United States       Prevention, Pesticides     EPA712-C-98-238
          Environmental Protection    and Toxic Substances     August 1998
          Agency         (7101)
&EPA   Health Effects Test
          OPPTS 870.6200
          Neurotoxicity Screening

     This guideline is one  of a  series  of test  guidelines that have been
developed by the Office of Prevention, Pesticides and Toxic Substances,
United States Environmental  Protection Agency for use  in the testing of
pesticides and toxic substances, and the  development of test data that must
be submitted to the Agency  for review under Federal regulations.

     The Office of Prevention, Pesticides and Toxic Substances (OPPTS)
has  developed this guideline through  a process of harmonization that
blended the testing  guidance  and requirements that  existed in the Office
of Pollution Prevention and  Toxics  (OPPT) and appeared in Title  40,
Chapter I,  Subchapter R of the Code of Federal Regulations  (CFR),  the
Office of Pesticide Programs (OPP) which appeared in publications of the
National Technical  Information Service (NTIS) and the guidelines pub-
lished by the Organization  for Economic Cooperation and Development

     The purpose of harmonizing these  guidelines  into a single set of
OPPTS guidelines is to minimize  variations among the testing procedures
that must be performed to meet the data  requirements of the U. S. Environ-
mental Protection Agency  under  the Toxic  Substances  Control Act  (15
U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7U.S.C. I36,etseq.).

     Final  Guideline Release: This guideline  is available from the U.S.
Government Printing Office,  Washington, DC 20402 on disks or paper
copies: call (202) 512-0132. This  guideline is also available electronically
in PDF (portable document format) from EPA's  World Wide Web  site
(http://www.epa.gov/epahome/research.htm) under the heading "Research-
ers and Scientists/Test Methods and Guidelines/OPPTS  Harmonized Test

OPPTS 870.6200   Neurotoxicity screening battery.
     (a) Scope—(1) Applicability. This guideline is intended to meet test-
ing  requirements   of both  the  Federal  Insecticide,  Fungicide,   and
Rodenticide Act (FIFRA) (7 U.S.C.  136, et seq.} and the Toxic Substances
Control Act (TSCA) (15 U.S.C. 2601).

     (2) Background. The source material  used in developing this har-
monized OPPTS test guideline  are 40 CFR 798.6050 Functional Observa-
tional Battery,  798.6200 Motor Activity,  and 798.6400 Neuropathology;
and  OPP  81-8 Acute Neurotoxicity—Rat, 82-7 90-Day Neurotoxicity—
Rat, and  83-1 Chronic Feeding—Two Species, Rodent  and Nonrodent
(Pesticide  Assessment  Guidelines,  Subdivision F—Hazard Evaluation;
Human and Domestic Animals, Addendum  10, EPA  report 540/09-91-
123, March 1991).

     (b) Purpose. This neurotoxicity screening battery  consists  of a func-
tional observational battery, motor activity, and neuropathology. The func-
tional observational battery consists of noninvasive procedures designed
to detect gross  functional deficits in animals and to better  quantify behav-
ioral or neurological effects detected in other studies.  The motor activity
test  uses  an automated device that measures the  level of activity  of an
individual animal.  The neuropathological  techniques are designed to pro-
vide data to detect and characterize histopathological changes in the central
and  peripheral nervous system.  This battery is designed to  be used in con-
junction with general toxicity studies and changes should  be evaluated in
the context of both the concordance between functional neurological and
neuropatholgical effects, and with respect to any other toxicological effects
seen. This test battery is  not intended to provide a complete  evaluation
of neurotoxicity, and additional functional and  morphological  evaluation
may be necessary to assess completely the neurotoxic potential of a chemi-

     (c) Definitions. The definitions in section 3 of the Toxic Substances
Control Act (TSCA) and the  definitions in 40 CFR Part 792—Good Lab-
oratory Practice Standards apply to this test guideline. The following defi-
nitions also apply to this test guideline.

     ED is effective dose.

     Motor activity is any movement of the experimental animal.

     Neurotoxicity is  any adverse effect  on  the structure or function of
the nervous system related to exposure to a chemical substance.

     Toxic effect is an adverse change in the structure or function  of an
experimental animal as a result of exposure to a chemical substance.

     (d) Principle of the test method. The test substance is administered
to several groups of experimental animals, one dose being  used per group.

The animals are observed under carefully standardized conditions with suf-
ficient frequency to ensure the detection and quantification of behavioral
and/or neurologic abnormalities,  if present. Various functions that could
be affected by neurotoxicants are assessed during each observation period.
Measurements of motor activity of individual animals are made in an auto-
mated device. The animals are perfused and tissue samples from the nerv-
ous system are prepared for microscopic examination. The exposure levels
at which significant neurotoxic effects are  produced are compared to one
another and to those levels that produce other toxic effects.

    (e) Test procedures—(1) Animal  selection—(i) Species. In general,
the  laboratory rat should be used. Under some  circumstances, other  spe-
cies, such as the mouse or the  dog,  may  be more appropriate, although
not all of the battery may be adaptable to other species.

    (ii) Age. Young adults (at least 42 days old for rats) should be used.

    (iii) Sex. Both males and females should  be used.  Females should
be nulliparous and nonpregnant.

    (2) Number of animals. At least 10 males and 10 females should
be used in each  dose  and control group  for behavioral testing. At least
five males and five females should be used in each dose and control group
for terminal neuropathology. If interim neuropathological evaluations are
planned, the number should be increased by the number of animals sched-
uled to be  perfused before the end of the  study. Animals should be  ran-
domly assigned to treatment and control groups.

    (3) Control groups, (i) A  concurrent (vehicle) control group is re-
quired. Subjects  should  be treated in the  same  way as for an exposure
group except that administration of the test substance is omitted.  If the
vehicle used  has known or potential toxic properties, both untreated or
saline treated and vehicle control groups are required.

    (ii) Positive  control data from the laboratory performing the testing
should provide evidence of the ability of the observational methods used
to detect major neurotoxic endpoints including limb weakness or paralysis,
tremor,  and autonomic signs. Positive  control data are also required to
demonstrate the sensitivity and reliability of the  activity-measuring device
and testing procedures. These data should demonstrate the ability to detect
chemically induced increases  and decreases in  activity.  Positive  control
groups exhibiting central nervous system pathology and peripheral nervous
system pathology are  also required.  Separate groups  for peripheral  and
central neuropathology are acceptable (e.g.  acrylamide and trimethyl  tin).
Permanently injurious substances  need not be used for the behavioral tests.
Historical data may  be used if the essential aspects  of the  experimental
procedure remain the same. Periodic  updating of positive control data is
recommended.  New  positive control  data  should also be collected when

personnel or  some  other  critical element  in  the  testing laboratory has

     (4) Dose level  and dose selection. At least  three  doses  should be
used in addition to the vehicle control group. The data should be sufficient
to produce a  dose-effect curve. The Agency strongly encourage the use
of equally spaced doses and a rationale for dose selection that will maxi-
mally  support detection  of dose-effect relations. For acute  studies, dose
selection may be made relative to the establishment of a benchmark dose
(BD).  That is,  doses  may be  specified  as  successive fractions,  e.g.
0.5,  0.25, ...n  of the BD. The BD itself may be estimated as the highest
nonlethal dose as determined in a preliminary range-finding lethality study.
A variety of test methodologies may be used for this purpose,  and the
method chosen may influence subsequent dose selection. The  goal is to
use a  dose level that  is sufficient to be judged a limit dose,  or clearly

     (i) Acute studies. The high dose need not be  greater than 2  g/kg.
Otherwise, the high dose  should result in significant neurotoxic effects
or other clearly toxic  effects, but not result in an indicence of fatalities
that  would preclude a meaningful evaluation of the  data. This dose may
be estimated by a BD procedure as described in paragraph  (e)(4) of this
guideline, with the middle and low dose levels chosen as fractions  of the
BD dose. The lowest dose  should produce  minimal effect, e.g. an ED 10,
or alternatively, no effects.

     (ii)  Subchronic and  chronic studies. The high dose need not be
greater than 1  g/kg. Otherwise, the high dose level  should result in signifi-
cant neurotoxic  effects or  other clearly toxic effects, but not produce an
incidence of fatalities  that would prevent a meaningful evaluation  of the
data. The middle and low doses should be fractions of the high dose. The
lowest dose should produce minimal effects, e.g. an ED 10, or alternatively,
no effects.

     (5) Route of exposure. Selection of route may be based on several
criteria including, the most likely route of human exposure, bioavailability,
the likelihood  of observing effects, practical difficulties, and the likelihood
of producing nonspecific effects. For many materials, it should be recog-
nized that more than  one  route  of exposure may be important and that
these criteria may conflict  with one  another. In order to save  resources,
initially only one route is  being  required for screening  for neurotoxicity.
The route that best meets these criteria should be selected. Dietary feeding
will generally  be acceptable for repeated exposures studies.

     (6) Combined protocol. The tests described in this screening battery
may be combined with any other toxicity  study, as  long as none  of the
requirements of either are violated by the combination.

     (7)  Study  conduct—(i)  Time of  testing.  All animals  should  be
weighed on each test day and at least weekly during the exposure period.

     (A) Acute studies. At a minimum, for acute  studies observations and
activity testing should be made before the  initiation of exposure, at the
estimated time of peak effect within 8 hours of dosing, and at 7 and 14
days after dosing. Estimation of times of peak effect  may  be made  by
dosing pairs of rats across a range  of doses and making regular observa-
tions of gait and arousal.

     (B) Subchronic and chronic studies. In a subchronic study, at a min-
imum, observations and activity measurements should be made before the
initiation of exposure and before the daily exposure, or for feeding studies
at the same time of day, during the 4th, 8th,  and  13th weeks  of exposure.
In chronic studies, at a minimum, observations and activity measurements
should be made before the initiation of exposure and before the daily expo-
sure, or for feeding studies at the same time of day, every 3 months.

     (ii)  Functional observational battery—(A) General conduct. All
animals in a given study should be observed carefully by trained observers
who are  unaware of the animals' treatment, using standardized procedures
to minimize observer variability.  Where  possible, it is  advisable that the
same observer be  used to evaluate the animals in a given study. If this
is not possible, some demonstration of interobserver reliability is required.
The animals should be  removed from the home cage to a standard arena
for observation. Effort  should be made  to  ensure that variations in the
test conditions are minimal and are not systematically related  to treatment.
Among the variables that can affect behavior are sound level,  temperature,
humidity, lighting, odors, time of day, and environmental distractions. Ex-
plicit, operationally defined  scales for each  measure of the battery are to
be used. The development of objective  quantitative measures  of the obser-
vational  end-points  specified  is encouraged. Examples of observational
procedures using defined protocols may be found in paragraphs (g)(6),
(g)(8), and (g)(ll) of this guideline. The functional  observational battery
should include a thorough description of the subject's appearance, behav-
ior, and functional integrity. This should  be assessed through  observations
in the home cage and  while  the rat is  moving freely  in an open field,
and through manipulative tests. Testing should proceed from the least to
the most interactive with the subject. Scoring criteria, or explicitly defined
scales, should be developed for those measures which involve subjective

     (B) List  of measures. The functional  observational battery should
include the following list of measures:

     (7) Assessment of signs of autonomic function, including but not lim-
ited to:

     (/) Ranking of the degree of lacrimation and salivation, with a range
of severity scores from none to severe.

     (//) Presence or absence of piloerection and exophthalmus.

     (Hi) Ranking  or count of urination and defecation, including polyuria
and diarrhea. This is  most easily conducted during the open field assess-

     (iv) Pupillary function such as constriction of the pupil in response
to light or a measure of pupil size.

     (v) Degree of palpebral closure, e.g., ptosis.

     (2) Description, incidence, and severity of any convulsions, tremors,
or abnormal motor movements, both in the home cage and the open field.

     (3) Ranking of the subject's reactivity to general stimuli such as re-
moval from the cage  or handling, with a range of severity scores from
no reaction to hyperreactivity.

     (4) Ranking of the subject's general level of activity during observa-
tions of the unperturbed subject in the open field, with a  range of severity
scores from unresponsive to hyperactive.

     (5) Descriptions  and incidence of posture and gait abnormalities ob-
served in the home cage and open field.

     (6) Ranking of any gait abnormalities, with a range of severity scores
from none  to severe.

     (7) Forelimb  and hindlimb grip strength measured using an objective
procedure,  e.g. that described by Meyer et al. under paragraph (g)(10) of
this guideline

     (8) Quantitative  measure of landing foot splay; the procedure de-
scribed in paragraph (g)(4) of this guideline is recommended.

     (9) Sensorimotor responses to stimuli of different modalities  will be
used to detect gross sensory  deficits. Pain perception may be assessed by
a ranking or measure  of the reaction to a tail-pinch, tail-flick, or hot-plate.
The response to a  sudden sound, e.g., click or snap, may be used to assess

     (10} Body weight.

     (11} Description and incidence of any unusual or abnormal behaviors,
excessive   or  repetitive  actions (stereotypies),  emaciation,  dehydration,
hypotonia  or hypertonia, altered fur  appearance,  red or crusty deposits
around the eyes, nose, or mouth, and any  other observations that may fa-
cilitate interpretation of the data.

     (C) Additional measures. Other measures may also be included and
the development and validation of new tests is encouraged. Further infor-
mation on the neurobehavioral integrity of the subject may be provided

     (7) Count of rearing activity on the open field.

     (2) Ranking of righting ability.

     (3) Body temperature.

     (4) Excessive or spontaneous vocalizations.

     (5) Alterations in rate and ease of respiration, e.g.,  rales or dyspnea.

     (6) Sensorimotor responses to visual or proprioceptive stimuli.

     (iii) Motor activity. Motor activity should be monitored by an auto-
mated activity recording apparatus. The device used must  be capable  of
detecting  both increases and  decreases in activity, i.e., baseline  activity
as measured by the device must not be so low  as to preclude detection
of decreases nor so high as to preclude detection of increases in activity.
Each device should be tested by standard procedures to ensure, to the ex-
tent possible, reliability  of operation across devices and across days for
any one  device. In  addition,  treatment groups must be  balanced across
devices. Each animal should be tested individually.  The test  session should
be  long enough for motor activity to approach asymptotic levels  by the
last 20 percent of the session  for nontreated control animals. All sessions
should  have  the   same  duration.  Treatment  groups  should   be
counterbalanced across test times. Effort should be made  to ensure that
variations in the  test conditions  are  minimal  and are not  systematically
related to treatment. Among the variables which  can affect  motor  activity
are sound level,  size and shape of the test cage,  temperature, relative hu-
midity, lighting conditions, odors, use  of the home cage or a novel test
cage, and environmental distractions.

     (iv) Neuropathology:  Collection, processing and  examination  of
tissue samples. To provide for adequate sampling as well as optimal pres-
ervation of cellular integrity for the detection  of neuropathological alter-
ations, tissue should be prepared for histological analysis using in situ per-
fusion and paraffin and/or plastic embedding procedures. Paraffin  embed-
ding is acceptable for tissue samples from the central nervous system. Plas-
tic embedding of tissue samples from the central nervous  system is  encour-
aged, when feasible. Plastic embedding is required for tissue samples from
the peripheral nervous system. Subject to professional judgment  and the
type of neuropathological alterations observed,  it  is recommended that ad-
ditional methods, such as Bodian's or Bielchowsky's silver methods, and/
or glial fibrillary  acidic protein (GFAP) immunohistochemistry be used
in conjunction with more standard stains to determine the lowest dose level

at which neuropathological alterations are observed. When new or existing
data provide evidence of structural alterations it is recommended that the
GFAP immunoassay  also be considered. A  description of this technique
can be found in paragraph (g)(12) of this guideline.

     (A) Fixation and processing of tissue. The nervous  system should
be fixed by in situ perfusion with an appropriate  aldehyde fixative. De-
tailed  descriptions  of vascular  perfusions may be found  in  paragraphs
(g)(7), (g)(13), (g)(19), and (g)(21) of this guideline. Any gross abnormali-
ties  should  be noted.  Tissue  samples taken should adequately represent
all major regions of  the nervous  system.  Detailed dissection  procedures
are described in paragraphs  (g)(19)(chapter 50), and (g)(13) of this guide-
line. The tissue samples should be postfixed and processed according to
standardized published  histological  protocols  under  paragraph  (g)(l),
(g)(2), (g)(3), (g)(14), (g)(19), or  (g)(20) of this guideline. Tissue blocks
and slides should be appropriately  identified when stored. Histological sec-
tions should be stained for hematoxylin and eosin (H&E), or a comparable
stain according  to standard published protocols under paragraphs (g)(l),
(g)(2), and (g)(14) of this guideline.

     (B) Qualitative  examination. Representative histological  sections
from the tissue samples should be examined  microscopically by an appro-
priately trained  pathologist  for evidence of neuropathological  alterations.
The  nervous system should be thoroughly examined for evidence of any
treatment-related neuropathological alterations. Particular attention should
be paid  to regions known  to be  sensitive to neurotoxic  insult or those
regions likely to be affected based on the results of functional tests. Such
treatment-related neuropathological  alterations  should be  clearly distin-
guished from artifacts  resulting from influences  other than exposure to the
test  substance. Guidance for both regions to be examined and the types
of neuropathological alterations that typically result from toxicant exposure
can be found in paragraph (g)(20) of this guideline. A stepwise examina-
tion  of tissue samples is recommended. In such a stepwise examination,
sections  from the high dose group are  first compared with those of the
control group. If no neuropathological alterations are observed in  samples
from the high  dose  group, subsequent analysis  is  not required.  If
neuropathological alterations are observed in samples from the high  dose
group, samples from the intermediate and low dose groups are then exam-
ined sequentially.

     (C) Subjective diagnosis. If any evidence of neuropathological alter-
ations  is found in the  qualitative examination, then a subjective diagnosis
will  be performed  for the purpose of evaluating dose-response relation-
ships.  All regions of the  nervous  system exhibiting any  evidenceof
neuropathological changes should be  included  in  this analysis. Sections
from all dose groups  from each region will be coded and examined in
randomized order without knowledge of the  code. The frequency of each
type and severity of each lesion will be recorded. After all samples from

all dose  groups including  all regions  have been rated, the code will be
broken and statistical analysis performed to evaluate dose-response rela-
tionships. For each type of dose-related lesion observed, examples of dif-
ferent degrees of severity should be described. Photomicrographs of typical
examples of treatment-related regions are recommended to augment these
descriptions. These examples will also serve to illustrate a rating scale,
such as 1+, 2+, and 3+ for the degree of severity ranging  from very slight
to very extensive.

     (f) Data reporting and evaluation. The final test report must include
the following information:

     (1)  Description of equipment  and  test methods. A description of
the  general design  of the experiment and any equipment used should be
provided. This should include  a short justification explaining any decisions
involving professional judgment.

     (i) A detailed description of the procedures used to standardize obser-
vations, including the arena and scoring criteria.

     (ii)  Positive control data  from the laboratory performing the test that
demonstrate the sensitivity of the procedures being used. Historical data
may be used if all essential aspects of the experimental  protocol are the
same. Historical control data can be critical in the  interpretation of study
findings. The Agency encourages submission of such data to facilitate the
rapid and complete  review of the significance of effects seen.

     (2)  Results. The following information must be arranged by test
group dose level.

     (i) In tabular form, data for each animal must be provided showing:

     (A)  Its identification number.

     (B)  Its body weight and score on each sign at  each observation time,
the  time  and cause of death (if appropriate), total session activity counts,
and intrasession subtotals for each day measured.

     (ii) Summary data for  each group must include:

     (A)  The number of animals at the start of the test.

     (B)  The number of animals showing each  observation score at each
observation time.

     (C)  The mean and standard deviation for each continuous  endpoint
at each observation time.

     (D)  Results of statistical analyses for  each measure,  where appro-


     (iii) All neuropathological  observations should be recorded and ar-
ranged by test groups. This data may be presented in the following rec-
ommended format:

     (A) Description of lesions for each animal. For each animal, data must
be submitted  showing its identification  (animal number, sex, treatment,
dose, duration), a list of structures examined as  well as the locations, na-
ture, frequency, and severity of lesions. Inclusion of photomicrographs is
strongly recommended for demonstrating typical examples of the type and
severity of the neuropathological alterations observed.  Any  diagnoses de-
rived from neurological signs and lesions including naturally occurring dis-
eases or conditions, should be recorded.

     (B) Counts and incidence of neuropathological  alterations by test
group. Data should be tabulated to show:

     (7) The number of animals used in each group and the  number  of
animals in which any lesion was found.

     (2) The number of animals affected by each different type of lesion,
the locations, frequency, and average grade of each type of lesion.

     (3) Evaluation of  data.  The  findings from the  screening  battery
should be evaluated in the context of preceding and/or concurrent toxicity
studies and  any correlated  functional and histopathological findings. The
evaluation should include the relationship between the doses  of the test
substance and the presence  or absence, incidence and severity, of any neu-
rotoxic effects. The evaluation should include appropriate statistical analy-
ses,  for example, parametric tests for continuous data and nonparametric
tests for the remainder.  Choice of analyses should consider tests appro-
priate to the experimental design, including repeated measures.  There may
be many acceptable ways to analyze data.

     (g) References. The following references should be consulted for ad-
ditional background material on this test guideline.

     (1) Armed Forces Institute of Pathology. Manual ofHistologic Stain-
ing Methods. McGraw-Hill, NY (1968).

     (2) Bennet, H.S. et al. Science and art in the preparing tissues embed-
ded in plastic  for light microscopy, with special  reference to glycol meth-
acrylate, glass knives  and simple   stains.  Stain Technology 51:71-97

     (3) Di  Sant Agnese, P.A.  and De Mesy  Jensen,  K. Dibasic staining
of large epoxy sections  and application to  surgical pathology. American
Journal of Clinical Pathology 81:25-29 (1984).

     (4) Edwards, P.M. and Parker V.H. A simple, sensitive and objective
method for early assessment of acrylamide neuropathy in rats. Toxicology
and Applied Pharmacology 40:589-591 (1977).

     (5)  Finger, F.W. Measuring  behavioral  activity.  In:  Methods in
Psychobiology Vol. 2, Ed. R.D. Myers. Academic,  NY. pp. 1-19  (1972).

     (6) Gad, S. A neuromuscular screen for use in industrial toxicology.
Journal of Toxicology and Environmental Health 9:691-704 (1982).

     (7) Hayat, M.A. Volume 1. Biological applications. In: Principles and
Techniques of Electron Microscopy. Van Nostrand Reinhold, NY  (1970).

     (8) Irwin, S. Comprehensive observational  assessment: la. A system-
atic quantitative procedure for assessing the behavioral physiological state
of the mouse. Psychopharmacologia 13:222-257 (1968).

     (9) Kinnard, E.J. and Watzman, N. Techniques utilized in the  evalua-
tion of psychotropic drugs on animals activity. Journal of Pharmaceutical
Sciences 55:995-1012 (1966).

     (10) Meyer, O.A. et al. A method for the routine assessment of fore-
and hindlimb grip strength of rats and mice. Neurobehavioral Toxicology

     (11) Moser V.C. et al. Comparison of chlordimeform and carbaryl
using a functional observational battery. Fundamental and Applied Toxi-
cology 11:189-206(1988).

     (12) O'Callaghan, J.P. Quantification of glial fibrillary acidic protein:
comparison of slot-immunobinding assays with  a novel sandwich ELISA,
Neurotoxicology and Teratology, 13:275-281 (1991).

     (13) Palay, S.L. and Chan Palay, V. Cerebellar Cortex: Cytology and
Organizatio. Springer Verlag, NY (1974).

     (14) Pender, M.P. A simple method for high resolution light micros-
copy of nervous tissue.  Journal of Neuroscience Methods  15:213-218

     (15) Ralis, H.M. et  al. Techniques in Neurohistology,  Butterworths,
London (1973).

     (16) Reiter, L.W. Use of activity measures in behavioral toxicology.
Environmental Health Perspectives 26:9-20 (1978).

     (17) Reiter, L.W. and MacPhail, R.C. Motor Activity: A survey of
methods with potential use in toxicity testing. Neurobehavorial Toxicology
1—Suppl. 1:53-66(1979).


    (18) Robbins, T.W. A critique of the methods available for the meas-
urement of spontaneous motor activity. Handbook of Psychopharmacology
Vol 7. Eds.  Iversen, L.L., Iverson, D.S.,  Snyder, S.H. Plenum, NY. pp.
37-82 (1977).

    (19) Spencer, P.S.,  Schaumburg, H.H. Eds., Experimental and Clini-
cal Neurotoxicology, Williams and Wilkins, Baltimore (1980).

    (20) World Health Organization. Principles  and Methods for the As-
sessment of Neurotoxicity Associated with Exposure to Chemicals (Envi-
ronmental Health Criteria 60), World Health Organizations Publications
Center USA, Albany, NY (1986).

    (21) Zeman, W. and Innes, J.R. Craigie's Neuroanatomy  of the Rat,
Academic, NY (1963).