mg/kg; po
(1 dose)
increased Rat^s
Swa rtzwe I de r
et a I.,1981
(Mouse)
3 mg/kg; ip
(1 dose)
Reduced Rate Wenger et ai,,
Altered Pattern 1982
Passive Avoidance
(Rat)
5, 6 or 7 mg/kg;
po (1 dose)
Impai red
Retention
wa Ish et a I.,
1982a
Hebb-WI11 lams Maze 7 mg/kg; po
(Rat) (1 dose)
Increased
Error
Swa rtzwe I.der
et a I., 1982
Alcohol Selection
(Rat)
7 mg/kg; po
(1 dose)
Reduced
Preference
Myers et a I.,
1982
Flavor Aversion
(Ret)
0.625, 1.25, 2.5
or s.O; ip
(1 or 3 doses)
Positive
MacPhaiI,
1982
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182
REITER AND RUPPERT
These differences in long-term toxicity have led to different stra-
tegies for the assessment of behavioral impairment. Evaluations of
TET have generally focussed on repeated testing throughout the pe-
riod of dosing, followed by a recovery period. Evaluations of spe-
cific behavioral impairments produced by TMT have generally
focussed on testing weeks to months after a single injection.
BEHAVIORAL EFFECTS OF TET
TET produces primarily a neuromotor impairment which is rever-
sible. Following subacute administration of 5 or 10 ppm TET in the
drinking water, decreases in locomotor activity were seen in
figure-eight mazes (Reiter et al., 1980); within 2 weeks after re-
moval of TET from the drinking water, the motor activity of
TET-exposed animals had returned to control levels. Squibb ec al.
(1980) used a different dosing regimen to evaluate the behavioral
effects of TET. With repeated exposures of either 0, 1, or 2 mg/kg
(po) to adult rats, dose-related decreases in both forelimb and
hindlimb grip strength were seen. As with motor activity, recovery
appeared to be complete by 4 weeks after dosing. The landing-foot
spread technique of Edwards and Parker (1977) indicated similar
neuromotor impairment and recovery (Reiter et al., 1980).
The startle response to both acoustic (Reiter et al., 1980;
Squibb et al., 1980) and tactile (air-puff) stimuli (Squibb et al.,
1980) was reduced during exposure to TET; these effects were also
reversible on termination of exposure. Since the motor component of
the startle reflex is the same for both acoustic and tactile
stimuli, the reduction in startle amplitude to both stimuli proba-
bly reflects neuromuscular weakness. Additional data of Fechter et
al. (1982) indicated that 30 ppm TET in the drinking water, an expo-
sure level specifically chosen to produce motor dysfunction,
reduced acoustic startle responding but did not produce a shift in
auditory thresholds. These data support the conclusion that re-
duced startle responding does not reflect specific sensory
impairment. Tilson and Burne (1981) reported a similar conclusion
for pain sensitivity. Responsiveness to painful stimuli was im-
paired during tail-flick and hot-plate testing but not on an operant
shock titration procedure requiring a nose-poke response. However,
while the tail-flick and hot-plate responses required large motor
movements, the nose-poke response required only a small head move-
ment. Recovery was seen by 2 weeks after dosing.
Both operant performance, conditioned flavor aversion, and ac-
tive avoidance learning have been assessed in TET-exposed animals.
Operant response rates on both fixed-interval and fixed-ratio
schedules of reinforcement were reduced when TET was injected in
rats immediately before the test session (DeHaven et al., 1982).
Conditioned flavor aversions have been consistently reported fol-
lowing acute and repeated exposures to TET (Leander and Gau, 1980;
MacPhail, 1982). When mice were exposed to 2 mg/kg TET for 27 days
and active avoidance training was begun 2 days after ths end of dos-
-------
BEHAVIORAL TOXICITY OF TRIALKYLTINS
183
ing (Gerren et al., 1976), no impairment was seen in the ability of
mice to turn a wheel to avoid footshock. Interestingly, while there
was no difference in the total number of wheel turns between control
and TET-exposed mice, spontaneous activity, measured daily 24 hr
after dosing, was decreased by approximately 65% in TET-exposed
mice. However, when shock avoidance was contingent on motor activ-
ity, TET animals were able to compensate for motor impairment.
To summarize, TET exposure produces decreases in motor activ-
ity, grip strength, operant response rates and startle response am-
plitudes as well as conditioned flavor aversions. These effects are
generally reversible 2-4 weeks after the termination of exposure.
TET does not appear to affect acquisition of an active avoidance
task or the threshold to pain.
BEHAVIORAL EFFECTS OF TMT
Swartzwelder et al. (1981) reported that rats dosed with 7
mg/kg TMT were three times more active than controls when tested in
an open field 40 days after exposure. Locomotor activity was also
assessed in figure-eight mazes at several time points following a
single exposure to 0, 5, 6 or 7 mg/kg TMT (Ruppert et al., 1982).
There were no differences in activity 2 hr after dosing, but animals
given 7 mg/kg TMT became progressively hyperactive^frora day 4 to day
16 after dosing. This time course parallels the development of lim-
bic system damage, which first appears 2 days after dosing and
becomes maximal within 3 weeks (Brown et al., 1979). These data il-
lustrate both the steep nature of the dose-effect curve for TMT (no
change in activity was seen at 5 or 6 mg/kg TMT) and the persistence
of TMT toxicity (animals remained hyperactive 50 days after
dosing).
Performance of a previously learned task (radial arm maze) and
learning of new tasks (Hebb-Williams mazes, active and passive
avoidance) have also been shown to be impaired by TMT. In a study
by Walsh et al. (1982b), rats previously trained to perform a radial
arm maze task were intubated with 0 or 6 mg/kg TMT. Animals rou-
tinely maximize accuracy on this task by entering all baited arms,
while avoiding arms which have been previously entered, until all
reinforcers are obtained. TMT-treated animals were less accurate
in selecting arms of the maze which still contained food, and re-
quired more arm entries to obtain all reinforcers; this deficit in
performance persisted for at least 70 days after dosing. Although
hyperactivity can be a confounding factor in interpreting learning
and performance deficits, there wera no differences in activity be-
tween groups on the radial arm maze 15-35 days after dosing.
Swartzwelder et al. (1982) alto evaluated the effects of TMT
exposure on performance in a Hebb-Williams maze. Animals receiving
7 mg/kg TMT made more errors than controls and also made more per*
severative responses, repeatedly entering blind alleys which did
not lead to reinforcement. In a subsequent study from this same
laboratory, alcohol consumption was reduced in rats tested 21 and
-------
184
REITER AND RUPPERT
150 days following 7 mg/kg TMT (Myers et al.t 1982). TMT exposure
has also been shown to impair retention of a passive avoidance task
(Walsh et al., 1982a) and to facilitate active avoidance perfor-
mance (Mactutus et al., 1981). These data are consistent in indi-
cating that TMT exposure impairs acquisition and/or performance of
tasks involving learning and memory.
Schedule-controlled performance is also affected by TMT expo-
sure. Response rates on a schedule with increasing fixed-ratio re-
quirements were increased by TMT (Swartzwelder et al., 1981); this
effect was directly related to the ratio requirements. In contrast,
Venger et al. (1982) reported a decreased rate of responding in
TMT-exposed mice performing on a multiple fixed-ratio,
fixed-interval schedule of reinforcement. Although a species or
schedule difference could account for this discrepancy, the time of
testing relative to exposure is probably a key factor contributing
to this difference. Mice were tested by Wenger et al. during the
acute phase of intoxication, 3-51 hours after dosing, while rats
were tested by Swartzwelder et al, at least 40 days after dosing.
Studies are needed to evaluate the cumulative toxicity of TMT.
MacPhail (1982), for example, showed that the dosages required to
produce conditioned flavor aversion were less following repeated
administration of TMT than following a single dosage.
In summary TMT produces locomotor hyperactivity, deficits in
learning and memory, conditioned flavor aversions and changes in
operant response rates. These effects persist after a single expo-
sure to TMT, and appear to be irreversible.
CONCLUSIONS
TET and TMT produce contrasting behavioral effects in adults
which probably reflect differences in their effects on the nervous
system. Both TET and TMT have been used as model compounds to in-
vestigate nervous system structure and function after defined neu-
rotoxic insult. TET has been studied as a model of cerebral edema
and neurodegenerative disorders, while TMT has been proposed as a
model of hippocampal lesions, minimal brain dysfunction, the septal
syndrome and kainic acid neurotoxicity. While these latter cat-
egorizations may be useful in guiding future research, they should
not be allowed to constrict our viewpoint. For example, dramatic
decreases in startle responding produced by TMT appear to reflect an
ototoxic effect (Howell et al., 1981; Reiter et al., 1983; and Young
and Fechter, 1983) and, therefore, a complete understanding of the
behavioral toxicity of this intriguing compound awaits further re-
search.
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BEHAVIORAL TOXICITY OF TRIALKYLTINS
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Fechter LD, Bierkamper GG, Young JS. Effects of triethyltin
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Fortemps E, Amand G, Bomboir A, Lauwerys R, Laterre EC. Tri-
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Gerren RA, Groswald DE, Luttges MW. Triethyltin toxicity as a
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Kimbrough RD. Toxicity and health effects of selected organotin
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Leander JD, Gau BA. Flavor aversions rapidly produced by inor-
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MacPhail RC. Studies on the flavor aversions induced by trialkyl-
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Reiter LW, Ruppert PH, Dean KF. Trimethyltin (TMT) disrupts
acoustic startle responding in adult rats. The Toxicologist
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Ross WD, Emmett EA, Steiner J, Tureen R. Neurotoxic effects of
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Soilmann T. A textbook of pharmacology. 2nd Edit. W.B. Saunders
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Tilson HA, Burne TA. Effects of triethyl tin on pain reactivity
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Walsh TJ, Miller DB, Dyer RS. Trimethyltin, a selective limbic
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Wenger GR, McMillan DE, Chang LW. Behavioral toxicology of
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NeurobehavioraI Toxicology and Teratology, Vol. 3, pp. 285-293, 1981. Printed in the U.S.A.
Developmental and Behavioral Effects of
Early Postnatal Exposure to
Trie thy ltin in Rats
LAWRENCE W. REITER, GLENDORA B. HEAVNER,
KAREN F. DEAN AND PATRICIA H. RUPPERT
Neurotoxicology Division, US Environmental Protection Agency, Research Triangle Park, NC 27711
Received 2 April 1981
REITER, L. W., G. B. HEAVNER, K. F. DEAN AND P. H. RUPPERT. Developmental and behavioral effects of early
postnatal exposure to triethyltin in rats. NEUROBEHAV. TOXICOL. TERATOL. 3(3) 285-293, 1981.—On Day 5 of
postnatal life, rat pups received a single injection of triethyltin and were later tested for a variety of developmental and adult
behaviors. A within-litter dosing design was used with one male and one female from each litter (N=8 pups/litter) receiving
either 0 (normal saline vehicle). 3,6 or a high dose of either 9 or 12 mg/kg triethyltin bromide (TET). The high doses of TET
produced 50% and 80% mortality, respectively. For the 3 and 6 mg/kg groups, TET-exposure resulted in a transient
decrease in body weight, and a permanent decrease in brain weight. Preweaning TET-exposed pups were less successful in
descending a rope, and were less active in both a homing orientation test and a figure-eight maze. When tested as adults,
however, these animals were consistently more active than controls in the figure-eight maze. These results indicate that a
single exposure to TET in the developing rat, unlike the adult, produces permanent alterations in both brain and behavior.
Acute postnatal exposure to toxicants may have general applicability as a model for developmental neurotoxicity.
Triethyltin Postnatal exposure Behavior Development Rats
THE development of the mammalian nervous system occurs
over a protracted period of time, beginning early in gestation
and extending into the postnatal period. Different compo-
nents of development, including cell division, migration, and
differentiation are vulnerable to disruption by a variety of
factors, including chemical exposure [32]. In order to assess
the consequences of toxicant exposure on the developing
nervous system, several exposure models have been em-
ployed [36] which include exposure during some portion of
the prenatal or early postnatal period. Considering the rapid
rate of neural development it is predictable that the functional
consequences of exposure will differ with the period of ex-
posure. In fact, this approach has been successfully em-
ployed to define the normal timing and sequence of func-
tional development associated with various brain structures
[12, 13, 25].
In the rat, the processes of neural cell division and mat-
uration are still active postnatally [1]. Dobbing [8] reported
that the rat brain undergoes rapid growth, termed the brain
"growth spurt", during the early postnatal period. Although
most postnatal cell division is associated with glia, postnatal
neurogenesis has been demonstrated in the hippocampus,
cerebellum, and olfactory bulbs [1]. Furthermore, quantita-
tive histological studies indicate that few dendrites are pres-
ent in the rat prenatally, whereas adult levels are reached by
postnatal Day 12 [9]. Early postnatal exposure to toxicants in
the rat should, therefore, evaluate the potential for a chemi-
cal to disrupt neural development.
Certain toxicants which produce long-lasting behavioral
deficits when administered to the developing rat also
produce hypoxia. Carbon monoxide produces hypoxia by
interfering with the oxygen carrying capacity of hemoglobin.
Culver and Norton [7] demonstrated that acute exposure to
carbon monoxide in 5-day-old rat pups produced hyperac-
tivity which was present between 4-6 weeks of age. Lead
disrupts a variety of mitochondrial functions including un-
coupling oxidative phosphorylation and inhibiting respira-
tion [3], and therefore high levels of exposure would produce
cytotoxic hypoxia. Several investigators have demonstrated
that early postnatal exposure to high levels of inorganic lead
also produces hyperactivity in the rat [20, 21, 26]. The pres-
ent study was designed to test the hypothesis that a toxicant
which disrupts energy metabolism during early postnatal de-
velopment will produce neurotoxicity characterized by
hyperactivity. Because of its known toxicity, triethyltin
(TET) was chosen as a prototype compound to test this hy-
pothesis.
TET inhibits both oxidative phosphorylation and coupled
respiration in isolated mitochondria [29] and inhibits glucose
oxidation both in vivo [5] and in vitro [16]. Exposure of adult
animals results in splitting of the myelin sheath at the inter-
period line which dilates and fills with fluid [17]; total myelin
content of the brain is also reduced [10]. In the neonate, the
degree of cerebral edema produced by TET exposure is less
than in the adult [30,31]. Since myelin formation begins be-
tween 7-10 days postnatally in the rat [14], TET administra-
tion at an early age would have only limited potential for
producing myelinopathy (vacuolation), although myelin de-
velopment could be impaired.
In the adult rat, TET produces behavioral changes which
285
-------
286
REITER ET AL.
are reversible following termination of exposure [24,28].
However, the behavioral toxicity of TET in the developing
rat had not been reported. In the present experiment, there-
fore, TET was administered to rat pups on postnatal Day 5
and pups were tested for physical maturation, development
of reflexes, motor coordination and motor activity. These
animals were also tested for motor activity as adults to de-
termine if behavioral impairment was reversible. A prelimi-
nary report of these findings has been published elsewhere
[23], and since then, the developmental toxicity of TET has
also been reported in a study using a similar postnatal expo-
sure [11].
EXPERIMENT I
METHOD
Animals
Pregnant CD rats (Charles River;Wilmington, MA) were
obtained 2 days after mating and housed singly in cages
measuring 45 x 24 x 20 cm. On the day of parturition (Day 0),
pups were randomized and each dam was assigned 4 male
and 4 female pups. At 21 days of age, animals were weaned
and littermates were housed by sex in groups of 3 or 4 in the
same size cages. Animals were maintained on a 12:12 hr
light:dark cycle beginning at 0600 hr, in a temperature
(22°C±2) and humidity (50%* 10) controlled animal facility.
Food (Purina Lab Chow) and water were available ad lib
throughout the experiment. All behavioral testing was con-
ducted during the diurnal period.
Dosing
On Day 5, each pup received a single intraperitoneal in-
jection of either triethyltin bromide as the salt (TET, Alfa
Products: Danvers, MA) or vehicle (sterile isotonic saline) in
a volume of 10 /xl/gm of body weight. Two groups of
animals, 10 litters/group, were used in this study. Group I
pups were injected with either 0, 3, 6, or 12 mg/kg TET. A
within-litter design was used for dosing: one female and one
male from each litter received each dose and therefore each
litter contained all treatments. Group I animals were subjects
for all tests except the development of open field activity.
For the developmental open field testing, a separate group of
animals. Group II, was subsequently used. Because all but 2
of the animals receiving 12 mg/kg TET(Group I) died within
3-8 days of dosing, pups in Group II received either 0, 3,6 or
9 mg/kg TET. Nevertheless, by Day 14, half of the 9 mg/kg
TET pups had died, and testing was, therefore, discontinued
for this group.
Body Weight
Rats were weighed at 5 days of age and every 3 days
thereafter until 20 days of age. After weaning, animals were
weighed approximately once a month throughout testing. At
1 year of age. animals were sacrificed and wet weights of
whole brains were recorded.
Developmental Testing
Several developmental tests were adapted from those re-
ported by Altman and Sudarshan [2]. Diagrams of the appa-
ratus, further descriptions of the tests, and developmental
trends of normal animals may be found in that article. For
this study, the age at testing and the criteria for each task
(A-G) are shown in Table I.
A. Negative geotropism: Each pup was placed on a 17°
inclined surface with its head pointed downward, and was
allowed 180 sec to turn 180°. If the pup did not complete the
turn, it was assigned the maximum latency of 180 sec.
B. Placing reaction (chin elicited): Each pup was held by
the nape of the neck and touched lightly on its chin with a
rod. No response to the rod was scored as 0; slight lifting of
the forelimbs without contact with the rod as 1; slow and
uncertain lifting of the forelimbs with extending paws and
spreading digits was rated 2; swift raising of the shoulders
and forelimbs with hindlimbs displaying treading movements
was scored 3.
C. Elevation of head: Each pup was observed for 1 min in
an empty clear plastic cage. The amount of time in which the
head was clearly raised above the surface of the cage was
recorded.
D. Ascending on a wire mesh surface: A 45x15 cm
wooden frame, covered with 6 mm wire mesh was con-
structed at a 70° angle with a platform at the top. This appa-
ratus was placed with the bottom resting in cold water (ap-
proximately 15°C). Three littermates were placed on the plat-
form and individual pups were then removed and placed on
the wire mesh with their hindlimbs touching the water. La-
tency to ascend to the platform was recorded, with a
maximum set at 180 sec.
E. Acoustic startle response: Each pup was held by the
nape of the neck, and a toy clicker was sounded immediately
behind the pup's head. Three daily trials were given until a
criterion was reached of a muscle jerk in any of the ex-
tremities on at least 1 trial on 2 consecutive days.
F. Eye opening: Pups were observed for complete open-
ing of both eyes.
G. Air righting reflex: Each pup was held in a supine
position and dropped from a height of 20 cm onto a bed of
wood shavings. Pups were tested until the ability to right in
mid-air and land on all four feet was demonstrated on 2 out of
3 daily trials for 2 consecutive days.
H. Descending on a rope: From 15-20 days of age, pups
were tested daily for the ability to descend a 30 cm x 17 mm
rope suspended above the home cage. Each pup was held by
the nape of the neck and placed against the rope with its head
pointing upward. Pups were allowed 180 sec to descend
without falling.
I. Open field: Pups were observed for 5 min at 10, 15, 20
and 25 days of age in an open field measuring 90x 90 x 60 cm
and divided into sixteen 22.5 cm squares. The pup was
placed in the center of the field and a crossover was counted
each time a square was entered with all four feet.
Postweaning Testing
A. Activity in a figure-eight maze: Locomotor activity
was measured at various ages in a figure-eight maze [22]. The
maze is a series of interconnected alleys (lQx 10 cm) con-
verging on a central arena and covered with transparent
acrylic plastic. Motor activity is detected by 8 phototransis-
tor/photodiode pairs. Rats were tested individually for 1 hr at
21,57,112-118 and 238 days of age, and for 23 hr at 258 days
of age.
B. Open field: At 110 days of age, behavior was measured
for 5 min in the open field described above. Activity was
measured as latency (time from the beginning of the test to
the first crossover), crossovers (counted each time an animal
entered a square with all four feet), central tendency (cross-
overs within the 4 central squares) and rears (counted each
-------
EFFECTS OF POSTNATAL TRIETHYLTIN
FEMALE
too
-MALE
0 CONTROL •
700 -
600
500
1 — 300
200
40
30 —
0 CONTROL
A3mfl/kg
~ 0 mg/kg
AGE. days
FIG. I. Body weights of control and TET-dosed rats. Values are
expressed as group means (N«8-l0/group). Values for the standard
error of the mean for preweaning body weights do not exceed the
size of the symbols. Acute exposure to TET on postnatal Day J
produced a slight growth depression during the preweaning period
but not during the postweaning period.
time an animal raised both front paws off the floor). Testing
was conducted under normal lighting, and activity was moni-
tored on closed circuit television by observers in an acjjacent
room.
Statistical Analysis
For body weight and behavioral tests with repeated
measurements, data were analyzed by a multivariate general
linear model [191; when significant differences were found,
individual data points were analyzed by ANOVA. For tests
with a single measurement, rope descent (scores collapsed
across days), adult open field, 23 hr maze activity and brain
weight, individual ANOVAs were performed. Values greater
than the critical value at p<0.05 were accepted as signifi-
cant. Post hoc comparisons were made using Tukey's (a) test
[34],
RESULTS
Body weights for Group I animals an shown separately
for preweaning and postweaning weights in Fig. 1. Prewean-
287
2.3
2.2
< 2.0
1.8
FIG. 2. Brain weights in one-year old control and TET-dosed rats.
Data are pooled for males and females and are presented as means =
SE (N»l8-20/group). TET exposure produced a dose-related de-
crease in brain weight.
ing TET-dosed rats weighed significantly less than controls.
F(12,96)=3.49, p <0,0003. At postweaning ages, males
weighed more than females, F(10,44)=55.71, p<0.0001, but
there were no differences due to treatment. Thus, TET ex-
posure resulted in a transient decrement in growth during the
preweaning period.
Brain weight (Fig. 2) at 12 months of age was significantly
decreased by TET, F(2,50)= 18.23, /?<0.0001. No independ-
ent sex effect or sex by treatment interaction was found, so
brain weights for males and females were combined in Fig. 2.
Although there was no significant treatment effect on body
weight at this time, there was a direct relationship between
brain weight and body weight i.e., body weight was a signifi-
cant covariate of brain weight, F(l,50)=25.6t, p<0.0001.
The 7.2% decrease in brain weight at 3 mg/kg, and the 14%
decrease at 6 mg/kg were both significantly different from
control brain weights (p<0.01).
Developmental Testing
Results of developmental testing are shown in Table 1.
For negative geotropism, on each of the 3 days of testing,
10-25% of the control pups and 5-15% of the TET-dosed
pups fell or rolled off the incline; this was an unexpected
finding. However, there were no statistically significant
differences between treatment groups, F(6,102)»0.882,
p <0.512. There were also no group differences in the placing
reaction or in the time spent with the head elevated. For the
task of ascending a wire mesh surface, most pups fell on the
first day, but all became more proficient with repeated test-
ing; there was no treatment effect. The age of development of
the acoustic startle, eye opening and air righting also showed
no indications of treatment effects.
-------
288
REITER ET AL.
TABLE 1
DEVELOPMENTAL MEASUREMENTS IN CONTROL AND TET-DOSED RATS
(N-17-20/DOSE)
Task Description
Measurement
Age (Days)
at Testing
0
Dose of TET (mg/kg)
3
6
Negative Geotropism
Latency to turn
6
108
-f-
15
129 £
13
142
10
180°(sec)
7
68
14
50 £
11
64
£
13
8
58
15
46 *
12
54
£
13
Placing Reaction
Strength of
9
1.0
0.3
1.6 -
0.6
1.3
£
0.3
(Chin Elicited)
Response (0-3)
Elevation of Head
Time (sec) spent with
9
0.8
£
0.4
4.6 ±
1.7
3.4
£
1.5
head elevated in 1 min
12
4.8
£
0.1
5.3 £
1.1
4.3
£
0.8
Ascending on a wire
Latency to ascend
11
162
£
10
174 £
6
180
£
10
mesh
to top (sec)
12
114
£
16
123 £
5
155
11
13
121
±
15
119 ±
14
98
£
14
14
88
15
76 £
13
52
£
3
Acoustic Startle
Age (days) at development
10-14
13.0
£
0.2
13.4 £
0.2
13.4
£
0.1
Eye Opening
Age (days) at development
—
15.4
£
0.2
15.4 £
0.2
15.4
£
0.1
Air Righting Reflex
Age (days) at development
16-19
18.2
£
0.2
18.4 £
0.2
18.6
£
0.1
Data on descending a rope are shown in Fig. 3. Pups were
scored as either successful (descended without falling) or
unsuccessful (fell). The data were combined for all test days
and a proportion was obtained. An arcsin transformation
was performed on these proportions and an ANOVA was
conducted. This analysis indicated a significant effect of
treatment, F(2,43)=9.84,p<0.0Q03. The 6 tug/kgTET-dosed
rats were less successful than either controls (p<0.01) or 3
mg/kg TET rats (p<0.05) in completing this task.
Ambulation increased with age in the open field as shown
in Fig. 4, but there was no significant treatment effect on
number of crossovers.
Postweaning Testing
Locomotor activity in the figure-eight maze is shown in
Fig. 5. Data analysis of log,0 scores showed an overall treat-
ment effect, F(2.53)»9.09, p<0.0005. The 6 mg/kg TET-
dosed rats were more active than both control rats
(p <0.0003) and 3 mg/kg TET rats (p<0.002). Also females
were more active than males, F( 1,53)=24.93, p <0.0005), but
there was no interaction of treatment with sex,
F(2,53)=1.19, p<0.314.
Hyperactivity persists over a 23 hr period as shown in
Fig. 6. Analysis of Iog,0 transformed total 23-hr counts
(shown in the insert) indicated that 6 mg/kg TET-dosed rats
were hyperactive, F(2,52)=7.38, p<0.01, in comparison to
both control (p<0.05), and 3 mg/kg TET-dosed rats
(p<0.05). Again, females were more active than males,
F(2,53)» 15.96, /><0.0l.
Measurements of activity in the open field are sum-
marized in Table 2. Activity counts in the figure-eight maze
for the first 5 min of a 1-hr test at 110 days of age are also
included. There was no treatment effect on activity meas-
ured for 3-min in either the open field or the figure-eight
maze. However, females were more active than males,
F(5,49)=5.69,p<0.0004. Univariate analysis showed the sex
100
S 70
<3" »o
•CONTROL
A 3 mg/kg
¦ 6 mg/kg
FIG. 3. Rope descending in control and TET-dosed rats. Data are
presented as the percentage of animals successfully descending the
rope at various ages (N »16-20/group). The mature response pattern
is shown in the insert (redrawn from Altman and Sudarshan. 1975).
TET exposure produced a significant decrement in the development
of this response.
-------
EFFECTS OF POSTNATAL TRIETHYLTIN
289
90—
80 —
O CONTROL (N ¦ 201
A 3 mg/kg IN • 191
Q 8 mg/kg IN - 15)
60 —
SO —
40 •
30-
20-
10 —
_ 15 26 28
AGE, davi
FIG. 4. Development of open field activity in control and TET-
dosed rats. There was a progressive increase in the number of cross-
overs recorded during a 5-min test period between 10 and 25 days of
age. Data are combined for males and females (N» 15-20/group).
TGT exposure did not significantly alter the development of open
field activity.
3.r
difference in crossovers, F(l,53)=4.58, p<0.035; rears,
F(l,53)=5.50, p<0.022; and 5-min maze activity, F(l,53)
=5.57, p<0.02.
EXPERIMENT 2
METHOD
In a second study, the effects of TET exposure on the
development of motor activity were further examined. To
simplify testing, each litter was reduced to 4 males at parturi-
tion and injected on Day 5 with either 0 or 6 mg/kg TET. A
total of 10 control and 12 TET-dosed litters were tested.
From 2 to 10 days of age, homing orientation was measured
in 4 control and 4TET-exposed litters; on Day 14, all 22 litters
were reduced to 3 pups and the development of 23-hr activity
in the figure-eight maze was measured on Days 15 through
21.
Developmental Testing
Homing orientation: Rats were tested for development of
nonambulatory homing orientation (2). This behavior was
measured in a circular arena (11 cm in diameter), as shown in
Fig. 7, which was positioned between two alleys (7.5 cm
long) leading to either the home cage containing mother and
littermates or to a cage containing only clean bedding. Pups
were tested daily for 180 sec and their orientation was scored
at 10 sec intervals. Rats were initially placed in the arena at a
right angle to both alleys. Direct orientation towards the
home cage or the empty cage was scored as +1 or -1, re-
spectively; intermediate orientations were scored as + '/: or
Since the development of motor activity confounded
orientation in this study, the 10 sec scores were also used to
estimate the development of ambulation. A crossover was
FEMALE
O CONTROL
O Sm^k|
¦Jg JoT
AO I, dtyt
FIG. 5. One hour figure-eight maze activity in control andTET-dosed rats. Log»» activity scores are
present^ sepwatelyfor males and female. * SE'
Acute exposure to 6 mg/kg TET on postnatal Day 5 resulted in a persistent hyperactivity.
-------
290
REITER ET AL.
TIT
m
Ml
00SK TET. m%!k«
m
. sss
|-
>
>
<
MALES
1.21
SI
QOSi TIT.
MM
LIGHTS Oil
FIG. 6. Figure-eight maze activity of control and 6 mg/kg TET-
dosed rats. Individual rats (258 days of age) were tested for 23 hr;
values are expressed as mean activity for I hr intervals for males and
females (N=9-IO/group). Group means for the total 23 hr activity
are shown in the insert.
O CONTROL IN-1B)
Q 6 mq/kg (N-161
EMPTY ( 1
/ —
TET EXPOSURE
TET EXPOSURE
AGE, days
FIG 7. Development of homing orientation and ambulation in control male rats and in those exposed
to TET on postnatal Day J (N = 16/group). No effect of TET on development of orientation was
observed (left panel). Ambulation, measured as the number of crossovers in the 180-sec test period,
was significantly depressed by TET (right panel). A diagrammatic representation of the test apparatus
is shown in the upper left insert.
-------
EFFECTS OF POSTNATAL TRIETHYLTIN
291
TABLE 2
EFFECTS OF TET EXPOSURE ON J-MIN OPEN FIELD BEHAVIOR AND FIGURE-EIGHT MAZE ACTIVITY
IN ADULT (110-117 DAY OLD) RATS. VALUES ARE EXPRESSED AS MEANS =SE (N-9-IO/DOSE)
Males
Control 3 mg/kg
6 mg/kg
Control
Females
3 mg/kg
6 mg/kg
Latency
(sec)
Crossovers
Rears
5 min
Figure-Eight
Maze
3.2 £ 0,7 4.2 £ 0.7 3.1 2: 0.9 4.3 £ 1.0 4.5 £ 1.0 5.5 £ 1.6
79 £ 9 68 £ 12 90 £ 8 99 £ 5 102 £ 14 111 £ 10
17 £ 2 13 £ 2 14 £ 4 24 £ 5 18 £ 2 17 £ 3
85 £ 10 79 £ 9 108 £ 9 100 £ 2 117 £ 9 110 £ 9
O CONTROL (N-10 UTTERS)
~ 8 rna/ka (N-12 LITTERS)
AuC, dcyt
FIG. 8. Development of figure-eight maze activity in control and
TET-dosed male rati (N-10-12 litters/treatment). Litters of 3 male
pups were tested between 15-21 days of age. Mothers were re-
stricted to the nest box. TET-exposure resulted in a transient delay
in the development of motor activity.
recorded when an animal's position in the arena changed on
succeeding observations. Therefore, a pup that moved to a
different wedge every 10 sec for the entire 180 sec would
receive a score of 18.
Nestbox study: A lactating female and three male litter-
mates were placed in a nestbox (20x20x 10 cm) containing
food and water ad lib and nesting material. The nestbox was
attached to the front alley of a figure-eight maze. A small
hole (2x3 cm) allowed pups access to the maze but restricted
the mother to the nestbox. In addition to the 4 control and
4 TET-dosed litters used in the homing orientation experi-
ment, the data were pooled with two previous replications of
the nestbox study (total N-10 control and 12 TET litters).
RESULTS
The mean orientation score of animals in both groups
increased to a maximum at 7-9 days and declined on Day 10
(Fig. 7, left panel). These results do not agree with the find-
ings of Altman and Sudarshan [2] who reported consistent
homeward orientation by Day 10. In the present study, the
decrease in orientation scores can be explained by the devel-
opment of ambulation (Fig. 7, right panel). Since animals
spend an increasing amount of time in locomotor activity
they spend less time in homeward orientation. A steady in-
crease in the number of crossovers is seen in the developing
rat pup between 2 and 10 days of age. TET-dosed rats show a
significant retardation in this development, F(8,23)»5.09,
/?<0.001.
The development of figure-eight maze activity is shown in
Fig. 8. Some measurable motor activity occurs on Day 13,
which corresponds to the age of eye opening, however little
activity outside the nest box occurs at this age. Emergence
clearly begins on Day 16; between 17 and 21 days of age
there is a 10-fold increase in the activity of normal animals. A
repeated measures ANOVA indicated that TET exposure
caused a significant delay in this developmental pattern,
F(1,16)« 12.93, p<0.003. This effect of TET on locomotor
activity is clearly seen between 18-20 days of age (p<0.02);
however, by 21 days of age, control and experimental litters
showed similar levels of activity.
-------
292
REITER ET AL.
DISCUSSION
These results demonstrate that TET produces both im-
mediate and long lasting effects when administered to the
developing rat. Following a single exposure on postnatal Day
5, TET produced a developmental sjelay in motor activity,
and a deficiency- in_ rope-descftiit^These early signs of
neurotoxicity were associated with hyperactivity which per-
sisted through_9.months_pf age. ;
In both experiments, the highest dose of TET caused
considerable mortality ; 12mg/kg produced 90% mortality vs
509c for the 9 mg/kg dose. These data suggest that the LD50
for TET in the 5-day-old rat pup is approximately 9 mg/kg.
However, the exposure regimen employed in this study is
somewhat unique in that the pups were randomly assigned to
litters at birth and a within-litter dose-response was deter-
mined for both sexes. The possibility exists that with this
design, the mortality curve would have an artifically steep
slope owing to competition of pups within a litter. In this
situation, the competition between pups for maternal care
may also interact with treatment to exaggerate the mortality.
This hypothesis is currently being tested in our laboratory.
Nevertheless, the mortality observed in this study is in close
agreement with that reported by Wender et al. [33] who
found SO percent mortality in 6-day old rat pups given 10
mg/kg TET.
Rat pups that received either 3 or 6 mg/kg TET showed a
slight growth retardation during the preweaning period. Al-
though growth retardation produced by undernutrition dur-
ing the early postnatal period has been associated with de-
layed physical and behavioral development [15], the level of
growth retardation is generally much greater than that ob-
served in the present study. This is supported by the normal
age of appearance of developmental landmarks including eye
opening, startle response, and righting response in TET-
treated pups. Other tests also revealed no differences in the
development of TET-dosed pups (Table 1). Other neurotoxi-
cants need to be tested, however, to determine if these tests
can be indicators of perinatal toxicity.
In several preweaning tests, however, behaviors were
disrupted by TET exposure. The ability of pups to descend a
rope v»as impaired by TET. This deficit may indicate im-
pairment in neuromotor development [2]. Simon and Volicer
[27] found a delay in the forelimb grasp reflex in rats sub-
jected to acute neonatal asphyxia. It is possible that similar
forelimb impairment may be reflected in rope descent and in
the grasp Teflex.
TET-dosed pups also difFered from controls in two pre-
weaning activity measurements. When tested for homing
orientation, TET-dosed pups were less active than controls
even though the direction of orientation (i.e. home cage vs
neutral cage) was not different. In addition, motor activity in
the figure-eight maze showed a developmental lag; at a time
when control pups began to move freely about the maze,
TET-dosed pups were less active.
In addition to producing developmental delays, neonatal
TET exposure resulted in hyperactivity which was present at
21 days of age and extended through the 9 month period of
testing. This sequence of initial hypoactivity and later
hyperactivity has also been reported following early expo-
sure to other toxicants [6,35]. The parallel nature of the 23
hour activity, measured at 9 months of age, suggests that the
rate of habituation to the test environment was not altered by
TET exposure, but rather that overall activity levels were
increased. This finding of hyperactivity following neonatal
TET exposure has been replicated by Harry and Tilson [11]
who used a similar exposure regimen. In addition to finding
hyperactivity in neonatal TET-dosed rats, these authors
found a decrease in the startle responsiveness and altered
shuttle box avoidance performance.
There were no significant differences in open field behav-
ior, either in animals tested during development or as adults.
The test period used (5 min) may not be sufficient to detect
treatment-related changes in behavior. Activity counts for
the first 5 min in the figure-eight maze are consistent in show-
ing no treatment effects (Table 2). Although activity in the 6
mg/kg treatment groups tended to be elevated in both test
situations, these differences were not significant.
The profile of neurobehavioral toxicity following early
postnatal exposure to TET differed from that previously
seen following adult exposure. First, rats exposed to TET on
postnatal Day 5 in this study were hyperactive as adults,
whereas rats exposed as adults were hypoactive when tested
in the same apparatus [24]. Second, the effect of neonatal
exposure on activity was essentially irreversible whereas
rats exposed as adults recover within 1 month after discon-
tinuation of TET exposure [24]. Third, neonatal TET expo-
sure permanently reduced brain weight whereas TET expo-
sure in the adult produces an increase in brain weight,
edema, and vacolation of myelin which is reversible follow-
ing termination of exposure [17, 24, 28]. These data demon-
strate that TET exposure produced long-term, irreversible
damage to the developing nervous system but not to the
mature nervous system.
Considerably more work is needed to establish whether a
direct relationship exists between toxicant-induced hypoxia
and developmental neurotoxicity. Although our results sup-
port this interpretation, no direct measurements of energy
metabolism were made in this study. Nevertheless, the resul-
tant hyperactivity produced by TET exposure is consistent
with the behavioral change following neonatal exposure to
carbon monoxide [7] and lead [20, 21, 26]. By disrupting
energy metabolism and therefore producing cytotoxic
hypoxia during early postnatal development, TET may inter-
fere with one of the basic organizational processes in the
developing nervous system. Bull et al. [4], for example, have
demonstrated a relationship between the effects of perinatal
lead-exposure on immature brain mitochondria and cyto-
chrome accumulation. This, in turn, was associated with de-
lays in synaptogenesis [18]. Therefore, the developmental
neurotoxicity of these compounds may share a common
mechanism which depends on their ability to interfere with
energy metabolism during critical periods of development.
Finally, the present study has successfully utilized an
acute postnatal exposure model to evaluate developmental
neurotoxicity. Since the basic processes of neural develop-
ment (i.e., ceil division, migration and differentiation) are
occurring during this time [32] in some brain areas, this ex-
posure model should prove useful in evaluating the devel-
opmental neurotoxicity of neurotoxicants acting by a variety
of mechanisms. This exposure regimen has several advan-
tages: (1) it is less likely to be confounded by other
teratogenic effects since other systems are more developed
at birth, (2) it eliminates in utero effects, and (3) it is rela-
tively easy to define and control exposure. Acute exposure
on Day 5 was adequate to demonstrate the neurotoxicity of
TET; the general utility of the model will be tested with other
compounds.
-------
EFFECTS OF POSTNATAL TRIETHYLTIN
293
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15. Leathwood. P. Influence of early undernutrition on behavioral
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Influences, edited by G. Gottlieb. New York: Academic Press,
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16. Lock, E. A. The action of triethyltin on the respiration of rat
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19. Morrison, D. Multivariate Statistical Methods. New York:
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34. Winer, B. J. Statistical Principles in Experimental Design. New
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36. Zenick, H. A review of the developmental models employed in
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nA
-------
BEHAVIORAL TOXICITY OF ACUTE AND SUBACUTE
EXPOSURE TO TRIETHYLTIN IN THE RAT
Lawrence Reiter, Karen Kidd, Glendora Heavner, Patricia
Ruppert
Neurotoxicology Division, Health Effects Research Laboratory,
U.S. Environmental Protection Agency, Research Triangle
Park, NC 27711
The behavioral effects of both acute and subacute triethyltin
(TET) exposure were examined in the rat. Animals acutely
exposed to TET at doses of 0, 1.5 and 3.0 mgfkg showed a
dose-related decrease in motor activity when tested between 2
and 4 hr following exposure. Subacute (3 wee/c) exposure to
TET in the drinking water (5 or 10 ppm) resulted in performance
decrements in the following: maze activity, open field behav-
ior, acoustic startle response and landing foot-spread. Early
signs of behavioral deficits were observed 2 weeks after 10
ppm TET. These effects were reversible within one month after
termination of exposure.
INTRODUCTION
Triethyltin (TET), the most toxic of the alkyltin compounds, is used
industrially as both a catalyst and a biocide (NIOSH, 1976). Stoner et a/.
(1955) determined the acute toxicity of a series of alkyltins and reported that in
the rat, the LD50 for TET was 5.7 mg/kg. Barnes and Stoner (1958) reported that
this toxicity was independent of the route of administration. Within 30 min of
exposure to 10 mg/kg, animals exhibited generalized weakness, especially in
the hindlimbs. In more severe cases, this condition progressed to both hind-
limb and forelimb paralysis.
Magee et a/. (1957) reported that TET produced a striking interstitial
edema in the white matter of both the brain and the spinal cord characterized
by split myelin sheaths which were dilated and filled with fluid (spongy
degeneration). The spongy degeneration produced by TET is similar to that
observed in rats with hexachlorophene intoxication (Cammer et a/., 1975) and
has also been reported in monkeys and cats (Hedges and Zaren, 1969). This
effect is reversible after termination of exposure (Magee et ai, 1957) and also
occurs in peripheral nerves (Graham and Gonatas, 1973). TET-induced brain
edema is associated with a reduction in total myelin content of the brain
Please send requests for reprints to Dr. Lawrence Reiter, Neurotoxicology Division (MD-74B), Environmen-
tal Protection Agency, Research Triangle Park, NC 27711
NeurotOTkotofy 2:97-113
Copyright © 19M by Mhotov Pufaifdiert, Inc.
-------
98
REITERFTAL
although the chemical composition of the myelin is normal (Eto et al., 1971).
No change in the blood-brain barrier to trypan blue has been found (Aleu et al.,
1963).
Several investigators have examined the biochemical consequences of
trialkyltin exposure. Stockdale etal. (1970) investigated a series of trialkyltins in
isolated mitochondria and reported inhibition of both oxidative phosphoryla-
tion and coupled respiration. TET in a concentration of 2.5 fiM inhibited
glucose oxidation by 50% in rat brain cortical slices (Lock, 1976). This effect
was also observed in rat brain following in vivo exposure (Cremer, 1970).
Although considerable research has focused on the anatomical and bio-
chemical toxicity of TET, little consideration has been given to the behavioral
consequences of TET exposure. The present series of experiments was per-
formed to evaluate the behavioral toxicity of both acute and subacute TET
exposure in adult rats. Results indicate that TET is an extremely potent be-
havioral toxicant producing impaired motor function and altered behavior
following 3 week exposure to levels of 5 ppm in the drinking water.
MATERIALS AND METHODS
General Methods
Animals Animals were male CD rats (90-120 days of age) obtained from
Charles River (Wilmington, MA). Purina Lab Chow and water were available
ad libitum throughout all experiments. All animals were housed in groups of 3
in cages measuring 45 x 24 x 20 cm. The animal room was maintained on a 12
hr: 12 hr light-dark cycle beginning at 0600 hr. All behavioral testing was
conducted during the diurnal period.
Dosing Triethyltin bromide (TET, Alfa Products; Danvers, MA) was used
in all experiments. For Exp. I, TET was dissolved in sterile isotonic saline and
injected subcutaneously in a volume of 1.0 ml/kg. For the subacute experi-
ments (II and III), TET was combined with 50% ethanol (by volume) to produce
a stock solution of 20 mg/ml TET (as the bromide salt); a control stock solution
of 50% ethanol was also prepared. Aliquots of these stock solutions were then
added to tap water to provide dose levels of 0,5,10,15 and 20 ppm TET in the
drinking water (mg/liter).
Body weight, food and water consumption For the subacute experi-
ments, body weights were obtained prior to exposure, and either once or twice
weekly for the duration of the experiment. Water consumption was measured
at weekly intervals by weighing water bottles on two consecutive days; the
difference in weight was calculated as water intake. Food consumption was
measured in Experiment Ilia by weighing food dishes containing ground Purina
Lab Chow. Since weekly estimates of food and water consumption were made
by cage, no statistical analyses of these data were performed.
Behavioral Testing
Figure-Eight Maze Motor activity was measured in a figure-eight maze
as previously described (Norton et al., 1975; Reiter et al., 1975). Briefly, the
-------
TRIETHYtTIN TOXICITY IN THE RAT
maze consists of a series of interconnected alleys (10x10 cm) converging on a
central open field and covered with transparent acrylic plastic. Motor activity
was detected by 8 phototransistor/photodiode pairs; one count was registered
each time a light beam was interrupted. Eight mazes were housed in a sound-
attenuated room maintained on a photoperiod identical to the animal quarters.
In all experiments, rats were individually tested for 2 hr and were transported to
the maze room 5 min prior to testing.
Open field Motor activity was also measured in a standard open field
measuring 90 x 90 x 60 cm, and divided into sixteen 22.5 cm squares. Activity
was measured as crossovers (counted each time an animal entered a square
with all four feet) and rears (counted each time an animal raised both front
paws). Testing was conducted under normal lighting, and all activity was
monitored on closed-circuit television by observers in an adjacent room. Each
animal was placed in the center of the open field and tested for 5 min.
Startle response Animals' response to an auditory stimulus, produced by
the sudden closure of a mousetrap, was measured on a force platform (Den-
enberg et a/., 1975). Vertical force exerted on the platform was detected by a
series of strain gauges. For each animal, a baseline reading was obtained as the
force exerted on the platform prior to startle (i.e., body mass). The baseline was
then subtracted from the force exerted at the peak of the startle response to
obtain the amplitude of the startle response. Each animal was tested once.
Drop technique Animals were tested for landing foot-spread as a mea-
sure of motor function according to the procedure of Edwards and Parker
(1977). The animals' hindpaws were dipped in ink, and the animal was
suspended in a horizontal position 30 cm above a table. Each animal was
dropped twice and the distance between the footprints produced by the
hindpaws was measured; for each animal, an average of the two trials was
used.
Statistical Analysis
Behavioral data were analyzed by either a univariate or a multivariate
general linear model (MGLM) procedure (Morrison, 1967). Post-hoc compari-
sons were made using Tukey's T-method.
Experiment I
Experiment I was conducted to determine the effects of acute exposure to
TET on motor activity in the figure-eight maze. Thirty-six male rats were
assigned to 3 dose groups with 12 animals per group. TET was injected
subcutaneously 2 hr prior to testing at doses of 0,1.5 and 3.0 mg/kg.
Results
Maze counts for the total 2 hr testing period are shown in Fig. 1. Total
activity counts differed significantly between treatment groups (F(2,32) -
3.354, p<.046); animals receiving the high dose of TET (3.0 mg/kg) were
-------
100
REITERETAl.
0 1.5 3.0
TET, mg /kg
FIGURE 1. Maze activity of mate rats following acute exposure to TET. Testing was performed 2 hr after sc
administration. Values are expressed as means (±SE) for 12 animals/group.
-------
TRIETHYLTIN TOXICITY IN THE RAT
101
significantly less active in the maze than control animals (p<.05). Therefore,
the effect of TET was to decrease locomotor activity.
Experiment II
Experiment II was performed to assess the effects of subacute exposure to
TET on behavior of adult male rats. Twenty-four rats were assigned to 4
exposure groups (N=6) that received either 0, 5, 10 or 20 ppm TET in the
drinking water for 28 days. On Day 22 open field activity was measured, on
Day 23 maze activity was measured and on Day 25 the startle response was
measured. All animals were sacrificed one week following testing, and body
weights, adrenal, brain and liver weights were obtained.
Results
Table 1 shows water consumption, body weights and both the average
daily dose of TET per week and the total dose of TET for all groups throughout
the first 3 weeks of TET exposure. Water intake for all TET groups was depres-
sed according to dose, and body weight gain was reduced in the 20 ppm dose
group. Animals receiving 20 ppm TET began to show signs of intoxication after
the first week of exposure. Initial signs were loss of body tone and altered gait in
the hindlimbs which developed into hind limb paralysis (i.e., animals would
drag their hind limbs while moving) after approximately 2 weeks of exposure.
These animals were not tested behaviorally.
Maze activity, magnitude of the startle reponse, and open field behavior
(rears and crossovers) are shown in Fig. 2. Total maze activity (N=5/group) for
the 2 hr test period differed significantly between treatment groups (F(2,12)=
10.316,p<.003); animals receiving either 5 ppm or 10 ppm TET were less
active than controls (p<.05). On the second test of activity, the open field, there
were no differences between groups in either crossovers or rears during the
5-mintest
Because of the variability in the magnitude of the startle response in
control animals, a logio transformation was performed on these data. The
magnitude of the startle response differed between treatment groups (F(2,15)=
7.358, p<.006); animals exposed to 10 ppm TET were less reactive to the
stimulus than controls (p<.01).
Organ weights at the time of sacrifice (adrenal, brain and liver weights) did
not differ among the treatment groups (Table 2).
Because animals receiving TET showed reduced water intake, and body
weight gain was reduced in the highest TET dose group (20 ppm) an additional
group receiving 0.025% quinine in the drinking water was included in a
control experiment to determine if reduced water consumption and weight
gain in the absence of TET exposure would in itself alter maze activity. For 12
control animals and 12 animals receiving 0.025% quinine in the drinking
water, food and water consumption were measured over a 3 week period,
followed by a test of maze activity.
These data, shown in fable 3, indicate that water consumption was
effectively reduced by quinine administration. Although body weights were
-------
102
REITER ET AL
TABLE 1. Water Consumption, Body Weight, Average Daily Dose of TET per Week (Mean
£ SE) and the
Total Dose of TET Following Subacute Exposure in the Drinking Water (N
»6 per Dose)
Exposure
W ater Consumption
Body Weight
Mean Daily
Total
Level
Week
Dose/Week
dose
(mg/kg)
0 ppm
0
.
388±11
-
1
57±5
3842:12
0
2
572:7
406*11
0
3
482:5
423 ± 10
0
0
5 ppm
0
-
383 ±8
-
1
39±8
399S8
0.49
2
302:1
4102:8
0.36
3
312:0
424±9
0.36
8.4
10 ppm
0
-
388± 11
-
1
2&± 1
3872:10
0.67
2
262:1
394±12
0.66
3
27s!
4142:13
0.65
13.9
20 ppm
0
-
384*9
-
1
112:10
381 ±8
0.58
2
22±2
379±11
1,16
3
142:1
384 ±9
0.73
17.3
also reduced In animals receiving 0.025% quinine (F(4,19)=5.58, p=.004),
maze activity was not significantly different from control levels. These data
indicate that the decreased activity shown by TET animals cannot be ac-
counted for by reduction in either water intake or body weight gain.
Experiment III
Experiment II demonstrated the subacute effect of TET on behavior fol low-
ing 3 weeks exposure. The purposes of Experiment III were: (1) to replicate the
behavioral results seen in Experiment II; (2) to determine the time course for the
appearance of TET effects on behavior; and (3) to determine the time course for
behavioral recovery following cessation of treatment. Two separate experi-
ments were performed, and the methods and results will be discussed separate-
ly.
Experiment Ilia
Thirty-six male rats were randomly assigned to 4 dose groups of 9 animals
each. The exposure levels of TET were 0,5,10 and 15 ppm. Since animals in
Experiment II receiving the highest dose of TET, 20 ppm, developed hind limb
paralysis, a 15 ppm TET dose was added in this experiment to specify the dose
level at which paralysis develops. In this experiment, food consumption was
also measured along with water consumption and body weight, to determine if
decreased food intake contributed to the weight loss previously seen in TET-
treated animals. TET was administered for 4 weeks, and open field activity and
-------
TRIFTHYITIN TOXICITY IN THE RAT
103
OPEN FIELD REARS, Y1 SE
3 5! s » g
c
9
m
r
m
<
Z
a
s
I
X
¦\V
J I L
T
MAZE ACTIVITY, COUNTS/2 hf,U ± SE
8 I § I i i
I
i—i—r
« a 8 3 8 8 g
OPEN FIELD CROSSOVERS,Tt St
AV
I
I
I I I I f-
b
Kl
HI
b w
AMPLITUDE, STARTLE RESPONSE. (lo»,Ti SE)
FIGURE 2. Maze activity, startle response, and open field behavior (rears and crossovers) following
subacute (3 week) exposure to TET in the drinking water (n-6 per dose).
maze activity were tested on Days 23 and 24, respectively. All animals were
sacrificed approximately one week following behavioral testing and body
weights, brain, kidney and seminal vesicle weights were obtained.
Results
Table 4 shows water consumption, body weights, average daily dose of
TET per week and total dose of TET during 3 weeks of exposure, combined for
both studies in Experiment III. Water intake was reduced in TET-exposed
-------
104
MITER ETAL
TABLE 2. Organ Weights Ig) in Control and TE7-Exposed Rats IN=6-9/Croup). Data is Presented for Both
Experiments II and III
Exposure Level
-------
TRIFTHYLTIN TOXICITY IN THE RAT
105
TABU 4. Water Consumption, Body Weight, Average Daily Do* of TET per Week, Total Dose of TET
(N=* 18 per Dose), and Food Consumption (N»9 per Dose) Following Subacute Exposure to TET in the
Drinking Water (Mean * 5E)
Exposure
Week
Water Consumption
Body Weight
Mean Daily
Total
Food
Level
Dose/Week
Dose
Consumption
(ml/Rat/Day)
(g)
(mg/kg/Day)
(mg/kg)
(g/Rat/Day)
Oppm
0
-
416+3
1
52=4
442*3
0
31*1
2
51 2:5
463*4
0
35*2
3
49*5
478*4
0
0
42*2
5 ppm
0
-
417±5
_
.
1
37*2
444*5
0.42
29*2
2
38* 1
465*7
0.40
35 = 1
3
46*2
474*8
0.51
9.3
38*1
10 ppm
0
-
418*4
.
.
1
31 ±2
434*4
0.71
29*2
2
35*3
450*5
0.76
31*4
3
35 ±2
451*6
0.96
17.0
26*2
15 ppm
0
-
417*6
.
_
1
22^:1
427*5
0.77
22*2
2(N
-9) 162:2
404*6
0.60
15*1
3'
48*2
415*7
-
31*3
'TET was removed from drinking water after week 2.
contrast to Experiment II, TET exposure significantly affected open field be-
havior. Crossovers in the open field differed between treatment groups
(F(2,24)=11.343, p<.0004), as did rears (F<2,24)»36.939, p<.0001>. In com-
parison to control animals, crossovers were reduced in both the 5 ppm
(p<.005) and in the 10 ppm (pc.001) exposure groups. Rearing activity was
also decreased in a dose-dependent manner: controls reared more than the 5
ppm exposure group (p<.005), and the 5 ppm group reared more than those
receiving 10 ppm (p<.001).
Brain weight was significantly increased in experimental animals
(F(2,24)*=14.179, p<.0001) in a dose-dependent manner (Table 2). Brains
from animals receiving 10 ppm TET weighed more than either those from the 0
ppm dose group (p<001) or those from the 5 ppm dose group (p<.001). This
increase in brain weight in the 10 ppm dose group is consistent with TET-
induced brain edema. No treatment related differences were observed in either
kidney or seminal vesicle weight.
Experiment lllb
In the second part of this experiment, both emergence and recovery of TET
toxicity were investigated. Thirty-six male rats were assigned to 4 dose groups
of 9 animals each, that received doses of 0,5,10or IS ppm TET. Animals were
tested for maze activity and landing foot-spread prior to exposure, then retested
for both at the end of the first, second and third weeks of TET exposure.
-------
106
reiteretal
EXPOSURE LEVEL Ippm IN DRINKING WATERI
FIGURE 3. Maze activity, and open field behavior (rears and crossovers) following subacute (3 week)
exposure to TET in the drinking water (N »9 per dose).
-------
TRIETHYtTIN TOXICITY IN THE RAT
107
Recovery of maze activity and landing foot-spread was evaluated at 2 and 4
weeks post-treatment.
Results
Activity data for the figure-eight maze are shown in Fig. 4. Because of the
week to week variation in activity levels, log,0 transformations were performed.
As in Experiment Ilia, animals receiving 15 ppm TET developed hind limb
paralysis and were not included in the behavioral testing. A MCLM analysis
indicated a significant effect of treatment on activity (F(12,38)=2.04, p<.047);
although there were no differences between groups in pretreatment maze
activity, by week 3 of maze testing treatment groups differed in activity
(F(2,24)=4.107, p<.029). Animals in both the 5 ppm and the 10 ppm TET
exposure groups were less active than controls (p<.05) by the end of the third
week of treatment. For maze activity there was also an interaction between
week of testing and dose of TET (F(10,40)=2.332, p<.028); this interaction
was significant between week 1 and week 2 of treatment (F(2,24)=6.761,
p<.005). Maze activity did not differ significantly between groups at either 2
weeks or 4 weeks following discontinuation of TET.
"• CONTROL
A S ppm
~ 10 ppm
>
Si
Ul
2.4 —
2.2 —
TET DISCONTINUED
TIMC IWIBKII
FIGURE 4. Maze activity in control and TET-expoied adult mate rats tested weekly during exposure and at 2
week intervals during recovery. Oose administered in drinking water.
-------
108
MITER ETAL
A significant effect of treatment was also found on the landing foot-spread
as measured by the drop technique (F(6,44=3.275, p<.01); data are shown in
Fig. 5. The difference between groups in the landing foot-spread was signifi-
cant both on week 2 (F(2,24)=8.848, p<.001) and on week 3 (F(2,24}=6.052,
p<.007) of TET exposure. On week 2, animals in the 10 ppm dose group
showed a narrower foot-spread than controls (p<.01), and on the third week,
10 ppm animals showed a narrower foot-spread than either controls (p<.01) or
those in the 5 ppm dose group
S*
i
a
<
LJ
AC
a.
-------
TRIETHYLTIN TOXICITY IN THE BAT
109
effects seen in the present study can be explained by brain edema. Torack etal.,
(1970) reported that no increase in brain wet weight occurred in rats receiving
4 mg/kg TET. With a dose of 9 mg/kg, no statistically significant increase in wet
weight occurred before 18 hr. Etoetal. (1971) reported that peak brain edema
occurs between 7 and 14 days following acute administration of 5 mg/kg TET.
Subacute exposure to TET produced decrements in maze activity, amp-
litude of the startle response, and landing foot-spread. Since TET exposure
produces myelinopathy (Magee et a/., 1957), these effects on motor behavior
would be predicted.
Open field activity did not provide a consistent measure of TET toxicity.
Whereas maze activity was repeatedly reduced by TET exposure, open field
activity was reduced in Experiment III but not Experiment II. Since the calcu-
lated weekly dose was equivalent in the two experiments, the maze may be a
more sensitive test of motor activity in TET-exposed animals.
The finding of hypoactivity agrees with Gerren et a/. (1976) who exposed
mice subacutely to 2 mg/kg/day of TET. When mice were tested for 5 min in a
proximity counter, decreased levels of activity were reported after 9 days of
exposure. They also reported that, when handled, TET-exposed mice were
hyper-reactive as compared to controls. In the present study, we did not
obUrve this hyper-reactivity in the rat. Specifically, TET exposure reduced the
amplitude of the acoustic startle response, a finding which suggests that ani-
mals are less responsive to stimuli, although muscle weakness would also
account for this effect
Although administration of TET in the drinking\water resulted in an
immediate and persistent decrease in water consumption, the behavioral
effects of exposure cannot be attributed to this effect alone. Addition of quinine
(0.025%) to the drinking water resulted in an equivalent fall in water consump-
tion but was without behavioral consequence. However, the decrease in food
consumption observed in the 10 ppm group near the end of the third exposure
week (Experiment III) probably represents a toxic effect of TET itself. Neverthe-
less, behavioral effects were observed in the 5 ppm exposure group which had
normal food consumption and body weights, indicating that the behavioral
effects occur at a lower threshold than effects on growth.
Administration of TET in the drinking water does not allow for the precise
determination of dose nor does it insure uniform dosing from experiment to
experiment. For example, rats receiving 10 ppm TET in Experiment III con-
sumed a total dose of 17.0 mg/kg which was almost identical to the 17.3 mg/kg
dose received by the 20 ppm group of Experiment II. We have no explanation
for why the former group did not develop the same signs of overt toxicity seen
in the latter group. Nevertheless, within a given experiment measurements of
water consumption and body weight did provide a close approximation of the
total dose. Differences in maze activity, observed after 3 weeks exposure to 5
ppm TET, correspond to an estimated total dose of 8.4 mg/kg (Experiment II).
Measurements of landing foot-spread indicate that motor function was affected
within 2 weeks of exposure to 10 ppm TET, corresponding to a total dose of
10.3 mg/kg (Experiment III). Therefore, under the conditions of the present
experiment, a behavioral threshold for TET is estimated to be between 8-10
-------
110
REITER ET AL
mg/kg for 3 week subacute exposure. This study provides a first approximation
of the threshold for the behavioral toxicity of TET. A more critical determination
of threshold will require more extensive investigation includinga more precise
determination of dose.
These data also suggest that the behavioral effects of TET are independent
of increases in brain weight. In Experiment II, hypoactivity occurred in the
absence of brain edema. In both Experiment II and lllb, 3 week exposure to 5
ppm TET produced a significant depression in maze activity whereas no
significant increase in brain weight was observed. It should be noted, however,
that the brain weights of the 5 ppm exposure group were increased over the
control group, although this effect was not statistically significant. However,
Marshall et a/. (1976) reported an increased water content of brains from rats
exposed for 4-5 weeks to 5 ppm TET.
It is interesting to note that the results of the landing foot-spread measure-
ments are qualitively different from acrylamide exposure reported by Edwards
and Parker (1977) and Jolecoeur et al. (1979). These investigators found that
subacute exposure to acrylamide in the rat resulted in an increased foot-spread
contrasted to the decrease observed in the present study. This qualitative
difference may reflect the difference between axonopathy, produced by
acrylamide (Fullerton, 1969) and myelinopathy, produced by TET. The appa-
rent sensitivity of this test, combined with its simplicity, support its continued
use as a potential screen for motor dysfunction.
Finally, these results indicate that the neuromotor effects of TET are largely
reversible. Measurements of both motor activity and motor function (landing
foot-spread) return to control levels within 1 month after discontinuation of
exposure. This behavioral recovery agrees with the morphological recovery
described by Magee et a I. (1957).
ACKNOWLEDGMENT
The excellent technical assistance of Ronnie McLamb and Charles Belser
is gratefully acknowledged. We also thank Mrs. Phyllis Keeter for preparing this
manuscript.
REFERENCES
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Barnes JM, Stomr HB. Toxic properties of some diaikyl and trialkyltin salts. Brit) Industr Med 1958:
15:15-22
Cammer W, Rote AL, Norton WT. Biochemical and pathological studies of myelin in hexachlorophene
intoxication. Brain Res 1975; 98:547-59
Owner |E. Selective inhibition of glucose oxidation by triethyltin in rat brain in vivo. Biochem ) 1970;
119:95-102
Denenbeif VH, Gartner |, Myers M. Absolute measurement of open field activity in mice. Physiol Behav
1975; 15:505-9
Edwardl fM, Parker, VH. A simple, sensitive and objective method for early assessment of acrylamide
neuropathy in rats. Toxicol Appl Pharmacol 1977; 40:589-91
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TRIETHYLTIN TOXICITY IN THE RAT
lit
Elo Y, Suzuki K, Suzuki K. Lipid composition of rat brain myelin in triethyl tin-induced edema.) Lipid Res
1971; 12:570-8
Fullterton PM. Electrophysiological and histological observations on peripheral nerves in acrylamide
poisoning in man.) Neurol Neurosurg Psychiatry 1969; 32:186-92
Cerren RA, Groctwaid DE, Luttge* MW. Triethyltin toxicity as a model for degenerative disorders. Pharm-
acol BiochemBehav 1976; 5:299-307
Graham 01, Gonata* NK. Triethyltin sulfate-induced splitting of peripheral myelin in rats. Lab Invest 1973;
29:628-32
Hedges TR, Zaien HA. Experimental papilledema: A study of cats and monkeys intoxicated with triethyltin
acetate. Neurol 1969; 19:359-66
)oiecoeur JB, Rondeau OB, Barbeau A, Wagner M|. Comparison of neurobehavioral effects induced by
various experimental models of ataxia in the rat. Neurobehav Tox 1979; l(Suppl. 1): 175-8
lock EA. The action of triethyltin on the respiration of rat brain cortex slices.J Neurochem 1976; 26:887-92
Magee PN, Stoner HB, Bamet JM. The experimental production of edema in the central nervous system of
the rat by triethyltin compounds. I Path Bact 1957; 73:107-24
Marshall LF, Bruce OA, Graham 01, Ungfttt TW. Alterations in behavior, brain electrical activity, cerebral
blood flow, and intracranial pressure produced by triethyltin sulfate induced cerebral edema. Stroke
1976; 7:21-5
Morrison O, Multivariate Statistical Methods. New York: McGraw-Hill. 1967
Norton S, Culver B, Mullenix P. Measurement of the effects of drugs on activity of permanent groups of rats.
Psychopharm Commun 1975; 1:131-8
NIOSH Criteria for a Recommended Standard. ...Occupational exposure to...organotin compounds.
DHEWPub. No. 77-115. Washington, D.C.: U.S. Govn't. Printing Office. Washington: 1976
Reiter LW, Andenon GE, Lakmy |W, Cahill DF. Developmental and behavioral changes in the rat during
chronic exposure to lead. Environ Health Perspect 1975; 12:119-23
Row MS, AkJridge WN. The interaction of triethyltin with components of animal tissues. Biochem) 1968;
106:821-8
StockdaJe M, Daw»on AP, Scfcwyn M|. Effects of trialkyltin and triphenylttn compounds on mitochondrial
respiration. Eur I Biochem 1970; 15:342-51
Stoner HB, Bame* |M, Duff |l. Studies on the toxicity of alkyltin compounds. Brit I Pharmacol 1955;
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Torack R, Gordon |, Prokop ). Pathology of acute triethyltin intoxication. Intern'l Review of Neurobiol
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NEUROTOXICOLOCY MS N-8 J
Received April 1. I960
Accepted June 23, 1980
-------
TOXJCOLOOY AND APPUHD PHARMACOLOGY If, 69-77 (1985)
Development of Locomotor Activity of Rat Pups
Exposed to Heavy Metals1
Patricia H. Ruppert, Karen F. Dean, and Lawrence W. Reiter
Seurotoxicology Division (MD-748), U.S. Environmental Protection Agency,
Research Triangle Park, North Carolina 27711
Received June 20,1984; accepted October 12, 1984
Development of Locomotor Activity of Rat Pup* Expoted to Heavy Metal*. Ruppert,
P. H., Dean, K. F„ and Reiter, L W. (1985). Toxicol. Appt. Pharmacol. 7S, 69-77.
Cadmium (Cd), triethyhin (TET), and trirnethyWn (TMT) an heavy metal* which are
neurotoxic to developing animals. In the present experiment, preweaning assessment of
locomotor activity was used to detect and differentiate between the developmental toxicity of
theae metal*. On postnatal day (PND) S, rat pup* received t single injection of either Cd. TET,
or TMT. A witbin-litter design wa* wed for dosing; 1 male and I female pup from each Utter
(N - 10 titten/compouad) received either the vehicle, low, medium, or tigb dotage of the
compound. Preweaning motor activity wai aseseMd in 30-min sessions in ftgure-eigtit maze*
ftom PND 13 to 21. Motor activity of control animate progressvely increased in the initial
days of toting, and then both within-KMkm and between session habituation developed. A
angle exposure to Cd, TET, and TMT produced hyperactivity by the end of the pnweaning
period but these metal* differed in the day of peak activity, the onset of hyperactivity, and the
development of habituation, e ists »r»nw rrm. lac.
Preweaning assessment of locomotor activity
is of interest in the behavioral evaluation of
agents which affect the development of the
nervous system since perinatal insult can
produce changes tn activity which are mani-
fest only at certain times (Culver and Norton,
1976; Reiter, 1977) or which change quali-
tatively (Zagon et aL, 1979) during develop-
ment During early postnatal life in the rat,
the nervous system is particularly vulnerable
to the neurotoxic e&cts of heavy metals.
This vulaerabMi is due to the rapid brain
growth which Spn at this time (Dobbing,
1968) and alio 9m incraaaed accumulation
'This paper hail
in nvfniwt fcjf tht HhMi BtfceH
VS.
Apaqr,
of heavy metals in the brains of suckling
animals (Jugo, 1977). Metals which produce
CNS pathology following acute postnatal ex-
posure include cadmium (Cd) (Gabfaiani et
aL. 1967; Wong and Klauien, 1982), trieth-
yltin (TET) (Suzuld, 1971; Wender et aL,
1974; O'Callaghan et at., 1983; Veronesi and
Bondy, 1983), and trimethyltin (TMT)
(Chang et al„ 1983; Miller and O'Callaghan,
1983). -
A common behavioral consequence of
heavy metal intoxication in developing ani-
mals is an alteration in locomotor activity.
Hyperactivity in figure-eight mans was re-
ported following postnatal exposure to Cd
(Wong and Klaaasett, 1982), TET (Reiter et
aI, 1981), and TMT (Ruppert et al„ 1983a)
when animals mm tested; as juvenfts or
m adultt Following TET exposute on Postnatal
Day (PND) S, tMa increaae In activity was
69
OOtl
-------
70
RUPPERT, DEAN, AND REITER
robust under several testing conditions: when
animals were tested only once on approxi-
mately PND 60 (Ruppert et al., 1983b),
when the same animals were tested repeatedly
from PND 21 to 238 (Reiter et al, 1981),
when individual animals were tested contin-
uously for a 2-week period (MacPhail et al,
1983), or when pairs of animals were tested
for 23-hr periods (unpublished data).
In contrast to this persistent hyperactivity
in TET-exposed animals tested in figure-eight
mazes after weaning, TET-exposed pups were
hypoactive when tested from PND 15 to 21
in figure-eight mazes attached to a nest box
(Reiter et al., 1981). TET-exposed pups were
also hypoactive in a test of homing orienta-
tion (Reiter et al., 1981), and over home-
cage bedding in an open field (Miller, 1984)
but not in open-field testing in the absence
of bedding (Reiter et al, 1981; Miller, 1984).
It is not clear whether this age-related differ*
ence in the effect of TET is related to the
time course of toxicity or to a difference in
arousal of TET-exposed pups to home-cage
cues. For example, hypoactivity in the pre-
weaning period could reflect acute toxicity,
or alternatively, a delay in maturation.
In the present experiment we compared
the development of locomotor activity in rat
pups exposed to Cd, TET, and TMT. Al-
though these metals reduce overall brain
weight following exposure on PND 4 or 5
(Reiter et al., 1981; Wong and Klaassen,
1982; Ruppert et ai.. 1983b), they produce
different patterns of pathology. In particular,
structures related to motor activity are differ-
entially affected. Lesions of the caudate-
puumen were produced by Cd (Wong and
Klaaam, 1982), while hippocampal weight
wKfmfcrcntially decreased by TET (Ruppert
et mbmm and TMT (Ruppert et al..
190^ exposure. Although acute postnatal
exposure to TMT produces necrosis of py-
ramidal neurons within the hippocampus
(Chang « «/-. 1983), the locus of ceil loss in
the hippocampus of TET-exposed pups has
not been established (Veronesi and Bondy,
1983). The purpose of this experiment was
to determine (1) if preweaning assessment of
activity in general would be predictive of
hyperactivity seen in juveniles and adults
and (2) if preweaning testing could differen-
tiate between metals which produce different
neurotoxic effects.
METHODS
Long-Evans female rats (Charles River) were obtained
3 days after matins and housed individually in cages
measuring 45 x 24 x 20 cm with pine shavings used as
bedding material. Animals were maintained on a 12 hr:
12 hr light:dark cycle (lights on at 0600 hr) in an animal
facility with controlled air temperature (22 ± 2°C) and
humidity (SO ± 10%). Purina Lab Chow and water were
available ad libitum throughout the experiment. One
day after parturition (day of birth ¦ PND 0), litters were
randomized and each dam was assigned four male and
four female pupa. Pups were tattooed on a paw to
provide unique identification within a litter (Avery and
Spyker. 1977).
Pupa were injected on PND 5; the vehicle was 0.9%
sterile saline for all compound!. A within-litter design
waa used for doting: I male and I female pup from each
litter
-------
BEHAVIORAL TOXICITY OF METALS
71
successive days at Mint) ww* examined. Activity during
5-min intervals for each day of teating was analyzed by
a repeated-measures A NOV A usin| kx and dose as
between-animal factors; ajc and time interval and inter-
actions with the* variables were used as within-animal
factors. When significant interactions with ace were
found, simple main effects tests were conducted. Post
hoc comparisons were made by Tukey's a test Data
were analyzed by programs on the Biomedical Data
Program (BMDP-4V). For all statistical tests, values
greater than the critical value at p < 0.05 were accepted
as significant.
RESULTS
Cadmium.* Exposure to Cd on PND 5
produced a triphasic effect on activity, with
initial hypoactivity followed by hyperactivity
(Fig. 1 A), This change was reflected in a dose
x age interaction for total activity [P(24,183)
- 3.71, p < 0.0001], with no overall sex
effect or interaction with sex. A dose effect
was seen on PND 13 to 15 {/T[3,70) • 3.34,
8.75, 9.36, respectively, all p's < 0.01] and
on PND 20 to 21 (/T3,70) - 11.91 and
23.13, respectively, p's < 0.00001], On PND
16, there was a dose X sex interaction (ft3,70)
- 5.29, p < 0.0024]. Pups of both sexes
receiving 4 mg/kg Cd were less active than
controls on PND 13 to 15, and males re-
mained hypoactive on PND 16. Pups of both
sexes receiving 4 mg/kg Cd were more active
than controls on PND 20 to 21.
Figure 2 shows within- and between-session
habituation for controls and 4 mg/kg Cd*
exposed pups. Cd-exposed pups did not de-
velop the patterns of habituation which are
seen in control animals. There was an age
x time x dose interaction 1/1120,93) - 1.56,
p < 0.0122]. On PND 13-ta 15, activity of
control pups was constant throughout the
test session, wMfc pups receiving 4 mg/kg Cd
showing lowsraetivity. On PND 16 to 21,
controls sho#'utoin session habituation
whereas pups receiving 4 mgftg Cd become
hyperactive but tbow ao within union ha-
bituation or between session habituation.
at tte Society fat
19S3.
}se
O MUNI
BOMfil.aiftt
IS !»
04
Nia0l.*/k| *1
III"''' 1°*
UMnadiin Annual
F». I. Preweantits motor activity for control and
metal-exposed rat pups. Data, combined for male and
fbmai* pupa. an fMNHMd aa photocell counts (£ ± SB)
in ftguwriifat °n«w 30-«
-------
72 RUPPERT, DEAN. AND REfTHR
>
P
—I 1—r—i—i 1—
mois
• SALINE
" t^rr-H "
i 1 r-~t " "T "IT
ma u
ii i. i—i—i
' i 1 1 1 1——>
moii
. i i i i i
ill l ) J
fv mo n
;h+^:
l„ L-.J 1 1 1
hIdh
.„i1—L—1 1 1
—i—:—|—i—r -i—
PN0 11
- "
1 1 1 1 -1 1
' ' 1 1 1 1 nU
, _
¦V-*" T 'f -T
' ' ' i i i
'' ' 1 1 PND 21
¦ —H -
- -
. 1, I .. 1 .. L—! 1
1,111
• "
11 1 I 1 1 ...
1*
1*
m mi ti a a #
TIMl, mi*
Fro. 2. Habituation of motor activity Tor controls and pup« exposed to 4 mi/kj cadmium. Data,
combined for male and female pupa, ate presented as photocell counts (x ± SE) for J-min intervals
durini the 30-min session on each day of testing.
weaning body weight 1/1(9,165) « 18.18, p
<0.00001]. An effect of dose was seen on
PND 10, 13, and 20 [/=T3,70) - 19.89, 22.34,
49.53, respectively, p's < 0.00001 ] and a dose
x sex interaction on PND 201/^3,70) - 3.41,
p < 0.0220}. Growth was reduced in pups
receiving 4 mg/kg cadmium, and this reduc-
tion was greater in males than in females on
PND 20y*r
TriethjijpK Exposure to TET produced
dose-depagittt hyperactivity which devel-
oped over time (Fig. IB). This hyperactivity
was reflected in a dose X age interaction for
total activity [/T24,180)« 3.17, p < 0.0001],
with no overall sex effect or interaction with
sex. A dose effect was seen on PND 14
1/13,69) - 3.29, p < 0.0255] and PND 16
to 21 [/T3.69) - 4.40, 33.44, 48.14, 33.53,
38.44, 38.77, and p's < 0.007], Pups receiving
4 mg/kg TET were less active than controls
on PND 14. On PND 16, pups receiving 5
and 6 mg/kg TET were more active than
controls, and by PND 17 to 21, pups receiving
all dosages of TET were more active than
controls.
Figure 4 shows within- and between-session
habituation for controls and TET-cxposed
pupa. Patterns of habituation differed between
controls and TET-exposed pups in a dose-
dependent manner. There was an age x time
x dose interaction [#1120,90) - 1.41, p
< 0.0437]. Simple main effects tests showed
a dose X time interaction for all ages. On
PND 13 to 15, TET-exposed pups were less
-------
BEHAVIORAL TOXICITY OF METALS
73
« -
M
S *
TIT
OOtAU, n|/k|
OUIIMI
04
©•
M
C
OlALWI
04
»•
01
Fio. 3. Prewcuiini body weight {i t SE), combined
for males ud females, fair control aad OMtal-expoMd
pup*. Body omfbt of doeed pop* wm different (torn
eonuoto on days indicated by an amrhlr, « double
aneriik indicates that body «efel* of oak pop* Motiving
4 mt/kt cadmium mm kmm tfcn that et fonalee
receiving tin am doaege. (A) Cadmium, (B) triethyltio,
(Q triiiuiili)M%
i
-
active Uia» iwtruli at later intervals during
the ten i—ion, Habituation devetoped in
controls on PND16 to 21 while TET-expoaed
pup* showed either a constant or increased
degree of activity within each session.
Preweaning growth was reduced by post-
natal exposure to all dosages of TET (Fig.
3B). There was a dose x age interaction for
preweaning body weight 1/^9,163) =¦ 16.64,
p < 0.00001]. An effect of dose was seen on
PND 10, 15, and 20 (^3,69) - 43.66, 24.49,
25.14; p's < 0.0001].
Trimethyltin. Exposure to TMT produced
hyperactivity which developed during testing
on different days for male and female pups
(Fig. 1C). This effect was reflected in a dose
X age interaction for total activity [F(24,186)
¦ 2.50, p < 0.0021] and a dose x sex inter-
action [/T3,71) - 3.28, p < 0.0258). An
effect of dose was seen on PND 16 and 17
for males only [F{3,71) - 5.62, 4.20, p's
<0.0016]. For PND 18 to 21, an effect of
dose was seen for both sexes [fT3,71) » 8.30,
12.67, 8.53, 9.60, all p's <0.0001]. Pups
receiving 6 mg/kg TMT were more active
than controls.
Figure 5 shows within- and between-session
habituation for control and 6 mg/kg TMT-
exposed pups. Habituation of activity was
parallel between the two groups. There was
no age X time X dose interaction [/^ 120,96)
» 1.23, p < 0.1422]. There was a change in
habituation over age ffl(40,32) - 8.02, p
<0.00001]. Activity on PND 13 to 15 re-
mained constant at low concentrations across
all time intervals while on PND 16 to 21
activity at later time intervals was lower than
initial activity.
Preweaning growth was reduced by post-
natal exposure to 6 mg/kg TMT (Fig. 3C).
There was a significant dose X age interaction
[^9,168) - 4.13, p < 0.0001]. An effect of
dose was seen on PND 10, 15, and 20
TO71) - $.88, 4.66, 2.73, p's < 0.05].
DISCUSSION
Cd, TET, and TMT all produced hyper-
activity by the end of the preweaning period,
but differed in the timing of the peak in
activity, in the onset of hyperactivity, and in
the pattern of habituation. These data dem-
onstrate that the age of the animal at testing
is an important variable in interpreting
-------
74
RUPPERT. DEAN, AND RE ITER
¦<
a
m
6
5
g
«
QQSA«(.a|At NI011
- . 1ALIM!
• 4
0 1
• 1
1 i( ill
r*o»
1 1 1 .J—1—1—
1 1 1 1 1
mo u
_.i i i—i—i—i—
1—1 1 1 1 1 I
non
:^£j:
' '—l 1 1 1—
' 1 (HM017
- ¦
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¦ -
—1— ' I 1 ' '
-t 1 ! 1 ! "Jo ii
i i i i .1 i—-
MOU
N——t
L_ 1 i.„_4 1 JL
mo 21
-
. .
—i—I—i—j i i
TIMS, Mia
Fw. 4. Habituation of motor activity for controls and pup* exposed to 4, 5, or 6 m|/1cf triethyltin.
Data, combined Cor male and female pupa, ait presented as photocell counu (i ± SE) for 5-min intervals
during tbe 30-min session on each day of icom*.
changes in prcweaning activity. It is likely
that differences in the neuropathology pro-
duced by these metals underlie the differences
in the pattern of developmental changes in
activity. Changes m prewetning activity were
seen at the suae dosages of these metals
which prattaMA hyperactivity when animals
were tested afljfWBiks and/or adults, which
tends supposcwtlM interpretation that they
are similar indicators of neurotoxicity.
Control groups in the present experiment
showed a peak is activity on PND 15 to 16,
similar to that originally repotted by Camp*
bell tt at. (1969). This padc in behavioral
arousal, which as been attributed to differing
rates of maturation of the noradrenergic and
cholinergic systems (Campbell et at., 1969),
was not present in the preweaning period for
Cd-exposed pups, was delayed for TET-ex-
posed pups, and occurred at the normal time
for TMT-expoaed pupa. Data are not available
on the development of neurotransmitter
functioning following exposure to these met-
als. However, stereotypy produced by apo-
morphine was altered in TET-exposed ani-
mals tested as adults which suggests a penis,
tent alteration in dopaminergic Amotion
(Hairy and Tilson, 1982).
The delayed onset of metal-induced hy-
peractivity, which developed from U to IS
days after (falsing for all metals, may reflect
the time course of toxicant-induced damap
-------
BEHAVIORAL TOXICITY OF METALS
75
«r
II ¦
21 ¦
It ¦
T 1 1 1 1 1
PM011
• MUMI
I I I I I
HI014
T—i—r
1—i—
mo i»
i—i
MO tl
l 1 1
MB II
mo u
mo i*
Flo. J. Habituation of motor activity for control! tod pup* expoeed to 6 ms/ks trimethyltin. Data,
combined for male and female pupa, an pmtntad ai photocell count! (S ± SE) for J-min interval!
durint the 30-min knob on each day of toting.
to the nervous system. For cadmium, brain
weight was progressively reduced from 4 days
(11%) to 19 days (26%) after dosing (Wong
and KJaassen, 1982), indicating progressive
neurotoxicity. High-dose Cd pupa were hy-
poactive on PND-13 to IS and kwMlose
TET pupa. Mm hypoactive on PND 14. A
similar bqjkmc effect on the development of
activity iris open Add was found for pups
expoaa^mVBT (Miller, 1984) or TMT
(Miller and 0*Callaghan, 1983) on PND 3.
This delayed onset of hyperactivity was not
due to a "masking* effisct resulting from the
rapid rate of increase in activity from PND
13 to IS; postnatal exposure to thyroxine,
which accelerates development, produced hy-
peractivity beginning on PND 13 when pupa
were tested under the same conditions (un-
published data). Also, hyperactivity was not
induced by repeated testing per se, since this
effect is also seen for older animals when
tested in figure-eight mazes for the first time.
Habituation of activity in - ftgure*ight
mazes changes during development (Ruppert
et ai., 1984b), and these developmental
changes were altered in pups exposed to Cd
and TIT. For controls, within-session habit-
uation was clearly seen on PND 16 but not
at earlier ages. Following the peak in activity
oe PND 16, overall activity was lower on
meamtm days of testing. From PND 13 to
21, tha activity of pups exposed to 4 mg/kg
Cd wasuniform throughout the 30»min test
(no within session habituation) andprogre*
-------
76
RUPPERT, DEAN, AND REITER
sivcly increased oveT each succeeding day of
testing (no between-session habituation).
While pups exposed to 5 mg/kg TET showed
a lack of habituation similar to that of
Cd-exposed pups, those receiving 6 mg/kg
TET actually increased their activity levels
throughout the session. The distinctive effects
of these metals on habituation may be pre-
dictive of more global deficits on tasks which
measure an animal's distractibility or ability
to inhibit responding.
At all dosages of metals which produced
alterations in locomotor activity preweaning
growth was reduced. An immediate effect of
these metals is an alteration in suckling during
the acute phase of toxicant exposure. The
size of the milk bands, which reflect the
amount of milk in the stomach, was reduced
following PND 5 exposure to Cd (unpub-
lished data), TET (Ruppert et ai., 1984a),
and TMT (Ruppert et ai, 1983a). Since even
2 hr of deprivation on PND S can produce
retardation of growth (Dean, 1983), it is
unlikely that neurotoxicity or behavioral tox-
icity would be produced at dosages lower
than those producing this acute toxicity.
However, the magnitude of growth reduc-
tion bears little relationship to the magnitude
of hyperactivity. At the highest dosage of Cd,
TET, and TMT, body weights on PND 20
were reduced by 29,24, and 12%, respectively,
yet TET produced a greater increase in activ-
ity than either Cd or TMT. In addition, both
4 and 5 mg/kg TET produced similar reduc-
tions in growth (8 and 11% on PND 20) but
different effects on activity. Although under-
nutrition in the postnatal period can produce
changes in behavior (Leathwood, 1978), the
growth retardation is generally much greater
thaa- that produced by a single exposure to
i1hb> irutaln
fiMneaning mortality in the present study
for Of was similar to that previously reported
(Wong and KJaassen, 1982). Although only
two pups died, growth retardation became
progressively more pronounced in the pre-
weaning period. Gross brain pathology, with
thinning of cortical tissue and accumulation
of fluid as described by Wong and Klaassen
(1982), was observed in many weanlings re-
ceiving the high dose Cd. No signs of poison-
ing were observed in pups receiving 2 mg/kg
Cd, which illustrates the steepness of dose-
response functions for these metals. No mor-
tality was observed in pups receiving TMT,
but three pups receiving 6 mg/kg TET died.
In several previous studies using CD rats
from the same supplier, we observed no
mortality at this dosage (Reiter et ai.. 1981;
Ruppert et al., 1983b, 1984a); therefore, LE
rats may be more sensitive to TET than CD
rats.
Preweaning assessment of motor activity,
as shown in the present experiment, does not
merely duplicate postweaning evaluation. Al-
though hyperactivity is seen both in juveniles
and in older animals as a result of postnatal
exposure to Cd, TET, and TMT, the dynamic
changes which occur during development in
this testing paradigm reveal additional aspects
of the toxicity of these metals (e.g., effects on
habituation) which can differentiate between
these compounds. Identification of the on-
togeny of behavioral differences produced b>
these metals provides a basis for identifying
the mechanism and progression of their spe-
cific neurotoxicity. This testing paradigm for
assessing the development of locomotor ac-
tivity offers several advantages as a method
for assessing the ontogeny of behavior follow-
ing alteration of nervous system development.
First, the peak in the development of loco-
motor activity in other types of apparatus
has been a useful landmark in assessing
effects of neurotoxicants such as 6-hydroxy-
dopamine (Erinoff et al., 1979) and lead
(Jason and Kellogg, 1981). Second, changes
in habituation provide an additional measure
of toxicant exposure (Shaywitz et ai., 1977).
Third, the size and configuration of the figure-
eight maze allows assessment of activity
throughout the life spaa (Norton, 1977), so
longtitudinal comparisons can be made in
the same apparatus.
ACKNOWLEDGMENTS
W« thank Ginpr Boncek aod Janice Brown for testing
the pupa. Partial support for thu *udy wai provided by
-------
BEHAVIORAL TOXICITY OF METALS
77
an interagency agreement with the Food and Drug
Administration.
REFERENCES
Avery, D. L., and Spyker, J. M. (1977). Foot tattoo
of neonatal mice. Lab. Animal So. 27, 110-112.
Campbell, B. a., Lytle, L. D., and Fibiger, H. C,
(1969). Ontogeny of adrenergic arousal and cholinergic
inhibitory mechanism} in the rat Science (Washington.
DC) 166, 635-637.
Chang, L. W„ Brown, D. a., and Dyer, R. S. (1983).
Different pattern of hippoeampal lesion induction in
rats aa a result of trimethyltin exposure at different
postnatal ages. Soc. Neurosci. Abs. 9, 1248.
Culver, B.. and NORTON, S. (1976). Juvenile hyper-
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Exp. Neurol. 50, 80-98.
Dean, K. F. (1983). Milk-band ratings: An index of
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DostiNC, J. (1968). Vulnerable periods in developing
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Erinoff, L., MacPhail, R. C„ Heller, a., and Seidcn,
L S. (1979). Age-dependem effects of 6-hydroxydo>
pamine on locomotor activity in the rat. Brain Res
64, 195-205.
OabBUNI, O., Baic, D., and DEZIEL. C. (1967). Toxicity
of cadmium for the nervous system. Exp. Neurol. It,
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Harry, O. J., and Tilson, H. a. (1982). Postpartum
exposure to triethyl tin produces long-term alterations
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Jason, K. M., and Ksllogg, C. K. (1981). Neonatal
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Jugo, S. (1977). Metabolism of toxic heavy metals in
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46.
Leathwood, P. (1978). Influence of tarty undernutrition
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In Studies on Urn Development of Behavior and the
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Academic PrasfcjNn* York.
MacPhail. It •^CfcorrON, K. M., and RirrtR,
L W. (1983). Hi of environmental challenges in
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Miller, D. a (1984), Pm- and poatweamng indicas of
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Appl. Pharmacol. 73,557-365.
Miller, D. B., and O'CallaOhan, j. P. (1983). Be-
havioral and Mrvow-tymm spaciAc protein changes
sascciatsd with eariy postnatal exposure to trimethyltin
(TMT). Soc Neurosci. Abs. 9,266.
Norton, S. (1977). Significance of sex and age differ-
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(I. Hanin and E. Usdin, eds.), pp. 17-25. Pergamon,
New York.
O'Callaghan, J. P., Miller, D. B., and Reiter,
L. W. (1983). Acute postnatal exposure to triethyltin
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RumRT, P. H. (1981). Developmental and behavioral
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(1983a). Development and behavioral toxicity following
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Neurobehav. Toxicol. Terasoi. S, 421-429.
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RumRT, P. H., Dean, K» F.. and Reiter, l W.
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RumRT, P. H., Dean, K. F., and Reiter. L. w.
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Zelterman, D. A. (1977), The effisct of 6-hydroxy-
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Veronem, B„ and Bondv, S, C (1983). Triethyltin-
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Winder, m., Muuuk, O., and Pmchowabu, a.
(1974), The effects of triethyltin intoxicatia* at the
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Neuropm. Pol. 1113-16.
Wono, K.-L.. andKLaaocn, C. D. (1982). Neurotoxic
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Pharmacol. 63,330*337.
Zaoon, 1.1, NcLauomun, P. ano Thompson,
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-------
-------
NturoToxicoloty S (4):9-22 (1984)
Copyright © 1984 by Intox Press, Inc.
Neurobehavioral Toxicity ol Triethyltln in Rats as a
Function of Age at Postnatal Exposure1*2
Patricia H. Ruppert, Karen F. Dean and Lawrence W. Reiter
Neurotoxicology Division (MD 74B), Health Effects Research Laboratory,
United States Environmental Protection Agency, Research Triangle Park,
North Carolina 27711
ABSTRACT: Trlethyltin (TET) has been shown to be neurotoxic
when injected on postnatal day (PND) 5. In the present experi-
ment we examined the toxicity of a single exposure to TET at
several postnatal ages. Rat pups were injected ip with 0 (saline),
1.5, 3.0 or 6.0 mg/kg TET bromide on PND 1, 5, 10 or 15. In
agreement with our previous data, PND-5 exposure to 6 mg/kg
TET produced behavioral toxicity and decreased adult brain
weight High dose pups were less successful in descending on a
rope at 20 and 21 days of age, and were hyperactive in figure-
eight mazes at 29-30 and 57-58 days of age. The spatial distribu-
tion of activity was also altered: photocell counts were increased
primarily in the figure-eight area of the maze. The size of the
milk bands was reduced in 8 mg/kg pups injected on either PND
1 or PND 5. Preweaning growth was decreased following all
injection ages; this reduction was most pronounced tor pups
exposed to TET on PND 1 and PND 5. Mating behavior was dis-
rupted in 6 mg/kg males irrespective of age at exposure. These
data demonstrate a differential sensitivity to the toxicity of TET
during postnatal life, with maximal susceptibility on PND 5.
Key words: Triethyltin, Postnatal Exposure, Developmental Toxicity
INTRODUCTION
Exposure of developing rat pups to
TET on PND 5 produces persistent neu-
robehavioral toxicity (Harry and Tilson,
1981; Reiter et a!., 1981; Harry and Til-
son, 1982; Ruppert et al„ 1983). This
exposure occurs during a period of
rapid brain growth which, in rats, occurs
during the first 3 wk of postnatal life.
Major events of this stage of neural
development are axonal and dendritic
growth, synaptogenesis, proliferation of
oligodendroglia and granule cells, mye-
PImm Mnd requests for reprints to Or. Patricia H. Ruppert.
'Presented at the International Confaranca on Neurotoxlcology of Selected Chemicals, September, 1982,
Chicago, Illinois (Neurotox/ee/3:134,1982).
This paper has been reviewed by tha Health Effects Research Laboratory, U.S. Environmental Protection
Agency, and approved for publication, Mention of trade names or commercial products does not constitute
endorsement or recommendation for use.
-------
10
RUPWEHT BT AL
linogenesis, and neurochemical natura-
tion (Davison and Dobbing, 1966; Dob-
bing, 1968; Ford, 1973; Cowan, 1979).
Sexual differentiation of the nervous sys-
tem, which is mediated by the action of
gonadal hormones, also occurs within the
first 2 wk of postnatal life in the rat (Goy
and McEwen, 1980). Dobbing has pro-
posed that this "brain growth spurt" is a
time of special vulnerability to environ-
mental disruption (Dobbing and Sands,
1971) because of the rapid growth and
development of the nervous system which
occurs at this time.
It has been repeatedly demonstrated
that the type of damage and resulting
behavioral deficits produced by a devel-
opmental neurotoxicant depend on the
time of exposure as well as the mecha-
nism of action of a toxicant (Rodier,
1980; Suzuki, 1980). TET is an interesting
neurotoxicant to pursue in defining peri-
ods of vulnerability during the brain
growth spurt because of its disruptive
effects on myelin and energy metabolism
(Cammer, 1980; Watanabe, 1980). In the
present experiment, we evaluated the
neurobehavioral toxicity resulting from a
single exposure to TET at several postna-
tal ages (PND 1, 5,10 or 15) which coin-
cide with different stages of rapid brain
growth in the rat (Gottlieb et ai, 1977).
We compared the effects of TET at these
exposure ages on mortality, body weight,
brain weight and behavioral functions
(rope descent, locomotor activity and the
acoustic startle response) which had pre-
viously been shown to be sensitive to TET
toxicity on PND 5 (Reiter et ai, 1981;
Ruppert et ai, 1983).
METHODS
Animals
Pregnant CD rats (Charles River)
were obtained 2 days after mating and
individually housed in cages measuring
45 x 24 x 20 cm. One day after parturition
(day of birth=day 0), pups were random-
ized and each dam was assigned 4 male
and 4 female pups. At 21 days of age,
animals were weaned and littermates were
housed by sex in groups of 4 in the same
size cages. Animals were maintained on a
12:12 hr light:dark cycle beginning at
0600 hr in an animal facility in which air
temperature (22°C±2) and humidity
(50%±10) were controlled. Except for
male sexual behavior, behavioral testing
was conducted during the diurnal portion
of the cycle (0900 hr-1700 hr). For pre-
weaning testing, all pups in a cage were
tested sequentially to prevent repeated
disturbance of litters, but testing was
counterbalanced for age at exposure. For
adult testing, animals within each test run
were counterbalanced for sex, home cage,
dose, and age at exposure. Food (Purina
Lab Chow) and water were available ad
libitum throughout the experiment.
Dosing
Rat pups were injected on PND 1,5,
10 or 15 (N=10 litters/age at exposure).
Each pup received a single intraperito-
neal injection of either 0 (sterile, 0.9%
saline), 1.5, 3.0 or 6.0 mg/kg triethyltin
bromide (Alfa Products; Danvers, Ma.)
calculated as the bromide. A within-litter
design was used for dosing: I male and 1
female from each litter received each dose
and, therefore, each litter contained all
treatments. The volume of injection was
10 fi 1/g of body weight for pups injected
on PND 1 and 5, and 5 jil/g of body
weight for pups injected on PND 10 and
15.
Data Analysis
The design model for this experi-
ment was a partial hierarchial four-way
factorial with no within-cell replication.
For all non-categorical data, analyses
were performed using litter as the unit of
analysis. Litter was assumed to be a ran-
dom factor nested within PND exposure
and crossed with treatment and sex.
-------
POSTNATAL TOXICITY OF TRIETHYLTIN
11
Treatment, sex and PND exposure were
considered fixed and completely crossed
factors.
Univariate repeated measures tech-
niques were used on BMDP (4V) to ana-
lyze pre-and post-weaning body weights,
and maze activity (total, figure-eight area
and blind-alley area). Age at exposure
was the between factor and sex, treatment
and age at testing were within factors.
Univariate repeated measures were also
used to analyze both the amplitude and
latency of the acoustic startle response.
Age at exposure was the between factor
and sex, treatment and background level
were within factors. Greenhouse-Geisser
adjustments were used where appropriate
with the overall level of significance set at
p<.005. Separate ANOVA's were used to
analyze organ weights. Age at exposure
was the between factor and treatment was
the within factor. The overall level of sig-
nificance was set at p<.01. There was one
missing value for preweaning body weight
from PND l exposure which resulted in
the deletion of the litter from the data.
Also, due to equipment failure there were
three missing values for the startle data
(one each from PND S, 10 and 15) which
resulted in deletion of the entire litters
from the analysis.
Due to the complexity of the exper-
imental design, the individual animal was
the unit of analysis of categorical data for
milk-band ratings, rope descent and male
sexual behayior. It was also difficult to
adequately assess interactions of expo-
sure age x treatment for categorical data.
Nevertheless, statistical methods which
were used for these measures are des-
cribed below.
Body Weight
Animals were weighed at 1,5,10,15,
20,30,44 and 38 days of age.
Milk Bands
As an estimate of milk consumption
during the acute phase of TET intoxica-
tion, pups injected on PND 1 and PND 5
were examined for the presence and size
of milk bands 24, 48, 72 and 96 hr after
injection. At these ages, the contents of
the stomach can be easily seen through
the ventral abdominal wall. Pups injected
on PND 10 and PND 15 were not rated
because the thickening of skin over the
abdomen prevented the viewing of milk
bands. Milk bands were rated according
to the following criteria: 0-no band vis-
ible; 1-small band visible on side of pup;
2-small band visible across pup's abdo-
men; 3-large band visible across pup's
abdomen. The rater was not aware of the
pup's treatment while performing the
observation. Milk-band ratings were ana-
lyzed using the Kruskal-Wallis test (chi2
approximation).
Descending on a Rope
On days 20 and 21, pups were tested
for the ability to descend a 30 cm x 17 mm
rope suspended above the home cage,
using a procedure modified from Ander-
son and Altman (1972). Each pup was
held by the nape of the neck and placed
against the rope with its head pointing
upward. Pups were allowed 180 sec to
complete the test; 1 trial was given per
day. Performance was scored as success-
ful if the pup displayed either motor
coordination or muscular strength by 1)
descending the rope in either the head-up
or head-down position, or 2) clinging to
the rope for the duration of the test. Per-
formance was scored as unsuccessful if
the pup fell or slid from the rope during
the test. The rater was not aware of the
pup's treatment while performing the
test. The proportion of successful ani-
mals/ group was transformed (arcsin) and
analyzed by repeated-measures ANOVA
using test day as the within-subjects fac-
tor.
Motor Activity
Motor activity of individual animals
was measured for I hr in a figure-eight
-------
12
RUPPERT ETAL
maze on days 29-30 and again on days
57-58. The maze (Reiter et al., 1975) is a
series of interconnected alleys (10 x 10
cm) converging on a central arena and
covered with a removable, transparent
acrylic-plastic top. Motor activity is
detected by 8 phototransistor/photodi-
ode pairs. Mazes (N= 16) were housed in 2
sound-attenuated rooms maintained on
the same lightrdark cycle as the animal
facility.
Startle Response
At approximately 70 days of age,
latency (time interval between stimulus
onset and the initiation of a response) and
amplitude (maximum force exerted on
the platform, corrected for the baseline)
of the acoustic startle response were mea-
sured. Testing was conducted in 4 sound-
attenuated chambers each containing a
acrylic plastic-framed wire cage (7.6 x 7.6
x 23 cm) mounted on a load cell/force
transducer assembly designed to measure
vertical force. Two speakers were mounted
on the ceiling of each chamber 30 cm
above the test cage. One speaker pres-
ented an acoustic stimulus (13 kHz, 120
dB(A), 40 msec tone with a 2.5 msec rise
time); the second delivered background
white noise at each of 3 intensity levels
(50, 65 and 80 dB(A)). These stimulus
parameters were adapted from Hoffman
and Searle (1965). Following a 10-min
period of adaptation to the chamber at
ambient noise levels, a total of 30 stimuli
were presented at an inter-stimulus inter-
val of 20 sec; 10 stimuli were presented at
each of 3 background noise levels which
were balanced across the session.
Male Sexual Behavior
Male rats received 3 weekly mating
tests starting at approximately 90 days of
age. Testing arenas were 37.5 x 47.5 x 20
cm acrylic-plastic cages with pine shav-
ings covering the bottom. Testing was
conducted during the nocturnal period
(1900-2300 hr); the rater was unaware of
the males'treatment during the test. Males
were allowed a 5-min adaptation period
in the arenas before a sexually receptive
female was introduced. Females were
made receptive by 3 daily injections of 12
/ug estradiol benzoate followed by 500 /ug
progesterone on the fourth day 4 hr
before testing. Components of male sex-
ual behavior (including frequency and
latency measures for mounts, intromis-
sions and ejaculation) were scored as des-
cribed by Beach and Jordan (1956). Cop-
ulatory activity was observed until either
1) the male ejaculated, or 2) 20 min (wk I
and 2) or 30 min (wk 3) had elapsed from
the introduction of the female and no
ejaculation had occurred. Since males
were inexperienced at the start of testing,
only data from the third week of testing
were used for statistical analysis. The
proportion of males ejaculating was ana-
lyzed using a 2 x 4 (performance x treat-
ment) chi2 test. The contingency coeffi-
cient, a measure of the strength of
association, was also obtained. Other
components of male sexual behavior were
analyzed using MANOVA.
Organ Weights
Males were sacrificed after tests for
mating behavior; body weight and wet
weights for brain, thyroid, adrenals, sem-
inal vesicles and testes plus epididymes
were obtained.
RESULTS
Body Weight
Growth was reduced in dosed pups
following all exposure ages (Fig. 1).
However, these body weight decreases
were most persistent following PND 1
and PND 5 exposure to TET. There was a
three-way interaction of treatment x age
x exposure for preweaning body weight
[F(13.18,153.75)=3.16, p<.0003]; there-
fore, tests of simple main effects and sim-
ple interactions were performed. There
-------
POSTNATAL TOXICITY OF TRieTHYLTIN
13
TET 0OSAGE, m«/k(
~ 1.5
A 3.0
0 6.0
mo i
m
s
IM
(9
i
>
a
o
M
PN01I
PNOIO
It «
ASC.dWl
PIG. 1. Pre weaning body weight* (X ± 8G) for control and TET-expoaed nit pupa. Arrow In
each panel Indicate* when TET exposure occurred. For all day* of expoaure, TET produced a
reduction In growth.
was a treatment x age interaction on
PND 1, 5, 10 and 15 [F(4.39,153.75)*
11.17,14.82,6.81,7.73 respectively; ail
p'sC.00001]. Tukey's (a) tests were used
to make post-hoc comparisons. For PND
1 exposure, there was no treatment effect
on day 1. On days 5,10,15 and 20 pups
receiving 6 mg/ kg TET weighed less than
all other groups. For PND 5 exposure,
there were no treatment effects on day 1
or day 5. On day 10, IS and 20 pupt
receiving 6 mg/ kg TET weighed less than
all other groups, and pups receiving 3
mg/kg TET weighed less than controls;
pups receiving 1.5 mg/ kg TET weighed
less than controls on day 15 and 20. For
PND 10 exposure, there were no treat-
ment effects on days 1,5 or 10. On days 15
and 20, pups receiving 6 mg/kg TET
weighed less than all other groups, and on
day 20 pups receiving 3 mg/kg TET
weighed less than controls. For PND 15
exposure, there were no treatment effects
on days 1, 5, 10 or 15. On day 20, pups
-------
14
RUPPERT ET AL
receiving all dosages of TET weighed less
than controls, and pups receiving 6 mg/ kg
TET weighed less than all other groups.
Decreases in body weight were not
persistent. At ages 30,44 and 58 the usual
sex effect on body weight was evident,
with males being heavier than females,
and body weight increased for both sexes
with age. There were no effects of treat-
ment or interaction with treatment.
Milk Bands
For pups injected on either PND I or
PND 5, there was an effect of treatment
on milk-band ratings 24 hr after dosing
[chi2(3)=27.53, p<.000l]. Pups injected
with 6 mg/kg TET on either PND 1 or
PND 5 (data combined) had smaller milk
bands than all other pups 24 hr after dos-
ing but not at later times (Fig. 2).
Rope Descent
There was an improvement in per-
formance for all rats from day 20 to 21
[F(l,260)=28.96, p<.0001), with no
treatment x day of testing interaction
[F(3,260)= 1.11, p<.3459]. There was a
treatment x exposure interaction [F(9,73)
=2.27, p<.0185]; therefore, separate ana-
lyses were conducted for each exposure
age. For PND-5 exposure only, there was
an overall treatment effect [F(3,73)=6.15,
p<.0005). The percentage of successful
animals was 87.5% for controls, 80.0%
for 1.5 mg I kg, 65.0% for 3 mg/ kg and
42.5% for 6 mg/ kg TET. Animals injected
with 6 mg/ kg on PND 5 were less success-
ful than controls in performing this task.
Figure-Eight Maze Activity
There was an interaction of treat-
TIT OOtAdt. mf/k«
PIQ. 2. Milk band rati ngs (0-3) measured 24-06 hr after Injection
for eontrola and pupa exposed to TET on PN01 or PND 5 (7 ±
SE). Pupa receiving 0 mg/kg TET on either PND 1 or PND S
(data combined) ahowad a reduction in tha size of the milk
banda 24 hr after dosing, indicating reduced milk consumption.
-------
POSTNATAL TOXICITY OF TRIETHYLTIN
15
ment x exposure [F(7.26,87.15)=10.72,
p<.00001] and sex x age [F(l,36)=25.84,
pC.OOOO I ]; therefore, tests of simple main
effects were performed. There was a
treatment effect for exposure on PND 5
[F(2.42,87.l5)=4l.78, pC.OOOO 1], but not
for other exposure ages. Tukey's (a) test
showed that activity of animals receiving
6 mg/kg TET on PND 5 was increased
compared to all other groups (Fig. 3).
There was a sex effect on PND 57-58 only
[F( l,36)=29.83, p<.00001], with females
more active than males and an age effect
for both males and females [F( 1,36)=28.22
and 159.74, respectively; p'sC.00001], with
activity on PND 57-58 higher than on
PND 29-30.
For activity in the figure-eight area
of the maze, there was a treatment x
exposure interaction[F(6.70,80.36)= 14.33,
pC.OOOO 1]; therefore, simple main effect
tests were performed. There was a treat-
ment effect for exposure on PND 5
[F(2.23,80.36)=56.88, p<.00001], but not
tor other exposure ages. Tukey's (a) test
showed that motor activity of animals
receiving 6 mg/kg TET on PND 5 was
higher in the figure-eight area of the maze
than all other groups (Fig. 4). There was
also an age effect [F( 1,36)=457.76,
p<.00001] with older rats (PND 57-58)
more active in the figure-eight area than
younger rats (PND 29-30).
For activity in the blind-alley area of
the maze, there was a sex x age interac-
tion [F( 1,36)=29.36, p<.00001 ]; therefore,
simple main effect tests were performed.
There was a sex effect on PN D 57-58 only
[F( 1,36)= 160.14, pC.OOOO 1], with females
more active in the blind alleys than males,
and an age effect for both males and
females [F( 1,36)=142.72,12.06 respec-
tively; p's<.001] with older rats (PND
57-58) less active in the blind-alley area
29-30 DAYS OF AGE
TET DOSAGE
S7MOAVSOF AGE
FIQ. 3. Figure-eight maze activity (or control and TET-exposed
rats. Data are expressed as photocell interruptions (7. ± SE)
during a 1-hr test session. Exposure to 6 mg/kg TET on PND 5
produced an increase in motor activity at both times of testing.
-------
16
RUPPERT 6T AL
BLIND ALLEY AGE 29-30OAVS FIGURE EIGHT
i———i nn 1—z—r
T— ' 1 _ 1 1
M ^ OMALE „
f>4 UOi «female
"1 " "I" " 1 -r
(g L
TET DOSAGE, mg/kg
PIQ. 4. Distribution of motor activity in figure-eight mazes for
PND-5 control and TET-exposad animals. Data are presented
as photocell counts (7± SE) in either the blind alleys or figure-
eight area of the maze. Young animals show a different pattern
of activity than adults. Exposure to 0 mg/kg TET increased
photocell counts primarily in the figure-eight area.
than younger rats (PND 29-30).
Acoustic Startle Response
For the amplitude of the startle
response, there was a sex x background
noise interaction [F(1.52,50.07)=48.49,
p<.00001]; therefore, simple main effect
tests were performed. There was an effect
of background level for both males and
females [F( 1,33)= 152.29,86.05 respec-
tively; p's<.00001], with the amplitude of
the response increasing with increasing
level of background noise. There was a
sex effect at all background noise levels
[F(l,33)=82.20,61.10,33.31 for 80,65 and
50 dB respectively; p'sC.OOOOl], with the
amplitude of the response greater in males
than in females. For latency to onset of
the response, there was an effect of back-
ground noise level [F(1.10,36.45)=8.25,
p<.0055], with latency of the response
decreasing with increasing background
noise levels. There were no effects of
treatment or interactions with treatment.
Male Sexual Behavior
Since a preliminary test indicated no
treatment x exposure age interaction for
the proportion of males ejaculating
[chiJ(9)=1.65, p<.9959], data were com-
bined across all days of exposure. There
was an effect of treatment on the propor-
tion of males ejaculating during the third
weekly mating test [chi2(3)= 16.258,
p<.001]. The percent of males ejaculating
was 92.3% for controls, 79.5% for 1.5
nig/ kg, 94.9% for 3 mg/ kg, and 64.1 % for
the 6 mg/ kg dose group. Exposure to 6
mg/kg TET decreased the number of
males ejaculating irrespective of age at
exposure. The contingency coefficient, a
measure of the strength of association,
was 0.307. There were no treatment effects
on latency or frequency for mounts or
intromissions.
Organ Weights
For brain weight, there was an
interaction of exposure x treatment
-------
POSTNATAL TOXICITY OF TRIETHYLTIN
17
2J
12
2.1
tr i.o
9
- 1.»
s
1.S
1.7
1.#
o moi
~ mot
a mo i*
onto it
i.s
3.0
TET DOSAGE,
».S
6.0
PIG. 8. Wet wight of tha whola brain (K ± SB) for control and TET-axpotad mala rats.
Expoaura to e mg/kg TET on PND S only producad a dacraasa in adult brain weight.
[F(8.20,98.37)=5.98, p<.00001]; therefore,
simple main effect tests were performed.
There was a treatment effect for exposure
on PND 5 [F(2.73,98.37)=3l.65,
pC.OOOOl], but not for other exposure
ages. Tukey's (a) test showed that brain
weight of animals receiving 6 mg/kg TET
on PND 5 was reduced compared to all
other groups (Fig. S). For seminal vesicle
weights, there was a treatment x exposure
interaction [F(7.81,93.75)=2.82, p<.0081 ];
therefore, simple main effect tests were
performed. There was a treatment effect
for exposure on PND 5 [F(2.60,93.75)
-5.22, p<.0035], but not for other expo-
sure ages. Tukey's (a) test showed that
seminal vesicle weights of rats receiving 6
mg/ kg TET were larger than those of rats
receiving 1.5 mg/ kg TET. there were no
effects on thyroid, adrenal or testes and
epididymes weights.
DISCUSSION
The toxicity produced by TET dur-
ing the preweaning period in the rat is
dependent on the day of exposure. TET
had its maximal effects as a developmen-
tal neurotoxicant following exposure on
PND 5. Behavioral changes following
acute exposure to TET only on PND 5
include preweaning deficits in rope des-
cent performance and adult hyperactiv-
ity. In animals exposed to TET as adults,
toxicity is reversible following termina-
tion of exposure (Magee et al., 1957;
Reiter et al, 1980; Squibb et al., 1980;
-------
11
RUPPERT ET AL
Tilson and Burne, 1981). Since the type of
disorder produced by toxicants can differ
as a function of the time during develop-
ment when exposure occurs (Rodier, 1980;
Suzuki. 1980), other behavioral tests
might detect disruption of function spe-
cific to exposure at other developmental
ages. However, brain weight was de-
creased only in animals exposed to TET
on PND 5. These behavioral and brain
weight data indicate that PND 5 is a time
of special vulnerability to the toxicity of
TET.
Milk bands were reduced when eval-
uated 24 hr afteT dosing with TET on
PND 1 and PND 5; this effect did not
persist beyond 24 hr. Milk bands were
reduced to a similar extent following 2 hr
of deprivation, and were correlated with
reduced stomach weight (Dean, 1983),
indicating decreased milk consumption.
Therefore, a decrease in the size of milk
bands reflects a transient lack of nutrition
during the acute phase of TET intoxica-
tion. Preweaning growth retardation is a
reliable indicator of TET toxicity (Reiter
et al., 1981). Since milk bands were
reduced only 24 hr after dosing and not at
later times, decreased preweaning body
weight may reflect an additional meta-
bolic effect of TET. Milk bands in pups
injected at later ages could not be evalu-
ated because of thickening of the skin
over the abdomen and the development
of hair. However, treatment effects on
body weight were most pronounced for
pups injected on PND 1 and PND 5.
Since body weight was transiently reduced
in pups injected at older ages (PND 10
and PND 15), food intake may have been
reduced in these older pups as well. Food
intake, and to a lesser extent water intake
is reduced in adult animals receiving TET
(Squibb et at., 1980; DeHaven et at.,
1982). Adult animals are also hypoactive
2 hr after TET injection (Reiter et ah,
1980). Since activity level influences suc-
kling in young rat pups (Blass et al.,
1979), decreased milk intake in rat pups
exposed to TET could be a result of
decreased activity. However, serotoner-
gic and cholinergic antagonists also sup-
press suckling behavior in young rat pups
(Caza and Spear, 1982; Spear and Ris-
tine, 1982), Acute administration of TET
to adult rats depletes several neurotrans-
mitters including serotonin (Moore and
Brody, 1961; Robinson, 1969). There-
fore, reduced milk bands and the resul-
tant preweaning growth reduction may
be specific effects of TET.
Hyperactivity was produced by TET
injected on PND 5 only. PND-5 TET also
altered the spatial distribution of activity
in developing and adult animals. Young
(day 30) control rats of both sexes dis-
tribute their activity in a figure-eight
maze unequally: photocell counts are
higher in the blind alleys than in the figu-
re-eight portion of the maze. As adults,
overall activity increases, but only in the
figure-eight area of the maze; photocell
counts in the blind alleys actually decrease.
At both ages of testing, hyperactivity
produced by 6 mg/ kg TET was primarily
due to an increase in photocell counts in
the figure-eight area of the maze. There-
fore, PND 5 TET animals remained
hyperactive while the distribution of
activity in control animals changed from
a juvenile to an adult pattern. In addition
to TET not producing hyperactivity fol-
lowing exposure at ages other than PND
5, the pattern of photocell interruptions
was consistent between these groups.
Other behaviors previously shown to
be disrupted by PND 5 TET were not
affected by dosing at other postnatal
ages. Pups exposed to 6 mg/kg TET on
PND 5 were less successful in descending
on a rope; this deficit persists with
repeated testing from days 15-20 (Reiter
et al., 1981). Although rope descent tests
motor coordination (Altman and Sudar-
shan, 1975), neuromuscular weakness
could produce deficits in this task. Neu-
romuscular weakness results from adult
exposure to TET (Barnes and Stoner,
-------
POSTNATAL TOXICITY OF TRNETHYLTIN
1959). However, performance of pups
exposed to TET on PND 5 was normal on
several tests of neuromotor function—
negative geotropism, placing reaction,
head elevation and ascending on a wire
mesh (Reiter et al., 1981). Also, Harry
and Tilson (1981) found no effects on grip
strength or negative geotaxis at 21,28,60
or 90 days of age. In this experiment,
PND-15 TET animals showed no impair-
ment of function when tested for rope
descent only 5 days after dosing.
The results of the present study do
not agree with previous findings (Harry
and Tilson, 1981; Ruppert et al., 1983)
that exposure to TET on PN D 5 decreased
the amplitude of the acoustic startle
response. This discrepancy was likely due
to the use of the litter as the unit of analy-
sis and a more conservative criterion of
significance in the present study (p<.00S),
as the data showed the same trends we
had reported previously (Ruppert et al.,
1983). Consistent relationships were found
for sensitization by background noise:
the amplitude of the response increased
and latency decreased with increasing
levels of background noise. Neither sen-
sitization, amplitude nor latency of the
response was affected by postnatal expo-
sure to TET at any age in the present
experiment.
Sexual behavior was altered in high-
dose males exposed to TET at all postna-
tal ages. A significant percentage of 6
mg/ kg TET males failed to ejaculate after
repeated pairing with a female in a res-
tricted (30 min) mating test. Neither sem-
inal vesicle nor testis + epididymis weight
was decreased by treatment, suggesting
that this behavioral deficit was not due to
a direct toxic effect on the gonads. Hex-
achlorophene, whose toxicity resembles
that of TET (Towfighi, 1980), produces a
similar deficit in mating behavior when
administered from day 1-8 postnataily
(Gellert et al., 1978). Following a dosage
which produced 25% mortality, all males
mounted and intromitted, but only 20-
40% ejaculated; this deficit was seen dur-
ing fertility tests as well as during a res-
tricted mating session. There were no
alterations in serum testosterone levels or
testes weight. Therefore, deficits in sexual
behavior seen in the present study may
indicate impaired fertility in TET males
as well. Since effects of TET were similar
following injection at all postnatal days,
toxicant exposure might be disrupting
processes involved in sexual differentia-
tion which can extend through the first 2
wk of postnatal life in the rat (Gorski et
al., 1977; Goy and McEwen, 1980).
Alternatively, TET may produce the same
mating deficits when given to adult males
because of its depletion of hypothalamic
neurotransmitters (Cook, 1983). Further
studies are needed to explore these
possibilities.
PND-5 TET produces persistent
effects on myelin (Wender et al, 1974;
O'Callaghan et ai, 1983; Padilla et al.,
1982), neuronal development (Veronesi
et ai, 1982), and other neurochemical
processes (Harry and Tilson, 1982) which
may be specific to that age of develop-
ment. TET is neurotoxic when given at
postnatal ages other than PND 5. Suzuki
(1971) reported that hemorrhage and
neuronal necrosis at lethal dosages of
TET were less severe in rat pups injected
on PND 8 than in newborns. Intramye-
linic vacuolation was seen in mouse pups
following exposure on PND 5, 10, 15 or
20, but few vacuoles remained 1-2 wk
after dosing (Watanabe, 1977). For other
heavy metals, such as cadmium, the
pathology produced by single dosages
changes substantially between PND 1
and PND 15 (Gabbiani et al., 1967;
Webster and Valois, 1981), These differ-
ences are predictable, given the complex
development encompassed within the
"brain growth spurt". It remains to be
determined why TET produces persistent
and specific changes in behavior and
reduction in brain weight following expo-
sure on PND 5.
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20
buppert rr al
ACKNOWLEDGEMENTS
We thank Jerry Tolson for her
assistance in conducting this study. Par-
tial support for this study was provided
by an interagency agreement with the
Food and Drug Administration. We also
thank two anonymous reviewers and Dr.
Keith Muller for their guidance in the
statistical analysis of this experiment.
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Anderson WJ and Altman J. Retardation of cere-
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Barnes JJ and Stoner HB. The toxicology of tin
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in rat pups. Tax Letters 1983; 18
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POSTNATAL TOXICITY OF TRIBTHYLTIN
21
Reiter LW, Anderson GE, Laskey JW and Cahill
DF. Developmental and behavioral changes in
the rat during chronic exposure to lead. Environ
Health Perspect 1975; I2:l 19-123
Reiter L, Kidd K, Heavner G and Ruppert P.
Behavioral toxicity of acute and subacute expo-
sure to triethyltin in the rat. Neurotoxicol 1980;
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Reiter LW, Heavner GB, Dean KF and Ruppert
PH. Developmental and behavioral effects of
early postnatal exposure to triethyltin in rats.
Neurobehav Toxicol Teratol 1981; 3:285-293
Robinson IM. Effects of some organotin com-
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Cosmet Toxic 1969: 7:47-52
Rodier PM. Chronology of neuron development:
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Develop Med Child Neurol 1980; 22:525-545
Ruppert PH, Dean KF and Reiter LW. Compara-
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split-litter and whole-litter dosing. J Toxicol
Environ Hlth 1983; 12:73-87
Spear LP and Rlstine LA. Suckling behavior in
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Squibb RE, Carmichaet NG and Tilaon HA.
Behavioral and neuro-morphologicai effects of
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Suzuki K. Some new observations in triethyl-tin
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Suzuki K. Special vulnerabilities of the developing
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NEUROTOXICOLOGY MS 1173
Submitted: May 16, 1983
Accepted: June 26, 19S4
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Toxicology Letters, 22 (1984) 33-38
Elsevier
33
TOXLett. 1231
TRIMETHYLTIN DISRUPTS ACOUSTIC STARTLE RESPONDING IN
ADULT RATS*
(Sensory dysfunction; neurotoxicity; behavioral toxicity)
PATRICIA H. RUPPERT, KAREN F. DEAN and LAWRENCE W. REITER
Neurotoxicology Division, US Environmental Protection Agency, Research Triangle Park, NC 27711
(U.S.A.)
(Received December 13th. 1983)
(Accepted March 3rd, 1984)
SUMMARY
Trimethyltin (TMT) is a limbic-system toxicant which also produces sensory dysfunction in adult
animals. In the present experiment, we examined the effects of TMT on the acoustic startle response.
Adult male, Long-Evans rats (N* 12/dose) received a single i.p. injection of either 0, 4.0, S.O or 6.0
mg/kg TMT hydroxide as the base. The number of responses, latency and peak amplitude of the startle
response to a 13 kHz, 120 dB tone were measured 2 h, 2 weeks, and 4 weeks after dosing. For each test
session, 10 stimuli were presented at each of three background noise levels (50, 65 and 80 dB). By 2 h
after dosing, the number of responses and response amplitude were decreased following 4.0-4.0 mg/kg
TMT; these treatment effects persisted through 4 weeks after dosing. Increases in latency were also seen
following all dosages of TMT. These data suggest that TMT produces disruption of function within the
acoustic-startle pathway.
INTRODUCTION
TMT is a limbic-system neurotoxicant which produces neuronal necrosis within
the hippocampus, amygdaloid nucleus and pyriform cortex following acute ex-
posures in adult rats [1, 2]. Behavioral effects include hyperactivity [3, 4] and
deficits in learning and memory (5-7]. TMT also produces sensory-system dysfunc-
tions including alterations in visual [8] and somatosensory [9] evoked potentials.
~Presented at the Society of Toxicology Annual Meeting, March, 1983, Lai Vegas (U.S.A.)
This paper Ins been reviewed by the Health Effect* Research Laboratory, U.S. Environmental Protec-
tion Agency, and approved for publication. Mention of trade names or commercial products does not
constitute endorsement or recommendation for use.
Abbreviations: IDPN, iminodipropionitrile; TMT, trimethyltin.
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34
The mechanism of this sensory-system toxicity is unknown, but may involve both
peripheral and central components. For example, changes in the visual-evoked
response indicate both reduced retinal output and increased cortical arousal [8].
TMT also produces swelling of hair cells within the organ of Corti in rats by 24
h after dosing, and hair-cell loss is seen by 30 days after dosing [10J. Preliminary
data have indicated decreases in acoustic startle responding [11], reduction or
elimination of prepulse inhibition of startle [12], and alterations in brainstem
auditory evoked potentials of rats exposed to TMT [13]. In the present study, we
determined both dose- and time-effect relationships of acute TMT exposure on the
acoustic startle response in adult rats. This response was selected because of its pro-
ven usefulness in assessing effects of drugs [14] and brain lesions on sensory-motor
reactivity.
METHODS
Male Long-Evans hooded rats (Charles River; Portage, MI), 60 days of age, were
housed individually in stainless steel cages measuring 24 x 20 x 18 cm with wire mesh
floors. Animals were allowed free access to both Purina Lab Chow and water. The
animal room was maintained on a 12 h light-dark cycle beginning at 06.00 h, and
testing was conducted during the diurnal period. On day 0, rats (N= 12/group)
received a single i.p. injection of either 0 (sterile, 0.9% saline), 4.0, 5.0 or 6.0 mg/kg
trimethyltin hydroxide calculated as the base; an injection volume of 1.0 ml/kg was
used. This close spacing of dosages was selected because steep dose-response func-
tions have been reported within this range for behavioral effects of TMT [4]. Time-
dependent changes in the acoustic startle response were assessed 2 h, 2 weeks and
4 weeks after dosing.
Testing was conducted in 4 sound-attenuated chambers each containing a plastic-
framed wire cage (7.6x7.6x23 cm) mounted on a load cell/force transducer
assembly designed to measure vertical force. Two speakers were mounted on the
ceiling of each chamber 30 cm above the test cage. One speaker (Motorola, 2 * x 5 *
piezo electric tweeter) presented an acoustic stimulus: a 13 kHz, 120 dB, 40 ms tone
with a 2.S ms rise/decay time. A second speaker (special design, full range 16 ohm)
delivered background white noise (produced by a Bruel and Kjaer Model 1403 noise
generator) at each of 3 intensity levels (50, 65 and 80 dB). These stimulus parameters
are similar to those described by Hoffman and Searle [IS]. Sound intensity (A scale)
was measured within the test chamber using a Bruel and Kjaer sound level meter
Model 2209.
Following a 10-min period of adaptation to the chamber at ambient noise level
(45 dB), testing was begun. Each rat was presented a total of 30 trials with an inter-
trial interval of 20 s; the animals were given 10 trials at each of the 3 background
noise levels. Each trial was initiated by presenting the rat with the selected
background noise level for 20 s prior to the stimulus presentation. The order of
-------
35
presentation of the 3 background noise levels was balanced across the session; se-
quences were randomly generated by computer. The analog response was sampled
at a rate of 1 kHz for 72 ms following the stimulus presentation, digitized and con-
verted into grams of force using a calibration constant. The following measures
were taken:
(1) Baseline. The average force exerted on the platform during the first 10 ms
following the stimulus presentation. This value reflects the body weight of the
animal.
(2) Number of responses. The number of trials per session in which a response
occurred (i.e., amplitude exceeded 4 S.D. of the baseline). If the force did not ex-
ceed the baseline by 4 S.D., 'no response' was recorded for that trial. Typically, the
coefficient of variability was 1% of the baseline; therefore, a change in baseline of
approx. 4% or greater was recorded as a startle response. Only trials in which a
response occurred were used to calculate session means for the latency to onset and
amplitude of the response (i.e., values of zero were not included for 'no response'
trials).
(3) Latency to onset. The interval from the stimulus onset to the time (ms) when
the force exerted on the platform was 4 S.D. above the baseline.
(4) Amplitude. The maximum force exerted on the platform during the response,
corrected for (i.e., minus) the baseline.
For the number of responses, a proportion of the total possible responses (pooled
across background noise levels) was obtained for each animal. The arcsin of the pro-
portion was analyzed by multivariate repeated-measures analyses [16], using time
after dosing as the within-subjects factor. Amplitude and latency of responses were
averaged across trials at each background noise level and analyzed using
multivariate repeated-measures analyses with background noise and time after dos-
ing as within-subjects factors. As a measure of sensitization, differences between the
amplitudes of the startle response at the 50 and 80 dB background noise levels were
calculated for each animal. These values were analyzed by repeated-measures
ANOVA using time after dosing and background noise as within-subjects factors.
When significant treatment interactions were found, simple main effects tests
(ANOVA) were conducted. Post-hoc comparisons were made using Tukey's (a) test
[17]. Analyses were performed using programs on the Statistical Analysis System
(SAS, 1982) and the Biomedical Data Program (BMDP, 1981).
RESULTS
The proportion of responses was decreased by TMT at all times after dosing
{F(9,102) *5.39; P< 0.0001]. Animals receiving all dosages of TMT made
significantly fewer responses than controls (Fig. 1A).
Latency to the onset of the response was increased by TMT [F(3,42)«6.16,
/> <0.0014]. AH dosages of TMT significantly increased latency (Fig. IB). There was
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36
30
M
1(0 ¦
300
100 S
m 1M
2 im
to 3
so
to
*0
1.0
IMTDO»»at. mj/kf
TMT DOMOt. mq/kt
Fig. I. Number of responses (A), latency io onset (B), amplitude (C), and amplitude difference (D) for
a 13 kHz, 120 dB stimulus in controls and TMT-treated rats. All values are mean ± SE. All dosages
of TMT produced a decrease in the number of trials in which the animals responded (A), an increase
in latency to respond (B), a decrease in response amplitude (C), and reduced amplitude facilitation by
increasing levels of background noise (D).
also a time by treatment interaction [F(6,82) = 2.88, P<0.0134]; latency increased
over time for the 6.0 mg/kg TMT-treated animals only [F{2,41)= 11.06,
P<0.0001], Latency decreased as a function of background noise [F(2,4l)= 16.08,
P <0.0001), but there was no interaction with treatment.
Response amplitude was reduced by TMT at all times after dosing
[F(3,42) =13.95, F<0.0001). There was an increase in amplitude over time
[F(2,41) = 3.90, P< 0.0282] but since there was no interaction of treatment with
time, data have been collapsed across times. Startle amplitudes were significantly
reduced in all TMT-treatment groups (Fig. 1C). There was an effect of background
noise by treatment interaction [F(6,82)=6.39, P<0.0001]. This interaction suggests
a difference in sensitization to background noise levels as a function of treatment.
An analysis of the change in amplitude from the SO to 80 dB background noise levels
indicated a significant effect of treatment at all times after dosing [F(3,44) = 14.41,
PC0.00011. All dosages of TMT significantly reduced sensitization (Fig. ID).
DISCUSSION
TMT produced both acute and persistent deficits in auditory startle responding
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37
following a single exposure in adult animals; following all dosages of TMT the
number of responses, response amplitude and sensitization by background noise
were decreased while response latency was increased. Effects of TMT on acoustic
startle were seen at lower dosages than those producing the TMT syndrome [18J or
behavioral changes associated with hippocampal dysfunction [3-7]. These deficits
were produced within 2 h of dosing and recovery was not seen by 4 weeks after
dosing.
The most dramatic treatment effect produced by TMT was the decrease in the
number of responses to the 120 dB tone stimulus. Although hippocampal lesions
characterize TMT toxicity [1,2], hippocampal lesions either have no effect [19, 20]
or increase startle responsiveness [21]. Likewise, lesions of the amygdala do not
disrupt startle responding [19], While drugs which affect a variety of neurotransmit-
ter systems can modulate the amplitude of the startle response, none of these drug
treatments abolish startle or even depress it markedly [14]. However, electrolytic le-
sions within the primary acoustic startle circuit also abolish or markedly attenuate
startle responding [22]. A similar decrease in the number of startle responses was
reported in rats following acute exposure to IDPN [23]. IDPN produces axonal le-
sions in the nucleus reticularis pontine caudalis, as well as damage to the nuclei of
the eighth nerve and motor neurons [24], Therefore, both electrolytic and neurotox-
ic lesions of the primary auditory startle circuit can produce a decrease in the
number of startle responses.
The startle reflex can be modified by stimulus factors, including the level of
background noise [15]. Sensitization, an increase in amplitude and decrease in laten-
cy of the response produced by increasing levels of background noise, was seen in
control animals. Following all dosages of TMT, the sensitization effect of
background noise on amplitudes but not latencies was significantly reduced. While
the neural mechanisms underlying sensitization are not understood, these data sug-
gest a differential effect on amplitude and latency. However, this lack of sensitiza-
tion for amplitudes at higher dosages could represent a 'floor' effect, so that further
amplitude decreases were classified as non-responses. For example, while the reduc-
tion in amplitude was comparable at all dosages of TMT, the number of responses
was dose-related. Also, sensitization effects on latency were maintained at all
dosages of TMT, while latencies to onset were increased. Since the neural circuit for
the startle reflex probably involves only 3 synapses plus the neuromuscular junction
[22], increases in latency produced by TMT are quite significant. Further studies at
lower dosages of TMT are needed to determine the threshold for these effects.
acknowledgements
We thank Jerry Tolson and Lee Bynum for assistance in conducting this study.
Partial support for this study was provided by an interagency agreement with the
Food and Drug Administration.
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38
REFERENCES
1 A.W. Brown, W.N. Aldridge, B.W. Street, and R.D. Veischoyle, The behavioral and
neuropathologies! sequelae of intoxication by trimethyltin compounds in the rat, Am. J. Pathol., 97
(1979) J9-82.
2 T.W Bouldin, N.D. Goines, C.R. BagneJI and M.R. Krigman, Pathogenesis of trimethyltin neuronal
toxicity. Ultrastructural and cytochemical observations. Am. J. Pathol., 104 (1981) 237-249.
3 H.S. Swartzwelder, R.S. Dyer, W. Holahan and R.D. Myers, Activity changes in rats following acute
trimethyltin exposure, Neurotoxicology, 2 (1981) 589-J94.
4 P.H. Ruppert, T.J. Walsh, L.W. Reiter and R.S. Dyer, Trimethyltin-induced hyperactivity: Time
course and pattern, Neurobehav. Toxicol. Teratol., 4 (1982) 135-139.
5 H.S. Swartzwelder, J. Hepler, W. Holahan, S.E. King, H.A. Leverenz, P.A. Miller and R.D. Myers,
Impaired maze performance in the rat caused by trimethyltin treatment: problem-solving deficits and
perseveration, Neurobehav. Toxicol. Teratol., 4 (1982) 169-176.
6 T.J. Walsh, M. Gallagher, E. Bostock and R.S. Dyer, Trimethyltin impairs retention of a passive
avoidance task, Neurobehav. Toxicol. Teratol., 4 (1982) 163-167.
7 T.J. Walsh, D.B. Miller and R.S. Dyer, Trimethyltin, a selective limbic system neurotoxicant, im-
pairs radial-arm maze performance, Neurobehav. Toxicol. Teratol., 4 (1982) 177-183.
8 R.S. Dyer, W.E. Howell and W.F. Wonderlin, Visual system dysfunction following acute
trimethyltin exposure in rats, Neurobehav. Toxicol. Teratol., 4 (1982) 191-195.
9 W.E. Howell, T.J. Walsh and R.S. Dyer, Somatosensory dysfunction following acute trimethyltin
exposure, Neurobehav. Toxicol. Teratol., 4 (1982) 197-201.
10 L.W. Chang and R.S. Dyer, Trimethyltin induced pathology in sensory neurons, Neurobehav. Tox-
icol. Teratol., 5 (1983) 673-696.
11 W.E. Howell, R.S. Dyer, W.F. Wonderlin, K. Kidd and L.W. Reiter, Sensory system effects of acute
trimethyltin (TMT) exposure, Toxicologist, 1 (1981) 43.
12 J.S. Young and L.D. Fechter, Trimethyltin disruption of reflex inhibition indicates an ototoxic effect,
Toxicologist, 3 (1983) 168.
13 R.S. Dyer, The use of sensory evoked potentials in toxicology. Fund. Appl. Toxicol., (1984) in press.
14 M. Davis, Neurochemical modulation of sensory-motor reactivity: Acoustic and tactile startle
reflexes, Neurosci. Biobehav. Rev., 4 (1980) 241-263.
15 H.S. Hoffman and J.L Searle, Acoustic variables in the modification of startle reaction in the rat,
J. Comp. Physiol. Psychol., 60 (1965) 53-58.
16 D. Morrison, Multivariate Statistical Methods, McGraw-Hill, New York, 1967.
17 B.J. Winer, Statistical Principles in Experimental Design, McGraw-Hill, New York, 1971.
18 R.S. Dyer, T.J. Walsh, W,F. Wonderlin and M. Bercegeay, The trimethyltin syndrome in rats,
Neurobehav. Toxicol. Teratol., 4 (1982) 127-133.
19 E.D. Kemble and J.R. Ison, Limbic lesions and the inhibition of startle reactions in the rat by condi-
tions of preliminary stimulation, Physiol. Behav., 7 (1971) 925-928.
20 R.N. Leaton, Habituation of startle response, lick suppression, and exploratory behavior in rats with
hippocampal lesions, J. Comp. Physiol. Psychol., 95 (1981) 813-826.
21 G.D. Coover and S. Levine, Auditory startle response of hippocampectomized tats, Physiol. Behav.,
9 (1972) 75-77.
22 M. Davis, D.S. Gendelman, M.D. Tischler and P.M. Gendelman, A primary acoustic startle circuit:
lesion and stimulation studies, J. Neurosci., 2 (1982) 791-805,
23 G. Wolff, E. Kunze, A. Rodden and H. Oepen, Loss of auditory startle-reflex in the iminodipro-
pionitrile (IDPN) treated rat, Life Sci., 20 (1977) 1163-1166.
24 S.M. Chou and H.A. Hartmann, Axonal lesions and waltzing syndrome after IDPN administration
in rats. Acta Neuropathol., 3 (1964) 428-450.
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COMPARATIVE DEVELOPMENTAL TOXICITY
OF TRIETHYLTIN USING SPLIT-LITTER
AND WHOLE-LITTER DOSING
Patricia H. Ruppert, Karen F. Dean, Lawrence W. Reiter
Neurotoxicology Division, U.S. Environmental
Protection Agency, Research Triangle Park,
North Carolina
Previous work In our laboratory suggested that toxicity resulting from acute postnatal
administration of triethyitin (TET) was influenced by the treatment condition of litter-
mates, To test this possibility, two dosing models were compared. For the split-litter
model (H = 20 litters/dose), 1 male and 1 female pup per Utter received a single dose
of 0 (saline), 3, 6, or 9 mg TET/kg on postnatal d 5; the remaining 6 littermates were
not injected. In the whole-litter model, all 8 littermates received 0, 3, 6, or 9 mg TET/kg
(H « s litters/dose). Differences between dosing models were found for preweanlng
body weight and adult figure>elght maze activity. Body weights were reduced in all
TET-dosed pups; for 3-mg/kg animals, the reduction in pre weaning growth was more
persistent for pups in the spilt-lltter gmup. Motor activity in a figure-eight mate was
increased in both 6- and 9-mg/kg animals; for the high dose, the increase in activity
was greater for animals in the split-litter group. There were no differences between
dosing models in mortality, brain weight, or postweaning body weight Approximately
5096 of the 9-mg/kg animals died; there was no treatment related mortality at lower
doses. Adult body weight also remoined decreased only in the 9-mg/kg animals. Brain
weight was reduced for all TET dose groups. These results indicate that developmental
toxicity produced by TET Is not primarily determined by the dosing regimen.
INTRODUCTION
In a previous study, we described the neurobehaviorai effects of acute
postnatal exposure to triethyitin (TET) in rats using a within-litter dosing
regimen (Reiter et al., 1981). Pups were removed from their dams on the
day of birth, and each dam was randomly assigned four male and four
female foster pups. One male and one female pup from each litter received
Ptrtitl support for thIt study wu provided by an Interagency agreement with the Food and
Drug Administration. We thank Bette Terrlll for assistance In dosing and weighing pups, and Jerry
Totson for assistance throughout this study.
Presented at the Behavioral Teratology Meeting, Palo Alto, California, June 1981.
This paper has been reviewed by the Health Effects Research Laboratory, U.S. Environmental
Protection Agency, and approved for publication. Mention of trade names or commercial products
does not constitute endorsement or recommendation for use.
Requests for reprints should be sent to Patricia Ruppert, Neurotoxieology Division (MD 748),
Health Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle
Park, North Carolina 27711.
73
Journal of Toxicology and Environmental Health, 12:73-97,1983
Copyright •1983 by Hemisphere Publishing Corporation
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74
P. H. RUPPERT ET AL.
the vehicle control, low, medium, or high dosage of TET. With this dosing
regimen, the contribution of genetic factors to behavioral development
is randomly distributed across litters. Also, postnatal litter effects pro-
duced by maternal influences or other environmental factors are distribu-
ted equally across all treatments. Experimental designs that incorporate
litter in the analysis are recommended for statistical treatment of data
from developmental studies (Weil, 1970; Abbey and Howard, 1973; Hase-
man and Hogan, 1975; Hughes, 1979). Therefore, within-litter dosing
provides several advantages.
However, experimental manipulations such as handling of pups or
disturbing litters can influence mother/pup interactions (e.g., Meier and
Schutzman, 1968; Lee and Williams, 1974; Villescas et al., 1977), sug-
gesting that interventions required for dosing pups may disrupt the normal
balance within the litter and influence the resulting toxicity. It is possible
that the influence of experimental manipulations could be maximized
using a within-litter design. For example, the dam may distinguish be-
tween the normal behavioral and physiological development of control
pups and the abnormal development of dosed pups within the litter.
Undernutrition or other environmental factors that put pups at a disad-
vantage can either enhance or diminish maternal care (Smith and Berkson,
1973; Cone, 1974; Lynch, 1976; Wiener et al., 1977; Misanin et al., 1979;
Piccirillo et al., 1979; Johanson, 1980; Fleischer and Turkewitz, 1981).
It is also possible that competition between littermates could affect the
expression of toxicity; this competition could be different depending on
whether a pup was competing with dosed or control littermates.
Using within-litter dosing, we had found significant mortality when
either 9 or 12 mg TET/kg was the high dose. This alerted us to the possibility
that mortality might be partially determined as a function of being the
high dose within the litter, irrespective of the dosage of the toxicant. In
some cases, however, within-litter dosing can be comparatively advantageous
for treated pups. For example, the behavioral toxicity of 6-hydroxydopa-
mine was attenuated when treated pups were reared with vehicle litter-
mates as compared to litters where all pups were treated (Pearson et al.,
1980). Also, growth was reduced in saline-injected pups compared to non-
injected pups dosed in a whole-litter design, but there were not differences
between these groups in weight gain using a split-litter design (Martin and
Moberg, 1981). In the present experiment, we investigated the influence
of litter composition on the developmental toxicity of TET by comparing
whole-litter and split-litter dosing. In our previous study, at the 6-mg/kg
dosage, TET produced preweaning deficits in body weight and rope
descent, and a decrease in brain weight and increase in activity that per-
sisted through adulthood (Reiter et al., 1981). Using TET, then, we could
compare the effects of dosing model on mortality, body weight, brain
weight, and behavior to obtain a range of variables that might be influenced
by the composition of the litter.
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EFFECTS OF DOSING MODEL ON TET TOXICITY
75
METHODS
Animals
Pregnant CD rats (Charles River, Portage, Mich.) were obtained 2 d
after mating and housed individually in cages measuring 45 X 24 X 20
cm. On the day of parturition (d 0), litters were culled to 8 pups (4 males
and 4 females) and distributed to foster mothers, with the restriction that
no pup was reassigned to its natural mother and only 1 pup per litter was
assigned to each foster mother. At 21 d of age, animals were weaned and
littermates were housed by sex in groups of 3 or 4 in the same size cages.
Animals were maintained on a 12:12 h light:dark cycle beginning at 0600
h in an animal facility controlled for temperature (22 ± 2°C) and humidity
(50 ± 10%). Food (Purina Lab Chow) and water were available ad libitum
throughout the experiment. All behavioral testing was conducted during
the diurnal portion of the cycle.
Dosing
On d 5, dosed pups received either a single intraperitoneal injection
of triethyltin bromide (TET, Alfa Products, Danvers, Mass.) or vehicle
(sterile isotonic saline) in a volume of 10 /Ltl/g body weight; some pups,
described below, received no treatment, but were left in the litter to
maintain a standard litter size. Each litter was assigned to one of two
models (dosing groups). For model I (split-litter group), 1 male and 1 fe-
male from each litter received 0, 3, 6, or 9 mg/kg; the remaining pups
(3 males and 3 females) were not dosed. This design was chosen as the
"worst case" to maximize within-litter differences and, therefore, to best
test the influence of typical within-litter designs which involve dose-response
evaluations upon littermates. Twenty litters per dose were used, providing
a total of 40 pups (20 males and 20 females) per dose. For model II (whole-
litter dosing), all pups in the litter (4 males and 4 females) received 0, 3,
6, or 9 mg/kg. This model was used to assess the influence of the typical
between-litter design. Five litters per dose were used, providing a total
of 40 pups (20 males and 20 females) per dose. In model I, therefore, 2
of 8 pups/litter were dosed, while for model II all 8 pups were dosed. Table
1 indicates the assignment of animals in this study. Due to technical or
mechanical problems, several animals were not tested or the data were
lost for the behavioral tests.
Body and Brain Weight
All rats were weighed at 3-d intervals until weaning, and then every
14 d through 60 d of age. A subgroup of animals was sacrificed at 22 d
of age and wet weight of brains was recorded; animals from the 9-mg/kg
dose were not included. Brains were then dissected (Glowinski and Iverson,
1966) and wet weights were obtained for hippocampus, cerebellum, ol-
factory bulbs, and hypothalamus.
-------
76
P. H. RUPPERT ET AL.
T ABLE 1. Number of Animals Assigned to Each Model and Variable4
Model /: TET dosage (mg/kg) Model II: TET dosage (mg/kg)
Variable
0
3
6
9
0
3
6
9
/V
40
40
40
40
40
40
40
40
Mortality
0/40
0/40
0/40
17/40
2/40
)/40
0/40
25/40
Prewearting, N
40
40
40
23
38
39
40
15
Body weight
X
X
X
X
X
X
X
X
Whole brain
20/40
20/40
20/40
-
15/38
16/39
16/40
-
Brain parts
9/20
10/20
9/20
-
8/15
8/16
8/16
-
Postweaning, N
20
20
20
23
23
23
24
15
Body weight
X
X
X
X
X
X
X
X
Activity
X
X
X
X
X
X
X
X
Startle
X
X
X
X
X
X
X
X
"An "X" indicates that all animals still remaining in the study were Included.
Figure-Eight Maze Activity
Locomotor activity was measured at 55-60 d of age in a figure-eight
maze (Reiter et al., 1975). The maze consists of a series of interconnected
alleys (10 X 10 cm) converging on a central arena and covered with trans-
parent acrylic plastic. Motor activity was detected by eight phototransistor-
photodiode pairs; data were collected by a microprocessor system. Animals
were tested individually for 1 h.
Acoustic Startle Response
Peak amplitude and latency of the startle response to an acoustic stimu-
lus were measured at 65-70 d of age. Testing was conducted in 4 sound-
attenuated chambers containing a plastic-framed wire cage (7.6 X 7.6 X
23 cm) mounted on a load cell/force transducer assembly {Gould Statham
Model UC3 with 5-lb adaptor Model UL4-5) designed to measure vertical
force. Two speakers were mounted on the ceiling of each chamber directly
above the test cage. One speaker (Phillips Model AD0163/T15) presented
an acoustic stimulus [13-kHz, 120-dB(A), 40-ms tone with a 2.5-ms rise
time]. The second (special design, full range 16 ohm) delivered background
white noise (produced by B&K Model 1405 noise generator) at each of 3
intensity levels (50,65, and 80 dB). These stimulus parameters were adapted
from Hoffman and Searle (1965). Following a 10-min adaptation period,
each rat was presented a total of 30 stimuli with an interstimulus interval
of 20 s; 10 stimuli were presented at each of 3 background noise levels.
The order of presentation of the 3 background noise levels was balanced
across the session; sequences were randomly generated by the computer.
Statistical Analysis
Since an original intent of this study was to examine the effects of
litter composition on mortality produced by TET, an equal number of
-------
EFFECTS OF DOSING MODEL ON TET TOXICITY
77
pups was assigned to each model. Therefore, all data were analyzed using
the animal as the statistical unit of analysis. All tests were conducted
using programs in the Statistical Analysis System (SAS, 1979). The general
format for the sources of variation and degrees of freedom were: model
(1), sex (1), model X sex (1), dose (3), dose X model (3), dose X sex (3),
dose X model X sex (3), and residual error (152). Mortality data were
analyzed using a Z-statistic for the difference between proportions. Mea-
sures of preweaning and postweaning body weight and brain weight were
analyzed using multivariate general linear model (MGLM) analyses (Mor-
rison, 1967); when overall significant differences were found, individual
measures were analyzed using a general linear model (GLM) regression
analysis. The amplitude and latency of the startle response were analyzed
using repeated-measures analysis of variance (ANOVA) (Kirk, 1968), with
background noise level as the within-subjects factor. Differences between
the peak amplitudes of the startle response at 80 and 50 dB(A) background
levels were calculated and analyzed using GLM. All post-hoc comparisons
were made using Tukey's (o)-test (Winer, 1971). For all statistical tests,
values greater than the critical value at p < .05 were accepted as signifi-
cant. All interactions with sex and model were tested and are only dis-
cussed when significant.
RESULTS
Mortality
For both exposure models, there were no treatment-related deaths
at doses of 0, 3, or 6 mg TET/kg. However, in the 9-mg/kg group, the
overall mortality was 52%. The majority of deaths occurred 8-11 d after
injection. A comparison of dosing models indicated that there was no
significant difference (Z = 1.8180, p = .065) in the proportion of deaths
between model I (42%) and model II (62%), nor was there a sex difference
in mortality.
Body Weight
TET produced a dose-related retardation in growth. Preweaning body
weights are shown separately for the two dosing models (Fig. 1), since
there was both a significant effect of model {F{5,242) = 10.15,p < .0001 ]
and a significant treatment-by-model interaction [F(15,668) = 2.99, p <
.0001). Individual regression analyses revealed that the treatment effect
was persistent across all preweaning ages (all p values less than .0001), At
ages 14-20 d, model I pups weighed less than model II pups. Pups injected
with either 6 or 9 mg TET/kg weighed significantly less than controls during
the entire preweaning period. The 3-mg TET/kg pups from both dosing
models weighed less than controls on d 8 and 11. Thereafter, 3-mg/kg
pups from model il did not differ from controls, while those from model
I still weighed less than controls on d 17 and 20. For postweaning body
-------
78
P. H. RUPPERT ET AL.
30 —
S so
MODEL II
30
FIGURE 1. Preweaning body weight for pups Injected on postnatal d S with TET or saline either
in a split-litter or whole-litter dosing model. Values are mean t SE. SE bars are not indicated when
they do not exceed the size of the symbols.
weights, there was no effect of model on body weight, so these data are
combined (Fig. 2). There was a sex effect [F{3,141) = 314.71 ,p< .0001 ]
and treatment effect (F(9,343) = 25.01 ,p < .0001 ] which was significant at
all postweaning ages (p < .0001). The 6-mg TET/kg animals were still signifi-
cantly different from controls on d 28, while for 9-mg TET/kg animals, body
weight decreases persisted to d 42 and 59.
Brain Weight
TET produced a dose-related decrease in whole brain weight (F(2,104) =
137.73, p < .0001], hippocampal weight (F(2,31) = 9.10, p < .0008]
and cerebellar weight [F{2,31) = 6.27, p < .0052] at 22 d of age. Since
there were no differences due to dosing model, these data have been com-
bined (Table 2). Whole brain weight was significantly reduced for both
the 3- and 6-mg TET/kg animals. Wet weight of the cerebellum and hippo-
campus was also significantly reduced in the 6-mg TET/kg animals, while
there were no differences in the weight of the olfactory bulbs or hypo-
thalamus.
-------
EFFECT OF DOSING MODEL ON TET TOXICITY
79
(•
i
>
FIGURE 2, Postweaning body weight for animals injected with TET or saline on postnatal d 5. Values
are mean ± SE.
TABLE 2. Wet Weight of Whole Brain Parts at 22 d of Age in Animals injected with
TET or Saline on Postnatal d 5
Dosage of TET (mg/kg)
Wet weight
0
3
6
Whole brain (g)
X
1.50
1.44
1.22
SE
0.01
0.01
0.01
Cerebellum (mg)
X
180.3
169.4
162.7
SE
2.7
3.2
5.1
Hippocampus (mg)
X
87.9
79.2
61.9
SE
6.7
2.5
3.7
Olfactory bulbs (mg)
X
66.2
65.6
62.6
SE
3.0
2.4
1.1
Hypothalamus (mg)
X
50.5
60.1
45.2
SE
3.0
3.3
3.2
D0M6E OF TET, n|/ll|
o •
iU i I 1 1
p it 5«•
ABE, tan
-------
80
P. H. RUPPERT ET AL.
Figure-Eight Maze Activity
TET produced a dose-related increase in motor activity [F(3,139) =
23.75, p < .0001] (Fig. 3). There was also a significant effect of sex
[FO ,139) = 15.60, p < .0001) and model 1,139) = 7.58, p < .0067],
Females were more active than males, as is typically seen, and model I
animals were more active than model II. Both 6- and 9-mg TET/kg animals
were significantly more active than both controls and 3-mg TET/kg animals.
Within the 9-mg/kg dose group, animals from model I were more active
than those from model II.
Startle Response
There was a significant effect of treatment on both the amplitude
and onset latency of the startle response. Since there were no effects or
interactions with dosing model on either measure, these data were com-
bined.
The amplitude of the startle response was greater in males than fe-
males (F(1,136) = 7.03, p < .0090], as was facilitation of amplitude by
background noise [F(2,271) = 3.33, p < .0372]. When data for males
and females were analyzed separately, there was a significant treatment
effect for males only (F(3,65) = 4.43, p < .0069]; this effect was con-
stant across all background noise intensities. However, TET produced
a nonlinear effect on startle amplitudes. At the 6-mg/kg dose, startle
amplitude was decreased when compared to control; for 9-mg TET/kg
males, amplitudes were increased to a level not different from control,
TIT Q0SAGE,mt/k«
$50
o%
4N
J10
2M
¥0011II
M00CO
TIMI, mi*
FIGURE 3. Figure-eight maze activity during 5-min intervals for adult animals injected with TET or
saline on postnatal d 5. Values are mean ± SE for photocell counts. The insert indicates the total
activity counts for the 1 -h test.
-------
EFFECTS OF DOSING MODEL ON TET TOXICITY
81
1M
2M
IN -
nt
Mi
1H
1M
1M
2M
2 jo.
1M
MALM
OOUMOf T»T,«VH
FIGURE 4. Amplitude of the startle response to a 13-kHz, 120-dB stimulus as a function of back-
ground noise intensity in adult animals Injected with TET or saline on postnatal d 5, Values are
mean t SE for 10 trials at each background noise level. The insert indicates startle amplitude col-
lapsed across background noise levels.
but significantly greater than the 6-mg/kg response (Fig. 4). The change
in the amplitude of the startle response from the 50-dB to the 80-dB
background noise intensity was increased by TET [F(3,119) = 4.91, p <
.00321. Th's effect was significant for the 9-mg/kg dose only (Fig, 5).
There was no sex effect on onset latency; however, latency decreased
as a function of increasing background noise levels [^(2,265) = 14.75,
p < .0001 ]. TET effects on latency were similar to those seen on ampli-
tudes. Again, treatment effects were seen only in males. However, for
latency, background noise intensity was a determinate in treatment ef-
fects [F(6,136) « 3.76, p < .0017). At all background noise levels, latency
was significantly decreased for 9-mg/kg males compared to controls and
6-mg/kg males. Latency for 6-mg/kg males was greater than control only
at the 50-dB background intensity (Fig. 6).
DISCUSSION
In general, these data are in agreement with our previous report on
TET toxicity using within-litter dosing (Reiter et al., 1981). Although
some differences did emerge between dosing models, these were not
major determinants of TET toxicity. TET produced mortality and defi-
cits in adult body weight at the 9-mg/kg dose only, increased activity at
the 6- and 9-mg/kg doses, and decreased preweaning body weight and
d 22 brain weight following all doses. However, some differences did
-------
1M
139
2 121
^ IIP
1
9
3
ft
0
DOSAGE Of TIT, rni/hf
FIGURE 5. Amplitude of the startle response to a 13-kHz, 120-dB stimulus as a function of back-
ground noise intensity in adult animals injected with TET or saline on postnatal d 5. Values are mean
± SE for the difference between responses at SO and 80 dB. Ten trials were presented at each back-
ground noise level.
BACKGROUND lEVtL. 41
- Oil
aw
AM
—»
«
15
I
i
2
OOtASI OF TIT,
FIGURE 6. Latency to onset of the startle response to a 13-kHz, 120-dB stimulus as a function of
background noise Intensity in adult animals injected with TET or saline on postnatal d 5. Values are
mean t SE for 10 trials at each background noise level.
82
-------
EFFECTS OF DOSING MODEL ON TET TOXICITY
83
emerge between the dosing models that indicate effects of litter composi-
tion on the expression of TET toxicity.
As shown previously, 9 mg TET/kg produced approximately 50% mor-
tality in rat pups injected on postnatal d 5 (Reiter et al., 1981). Although
there was a 20% difference in mortality (42 versus 62%) between dosing
models, this was not statistically significant. Neither the 6-mg/kg nor the
3-mg/kg dose of TET produced any treatment-related mortality in either
dosing model. Thus, our original hypothesis that the dosing model would
be a determinant of mortality was not supported. Survivors of the high
dose, however, were severely affected by treatment. Body weight was
decreased throughout the experiment for the 9-mg/kg animals; at 59 d
of age, body weight was still decreased to 74% of control. In agreement
also with previous data (Reiter et al., 1981), body weights were decreased
at the 3- and 6-mg/kg dosage but this difference did not persist to adulthood.
There was an effect of dosing model on preweaning body weight and
a model-by-treatment interaction during the third week of postnatal life.
Differences indicated lower body weights for TET-dosed animals in the
split-litter model. Saline injections decreased body weight only in whole
litters (Martin and Moberg, 1981) or homogeneous litters (Pearson et al.,
1980); in the present study, there were no differences in body weights
of saline-dosed animals between the two models. It is interesting that
effects of dosing model on body weight did not appear until d 15 follow-
ing injection of 6-OHDA on d 5 (Pearson et al., 1980), and until d 7-14
when saline was injected on d 2-12 (Martin and Moberg, 1981). Since
effects of dosing model on body weight were not seen earlier in develop-
ment in either of these studies or in the present experiment, differences
may reflect a disturbance during a transition in the regulation of suckling
(Hall et al., 1977).
Brain weight at 12 mo of age was reduced by 14% in animals injected
with 6 mg TET/kg on postnatal d 5, and by 7.2% in animals injected with
3 mg TET/kg (Reiter et al., 1981). In the present experiment, when ani-
mals were sacrificed at 22 d of age, brain weight was decreased by 19%
in 6-mg/kg animals and by 4% in 3-mg/kg animals. Thus, decreases in brain
weight produced by TET are achieved by d 22 and remain constant as the
brain grows in size. Different brain areas, however, were differentially affected
by TET. The most severe decrease in wet weight was in the hippocampus
(30%) of 6-mg TET/kg animals, followed by the cerebellum (10%). There
were no differences in wet weight in the other brain areas measured, the
olfactory bulbs and the hypothalamus. The hippocampus, cerebellum,
and olfactory bulbs were measured because neurogenesis of microneurons
continues in these structures through early postnatal life (Altman, 1970).
Thus, if TET were interfering with dividing neurons, these areas should
be preferentially and similarly affected. This hypothesis was not supported,
since the olfactory bulbs were unaffected while the cerebellum, at the
peak of its "growth spurt" on d 5, was less affected than the hippocampus,
-------
84
P. H. RUPPERT ET AL.
which is in a relatively quiescent state on d 5 (Dobbing et alM 1970, 1971;
Fish and Winick, 1969).
Again in agreement with our previous report (Reiter et al., 1981),
animals injected with TET on postnatal d 5 were hyperactive as adults.
The highest dose of TET previously tested for motor activity was 6 mg/kg.
Although TET at both 6 and 9 mg/kg produced equivalent increases in
total activity, the typical habituation curve was not seen for 9-mg/kg
animals. There was also an increase in variability at 9 mg/kg and a signifi-
cant effect of model at this dose only. Animals from our previous study
had been repeatedly tested during development and repeatedly tested in
figure-eight mazes (Reiter et al., 1981), while in the present experiment
animals were only tested once in the maze. The magnitude of the increase
in activity at comparable ages was similar in both experiments. This con-
stancy stands in contrast to 6-OHDA hyperactivity, where rearing in a
between-litter dosing model shifted the curve and prolonged heightened
activity ievels to a period when behavior would have recovered (Pearson
et al., 1980).
TET altered the startle response for males regardless of dosing model.
As previously reported for male rats (Hoffman and Searle, 1965; Ison
and Hammond, 1971; Davis, 1974), the amplitude of the response in-
creased as a function of increasing background noise levels. This facilita-
tion was also observed for female rats in the present study. The decrease
in the latency to onset of the response in both sexes with increasing back-
ground noise levels provides further support for the facilitative effect of
background noise.
Harry and Tilson (1981) reported decreased acoustic startle response
in rats injected with TET at 3 mg/kg on postnatal d 5 when tested on d
60 but not on d 21, 28, or 90. The results of the present study show a
trend toward decreased amplitude at the 3-mg/kg dose that was not signifi-
cant. Doses ranging from 3 to 9 mg/kg produced a biphasic response in
males for both amplitude and latency; the response for females showed
the same trend but was not significant. At the 6-mg/kg dose, amplitude
of the response was decreased and latency increased. These results parallel
increased latencies and decreased amplitudes reported for brain stem
auditory-evoked potentials in young adult rats ingesting TET via their
water supply (Amochaev et al., 1979). At the 9-mg/kg dose, the ampli-
tude of the startle response was not altered, while latency was decreased.
This apparent recovery in response amplitude and acceleration of the re-
sponse seems paradoxical. However, these findings, coupled with the change
in the slope of the response at the 9-mg/kg dose, suggest an increase in
sensitization.
Horlington (1970) reported decreased latency and increased ampli-
tude of the startle response during the nocturnal phase of the light cycle
compared to the diurnal. Hoffman and Ison (1980) have reported decreased
latency to startle with prepulse stimulation that was independent of changes
-------
EFFECTS OF DOSING MODEL ON TET TOXICITY
85
in amplitude. Both studies concluded that decreased latency reflects in-
creased activation in neural pathways regulating the startle reflex. The
balance of neurotransmitter systems and environmental conditions that
modulate the startle response have been studied extensively (Davis,
1980); for example, serotonin depletion and dopamine agonists both
produce biphasic effects on the startle response. High doses of TET in
adult animals severely deplete several neurotransmitters, including sero-
tonin and norepinephrine (Moore and Brody, 1961; Robinson, 1969);
brain weight decreases in postnatal d 5 TET-dosed animals, and be-
havioral alterations could be associated with interference with postnatal
development of these systems (Harry and Tilson, 1981).
Littermate control designs have been extensively used for studies of
hormone, drug, and toxicant action during postnatal development. In
the present study, differences between dosing models involved the magni-
tude but not the occurrence of toxicity. Handling effects are seen with as
little as 3 min removal of pups from litters (e.g., Levine et al., 1967); in
toxicology studies, removal of pups for weighing is a minimal requirement.
In the within-litter design, handling and testing of pups are distributed
equally across ail treatments in a litter. Support for the within-litter design
is gained from these advantages, coupled with minimization of genetic
differences in assessing dose response relationships and economy of animal
usage.
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Smith, L., and Berkson, G. 1973. Litter stimulus factors in maternal retrieval (Rattus rattus). Anim.
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Vlllescas, R., Bell, R. W., Wright, L., and Kufner, M. 1977. Effect of handling on maternal behavior
following return of pups to the nest. Dev. Psychobiol. 10:323-329.
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EFFECTS OF DOSING MODEL ON TET TOXICITY
87
nation in toxicological studies involving reproduction, teratogenesis or carcinogenesis. Food
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Winer, B. J. 1971. Statistical Principles in Experimental Design. New York: McGraw-Hill.
Received December 23, 1982
A ccepted A pril 3, 19S3
-------
NeurobehavioraI Toxicology and Teratology, Vol. 5, pp. 421-429, 1983. c Ankho International Inc. Printed in the U.S.A.
Developmental and Behavioral Toxicity
Following Acute Postnatal Exposure
of Rat Pups to Trimethyltin1,2
PATRICIA H. RUPPERT,3 KAREN F. DEAN AND LAWRENCE W. REITER
Neurotoxicology Division, US Environmental Protection Agency, Research Triangle Park, NC 277/1
Received 3 January 1983
RUPPERT, P. H., K. F. DEAN AND L. W. REITER. Developmental and behavioral toxicity following acute postnatal
exposure of rat pups to trimethyltin. NEUROBEHAV TOXICOL TERATOL 5(4) 421-429, 1983.—The purpose of this
study was to extend our investigations on the developmental neurotoxicity of trialkyltin compounds. On postnatal day J
(PND 5). rat pups received a single intraperitoneal ii\jection of either 0 (saline), 4, 5 or 6 mg/kg trimethyltin hydroxide
(TMT) calculated as the base. The size of the milk bands was decreased in 6 mg/kg TMT pups 48-% hr after dosing, while in
5 mg/kg TMT pups, milk bands were reduced 96 hr after dosing only. Dosages of 5 and 6 mg/kg TMT reduced growth and
impaired performance in rope descent during the preweaning period. As adults, motor activity in figure-eight mazes was
increased for 6 mg/kg TMT animals. The startle response to an acoustic stimulus (a l3 kHz. 120 dB tone) was also affected
by TMT when measured both during ontogeny and in adulthood. During development, on days 10-21. both 5 and 6 mg/kg
TMT reduced the number of responses during 30-trial sessions for both males and females. Amplitudes were decreased for
the 5 and 6 mg/kg dose on days 12-13. and for all dosages on days 18-19 and 20-21. Startle amplitude of adults was
decreased at all dosages for males but not for females. These behavioral changes were accompanied by decreases in adult
brain weight for both sexes. Whole brain weight and weight of the olfactory bulbs were decreased following all dosages of
TMT. while hippocampal weight was decreased following both 5 and 6 mg/kg TMT. These results indicate that acute
postnatal exposure to TMT produces long-term effects on the nervous system and behavior.
Trimethyltin Postnatal exposure Behavior Development Rats
TRIMETHYLTIN (TMT) is a neurotoxic organotin com-
pound which produces neuronal necrosis primarily in the
hippocampus, pyriform cortex, amygdaloid nucleus and
neocortex [8.9]; these neuropathological changes are
produced by single exposures in adult animals. The
neurobehavioral effects of this compound have been exten-
sively studied in adults [18], but not in developing animals. It
has been repeatedly demonstrated that the nervous system
can be particularly vulnerable to the action of toxic agents
during development, and that the type of damage and result-
ing functional deficits depend not only on the toxicant's
mechanism of action but also on the time of exposure
[42,52]. During postnatal life in the rat, the brain undergoes a
period of rapid growth characterized by neuronal maturation
and myelination [16]; postnatal neurogenesis also continues
>n the hippocampus, cerebellum and olfactory bulbs [1].
Since neuroendocrine differentiation also continues
Postnatally in n:- rat [22], postnatal exposure to toxicants
can also permanently alter endocrine functioning.
Triethyltin (TET) is a neurotoxic organotin compound
which, unlike TMT, produces vacuolation of myelin in
adult animals [54]. Whereas toxicity following adult expo-
sure is reversible [42,51], persistent neurobehavioral effects
result from exposure to TET on PND 5 [23, 24, 43. 44, 48].
These changes include impaired rope descent performance,
hyperactivity in figure-eight mazes, a reduction in the ampli-
tude of the acoustic startle response, deficits in male sexual
behavior and decreased brain weight, particularly of the hip-
pocampus [43, 44,48}. The purpose of the present study was
to evaluate the neurobehavioral toxicity of TMT in develop-
ing animals using the same dosing model (acute exposure on
PND 5) and the same dependent variables (behavior, brain
weight and endocrine organ weights) previously used to
assess the developmental toxicity of TET.
METHOD
Animals
Pregnant Long-Evans rats (Charles River) were obtained
2 days after mating and individually housed in cages measur-
1 Presented at the Society for Neuroscience Annual Meeting, Minneapolis, MN, 1982.
This paper has been reviewed by the Health Effects Research Laboratory, U. S. Environmental Protection Agency, and approved for
Publication. Mention of trade names or commercial products does not constitute endorsement or recommendation for u";
'Requests for reprints should be addressed to Patricia Ruppert, Neurotoxicology Division (MD 74B), Environmental Protection Agency,
Research Triangle Park. NC 27711.
421
-------
422
RUPPERT, DEAN AND REITER
ing 45x24x20 cm. One day after parturition (day of birth
=day 0). pups were randomized and each dam was assigned
4 male and 4 female pups. At 21 days of age, animals were
weaned and littermates were housed by sex in groups of 4 in
the same size cages. Animals were maintained on a 12:12 hr
light.dark cycle beginning at 0600 hr in an animal facility in
which air temperature (22°C±2) and humidity (50%±10)
were controlled. Food (Purina Lab Chow) and water were
available ad lib throughout the experiment. Behavioral test-
ing was conducted during the diurnal portion of the cycle
except where noted.
Dosing
On PND 5, each pup received a single intraperitoneal
injection of either 0 (vehicle, sterile isotonic saline), 4, 5 or 6
mg/kg TMT-hydroxide (ICN Pharmaceuticals, Inc.; Plain-
view, NY) calculated as the base. A within-litter design was
used for dosing: 1 male and 1 female from each of 20 litters
received each dose and, therefore, each litter contained ail
treatments. The volume of injection was 10/il/g body weight.
Testing Procedures
The assignment of animals to test groups is shown in
Table 1. For all animals (N=I60) milk bands, body weight
and adult acoustic startle response were measured. All
animals were weighed at 5, 10, 15, 20, 30, 45, 55, 90 and
approximately 120 days of age. One group of litters (N=80
animals) was used to assess development of the startle re-
sponse, adult mating behavior, and weights for endocrine
organs. The second group of litters (N=80 animals) was
tested for preweaning rope descent, adult motor activity, and
weights for whole brain and brain parts. For tests requiring
direct observation of the animals (i.e., milk bands, rope des-
cent and male sexual behavior), the observer was not aware
of the animal's treatment group while performing the test.
Data Analysis
All data were analyzed using programs on the Statistical
Analysis System [49]. When treatment effects were found for
multivariate (MANOVA) tests [34], individual variables
were analyzed by analysis of variance (ANOVA). When
significant interactions with the within-subjects factor were
found for repeated-measures ANOVA [28], simple main ef-
fects were examined at each level of the factor. Post-hoc
comparisons of treatment means were made using Tukey's
(a) test [55], All values greater than the critical value at
p<0.05 were accepted as significant. MANOVA was used to
analyze treatment effects on preweaning body weight. Sepa-
rate MANOVAs were performed for postweaning body
weight, organ weights and brain weights. Other statistical
methods for specific variables are described below.
Milk Bands
As an estimate of milk consumption during the acute
phase of TMT intoxication, all pups were examined for the
presence and size of milk bands 24, 48, 72 and 96 hr after
injection. At these ages, the contents of the stomach can be
easily seen through the ventral abdominal wall. Milk bands
were rated according to the following criteria: 0—no band
visible; 1—small band visible on side of pup; 2—small band
visible across pup's abdomen; 3—large band visible across
pup's abdomen. This rating scale can distinguish between
stages of deprivation in rat pups; milk-band ratings correlate
TABLE 1
ASSIGNMENT OF ANIMALS TO EXPERIMENTAL VARIABLES
Group 1
Group 2
Variable
(N = 10 litters)
(N = 10 litters)
Milk bands
X
X
Body weight
X
X
Rope descent
—
X
Developmental startle
X
—
Adult startle
X
X
Maze activity
—
X
Male sex behavior
X
—
Organ weights
X
—
Brain weight
—
X
with stomach weight in pups which were removed from the
dam for periods from 2-24 hr [15]. Milk-band ratings were
analyzed by the Kruskal-Wallis test [30], Spearman rank-
order correlations [20] were obtained to examine the rela-
tionship between milk-band ratings and body weight.
Rope Descent
On days 20 and 21, pups were tested for the ability to
descend a 30 cmxl7 mm rope suspended above the home
cage, using a procedure modified from Anderson and Altman
[5]. Each pup was held by the nape of the neck and placed
against the rope with its head pointing upward. Pups were
allowed 120 sec to complete the test. Performance was
scored as successful if the pup displayed either (1) motor
coordination by descending the rope in either the head-up or
head-down position, or (2) muscular strength by clinging to
the rope for the duration of the test. Performance was scored
as unsuccessful if the pup fell or slid from the rope during the
test. These data were analyzed using chi square. Post-hoc
comparisons between proportions were made using the
Scheffe test.
Acoustic Startle Response
Testing was conducted in 4 sound-attenuated chambers
each containing a plastic-framed wire cage (7.6x7.6x23 cm)
mounted on a load cell/force transducer assembly designed
to measure vertical force. Two speakers were mounted on
the ceiling of each chamber 30 cm above the test cage. One
speaker presented an acoustic stimulus. 13 kHz. 120 dB(A>.
40 msec tone with a 2.5 msec rise time; the second delivered
background white noise at each of 3 intensity levels (50. 65
and 80 dB). These stimulus parameters were obtained from
Hoffman and Searle [25]. Following a 10-min period of adap-
tation to the chamber at ambient noise levels, a total of 30
stimuli was presented at an inter-stimulus interval of 20 sec ;
10 stimuli were presented at each of 3 background noise levels.
The order of presentation of the 3 background noise levels
was balanced across the session.
Three components of the startle response were measured:
the number of responses, latency to onset, and amplitude.
The response (an analog signal from the load cell) was sam-
pled at a rate of 1 kHz for 72 msec following the presentation
of the stimulus, and then converted into grams of vertical
force. Baseline was defined as the force exerted on the plat-
form during the first 10 msec following the stimulus presen-
-------
EFFECTS OF POSTNATAL TRIMETHYLTIN
423
tation. These 10 values were averaged and reflect the body
weight of the animal. The sensitivity of the platform was
calibrated separately for small (preweaning) and large (adult)
animals. Latency to onset was defined as the interval from
the stimulus onset to the time (msec) when the force exerted
on the platform was 4 standard deviations above the
baseline. Amplitude was defined as the maximum force
exerted on the platform during the response, corrected for
the baseline. If the force did not exceed 4 standard devia-
tions above the baseline, the latency to onset was zero and
no response was recorded.
For assessing the development of the startle response,
since all animals could not be tested in a single day, 10 litters
(N=80 animals) were divided into 2 groups of 5 litters, each
containing an equal number of pups/sex/dose. One group
was repeatedly tested on days 10. 12. 14, 16, 18 and 20; the
other group was tested on days II, 13, 15. 17, |9and21.Data
from the 2 groups were combined (e.g., days 10and 11, days
12 and 13. etc.) for graphic presentation and statistical
analysis. For assessing the adult startle response, all animals
(N=I60) were tested to determine if developmental treat-
ment effects would persist, and also to compare the startle
response of animals repeatedly tested during development
with that of naive animals (not previously tested for startle).
Each adult animal was tested during a single session between
70-107 days of age using the same stimulus conditions de-
scribed above.
For startle response amplitudes and latencies, a mean
value for each animal per test day was computed for trials
during which a startle response occurred. Number of re-
sponses, amplitude and latency were analyzed by
repeated measures ANOVA using age as the within-subjects
factor.
Motor Activity
The motor activity of individual animals was measured
for I hr in a figure-eight maze at 57 days of age to assess
exploratory activity and again for 23 hr at 120-122 days of
age to assess activity over the circadian cycle. The maze [41]
is a series of interconnected alleys (lOx 10 cm) converging on
a central arena and covered with transparent acrylic plastic.
Motor activity is detected by 8 phototransistor/photodiode
pairs. A total of 16 mazes was housed in two sound-
attenuated rooms maintained on the same light:dark cycle as
the animal room. Total photocell counts for each test were
analyzed using individual ANOVAs. For the 1-hr test, the
number of counts/photocell was calculated for controls and 6
mg/kg TMT animals in both the figure-eight and the blind-
alley areas of the maze, and the photocell counts in these
areas were analyzed using ANOVA.
Male Sexual Behavior
Male rats were paired with a receptive female for 3
weekly tests from 90-120 days of age. Testing arenas were
37.3x47.5x20 cm plastic cages with pine shavings covering
the bottom. Testing was conducted 2-6 hr after the beginning
of the nocturnal portion of the cycle. Following a 5-min ad-
aptation period, a sexually receptive female was placed in
the arena. Females were made receptive by 3 daily injections
of 12 (xg estradiol benzoate followed by 500 ng progesterone
on the fourth day 4 hr before testing. Components of male
sexual behavior (including frequency and latency measures
for mounts, intromissions and ejaculations) were scored as
described by Beach and Jordan [7}. Copulatory activity was
observed until either: (I) the male ejaculated, or (2) 30 min
had elapsed from the introduction of the female and no ejacu-
lation had occurred. The first 2 sessions were considered
pretests, and data from the third week of testing were used
for statistical analysis. The proportion of males ejaculating
was analyzed using a chi-square test. Other components of
male sexual behavior were analyzed using MANOVA.
Organ Weights
Animals were decapitated, and the wet weight of the
whole brain was obtained as a gross measure of TMT
neurotoxicity. Brains were then dissected [21]. and wet
weights were obtained for brain areas in which postnatal
neurogenesis has been demonstrated: cerebellum, hip-
pocampus and olfactory bulbs. Wet weights were obtained
from a separate group of animals for thyroid and adrenal
glands (males and females) and testes. Hypothalamic and
endocrine organ weights were obtained in conjunction with
male sexual behavior as indicators of neuroendocrine devel-
opment.
RESULTS
Body Weight
TMT produced a dose-related retardation in growth dur-
ing the preweaning period, F(12.293)=8.5l,p<0.0001. Body
weights were reduced at 10, 15 and 20 days of age in pups
receiving 5 or 6 mg/kg TMT, but by day 30 these differences
were no longer significant (Fig. 1).
Milk Bands
TMT produced a treatment effect on the size of the milk
bands at 48, 72 and 96 hr after dosing, *-(3)= 10.66; 10.89;
18.95. allp's<0.01. No differences were present 24 hr after
dosing, whereas milk bands in 6 mg/kg pups were decreased
at all later times (Fig. 2). This effect was delayed in the 5
mg/kg TMT group and was only different from controls 96 hr
after dosing. Milk-band ratings at all intervals after dosing
were correlated with body weight at 10,15 and 20 days of age
but not at 30 days of age (Table 2).
Rope Descent
While all pups improved from day 20-21. TMT produced
a dose-related deficit in the pups' ability to perform this task
(Fig. 3). On day 20, x2(3)m 13.03, p<0.0046, 6 mg/kg pups
were less successful than controls (25% vs. 65%). On day 21,
Xs(3)« 18.02, p<0.0004, both 5 and 6 mg/kg TMT pups were
less successful than controls (45% and 40% vs. 90%).
Acoustic Startle Response
During the development of the startle response, both the
number of responses and the amplitude of the startle re-
sponse increased, and response latency decreased in control
animals (Fig. 4). Since there were no sex differences during
the preweaning period, these data were combined. TMT de-
creased the number of responses throughout developmental
testing, F(3,72)=»20.65, p <0.0001; this effect was significant
for 5 and 6 mg/kg TMT. There was also a treatment effect on
startle amplitude, F(3,72)-8.08,/><0.0001, and a treatment
by age interaction, FU5,334)«2.01,p<0.0t41. For both the 5
and 6 mg/kg TMT groups, amplitude was decreased on days
12-13. On days 18-19 and 20-21, amplitudes were reduced at
-------
424
RUPPERT, DEAN AND REITER
• 0 mi/kg TMT
O * mg/kg TMT
4 9 nig/kg TMT
» « m«/ki TMT
AOS, davi
FIG. 1. Body weight for control and TMT-exposed rats
(N-40/doseV Data are expressed as mean values for preweaning
and postweaning (insert) body weight. SE bars are too small to be
represented on the graph.
TABLE 2
CORRELATIONS BETWEEN MILK BAND RATINGS AND BODY
WEIGHTS FOR ALL PUPS (N-160). VALUES ARE CORRELATION
COEFFICIENTS (r) AND ASSOCIATED PROBABILITY VALUES (p)
Milk Band Rating
Body Weight
Time after dosing
Age (days)
(hr)
10
15
20
30
24
r
0.17666
0.19926
0.21345
0.02017
P
0.0255
0.0115
0,0067
0.8001
48
r
0.46108
0.41143
0.42049
0,01002
P
0.0001
0.0001
0.0001
0.8999
72
r
0.34688
0.24939
0.29173
-0.07026
P
0.0001
0.0015
0.0002
0.3773
96
r
0.30315
0.35175
0.38751
-0.03491
P
Q.OOQl
0.0001
0.0001
0.6612
• 0 mg/kg TMT
04 mg/kg TMT
* 5 mg/kg TMT
» 6 TMT
TIME AFTER DOSING hr
FIG. 2. Milk-band ratings for control and TMT-exposed pups meas-
ured 24-96 hr after injection (N=40/dose). Values are expressed as
mean±SE, using a rating scale of 0-3 (see the Method section).
100
60
40
20
• OA Y 20
O DAY 21
0 4 s 6
OOS&.GE Of TWt tm»/V«>
FIG. 3. Rope performance of control and TMT-exposed pups at 20
and 21 days of age (N=»20/dose). Data are presented as percentage of
successful animals (see the Method section) during a 2-min test.
all dosages (allp's<0.00l). There were no treatment effects
on response latency (Fig. 4).
When tested as adults, there were no differences in re-
sponse amplitudes between ivaive animals and animals tested
for development of the startle response. For both males and
females, there was an increase in response amplitude with
increasing background noise level, F(2.308)»65.86.
p <0.0001, but no interaction with treatment. Data therefore
were combined for experienced and naive animals and col-
lapsed across background noise levels. For all dosages of
TMT, startle response amplitude was reduced for adult
males but not females (Fig. 5). This was seen as an effect of
-------
EFFECTS OF POSTNATAL TRIMETHYLTIN
425
DOSAGE OF TMT. mg/kg
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FIG. 4. Development of the acoustic startle response in control and
TMT-exposed pups (N-20/dose). Data are presented as mean±SE
for number, amplitude and latency of responses.
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FIG. 5. Acoustic startle response in adult rats exposed to TMT on
PND 5 (N-40/dose). Data for number of responses, amplitude and
latency are presented as mean±SE.
treatment. F(3.I54)«7.02, p<0.0002, sex, F(M54)-25.72.
p<0.001, and a treatment by sex interaction, F(3,I54)«6.82,
p<0.0003. There were no treatment effects on number of
responses or latency in adults.
Motor Activity
TMT produced a dose-related increase in figure-eight
maze activity of males and females during both a 1-hr test,
F(3,64)=»3.05, p <0.034, and during a subsequent 23-hr test,
F(3.64)-2.83, p<0.045. Since there was no treatment by sex
interaction, data were combined for males and females. For
both tests, 6 mg/kg TMT animals were more active than
controls (Fig. 6), However, there was no difference in the
spatial distribution of activity (figure eight vs. blind alleys)
between controls and 6 mg/kg TMT animals.
Male Sex Behavior
All control and 4 mg/kg TMT males (100%) ejaculated on
the third mating test, whereas 80% of the 5 mg/kg males and
70% of the 6 mg/kg males ejaculated. This was not a signifi-
cant difference, nor were there treatment effects on other
measures of mating behavior.
Organ Weights
TMT produced a dose-related decrease in whole brain
weight. F(3,71)»32.55, p<0.0001, and weights of olfactory
bulbs, F(3,7l)-4.1l. p<0.0l, and hippocampus,
F(3,70-28.33, p<0.0001. in adult animals. Decreases in
whole brain and olfactory bulb weights were significant for
all dosages of TMT, whereas the wet weight reduction for
the hippocampus was significant for the S and 6 mg/kg dose
-------
426
RUPPERT, DEAN AND REITER
>-Fi
DOSAGE Of TMT mq/k«
FIGURE EIGHT
Q] BUND ALLEYS
A
tkl_
FIG. 6. Figure-eight maze activity for control and TMT-exposed rats
(N = 20/dose). Data are expressed as photocell interruptions
(meamrSE) for a 1-hr and 23-hr (insert) test.
TABLE 3
WET WEIGHT OF WHOLE BRAIN AND BRAIN PARTS FOR CONTROL
AND TMT-EXPOSED RATS females)
in this brain area only. F( 1.71 )=7.77. p<0.006. There were
no treatment effects on adrenal, thyroid or testes weights.
DISCUSSION
Acute postnatal exposure to TMT impaired development
and produced long-term neurobehavioral deficits. Following
dosages of 4. 5, or 6 mg/kg TMT. responsiveness to an
acoustic startle stimulus was decreased, and brain weight
was reduced. Following both 5 and 6 mg/kg TMT, the size of
pups' milk bands and preweaning growth were reduced, and
the ability of pups to descend a rope was impaired. Finally,
motor activity in a figure-eight maze was increased following
the high dose only. No measures of neuroendocrine devel-
opment were affected by treatment. These results indicate
that various neurobehavioral functions are differentially af-
fected by PND-5 exposure to TMT, and. as with previous
studies on the adult [18], that these graded responses occur
over a relatively narrow range of dosages. As discussed be-
low, it is likely that these differential effects are the result of
multifocal damage.
The size of the milk bands provides an estimate of the
nutritional status of pups during the acute phase of toxicant
exposure. Within 48 hr, the size of the milk bands was re-
duced in pups receiving 6 mg/kg TMT; the reduction in 5
mg/kg TMT pups was less severe and was significant 96 hr
after dosing only. Positive correlations between milk-band
ratings and preweaning body weight indicate that changes in
milk bands were predictive of changes in body weight, and
support the interpretation that alterations in suckling con-
tribute to growth retardation in pups dosed with organotins.
The delayed effects of TMT on milk bands (48-96 hr after
dosing) contrast with the effects of TET on PND 5 which
were seen 24 hr after dosing only [44], However, the growth
retardation produced by TMT and TET was neither large
(13-18%) nor persistent beyond weaning.
Animals receiving either 5 or 6 mg/kg TMT were deficient
in their ability to descend on a rope. Although all animals
improved from day 20 to day 21. performance was impaired
on both days of testing for 6 mg/kg TMT pups. Pups dosed
with TET on PND 5 were also deficient on this task [43],
even following the experience of repeated testing (days 15-
20). Rope descent was developed as a test of motor coordi-
nation to assess functional impairment following brain dam-
age in young animals [4). Performance shows a devel-
opmental progression during the preweaning period [3.4].
Younger animals cling to the rope with their tbrelimbs. and
then eventually fall. Later, the hindlimbs provide synergistic
support, and pups descend the rope in the head-up position.
By day 19-22, control animals descend with the head in the
leading position. Pups receiving TMT which were unsuc-
cessful generally fell off the rope immediately after being
placed in position; pups did not fall off the rope while cling-
ing, but actually seemed to jump off the rope. Pups which
were successful on day 20, or which improved their perform-
ance on day 21, immediately began to descend after being
positioned against the rope. The deficit shown by high-dose
TMT pups was not simply a delay in development; this defi-
cit resembles motor incoordination rather than muscular
weakness.
The acoustic startle response also shows clear devel-
opmental changes during the preweaning period in control
animals; the number of responses and response amplitude
increase, while latency to respond decreases [ 10. 31. 40, 50].
In this experiment, the developmental profiles for the
number of responses and response amplitude in control
animals were biphasic with an initial rapid increase between
10-13 days of age, and a second increase from 18-21 days of
age. Response latency decreased monotonically throughout
development. TMT reduced the number of trials per session
in which a response occurred, and also decreased the ampli-
tude of the startle response. TMT did not alter the biphasic
pattern of development for these measures, but decreased
the level of responding at each phase. Since amplitudes were
calculated only for trials in which a response occurred, de-
creases in response amplitude were independent of the
number of missed responses. The developmental decrease in
response latency was not affected by TMT.
The startle response continues to develop after weaning:
-------
EFFECTS OF POSTNATAL TRIMETHYLTIN
427
adult latencies and amplitudes were still not seen at 21 days
of age. Also, preweaning animals in the present study, con-
trary to the findings of Kellogg et al. [26], did not show
response facilitation with increasing levels of background
noise. However, response facilitation was present in adults
of both sexes, but was not affected by treatment. Although
the startle response was disrupted in adult animals, there
were differences in comparison to the developmental defi-
cits. While TMT decreased the number of responses during
development, as adults, all dose groups showed essentially
the maximum number of responses per session. Latencies
were also unchanged but amplitudes were decreased for all
dosages. However, decreased amplitudes were seen in adult
males but not females. This sex difference in the toxicity of
TMT was not a generalized finding, since both motor activity
and brain weights were equally affected in adults of both
sexes. These differences between the developmental effects
of TMT and those seen in adults may represent recovery of
function in the neural pathways regulating the startle re-
sponse. Based on published data, several mechanisms of ac-
tion can be eliminated whereas others warrant further con-
sideration.
Neuronal damage produced by TMT in the hippocampus,
amygdala and neocortex in adult animals [8.9] does not ac-
count for the deficits in auditory startle. Hippocampal le-
sions are reported to either increase startle responsiveness
[12] or have no effect [27.29]; lesions of the amygdala
likewise do not disrupt startle responding [27], These data
suggest that TMT produces direct damage within the primary
acoustic startle pathway. This circuit consists of acoustic
sensory organ, auditory nerve, ventral cochlear nucleus,
nuclei of the lateral lemniscus, nucleus reticularis pontis
caudalis, spinal interneuron. lower motor neuron and mus-
cles [14].
Since the auditory system of the rat develops extensively
during the postnatal period [46], TMT could disrupt the de-
velopment of this circuit in several different ways. For
example, granule cells in the ventral cochlear nucleus con-
tinue to divide postnatally [2], and the volume of this nucleus
increases more than three-fold from 10 to 70 days of age with
the greatest increase occurring between 10 and 16 days of
age [11]. This continued cell proliferation and rapid devel-
opment could make this nucleus preferentially vulnerable to
TMT. It is also possible that TMT produces a developmental
impairment in the ability of pups to respond to the high fre-
quency 13 kHz stimulus used in the present study. Auditory
sensitivity, measured by the acoustic startle response, de-
velops later to high frequencies than to low frequencies for
both rat [10,31] and mouse pups [50]. The cochlear mi-
crophonic also develops later for high frequency sound [13],
Rubel [46] has suggested that there is a functional shift dur-
ing development, so that hair cells at the base of the cochlea
initially code low frequencies but with maturation, these
same cells code high frequencies. If TMT exposure during
early development produced specific damage within the
cochlea or the ventral cochlear nucleus, recovery could
occur if coding for these frequencies shifted to an undam-
aged area in the adult. Furthermore, the sex difference in
treatment seen in adults might reflect either a difference in
the ability to affect this shift or a difference in time course. In
the latter case, measurements taken at an older age in the
males should reflect a recovery in response amplitude.
Further studies are needed to explore these possibilities.
Motor activity in a figure-eight maze was increased in
animals receiving 6 mg/kg TMT. Similar increases in activity
were seen in both the I hr (22%) and 23 hr (35%) test ses-
sions. Miller (personal communication) found a 300% in-
crease in motor activity in pups receiving 7 mg/kg TMT on
PND 5 and tested at 21 days of age in a Motron. Acute
exposure to TMT in adult rats also produces hyperactivity;
although 6 mg/kg had no effect. 7 mg/kg produced a 190%
increase in activity [47], These data agree with the steep
dose-response functions reported for other neurobehavioral
effects of TMT [18],
Hyperactivity in figure-eight mazes is produced by
PND-5 dosing with TMT, PND-5 dosing with TET [43] and
adult dosing with TMT [47], All three of these treatments
affect the hippocampus, as indicated by decreased hip-
pocampal weight in PND-5 dosed animals (this study; [48])
and altered hippocampal morphology in adult animals [8. 9.
17], This agrees with the general finding that hippocampal
lesions produce increases in motor activity [37].
Spatial patterning is inherent in the locomotor activity of
rats in figure-eight mazes [35 , 36, 47]. In the present study,
the spatial distribution of motor activity was not altered by
TMT. i.e.. photocell counts were increased in all areas of the
maze (see Fig. 6). In contrast, both PND-5 TET [44] and
adult TMT [47] alter the spatial distribution of activity by
increasing activity primarily in the figure-eight area of the
maze. These effects on spatial patterning of activity parallel
the effects on accuracy of performance in a radial-arm maze
which requires the use of spatial memory [38.39]. Both
PND-5 TET [33] and adult TMT [53] decrease accuracy,
whereas PND-5 TMT does not [32]. Presumably, animals
whose spatial pattern of activity is disrupted would be unable
to use spatial cues to perform a task requiring remembrance
of locations in a maze previously entered. In figure-eight
mazes, overall activity can be increased either with or with-
out a disruption of spatial patterning. This measure, then,
distinguishes between different types of hyperactivity, and
may be predictive of an animal's ability to use spatial cues in
a learning task to obtain food or water reinforcement.
The reduction in whole-brain weight seen following PND
5 exposure to TMT parallels that produced by a similar ex-
posure to TET [43,48]. For both organotins, the hippocam-
pus is more severely affected than the whole brain, suggest-
ing some regional selectivity in the effects of trialkyltin com-
pounds during development. Additional work is needed to
characterize the nature of this effect, since a reduction in
hippocampal weight might be produced by several mech-
anisms. For example, based on adult toxicity, TMT might be
producing neuronal necrosis [8.9], while TET might be de-
creasing the amount of myelin [19] in fiber tracts associated
with the hippocampus. Alternatively, both compounds might
interfere with proliferation of granule cells which continue to
increase in the rat dentate gyrus into adult life [6]. Regardless
of the mechanism, both compounds are acting on the nerv-
ous system during the same time in development. TET does
not produce the same toxicity when animals are exposed on
either PND 1, 10, or 15 [44]. Therefore, a critical factor
contributing to this developmental toxicity is the stage of
development on PND 5.
acknowledgements
We thank Jerry Tolson and Lee Bynum for their assistance in
conducting this study. Partial support for this study was provided by
an interagency agreement with the Food and Drag Administration.
-------
428
RUPPERT. DEAN AND REITER
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Neurobehavioral Toxicology and Teratology, Vol, 4, pp. 135-139, 1982. Printed in the U.S.A.
Trimethyltin-Induced Hyperactivity:
Time Course and Pattern1,2
PATRICIA H. RUPPERT, THOMAS J. WALSH, LAWRENCE W. REITER
AND ROBERT S. DYER
Neurotoxicology Division (MD-74B), Health Effects Research Laboratory
United States Environmental Protection Agency, Research Triangle Park, NC 27711
RUPPERT. P. H.. T. J. WALSH, L. W. REITER AND R. S. DYER. Trimethyltin-induced hyperactivity: Time course and
pattern. NEUROBEHAV. TOXICOL. TERATOL. 4(2) 135-139, 1982.—Adult male Long-Evans rats were intubated with
either 0,5,6 or 7 mg/kg trimethyltin chloride. Activity was measured for 1 hr in a figure-eight maze 2 hr after dosing (day 0)
and again on days 4, 8, 16 and 32 after dosing. On days 49-51, activity was measured in a figure-eight maze over a 23-hr
period. There were no differences in activity on the day of dosing, but on all subsequent test days the 7 mg/kg TMT animals
were hyperactive. TMT also altered the spatial pattern of activity: activity was increased in the "figure-eight" portion of
the maze but not in the blind alleys. Activity of the 7 mg/kg TMT animals was increased during all periods in the 23-hr test.
Decreases in the length of the pyramidal cell line (CAl to CA3c of the hippocampus) confirmed neuronal cell loss in
TMT-dosed rats.
Trimethyltin Hyperactivity Hippocampal damage Behavioral toxicology
TRIMETHYLTIN (TMT), a neurotoxic organotin com-
pound, produces a selective pattern of neuronal damage
most evident in the hippocampus, although some alterations
are also observed in the pyriform cortex, amygdala and
neocortex [2J. Limited neuropathological changes are evi-
dent as early as 2 days following a single dose of TMT and
increase in severity over time with maximal damage seen 21
days following dosing 12]. Within the hippocampus, loss of
pyramidal cells primarily in CA4 and CA3 is a conspicuous
feature of TMT toxicity [2,6], although the earliest ultra-
structural changes may appear in the dentate granule cells
[1]. Due to this relatively selective neurotoxicity, TMT may
be useful for investigating hippocampal physiology and
function.
A commonly reported behavioral change following dam-
age to the hippocampal complex is hyperactivity [9, 14, 21].
Since systemically administered TMT damages the hip-
pocampus, it is likely that this neurotoxicant also affects
motor activity. Swartzwelder et al. [25] have recently re-
Ported that TMT-dosed rats were hyperactive in a 2-min test
of open field activity 40 days following dosing* The purpose
of the present study was to delineate the time course, dose-
dependence and circadian distribution of hyperactivity in
TMT-dosed rats. In addition, the length of the pyramidal cell
line of the hippocampus was measured as an index of hip-
pocampal damage.
METHOD
Male Long-Evans hooded rats (Charles River), 60 days of
age at the start of the study, were housed individually in
cages measuring 21x23x21 cm. Animals were allowed free
access to both Purina Lab Chow and water, and were
weighed daily throughout testing. The animal room was
maintained on a 12 hr light/dark cycle beginning at 0600 hr;
behavioral testing was conducted during the diurnal period,
except where noted.
On day 0, rats (N =» 10/group) were intubated with either 0
(physiological saline), 5, 6 or 7 mg/kg trimethyltin chloride
(ICN, Plainview, NY) measured as the base and dissolved in
saline in a volume of 1.0 ml/kg. Time-dependent changes in
locomotor activity were determined during 1 hr test sessions
in a figure-eight maze 2 hr after dosing, and again 4, 8, 16,
and 32 days after dosing. To determine whether TMT altered
the circadian pattern of activity, each animal was tested for
23 hr activity in the maze on days 49-51 following dosing.
The maze (Fig. 1) consists of a series of interconnected al-
leys (10x10 cm) converging on a central open arena, and
covered with transparent acrylic plastic [22]. Motor activity
was detected by 8 phototransistor/photodiode pairs; a count
is registered each time a light beam is interrupted. A total of
sixteen mazes were housed in two sound attenuated rooms
maintained on a photoperiod identical to the animal quarters.
Each rat was tested individually in a maze.
For assessment of hippocampal damage, 10 turn paraffin
sections cut in the sagittal plane wen stained with cresyl
violet. Sections were projected onto plain white piper for
viewing (1 mm*37 pm). Nine slides were prepared from
sequential sections beginning at 2 mm from the midline, and
samples for measurement were obtained from alternate
slides. The length of the pyramidal cell line was measured to
the nearest mm from the CAl subiculum border until the
'This paper has been reviewed by the Health Effects Research Laboratory, U. S. Enviommantal Protection Agency, and approved for
Publication. Mention of trade names or commercial products does not constitute iendorsement or recommendation fer use.
'Presented at Society for Neurosctence Annual Meeting, Los Angeles. CA> 1981.
-------
136 RUPPERT, WALSH, REITER AND DYER
FIG. I. Figure-eight maze. The location of photocells
H
50
3
25
O
>
~—
>
100
K
O
<
75
50
25
eS3
I I CONTROL
7 mg/kg TMT
[ j
—'— ^ |
+1
-
||§
r
ik
-f
1 1
wz/M
1 r
r
4 8 16
TIME POST-DOSING. d*yi
FIG. 3. Spatial distribution of activity in the figure-eight maze for
control (N= 10) and 7 mg/kg TMT-dosed rats (N=9) during hourly
test periods. Values are meanirSE for photocell counts in areas A. B
and C of the maze.
blind alleys (Area A), the right arm of the maze (area B), and
the left arm of the maze (area C). Animals exposed to 7
mg/kg TMT increased their activity in the "figure-eight"
portion of the maze but not in the blind alleys. This increase
in activity was symmetrically distributed in the 2 sides (B
and C). For photocell counts on the right side of the maze,
there was a significant effect of treatment, F(1,68) = 2I.52,
p<0.0002, day of testing, F(4,68)=5.08, p<0.0013, and a
treatment-by-day interaction, F(4,68)=8.79, p<0.0001. On
day 0, the 7 mg/kg animals were slightly less active than
controls, F(l,17)=4.86, p<0.04. On days 4-32, 7 mg/kg TMT
animals were more active than controls, F(l,17)=7.33, 8.65.
30.63 and 12.49, respectively, p<0.01. For photocells on the
_ 600
Ui
H
500
CC
X
£ 400
z
3
O
U 300
>*
H
2 200
O
<
100
0 4 8 16 32
TIME POST-DOSING, days
FIG. 2. Figure-eight maze activity during l-hr-test periods in control
and TMT-dosed rats(N=9-IO/group). Values are mean±SE for total
photocell counts at each time post-dosing.
o CONTROL
~ S mg/kg TMT
& 6 mg/kg TMT
• 7 mg/kg TMT
-------
TRIMETHYLTIN HYPERACTIVITY
137
i—i—nr
DAY II
I I I I I I
DAV 4
• CONTROL
a 7 ma/kg TMT
70
SO
s 50
h 40
e.
DAY 32
DAY 8
"1 1 1
TOTAL
60 S
TIME. minulM
FIG. 4. Temporal distribution of activity in the figure-eight maze for
control (N = 10) and 7 mg/kg TMT-dosed rats (N=9) during test
periods when hyperactivity was present. Values are meaniSE for
5-min intervals during hourly test periods.
I I 7 m«/lig TMT
CONTROL
J1 500
u 300
LIGHTS OFF
TIME, hour
0000
>
LIGHTS ON
FIG. 6. Figure-eight maze activity for hourly intervals during a 23-hr
test for control <0.0005. This measurement was decreased
in the 5 mg/kg (p<0.05), 6 (p<0.0l) and 7 mg/kg (p<0.01)
TMT animals.
DISCUSSION
A single dose of 7 mg/kg TMT produced a persistent
hyperactivity in adult rats. Hyperactivity was not present 2
hr after dosing, but was apparent by day 4 and reached
asymptote on day 16. This time course parallels previously
reported neuropathological changes which first appear 2
days after dosing and are maximal within 3 weeks [2]. Dyer
et al. [7] have suggested that the neurotoxicity of TMT is
similar to that of kainic acid, which also produces cell loss in
the pyramidal layer of the hippocampus and hyperactivity
[8,16],
Increased activity in animals with hippocampal lesions
has been described as a stereotypic running rather than in-
creased exploration [13,24]. Most studies investigating re-
-------
138
RUPPERT, WALSH, REITER AND DYER
7000
Q 5000
3000
1000
DOSE OF TRIMETHYLTIN (mg/kg}
FIG. 7. Length of the pyramidal cell line of the hippocampus for
control and TMT-dosed rats (N»7-9/group). Values are mean±SE.
sponse perseveration of activity have used either a T maze or
a Y maze [3, 4, 15, 18, 23] which produces a decrease in
spontaneous alternation in animals with hippocampal le-
sions. In the figure-eight maze, TMT produced a clear dis-
sociation of activity patterns between the continuous circu-
lar portion of the maze and the blind alleys. Whereas control
animals show an equal distribution of activity in all photo-
cells, hyperactive TMT animals show a selective increase in
activity in the circular alleys. This suggests that TMT
produced an alteration in the pattern of activity rather than
an increase in exploration. This spatial patterning is not
unique. For animals with lesions in the globus pallidus,
which also produce hyperactivity, increased activity is more
pronounced in the continuous corridors of the maze than in
the blind alleys [19].
It is controversial whether hippocampal lesions produce
an increased initial activity in a specific test apparatus, or
whether activity changes reflect a decrease in the rate of
habituation (4,17,23,24,26]. In 7 mg/kg TMT animals initial
activity was clearly increased. The rate of habituation re-
mained relatively stable (only on day 32 did this pattern shift
.Heating both increased initial activity and a slower decline
of activity within the test session for 7 mg/kg TMT animals)
producing dramatically increased overall activity. The
curves show some trend towards differences in habituation
on other days which may be masked by variability. This
persistent hyperactivity is in agreement with data of
Swartzwelder et ai. [25].
Several studies have indicated that hyperactivity of
animals with hippocampal lesions is more pronounced during
the nocturnal period [10, 11, 12]. In the present study, when
activity was measured over 23 hr, the increase in activity of
the 7 mg/kg group was equivalent during the diurnal and
nocturnal periods. However, for this single 23-hr period,
there was no circadian pattern of activity for any group. Only
when animals are tested in the maze over several consecu-
tive days does a clear circadian rhythm of activity develop
[20,22]. The overlap in diurnal activity levels in the 3 hr
following the nocturnal period (Fig. 6) suggests that possibly
diurnal activity on subsequent days may not differ between 7
mg/kg TMT animals and controls.
The decrease in the length of the pyramidal cell line found
in this study, which reflects a decreased number of pyrami-
dal cells in area CA3 [6], confirms that definite hippocampal
damage was present in TMT-dosed animals tested for loco-
motor activity. However, TMT produced significant mor-
phological damage after all doses, but hyperactivity only
after the highest dose. One interpretation of this discrepancy
is that hippocampal morphology is a more sensitive indicator
of TMT toxicity than locomotor activity, e.g., a threshold
number of pyramidal cells may need to be damaged before
behavior is disrupted. An alternative interpretation is that, at
the highest dose of TMT (7 mg/kg), additional
neuropathological damage occurs in another brain area
which in turn is responsible for the hyperactivity. Although
all doses of TMT produced a significant loss of pyramidal
cells the degree of damage between doses was not large. Yet
TMT induced hyperactivity was an all or nothing effect, i.e.,
it was not graded with dose, but when present, it was not a
marginal effect. Thus, while systemically administered TMT
clearly damages the hippocampus and clearly produces be-
havioral changes, caution is required in attributing this be-
havioral toxicity solely to disruption of hippocampal func-
tion.
ACKNOWLEDGEMENTS
We would like to thank Ann Brady for performing the histology.
Jerry Tolson for obtaining the hippocampal measurements and
Phyllis Keeter for typing the manuscript.
t. Bouldin, T. W„ N. D. Goines, C. R. Bagne!) and M. R. Krig-
man. Pathogenesis of trimethyltin neuronal toxicity: Ultrastruc-
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TRIMETHYLTIN HYPERACTIVITY
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/vtt &7~
*>=unatal Exposure to Triethyltin Disri^ts Olfactory Discrimination
Learning in Preweanling Bats
Mark E. Stanton
Neurotoxicology Division (MD-74B), Health Effects Research laboratory
U.S. Environmental Protection Agency, Research Triangle Park, NC 27711
Disclaimer: This manuscript has been reviewed by the Health Effects
[Research laboratory, U.S. Environmental Protection Agency, and approved
for publication. Mention of trade names or cooroercial products does not
constitute endorsement or recommendation for use.
Address for correspondence: Mark E. Stanton, Ri.D.
HERL (MD-74B)
U.S. EPA.
FTP, NC 27711
Running head: IViethyltin and infant odor conditioning.
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Abstract
STANTON, M.E. Neonatal Exposure to Triethyltin Disrupts Olfactory Discrim-
ination Learning in Preweanling Rats. NEUPQTOXICOL TERATOL Jflf, ppp-ppp, 19 9x.
Triethyltin is an organotin canpound which is known to produce neurotoxicity in
both adult and developing organisms. Although this neurotoxicity has been docu-
mented with a variety of behavioral and biological measures, the effects of
this canpourid on learning during early development have been less systematic-
ally studied. The present study reports four experiments that examined this
question with an odor aversion learning paradigm in which pups received
presentations of one odor paired with footshock and an alternate odor without
shock. In Experiment 1, long-Evans rat pups were injected i.p. on postnatal
day 5 (RJD5) with either 0, 3 or 5 mg/kg TET and then tested for olfactory
discrimination learning on FND18. Only the 5 mg/kg dose impaired discrimina-
tion learning. In Experiment 2, FND5 exposure to TET (5 mg/kg) disrupted
olfactory learning an PND18 but not on FND12 whereas exposure on FND10 disrupt-
ed learning at both ages of testing. In Experiment 3, PND16 exposure to TET (5
mg/kg) also disrupted acquisition of olfactory learning on FND18 but had no
effect on retention of an olfactory discrimination that was acquired prior to
TET exposure (ie. on FND14 and RTO15). Unconditioned responses to footshock
were also unaffected by TET (Experiment 4). These findings indicate that
neonatal exposure to TET impairs associative learning in developing rats and
are discussed in relation to other studies of the developmental neurotoxicity
of this coipound.
KE¥ WCRD6: DEVELOPMENTAL NEUHCODXIOOIJOGY, INFANT RATS, TRIEBWIirDf, OLFACTORY
SYSTEM, ASSOCIATIVE CONDITIONING, EARLY LEARNING, QRGANCMETALS,
MENTAL RETARDATION.
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Triethyltin and infant odor conditioning
Page 3
The vulnerability of the developing brain to neurotoxic insult is widely
recognized and growing interest in this problem in recent years has led to the
emergence of developmental neurotaxioology as a distinct area of inquiry (38,
59 , 64). Despite recent progress, there are a number of frontiers in this
emerging field that remain relatively unexplored. One of these is the assess-
ment of learning and memory during early development. Although behavioral
ontoaenv is emphasized in developmental neurotoxicology, and learning is a
widely-used endpoint in this field, assessments of learning and memory are
typically carried out relatively late in development. With the exception of
fetal-alcohol studies with rodents (3, 9, 39, 63) most assessments of learning
are carried out during the juvenile period or early adulthood. As a result,
animal studies shew an imbalance in the age at which different behavioral
functions are assessed, with tests of sensory and motor function conducted
early and tests of learning and memory conducted late (51). This imbalance
creates a number of problems for the field. To take just a few examples, it
may underestimate the extent to which learning is inpaired by developmental
neurotoxicants because of recovery of function (7). Second, it confounds
analysis of how different functional endpoints are affected by early neuro-
toxicant exposure (54). And, thirdly, it makes it difficult to catpare data
frcm animal studies with those frem human studies because cognitive effects are
typically aaaowsorl during development in humans (54).
The relatively infrequent use of early learning in (animal) developmental
neurotoxicology is not owing to an unavailability of methods, the explosion of
basic latieauh in developmental peydhobiolcgy in the last 10-15 years has
yielded ouch detailed information on the ontogeny of learning in animals (16,
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Triethyltin and infant odor conditioning
Page 4
17, 53). This has led a number of developmental neurotoxioologists to urge the
use of early learning in toxicity assessment (1, 24, 40). The present report
sakes a start in this direction by examining the effects of the neurotoocicant,
triethyltin (TET), on olfactory discrimination learning in prewaanling rats.
The olfactory learning paradigm used in the present study was an adaptation
(48) of the method of Rueharski and Spear (19). The use of olfactory learning
offers several general advantages. First, olfaction is an early-developing
sensory modality in the rat which can support conditioning at very early ages
(eg., 10). Second, their is already an extensive literature on the behavioral
properties of olfactory learning in developing rats (12, 14, 15, 19, 21, 26,
47, 49). And third, there is much information available concerning the neuro-
biology of olfactory learning in both adult (8, 23, 55) and developing rats
(11, 18, 20, 22, 48, 56). There are also several methodological advantages of
the particular odor conditioning preparation used in this study. In this prep-
aration, rat pups are presented with a CS+ odor with footshock and a CS- odor
without shock, and then given a preference test between the CS+ and CS- odors.
The advantages of this paradigm are: (1) rapid acquisition: conditioning
typically approaches asynptotic levels after 4-6 trials; (2) a discrimination
design: this affords a within-subjects control for nonassociative effects of
the training experience—only an association between CS+ and shock can account
for odor preference Airing testing; (3) low response requirement: as with all
forms of Pavlcwian conditioning, responding is not necessary Airing training
and the minimal response requirements during testing are wall within the motor
capabilites of pups at all ages tested; and (4) the footshock unconditioned
stimulus (US): use of this stiaulus also facilitates age ocnparisons because
sensitivity to footshock does not change during development (12, 19).
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Triethyltin and infant odor conditlcning
Page 5
The neurotaxicant enployed in the present stud/ was triethyltin (TET).
Uiis organcmetal ocnpcund was chosen for a number of reasons. First, the
neurotoxicity of TET following adult exposure has a long history of study and
its properties and mechanisms of action are relatively well characterized (61).
Second, there has also been much recent attention paid to the developmental
neurotoxicity of TET (see 37, 43 for reviews) at both the neural (4, 25, 32,
33, 34, 62) and behavioral levels (13, 28, 31, 36, 44, 45, 46, 58). Third, the
distribution of tin in the adult and developing brain produced by TET exposure
is kncwn (5). Fourth, this carpound has sane interesting developmental prop-
erties in that same of the neural effects of early exposure to TET appear to
differ qualitatively from those found after adult exposure (33, 34), and sane
of the behavioral effects of this carpound depend on age of exposure (45).
Finally, despite this recent developmental research (and consistent with the
theme raised previously), there is very little information on the effects of
TET on early learning. To my knowledge, only a single experiment has been
published which claims to have examined learning in preweanling rats (28) and a
number of issues remain to be addressed. For example, there have been no
studies that have employed the same test of learning at different stages of
development or that have examined early learning as a function of age of TET
exposure.
Here I report four experiments on the effects of developmental exposure to
TET on olfactory conditioning in preweanling rats that begin to address sane of
these issues, in Experiment 1, SND5 exposure to 5 ng/kg IBS (but not 3 mg/kg
TET or saline vehicle) produced performance deficits on a test of learned odor
preference in 18-day-old rat pqps. In Experiment 2, these TOT-inAioed deficits
were assessed as a function of age of exposure (WDS vs 10) and age of testing
(FND12 vs It). Experiments 3 and 4 we designed to further examine the role
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Triethyltin and infant odor conditioning
Page 6
of age of exposure and showed that the effects of TEI could not be attributed
to deficits in nonassociative (sensory, motor and motivational) processes
necessary for learned performance.
jSESRPgyr x
In Experiment 1, rat pups recieved a single i.p. injection of saline
vehicle or 3 or 5 ng/kg TET sulfate on H05 and were then tested for olfactory
discrimination learning on FND18. The choice of this age of exposure and these
doses of TET was based an previous studies of the developmental neurotoxicity
of this compound (5, 44, 45, 46). Similarly, FND18 was chosen as the age of
testing because it has canmsnly been used in previous studies enploying this
olfactory learning paradigm (eg., 19, 48).
Subjects
Sixty-four 18-day-old Long-Evans rats drawn fron 8 litters were the sub-
jects. The rats were offspring of time-bred females obtained fron Charles
River (Raleigh, N.C.) on either the second or fourteenth day of gestation.
Offspring were raised in the animal colony at the Neurotoxioology Division,
U.S. Environmental Protection Agency, Research Triangle Park, North Carolina.
This facility is certified by the American Association for Accreditation of
Laboratory Animal Care. All subjects were maintained on a 12-hr light, 12-hr
dark photcperiod, with light onset at 7:00 a.m.. (All experimental procedures
took place during the light phase of this cycle.) The pqps were born and
housed with their mothers on a bad of wood shavings in plastic cages measuring
48 x 27 x 20 cm. All litters were culled to 4 males and 4 females by the third
day after birth. Age of the pups was determined fay checking for birth during
the light phase of the light-dark cycle and designating that day as postnatal
day 0 (FNDO). Subjects remained undisturbed with their mothers exoapt for
periodic cage cleaning and experimental manipulations (dosing and tasting).
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Triethyltin and infant odor conditioning
Rage 7
Littermates were assigned to each of the six ejqperimental gitxps (see below) in
this study with the restriction that no more than one female and one sale frcm
each litter was assigned to a given group (n - 8-14/group).
Apparatus
The apparatus was similar to that used by Saperstein, Rxharski, Stanton,
and Kail (48) and had two components, one for training and another for testing.
Training (odor-f ootshock pairings) occurred in a 26.5 x 19 x 12.5 an Plexiglas
apparatus divided into three oarpartjnents (left, middle, and right) of equal
size. Odorants (3 oc of lemon oil or methyl salicylate [Humco laboratories])
were spread evenly on pieces of fur attached to wooden rollers at opposite ends
of the the left and right compartments. The middle ccrpartment served to
minimize diffusion of odors between the two end chambers. The entire apparatus
was placed over a shock grid (.5 mm between bars) which served as the floor
during training. Footshock (1.6 mA AC) was delivered by a Colbourn shock
generator (Model # E13-35). Animals were placed in and out of the odorized
corpartments through a hinged Plexiglas lid. 'Pasting for odor preference took
place in an apparatus that was similar to the training apparatus except that it
was larger (55 x 14 x 15 cm), had a wire mesh floor, and housed a single con-
tinuous section (rather than three oonparbnents). This left subjects free to
loccmote throughout the entire length of the apparatus during testing. Like
the training apparatus, the test apparatus was fitted with pieoes of fur
attached to wooden rollers at opposite ends that were odorized in the same
way. This produced two odor gradients and the apparatus was bisected with a
black line on the mesh floor, to indicate lemon and methyl sides. Odor
preference was defined as the amount of time spent over the l«non or methyl
side during the 3-otinute testing period and was recorded with a stop watch.
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Triethyltin and infant odor conditioning
Page 8
Dosing
On RJD5, pups were taken nonentarily from the hone nest, individually ident-
ified with paw tatoo6, and injected (i.p.) with physiological saline or with 3
or 5 rag/kg bis-triethyltin sulfate (Strem Chemicals, Newburyport, MA) dissolved
in saline. Doses of TET are expressed as base and were administered in a
volume of 10 ul/g body weight. A split litter design was used in which all
three doses were administered to pups in a given litter. Usually one and no
more than two same-sex littermates received a given dose. When two same-sex
littermates received the same dose, they were assigned to different behavioral
conditions (see below).
Pegiqn
At each of the three doses of TCI, half the animals received a test of odor
preference following acquisition training (Group Acq) and half received the
test without such training (Group Naive). Thus, the design was a 3 X 2
between-groups factorial, involving the factors of TET (0, 3, and 5 mg/kg) and
groups (Naive vs Acq).
Behavioral Procedure
On PND18, pups were taken from the heme nest, placed in individual com-
partments (8 x 9 x 13 cm) of a Plexiglas incubator (37 x 29 x 13 cm), and
transferred to the rocn where testing took place. This roam was sound atten-
uated and testing was conducted under reduced light (in order to minimize
distraction by cues outside the apparatus). The incubator contained wood
Savings and was maintained at 30-33 degrees C by an electric heating pad
(General Electric) placed underneath. Incubator compartments served as each
pup's "heme cage" until behavioral procedures were ocrpletad.
All ptqps were weighed just prior to training ard/ar testing. Pups in Group
Naive then received a standard odor preference test (see below) whereas those
in Group Acq were first given four conditioning trials. Training anVor
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Triethyltin and infant odor conditioning
Page 9
testing odor was counterbalanced within both the Acq and Naive grcups, that is,
half the animals were tested with the lemon odor and half with the methyl odor
designated as the positive conditioned stimulus (CS+). Conditioning trials
consisted of placing a pip in the odorized compartments of the training
apparatus. In one ccnpartanent, animals were exposed to the CS+ odor (paired
with footshock) and in the other they were exposed to the CS- odor (without
footshock). On each trial the animal was placed for 20 seconds in both the CS+
and CS- ccnpartments (the order of placement was determined by a latin Square
Design). Footshock was administered during seconds 8-10 and 18-20 of each 20-s
CS+ exposure. Placement in one oatpartraent (CS+ or CS-) was followed
immediately by placement in the other. The rat pup was then removed from the
training apparatus and returned to its incubator oanpartment during the
30-second intertrial interval. This sequence was then repeated until all four
trials had been run. One minute later, the odor preference test was conducted.
Preference tests were conducted in the testing apparatus as follows: A pup
was introduced into the apparatus on the side bearing the CS+ odorant and the
amount of time spent over the lemon or methyl side was recorded for the next 90
seconds. The animal was then momentarily removed from the apparatus and then
placed on the side bearing the CS- odorant and the measurement was repeated for
another 90 seconds. Animals were judged to be on the lemon or methyl sides of
the apparatus when their snouts crossed the midline and into that half of the
apparatus. The measure of conditioning was the cumulative time (out of the 180
total seconds of preference testing) that a pup spent on the CS+ side of the
preference chanter. Conditioning is indicated by lew values of this measure
whereas no preference (abeense of conditioning) is indicated lay values approach-
ing 90 seconds.
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Triethyltin and infant odor conditioning
Page 10
Following behavioral procedures, animals were decapitated and their brains
were removed and weighed. This brain-weight measure was used as a sisple and
effective way to confirm previous, more detailed studies of the central nervous
system effects of developmental exposure to TET (eg., 33, 44).
RESULTS WP PISCTSSIW
The behavioral results appear in Figure 1. These data were analyzed with a
3 (drug) X 2 (groups) analysis of variance (ANOVA). Neonatal exposure to TET
produced an impairment in performance on the olfactory preference test. This
is indicated by a reduced preference for CS+ in trained animals (Group Acq)
relative to untrained ones (Group Naive) following exposure to saline or to the
3 mg/kg dose but not following the 5 mg/kg dose of TET. This was confirmed
statistically by a significant interaction of TET x Groups, f (2, 58) * 3.85,
p < .028. Post hoc mean ocnparisons (Newman-Keuls) indicated that following
SAL and 3 mg/kg TET, animals in Group Acq differed from their Naive counter-
parts (p < .01) and from 90-second control levels (p < .01 for SAL, p < .05 for
3 mg/kg TET). In the 5 mg/kg TET condition, Group Acq did not differ from
Group Naive or fran 90-second control levels; and showed a greater preference
for CS+ than its SAL or 3 mg/kg counterparts (p < .01) which did not differ
between themselves. None of the Naive groups differed fran 90-second control
levels, indicating that 90 seconds provides a good estimate of the performance
of untrained animals.
Body and brain weight data appear in Table 1. (These data are discussed in
detail in a separate section of this manuscript [see below]). TET exposure
resulted in dose-related reductions in both brain and body weight. This agrees
with previous studies on the neurotoxicity of TET in developing cedents that
have used a split-litter dosing procedure (44).
The principal finding of this experiment ia that 18-day-old rat pups
exposed to TET on RIDS fall to show reduced preferences for an odor previously
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Triethyltin and infant odor conditioning
Page 11
paired with footshoc.k. Exposure to TET did not alter the performance of naive
animals in the odor preference test. Disruption of performance in trained
animals occurred only at the highest dose (5 mq/kg) employed. The ineffective-
ness of the 3 mg/kg dose is consistent with the steep dose response curve that
has been observed with other measures of the developmental neurotoxicity of
this ocnpcund (eg. 33, 44, 45, 46). The present finding suggests that neonatal
exposure to TET iupairs associative learning during infancy. However, the
effects of this oanpound on nonassociative processes remains to be examined
(see Experiments 3 and 4).
EEFSFPffiyr 2
An important general principal in developmental neurotoxicology is that age
of exposure and age of assessment can importantly influence neurotoxic outcane
(42, 50). This principal has been borne out in studies of the behavioral tox-
icity of neonatal exposure to TET. Exposure on FND5 produces hyperactivity in
weanling and adult rats whereas exposure on FND1, 10 or 15 fails to alter activ-
ity at these ages (45). The present experiment sought to determine whether
olfactory learning might also be influenced by the variables of age of exposure
and assessment. Separata groups of rat pips were exposed to TET on either HTO5
or 10 (in separate experiments) and tested for olfactory discrimination learn-
ing an either FND12 or FND18.
METHOD
Experiment 2a
Subjects. Subjects were Long-Evans rats pqps derived from 9 litters that
vera obtained, culled, housed and maintained as in Experiment 1. On FND5, half
of the pups in each litter were injected i.p. with 5 mg/kg TET and half were
dosed with saline vrfiicle as described previously, initially, 72 pqps were
available as sifcjects, however, 31 pups fixn 4 litters ware tasted can R4D12
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Triethyltin and infant odor conditioning
Page 12
(one treated pip died prior to testing) and 34 pups from 5 litters were tgstad
on on FND18 (3 treated and 2 control pups from one litter, and 1 treated pup
from another litter were not tested because of poor health).
Apparatus and Procedure. The apparatus and training procedure were the
same as in Experiment 1. The experimental design differed in that all animals
received acquisition training—ie., there was no Group Naive. Conditioning in
trained animals was assessed relative to 50%-preference levels rather than to
preference of naive animals, since these two estimates of control performance
were oaiparable in Experiment 1. Within each litter, half the pups (one male
and one female) fran each treatment group were trained with the methyl odor as
CS+ and half were trained with the lemon odor as CS+.
Results. The results of Experiment 2a appear in Figure 2 (lower panel).
Data were analyzed by means of a 2 (SAL vs Id) X 2 (RJD12 vs FND18) between
groups ANOVA. As in Experiment 1, a single injection of TET on 5ND5 disrupted
performance of rat pups trained and tested at IB days of age. However, there
was no disruption of performance in their 12-day-old counterparts. This was
confirmed by ANOVA which revealed a significant interaction of TET x Age,
£(1,61) « 5.34, p < .025? and by Newman-Keuls tests which indicated a reliable
difference (p < .01) between TET and SAL animals tested at 18 days but not at
12 days. In addition, Newman-Keuls tests shewed that all groups showed odor
aversions except for the TET-treated group tested at 18 days. That is, mean
preferences were reliably below 50%-control levels at 12 days (p < .01)
regardless of TET condition, and at 18 days (p < .01) only in the SAL-txeated
animals.
Experiment 2b
Subjects. Long-Evans vats pups, derived frcn 8 litters that were obtained,
culled, housed and maintained as described previously, served as subjects. On
IM&O, halt of the pups in aach litter (2 of mch sex) ware injected i.p. with
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Triethyltln and infant odor conditioning
Page 13
either 5 ng/kg TET or saline vehicle as described above. Of the 64 pipe that
were initially available, 2 TCP-treated pups from one litter and a TCT-treatad
and saline-treated pup from another litter died. This left €0 animals for
testing, 28 from 4 litters on HTO12 and 32 from 4 litters on R4D18.
Apparatus and Procedure. The apparatus, training procedure, experimental
design, and data analysis were the same as in Experiment 2a.
Results. The results of Experiment 2b appear in Figure 2 (upper panel).
In contrast to what was seen after FND5 administration (Experiment 2a), in-
jection of TET on FND10 produced performance deficits during the olfactory
preference test at both ages of testing. This was^confirmed by ANCJVA which
revealed significant main effects of TET, S(1,56) - 40.85, p < .0001; and Age
of testing, f(l,56) » 10.34, p < .003? but no interaction of these two factors
(F < 1). Newman-Keuls tests shewed that pups in Group SAL showed odor aver-
sions whereas those in Group TET did not, regardless of age of testing. That
is, following SAL treatment, mean preferences were reliably below 50%-oontxol
levels at both 12 and 18 days of age (p < .01), whereas, following TET
treatment, mean preferences failed to differ from 50%-oontrol levels, again
regardless of age of testing.
TET produced significant reductions in body and brain weights in the
present experiment (Tfeble 1). Hcwaver, on a percentage basis, brain (but not
body) weight reductions ware greater after FND5 exposure than after RJD10
exposure. This suggests • dissociation of the behavioral, neural and somatic
effects of 1ST (see results and discussion of Table X below).
wsoasum
Together these experiments replicate and extend the findings of feperi-
mant 1. They again Acw that monatal exposure to TBt disci*** performance of
learned odor aversicm in preweanling rats twt they indicate that this effect
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Ttiethyltin and infant odor conditioning
Page 14
depends on age of exposure and age of testing. Administration of TET on RTO5
disrupts performance on R4D18 but not on IVD12 whereas injection of this com-
pound on FND10 disrupts performance at both ages of tasting. This pattern of
results confirms the general principal that age of exposure and testing can
influence neurotoxic outcome. However, the pattern revealed here differs fran
that shown in the above mentioned study of locomotor activity (45) which
revealed effects only following RJD5 exposure. This suggests a dissociation of
the effects of neonatal TET on olfactory learning and motor activity and may be
instructive regarding the (probably different) mechanisms underlying the two
behavioral effects (see General Discussion).
Bg£KMB*T 3
Experiment 3 sought to extend the previous ones in two ways. First, it
reexamined the deficits shown by 18-day-olds with this olfactory learning
paradigm as a function of yet another, even later age of exposure: FND16.
Second, it sought to determine the possible role of two nonassociative effects
of TET that could account for performance deficits on this task. One of these
is a sensory deficit. It is possible that TET produces an impairment of
olfaction rather than of learning. Although there is other evidence indicating
that pups exposed to TET neonatally are not anosmic (28), it is possible that
some more subtle olfactory deficit involving the odors employed in these
experiments could produce a lack of odor preference during testing. The other
possible nonassociative effect is altered motor activity. Effective odor
preference during tasting requires subjects to locomote to one side of the
preference chanber and remain there during the majority of the test period.
Gross increases or decreases in locomotor activity caused by neonatal TIT would
result in an apparent lack of odor preference. Although such gross motor
effects were not evident by direct observation, additional evidence against
this possibility seemed warranted.
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Triethyltin and infant odor conditioning
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One way to experimentally distinguish the associative versus sensory or
motor effects of TET is to shew that learned odor preferences axe impaired
whereas unlearned or previously-learned preferences are not. Experiment 3
examined this possibility by capering acquisition of odor preferences estab-
lished following TET exposure with retention of preferences established prior
to TET exposure. A selective effect of TET on acquisition would rule out
sensory and motor interpretations of performance failure during the preference
test. Experiment 3 examined the effects of RJD16 exposure to TET on olfactory
preference on FND18 in two groups of rat pups. One (Group RET) was trained
prior to TET exposure (on R4D14 and 15) whereas the other (Group AOQ) was
trained following TET exposure (on R4D18).
Subjects
One hundred and sixty Long-Evans rat pups derived frcm 20 litters served as
subjects. Litters were obtained, culled, housed, and maintained as described
previously.
Afpwratvg and Praceflua
The apparatus was the same as described previously. Except as noted below,
the general training and testing procedures were the same as in the previous
e>qperimants. On FND14 and again on FND15, half (2 males and 2 females) of each
litter received 4 odor discrimination training trials (Grcqp RET) and half were
not trained (Groqp CIL) but were otherwise treated similarly (taken frcm the
nest, housed in incubator oanpartmenta for a conparable period, etc.). On
FND16, half of each of these tw> grcupe (l male and 1 female) wear* injected
i.p. with TET (5 vq/kq) and half with SAL vehicle as described previously. On
FND18, all animals received a standard test of odor prefer***. For BET
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Triethyltin and infant odor conditioning
Page 16
animals, this Mas a (retention) test of the discrimination learned on FND14 and
15. For CTL animals, this was a test of odor preference in the naive or
untrained state. Uiis preference test in CTL animals was followed immediately
by 4 odor discrimination training trials and, 1 minute later, by a second
standard odor preference test. Acquisition was defined as the change in odor
preference on this second test relative to the one given prior to training.
Pesjqp
The purpose of Experiment 3 was to determine whether END16 exposure to TET
would differentially affect odor preferences in 18-day-old rats based on
acquisition training following exposure versus retention of training that
occurred prior to exposure. Acquisition and retention were addressed in two
separate, but overlapping, designs. Acquisition was assessed with a 2 x 2
between-within design involving the factors of TET (SAL vs TET) and test (pre-
vs post-training). Retention was assessed with a 2 x 2 between-between design
involving the factors of TET (SAL vs TET) and groups (CTL vs RET).
PEEWITS m> PTSfflSSICH
Acquisition
Ihe effect of HTO16 administration of TET on acquisition of the odor
discrimination in 18-day-olds is shown in Figure 3. As was seen with earlier
ages of exposure in the previous experiments, TOT appeared to disrupt olfactory
learning. Animals injected with this ccqpound did not show the reduction
across tests in preference for the odor paired with footahock (CS+) that was
evident in their vehicle-injected counterparts. This was confirmed by AN0VA
whidi revealed a significant TET x lest interaction, £(1,78) - 5.30, p < .025.
Analysis of this interaction (Newman-Hauls) indicated a nsliable different*
(B < .01) between the 8ALrACQ mean and the remaining three group means which
did not differ among theneelves.
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Triethyltin and infant odor conditioning
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Retention
The effect of TET on retention of odor preferences established prior to
exposure to the ocrpound is shewn in Figure 4. In contrast to its effect on
acquisition, TET did not alter performance on the retention test. Pups given
odor discrimination training on FND14 and 15 (RET) showed reduced preferences
for CS+ relative to naive controls (CTL) regardless of intervening TET exposure
on FND16. This was confirmed statistically by a groups main effect, f( 1,156) *
21.26, p < .0001. Then was no main effect of TET (£ < 1.8) nor was there a
TET x Groups interaction (f < 1).
Exposure to TET in this study did not affect body weight but did produce a
small but significant reduction in brain weight (see Table 1). These data
support previous reports indicating that the effects of this cctpound on brain
and body weights can be dissociated (33, 44) and indicate that TET can affect
the nervous-system within 48 hours after exposure on FND16.
The results of Experiment 3 add to those of the previous experiments in a
number of ways. First, they extend the age of TET exposure that can cause
olfactory learning deficits up to FND16 and indicate that such deficits can
appear in 18-day-olds as soon as two days after exposure. Second, they indi-
cate that TET toxicity Basra to act selectively on the process of acquisition,
since learning following exposure is disrupted but that preceding exposure is
spared, Third, this- selectivity supports ths view that TET impairs associative
processes and not sensory or rotor processes that are necessary tor learned
performance during the pesfaranot test. For exanple, it oould be argued that
TET impairs olfaction itself car alters motor brtaviar in ways that make it
difficult for pups to demonstrate * preference during testing. However, these
arguments do not fit nail with the present findings, for such iafaeinnents would
to deficits on both the retention and acquisition tests rather than on the
acquisition test alone.
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Triethyltin and infant odor conditioning
Page 18
BBRPffiOT 4
The purpose of Experiment 4 was to examine the possible role of a
nonassociative factor that was not addressed in the previous experiment:
sensitivity to foctshock. Evidence fran studies involving adult exposure
suggest that shock sensitivity is unaffected by 1ET (58). However, the effects
of neonatal TET exposure cn pain perception in infant rats have apparently not
been studied and it is possible that reduced sensitivity to footshock could
account for the apparent deficits in odor aversion learning shown previously.
In Experiment 4, the effect of FND16 exposure to TET on an unlearned response
to footshock, shock-elicited vocalization, was determined in order to assess
this possibility.
subjects
Twenty-three Long-Evans rat pups derived from 3 litters served as
subjects. Litters were obtained, culled, housed, and maintained as described
previously.
flfparatug and Prwyfcgs
The apparatus was the same as described previously. On RJD16, half of the
pups in each litter (2 males and 2 females) were injected i.p. with TET
(5 mq/kg) and half with SAL vehicle as described previously. On FND18, all
animals received two standard conditioning trials (4 shocks) as described
previously except that no odors were present. The number of vocalizatior® to
each shock was recorded and an average vocalization score (per shock) was
determined for each subject. Differences between the TET (n - 11) aid SAL (n -
12) groups were tested statistically by a students t-test.
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Triethyltin and infant odor conditioning
Page 19
rasmirs amp discission
Rqps typically emitted 8 vocalizations to each shock and there was no
effect of TET on the number of elicited vocalizations. Mean (± SEM) vocal-
ization scores for the SAL and TE7T groqs were 8.62 ± .50 and 7.72 + .39,
respectively (p > .173). It is therefore unliJcaly that TET-induced differences
in sensitivity to the footshock unconditioned stimulus that was errployed in
these experiments can account for deficits in odor aversion learning.
effect of tet cn body and brain weights
Table 1 presents a sutimaxy, combined across all experiments in this study,
of the effects of TET on brain and body weight as a function of age of dosing
and testing. Exposure to TET on FND5 produced dose-related reductions in both
measures on FND18. All pair-wise comparisons (Student's t-tests) of the three
doses (0, 3, and 5 mg/kg) were significant (p < .01). FND5 exposure to the 5
wg/kg dose also reduced brain and body weights on FND12. On a percentage basis,
this reduction was similar in magnitude to that observed on FND18. Exposure to
TCI on FND10, also reduced body and brain weights at both ages of testing (all
SS < •01). On a percentage basis, the reduction in body weight was similar;
but brain weight reductions were significantly less (p < .01) than those
produced by RJD5 exposure to the ccnpound. Exposure to TET on FND16 did not
change body weight at 18 days of age but did produce a snail but significant
(p < .01) reduction in brain weight. This reduction was significantly less
(p < .01) than that observed at this age after exposure on FND6 or BTO10.
These results confirm previous studies on the developmental toxicity of TET
that have enployed these measures (33, 44). They suggest, however, that body
and brain weight changes par & cermot account for the observed behavioral
deficits. These changes tended to become less rather than mora pervasive with
increasing age of exposure whereas, if anything, the opposite trend was observ-
ed for the olfactory learning deficits.
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Triethyltin and infant odor conditioning
Page 20
GEMERAL DTSOISSICM
The present experiments indicate that neonatal administration of TET
produces an impairment of associative learning involving odors and footshock in
infant rat pups. This impairment is found at a dose of 5 but not 3 nq/kg of
TET (Experiment 1), interacts with age of exposure and age of testing (Exper-
iment 2), and appears to reflect a deficit of associative processes rather than
of sensory, motor, and motivational capacities that are necessary for learned
performance in this learning paradigm (Experiments 3 and 4).
These findings add to previous behavioral studies on the developmental
neurotoxicity of TET. These studies have oiployed a wide range of behavioral
measures at different points in development. Different exposure conditions
have been used as well, including repeated administration of low doses fron
RJD3-29 (31) and single administrations of higher doses at different postnatal
ages (45). However, the majority of studies have used a single injection of
TET on IND5 (13, 28, 36, 44, 46). Behavioral effects shown in preweanling rats
include: altered rope descent (36) and heme orientation in a straight alleyway
(28); and in adult rats include: altered male sexual behavior (36, 45), active
avoidance (13) and radial-arm-maze performance (28, 31). Perhaps the most
prominent behavioral effect of developmental TBI exposure is hyperactivity.
This is found after both chronic (31) and acuta exposure (13, 28, 36, 44, 46),
and occurs because activity levels do not decrease (habituate) over the test
period. Hyperactivity produced by FND5 exposure to TET emerges with develop-
ment, first appearing at approximately 17 days of age (36, 46), arri persists
into adulthood (13, 28, 36, 44, 46). These behavioral studies (arid more recent
neurobiological ones, 33) indicate that developmental TET produces
early and nonreooverable damage to the nervous sysUn. This ocntcasts with the
transient neurotoxicity produced by adult exposure to the oonpound (36). The
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Triethyltin and infant odor conditioning
Page 21
present study shows that impaired olfactory learning may serve as another early
indicant of the developmental neurotoxicity of TET. Indeed, impairment of this
learning on FND12 (Experiment 2) is, to my knowledge, the earliest age at which
the behavioral toxicity of this compound has been demonstrated. The present
data, however, do not indicate whether this behavioral impairment persists into
adulthood. This is an interesting question for future investigation.
Impairment of olfactory learning was not found with every combination of
exposure and test age enployed in this study. Deficits were found in 18-day—
olds after exposure on END5, 10, and 16; whereas 12-day-olds showed deficits
after exposure on PND10 but not ENDS. Such interactions are sore often the
rule than the exception in developmental neurotoxicology (42, 50) and may be
instructive regarding the mechanisms of the observed effects. For example, one
could ask whether learning impairments were caused by the presence of TET in
the brain during testing or by brain damage and/or alterations in neural devel-
opment that would persist after TET was eliminated from the nervous system.
Brain levels of tin were not measured in the present study but previous work
describing brain concentrations of tin following adult and RIDS exposure to TET
(5) implies that tin was present in the brain at the ages of testing enployed.
However, it is unlikely that these concentrations EST S§ can aooount for the
observed learning deficits. Eighteen-day-old rat pups shewed the same learning
deficit after three ages of exposure (ENDS, 10, and 16) that would have pro-
duced dramatically different brain levels of tin at the time of testing (about
8, 50, and 95% of peak concentrations, respectively). Moreover, FND5 exposure
produces brain concentrations in 12-day-olds that are at least 5 fold higher
than in 18-day-olds and yet learning iapaixnents were only evident at this
latter age (Experiment 2a). Xn addition, neuropathologioal effects have been
observed within 24 hours of neonatal TBT exposure (60) and sane biochemical
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Triethyltin and infant odor conditioning
Page 22
indicants of the persistant brain damage that occurs following FND5 exposure
are evident at least by IND13 (the earliest tine point examined, 33). It is
therefore likely that the deficits reported here reflect TCP-induced brain
damage rather than sane other physiological effect of the presense of tin in
the brain.
The age of dosing by testing interaction in the present study differs from
that obtained by Ruppert et al. (45) who reported hyperactivity following TET
exposure on FND5, but not on RTOl, 10, or 15. These different interactions
suggest that neonatal TET probably alters motor activity and olfactory learning
thrcugh different mechanisms. For exarrple, it is often suggested that hyper-
activity reflects early damage to the hippocaupal formation (eg., 42) and there
is indeed direct evidence of such damage following R1D5 exposure to TET (33).
In contrast, it is unlikely that hippocaitpal damage accounts , for the learning
impairments in the present stud/ because experimental lesions of the septo-
hippocaiTpal system do not alter acquisition in rat pups trained with this
procedure (48). lhe (liJcely) differing mechanisms of these two behavioral
effects of neonatal TET further supports the claim (Experiment 3) that the
inpairments of learned performance obtained in the present study are not by-
products of the motor effects of this oaqpound. That different behaviors are
differentially affected fay age of TET exposure indicates that the developmental
neurotoxicity of TET is a eultifaceted phenomenon that cannot be reduced to a
single underlying mechanism (eg., interference with the brain growth spurt l*h
or limbic damage (28}).
Studies at the neural leval have also ccne to this conclusion (33). In
contrast to its effects after adult exposure (primarily denylination [57, 61)),
neonatal TET exposure has ml triple effects (33, 34). Elegant experiments
examining gross brain regions Cor neuronal and glial proteins have demonstrated
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Triethyltin and infant odor conditioning
Page 23
diverse region- and cell-specific effects of neonatal TET administration. For
exanple, the ccnpcund reduced concentrations of myelin basic protein (a protein
associated with oligodendroglia, the cell type responsible far myelinaticn) in
the hippocanpus and cerebellum but not the forebrain; and these effects showed
a delayed emergence with age (ie., tine since exposure). In contrast, con-
centrations of glial fibrillary acidic protein (a protein associated with
astrocytes which increases in response to neural damage), were elevated in
hippocairpus and forebrain but not the cerebellum; and these effects appeared
early (FND13) and then became generally smaller with age. Concentrations of
proteins associated with synaptogenesis also shewed diverse region- and
age-specific effects (33, 34). These and other studies (30, 33, 34) tenta-
tively suggest two principles: (1) in contrast to what is seen with adult
exposure, the developmental neurotoxicity of TET shares some properties with
trimethyltin (TMT, a similar alkyltin oatpound); and (2) exposure to these
aUcyltin compounds during cell proliferation produces delayed neurotoxic
effects whereas exposure following this stage of neural development produces
more immediate damage.
The manner in which age of exposure and testing interacted to produce
learning impairments in the present study cannot be explained on the basis of
available data but it is possible to speculate. As mentioned previously,
hippocanpal damage probably does not produoe these inpairraents (48). More
recent studies with neonatal rat pups suggest that Favlovian odor conditioning
is Mediated at the level of the olfactory bulb (22, 56} or the anterior
olfactory nucleus (18, 20). It is currently not known whether early exposure
to organotin compounds ilwmwgas these regions of the olfactory systen; although
adult exposure to THT does indeed damage these areas (2) • Zf developmental
exposure conveys nsurotcodc properties to TET that rasenble those of HO (the
first principle mentioned above), it is not iaplausible that HT dmwgsrt these
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Triethyltin and infant odor conditioning
Page 24
areas of the olfactory system. Indeed, in light of the postnatal neurogenesis
that occurs in this system (41) one could imagine (by extrapolation from the
second principle mentioned above) that early exposure would produce delayed
neurotoxicity whereas later exposure would produce more severe and ismediate
damage to this system. This highly speculative explanation of the effects of
age of exposure on the behavioral toxicity shown in the present study (Experi-
ment 2) could be tested by examining neural and glial proteins associated with
specific developmental processes in discrete forebrain areas as a function of
age of TET exposure.
In the present study, care was taken to determine whether performance
deficits resulted fran impairment of associative or nonassociative processes.
The learning deficits shown here cannot be easily attributed to impairment of
sensory (olfaction), motivational (footshock sensitivity), or motor effects of
the oonpcund. These (negative) results are consistent with reports that nest
odors can influence the behavior of TET-axpoeed rat pups (28) and with the
results of operant shock-titration experiments in adult rats (58). These
results are important because neurotoodcants often disrupt a range of behavior-
al processes nonspecifically (27) and lack of behavioral analysis makes it hard
to interpret many neurotoxioological studies of learning and memory (29).
The present findings indicate that olfactory conditioning may serve as a
useful rodent model far the early aaoonnment of learning deficits produced by
early nsurotoodcant exposure. Olfactory learning deficits have also been found
after developmental exposure to ethanol (3) and cocaine (52). The success of
this paradigm supports the view (1, 40, 24 , 51) that recent advances in develop-
mental psychcbiology can be profitably used to redress the relative paucity of
animal models of infant Muocy in developmental neurotaxicology.
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Triethyltin and infant odor conditioning Page 25
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Nteurobehav. Toxicol. Teratol., in press.
52. Spear, L.P.; Bell, K.J.; Yoottanasunpun, V.; Greenbaum, R.; O'Shea, J.;
Hoffman, H.; Spear, N.E. Effect of prenatal cocain exposure cn behavior
during the early postnatal period. Neurotoodcol. Teratol. 11:57-63; 1988.
53. Spear, N.E.; Canpbell, B.A. Ontogeny of learning and menory. Hillsdale, NJ:
lawrenoe Erlbaum; 1979.
54. Stanton, M.E.; Spear, L.P. Workshop on the Qualitative and Quantitative
ocrparibility of Hunan and Animal Developmental Neurotoxicity, Work Group I
Report: Oanparability of Measures of Developmental Neurotoxicity in Humans
and Laboratory Animals. Neurotoxiool. Teratol. 12:261-268; 1990.
55. Staubli, U.; Ivy, 6.; lynch, 6. Hippocanpal denervation causes rapid forget-
ting of olfactory information in rats. Proc. Nat. Acad. Sci. 81:5885-5887;
1984.
56. Sullivan, R.M.; iaon, M. Gne--trial olfactory learning enhances olfactory
bulb responooa to an appetitive conditioned odor in 7-day-old rats.
Develop. Brain Res. 35:307-311; 1987.
57. Suzuki, K. sans new observations in triethyltin intoxication of rats. Exp.
Neurol. 31:207-213; 1971.
-------
Triethyltin and infant odor cxnditioning
Efcge 31
58. Tilson, H.A.; Burns, T.A. Effects of triethyltin cn pain reactivity anfl
neuromotor function of rats. J of Toxicol. Environ. Hlth. 8:317-324; 1981.
59. Tilscn, H.A.; Cranmer, J.M. Neurotoxioology in the fetus and child.
Neurotoxioology 7: 1986.
60. Watanabe, I. Effect of triethyltin on the developing brain of the souse.
In L. Roizin, H. Shiraki, N Grcevic, eds., Neurotoxioology, New York:
Raven; 1977:317-326.
61. Watanabe, I. Organotins (Triethyltin). Ftraa P.S. Spencer & H.H. Scftaumburg,
Experimental and Clinical neurotoxioology. Baltimore: Williams and Wilkins?
1980:545-557.
62. Wender, H.; Mularek, O.; Peechowski, A. The effects of triethyltin
intoxication at the early stage of extrauterine life on cerebral
myelinaticn. Neurcpat. Pol. 12:13-16; 1974.
63. Wigal, T.; Arnsel, A. Behavioral and neuroanatcnical effects of prenatal,
postnatal, or combined exposure to ethanol in weanling rats. Behav.
Neurosci. 104:116-126; 1990.
64. Yanai J., ed. Neurobehavioral teratology. New York: Elsevier; 1984.
-------
Triethyltin and infant odor conditioning Page 32
TABLE 1
Mean (± SEM) body and brain weictfits (in grams) as a function of postnatal day
(HJD) of dosing and testing, and of dose of triethyltin (TET). Numbers in
parentheses are peroent-of-vehicle control. Data are the combined results from
all experiments.
Age of Testing (Sacrifice)
R4D12 FND18
Age of
Dosing
Dose
(mg/kg)
Body Height
Brain Weight
Body Weight
Brain Weight
FND5
0
26.6 ± 0.82
1.12 + 0.03
40.1
+
1.03
1.34 ± 0.01
3
37.1
+
1.14
1.21 + 0.02
(92.6
±
2.81)
(89.8 + 1.48)
5
18.7 +
0.69
0.74 +
0.02
31.6
±
0.65
0.89 ± 0.02
(82.6 +
2.44)
(66.7 +
1.36)
(79.0
±
1.62)
(66.0 ± 1.94)
FND10
0
31.3 ±
0.26
1.17 ±
0.02
41.4
±
0.93
1.36 ± 0.02
5
24.6 ±
0.63
1.03 +
0.02
33.5
±
1.05
1.14 ± 0.02
(78.5 ±
2.01)
(88.3 ±
1.59)
(80.8
+
2.55)
(84.2 ± 1.32)
FND16
0
34.0
+
mm
0.35
1.39 + 0.01
5
34.2
±
0.32
1.34 ± 0.01
(100.1
±
1.04)
(96.1 ± 0.71)
-------
Kriethyltin and infant odor conditioning
Page 33
p»pHena
Figure 1. Mean (± SEM) time spent over the CS+ odor during the 180-sec test of
preference for the CS+ versus CS- odors. The dashed line at 90 sec
indicates lade of preference whereas values below this level indicate
aversion learning. Rat pups were injected with saline vehicle (SAL),
oar 3 or 5 mg/kg triethyltin (TET) on FND5 and then tested on FND18
for odor preference either after 4 discrimination training trials
(ACQ) or without such training (Naive).
Figure 2. Mean (+ SEM) percent tine spent over the CS+ odor during the 180-sec
odor preference test. The dashed line at 50% indicates control (CTL)
levels of perfonnanoe (ie., lack of odor preference) whereas mean
values below this level indicate aversion learning. Rat pups were
given saline (SAL) or TET on FND5 (Lower panel) or FND10 (Upper
panel) and then trained and tested on the olfactory discrimination
task on FND12 or RTO18.
Figure 3. Mean (+ SEM) percent tine spent ever the CS+ odor during the 180-sec
odor preference test. The dawherl line at 50% indicates lack of odor
preference whereas mean values below this level indicate aversion
learning. Rat ptqps were given saline (SAL) or TET (5 mg/kg) on FND16
followed, on FND18, by an initial preference test (CTL), 4 discrimina-
tion training trials, and a second preference teat (ACQ).
Figure 4. Mean (± SEM) percent time spent over the CS+ odor during the 180-sec
odor preference test. The rtiwhert line at 50% indicates lack of odor
preference whereas mean values below this level indicate aversion
learning. Rat pups were given 4 disrimination training trials per day
on PND14 and IVD15 (RBI) or similar treatment without training (CXL) •
On IND16 they were injected with saline (SAL) or 5 ag/kg TET followed
lay the odor preferanoe test on M30J.
-------
Triethyltin and infant odor conditioning
Page 34
HOtes
Thanks are due to Cfcrisley V. Pickens and Craig Barry for expert technical
assistance, and to Des. Diana B. Miller and Linda P. Spear for reviewing an
earlier version of this manuscript.
Disclaimer: This manuscript has been reviewed by the Health Effects
Research laboratory, U.S. Environmental Protection Agency, and approved for
publication. Mention of trade names or ocramercial products does not consti-
tute endorsement or reoanmendation for use.
Address correspondence to: Marie E. Stanton, Neurotcxicology Division
(MD-74B), U.S. Environmental Protection Agency, Research Triangle Park, NC
27711.
-------
(-.«re 3-
J
18 DAY-OLDS
+
M
O
Of
111
o
U1
140
120
100
80
60
40
20
0
I
yyj
NAIVE
ACQ
T
SAL
TET (3 mg/kg) TET (5 mg/kg)
-------
if*
ACQUISITION AFTER PND10 EXPOSURE
LZD sal
EZ3 TET(5mg/kg)
w 20
m
12 DAYS
18 DAYS
ACQUISITION AFTER PND5 EXPOSURE
50 CTL
+
10
o
ec
ui
2
F
40--
SO--
20 •
10-.
0
1
CU SAL
EZ3 TET(5mg/kg)
1
12 DAYS
18 DAYS
-------
PND18 ACQUISITION AFTER PND16 EXPOSURE
60
SAL
TET
50
40-
3 30-
20-
10-
CTL
ACQ
CTL
ACQ
-------
PND18 RETENTION AFTER PND16 EXPOSURE
CTL RET CTL RET
-------
Neonatal Exposure to Trimethyltin Disrupts Spatial Delayed Alternation
Learning in Preweanling Bats
Mark E. Stanton
Neurotoxioology Division (MD-74B), Health Effects Research Laboratory
U.S. Environmental Protection Agency, Research Triangle Park, NC 27711
Karl F. Jensen
Neurotoxioology Division (MD-74B), Health Effects Research Laboratory
U.S. Environmental Protection Agency, Research Triangle Park, NC 27711
Chrisley V. Pickens
NSI Technologies, Inc., Research Triangle Park, NC 27709
Disclaimer: This manuscript has been reviewed by the Health Effects
Research Laboratory, U.S. Environmental Protection Agency, and approved
for publication. Mention of trade names or commercial products does not
constitute endorsement or recatmendation for use.
for correspondence: Mark E. Stanton, Fh.D.
HERL (MD-74B)
U.S. EPA
RIP, NC. 27711
Punning head: TMT and w<#ang memory in infant rats
-------
Abstract
STANTCN, M.E., JENSEN, K.J., AND PICKENS, C.V. Neonatal Exposure to
Trimethyltin Disrupts Spatial Delayed Alternation Learning in Preweanling
Pats. NEUROTOXIOOL TERATOL jflf, ppp-ppp, 199x. Trimethyltin is an organotin
compound that produces potent neurotoxicity in both adult and developing
animals. The limbic system is a primary CNS target site for this toxicity and
a proninent behavioral effect of TMT is disruption of learning and memory.
Impairment of cognitive development has also been suggested by studies showing
that rats neonatal ly expoosed to TMT cannot perform spatial vorking memory
tasks during adulthood. However, the question of hew early in ontogeny such
deficits can be detected has not been addressed. The present study examined
this question with a T-maze delayed alternation learning paradigm. Long-Evans
rat pups, injected i.p. on postnatal day 10 (HJD10) with 6 mg/kg IMT and tested
on RJD18, were unable to learn delayed alternation in the manner shown by
vehicle control pups. However, TMT- and vehicle-treated groups were both able
to learn a siitple position discrimination. These findings indicate a selective
impairment of spatial working memory by neonatal TMT exposure and shew that
this impairment can be demonstrated during the preweanling period in the rat.
KEY WORDS: DEVELOPMENTAL NEUROTOXIOOLDGY, INFANT RMS, TRIMEIHYUITN, LIMBIC
SYSTEM, SPATIAL WORKING MEM3RY, EARLY LEARNING, ORGANCMEIALS,
MENIAL FEEARDASTCN.
-------
TMT and working memory in infant rats
Page 3
The purpose of this report is to describe briefly the successful applica-
tion to developmental neurotoxioology of a technique for studying spatial
working memory in preweanling rats. With few exceptions (3,19), most animal
studies examining the cognitive effects of developmental exposure to neurotox-
icants have involved testing of juvenile or adult animals while relatively
less attention has been paid to assessment during early development (18 ,23,
24). As a result, cognition has generally not been examined in the true
developmental sense, something which seriously weakens the interpretation and
power of animal models in developmental neurotoxioology (23,24). Moreoever, in
those cases where learning has been assessed at different stages of ontogeny,
age and task are often confounded. For example, in one study, approach, learn-
ing in a runway was used during the preweanling period and the radial-arm maze
was used during adulthood (12). The underlying assunption appears to be that
infant animals are incapable of learning the more demanding cognitive tasks
that are commonly used in adult rats. Here we report that preweanling rats can
learn a spatial working memory problem in a T-maze and that this form of memory
is impaired by developmental exposure to a known neurotoxicant, trimethyltin
(TMT).
TMT is an organometal ocrpound that produces potent neurotoxicity when
administered acutely to both adult and developing organisms (for reviews, see
4,6,16,17,20). At either age of exposure, TMT administration produces neural
damage in a number of brain regions, although the limbic system, in particular
the hippocanpus, appears to be the most severely and consistently affected
(1,4,12). Memory impairments associated with damage to this system are
-------
TMT and working memory in infant rats
Page 4
found after exposure to BO (3,12,13). For example, previous studies have
shown that developmental exposure to TMT produces spatial working memory
deficits in juvenile or young adult rats (12,13). The present study employed a
recently developed procedure for studying this form of memory in developing
rats (8) to determine whether these TMT-iriduced deficits can be demonstrated
during the prewearvling period.
METHODS
Subjects. Sixty-three 18-day-old long-Evans rats drawn from 22 litters
served as subjects. The rats were offspring of time-bred females obtained from
Charles River (Raleigh, N.C.) on either the second or fourteenth day of gest-
ation. Offspring were raised in the animal colony at the Neurotoxicology
Division, U.S. Environmental Protection Agency, Research Triangle Park, North
Carolina. This facility is certified by the American Association for Accredit-
ation of Laboratory Animal Care. The colony room was illuminated on a 12-hr
li#it, 12-hr dark photcperiod, with light onset at 7:00 a.m.. The pups were
bom and housed with their mothers in plastic cages measuring 48 x 27 x 20 cm
that contained wood shavings and were continuously supplied with Purina Pat
Chcv and water. Litters were checked daily at the mi of the light phase of
the light-dark cycle and, if newborn pups were found, that day was designated
as postnatal day 0 (HtfDQ). All litters were culled to 4 males and 4 females by
the third day after birth. Subjects remained undisturbed with their mothers
except for periodic cage cleaning and experimental manipulations (dosing and
testing).
Apparatus. The apparatus was the same Plexiglas T-ma2e vised in a previous
study [see (8) for a detailed ctosctiption]. it consisted of a start box,
choice point, and left and rjflftt maze (goal) arms fitted with hinged tops made
of clear Plexiglas. Manually-operated guillotine doors separated the start box
-------
TMT and working memory in infant rats
Page 5
from the choice-point and the choice-point firm both naze arms. Chambers that
could accomodate an anesthetized dam were attached to the end of each maze arm.
Removable Plexiglas doors at the end of each maze arm provided access to the
dam-reward. All Plexiglas walls of the apparatus were covered on their
external side with opaque brcwn paper to prevent animals from being distacted
by extraneous visual stimuli outside of the naze. A 25-W amber-colored light
bulb, located 25 can above the choice-point was the only source of illumination
during testing. Subjects spent the period between trials in 17 x 10 x 10 cm
Plexiglas "ITI" (inter-trial interval) oarpartments with hinged tops and floors
which were covered with paper tcwels. The floor of the entire T-maze, the dan
chambers and the ITI compartments were warmed to 30-33*C with conventional
electric heating pads.
Dosing. On IND10, pups were taken fran the heme nest, identified irxiivid-
ually with paw tatoos, and injected (i.p.) with either saline (Bacteriostatic
0.9% Sodium Chloride Inj., Abbott labs, North Chicago, IL), or 6 mg/kg tri-
methyltin hydroxide (ICN Biomedicals, Plainview, NY) dissolved in saline. Dose
of TMT is expressed as base and was administered in a volume of 10 ul/g bod/
weight. The 6 mg/kg dose was chosen in order to facilitate comparison with
previous research (12) and a single dose was used because this research has
shewn that the dose-response curve for this compound is steep and seldom yields
"graded" effects. A split-litter prooedure (21) was used in which half the
pups (2 males and 2 females) in a given litter were injected with saline (SAL)
am half with TMT.
Design. On R4D18, rat pups fran the SAL and TMT conditions received five
12-trial blocks of training oruorie of two T-maze tasks: Discreibe-trials Delayed
Alternation or Position HabitJT(see belcw). For each of these tasks, some pups
were assigned to an experimental group (EXP), in which reward was contingent on
-------
TMT and working memory in infant rats
Page 6
correct choice; and others were assigned to a nonccntingent control group (NC),
in which reward was delivered on every tried irrespective of ctoice behavior.
For each of the tasks, this yielded a 2 X 2 X 5 design involving the factors of
TMT (SAL vs. 7MT), groups (EXP vs. NC), and trial blocks.
Procedure. A more detailed account of the procedure has been provided
elsewhere (8). Pups were deprived of food and social oontact approximately 24
hours prior to the start of training. Rjps were removed from their hone cages,
weighed to the nearest 0.1 g, and housed individually in 9 x 9 x 12.5 cm com-
partments of a Plexiglas holding box, which was heated to approximately nest
temperature (33 *C) by ccttmercially available electric heating pads placed
beneath the floor. Within a few hours, pups were removed for 2-3 minutes fran
their compartments and fitted with oral cannulas according to the technique of
Hall and Rosenblatt (9).
The following morning, 2 lactating dams with pups approximately the same
age as the experimental animals (18 ± 4 days postpartum) were anesthetized with
Nembutal (32.5 mg/kg, Abbott laboratories) and placed in the "dam chambers" of
the apparatus. (Anesthesia was maintained throughout the experiment via sup-
plementary doses as necessary). Each pup then had its bladder voided, was
weighed, and was placed in an m caipartment, where it remained between trials
during training sessions. Iitroediately prior to the first training session,
pups were acclimated to the maze and taught to consume the reward (nutritive
suckling on the anesthetized dam): each animal was placed on one of the dams,
allowed to attach to a nipple, and then received .03 ml light cream (Half &
Half) via the oral cannula. This procedure occurred twice on each dam. The
animal was then placed in each the goal arms of the maze and allowed to run
dcwn the arm and attach to fhJFanesthetized dam. This procedure continued
until the running latency fell below approximately 5 sec, which usually
required 3-4 runs in eadi maze arm.
-------
TMT and working memory in infant rats
Page 7
The first session ocmnenoed at 0800-0900 hcurs, followed by two more ses-
sions, each starting at 6 hr intervals. These three sessions consisted of 12,
24, and 24 trials, respectively (yielding five 12-trial blocks). on a given
day, an average of 3 pups from a litter were ran as a squad (the remaining
littermates were used in a different, unrelated study). Pups in each squad
were assigned to a different experimental group and/or task. Across squads
(litters), this yielded the following group sizes: Delayed Alternation, Group
SAL-EXP (n-10), SAL-NC (n=9), TMT-EXP (n-9) and TMT-NC (n-5); Position Habit,
Group SAL-EXP (x*-8), SALrNC (n-7), TMT-EXP (n-9) and TMT-NC (n-6).
Pups in each squad were run in rotation. The delayed alternation task
involved a discrete-trials (paired-run) procedure. Each trial consisted of two
runs: a forced run immediately followed by a free-choice run. The forced runs
followed an irregular, counterbalanced sequence of left and right maze arms
(7). Pups in the EXP condition were trained as follcws: at the beginning of
each trial, the pup was removed fran its ITT carpartment and placed in the
start box for the forced run. The guillotine door separating the start box and
choice-point was then raised and a stopwatch started. After 2 sec had elapsed,
the guillotine door to one of the two maze arms was raised, thereby forcing the
animal to that maze arm. An arm choice and choice latency were recorded when
the pip ran to the end of an arm (past a black line on the hinged top of the
maze arm). All farced runs were followed by the nutritive-suckling reward.
The animal was then immediately placed back into the start box for the free-
choice run. On the free-choice run, the animal was given a choice of arms by
raising both maze arm doors simultaneously, 2 sec after the start box door was
raised. If the pup chose the alternate arm to that entered on the forced run,
the door to the dam chamber v*s quickly opened, and the pup received the
nutritive suckling reward. If the pup failed to alternate, this door was not
-------
TMT and working memory in infant rats
Page 8
raised and the pip was detained in the naze arm without reward for a tine
period equivalent to that of a rewarded trial (approximately 20-30 sec). At
the end of this second run, the pep was placed back into the rn ccnpartment
for a period that was determined by the time required to run trials for the
other animals in its squad (120-160 sec). Pups in the NC condition were
treated as just described except that they were rewarded on the free-choice run
regardless of the arm chosen.
The task was a simple spatial discrimination. Each trial
consisted of a single free-choice run, as described above. Animals in the EXP
condition were rewarded if they consistently chose one of the two arms of the
maze (randomly preassigned as left for half the subjects and right for the
other half) whereas pips in the NC condition were rewarded regardless of
choice.
At the end of each session, pips were placed with one of the anesthetized
dams in a chamber away frcm the T-maze and given supplementary diet. The
amount given was determined so as to match all pips for total daily intake,
(correcting for differences in deprivation associated with rate of reward for
different conditions (ie., EXP vs NC) or individual pups and ensuring that the
animals did not fall below 85% of their body weight at the time of deprivation.
Histology. Within 72 hours of the end of training, subjects received a
lethal overdose of Nembutal (Abbott labs, North Chicago, IL) and were perfused
intranatal ally with a 0.9% saline flush followed by 10% phosphate-buffered
formalin (Fisher Scientific). Their brains were removed and postfixed in 10%
formalin. Histology was performed on a randomly selected subset of subjects
from the SAL (17=5) and TMT (n-SL renditions. Brains were frozen, sectioned
coronally at 20 microns, and pcained with cresyl violet.
-------
TMT and working memory in infant rats
Paige 9
RESULTS
Behavioral
Two separate analyses of variance (ANOVAs) were performed on the delayed
alternation and position habit data shewn in Figure l. For each task, a 2 X 2
X 5/ betveen-between-within ANOVA was performed involving the factors of
TMT (SAL vs. TMT), Groups (EXP vs. NC), and Trial Blocks.
Neonatal exposure to TMT totally abolished acquisition of discrete-trials
delayed alternation learning (Figure 1, left panels). Experimental animals
exposed to saline vehicle on PND10 (Grap SAL-EXP) improved across trial
blocks, whereas their noncontingently-rewarded controls (Group SAL-NC) did not
(lower left panel). in contrast, experimental animals injected with TMT on
FMD10 (Group TMT-EXP) failed to show this inprcvament and never differed fran
their nanoontingently rewarded counterparts (Group 3M3HJC, upper left panel).
This was confirmed by ANOVA which yielded main effects of TMT, 1(1,116) *
18.76, p < .0002y groups, £(1,116) » 4.82, p < .037; and a TMT x Groups inter-
action, f (1,116) « 7.88, B < .009. Analysis of this interaction with post hoc
tests (Newman-Keuls) indicated that Group SAL-EXP outperformed (p < .01) its
control group (Group SAI/-NC) whereas Group TMT-EXP did not. Thus, END1Q
exposure to TMT iapaired delayed alternation learning in rat pups tested at 18
days of age under the conditions of the present experiment.
In contrast to the results with delayed alternation, TNT did not alter
acquisitioinjbf the position habit task (Figure 1, right panels). ANOVA on the
animals trained on this task revealed main effects of groups, £(1,104) * 21.64,
p < .0001; and a Groups x Blocks interaction, £(4,104) - 6.18, p < .001. This
indicates that EXP animals peefi»emad at higher levels than their NC controls,
and that the performance of WQ? animals inproved with training whereas perfor-
mance of NC controls did not. There was an indication that TMT-EXP animals
-------
TMT and working memory in infant rats
Page 10
performed at lower levels than SALr-EXP animals on the first training block but
this was not confirmed statistically fay ANOVA. The absence of interactions of
TMT with groups, blocks, or Group x Blocks (all £§ < 1.61), indicates that
FND10 exposure did not alter position discrimination learning under the
conditions of this experiment.
Histology
Histological examination of cresyl-violet stained sections frcm TMT- and
SAL-treated animals (Figure 2) revealed the characteristic pattern of hippo-
campal damage that has been reported in previous studies (4,12). There was
clear reduction in size of the hippocainpus, and loss of pyramidal cells in the
CA3-CA4 subfields, in TMT-treated rats (TMT), relative to SAL-treated controls
(SAL). This damage was found in all the TMT-treated subjects that were examin-
ed, although it varied somewhat, being more severe in sane and moderately less
severe in others.
DISCUSSION
The present findings indicate that neonatal exposure to TMT produces a
selective impairment of spatial working memory in 18-day-old rat pups. Pups
treated with OCT were unable to learn discrete-trials delayed alternation in
the manner shewn by their vehicle-injected counterparts. However, both treat-
ment groups were able to learn a simple position discrimination. This pattern
of results resembles that observed in adult rats with damage to the limbic
system (15), including that associated with exposure to TMT (3,12,13). This
aspect of TMT-induced brain damage may not be the exclusive determinant of the
impairments reported here, but it is certainly sufficient to account for than.
The present finding extendiwevious work examining the cognitive effects of
neonatal TMT exposure in a number of ways. First, when combined with existing
reports (12), it provides the first indication of how TMT effects the «***>
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HIT and working memory In infant rats
Page 11
learning and memory phenomenon in both preweanling and adult rats. To our
knowledge, previous studies have confounded task with age; for example,
contrasting preweanling runway performance with weanling swim escape behavior
(14) or with adult spatial working memory (12). These studies have therefore
failed to indicate whether the early learning deficits produced by TMT persist
into adulthood or, oonversely, whether deficits shown in adulthood can be
detected during the preweanling period. The present study shows that adult
spatial working memory deficits can indeed be predicted by preweanling testing.
Second, the use of two T-maze tasks to reveal a selective effect of TMT in-
dicates that this compound produces an impairment of learning and memory rather
than of performance. Previous studies have provided little or no evidence on
the question of whether perinatal TMT exposure specifically disrupts learning
rather than other sensory, motor, or motivational processes contributing to
performance on tests of early learning [see (10,11) for discussions of this
issue in neurotoxicology]. For example, it has been acknowledged that poorer
runway learning (increased latencies) of preweanling rats treated with TMT may
be a byproduct of hypoactivity, as measured in a separate test [see p. 747 of
(12) ]. Because the two T-maze tasks—delayed alternation and position discrim-
ination—employed in the present study involve the same sensory, motor, and
motivational capacities, but differ with respect to the memory processes requir-
ed, the selective effect of TMT on delayed alternation suggests that memory is
indeed the bdiavioral process that is impaired.
Third, the present experimental model extends previous ones by virtue of
its potential for extrapolation to (ncnfcuman) primates and humans. The two
tasks employed in this study isrfwesent one of a family of memory distinctions
that, on operational and neurODiological grcunds, are actively being studied in
humans and a variety of maaaaalian species by behavioral neuroscientists [see
(3,10) for further discission of this issue in relation to neurotaxicology].
-------
TMT and vrorkirig memory in infant rats
Page 12
Moreover, this research tradition is being rapidly extended to the problem of
cognitive development (5). Thus, the animal model described in the present
report shews premise of becoming cast in a such larger context of research on
the neurobiology and toxicology of memory in rodents, monkeys, and humans at
various points in the lifespan.
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TMT and working memory in infant rats
Page 13
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5. Diamond, A. ,ed., The development and neural basis of higher cognitive
functions. New York: New York Acadeny of Sciences Press, in press.
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-------
TMT and working memory in infant rats
Page 14
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11. Miller, D.B.; Eckarman, D.A. Learning and memory measures. In Z. Annau,
ed. Neurcbehavioral toxicology. Baltimore: Johns Hopkins University Press;
94-149, 1986.
12. Miller, D.B.; O'Callaghan, J.P. Biochemical, functional and morphological
indicators of neurotoxicity: Effects of acute administration of trimethyl-
tin to the developing rat. J. Riarmacol. Exp. Iher. 231:744-751; 1984.
13. Miller, D.B.; Eckerman, D.A.; Krigman, M.R.; Grant, L.D. Chronic neonatal
organotin exposure alters radial-arm maze performance in adult rats.
Neurobehav. Toxicol. Teratol. 4:185-190, 1982.
14. Noland, E.A.; Taylor, D.H.; Bull, R.J. Moncmethyl and trimethyl tin
compounds induce learning deficiencies in young rats. Neurobehav. Toxicol.
Teratol. 4:539-544, 1982.
15. 01 ton, D. S. Memory functions and the hippocairpus. in W. Seifert, ed.,
Neurobiology of the hippocampus. Iondon: Academic Press, 1983:335-373.
16. Re iter, L.W.; Ruppert, P.H. Behavioral toxicity of trialkyltin compounds: A
review. Neurotaxioology 5:177-186, 1984.
17. Reuhl, K.R.; Cranmer, J.M. Developmental neuropathology of organotin
compounds. Neurotaxioology 5:187-204, 1984.
18. Riley, E.P.; Hannigan, J.H.; Balaz-Hannigan, M.A. Behavioral teratology as
the study of early brain damage: Considerations for the assessment of
neonates. Neurobehav. Tnsdcor. Teratol. 7: 635-638, 1985.
19. Riley, E.P.; Barron, S.; #riscoll, C.D; Chen, J.S. Taste aversion learning
in preweanling rats exposed to aloohol prenatally. Teratology 29: 325-331,
1984.
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TMT and working memory in infant rats
Page 15
20. Ruppert, P.H. Postnatal exposure. In: Z. Armau, ed. Neurobehavioral
Toxicology. Baltimore: Johns Hopkins University Press; 1986:170-189.
21. Ruppert, P.H. ; Dean, K.F.; Reiter, L.W. Oarparative developmental toxicity
of triethyltin using split-litter and whole-litter dosing. J. Toxicol.
Environ. Health 12:73-87, 1983.
22. Ruppert, P.H.; Dean, K.F.; Reiter, L.W. Development of locomotor activity
of rat pups exposed to heavy metals. Toxicol. Appl. Riarmacol. 78:69-77,
1985.
23. Spear, L.P. Neurobehavioral assessment during the early postnatal period.
Neurcbehav. Toxicol. Teratol., in press.
24. Stanton, M.E.; Spear, L.P. Workshop on the Qualitative and Quantitative
camparibility of Human and Animal Developmental Neurotoxicity, Work Group I
Report: Comparability of Measures of Developmental Neurotoxicity in Humans
and laboratory Animals. Neurotoxiool. Teratol., 12:261-267, 1990.
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TMT and working memory in infant rats
Page 16
Figure Captions
Figure 1. Mean (± SEM) percent-correct choice scores for 18-day-old pups
tested on either delayed alternation (left panels) or position
habit (right panels) who ware previously exposed on FND10 to
either saline vehicle (SAL, lower panels) or 6 wq/kg TMT (TMT,
upper panels). Reward was presented either contingently (EXP) or
noncontingently (NC) with regard to choice. Learning is indicat-
ed by a difference in percent-correct scores between EXP and NC
groups that emerges across training blocks. The absence of error
bars on sane data symbols indicates SEM was smaller than symbol.
Figure 2. Cresyl-violet stained, coronal sections through the dorsal hippo-
campus of representative vehicle-treated (SAL, lower panel) and
TMT-treated (TMT, upper panel) rats. A reduction in the size of
the hippocampus and loss of pyramidal cells in areas CA3-CA4 (ar-
rows) are evident following FND10 exposure to 3MT. Scale « 1 mm.
-------
TMT and working memory in infant rats
Page 17
Noteff
Thanks are due to Craig Barry, Julia Davis, and Qiris Hurchison for expert
technical assistance, and to Drs. Fhillip J. Bushnell and W. G. Hall for
reviewing an earlier version of this manuscript.
Disclaimer: This manuscript has been reviewed by the Health Effects
Research Laboratory, U.S. Environmental Protection Agency, and approved for
publication. Mention of trade names or commercial products does not consti-
tute endorsement or recommendation for use.
Address correspondence to: Mark E. Stanton, Eh.D., Neurotoxicology
Division (MD-74B), U.S. Environmental Protection Agency, Research Triangle
Park, NC 27711.
-------
100
90
80
70
60
50
40
100
90
80
70
60
50
40
\ \ j X
DELAYED ALTERNATION
POSITION HABIT
A- - A TMT-NC
A A TMT-EXP
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na' I
i
I I
I I
tn
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12-TRIAL BLOCKS
-------
-------
TOXICOLOGY AND APFUED PHARMACOLOGY 94, 394-406 (1988)
Atkyltin inhibition of ATPase Activities in Tissue Homogenates and
Subcellular Fractions from Adult and Neonatal Rats'
Karen E. Stine,*-2 Lawrence W. Reiter,*^ and John J. Lemasters**
'Curriculum in Toxicology, University of North Carolina. Chapel Hill, North Carolina J 7 5 99: 'rSeurotoxtcoiony
Division. U.S. Environmental Protection Agency, Research Triangle Park. North Carolina 27"! I.
and XLaboratariesfor Cell Biology. Department of Cell Biology and Anatomy.
University of North Carolina. Chapel Hill. North Carolina 27599
Received August 10,1987; accepted December 16. 1987
Atkyltin Inhibition of ATPase Activities in Tissue Homogenates and Subcellular Fractions
from Adult and Neonatal Rats. Stine, K.. E., Reiter, L. W.. and Lemasters, J. J. (1988).
Toxical. Appl. Pharmacol. 94, 394-406. Inhibition of ATPase activities by triethyltin (TET),
diethyltin (DET), monoethyltin (MET), and trimethyltin (TMT) was studied in homogenates
of brain and liver from adult and neonatal rats. In the adult, sensitivities were as follows: mito-
chondrial ATPase of liver » Na',K.*-ATPase of brain a mitochondrial ATPase of brain > non-
specific ATPase of brain and liver. MET did not produce significant inhibition. ATPase activities
in brain and liver homogenates from TET-trtated adult rats did not differ from controls. Mito-
chondrial ATPase in brain homogenates from J-day-old rats was two orders of magnitude more
sensitive to TET than brain homogenates from adult rats (ICM of 2.5 «m in the J-day-old neonate
vs 260 »M in the adult). By contrast, isolated mitochondria and synaptosomal fractions from
adult and neonatal brains were equally sensitive to TET (IC» " 1-3 mm). At 10 days of age.
following the onset of myelination, the IC*> for TET inhibition of brain mitochondrial ATPase
increased to 71 «M. Myelin added directly to isolated mitochondria also reduced TET-induced
inhibition. It it concluded that In viva brain tin concentrations in 5-day-oId rats following a
neurotoxic does of TET are sufficient to inhibit brain mitochondrial ATPase. whereas in adults,
tin concentrations an insufficient for inhibition. In the adult rat, TET binding to myelin appears
to prevent inhibition of brain mitochondrial ATPase, and the target of toxic action may be
myelin. In the neonateal rat, TET may inhibit oxidative phosphorylation in unmyelinated brain
tissue, leading to neuronal ceU death. •iMlAadmricftmiK.
Triethyltin (TET) and trimethyltin (TMT)
are potent neurotoxicants. In adult rats, sub*
acute administration of TET produces cen-
tral nervous system edema characterized by
intramyelinic variolation (Scheinberg et al.,
1966) and alters nerve conduction (Graham
1 This paper has been reviewed by the Health Eflhcta
Reeaarch Laboratory, U.S. Environmental Protection
Agency, and approved for publication. Mention of trade
names or commercial products does not commute en-
donemem or recommendation for use.
1 Prseeat addnes: Department of BMogy, Radford
University, Radford, Virginia 24142.
et al., 1976). Chronic exposure to TET alters
muscle contraction (Millington and Bier-
kamper, 1982), and decreases in brain neuro-
transmitter levels follow acute exposure
(Moore and Brody, 1961). Acute or subacute
administration of TMT produces different
neuropathological effects, consisting primar-
ily of neuronal necrosis (Brown et al., 1979).
Early postnatal administration of TET
however, produces neuropathological changes
which differ from those following adult expo-
sure. In TET-treated neonates, synthesis of
myelin components decreases and gross
004l-OOSX/88 S3.00
CopyitaMC IMS by Aeedwk Km Inc.
AS rtefcwef mmntoaim m mi fctai i—rni
394
-------
ALKYLT1N INHIBITION OF ATPases
395
edema is absent (Blaker et al, 1981). In neo-
nates treated with TET on Postnatal Day 5,
development of motor activity is initially de-
layed and is followed by hyperactivity which
persists into adulthood. When animals are
treated on Postnatal Day 10 or 15, however,
hyperactivity does not develop (Ruppert et
al., 1984), and TET-treatedadult rats, in con-
trast, display a reversible hypoactivity (Reiter
et al., 1981). Brain tin leveis in adults and ne-
onates following administration of TET are
comparable, however, indicating that dispo-
sitional factors are not responsible for ob-
served differences (Cook et al., 1984a).
In vitro, TET, TMT, and other alkyltins
are effective inhibitors of both mitochondrial
and Na7K* ATPase activities in subcellular
fractions from brain (Wassenaar and Kroon,
1973) and liver (Aldridge and Street, 1971).
Although TET binds with high affinity to the
liver mitochondrial ATPase (Aldridge and
Street, 1970; Farrow and Dawson, 1978), it
also binds to other tissue constituents includ-
ing hemoglobin (Elliott et al., 1979) and my-
elin (Lock and Aldridge, 1975). TET is also a
potent inhibitor of ATPases in hepatic sub-
cellular fractions in vitro (Aldridge and Street,
1970) and is toxic to hepatocyte cell cultures
at a concentration of 100 mm (Wiebkin et al.,
1982). TET and other alkyltins are dealky-
lated by liver microsomes to di- and monoal-
kyl derivatives by a cytochrome P-450-de-
pendent process (Casida et al., 1971). How-
ever, liver damage has not been reported to
follow TET administration.
The aim of the present study was to assess
alkyltin-induced inhibition of ATPase activ-
ities in brain and liver homogenates, and to
compare concentrations necessary for inhibi-
tion with reported tissue tin leveis developed
following in vivo exposure to neurotoxic dos-
ages of TET and TMT. The effects of TET on
ATPases from adult and neonatal homoge-
nates and subcellular fractions were com-
pared in order to investigate a potential bio-
chemical basis for age-related differences in
toxicity.
METHODS
Animals. Maie CD rats were obtained from Charles
River Breeding Labs (Wilmington, MA) « 60 days of
age. Rats were housed three to a cage, and food (Purina
Lab Chow) and water were available ad libitum through-
out the experiments. Neonatal studies utilized offspring
of timed pregnant females obtained from Charles River
Breeding Labs. Dams with litters were housed individu-
ally, and food (Purina Lab Chow) and water were avail-
able ad libitum throughout the experiments. At appro-
priate times the animals were killed by decapitation.
Subcellular fractionation. Subcellular fractions were
prepared by a modification of the method of Gurd et al.
(1974). Brains were removed and homogenized in 2 vol
of 0.25 M sucrose, I mM EDTA. After bringing the vol-
ume to 10 ml/g wet wt, samples were centnfuged at
2000; for 10 min. The supernatant was then centnfuged
at 12,000# for 15 min. The resulting pellet was resus-
pended in 0.4 m sucrose and centnfuged at 60.000? for
60 min over a discontinuous Ficoll gradient 18 and 14%
(w/v) Ficoll in 0.4 M sucrose). Material at the 0.8% i nter-
face was taken as the crude synaptosomal fraction, and
the pellet as the mitochondrial fraction. Each fraction
was resuspended and washed at 10.000; for 45 min and
resuspended in 0.4 m sucrose. The ratio of the yield of
mitochondrial protein to myelin protein in the prepara-
tion was approximately 1:5.
ATPase assays. Brains and liven were removed and
homogenized in 4 vol of 0.25 m sucrose, 2 mM K-Hepes
buffer, pH 7.4, I-4'C. Tissues from a total of eight adult
and sixteen neonatal rats were pooled for each organ ho-
mofenate. Homogenates were frozen and stored at
-60*C prior to analysis. ATPase assays were based on a
modification of Sortnson and Mahler 11981), and AT-
Pase activity was determined by formation of inorganic
phosphate from ATP using 5 mM azide as a specific in-
hibitor of the mitochondrial ATPase and 2 mM ouabain
as an inhibitor of the Na 7K* ATPase. Homogenized tis-
sue wis diluted and treated with 0.16 mg Lubrot/mg pro-
tein as an alternative to sonication to increase measur-
able mitochondrial ATPase activity (Chan et al.. 1970).
Homogenate was incubated with the appropriate inhibi-
tors (including TET) in a medium consisting of 20 mM
Tris-HCl buffer (pH 7.4, 37*Q, 150 mM NaCl. 50 mM
KCl and 5 mM MgClj (total volume 0.4 ml). The
amount of protein in each homogenate incubation was
0.23 nag. The reaction was started by the addition of 0.1
ml of0.025 m ATP and was stopped after 5 min by the
addition of 0.1 mi of 10% sodium dodecyl sulfate. Inor-
ganic phosphate was determined by the method of Peter-
son (1978). Proteins were determined by a biuret method
(GoraallAo/.. 1949) with crystalline bovine serum albu-
min as a standard. ATPase activities obtained by this
method wen comparable to values obtained utilizing a
coupled enzyme may method (Lemaiten and Billica.
1981). Activities from fiesh and frozen tissue were also
-------
396
STINE, REITER. AND LEMASTERS
comparable. Reactions were linear with respect to time
and protein concentration over the ranges studied, and
substrate conditions were saturating.
For in vivo exposures to alkyltins. 6.0 mg/kg TET-Br
(in saline) was injected ip into the rats. They were killed
1 to 24 hr postadministration. Tissues were removed and
treated as described above. Triethyltin bromide (TET),
trimethyltin bromide (TMT). diethyltin dibromide
(DET), and monoethvltin trichloride (MET) were all
purchased from Organometallics. Inc. lEast Hampstead.
NH). Organotin compounds were spectfied by Organo-
metallics. Inc.. as 97-99% pure, and were used without
further purification. All compounds were dissolved in
water with the exception ot" the TET-Br. which was ini-
tially solubilized in a 0.9<% saline solution. Other reagents
were obtained from standard commercial sources.
Calculations. Enzyme activities are expressed either as
nmol/min/mg of protein or as percentage of control. ICjo
values were taken from plots of log (//!-<) vs /, where i
is percentage inhibition and / is inhibitor concentration.
The x intercept of the least-squares regression line de-
fined the ICjo- Slopes of the regression lines were calcu-
lated with the SAS general linear models procedure (SAS,
1982). Tissue tin levels as determined in an earlier study
by atomic absorption spectroscopy (Cook et al„ 1984a.b)
were used to calculate tissue tin concentrations. This
method does not discriminate between the various tin-
containing metabolic breakdown products of the admin-
istered alkyltins. Water content was taken as 0.78 ml/g
wet wt of brain and as 0.6S ml/g wet wt of liver as deter-
mined gravimetrically after oven drying of the organs for
2 days at 45'C.
RESULTS
Adult Brain Homogenates
Three ATPase activities were distinguished
in adult rat brain: mitochondrial (azide-sensi-
tive), Na7K* (ouabain-sensitive), and non-
specific (ouabain- and azide-insensitive). All
were inhibited by TET (Fig. 1A). IC» values
of the Na7K* ATPase (250 and the mi-
tochondrial ATPase (260 mm) were compara-
ble, and both activities were much more sen-
sitive to inhibition than nonspecific ATPase
(ICjo ¦ 1100 mm) (Table I). IC» values for
all ATPase activities in brain were more than
tenfold greater than peak tin concentrations
in brain following a neurotoxic dose of TET
(Table 1).
TMT also inhibited all brain ATPase activ-
ities (Fig. IB). Relative sensitivities were as
follows: brain Na7K+ ATPase (IC50 = 780
mM) > brain mitochondrial ATPase (ICSo
= 1400 mM) > nonspecific ATPase (IC50
= 3000 jiM) (Table 1). Both the mitochon-
drial and Na'/K* ATPases were much less
sensitive to TMT than to TET. This de-
creased sensitivity, in combination with
lower tin levels in brain (7.6 mm) following in
vivo exposure to TMT (Table I), indicates
that TMT is even less likely than TET to pro-
duce in vivo inhibition of brain ATPases.
The initial putative metabolite of TET.
DET, was more effective than TET as an in-
hibitor of the Na"/K* ATPase, and was
equally effective in inhibition of the brain mi-
tochondrial ATPases (Fig. 1C. Table 1).
However, further dealkylation produced an
apparent decrease in effect: at concentrations
up to 5 mM, MET was not an effective inhibi-
tor of any of the ATPases studied (Fig. ID.
Table 1).
Adult Liver Homogenates
In homogenates from the adult liver, two
ATPase activities were identified: mitochon-
drial (azide-sensitive) and nonspecific (oua-
bain-and azide-insensitive). Na7K* ATPase
activity in liver homogenates was below de-
tectable limits. Mitochondrial ATPase (IC50
» 2.3 mm) was much more sensitive to TET
inhibition than nonspecific ATPase (IC30
* 6200 #iM) and more sensitive than the com-
parable activity in the adult brain (Fig. 2A.
Table 2). As in brain, TMT was a less effective
inhibitor of mitochondrial ATPase than TET
(Fig. 2B, Table 2), and the effects of DET
were comparable with TET (Fig. 2C, Table
2). Again, MET was the least effective inhibi-
tor (Fig. 2D, Table 2).
ATPase Activities in Homogenates of Brains
and Livers from TET-Treated Adult Rats
A comparison oflC» values with tin levels
in liver following 6.0 mg/kg dose of TET-Br
indicates that if all tin measured by atomic
-------
ALKYLTIN INHIBITION OF ATPases 397
TET
TMT
100
¦3
^ SO
o
o
*
T
MET
C
0
SO
10-«
Alkyttln (M)
Fig. 1. Inhibition of brain homogenate ATPases by alkyltins. (A) TET. (B) TMT. (C) DET. and (D)
MET. Each point represents the mean ± SE for 6-12 replicates. O, mitochondrial ATPase (control value
- 21.8 + 0.9 nmol/min/mg protein; ~, Na*/K* ATPase (control value - 124.9 x 18.4 nmol/min/mg
protein); A, nonspecific ATPase (control value » 81.9 ± 7.4 nmol/min/mg protein).
absorption spectroscopy were in the molecu- cur. To test for inhibition after in vivo admin-
lar form of TET, substantial in vivo inhibition istration of TET, animals were treated with
of the liver mitochondrial ATPase should oc- a neurotoxic dose (6.0 mg/kg) of TET. and
TABLE!
ICjo Values for alkyltin Inhibition of Neural ATPases, Based on the addition
OF ALKYLTINS TO ADULT BRAIN HOMOGENaTES
Compound
ICm (mm) in brain homogenate
NaVK"
ATPase
Mitochondrial
ATPase
Nonspecific
ATPase
Tin
concentration
UM)in adult
brain 24 hr after
administration
Triethyltin
Trimethyltin
Diethyltin
Monoethyltin
250
780
72
>1000
260
1400
260
>1000
1100
3000
250
>1000
17.1"
7.6®
' Male CD rats, given 6.0 mg/kg TET-Br, ip (Cook et at., 1984a).
" Male Long-Evans rats, given 6.0 mg/kg TMT-OH, ip (Cook tied.. 1984b).
-------
398
STINE, REITER, AND LEMASTERS
TABLE 2
IC50 Values for alkyltin Inhibition of Hepatic ATPases. Based on the addition
OF ALICYLTINS TO ADULT LlVER HOMOGENATES
Tin
[Cjo (mm) in liver homogenale concentration
Compound
Mitochondrial
ATPase
Nonspecific
ATPase
(nM) in adult
liver 24 hr after
administration
Triethyltin
2.3
6200
12i.5J
Trimethyltin
83
6300
66.8"
Diethyltin
2.3
2100
—
Monoethyltin
>1000
>1000
—
" Mate CD rats, given 6.0 mg/kg TET-Br. ip(Cook etal.. 1984a).
" Male Long-Evans rats, given 6.0 mg/kg TMT-OH, ip (Cook et at., 1984b).
ATPase activities of brain and liver horaoge- Pase activities differed from controls at either
nates were compared with those of controls. I or 24 hr postexposure (Fig. 3). In liver ho-
As anticipated, none of the three brain AT- mogenates the predicted in vivo inhibition
TMT
T6T
100
OtT
MET
0
100
<
r*
r«
10''
Alkyltin
i-j
Fta. 2. Inhibition of liver homoftnate ATPiaa by aikyWu. (A) TET, (B) TMT, (C) DET, and (D)
MET. Each point npmmti the mean * SE tor 6-12 npiteataa. O, mitochondrial ATPue (control value
- 49.4 ± 4.1 nmol/tntn/mj protein); A, nonspecific ATPaie (control value - 41.3 ± 5.1 nraol/min/mg
protein).
-------
ALKYLT1N INHIBITION OF ATPases
399
Control
wa K M(to Nonio«c
uvea
Mito Nontptc
1 Hour
Mito Nonsptc
24 Hours
Fio. 3. ATPase activities in brain and liver homogenates from control and TET-treated rats. Male CD
rats were injected ip with 6.0 mg/kg TET-Br, and lulled I or 24 hr postinjection. Values given are the
means x SE from groups of 6 rats.
was not observed, as ATPase activities in ho-
mogenates from TET-treated animals (6.0
mg/kg TET) did not differ from controls at
either 1 or 24 hr postexposure.
Postnatal Day 5 Brain Homogenates
Three ATPase activities were measured in
brain homogenates from 5-day-old rats: mi-
tochondrial (azide-sensitive) ATPase, Na+/
K* (ouabain-sensitive) ATPase, and nonspe-
cific (ouabain- and azide-insensitive) AT-
Pase. All three activities were inhibited by
TET (Fig. 4). The ICjo for neonatal mito*
chondrial ATPase was two orders of magni-
tude lower than the corresponding activity in
the adult (2.5 pM vs 260 mm), whereas the ICso
value for the Na*/K+ and nonspecific AT-
Pases in neonatal tissue were comparable to
the adult (Table 3). Tin concentrations in
brain following a Postnatal Day 5 dose of 6.0
mg/kg were more than two times more than
the ICso for mitochondrial ATPase (Table 3).
Postnatal Day 5 Liver Homogenates
Two ATPase activities were measured in
liver homogenates from 5-day-old rats: mito*
chondrial (azide-sensitive) ATPase and non-
specific (ouabain- and azide-insensitive) AT-
Pase. ICso values in neonatal liver tissue were
comparable with those in the adult (Fig. 5.
Table 3). These values were also comparable
with ICjo values for neonatal brain homoge-
nates.
Postnatal Day 10 Brain Homogenates
If the binding of TET to myelin competes
with binding to mitochondrial ATPase, then
the ICjo in older pups, following the onset of
myelination, should begin to resemble those
of adults. ATPase activities measured in
brain homogenates from 10-day-old rats fol-
lowed this pattern with the IC50 for mito-
chondrial ATPase (71 mm) beginning to ap-
proach that of the adult (260 mm). The IC5o
for the Na*/K.* ATPase in the brain homoge-
nate was 410 pM, and for the nonspecific AT-
Pase was > 1 mM (Fig. 6).
Inhibition of the Mitochondrial ATPase in
Subcellular Fractions from Adult and Neo-
natal Brains
To determine whether there are intrinsic
differences in sensitivity of adult and neona-
-------
400
STINE, REITER, AND LEMASTERS
100
O MMocrtOrrdrtif
~ *•* K-
Merttptetfle
i
J
U
<
I
TET (M)
Fig. 4. TET-induced inhibition of mitochondrial, Na'/K.*, and nonspecific ATPascs in brain homoge-
nates from Postnatal Day 5 rats. Each point represents the mean t SE for 9-12 replicates. Control values
are 36.9 s 4.2 nmol/min/mg protein for mitochondrial ATPase, 91.7 ± 9.7 nmol/min/mg protein tor
Na7K" ATPase. and 126.7 = 8.9 nmol/min/mg protein for nonspecific ATPase.
tal mitochondrial ATPascs to TET, inhibi-
tion of ATPase activities in brain subcellular
fractions from adult and neonatal (3-day-old)
rats were measured. Following subcellular
fractionation, mitochondrial ATPase activity
was identified in the synaptosomal and mito-
chondrial fractions, with mitochondrial AT-
Pase activity in the myelin fraction constitut-
ing less than 3% of the total activity in all
three fractions (expressed as nmot/mg pro-
tein). Although there were some differences
in sensitivity between the synaptosomal and
mitochondrial fractions in the adult (2.7 vs
1.5 mm), both were much more sensitive to
TET inhibition than the ATPases in adult
brain homogenate (260 ^m) (Fig. 7). In addi-
tion, ICjo values for the ATPase activities in
subcellular fractions were comparable be-
tween neonates and adults (Fig. 7, Table 4).
Thus, differences in sensitivity to TET be-
tween adults and neonates do not arise from
differences in the mitochondria themselves.
TABLE 3
ICjo Values for TET Inhibition of Neonatal ATPases Based on the addition of TET to Brain
and Liven Homogenates from Postnatal Day 5 Rats
ICjo (mm) in homogeiiate
Peak tin
concentration in
(»M) in organ 24 hr
after administration"
Tissue
NaVK*
ATPase
Mitochondrial
ATPwe
Nonspecific
ATPase
Braia
Liver
420
n.d.*
2.3
2.1
1300
>10.000
6.9
* CD rats were injected with 6.0 mg/kg TET-Br, ip on Poctnatal Day J and tin levels measured at 24 hr postadminis-
tration (Cook tt ai, 1984«).
'Notdewctabfe.
-------
ALKYLTIN INHIBITION OF ATPases
401
O 100 .r-
S0
TET (M)
Fig. 5. TET-induced inhibition of mitochondrial and nonspecific ATPases in liver homogenates from
Postnatal Day 5 rats. Each point represents the mean a: SE for 6-9 replicates. Control values are 40.5 r 5 8
nmol/min/mg protein for mitochondrial ATPase and 41.5 ± 5.1 nmol/min/mg protein for nonspecific
ATPase.
If binding of TET to myelin in the adult rat
brain is a factor in the decreased sensitivity of
adult brain mitochondrial ATPases to TET,
then addition of myelin to isolated mitochon-
dria would be expected to confer similar pro-
tection. To test this, varying amounts of
crude myelin were added to a constant
amount of mitochondria. Increases in the ra-
tio of myelin to mitochondria led to reduc-
tions in the degree of ATPase inhibition ob-
O MU0Cft©m»n»l
i
^ 100
*
>»
s
>
<
10 -•
TET (M>
FIO. 6. TET-induced inhibition of mitochondrial, Na*/K\ and nonspecific ATPases in brain homoge-
nates from Postnatal Day 10 rats. Each point represents the mean ± SE for 6-9 replicates. Control values
are 28.4 ± 3.5 nmol/min/mg protein for mitochondrial ATPase, 121.1 ± 1S.0 nmol/min/mg protein for
Na*/K." ATPase, and 169.9 ± 6.2 nmol/min/mg protein for nonspecific ATPase.
-------
402
STINE, REITER, AND LEMASTERS
"5
100
0 Mit
Fraction
& S
Fraction
TET (M)
»
I
a
£
5
Fig. 7. (A) TET-induced inhibition of the mitochondrial ATPase in the synaptosomal and mitochon-
drial fractions from adult rat brain. Each point represents the mean ± SE for 6 replicates. Control values
are 336.2 s 17.9 nmol/min/mg protein for synaptosomal fraction ATPase and 632.6 * 38.9 nmol/min/
mg protein for mitochondrial fraction ATPase. (B) Inhibition of the mitochondrial ATPase in the synapto-
somal and mitochondrial fractions from Postnatal Day 5 rat brain. Each point represents the mean s SE
for 6 replicates. Control values are 202.6 ± 21.4 nmol/min/mg protein for synaptosomal fraction ATPase
and 361.3 s 23.7 nmol/min/mg protein for mitochondrial fraction ATPase.
served, producing ICjo values comparable to
those observed in adult brain homogenates
(Fig. 8).
DISCUSSION
The relative sensitivities to inhibition by
TET for ATPases in tissues from the adult rat
were as follows: liver mitochondrial ATPase
> brain Na+/K+ ATPase a brain mitochon-
drial ATPase > brain and liver nonspecific
TABLE4
ICjo Values for TET Inhibition of Mitochon-
drial aTPascs in Brain Subcellular Fractions
Based on Addition or TET to Mitochondrial and
Synaptosomal Fractions Isolated from Adult
and Postnatal Day S Rat Brains
ICjo (mM)
Mitochondrial Synaptosomal
Tissue
fraction fraction
Adult
1.5 2.7
Postnatal Day 5
1.3 1.3
ATPase (Tables 1 and 2). The IC$o value for
brain NaVK.+ ATPase was comparable with
literature values for the same activity in rab-
bit brain microsomal fractions, approxi-
mately 2 X IQ"4 M (Wassenaar and {Croon.
f
so
Flo. 8. Effect of myelin content on TET inhibition. M.
isolated brain mitochondria; H. adult brain homogenate.
Increasing amounts of crude myelin wert added to a con-
stant amount of mitochondria and inhibition was as-
smmI at 10"' m TET. Numerical values represent the
ratio of mitochondrial protein to myelin protein in each
experiment Values represent the mean ± SE for 6 to 12
replicates.
[T1T1-10-* M
M 1:S 1:10 V.I0 H
Mitochondria: Mytllri Ratio
-------
ALKYLTIN INHIBITION OF ATPases
403
1973), whereas the ICjo value for brain mito-
chondrial ATPase was much higher than that
reported for isolated rabbit brain mitochon-
dria, on the order of 10~6 m (Wassenaar and
Kroon, 1973) or for the same activity in liver
homogenates. One factor which may contrib-
ute to differences in sensitivity between AT-
Pases in liver and brain is the high content of
myelin and other hydrophobic components
in the adult brain. The combination of TET
binding sites in myelin (Lock and Aldridge,
1975) and the partitioning of TET into brain
lipids may prevent TET from binding to and
inhibiting ATPases.
Peak tin concentrations in adult rat brain
24 hr following 6.0 mg/kg TET or TMT are
much lower than the IC50 values of any of the
ATPases studied (Cook et al., 1984a,b).
These dosages, however, were sufficient in
both cases to produce neurotoxic effects
(Reiter et al., 1979; Dyer et al., 1982). This
indicates that unless local concentration is
raised by active partitioning or other mecha-
nism, ATPase inhibition is not a factor in ei-
ther TET or TMT intoxication in adult rats.
These data support suggestions by Cremer
(1981), Lijinsky and Aldridge (1975), and
Katzman et al. (1963) that ATPase inhibition
is not a major factor in the production of my-
elin edema.
Following administration of 6.0 mg/kg
TET, tin levels are sufficiently high in the
liver that in vivo ATPase inhibition would be
expected to occur (Cook et al., 1984b). The
failure to detect inhibition, even taking into
account the sample dilution inherent in per-
formance of the assay, was unexpected based
on the in vitro homogenate data. These data
are consistent, however, with the absence of
reports of TET-induced hepatotoxicity. The
liver has many mechanisms which protect it
against injury by xenobiotics and which
might account for the failure of TET to pro-
duce ATPase inhibition. Inorganic tin is an
inducer of metallothionein (Yoshikawa and
Ohta, 1982), and TET may interact with this
or other binding proteins. However, peak
synthesis of metallothionein in liver does not
occur until 8-12 hr postexposure (Shaikh and
Smith, 1977). Also, since the technique of
atomic absorption spectroscopy permits only
measurement of total tin without providing
any information on speciation, measured tin
could be in any of several forms. TET is rap-
idly dealkylated in vitro by a cytochrome P-
450-dependent process (Wiebkin et al..
1982). The rate of production of ethane and
ethylene by isolated hepatocvtes incubated
with 100 TET is 0.43 ± 0.06 nmol/hr/106
cells (Wiebkin et al., 1982). On the basis of
9.8 ± 1.5 x 107 parenchymal cells/g wet wt
liver (Zahlten et al., 1973), the rate of metab-
olism in vitro can be extrapolated to 42 nmol/
hr/g liver wet wt. If metabolism occurs in vivo
at a similar rate, then the 76 nmol tin/g wet
wt found in liver 24 hr after a 6.0 mg/kg dose
of TET (Cook et al.. 1984b) could be dealky-
lated in a few hours. Since monoethyltin is
not an effective inhibitor of ATPases, rapid
deaikylation would have a protective effect in
the liver.
There are major differences between the
neurotoxic effects of TET in the neonate and
in the adult; adult exposure leads to reversible
myelinopathy (Blaker et al., 1981) and neo-
natal exposure leads to neuronopathy and
cell death (Veronesi and Bondy, 1986). One
purpose of this study was to investigate a po-
tential biochemical basis for those differences
in terms of differential sensitivities of neural
ATPases. This study has demonstrated a
higher sensitivity of mitochondrial ATPase to
TET in neonatal as compared to adult brain
homogenates (IC50 ¦ 2.5 nM vs 260 nM).
while brain tin levels are comparable (Cook
et al., 1984a). This indicates that, in contrast
to the adult, TET levels in neonatal brain are
sufficient to produce significant in vivo AT-
Pase inhibition. Interference with neuronal
energy metabolism may be particularly sig-
nificant during critical periods in neural de-
velopment such as the early postnatal
"growth spurt" (Dobbing, 1968) and could
contribute to the neuronopathy which fol-
lows Postnatal Day 5 exposure to TET. It is
interesting, though, that these dramatic
-------
404
STINE, REITER, AND LEMASTERS
differences in TET sensitivity between adult
and neonate are not seen in the case of the
Na+/K* ATPase. In either case, though, brain
tin levels are insufficient to produce in vivo
NaVBC ATPase inhibition.
This study has also shown that the ob-
served difference in sensitivities between mi-
tochondrial ATPases in adult and neonatal
brain homogenates is not due to any differ-
ence in the sensitivities of adult and neonatal
brain mitochondria (Fig. 4). Although the
mitochondrial ATPase of the adult synapto-
somal fraction was slightly less sensitive than
that of the mitochondrial fraction, this small
difference cannot account for the much lower
total sensitivity of the brain homogenate.
Therefore, the difference in apparent sensitiv-
ity between adult and neonatal brain homog-
enate ATPases must be due to the composi-
tion of the homogenates. Further evidence
that this difference in composition is related^
to development is the decreasing sensitivity
to TET of mitochondrial ATPase from brain
homogenates in the 10-day-old pup (Fig. 3).
One major difference betweea the adult
brain and the 5-day-old brain is the smaller
amount of myelin in the neonate. TET binds
to a high-affinity site on myelin (Lock and Al-
dridge, 1975), and binding of TET to this site,
as well as nonspecific partitioning of lipo-
philic TET into myelin lipids, may be respon-
sible for the decreased sensitivities of adult
brain homogenate mitochondrial ATPase to
TET. The association of increased myelin
content with the decreased sensitivity of mi-
tochondrial ATPase is supported by the ob-
servation that the experimental addition of
myelin to isolated brain mitochondria dupli-
cated the effect of reduced ATPase sensitiv-
ity. Although, myelination does not begin un-
til 7 to 10 days postoatally (Jacobson, 1963),
total lipid does increase between Postnatal
Days 5 and 10 (Kishimoto et ai„ 196S),
which may explain the decrease in sensitivity
of mitochondrial ATPase in the 10-day-
old pup.
In conclusion, higher concentrations of
TET are necessary to inhibit mitochondrial
ATPase in adult brain homogenates than in
adult liver homogenates, neonatal brain and
liver homogenates, or isolated brain mito-
chondria. Tin concentrations in adult brain
following a neurotoxic dosage of TET are in-
sufficient to inhibit any of the brain ATPases
measured. Thus, ATPase inhibition does not
appear to be a factor in the neurotoxicity of
TET in the adult. Instead. TET may bind to
myelin, producing the observed reversible
myelinopathy. Tin concentrations in adult
liver and neonatal brain, however, should be
sufficient for inhibition, although lack of ap-
parent inhibition of liver mitochondrial AT-
Pase in TET-treated adults indicates that
TET in liver may be bound, metabolized, or
otherwise rendered incapable of ATPase in-
hibition. In the neonate, TET may bind to
and inhibit mitochondrial ATPase, interfer-
ing with energy-dependent processes. Thus,
the binding of TET to myelin in the adult
brain provides a framework for explaining
age-related differences in TET neurotoxicity
in the adult and neonate in terms of differ-
ences in mechanism of action.
ACKNOWLEDGEMENTS
This work was supported in pan by NIH AM-3"034,
N1H HL-35490, and EPA CR809644-02. Portions of this
work were presented at the Third International Congress
on Toxicology (Jacobs etal.. 198 3), Federation of Ameri-
can Societies for Experimental Biology (Stine et at..
1983a), and Society of Toxicology (Stine et ai.. 1985b).
J.J.L. is an Established Investigator of the American
Heart Association.
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SeuroToxicology 7(1): 69-80 (1986)
Copyright © by Intox Press, Inc.
Triethyltin-lnduced Neuronal Damage in Neonatally Exposed Rats
BELLINA VERONESr AND STEPHEN BONDY2
'U.S. Environmental Protection Agency, Health Effects Research Laboratories,
MD-74B, Research Triangle Park, NC 27711,'National Institute of Environmen-
tal Health and Science, Laboratory of Behavioral and Neurological Toxicology,
Research Triangle Park, NC 27709
ABSTRACT: Neuropathologies and biochemical affects of neona-
tal exposure to the alkyl metal triethyltin were examined in juvenile
male Long Evans rats. Rats were injected intraperitoneally on post-
natal day 5 with 6 mg/kg of triethyltin bromide and sampled on day
20. The brains of tin-treated animals weighed significantly less than
either saline or starved controls and exhibited a marked caviation
of Vie ventrolateral surfaces. Histologically, neuronal necrosis was
noted in the entorhinal and transitional cortex, an observation con-
firmed by immunocytochemical staining of astrocytes. Hippo-
campal involvement was further evidenced by a protrusion of the
molecular layer of the dentate gyrus, and an abnormal histo-
chemical staining pattern of acetylcholinesterase in this layer. Sec-
tions stained by the Timm's method for the deposition of
heavy metals showed a marked reduction in the staining of
the hippocampal CA4,3,2 sectors and an absence of stained
laminae in the outer molecular layer of the dentate gyrus. Recep-
tor binding assays indicated a selective depression of the ben-
zodiazepine receptor in the hippocampus of tin-treated pups com-
pared to starved controls. Taken in concert, these data indicate
that neonate/ exposure to triethyltin produces severe neuronal
damage in the posterior cortex and a derangement of hippo-
campal afferent circuitry.
Key Words: Triethyltin, Neuronal Neurotoxicity, Postn'atal Toxicity
INTRODUCTION
Triethyltin (TET) is a classic myelino-
toxicant which produces severe central
nervous system (CNS) edema in the adult
after acute exposure (Magee et al., 1957;
Aleu et al., 1963; Watanabe, 1981). Bio-
chemical and morphological studies indi-
cate that the myelin sheath is the primary
target tissue (Locke and Aldridge, 1975;
Jacobs etal., 1977). Although the underly-
ing biochemical lesion is presently un-
resolved, several possibilities have been
proposed involving inhibition of Na*/K*
ATPase, (Torak 1965; Jacobs et al., 1983),
impairment of energy related metabolism,
specifically glucose oxidation (Cremer
1970; Locke, 1976), and uncoupled
mitochondrial oxidative phosphorylation
(Aldridge, 1958; Stockdale, et al., 1970). A
variety of pathogenic changes results from
Please send requests for reprints to Or. BelHna Verenesl.
'Present address: Stephen Bondy, University of California of Irvine, Department of Community and Environmental
Medicine, Irvine, CA 92717
-------
70
VERONESI AND BONDY
acute or chronic neonatal exposure to the
aikyl metal including hemorrhagia, cellular
necrosis and myelin edema (Suzuki, 1971;
Watanabe, 1977). In addition, disruption of
myelinogenesis (Blaker et al., O'Callaghan
etal., 1983), behavioral (Reiter^f al., 1981;
Harry and Tilson, 1981) and electrophysio-
logical (Dyer et al., 1981) alterations have
been demonstrated. In this study, using
conventional and histochemical light mi-
croscopic techniques and receptor binding
assays, we have documented TET-induced
neuronal alterations in the posterior cortex
of neonatally exposed juvenile rats.
EXPERIMENTAL PROCEDURES
Animals
Randomly selected Long-Evans, male
- pups (n=60) (Charles Rivers, Portage, MI.)
were injected intraperitoneally on post-
natal day (PND) 5 with 6 mg/kg TET-
bromide (Alfa Chemicals, Danvers, MA) or
phosphate-buffered saline vehicle in a vol-
ume of 10(j.l/gm. Starved controls were
also included in our study to provide body
weight-corrected samples. For this, saline-
treated pups were separated from their
mother for 8 hrs and then returned to over-
sized litters (n=16). This procedure was
repeated for 3d which approximated the
length of time TET-treated pups showed a
reduced or absent milk band. Eight litters,
composed of TET (n=3), starved controls
(n=3) and saline controls (n=2), were used
for histological examination and brain
weight data. For receptor binding assays, 12
randomly selected TET-treated pups (n=6)
and starved controls (n=6) were used.
Pathology
Tissue Preparation: Whole litters
(n=8) were killed at PND-20. Animals
were weighed, anesthetized with Chlo-
ropent containing 1 % heparin (50 units/ml)
and perfused through the heart with 3.0%
Sorensen's-buffered glutaraldehyde. The
brains were allowed to fix in situ overnight
at 4°C before being removed from the crani-
al cavity. After gross examination, brains
were sectioned in the mid-horizontal plane
to give an overview of the entorhinal cortex
(ERC) and hippocampus.
Histology: Paraffin-embedded tis-
sues, cut on a rotary microtome at 6-8
microns, were stained with either 1% cresyl
violet acetate or hematoxylin-eosin. Six-
teen (TET=6n; starved=6n; saline=4n)
pups were randomly selected from 8 liters
and furnished tissue for each of the follow-
ing histochemical procedures: the Timm's
stain for heavy metal deposition (Danscher,
1981); a modified Friedenwald-Koelle tech-
nique for acetylcholinesterase (Lynch et al.,
1972) and the Rapid Golgi stain for metal
impregnation of the neuronal cell body and
dendrites (Kemali, 1976). In addition, 8
pups were reserved for a commercially ob-
tained (Dako Corp., Santa Barbara, CA.)
peroxidase-antiperoxidase (PAP) stain,
(Sternberger et a., 1970) which uses
antiserum specific for the glial acidic fibril-
lary protein contained in astrocytes (Eng
and Bigbee, 1978) and provides a specific
immunocytochemical stain for astroglial
cells. Areas of interest were photographed
with Kodak Ectachrome Professional film
(ASA 50) using either a Wild macroscope
or Leitz Ortholux microscope.
Biochemistry
Brain Weights and Dissection: Eigh-
teen (TET=6n, starved=6n, saline=6n)
randomly selected pups were weighed, de-
capitated and the whole brain removed and
weighed to give relative brain weights
among test groups (Table 1). For regional
brain weights, the brain was hemisected
coronally using the midpoint of the in-
fundibulum as a reference. The anterior
brain contained frontal and parietal regions
-------
T^T-INDUCED NEURONOPATHY
71
and the posterior brain included the tempo-
ral, entorhinal and occipital components of
the cortical mantel.
Receptor Binding Assay: Since
perinatal undernutrition is reputed to
produce profound effects on CNS neuro-
transmitters and their receptors (Shoemaker
and Bloom, 1977; Telang et al., 1984), we
elected to use only body weight-corrected
starved controls in the receptor binding
assays. PND-20 TET-treated (n=6) and
starved-control (n=6) pups were decapi-
tated and the hippocampus removed and
dissected according to Iverson and
Glowinski (1966) methodology. The pieces
were weighed and homogenized in a glass-
Teflon homogenizer in 19 volumes 0.32 M
sucrose. A total membrane fraction was pre-
pared by centrifugation (20,000 g, 10 min)
of a 5% (w/v) tissue homogenate in 0.32 M
sucrose. Fractions were stored frozen
(-20°C) and membranes were prepared
from them by homogenizing precipitates in
cold distilled water corresponding to 19
times the original tissue wet weight. Cen-
trifugation (20,000 g, 10 min) was followed
by a further homogenization in 40 mM tris-
HC1 pH 7.4. Protein was estimated by the
method of Lowry (Lowry et al., 1951) and
membrane suspensions were diluted with
tris buffer to a final concentration of 1-2.S
mg/ml.
Quinuclidinyl benzilate (QNB) bind-
ing was used to measure muscarinic sites
with atropine as a competitor. The binding
assay, using 10~*M dl-[benzilic-4, 4'-5H]
(Magee et al., 1957) (Ci/mmol New
England Nuclear Corp., Boston, MA) was
performed by a glass fiber disc filtration
method previously described (Bondy,
1982). Saturability, specificity, regional
distribution, and characteristics of ligands
were delineated prior to this study. Briefly,
the method consisted of incubating the
washed membranes (100-250 |Ag protein)
together with 10~* M of a tritiated ligand in
40 mM tris HC1 pH 7.4 for 15 min at 37°C.
The receptor-ligand complex was separated
from free ligand by filtration through 0.3(x
pore size glass fiber discs (Gelman Inc.,
Ann Arbor, Michigan) and washed three
times with 4 ml Tris buffer. Dried discs
were then counted in a scintillation counter
(38-43% efficiency) in order to determine
the total amount of bound ligand. Non-
specific binding was also determined in a
series of parallel incubations in the presence
of 10~4 M atropine sulphate. Those counts
which remained in the presence of this ex-
cess of the competitor were taken to repre-
sent nonspecific binding.
Benzodiazepine (BZD) receptor sites
were similarly estimated with 10" M
[methyl-3H]diazepam (73 Ci/mmol, New
England Nuclear Corp., Boston, MA.) and
the unlabeled compound at 10~* M was
used as a competitor. Incubation was for 15
min at 0°C.
Statistical Analysis: The effect of
treatment was evaluated by analysis of vari-
ance (ANOVA). This was individually
performed for each brain parameter since
assays were carried out on separate days.
Appropriate pair-wise comparisons were
performed with a Fisher's Least Significant
Difference (LSD) Test. Acceptable statisti-
cal significance was established as P <
0.05.
RESULTS
Macroscopic examination: Grossly,
the brains of both saline and starved con-
trols were indistinguishable from each other
but differed from the tin-treated pups. There
was a highly variable response within litters
to TET with some brains more severely af-
fected macroscopically than others. The
whole brains of severely affected TET-
treated pups were characterized by a
cavitated ventrolateral surface (Fig. 1). Fo-
cal areas of petechial hemorrhages were
seen along the meninges of fresh brains and
were especially prominent in the posterior
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72
VERONESI AND BONDY
FIG 1. Whole brain of severely affected TET-treated rat showing cavitated ventral-lateral surface. X3.5
FIG 2. Cresyl violet-stained paraffin section of starved control showing normal cytology of the posterior brain. X24
FIG 3. TET-treated cresyl violet-stained section of posterior cortex of TET-treated pup showing compacted laminae of
the entorhinal cortex and parasubiculum. Note protrusion of molecular layer in dentate gyrus (arrow). X24
region. When examined in the horizontal
plane, the lateral ventricles appeared mod-
erately distended.
Microscopic Examination: Histolog-
ically, saline and starved-control tissues
showed the well-defined laminated cortex
described elsewhere (Swanson, 1979). In
these animals, the ERC, located adjacent to
the rhinal fissure had a more defined cellu-
lar lamination than its neighboring para-
and presubicular regions, which were
marked by a dense fiber plexus and a com-
pressed layer of granular cells (Fig. 2). The
subiculum lacked this laminated appear-
ance and its cells appeared scattered in an
apparently unsystematic fashion.
The histopathology of TET-treated
animals, was generally confined to the pos-
terior brain, specifically the transitional
cortex (i.e., the pre- and parasubiculum),
and the hippocampal complex. In contrast
to control tissues, where the highly vascular
pia mater closely outlined the cortical con-
tour, the meninges of TET-treated animals
were retracted from the cortex and appeared
-------
INDUCED NEURONOPATHY
73
^oiien, hemorrhagic and infiltrated with
darkly stained cells. There appeared to be a
direct relationship between regions of
meningial disruption and areas of cortical
necrosis. In passing from the pial surface,
the molecular layer appeared irregular in
width, cell-infiltrated and extremely
basophilic. In general, the ERC's lamina-
tion pattern appeared severely compressed
(Fig. 3).
FIG 4. Golqi-stained material of saline-treated posterior cortex showing neurons with well-developed apical and
basilar dendritic arborization. X24
FIG 5. Golgi-stained section of TET-treated animal showing necrosis of the upper level neurons and severely
withered dendritic arborization in the surviving population of pyramidal neurons. X24
FIG 6. Horizontal section of TET-treated pup brain immunocytochemically stained for astrocytes indicated a replace-
ment of outer-layer neurons of the entortiinal and transitional cortex with astroglial cells. X40
FIG 7. Higher magnification of cortical tissue stained by the PAP technique which imparts a rose-red color to the
astrocytic processes and the cytoplasm of their cell bodies. X820
-------
74
Below the molecular layer, evidence
of severe cortical necrosis of the outer layer
(II, EI) neurons was present. The cell bod-
ies of the inner cortical layers (i.e., the in-
ternal pyramidal layer), which consisted of
medium and large pyramidal neurons, ap-
peared largely intact although occasional
aggregates of microglial were noted. Golgi-
impregnated material revealed these inner-
level neurons to have severely withered
VERONESI ANO BONOY
dendritic arborization in contrast to the well
developed apical and basilar dendrites seen
in saline and starved control tissues (Figs.
4, 5). The upper cellular layers of the ERC
and transitional cortex of TET-treated
brains were replaced largely by astrocytes
(Fig. 6). Astrocytes stained by the PAP im-
munocytochemical technique developed a
rose-red color to their fibrillary structures
and were easily identified by light micros-
FKS 8. Timm's stain of starved-control tissue showing heavy metal deposits of the entorhinal and the CA4-2 hippo-
campai complex. Note laminae formation (arrow) around the dentate gyrus. X24
FKJ 9. Timm's stain of severely affected TET-treated animal showing restricted staining in the hippocampal prop
and complete loss of laminae staining. X24
FIG 10. Starved-controt section of posterior brain stained for acetylcholinesterase showing normal pattern ol precipi
tate. X24
FKJ 11. TET-treated section showing intense band (arrow) of acetylcholinesterase staining on the outer rim o
molecular layer of the dentate gyrus. X24
-------
^-INDUCED NEURONOPATHY
75
copy (Fte- 7)-
In both saline and starved control
animals, sections stained by Timm's meth-
od showed a heavy precipitation in the
ERC, dentate gyrus, and the hippocampus
proper extending from CA4,3, and 2. Dis-
tinct laminae, corresponding to the terminal
fields of various afferent systems (Zimmer,
1973), were especially prominant in the mo-
lecular layer of control tissues (Fig. 8). Sec-
tions from severely affected TET-treated
pups, sampled at the same horizontal level
as controls and processed identically for the
Timm's stain, showed a marked reduction
in the staining of the CA4,3, and 2 sectors,
with these regions achieving only a fraction
of their normal width. A conspicuous ab-
sence of those laminae which identified the
perforant path zones in the outer parts of the
dentate molecular layer of control tissues
was also evident (Fig 9).
Control sections of the posterior cor-
tex, stained for AChE, showed precipitate
localized in discrete layers of the hippo-
campus and dentate gyrus (Fig. 10). In sec-
tions of TET-treated brain, a similar stain-
ing pattern was noted and in addition, there
occurred an intense band of AChE precipi-
tation in the outer rim of the molecular layer
of the dentate gyrus, an area which gave
only a slight reaction in control material
(Fig. 11).
The major myelinated tracts of the pos-
terior cortex (e.g., external, internal cap-
sule, fimbria) were examined in the hori-
zontal plane. Delayed myelination was
suggested by an ambundance of oligoden-
droglia with round, pale nuclei as opposed
to the mature oligodendrocytes with small,
dark, disc-like nuclei arranged in rows
TABU 2. Receptor Binding Assays.
TABLE 1. Brain Weights.
BODY AND BRAIN WEIGHTS (gm)
Group
Whole Body Wt. Brain Wt.
Saline
46.7 + 1.9 1.42 + 0.03
Starved
•34.0 + 4.2 1.38 + 0.02
TET
'31.9 + 0.9 '1.10 + 0.04
BRAIN REGIONAL WEIGHTS (mg)
Anterior Posterior
Cortex Cortex Hippocampus
Starved
283 + 37 410 + 15 99 + 4
TET
•199 + 20 '242 + 9 '74 + 9
Body and brain weight data derived from 18 pups (n=6/group).
Brain regional weights are expressed as the mean of n=6+SE.
(*) indicates TET value is statistically different from correspond-
ing starved or saline value (p < 0.05), Fishers least significance
test for body and whole brain weights, students' 2-taiied t-test
for regional weights.
within the myelinated tracts. The effects of
neonatal exposure to TET on myelin will be
discussed in a companion paper.
Brain weights: Table 1 indicates that
although the body weights of TET-treated
pups did not differ significantly from
starved controls, their brains (including ol-
factory bulbs) weighed approximately 80%
of starved and 77% of saline controls. In ad-
dition, the regional brain weights of TET-
treated pups were significantly lower than
the starved controls, the magnitude of this
loss being greatest in the posterior cortex.
Receptor Binding Assay: The status
of cholinergic circuity was examined in the
hippocampus using [3H]-quinuclidinyl
benzilate (QNB) as a muscarinic choliner-
gic receptor ligand. QNB binding was de-
pressed on a per-region basis in TET-treated
QNB Diazepam
Group /5 mg tissue/region P moiee bound 5/mg tiseue/region
Starved 0.284+ .007 5.64+ .38 0.177+ .013 4.08+.47
TET 0.264 + .011 *4.05 + .28 *0.143 + .005 *240+.12
Values are means from fln animals+ S£. (>p <0.06 that TET values dWer from correspond-
ing starved vaiue. (p < 0.06, Students, t-teet)
-------
78
VERONESI ANO BONOY
rats but was identical to starved control
values on a tissue-weight basis. Hippo-
campal benzodiazepine receptor sites were
also assayed using [3H]-diazepam. Binding
capacity was reduced both on a per tissue
basis and on a per unit wet-weight basis
(Table 2).
DISCUSSION
The present study provides evidence
that neonatal exposure to an acute dose of
TET results in damage to neuronal popula-
tions of the ERC and transitional cortex and
a derangement of hippocampal circuitry.
This is in line with the development status
of the entorhinal-hippocampal circuitry of
the altricial rodent at PND-5, a time when
the processes of hippocampal neurogenesis
(Altman and Bayer, 1970) and more specifi-
cally synaptogenesis (Gall and Lynch,
1980; Steward and Scoville, 1976) are
largely unfinished, rendering these events
especially vulnerable to neurotoxic insult.
In normai hippocampal development, major
afferents arrive in the dentate gyrus during
the first week of life and quickly restrict to
their appropriate levels on the granule cell
dendrites. These fibers, originating from
the ipsilateral ERC cortex and the pyrami-
dal cells in both the ipsi- and contralateral
Amnion's horn, terminate in the molecular
layer on the granule cells' dendrites in a
strictly ordered fashion, with the entorhinal
afferents synapsing on the distal two-thirds
of the dendrites (Hjorth-Simonsen and
Jeune, 1972) and the commissural (con-
tralateral) and associational (ipsilateral)
fibers terminating on the proximal one-third
(Zimmer, 1971; Gottlieb and Cowan, 1972).
A third afferent fiber system, the cholin-
ergic septohippocampal tract, terminates in
a stratum between the granule cell bodies
and the zone of the commissural fiber
synapses (Matthews et al., 1974; Raisman
et al., 1965). Synaptogenesis in this region
is most active between PND 4-11, a time pe-
riod when the number of synapses doubles
every day (Crain et al., 1973). In view of
tin's exposure time and its 7.3d half-life
(Cook et al., 1984), synaptogenesis might
be expected to be severely affected and may
actually represent the crucial target-event in
the neonatal model of TET-encephalopathy.
Disruption of hippocampal synap-
togenesis in the immature rat has been
achieved experimentally by surgical abla-
tion of the ERC, a procedure which results
in vacant synaptic fields in the molecular
layer of the dentate gyrus. These sites are
avidly filled by commissural, septohippo-
campal and associational fibers which
travel beyond their usual proximal place-
ment on the dendritic tree and extend to-
ward the distal surface of the molecular lay-
er to occupy the entire height of the granule
cell's dendritic tree (Lynch etal., 1973; Gall
and Lynch, 1980; Zimmer, 1973). Pertinent
to our data is the histochemical demonstra-
tion of AChE-containing septohippocampal
terminals in the outer rim of the molecular
layer, a location normally reserved for the
entorhinal afferents. This staining pattern
has been produced by others in surgically
de-entorhinalized rat neonates (Lynch ^
al., 1973; Cotman et al., 1973) and reflects
the aberrant hippocampal circuitry pr°"
duced by the excessive axonal sprouting
the cholinergic fibers. Additional evidence
of perforant path disruption comes from the
Timm's stain for heavy metal deposition
which not only delimits the granule cells
mossy fibers but acts as a potential marker
for synaptic fields of the laminated afferent
zones of the hippocampus and dentate
gyrus. The differential staining pattern seen
in TET-treated pups compared to controls is
reminiscent of that seen in surgically d®"
entorhinalized neonates (Zimmer, 19'3|
and indicates an absence of perfc^1.
synapses on the zones normally occupied by
terminals from the lateral and medial pef'
forant paths of the dentate gyrus. ,fl
Tin's persistance in the juvenile br&
(Cook et al., 1984) impacts on other crittf
events namely hippocampal neurogene*
(Altman and Bayer, 1970) and naye
-------
TET-JNOUCED neuronopathy
oogenesis (Jacobsons, 1963). Insult to these
processes was evidenced by the pyknotic
granule ceils noted two weeks post-expo-
sure and the delayed differentiation of the
oligodendroglial populations. In view of
these data, the overall definition of TET
neonatal-neurotoxicity should be expanded
to include not only the effects of synaptic
plasticity and reorganization of the hippo-
campal circuitry which were suggested by
the histochemical and biochemical data, but
also the consequences of neuronal and
myelin loss.
In view of the critical interdependence
that exists between pre- and post-synaptic
elements in their development and mainte-
nance (Hamori, 1973), the diminished or
absent entorhinal input could expectedly re-
sult in an aberrant dendritic arborization of
the hippocampal granule and pyramidal
neurons. Evidence of dendritic involvement
was indicated by the benodiazepine recep-
tor binding data of Table 2. The benzodiaze-
pine receptor has been reported to be post-
synapticaily localized to the somata and
dendritic tree of granule cells (Valdes et al.,
1982). In our study, benzodiazepine bind-
ing was severely and selectively depressed
in the hippocampus of TET-treated rats, da-
ta which suggests an actual loss of the gran-
ule cell population or a disorganization of
its dendritic arborization. The muscarinic
receptor, which in the hippocampus is
largely post-synaptic (Yamamura and
Snyder, 1974; Ben-Barak and Dudai,
1979), appeared less affected by TET. Our
data report that the binding of [3HJ-QNB in
the hippocampus was depressed on a per
region basis, but not on a tissue weight ba-
sis. This indicated that QNB receptor
proteins were not deficient relative to the to-
tal protein in this region, an apparent nor-
malcy that may reflect on a down-regulation
in response to the increased cholinergic in-
put to the dentate gyms.
In summary, we have demonstrated
that neonatal exposure to TET severely
damages the ERC and transitional cortical
neurons and disrupts synaptogenesis in the
77
afferent layer of the dentate gyrus. Because
of the developmental status of this model, it
can be argued that this damage is a compos-
ite of both the direct neurotoxic effects of
the alkyl tin and the consequences of inter-
rupted or aborted developmental events. In
either case, the described changes represent
permanent alterations in the neuronal cir-
cuitry of the posterior brain and may under-
lie the reported persistent abnormalities
associated with this model.
ACKNOWLEDGEMENTS
The author thanks Dale Newland and
Al Inman for their excellent technical assist-
ance and Ava Hinton for her preparation of
this manuscript.
This manuscript has been reviewed by
the Health Effects Research Laboratory,
U.S.E.P.A., and approved for publication.
Mention of trade names and commercial
products does not constitute endorsement of
recommendation for use.
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NEUROTOXICQLOGY MS 1324
Submitted: March IS. I9HS
Accepted: August 20. 1985
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"stf
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f)tMo**havu>rttl Toxicology and Teratology. Vol. 4, pp. 177-183, 1982. Printed in ltw U.S.A.
Trimethyltin, a Selective
Eimbic System Neurotoxicant,
Impairs Radial-Arm Maze Performance1
THOMAS J. WALSH,8 DIANE B. MILLER AND ROBERT S. DYER
Neurotoxicology Division (MD-74B), Health Effects Research Laboratory
U. 5. Environmental Protection Agency. Research Triangle Park, NC 27711
WALSH. T. J.. D. B. MILLER AND R. S. DYER. Trimethyltin. a selective limbic system neurotoxic am impairs
radial-arm maze performance. NEUROBEHAV. TOXICOL. TERATOL. M2) 177-183, 1982.—Rats were trained for
fifteen sessions in an automated eight aim radial maze prior to treatment with 6 mgfcg trimethyltin chloride. This compound
is a neurotoxicant which primarily damages the limbic system, in particular pyramidal cells in the CA3 region of the
hippocampus. Following treatment the animals exhibited a marked and persistent impairment of maze performance charac
terized by decreased selection accuracy and an altered spatial pattern of responding within the maze. These results offered
additional evidence that CA3 pyramidal neurons or their connections play an important, if not essential, role in radial-arm
maze performance. It was suggested that trimethyltin might be a useful tool for elucidating the neural substrates of K>ih
radial maze performance and learning and memory processes.
Trimethyltin Radial-arm maze Organotins Memory Spatial memory Neurotoxicology
Limbic system Hippocampus
TRIMETHYLTIN -< .. -en-
sitive indicator of hippocampal dysfunction and i> x^cd
to be particularly useful in assessing deficits in >hon k-m .>r
"working" memory (for review see [26)).
METHOD
Subjects
Eighteen male Long-Evans hooded rats obtained trum
Charles River Breeders (Wilmington. MA) were u<«d n :n«
present study. All rats were individually housed ¦" c'^ik:
cages with wood chip bedding in a temperature and humidity
controlled animal colony under a 12-hr 12-hr !t*m j.irk *<¦
cle. Laboratory chow and water were freely a* ui.irn- „ntil
the start of behavioral testing.
Apparatus
The apparatus used was an automated :
cm dia./7.5 cm side) from which radiated ci*"' vguaiiv
spaced alleys (26 cm long x 10.5 cm wide •
(Fig. I). At the end of each alley was a trough •>> >
Gerbrands pellet dispenser which delivered 4< ' -^i pel-
lets (Noyes Co.. Lancaster. NH). The base »t 'f* if iw>
was constructed of black Plexiglas and the u>r •'
'This manuscript has been reviewed by the Health Effects Research Laboratory. U- S. Environmental Protection Ageno -¦
publication. Mention of trade names or commercial products does not constitute endorsement or recommendation tor n«c
To whom requests for reprints should be addressed.
177
-------
178
WALSH. MILLER AND DYER
FIG. I The automated eight arm radial maze used in this experiment. See text for further details and dimensions
clear Plexiglas. Entry into the arms was controlled by motor
driven guillotine doors. The lime of each feeder entry and the
rate of locomotor activity within the maze were detected by
LED/photodiode circuits in the following locations: 8 in the
central arena. I at the entry to each alley and I in each pellet
trough.
Operation of the doors and pellet dispensers and the col-
lection of experimental data was controlled by a pro-
grammed North Star Horizon microprocessor.
Procedure
Baseline acquisition. Prior to acquisition animals were
gradually reduced to 859c of their free-feeding body weight
by limiting their daily ration of food. During each test session
a rat was placed into the central arena of the maze with the
doors closed and access to the arms and feeders blocked.
Within ten seconds thereafter the program was initiated, the
entry arms automatically opened and a food pellet was deliv-
ered to each of the feeders. A trial was ended following entry
into all eight feeders or after ten minutes, whichever came
first. The following dependent measures were automatically
recorded: (1) number of correct (i.e.. non-repeated) feeder
selections within the first eight choices, (2) total number of
feeder selections necessary to obtain all eight pellets. (3) a
measure of temporal efficiency or rate of correct selections,
and 14) averaged locomotor activity (responses minute)
within the session. Immediately following each test session
the rats were given a ration of food to maintain 'heir
weight. The rats were trained for fifteen du\s in ;he maze
Testing was performed approximately the same dme cen
day (Monday-Friday). Following these acquisition *e»Mons
the animals were divided into two groups in = « per group",
which were equated for selection accurac> and loci of lo-
comotor activity in the maze during the test session* < in the
following Monday animals were intragastric! K mtur.ited
with either 0 (physiological saline) or 6 mg. kg FMT i Jo*
age calculated as the base). TMT chloride *as obtained from
K&K Laboratories (Plainview, NY) and admini-rccd n a
volume of I ml/kg body weight. Two weeks to 11. •*«.¦ J doling
before the resumption of behavioral testing mi 'h.i: rats
could recover from the acute phase of TMT-induce J \uit\
[8|. During this tune all rats had ad lib access .>u .md
water.
Reacquisition I. Prior to the beginning of ihc • caw
quisition phase (days 15-35 post-dostng) animal n"e main
reduced to 85% of their newly acquired free feed.r* weight
During Reacquisition I the rats were again trained • < . :otal
of fifteen sessions in the maze using the same d.u.. ^ol
as dunng acquisition.
Reacquisition 2. To determine whether detui - ~ i.i^e
performance following TMT were transient and •.•.crMrle
with either extended training or time the rats *e-c - nned
in the maze for another fifteen sessions fourtoe-
ing the completion of Reacquisition I idav ;• ro-t-
dosing).
-------
tmt AND radial arm maze performance
179
HO 11 It t I
HUTMATNINT UStKM
• i« tn» ii~
«ucau4itiou i
• 10 ini
M ACQUISITION HQ J
FIG. 2. Mean number (sS.E.M.) of non-repeated feeder entries in
the first eight choices for the three phases of this experiment. The
data for this figure and Figs. 3. 4 and 5 were summarized for each
block of five sessions (i.e.. 1-5. 6-10. 11—15).
o mr
Ti 4*1 miITi Til nil II nr^
•MTftlATIHNTIAIIllNI MACQUtflTIO* 1 MACQUISITION i|Q 2
FIG. 3. Mean number (±S.E.M.) of total feeder selections
sary to obtain ail eight pellets within a session.
Data Analysis
Data from the three phases of behavioraJ testing I Baseline
Acquisition. Reacquisition 1 and Reacquisition 2) were
treated as independent data sets. Each data set was analyzed
for overall treatment, time (weeks) and treatment < week
interaction effects by multivariate analysis of variance
(MANOVA) procedures using Wilk's Criterion. Significant
Bonferroni-corrected MANOVAs (at=0.05/3=0.016) were
followed by repeated measures ANOVAs using weeks as the
repeated factor. Signftcant ANOVAs were followed by ap-
propriate group by group comparisons using Duncan's Mul-
tiple Range test. The experiment-wise error rate was main-
tained at 0.05.
Histology
Thirty-five days following the completion of behavioral
testing (i.e.. 105 days after dosing) all rats were perfused
through the heart with saline followed by neutral buffered
Formalin. Frozen sagittal sections (40 **), obtained from one
hemisphere, were stained with cresyl violet for histological
evaluation. In this report the only measure of hippocampal
morphology to be presented will be the length of the pyrami-
dal cell line measured from the CAl-Subiculum border to the
point where the organized line of pyramidal cells was no
longer readily discriminate (i.e., CA3c in normal tissue).
This measure was chosen since it has been shown to be the
most dose-related indicator of hippocampal pathology fol-
lowing TMT [71. A more complete description of the hip-
pocampal damage in the TMT-treated rats used in this exper-
iment can be found in Dyer ei al. [7].
RESULTS
Baseline Acquisition
During the pretreatment baseline period there were no
significant between group differences, F(4.l 1)«0.26.
p>0.10, or group x week interactions, F(8.50)»0.78,
p >0.10; however, there was a significant week effect,
F(8.50)-9.40, p<0.0001. indicating that maze performance
did in fact change over time. Univariate ANOVAs revealed a
O TMT
ii ft ii ti i i I in u it i i »o
*ITM*mMTMUMM MACQUtSlTIMM * MACOM'O**® J
FIG. 4. Mean (sS.E.M.) index of temporal efficient of maze per
formance. The index was calculated as the rate of correct elections
per minute (number of correct choices/time in maze immn
signficant increase in the number of correct arms in the first
eight choices, temporal efficiency and locomotor .ictiv itv i all
F's(2.28)> 15.62, all p's<0.Q00l) but not number of total
selections. F(2.28H1.11, p>0.10, over week*. These data
demonstrate that prior to treatment both groups of rats were
comparable on all measures of maze performance und loco-
motor activity within the maze. Furthermore, the Mgnificunt
main effect of weeks indicates, as can be seen in Figs ;. 3.4
and 5, that there was a progressive increase in selection accu-
racy and temporal efficiency coincident with an increased
level of intramaze motor activity. By the end of the third
week of testing both groups made approximately seven cor-
rect responses within the first eight arm choices unu obtained
-------
180
WALSH, MILLER AND DYER
" o r«T
m
11#
FIG. 5. Mean number (rS E.M.) of photocell interruptions per
minute in the maze.
all pellets within ten to eleven entries. It was also evident
that response patterns were similar between groups. Both
groups of animals exhibited a marked tendency to enter an
arm immediately adjacent to the one just exited (Fig. 6). The
lack of a signficant group x week interaction suggests that
the rate of improvement in maze performance and the in-
crease in activity were similar or parallel in both groups be-
fore dosing. The two groups acquired efficient maze per-
formance at a similar rate and with the use of comparable
response strategies.
Reacquisition I
Following dosing the TMT-treated rats exhibited a tran-
sient period of weight toss and hyperexcitability. At the re-
sumption of behavioral testing however, the treated animals
were comparable in ail overt respects to the controls.
In contrast to the baseline period, the maze performance
of the two groups differed markedly during Reacquisition 1.
The MANOVAs demonstrated signficant effects of both treat-
ment, F<4.in-13.32. p<0.0003, and weeks, F<8.50)=5.31.
P <0.0001. but no treatment x week interaction,
R8.50)* 1.45. p>0.10. Therefore, although the groups were
different on certain measures of performance and/or locomo-
tor activity, their rate of improvement was similar over time.
ANOVAs revealed a significant treatment effect on total
selections, number of correct arms in first eight choices and
temporal efficiency (all F's(l.l4)>7.lO. all ps<0.0l). An
important finding of the present experiment was that no sig-
nificant between group differences were evident on the ac-
tivity measure. F(l.l4)-0.44. p>0.10. Therefore, impaired
maze performance was not secondary to TMT-induced ac-
tivity changes.
The spatial pattern of responses was different for the two
groups. The rats treated with TMT did not prefer the im-
mediately adjacent arm but shifted their modal preference to
the second arm away from the one just exited (Fig. 6).
ANOVAs demonstrated a significant effect of weeks on
SAIINC
O (ASHINt
~ RCACQUISITI0NN0 1
A Rf ACQUISITION NO. 2
«*
m
S
3
u
n»T
OlSTANCi TO Nf XT CHOICC iunMr ol <>m,
FIG. 6. The relative frequency of selecting feeder', di*ianctf>
(clockwise or counter-clockwise) from the one jum Arm u>
number of correct arms in the first eight choice* temporal
efficiency and locomotor activity (all F'mM*' 54. all
/>'s<0.0007). These data again demonstrate that here were
improvements in maze performance over time which were
coincident with increased levels of activity The r.iie* ot im-
provement were similar across the two grourv ,i« indicated
by the lack of a significant treatment • week interaction.
The TMT-treated animals, however, required mure flec-
tions to obtain the pellets, were less accurate :n heir elec-
tion of baited food arms and exhibited a le>* ctVioent tem-
poral pattern of performance in the maze (he-e Jau
demonstrate that while TMT markedly imp .i - -.ize
formance it did/not prevent improvement w,;r • .-»er train-
ing.
-------
Tjff AND radial arm maze performance
181
TABLE 1
correlation between length of pyramidal cell line
AND INDICES OF MAZE PERFORMANCE
Performance
R*
Measure
R'~
P
Condition
Total Selections
-.48
0.0009
.23
Reacquisition 1
-.32
0.0306
10
Reacquisition 2
Correct in First
.46
0.0015
.21
Reacquisition 1
Eight
.46
0.0013
.21
Reacquisition 2
Temporal
-.36
0.0150
.13
Reacquisition 1
Efficiency
-.16
0.29
.025
Reacquisition 2
Activity
.05
0.73
.0025
Reacquisition 1
-.33
0.028
.108
Reacquisition 2
Reacquisition 2
During Reacquisition 2. the rats treated with TMT re-
mained impaired, as during Reacquisition 1, but developed in
addition a pronounced hyperactivity.
An overall MANOVA demonstrated a significant effect of
treatment, F(4,10)»6.22.p<0.0088; however, neither the ef-
fects of week, F(8.46)» 1.98. p>0.03, nor the treatment x
week interaction, F(8.46)= 1.67, p >0.10. were statistically
significant. The absence of a week effect indicates that
animals were already performing at an asymptotic level. Re-
sults of the ANOVAs indicated a significant treatment effect
on total selections, number of correct responses in the first
eight choices and locomotor activity (all F's(l.l3)>13.55. all
p's<0.0028).
Histology
Histological evaluation indicated extensive loss of hip-
pocampal pyramidal cells, especially those of CA3. some
loss of granule cells and shrinkage of the intrahilar space of
the fascia dentata. Table 1 shows the Pearson Product Mo-
ment Correlations between one measure of hippocampal
damage (i.e.. length of the pyramidal cell line) and perform-
ance measures in the maze. The results indicate that al-
though there were significant correlations between this
measure of damage and performance in the maze, the per-
centage of variance accounted for by the length of the pyram-
idal cell line was relatively small. For example, although the
correlation between hippocampal damage and number of
correct arm selections in the first eight choices was highly
significant (i.e., R«.46,p<0.0013), differences in the pyram-
idal cell line accounts for only about 21% of the varia-
bility in this behavioral measure.
DISCUSSION
The purpose of the present experiment was to examine
the effects of trimethyltin, a neurotoxic organometaJ. on the
performance of rats previously trained in a radial-arm maze
task. The results of this experiment demonstrated that rats
treated with TMT exhibited a marked and persistent impair-
ment of radial-arm maze performance which was evident
throughout the period of testing (i.e.. up to 70 days post-
treatment). This impairment was characterized by more re-
peated feeder entries in the first eight choices and a greater
total number of selections needed to obtain all pellets within
a given session. During the first reacquisition period the
deficits in maze performance were not secondary to changes
in locomotor activity, the ability to execute the appropriate
responses or alterations in motivation. The rats treated with
TMT performed the necessary responses and consumed the
pellets following a correct arm entry. These data could indi-
cate that the treated rats were unable to inhibit responding to
already entered arms. The treated animals "remembered"
the reference memory aspects of the task (i.e.. leave central
arena, enter arm, consume pellet in food trough, exit arm.
etc.) however, they could not differentiate between previ-
ously entered and non-entered arms during a test session.
That is, they appeared unable to perform the short-term
working memory components of the task (i.e.. remembering
which arms were chosen during a session). This hypothesis
is consistent with one advanced by Olton and colleagues
[26,27] to explain the behavioral deficits seen in maze per-
formance folldwing hippocampal lesions.
Analysis of response patterns in TMT-treated rats
suggests that these animals utilized a different behavioral
strategy than controls to solve the maze. For example, it is
commonly reported (9,291 that the response preference of
control animals is to select an arm immediately adjacent to
one they have just exited (see Fig. 6). In the present study
the controls entered the adjacent arm on approximately -UX7
of their selections. While this is a preferred strategy over
time it is not essential for effective maze performance since
the untreated rats varied their daily choice preferences with-
out altering their accuracy in the maze. Therefore, the re-
sponse pattern of control animals exhibits a certain degree of
plasticity. Animals with damage to the fimbia-fomu ; 2^1. the
septal nucleus [2], or to specific hippocampal regions, as
reported here, exhibit a different spatial pattern of responses
such that they shift their selections to more distant arms u.e..
2-4 arms away). Such a different pattern could indicate that
rats with hippocampal damage adopt a less adaptive strategy
for solving the maze. Furthermore, it might indicate that
their behavior comes under the control of different, and
perhaps, less relevant situational cues (i.e.. intra- ^ extra-
maze or intero- vs exteroceptive stimuli, etc.) than (he un-
treated rats [37]. If, for example, the rats treated with TMT
developed a response algorithm of "choose ever. second
arm" they would ultimately repeat sequences of arm entries
and perseverate their responding to no-longer baited feeders.
Since animals in the present experiment were not confined
following arm selections, a procedure which disrupts the
spatial patterning of responding without altering accuracy of
maze performance (see [29]) the underlying cognitive deficits
responsible for the impaired maze performance cannot he
defined with certainty. Regardless of the interpretation how-
ever, the results presented here clearly demonstrate that
acute TMT administration produces a long-term, and
perhaps permanent, impairment in the performance of a
radial-arm maze task.
An unexpected finding during the second re.icquiMtion
period was a pronounced and persistent hyperacid n> m the
TMT-treated group. This delayed onset of hvpenctiwtv is
unlike the reported time-course of activity changes follow ing
hippocampal lesions [19] or TMT administration!; These
activity changes could reflect the consequences oi some un-
specified neural reorganization (see (1|) follow mw hirivcam-
pal insult or the animals adopting a new behavior..i >u..i*gv
-------
182
WALSH, MILLER AND DYER
to contend with the demands of the task. It was observed
during the second reacquisition period that temporal effi-
ciency was not different between groups. Although the
treated animals werejess accurate and required more selec-
tions they were able to obtain all the pellets within a session
in about the same amount of time as the controls. The in-
creased activity of the TMT-treated animals might therefore,
be a behavioral strategy which compensated for their im-
paired cognitive abilities.
In the present study we observed a specific distribution of
hippocampal damage, localized primarily in CA3, in all rats
treated with TMT. While the length of the pyramidal ceil line
was significantly correlated with maze performance it ac-
counted for only a portion of the variability in the behavioral
measures. These findings could indicate that (1) length of the
pyramidal cell line did not accurately reflect the extent of the
hippocampal damage produced by TMT in these rats or (2)
damage to structures other than the hippocampus contrib-
utes to the performance deficits observed here. Amygdala
damage, which has been observed following TMT [5], is not
reported to disrupt radial-arm maze performance 13]. How-
ever. Mishkin and colleagues [23.24] have observed a
marked potentiation of memory impairments in monkeys ex-
posed to combined hippocampal-amygdala insult. In light of
these observations it might be useful for future studies to
address whether combined damage to these limbic regions
contributes to the behavioral deficits observed here.
The results of the present study are consistent with an
extensive literature demonstrating that hippocampal damage
produces long-term impairments in a variety of maze situa-
tions. Experimental destruction of the hippocampus disrupts
the acquisition and performance of rats in a Hebb-Williams
maze [12.25], Lashley III [14] and other complex spatial
mazes [18] in addition to the radial-arm maze. Furthermore,
humans with bilateral temporal lobe damage, involving the
hippocampus are deficient in performing a visual maze task
[221. The results of these studies support the hypothesis that
the hippocampus plays an important role in learning and
memory processes. Moreover, there is now evidence that
the hippocampal cell fields are differentially involved in the
mediation of certain behaviors (see [15.20]). For example.
Jarrard [16.17] reported that selective damage to either the
fimbia or dorsal fornix, which would disconnect the dorsal
hippocampus (CA3-CA4) from subcortical regions, selec-
tively impaired post-operative performance in the radial-arm
maze. Similar deficits were not observed following lesions to
the CA1 cell field or the alveus. A report by Handelman and
Olton [13] demonstrated that selective destruction of CA3
pyramidal neurons with intrahippocampally applied kamic
acid produced impairments in radial-arm maze performance.
Taken together, these studies indicate that pyramidal cells in
CA3 or their connections play an important role in the ac-
quisition and retention of radial-arm maze performance. A
problem associated with attempts to isolate the neural sub-
strates of behavior by using conventional lesion techniques,
however, is that damage cannot be restricted to cir-
cumscribed regions and therefore incidental damage to fibers
of passage and surrounding areas must be considered in any
analysis of lesion-induced behavioral changes. Since system-
ically administered TMT exerts a regionally specific effect
on the hippocampus it might be a useful tool for elaborating
the differential involvement of hippocampal cell fields and
their associated fiber systems in learning and memory. It has
already been reported, for example, that TMT administra-
tion disrupts performance in both the radial-arm maze. a>
reported here, and a Hebb-Williams maze (331. and impairs
the acquisition and retention of both passive |?ft| .ind active
[21] avoidance responses. Thus, futher studies on the
biochemical and neural correlates of TMT-indueeU behav-
ioral changes are likely to provide important insight* mo the
neurobiology of learning and memory.
ACKNOWLEDGEMENT
The authors would like to thank Drs. David niton David H.K-
erman. Michael Gage and Robert MacPhail for their >n>:ruwi^
comments on this manuscript.
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36. Walsh. T. J., M. Gallagher, E. Bostock and R. S Oyer Tn-
methyltin impairs retention of a passive avoidance task
Neurobehav. Toxicol. Teratol. 4t 163-167, 1982.
37. Winocur, G. The hippocampus and cue utilization
Psychol. »: 2Sft*2M, 1980.
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Seurobehavioral Tosicohgy and Teratology. Vol. 4, pp. 163-167, t982. Printed in the U.S.A.
Trimethyltin Impairs Retention of a
Passive Avoidance Task1
THOMAS J. WALSH.* MICHELA GALLAGHERS ELIZABETH BOSTOCK+
AND ROBERT S. DYER*
*Neurotoxicology Division (MD-74B)
U. S. Environmental Protection Agency, Health Effects Research Laboratory
Research Triangle Park, NC 27711
and +Department of Psychology and the Neurobiology Program
University of North Carolina at Chapel Hill, Chapel Hill, NC 27514
WALSH. T. J.. M. GALLAGHER. E. BOSTOCK AND R. S. DYER. Trimethyltin impairs retention of a passive
avoidance task. NEUROBEHAV. TOXICOL. TERATOL. 4<2) 163-167, 1982.—Trimethyltin is a neurotoxic organometai
which produces neuronal damage in several limbic regions including the hippocampus, amygdala and the pyriform cortex.
One administration of trimethyltin <3, 6 or 7 mg/kg) twenty one days prior to passive avoidance conditioning produced an
impairment of retention when animals were tested 24 hours after training. Rats treated with trimethyltin exhibited shorter
step-through latencies and freezing durations during the retention test. It was observed that the three dosages of trimethyl-
tin were equally effective in disrupting retention performance. These retention deficits were not secondary to alterations in
footshock sensitivity. The data presented here indicate that acute trimethyltin administration disrupted learning and
memory. This compound might be a useful tool for examining the role of the limbic system in associative processes.
Trimethyltin Memory Organotins Seurotoxicology Limbic system Passive avoidance
ALKYLTINS are used for a variety of industrial and agricul-
tural purposes such as polymer stabilization and pest control
[21,22]. Their increasing use and potential health-related ef-
fects has prompted our laboratory to investigate their
neurobehavioral toxicity.
Trimethyltin (TMT) is a common intermediate and
byproduct in the synthesis of the more commonly used al-
ky itins (6,14). This compound has been implicated in several
episodes of human poisoning and has also been shown to
produce a complex spectrum of both neuropathological and
behavioral changes in laboratory animals. For example,
acute or chronic exposure to TMT in the rat produces a
relatively selective pattern of limbic system damage involv-
ing the hippocampoa, amygdala and pyriform cortex [1,2].
Furthermore, pyramidal neurons in the CA3 cell field of the
hippocampus appear to be particularly vulnerable to the
toxic effects of TMT (4], Acute administration of TMT also
produces a transient behavioral syndrome consisting of
anorexia, spontaneous seizures, self-mutilation and
hyperexcitability (3]. While these behavioral effects are typi-
cally gone within fourteen day* of dosing, recent work in our
laboratory has characterized more subtle and persistent
neurobehavioral consequences of TMT exposure (see
[17,201).
Accidental exposure to TMT is the workplace has been
'This manuscript has been reviewed by the Health Effects Reseaicl
publication. Mention of trade names or commercial products does
requests to first author.
reported to produce a neurologic syndrome consisting of
spontaneous seizures, emotional lability, anorexia and learn-
ing and memory impairments [8,16]. Since cognitive deficits
were prominent symptoms in both of these incidents we
were interested in whether TMT impaired memory proc-
esses. This hypothesis was supported by previous reports on
the adverse consequences of limbic system damage on learn-
ing and memory [11,15].
In the first experiment we examined the dose-dependent
effects of TMT on retention of a one-trial passive avoidance
response.
EXPERIMENT 1
METHOD
Subjects
Forty-two male Long-Evans hooded rats obtained from
Charles River Breeders (Wilmington, MA) were used in the
present study. All rats wen individually housed in hanging
metal cages in a temperature and humidity controlled animal
colony under a 12 hr/12 hr light-dark cycle. Laboratory chow
and tap water were freely available throughout the experi-
ment. At the start of the experiment the animals were 90-120
days of aga and weighed between 230-300 grams.
abontory, U. S. Environmental Protection Agency and approved for
t constitute endorsement or recommendation for use. Send reprint
163
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164
WALSH ET AL.
CONTROL * i***7
o*ou»
FIG. 1. Effectsof5mg/kg(n->8),6mg/kg0.10). Two-tailed
Mann-Whitney U tests performed on the experimental groups re-
vealed significantly shorter latencies for the 5 mg/kg (p<0.02). 6
mg/Vg <0.001> groups as com-
pared to the controls.
removed immediately following a step-through response oc-
curred 24 hours prior to training. During this pre-training
session and the subsequent training trial the guillotine door
remained open. Twenty-four hours later each rat was trained
in a single trial passive avoidance task. Passive avoidance
conditioning consisted of placing the rat into the illuminated
start chamber and recording the latency to step-through into
the darkened section. Immediately following step-through
(defined as the passage of the hind limbs beyond the
threshold) the door was closed and the rat given a 0.6 mA
footshock for 1 sec. The retention test was performed
hours later during which the rat was again placed into the
start box and the step-through latency recorded to an arbi-
trary maximum of 600 sec. The step-through latency served
as the dependent measure and was taken to indicate the de-
gree of retention of the task. The duration of time the animal
remained immobile following placement into the apparatus
during the retention test was also measured (i.e., freezing
duration).
RESULTS
Some of the animals treated with 6 or 7 mg/kg TMT ex-
hibited transient weight loss and hyperexcitability. By the
time of training however, they were generally comparable to
the controls.
A Kruakal-Wallis nonparametric analysis of variance
demonstrated a significant treatment effect on initial step'
through latencies during the training trial, H<4)» 12- »•
p<0.02. Post-hoc group by group comparisons with a two-
tailed Mann-Whitney U test demonstrated that the latency 0'
the 7 mg/kg group was significantly longer than the latencies
of the other groups (p<0.005). There were no other signifi-
cant between-group differences. The median initial steP*
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TMT IMPAIRS PASSIVE AVOIDANCE RETENTION
I6J
through latencies of the 0. 5. 6 and 7 mg/kg groups were 19,
16, 21 and 68 sec, respectively.
The data from the first experiment revealed that acute
administration of TMT produced impaired retention of a
single-trial passive avoidance task (see Figs. 1 and 2). A
Kruskal-Wallis nonparametric analysis of variance demon-
strated a significant treatment effect on both step-through
latencies. H(4) = 24.57, p<0.001. and freezing durations,
H<4)= 16.3. p<0.01, during the retention test. As can be seen
in the figures the retention impairment was coincident with
shorter freezing durations in the treated animals.
While the TMT-treated rats exhibited poor retention of
the task, this was not a dose-related phenomenon. A
Kruskal-Wallis analysis of variance performed on the step-
through latencies and freezing durations of the groups
treated with TMT revealed no significant between-group
differences on these measures. H<2)=»5.04, p>0.05, for step-
through latencies: H<2)=3.59. p>0.05, for freezing dura-
tions. Therefore, 5.6 or 7 mg/kg TMT produced comparable
deficits in passive avoidance retention.
In the second experiment we examined whether the re-
tention impairments were secondary to alterations in reac-
tivity to footshock.
EXPERIMENT 2
TMT has been reported to alter the responsiveness of
several sensory systems. For example, acute TMT alters the
visual-evoked potential [3] and attenuates the acoustic star-
tle response (9j. Of greater relevance to the interpretation of
the results of the first experiment is the report that TMT
compromises behavioral reactivity to noxious stimuli; We
recently reported that acute TMT administration produced
time-dependent changes in hot-plate latencies indicative of
somatosensory dysfunction [10]. Taken together, these re-
ports could indicate that TMT changes sensitivity to sensory
stimulation in general. If the TMT-treated rats were less
sensitive to the footshock during the training session, then
the resulting retention deficits might be secondary to
changes in perception of footshock and not to disrupted
memory processes. To investigate this possibility we exam-
ined the sensitivity ofTMT-treated rati to electric footshock
using a modified Evans [7] flinch-jump procedure.
METHOD
Subjects
The rats described for Experiment 1 were retested in this
experiment.
Apparatus
The apparatus was a transparent Plexiglas operant
chanber with a grid floor. The maniptulanda had been re-
moved prior to the start of the experiment. Scrambled AC
footshock was delivered to the grid floor by a Coulbourn
Instruments Solid State Shocker identical to the one used in
Experiment 1 for avoidance training.
Procedure
Approximately 1 week following the retention test, the
jump thresholds were determined for the rats used in Exper-
iment t. At the beginning of testing each rat was allowed a 60
sec shock-free period to acclimate to the apparatus. Follow*
ing this period, six series of unavoidable footshocks were
TABLE 1
MEDIAN JUMP THRESHOLDS OF RATS TREATED WITH
0. 5. 6 OR 7 mg/kg TMT CI
Jump Threshold
Interquartile
Group
n
(mA)
Range
Saline
8
0.36
0.33-0.41
5 mg/kg TMT
8
0.41
0.33-0.47
6 mg/kg TMT
II
0.4:
0.:»-0.4J
7 mg/kg TMT
5
0.48
"
•Interquartile range not computed due to the small experimental n.
presented in alternating ascending and descending inten-
sities. The shock generator was set at 0.05. 0.10. 0.20, 0.30,
0.40, 0.50, 0.60, 0.70, 0.80 or 0.90 mA. Footshock duration
was 1 sec and there was a 15 sec interstimulus interval. A 60
sec shock-free period was allowed between alternating
series. Shock presentation in the first series was always in
ascending order.
The presence or absence of a jump response, defined as
the simultaneous removal of all 4 paws from the grid floor,
was determined at each shock intensity. The threshold for
each animal was computed as the mean shock level at which
a jump response was reliably recorded. In the present exper-
iment we chose not to measure flinch thresholds since we
were primarily interested in how the treated rats reacted to
footshock intensities comparable to those used in Experi-
ment 1 (i.e., 0.6 mA for 1 sec).
RESULTS
The rats treated with TMT had jump thresholds which
were comparable to those of the controls. The median jump
thresholds for the groups treated with 0,5,6 or 7 mg/kg TMT
are presented in Table 1. A Kruskal-Wallis nonparametric
analysis of variance demonstrated that there were no signifi-
cant differences between groups on this measure,
H(3)«3.44, p>0.10. These data indicate that TMT did not
impair responsiveness to footshock, as assessed by this
method, and further suggest that the retention deficits ob-
served in Experiment 1 were not secondary to changes in
footshock sensitivity.
GENERAL DISCUSSION
The results of the first experiment demonstrated that
TMT produced an impairment of retention for a one-trial
shock-motivated passive avoidance task. The groups ad-
ministered S, 6 or 7 mg/kg TMT twenty one days prior to
training had shorter step-through latencies and freezing du-
rations than the controls during the 24 hour retention test.
While these data suggest that TMT impaired either the initial
acquisition or consolidation of the task several other alter-
natives should be considered. For example, it could be
argued that the acute toxic effects of TMT, which include
weight loss and hyperexcitability indirectly contributed to
the shorter step-through latencies during the retention test. It
was observed however, that at the time of training (i.e.. 21
days after dosing) there were no differences in body weight
or apparent difficulty of handling between groups. Further-
more, the group administered 3 mg/kg never did lose weight
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166
WALSH ETAL.
or exhibit enhanced excitability although they were impaired
in retaining the task. Therefore, it is unlikely that the reten-
tion impairments seen in Experiment 1 were secondary to
the nonspecific toxicity of TMT. It was observed during
training however, that the 7 mg/kg TMT group had longer
initial step-through latencies than any of the other groups.
This could indicate that toxicant-induced alterations in lo-
comotor activity, attention or emotionality might have con-
tributed. in some unspecified way. to the retention deficits
seen in this group. Since the other TMT-treated groups did
not exhibit longer training latencies than the controls how-
ever, this explanation cannot serve as a global interpretation
of TMT's effects on passive avoidance retention.
The second experiment demonstrated that rats treated
with TMT were behaviorally just as reactive to footshock as
the controls. There were no differences between any of the
groups in jump thresholds following presentation of a series
of unavoidable footshocks. Caution must be exercised in ex-
tending the generality of these results because the physical
characteristics of the chambers (i.e., dimensions, surface
area, grids vs metal plates, etc.) in which shock was deliv-
ered in Experiments 1 and 2 were different. While the lack of
a TMT effect on shock sensitivity might seem contrary to
previous results from our laboratory demonstrating time-
dependent increases in hot-plate latencies following TMT,
several explanations might be offered to account for the ap-
parent discrepancies. For example, the jump-flinch proce-
dure and the hot-plate test might measure behavioral re-
sponses which are organized at different levels of the
neuraxis. The jump-flinch procedure seems to assess
spinally-mediated reflexes whereas the behavioral response
to the hot-plate could be mediated by a more central integra-
tive process. Our data indirectly support this hypothesis
since we observed no differences in peripheral nerve func-
tion following TMT but did find changes in central
somatosensory processes [10]. Another alternative is that
TMT produced alterations in neuromotor function which
confounded the analysis of pain sensitivity. Tilson and Buroe
[19] reported that triethyltin-induced changes in pain sen-
sitivity were the consequence of neuromotor impairment
rather than altered behavioral reactivity to noxious stimuli.
While TMT-treated rats exhibit tremor which might con-
tribute to impaired neuromotor capacity this effect is seen
only with higher doses and is typically gone within ten to
fouiteen days. This time course might coincide with the
changes in hot-plate latencies observed four days after dos-
ing and with the apparent lack of effect on shock sensitivity
thirty five days, following dosing. In light of the apparent
t. Bouldin, T. W., N. D. Goinm, C. R. Bagnell and M. R. Krig-
man. Patbogmeais of trimethyltin neuronal toxicity: Ultraatnic-
tural and cytochtmkai observations. Am. J. Path. 104s 237-249,
1981.
2. Brawn. A. W., W. N. Aldridge, B. W. Street and R. D. Ver-
se hoy ie. The behavioral and oeuroptthologic sequelae of intox-
ication by trimethyltin compounds in the rat. Am. J. Path. 97:
3»-82, 1979.
3. Dyer, R. S., W. E. Howell and W. F. Wooderiin. Visual system
dysfunction following acute trimethyltia exposure in rats.
Neurobthav. Toxicol. Teratol. 4t 191-195, 1982.
4. Dyer, R. S., T. L. Deshields and W. P. Wonderiin.
Trimethy Kin-induced changes in gross morphology of the hip*
pocampus. Neurobthav. Toxicol. Teratol. 4j 141-147, 1982.
3. Dyer. R. S., T. J. Walsh, W. F. Wooderiin and M. Bercegeay.
The trimethyltin syndrome in rats. Neurobthav. Taxciol.
Ttratoi. 4: 127-133, 1982.
discrepancies a more complete characterization of the TMT-
induced changes in behavioral and electrophysiological cor'
relates of pain sensitivity is warranted. For the purpose of
interpreting the present experiment, however, it is clear thai
the treated animals were as reactive to the footshock as the
controls. Therefore, it is reasonable to conclude that TMT
administration altered retention of the task by disrupting
neural processes or events which participate in learning and
memory.
While histology was not performed on the animals used in
these experiments several comments on the time course and
characteristics of the neuropathology associated with similar
dosing regimens seem appropriate. Brown and coworkers l-l
observed cell loss in the hippocampus within ten days ot a
single 10 mg/kg dose of TMT. Maximal neural damage, evi-
dent at 21 days after dosing, was characterized by extensive
cell loss in the hippocampus, pyriform cortex, dentate gyrus
and amygdala. More recently. Dyer and colleagues [4| re-
ported a dose-related decrease in both the length of the
pyramidal cell line and the thickness of the dentate gyrus 30
days following a single dose of 5. 6 or 7 mg,kg or IMT-
Furthermore, administration of either of the higher doses
reduced the density of cells in the CA3 portion of the pyram-
idal cell line. While these reports have established the
preferential sensitivity of limbic forebrain regions to TMT-
induced neurotoxicity, it will be important for future studies
to correlate the neuropathology with the behavioral changes-
In conclusion, the experiments reported here demon-
strated that acutely administered TMT produces retention
impairments for a passive avoidance task. This observation
is consistent with the extensive literature on the adverse
effects of limbic system damage on avoidance learning. F°r
example. Isaacson [11] reported that lesions of either the
amygdala or the hippocampus were capable of affecting
aversively-motivated learning in a variety of test situation*-
Since TMT produces a relatively selective limbic system tox-
icity it might be used to investigate the physiology and func-
tion of this neural system. Furthermore, recent evident*
indicates that TMT might be a particularly useful tool f°r
elucidating the neurobiological substrates of memory vtoC'
esses. For example, it has been reported that TMT impair*
the acquisition and performance of complex spatial task*
such as the radial-arm maze [20] and the Hebb- William*
maze [18] and also disrupts retention of two-way [131 ano
passive avoidance (present data) responses. Therefore,
studies examining the biochemical and neural correlates
TMT-induced behavioral changes might provide further
sights into the biology of learning and memory.
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