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Dear Doctor

Enclosed are six copies of cur final rcport on "Studies of Screening of Chemical Compounás for Detection of Behavioral Effects."

Please note that only the title page bears the letterhcad of our laboratory and the appropriate sigrature. This page can.bc removed.from any or all copios of tho report at your discretion.

If there arc any gucstions or comments, ploase do not hesitate to let us know.

Sincerely ysucs,

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DR.TASK OPDER :O.CONTPACT HO. 一

STUDIES OF SCREEN!NG OF CHEIIICAL COMPOUNDS FOR DETECTION OF BEHAVIORAL EFFECTS

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FIVAL REPORT STUDIES ON SCREEIING OF CHE:IICAL COHPOLNDS FOR DETECRION OF BERAVIOPAL EFFECTS

INTRODUCTloN: This report-encompasses three phasas ot work. Initially, the task was to selsot fron nuπerous publishn procedures the best techniques for defining the behavioral and neurological profilo ot a chemical, In addition, a literature review on musclo ceceptors and drugs affectii) these sensory organs was prepared.

Potentially usoful ssreening methods were tested utilizini four modal compounds

Test procedures deamad wôrthy of fur. ther study were chosen on the basis of rosults obtained with these four compounds. These pro:adures were applig:l to a further series of compounds tor classification purposes and to gain further informaslon about the tests.

The second phase of work undarrook a study of the behavioral profile of a series or well known clinically and experimentally used compounds, as well as ot 10 relatively unknown experimentai materials submitsed for screening. The screening tos:s selected in the initial phase ot shis work were used for this purpose.

A third aspect concerned in-depth studies of interesting materials selected on the basis ot screening results. A particular compound, which showed unusual and sustained behavioral effects was subjected to detailed invastigation using pharmacologis inhibitors and potentiators to elucidate the mechanism of action. A method was developed for studying drug effects on mosivation and then put into practice using the l0 experimentai compounds submitted for

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PHASE!

A. LITEPATUPE PEVIRR-PHYSIOLOSY ASD PHAPHACOLOOY OF THE PEGULATION OF POSTURE AND HOVENENT! An OXtOnSIVe rOVIeW Of tho literature was rado in the physlolo)y and phatnisols)y of propricceptors, and on the transnission of Irpulsos trcs theso receptors and tho integrstion of sonsory slgnsls In . the centcal nervous systen. This rovicw was sutnitted with the first interin report.

B. SYSTFHATIC STUDY OF THE EFFECTS OF FOUR COMPOU:OS IN VARIOUS TEST PFOCEOURES: Compounds tostod woro tho

1. In Cats: On Induced Rago and on Aggrossivoness to Mico: Cats of oach sox recoived 1 .and in lator tosts Ito induce a rago reaction

ability of each of tho four compounds to subduo this behavior was tested. Additionally, tho behavior of the subjcct cats to mico was tested. Tho dotails wore submitted in the first intorim rcport. Results are summarized in a lator scction of this report.

2. On the Maze Performance of the Rat: Long-Evans strain hcoded male rats were trained to run a Lashly III alley maze for a food reward. The procedure and maze were as described by Carlini, E. A., and Kramer, D., Effects of on Maze Performance

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of the Rat,

The effect of each of the four compounds on the performance of a trained rat was determined by the time and the number of errors required to complete the maze.

3. On Shock-Induced Fighting Response (Mice): Fighting in mice was exaained. Pairs of male mice selected because they were previously shown to fight were intravenously injected with graded doses of the test compound 30 minutes before testing. Five pairs of mice were used per dose level. The results are summarized below. っ

4. Spontareous Activity (Mice): Spontaneous activity of mice was studied. The test compounds were administered intravenously at graded dose levels (three mice per level). The mice from each level were immediately placed in a The activity (number of times a beam of light was interrupted) was recorded for 60 minutes after injection. The results are summarized below.

5. Sequential Response Performance (Rat): Male Long-Evans hooded rats were trained to a criterion of performance on a B-D schedule of reinforcement (water) in a Polidora Apparatus (J. exper. anal. Behav. 6, 271-277, 1963). (Graded doses of test compounds were administered intravenously and the effects on rewards and total responses were recorded for an interval of l0 minutes immediately afterward and 2, 4, and 6 hours post-treatment. The results wer? reported in the first interim report.

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DISCUSSION: This battery of behavioral tests was used to determine minimal (MED) and median $( E D _ { 5 0 } )$ effective doses of the test drugs and differences in patterns of response. From the results summarized above, it may be seen that yield similar effects, as do. 1 but that differences in dosage and spectrum of activity can. be discerned between these pairs.

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COMPARISON OF COMPOUNDS IN BEHAVIORAL TESTS

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The most sensitive tests are those for Grouped Activity of Mice and the Polidora Sequential Behavior for rats. Both were effective in increasing
activity of mice and in inhibiting responses of rats in these tests at a level of However,
increased responses in the sequential response (polidora) apparatus ati and occasionally at lower levels: was much more potent in inhibition of mouse fighting behavior than was
]. Similarly more effectively inhibited learned goal-oriented behavior of rats in the Lashley III maze at lower dosage levels. 1

. Both ieffectively inhibited the linduced rage response of cats , whereas even at the largest doses given, did not fully

Three compounds tested Iblocked cats' aggression to nice at different
minimally effective dosage levels was not
tested in this fashion).

were active in inhibiting rats tested in the Polidora apparatus and the Lashley III maze, but the effective doses were higher than those for the other two compounds.

With \grouped mouse spontaneous activity showed similar biphasic responses of lat similar dose levels. However, effectively inhibited

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cats' aggression to a mouse at whereas was necessary in this test. In mice, induced fighting was effectively inhibited by a smaller dose of 1 than of

than did could be distinguished from lon the basis of a stimulant effect in which low doses effectively increased responses in the Polidora apparatus. Other tests were necessary to distinguish 1 一

C. ELECTROENCEPHALOGRAPHIC RESPONSE TO AROUSAL (RATS AND RABBITS) A technique for monitoring EEG response to stimuli (sound or light) was investigated in rabbits and rats. The use of the rat with this procedure appeared to be of possible value for incorporation into a primary screen. Samples of recording were included in the first interim report.

PHASE 2

On the basis of the preliminary work described in the preceding section, a series of screening procedures were established for the study of candidate compounds.

The screening proceaures included:

A. ACUTE TOXICITY AND PRELININARY SCREENING: Mice (2 per dosage level) received test compounds orally or intravenously at 1/2-log dosage intervals. The pharmacological signs which were sought were listed on a sample work sheet which was appended to the first and second interim reports. For this test, signs which were observed at each dose, the number of mice reacting, the

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time of onset of these signs, the degree of their severity, and the time for recovery were recorded. The median lethal dose $( I D _ { 5 0 } )$ and the median effective dose $( E D _ { 5 0 } )$ were estimated. This information was tabulated by electronic data prócessing equipment, and was presented in tabular form. An explanatory sheet, which is a guide to the tables, was also appended in the . second interim report.

B. LOCOMOTOR ACTIVITY: Mice (male, 3 per dose level) received graded doses of test compounds intravenously. The mice of each dosage group were then placed together in the The\_activity (number of times a beam of light was interrupted) was recorded by digital accumulation and printing equipment at five minute intervals for 60 minutes after injection. The tabulated data (number of counts for each five-minute interval) were also presented graphically in the second interim report.

C.PHYSICAL AND NEUROLOGICAL EXAMINATION·OF CATS: The pUrpOSe. of this screen was to determine effects of test compounds on physical status, proprioceptive function, and other behavioral and neurological parameters. Cats (2 per dosage level) received test compounds orally or intravenously, and were examined periodically after treatment. Doses were determined usually by levels which yielded significant effects in the mouse toxicity screen. Because of limited quantities of test compounds, only a few animals could be used for each.

Observations included determination of heart rate, respiration rate, body temperature, pupillary size and reaction to licht, and neurological signs and reflexes elicited as described by McGrath (Neurological Examination of the Dog.

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Lea & Febiger, Phila., 2nd Ed. 1960), modified as appropriate for the cat. A list of the test reflexes and signs was attached to the.second interim report. An interval of at least two weeks was allowed for recovery from drug effects before using any one cat again. The data were included in the first and second interim reports.

One monkey was also tested with one compound in a preliminary experiment and reported in the second interim report.

D. EFFECTS ON THE AUTONOMIC NERVOUS SYSTEM (DOGS): MOngrel dogs, anesthetized with were used in a test system in which baseline recordings of the electrocardiogram, arterial blood pressure, respiratory movements, gastrointestinal activity, and surface electroencephalogram were obtained. The responses to. 1

preparation were noted. Selected test compounds were injected into the femoral vein until an effect was noted. The responses to previously fixed doses of 1 after the administration of the test compound were noted. The data were submitted in the first interim report.

E. POLIDORA SEQUENTIAL RESPONSE APPARATUS: The technique was utilized as described in the preceding section. The data were submitted with the first and secondiinterim reports.

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Code No.

TEST COMPOUNDS

Compounds tested were assigned code numbers in order of receipt as follows:

Compound

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PHASE III

SPECIAL STUDIES

A. The compound Ishowed interesting pharmacological effects and was a subject of special study. The studies include.l prolonged investigation of its effect on locomotor activity of mice, on behavior of rats in the Sequential Response Behavior apparatus, and interaction with possible potentiators and inhibitors. The details were presented in the second interim report.

It was concluded that the behavioral effects of compound were mediated

B. DRUG EFFECTS ON MOTIVATION: To supplement the various testing procedures employed in this program, a method for the study of drug effects on motivation was tested.

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C. To further screen candidate compounds,

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