Pharmacology 13: 297-308 (1975)
Effects of A9-Tetrahydrocannabinol on Active Avoidance Acquisition and Passive Avoidance Retention in Rats with Amygdaloid Lesions Robert J. Pandina and Richard E. M usty1 Neuropsychology Laboratory, Department of Psychology, University of Vermont, Burlington, Vt.
Key Words. Active avoidance acquisition • Basolateral amygdala • Passive avoidance retention • Rodents • A*-Tetrahydrocannabinol Abstract. A 9 -Tetrahydrocannabinol was administered to rats with basolateral amyg daloid lesions, control rats, and normal rats in doses of 0.75, 1.5, and 3.0 mg/kg i.v. They were trained in a one-session two-way active avoidance task. A 9 -Tetrahydrocannabinol in creased the percentage of avoidances and the intertrial crossing rates in all groups, regardless of lesion treatment. Rats with basolateral amygdaloid lesions were not different from controls on any measure. In a second experiment, A 9 -tetrahydrocannabinol was admin istered to rats with basolateral amygdaloid lesions and control rats in doses of 0.75 and 3.0 mg/kg 24 h after learning of a one-trial passive avoidance task, and retention was mea sured. No differences were found as a function of drug treatment or lesion condition. It was concluded that the basolateral amygdala is not a necessary condition for the action of A 9 -tetrahydrocannabinol on active avoidance acquisition, that the drug has no effect on passive avoidance retention, and the basolateral amygdala is not necessary for two-way active avoidance acquisition or passive avoidance retention. Active avoidance results are discussed in terms of a possible relationship between A 9 -tetrahydrocannabinol, ACTH, and avoidance learning.
1This research was supported, in part, by an NSF-Student Originated Studies Grant (NSF-GY 9682) to William R. Saxby (R.J.P'. was a coinvestigator) and from funds to R.E.M. from an NSF Institutional Grant (1968-82) and from the Department of Psychology, University of Vermont. Portions of this manuscript were prepared during the sabbatical leave of R.E.M. at Escola Paulista de Medicina, Sao Paulo. The authors wish to thank William R. Saxby for help in design; Michaela Zetumer, Joseph Aloia, and Mordecai Tyson for assisting in the experiments; Joel Najman for statistical analysis: and E.A. Carlini for critical reading of the manuscript.
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Received: July 19, 1974.
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A9-Tetrahydrocannabinol (A9-THC) has been shown to disrupt both acqui sition and performance of two-way avoidance response in rats (7, 9, 14), but appears to have no effect on retention of a passive avoidance response (6, 15). Autoradiographic studies have shown that A9-THC is taken up in the amygdala (13, 21). Electrophysiological changes have been observed in the amygdala after A9-THC administration (8). The amygdala seems to have an important regulatory function in the acquisition of various behaviors including two-way avoidance. Amygdala lesions in rats seem to disrupt acquisition of the two-way avoidance response (2). Simi larly, the amygdala is apparently involved in the acquisition of passive avoidance (17). The present studies were designed to test the hypothesis that the amygdala is a site of action of A9-THC by testing the effect of this drug on animals with lesions of the amygdala in active and passive avoidance.
Experiment I Since A9-THC is taken up by the amygdala (13, 21) and changes the electrophysiological responses of the amygdala (8), it would appear that the amygdala is one site of action of the drug. If the amygdala is removed and disruption of active avoidance acquisition occurs, and if A9-THC has little or no effect on animals with such lesions, one would be led to infer that the amygdala is a necessary and sufficient site of action for the disruptive effects of A9-THC on avoidance behavior. Experiment I was designed to test this possibility. Methods and Procedure
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Subjects. 119 male Long-Evans hooded rats (Blue Spruce Farms), 290-390 g were individually caged and maintained with food and watered libitum. They were placed on an habituation stand, in groups of 12-20. for 2 -4 h/day for 5 -7 days prior to the start of the experiment. 36 rats were randomly selected to receive bilateral lesions of the basolateral amygdala. Surgery was performed with subjects under pentobarbital (Nembutal, 50 mg/ml i.p., administered at a dosage of 1.1 ml/kg). After surgery 40,000 IU penicillin were given intramuscularly. Lesions were made with a Grass Radio Frequency Lesion Maker (model LM-3), a formvar-coated stainless steel wire (0.25 mm), with tip bare, was used as the electrode. The stereotaxic coordinates were 0.6 mm posterior to bregma, 5.0 mm lateral to the mid-line and 7.0 mm below dura with the nose bar elevated 5 mm above the zero plane. 38 additional rats were randomly selected to receive control lesions. All procedures were the same as for rats receiving amygdaloid lesions except the electrode was lowered to 5.5 mm below dura, and was removed without current being passed. The remaining 45 subjects served as normal subjects. All operated subjects were allowed to recover from surgery for 9 -1 4 days before testing. Drug. Desired amounts of A’ -THC (kindly supplied by NIMH) were extracted from the ethanol solution following the procedure used by Domino (5) and suspended in a 0.9 %
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Tween 80 (Fisher Scientific) and saline solution. The suspension was frozen (—10 °C) until needed. No suspensions were used which had been prepared more than 4 days prior to use. Apparatus. Two identical plexiglass shuttle boxes, 50 X 20 X 20 cm styled after Lehigh Valley (model 146-02) were used. Each box was contained in a separate ventilated sound proof chamber illuminated by a centrally placed 28 V 0.075 A lamp. Each shuttle box was divided into two equal sections by a metal barrier 2.5 cm in height. A waveform oscillator was used to drive a speaker which delivered an 800 Hz 70 dB tone. A Grason-Stadlcr shock generator (model E1064) was programmed to deliver a 0.5 mA scrambled electric shock to the grid floor of the shuttle box. Behavioral procedure. The subjects were assigned to serve in 1 or 4 drug treatment conditions as follows: 1.0% Tween 80 saline placebo (9 subjects with amygdala lesions, ABL; 10 surgical controls, S, and 17 normals, N), 0.75 mg/kg A’ -THC (9 ABL, 9 S, and 8 N), 1.5 mg/kg A9-THC (9 ABL, 9 S, and 11 N) and 3.0 mg/kg A9-THC (9 ABL, 10 S, and 9 N). At the beginning of the experimental session, a subject was removed from the home cage and received an intravenous injection (tail vein) of the appropriate solution. Drug administration followed double-blind procedures. After the injection, the subject was placed in a dark holding cage for 20 min prior to being placed in the shuttle box. Following the 20-min holding period, each subject was placed in the shuttle box and allowed 5 min of habituation prior to the beginning of the first trial. The shuttle boxes were programmed such that the light and the tone (combined CS) were presented, simultaneously, to signal the initiation of a trial. If the subject failed to cross to the alternate side of the box within 8 sec of the CS initiation, 0.5 mA scrambled electric shock (UCS) was delivered through the grid Door to the subject. The CS remained and UCS remained on until the subject crossed, or until 30 sec of shock had been delivered. Crossings during the CS, pre-shock period, were scored as avoidances, while crossings during the shock period were scored as escapes. Ter mination of the CS during the pre-shock period, or of the CS-UCS combination constituted the end of a trial. Trials were programmed to occur on a VI 30 sec schedule (range 15-45 sec). The learning criterion was 9 of 10 consecutive avoidances or training was terminated after 300 trials had elapsed, whichever occurred first. The number of escapes, avoidances and intertrial crossings was recorded. Histological procedure. Following testing, operated subjects were sacrificed and per fused with a 10 % formalin-saline solution. Brains were removed and stored in the formalin solution for 48 h. Each brain was sectioned at 30 Mm (on a cryotome) and every fifth section was stained with thionin.
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Results Histological results. Reconstructions of the minimum (darkened), maximum (stipled), and typical (striped) extent of the amygdala lesions of subjects are shown in figure 1. The lesions were confined to the basolateral nucleus, the basomedial nucleus, and the central nucleus of the amygdala. Some damage to the claustrum occurred in most of the rats. Animals whose lesions extended to the corticomedial nuclei, piriform cortex, stria terminalis, optic tract, or ventral hippocampus were excluded from the study. Determination of a subject’s ac ceptability was made independently of knowledge of behavioral findings. Behavioral results. Three parameters of active avoidance behavior were examined: (1) trials to criterion; (2) percent correct avoidances, the percentage
300
Fig. 1. Reconstruction diagrams of the minimum (darkened), maximum (stippled), and typical (striped) extent of basolateral amygdaloid lesions. Plates from Pellegrino and Cushman (18).
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of avoidances made in a test session; (3) intertrial crossing rate (ICR), the ratio of total intertrial crossings to number of trials to criterion. Figure 2a presents the mean number of trials to criterion for all groups at each drug condition: two-way analysis of variance indicated that there was no significant effect of drug treatment, surgical treatment, or drug by surgical treatment interaction. The number of animals not reaching criterion (i.e., ran 300 trials) ranged from 0 to 2 per group and was not related to lesion condition or drug treatment. Figure 2b shows the mean percent of avoidance response for all groups at each drug condition. As drug dose was increased, the percentage of avoidance re sponses increased significantly (F = 3.77; d.f. = 3.10; p < 0.01) but there was neither a significant effect of lesion condition or interaction. The mean ICRs for all groups at each drug level are presented in figure 2 c. ICRs increased signifi cantly as dnig dose was increased (F = 10.05; d.f. = 3.107; p < 0.001) but no effect of surgical treatment or interaction was found. The apparent rise in per centage avoidances and ICRs in the operated control group at the 1.5-mg/kg dose was due to two animals whose ICRs and avoidances were 3 SD above the means. Thus, if their data were removed the curves would look similar to the other two curves.
Experiment / / Pellegrino (17) demonstrated passive avoidance deficits in rats with basolateral amygdaloid lesions. Experiment II was designed to see whether or not A9-THC would have an effect on passive avoidance retention deficits in rats with amygdaloid lesions. Thus, if A9-THC increased or decreased retention deficits in animals with amygdaloid lesions, one would be led to infer that the A9-THC effects on these rats was extra-amygdaloid. If A9-THC has no effect on passive avoidance retention, however, one would infer that the effects of A9-THC were on the amygdala.
Subjects. 95 adult male Long-Evans hooded rats (Blue Spruce Farms), 250-350 g, were used in this experiment. All rats were caged and handled as in experiment 1. Surgical and histological procedures were identical to those used in experiment I. 31 subjects were given bilateral basolateral amygdaloid lesions and 32 subjects were given control lesions. 32 remaining subjects served as unoperated controls. Apparatus. A plexiglass step-through passive avoidance box, 70 X 8 X 30 cm, patterned after Schneider et al. (20), was used in this experiment. The box was divided by a guillotine door into two chambers, one large and one small. A dim lamp positioned over the small chamber provided illumination. The outside of the large chamber was blackened such that the large chamber was darker than the small chamber. The floors and walls of both cham bers were covered with stainless steel sheets. A Grason-Stadler shock generator (model E l064) was used.
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Methods and Procedure
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Fig. 2. a Mean number of trials to criterion for each treatment group (see legend above) across drug treatments which are labelled on the abscissa, b Mean percent avoidances for each treatment group across drug treatments, c Mean intertrial crossing rates for each treat ment group across drug treatments. 0.5 SD above and below the mean is plotted for each group. • = Unoperated normals; ■ = control lesions; o = amygdaloid lesions.
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Behavioral procedure. 73 of the 95 subjects were assigned to serve in 1 of 3 drug treatment conditions (10 % Tween 80 saline placebo, 0.75 mg/kg body weight A’ -THC, or 3.0 mg/kg body weight A’ -THC) according to surgical treatment (basolateral amygdaloid lesions, surgical controls, unoperated normals). The number of subjects in each condition is presented in table 1. On day I (habituation day), the subject was removed from the home cage and placed into the smaller chamber of the apparatus facing away from the open guillotine door. When the subject stepped into the larger chamber the guillotine door was lowered and the subject immediately removed from the larger chamber. On day 2 (training day) the subject was removed from the home cage and placed in a dark holding cage for 20 min prior to the behavioral test. After the holding period, the subject was placed into the smaller chamber as on day 1, and the latency of the subject to cross into the large chamber with all four paws was recorded. When the subject crossed into the larger chamber, the guillotine door was lowered and a 0.8 mA electric shock was delivered for 2.0 sec. Imme-
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2b
----1------------- 1------------- 1----------- 1----------Placebo
0.75
15
30 mg/kg iv
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2c
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Table l. Mean latencies and retention scores (SEC) for passive avoidance performance n
Day 2 (training)
Day 3 (retention test)
Retention scores
Normal groups Saline 0.75 3.0 3.0 (motor control)
8 8 8 8
12.7 7.0 6.4 8.1
357.2 593.0 441.1 15.3
344.6 S85.9 434.7 7.2
Surgical control groups Saline 0.75 3.0 3.0 (motor control)
8 8 8 8
10.4 7.2 17.7 6.9
400.0 404.5 512.1 14.9
389.7 397.2 494.4 8.0
Amygdaloid lesion groups Saline 8 9 0.75 8 3.0 3.0 (motor control) 6
8.4 7.8 11.1 14.2
386.7 300.4 471.2 14.2
378.2 292.7 460.1 0.1
diately following the shock, the subject was removed to the home cage. On day 3 (retention test day) the subject was removed from the home cage, injected with the appropriate substance, and placed in the holding cage for 20 min. After the holding period, the subject was placed in the smaller chamber and the latency to cross into the larger chamber was recorded. No shock was given during retention tests. A maximum of 600 sec was allowed in the retention test. If the subject had not crossed into the shock chamber after this time, he was returned to his home cage. The remaining 22 subjects served in a motor control treat ment group and were divided into surgical treatment groups as with the experimental subjects (table I). In this condition, the subjects underwent the same behavioral regime as the previous subjects except that they are not shocked on day 2. Subjects in this group received 3.0 mg/kg body weight A9-THC i.v. on day 2. The purpose of this group was to test for possible motoric ataxic effects of the THC which might interfere with the passive avoidance response.
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Results Histological results. The amygdala lesions in this study were similar to those reported for experiment I in all respects. Behavioral results. Retention scores were computed for each subject by subtracting training day latency from the retention day latency in order to correct for minor variations in individual subject’s performance on training day. Table I presents mean latencies for training and retention days and retention scores for all groups. Analysis of retention scores indicated no significant effects
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of either drug treatments or lesions. Kruskal-Wallis tests (1) applied to retention scores within each surgical treatment, across drug treatments (placebo, 0.75 and 3.0 mg/kg A9-THC), indicated that subjects within any surgical treatment group did not significantly differ as a function of drug treatment. Comparisons were also made to determine possible retention score differences across surgical treat ments within each drug treatment. Kruskal-Wallis tests indicated no significant differences across surgical treatments for any drug treatment. No significant differences were found between motor control groups in either training day latencies, retention day latencies or retention scores.
In experiment I, A9-THC produced a dose-related increase in the percentage of avoidances and in the intertrial crossing rate, but no effect on the number of trials to criterion. These results are contrary to the findings of Henriksson and Jarbe (9), who found that A9-THC disrupted acquisition of a shuttle box re sponse, and to the findings of Orsingher and Fulginiti (14), who found no effect of A9-THC on the acquisition of the response. Both of these investigators used distributed trial procedures, while, in the present study, massed trials were used. The increased intertrial crossing rate confirms the earlier report of Saxby and Musty (22) and extends the findings by the demonstration of increased avoid ance responding under the drug. In addition to differences in behavioral procedure, the discrepancy between the present findings and those of others may be due to route of administration. Henriksson and Jarbe (9), Orsingher and Fulginiti (14), and Grunfield and Edery (7), all used intraperitoneal injections while the present studies employed the intravenous route. Dose differences are unlikely to account for the differences among these studies since the EDS0 to induce sleep is 4 .0 -5 .0 mg/kg i.v. {Musty, unpubl. report). Thus, we used a dose range which is probably equivalent to doses of approximately 2 -1 0 mg/kg of A9-THC injected intraperitoneally. Pirch et al. (19) reported enhancement of the percent avoidances made by rats being trained in a shuttle box after oral administration of a marijuana extract (17.1 % A9-THC). They obtained tire enhancement with a dose equi valent to 3.4 mg/kg A9 THC. Avoidance did not increase in the first day of administration (80 trials) but increased about 10% on the second day of 80 trials, which is approximately the same magnitude of increase observed in the present study. When testing their animals for an additional 8 days, they observed a 40 % increase in the percentage of avoidances. Within the marijuana group, Pirch et al. (19) reported that the increase in avoidance responding was due to some animals which had high initial performance levels, while no such effect occurred in the present study.
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Discussion
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It is possible that the facilitation of avoidance behavior observed in this study is due to the increase in intertrial crossings. Investigators have invoked such explanations for the facilitative effects of amobarbital on shuttle box learn ing (10). It may be naive to assume that increased intertrial activity is the cause of improved avoidance learning, since Saxby and Musty (22) found increases in intertrial crossing rates but no facilitation of avoidance responding with A9-THC and Cooper et al. (3) have obtained similar results by depleting brain dopamine. Until such time that the relationship between intertrial crossings and avoidances are understood, such theorizing is not very useful. The observed facilitation may be due to depletion of norepinephrine (NE), since Cooper et al. (3) reported increases in avoidance responses and intertrial crossings when brain NE was preferentially decreased. This seems unlikely be cause investigators have not found changes in brain NE levels after administra tion of A9 -THC (11; Carlini, personal commun.). Injection of ACTH is known to facilitate the acquisition o f shuttle box avoidance and intertrial crossing rates (16) at a dose of approximately 0.1 mg/kg. Since A9-THC causes a release of adrenocortical steroids at 2 mg/kg and above (4, 12) it is conceivable that the facilitation observed in the present study is due to increased output of ACTH and/or adrenocorticosteroids. The study of Kubena et al. (12) further demonstrated that the action of A 9-THC was on the brain and not the adrenal gland itself: hypophysectomized rats showed no increases in output of corticosteroids after A9-THC treatment. In experiment II, no effects of A9-THC on retention of a passive avoidance response were observed among any groups. These data replicate the findings of Miller et al. (6) and Pandina and Musty (15). (These experiments were com pleted after the present study.) In addition to the lack on effect of A9-THC on passive avoidance retention, it is surprising that amygdala lesions had no effect on the passive avoidance behavior used in experiment II. Basolateral amygdaloid lesions identical to those of tire present study were made by Pellegrino (17) in rats. Such animals showed severe deficits in the acquisition of a passive avoid ance response in which the rat was required to inhibit water drinking. Pellegrino (personal commun.) suggested that animals with basolateral amygdaloid lesions have difficulty inhibiting responses which are well learned or have a strong approach gradient. In the present passive avoidance task learning may not be strong and it is unlikely that there is a strong approach gradient to the dark chamber. In summary, no differences among groups were found as a function of the lesion conditions, showing that A9-THC does not differentially affect rats with basolateral amygdaloid lesions on two-way active avoidance learning task or passive avoidance retention. Since the results are similar for normal and operated subjects, it appears that the basolateral amygdala is not a necessary or sufficient condition either for the behavioral changes produced by A9-THC on active
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avoidance, or for the acquisition of active avoidance or retention of passive avoidance. Bush et al. (2) found amygdaloid lesions that were placed more ventrally and more posteriorly which invaded lateral amygdala, basolateral amygdala, cor tical amygdala, basomedial amygdala, zona transitionalis and pyriform cortex produced deficits in the acquisition of shuttle box avoidance. ACTII treatment abolished these learning deficits in another group of similarly amygdalectomized rats. Thus, if nuclei of the amygdala, other than the basolateral nucleus, are a site of action of A9-THC, one would expect that it would have no effect on acquisition of active avoidance in animals with lesions similar to those of Bush et al. (2). If, however, A9-THC had the effect of cancelling out learning deficits in such animals, one would hypothesize that the drug was acting on hypothalamic structures. Further research is necessary to test these hypotheses.
References
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1 Bradley. J. V.: Distribution-free statistical tests (Prentice-Hall, Englewood Cliffs, 1968). 2 Bush, D.F.; Lovely, R.H., and Pagano, R.R.: Injection of ACTH induces recovery from shuttle-box avoidance deficits in rats with amygdaloid lesions. J. comp, physiol. Psy ch o l.« .' 168-172 (1973). 3 Cooper, B.R.; Breese, G.R.; Howard, J.L., and Grant, L.D.: Effect of central cate cholamine alterations by 6-hydroxydopamine on shuttle box avoidance acquisition. Physiol. Behav. 2: 121-721 (1972). 4 Dewey, N.L.; Peng, T.C., and Harris, L.S.: The effect of 1-trans-A9-tetrahydro cannabinol on the hypothalamo-hypophyseal-adrenal axis of rats. Eur. J. Pharmacol. 12: 382-384 (1970). 5 Domino, E.F.: Neuropsychopharmacologic studies of marijuana: some synthetic and natural THC derivatives in animals and man; in Singer Marijuana: chemistry, pharma cology, and patterns of social use. Ann. N.Y. Acad. Sci. 191: 166-191 (1971). 6 Miller, L.L.; Drew, W.G., and Joyce, P.: A’ -THC: effect on acquisition and retention of one-trial passive avoidance response. Behav. Biol. 8: 421-426 (1973). 7 Grunfield, Y. and Edery, H.: Psychopharmacological activity of the active constituents of hashish and some related cannabinoids. Psychopharmacologia 14: 200-210 (1969). 8 Heath, R.C.: Marihuana: effects on deep and surface electroencephalograms of rhesus monkeys. Neuropharmacology 12: 1—14 (1973). 9 Henriksson, B.G. and Jarhe, T.: The effect of two tetrahydrocannabinoids on condi tioned avoidance learning in rats and its transfer to normal state conditions. Psycho pharmacologia 22: 23-30 (1971). 10 Kamano, D.K.: Martin, L.K., and Powell, B.J.: Avoidance response acquisition and amobarbital dosage levels. Psychopharmacologia 8: 319-323 (1966). 11 Kilbey, M.M. and Moore, J.W., jr.: A9-Tetrahydrocannabinol-induced inhibition of predatory aggression in the rat. Psychopharmacologia 31: 157-166 (1973). 12 Kubena, R.K.: Perchach, J.L., and Barry, H., Ill: Corticosterone elevation mediated centrally by A1-tetrahydrocannabinol in rats. Eur. J. Pharmacol. 14: 89-92 (1971). 13 Mclsaac, W.M.; Fritchie, E.G.; Idanpaan-Heikkila, J.E.; Ho, B.T., and Englert, L.F.:
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16 17 18 19 20
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Distribution of marihuana in monkey brain and concomitant behavioural effects. Na ture, Lond. 230: 593-594 (1971). Orsingher, O.A. and Fulginiti, S.: Effects of Cannabis saliva on learning in rats. Pharmacology 3: 337-344 (1970). Pandina, R.J. and Musty, R.E.: Effects of A 9 -tetrahydrocannabinol on retention of a passive avoidance response. Proc. 81st Ann. Convention Amer. Psychol. Ass., vol. 8, pp. 993-994 (1973). Pagano, R.R. and Lovely, R.H.: Diurnal facilitation of shuttle box avoidance behavior. Physiol. Behav. 8: 721-723 (1971). Pellegrino, L.J.: Amygdaloid lesions and behavioral inhibition in the rat. J. comp, physiol. Psychol. 65: 483-491 (1968). Pellegrino, L.J. and Cushman, A.J.: Stereotoxic atlas of the rat brain (AppletonCentury-Crofts, New York 1967). Pirch, J.H.; Osterholm, K.C.; Barratt, E.S., and Cohn, R.A.: Marijuana enhancement of shuttle-box performance in rats. Proc. Soc. exp. Biol. Med. 141: 590-592 (1972). Schneider, A.M.: Kapp, B.: Aron, C., and Jarvik, M.E.: Retroactive effects of transcorneal and transpinnatc ECS on step-through latancies of mice and rats. J. comp, physiol. Psychol. 69: 506-509 (1969). Shannon, M.E. and Freid, P.A.: The macro- and microdistribution and polymorphic electroencephalographic effects of A 9 -tetrahydrocannabinol in the rat. Psychopharmacologia 27: 141-156 (1972). Saxby, W.R. and Musty, R.E.: Effects of intravenous trans-1- A 9 -tetrahydrocannabinol on the acquisition of a two-way active avoidance response. Proc. 81st Ann. Convention Amer. Psychol. Ass., vol. 8, pp. 991-992 (1973).
Dr. R.E. Musty, Neuropsychology Laboratory, Department of Psychology, John Dewey Hall, University of Vermont, Burlington, VT 05401 (USA)
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Effects of A9-Tetrahydrocannabinol on Active Avoidance Acquisition and Passive Avoidance Retention in Rats with Amygdaloid Lesions Robert J. Pandina and Richard E. M usty1 Neuropsychology Laboratory, Department of Psychology, University of Vermont, Burlington, Vt.
Key Words. Active avoidance acquisition • Basolateral amygdala • Passive avoidance retention • Rodents • A*-Tetrahydrocannabinol Abstract. A 9 -Tetrahydrocannabinol was administered to rats with basolateral amyg daloid lesions, control rats, and normal rats in doses of 0.75, 1.5, and 3.0 mg/kg i.v. They were trained in a one-session two-way active avoidance task. A 9 -Tetrahydrocannabinol in creased the percentage of avoidances and the intertrial crossing rates in all groups, regardless of lesion treatment. Rats with basolateral amygdaloid lesions were not different from controls on any measure. In a second experiment, A 9 -tetrahydrocannabinol was admin istered to rats with basolateral amygdaloid lesions and control rats in doses of 0.75 and 3.0 mg/kg 24 h after learning of a one-trial passive avoidance task, and retention was mea sured. No differences were found as a function of drug treatment or lesion condition. It was concluded that the basolateral amygdala is not a necessary condition for the action of A 9 -tetrahydrocannabinol on active avoidance acquisition, that the drug has no effect on passive avoidance retention, and the basolateral amygdala is not necessary for two-way active avoidance acquisition or passive avoidance retention. Active avoidance results are discussed in terms of a possible relationship between A 9 -tetrahydrocannabinol, ACTH, and avoidance learning.
1This research was supported, in part, by an NSF-Student Originated Studies Grant (NSF-GY 9682) to William R. Saxby (R.J.P'. was a coinvestigator) and from funds to R.E.M. from an NSF Institutional Grant (1968-82) and from the Department of Psychology, University of Vermont. Portions of this manuscript were prepared during the sabbatical leave of R.E.M. at Escola Paulista de Medicina, Sao Paulo. The authors wish to thank William R. Saxby for help in design; Michaela Zetumer, Joseph Aloia, and Mordecai Tyson for assisting in the experiments; Joel Najman for statistical analysis: and E.A. Carlini for critical reading of the manuscript.
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Received: July 19, 1974.
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A9-Tetrahydrocannabinol (A9-THC) has been shown to disrupt both acqui sition and performance of two-way avoidance response in rats (7, 9, 14), but appears to have no effect on retention of a passive avoidance response (6, 15). Autoradiographic studies have shown that A9-THC is taken up in the amygdala (13, 21). Electrophysiological changes have been observed in the amygdala after A9-THC administration (8). The amygdala seems to have an important regulatory function in the acquisition of various behaviors including two-way avoidance. Amygdala lesions in rats seem to disrupt acquisition of the two-way avoidance response (2). Simi larly, the amygdala is apparently involved in the acquisition of passive avoidance (17). The present studies were designed to test the hypothesis that the amygdala is a site of action of A9-THC by testing the effect of this drug on animals with lesions of the amygdala in active and passive avoidance.
Experiment I Since A9-THC is taken up by the amygdala (13, 21) and changes the electrophysiological responses of the amygdala (8), it would appear that the amygdala is one site of action of the drug. If the amygdala is removed and disruption of active avoidance acquisition occurs, and if A9-THC has little or no effect on animals with such lesions, one would be led to infer that the amygdala is a necessary and sufficient site of action for the disruptive effects of A9-THC on avoidance behavior. Experiment I was designed to test this possibility. Methods and Procedure
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Subjects. 119 male Long-Evans hooded rats (Blue Spruce Farms), 290-390 g were individually caged and maintained with food and watered libitum. They were placed on an habituation stand, in groups of 12-20. for 2 -4 h/day for 5 -7 days prior to the start of the experiment. 36 rats were randomly selected to receive bilateral lesions of the basolateral amygdala. Surgery was performed with subjects under pentobarbital (Nembutal, 50 mg/ml i.p., administered at a dosage of 1.1 ml/kg). After surgery 40,000 IU penicillin were given intramuscularly. Lesions were made with a Grass Radio Frequency Lesion Maker (model LM-3), a formvar-coated stainless steel wire (0.25 mm), with tip bare, was used as the electrode. The stereotaxic coordinates were 0.6 mm posterior to bregma, 5.0 mm lateral to the mid-line and 7.0 mm below dura with the nose bar elevated 5 mm above the zero plane. 38 additional rats were randomly selected to receive control lesions. All procedures were the same as for rats receiving amygdaloid lesions except the electrode was lowered to 5.5 mm below dura, and was removed without current being passed. The remaining 45 subjects served as normal subjects. All operated subjects were allowed to recover from surgery for 9 -1 4 days before testing. Drug. Desired amounts of A’ -THC (kindly supplied by NIMH) were extracted from the ethanol solution following the procedure used by Domino (5) and suspended in a 0.9 %
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Tween 80 (Fisher Scientific) and saline solution. The suspension was frozen (—10 °C) until needed. No suspensions were used which had been prepared more than 4 days prior to use. Apparatus. Two identical plexiglass shuttle boxes, 50 X 20 X 20 cm styled after Lehigh Valley (model 146-02) were used. Each box was contained in a separate ventilated sound proof chamber illuminated by a centrally placed 28 V 0.075 A lamp. Each shuttle box was divided into two equal sections by a metal barrier 2.5 cm in height. A waveform oscillator was used to drive a speaker which delivered an 800 Hz 70 dB tone. A Grason-Stadlcr shock generator (model E1064) was programmed to deliver a 0.5 mA scrambled electric shock to the grid floor of the shuttle box. Behavioral procedure. The subjects were assigned to serve in 1 or 4 drug treatment conditions as follows: 1.0% Tween 80 saline placebo (9 subjects with amygdala lesions, ABL; 10 surgical controls, S, and 17 normals, N), 0.75 mg/kg A’ -THC (9 ABL, 9 S, and 8 N), 1.5 mg/kg A9-THC (9 ABL, 9 S, and 11 N) and 3.0 mg/kg A9-THC (9 ABL, 10 S, and 9 N). At the beginning of the experimental session, a subject was removed from the home cage and received an intravenous injection (tail vein) of the appropriate solution. Drug administration followed double-blind procedures. After the injection, the subject was placed in a dark holding cage for 20 min prior to being placed in the shuttle box. Following the 20-min holding period, each subject was placed in the shuttle box and allowed 5 min of habituation prior to the beginning of the first trial. The shuttle boxes were programmed such that the light and the tone (combined CS) were presented, simultaneously, to signal the initiation of a trial. If the subject failed to cross to the alternate side of the box within 8 sec of the CS initiation, 0.5 mA scrambled electric shock (UCS) was delivered through the grid Door to the subject. The CS remained and UCS remained on until the subject crossed, or until 30 sec of shock had been delivered. Crossings during the CS, pre-shock period, were scored as avoidances, while crossings during the shock period were scored as escapes. Ter mination of the CS during the pre-shock period, or of the CS-UCS combination constituted the end of a trial. Trials were programmed to occur on a VI 30 sec schedule (range 15-45 sec). The learning criterion was 9 of 10 consecutive avoidances or training was terminated after 300 trials had elapsed, whichever occurred first. The number of escapes, avoidances and intertrial crossings was recorded. Histological procedure. Following testing, operated subjects were sacrificed and per fused with a 10 % formalin-saline solution. Brains were removed and stored in the formalin solution for 48 h. Each brain was sectioned at 30 Mm (on a cryotome) and every fifth section was stained with thionin.
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Results Histological results. Reconstructions of the minimum (darkened), maximum (stipled), and typical (striped) extent of the amygdala lesions of subjects are shown in figure 1. The lesions were confined to the basolateral nucleus, the basomedial nucleus, and the central nucleus of the amygdala. Some damage to the claustrum occurred in most of the rats. Animals whose lesions extended to the corticomedial nuclei, piriform cortex, stria terminalis, optic tract, or ventral hippocampus were excluded from the study. Determination of a subject’s ac ceptability was made independently of knowledge of behavioral findings. Behavioral results. Three parameters of active avoidance behavior were examined: (1) trials to criterion; (2) percent correct avoidances, the percentage
300
Fig. 1. Reconstruction diagrams of the minimum (darkened), maximum (stippled), and typical (striped) extent of basolateral amygdaloid lesions. Plates from Pellegrino and Cushman (18).
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of avoidances made in a test session; (3) intertrial crossing rate (ICR), the ratio of total intertrial crossings to number of trials to criterion. Figure 2a presents the mean number of trials to criterion for all groups at each drug condition: two-way analysis of variance indicated that there was no significant effect of drug treatment, surgical treatment, or drug by surgical treatment interaction. The number of animals not reaching criterion (i.e., ran 300 trials) ranged from 0 to 2 per group and was not related to lesion condition or drug treatment. Figure 2b shows the mean percent of avoidance response for all groups at each drug condition. As drug dose was increased, the percentage of avoidance re sponses increased significantly (F = 3.77; d.f. = 3.10; p < 0.01) but there was neither a significant effect of lesion condition or interaction. The mean ICRs for all groups at each drug level are presented in figure 2 c. ICRs increased signifi cantly as dnig dose was increased (F = 10.05; d.f. = 3.107; p < 0.001) but no effect of surgical treatment or interaction was found. The apparent rise in per centage avoidances and ICRs in the operated control group at the 1.5-mg/kg dose was due to two animals whose ICRs and avoidances were 3 SD above the means. Thus, if their data were removed the curves would look similar to the other two curves.
Experiment / / Pellegrino (17) demonstrated passive avoidance deficits in rats with basolateral amygdaloid lesions. Experiment II was designed to see whether or not A9-THC would have an effect on passive avoidance retention deficits in rats with amygdaloid lesions. Thus, if A9-THC increased or decreased retention deficits in animals with amygdaloid lesions, one would be led to infer that the A9-THC effects on these rats was extra-amygdaloid. If A9-THC has no effect on passive avoidance retention, however, one would infer that the effects of A9-THC were on the amygdala.
Subjects. 95 adult male Long-Evans hooded rats (Blue Spruce Farms), 250-350 g, were used in this experiment. All rats were caged and handled as in experiment 1. Surgical and histological procedures were identical to those used in experiment I. 31 subjects were given bilateral basolateral amygdaloid lesions and 32 subjects were given control lesions. 32 remaining subjects served as unoperated controls. Apparatus. A plexiglass step-through passive avoidance box, 70 X 8 X 30 cm, patterned after Schneider et al. (20), was used in this experiment. The box was divided by a guillotine door into two chambers, one large and one small. A dim lamp positioned over the small chamber provided illumination. The outside of the large chamber was blackened such that the large chamber was darker than the small chamber. The floors and walls of both cham bers were covered with stainless steel sheets. A Grason-Stadler shock generator (model E l064) was used.
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Fig. 2. a Mean number of trials to criterion for each treatment group (see legend above) across drug treatments which are labelled on the abscissa, b Mean percent avoidances for each treatment group across drug treatments, c Mean intertrial crossing rates for each treat ment group across drug treatments. 0.5 SD above and below the mean is plotted for each group. • = Unoperated normals; ■ = control lesions; o = amygdaloid lesions.
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Behavioral procedure. 73 of the 95 subjects were assigned to serve in 1 of 3 drug treatment conditions (10 % Tween 80 saline placebo, 0.75 mg/kg body weight A’ -THC, or 3.0 mg/kg body weight A’ -THC) according to surgical treatment (basolateral amygdaloid lesions, surgical controls, unoperated normals). The number of subjects in each condition is presented in table 1. On day I (habituation day), the subject was removed from the home cage and placed into the smaller chamber of the apparatus facing away from the open guillotine door. When the subject stepped into the larger chamber the guillotine door was lowered and the subject immediately removed from the larger chamber. On day 2 (training day) the subject was removed from the home cage and placed in a dark holding cage for 20 min prior to the behavioral test. After the holding period, the subject was placed into the smaller chamber as on day 1, and the latency of the subject to cross into the large chamber with all four paws was recorded. When the subject crossed into the larger chamber, the guillotine door was lowered and a 0.8 mA electric shock was delivered for 2.0 sec. Imme-
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2b
----1------------- 1------------- 1----------- 1----------Placebo
0.75
15
30 mg/kg iv
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Table l. Mean latencies and retention scores (SEC) for passive avoidance performance n
Day 2 (training)
Day 3 (retention test)
Retention scores
Normal groups Saline 0.75 3.0 3.0 (motor control)
8 8 8 8
12.7 7.0 6.4 8.1
357.2 593.0 441.1 15.3
344.6 S85.9 434.7 7.2
Surgical control groups Saline 0.75 3.0 3.0 (motor control)
8 8 8 8
10.4 7.2 17.7 6.9
400.0 404.5 512.1 14.9
389.7 397.2 494.4 8.0
Amygdaloid lesion groups Saline 8 9 0.75 8 3.0 3.0 (motor control) 6
8.4 7.8 11.1 14.2
386.7 300.4 471.2 14.2
378.2 292.7 460.1 0.1
diately following the shock, the subject was removed to the home cage. On day 3 (retention test day) the subject was removed from the home cage, injected with the appropriate substance, and placed in the holding cage for 20 min. After the holding period, the subject was placed in the smaller chamber and the latency to cross into the larger chamber was recorded. No shock was given during retention tests. A maximum of 600 sec was allowed in the retention test. If the subject had not crossed into the shock chamber after this time, he was returned to his home cage. The remaining 22 subjects served in a motor control treat ment group and were divided into surgical treatment groups as with the experimental subjects (table I). In this condition, the subjects underwent the same behavioral regime as the previous subjects except that they are not shocked on day 2. Subjects in this group received 3.0 mg/kg body weight A9-THC i.v. on day 2. The purpose of this group was to test for possible motoric ataxic effects of the THC which might interfere with the passive avoidance response.
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Results Histological results. The amygdala lesions in this study were similar to those reported for experiment I in all respects. Behavioral results. Retention scores were computed for each subject by subtracting training day latency from the retention day latency in order to correct for minor variations in individual subject’s performance on training day. Table I presents mean latencies for training and retention days and retention scores for all groups. Analysis of retention scores indicated no significant effects
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of either drug treatments or lesions. Kruskal-Wallis tests (1) applied to retention scores within each surgical treatment, across drug treatments (placebo, 0.75 and 3.0 mg/kg A9-THC), indicated that subjects within any surgical treatment group did not significantly differ as a function of drug treatment. Comparisons were also made to determine possible retention score differences across surgical treat ments within each drug treatment. Kruskal-Wallis tests indicated no significant differences across surgical treatments for any drug treatment. No significant differences were found between motor control groups in either training day latencies, retention day latencies or retention scores.
In experiment I, A9-THC produced a dose-related increase in the percentage of avoidances and in the intertrial crossing rate, but no effect on the number of trials to criterion. These results are contrary to the findings of Henriksson and Jarbe (9), who found that A9-THC disrupted acquisition of a shuttle box re sponse, and to the findings of Orsingher and Fulginiti (14), who found no effect of A9-THC on the acquisition of the response. Both of these investigators used distributed trial procedures, while, in the present study, massed trials were used. The increased intertrial crossing rate confirms the earlier report of Saxby and Musty (22) and extends the findings by the demonstration of increased avoid ance responding under the drug. In addition to differences in behavioral procedure, the discrepancy between the present findings and those of others may be due to route of administration. Henriksson and Jarbe (9), Orsingher and Fulginiti (14), and Grunfield and Edery (7), all used intraperitoneal injections while the present studies employed the intravenous route. Dose differences are unlikely to account for the differences among these studies since the EDS0 to induce sleep is 4 .0 -5 .0 mg/kg i.v. {Musty, unpubl. report). Thus, we used a dose range which is probably equivalent to doses of approximately 2 -1 0 mg/kg of A9-THC injected intraperitoneally. Pirch et al. (19) reported enhancement of the percent avoidances made by rats being trained in a shuttle box after oral administration of a marijuana extract (17.1 % A9-THC). They obtained tire enhancement with a dose equi valent to 3.4 mg/kg A9 THC. Avoidance did not increase in the first day of administration (80 trials) but increased about 10% on the second day of 80 trials, which is approximately the same magnitude of increase observed in the present study. When testing their animals for an additional 8 days, they observed a 40 % increase in the percentage of avoidances. Within the marijuana group, Pirch et al. (19) reported that the increase in avoidance responding was due to some animals which had high initial performance levels, while no such effect occurred in the present study.
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It is possible that the facilitation of avoidance behavior observed in this study is due to the increase in intertrial crossings. Investigators have invoked such explanations for the facilitative effects of amobarbital on shuttle box learn ing (10). It may be naive to assume that increased intertrial activity is the cause of improved avoidance learning, since Saxby and Musty (22) found increases in intertrial crossing rates but no facilitation of avoidance responding with A9-THC and Cooper et al. (3) have obtained similar results by depleting brain dopamine. Until such time that the relationship between intertrial crossings and avoidances are understood, such theorizing is not very useful. The observed facilitation may be due to depletion of norepinephrine (NE), since Cooper et al. (3) reported increases in avoidance responses and intertrial crossings when brain NE was preferentially decreased. This seems unlikely be cause investigators have not found changes in brain NE levels after administra tion of A9 -THC (11; Carlini, personal commun.). Injection of ACTH is known to facilitate the acquisition o f shuttle box avoidance and intertrial crossing rates (16) at a dose of approximately 0.1 mg/kg. Since A9-THC causes a release of adrenocortical steroids at 2 mg/kg and above (4, 12) it is conceivable that the facilitation observed in the present study is due to increased output of ACTH and/or adrenocorticosteroids. The study of Kubena et al. (12) further demonstrated that the action of A 9-THC was on the brain and not the adrenal gland itself: hypophysectomized rats showed no increases in output of corticosteroids after A9-THC treatment. In experiment II, no effects of A9-THC on retention of a passive avoidance response were observed among any groups. These data replicate the findings of Miller et al. (6) and Pandina and Musty (15). (These experiments were com pleted after the present study.) In addition to the lack on effect of A9-THC on passive avoidance retention, it is surprising that amygdala lesions had no effect on the passive avoidance behavior used in experiment II. Basolateral amygdaloid lesions identical to those of tire present study were made by Pellegrino (17) in rats. Such animals showed severe deficits in the acquisition of a passive avoid ance response in which the rat was required to inhibit water drinking. Pellegrino (personal commun.) suggested that animals with basolateral amygdaloid lesions have difficulty inhibiting responses which are well learned or have a strong approach gradient. In the present passive avoidance task learning may not be strong and it is unlikely that there is a strong approach gradient to the dark chamber. In summary, no differences among groups were found as a function of the lesion conditions, showing that A9-THC does not differentially affect rats with basolateral amygdaloid lesions on two-way active avoidance learning task or passive avoidance retention. Since the results are similar for normal and operated subjects, it appears that the basolateral amygdala is not a necessary or sufficient condition either for the behavioral changes produced by A9-THC on active
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avoidance, or for the acquisition of active avoidance or retention of passive avoidance. Bush et al. (2) found amygdaloid lesions that were placed more ventrally and more posteriorly which invaded lateral amygdala, basolateral amygdala, cor tical amygdala, basomedial amygdala, zona transitionalis and pyriform cortex produced deficits in the acquisition of shuttle box avoidance. ACTII treatment abolished these learning deficits in another group of similarly amygdalectomized rats. Thus, if nuclei of the amygdala, other than the basolateral nucleus, are a site of action of A9-THC, one would expect that it would have no effect on acquisition of active avoidance in animals with lesions similar to those of Bush et al. (2). If, however, A9-THC had the effect of cancelling out learning deficits in such animals, one would hypothesize that the drug was acting on hypothalamic structures. Further research is necessary to test these hypotheses.
References
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1 Bradley. J. V.: Distribution-free statistical tests (Prentice-Hall, Englewood Cliffs, 1968). 2 Bush, D.F.; Lovely, R.H., and Pagano, R.R.: Injection of ACTH induces recovery from shuttle-box avoidance deficits in rats with amygdaloid lesions. J. comp, physiol. Psy ch o l.« .' 168-172 (1973). 3 Cooper, B.R.; Breese, G.R.; Howard, J.L., and Grant, L.D.: Effect of central cate cholamine alterations by 6-hydroxydopamine on shuttle box avoidance acquisition. Physiol. Behav. 2: 121-721 (1972). 4 Dewey, N.L.; Peng, T.C., and Harris, L.S.: The effect of 1-trans-A9-tetrahydro cannabinol on the hypothalamo-hypophyseal-adrenal axis of rats. Eur. J. Pharmacol. 12: 382-384 (1970). 5 Domino, E.F.: Neuropsychopharmacologic studies of marijuana: some synthetic and natural THC derivatives in animals and man; in Singer Marijuana: chemistry, pharma cology, and patterns of social use. Ann. N.Y. Acad. Sci. 191: 166-191 (1971). 6 Miller, L.L.; Drew, W.G., and Joyce, P.: A’ -THC: effect on acquisition and retention of one-trial passive avoidance response. Behav. Biol. 8: 421-426 (1973). 7 Grunfield, Y. and Edery, H.: Psychopharmacological activity of the active constituents of hashish and some related cannabinoids. Psychopharmacologia 14: 200-210 (1969). 8 Heath, R.C.: Marihuana: effects on deep and surface electroencephalograms of rhesus monkeys. Neuropharmacology 12: 1—14 (1973). 9 Henriksson, B.G. and Jarhe, T.: The effect of two tetrahydrocannabinoids on condi tioned avoidance learning in rats and its transfer to normal state conditions. Psycho pharmacologia 22: 23-30 (1971). 10 Kamano, D.K.: Martin, L.K., and Powell, B.J.: Avoidance response acquisition and amobarbital dosage levels. Psychopharmacologia 8: 319-323 (1966). 11 Kilbey, M.M. and Moore, J.W., jr.: A9-Tetrahydrocannabinol-induced inhibition of predatory aggression in the rat. Psychopharmacologia 31: 157-166 (1973). 12 Kubena, R.K.: Perchach, J.L., and Barry, H., Ill: Corticosterone elevation mediated centrally by A1-tetrahydrocannabinol in rats. Eur. J. Pharmacol. 14: 89-92 (1971). 13 Mclsaac, W.M.; Fritchie, E.G.; Idanpaan-Heikkila, J.E.; Ho, B.T., and Englert, L.F.:
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16 17 18 19 20
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Distribution of marihuana in monkey brain and concomitant behavioural effects. Na ture, Lond. 230: 593-594 (1971). Orsingher, O.A. and Fulginiti, S.: Effects of Cannabis saliva on learning in rats. Pharmacology 3: 337-344 (1970). Pandina, R.J. and Musty, R.E.: Effects of A 9 -tetrahydrocannabinol on retention of a passive avoidance response. Proc. 81st Ann. Convention Amer. Psychol. Ass., vol. 8, pp. 993-994 (1973). Pagano, R.R. and Lovely, R.H.: Diurnal facilitation of shuttle box avoidance behavior. Physiol. Behav. 8: 721-723 (1971). Pellegrino, L.J.: Amygdaloid lesions and behavioral inhibition in the rat. J. comp, physiol. Psychol. 65: 483-491 (1968). Pellegrino, L.J. and Cushman, A.J.: Stereotoxic atlas of the rat brain (AppletonCentury-Crofts, New York 1967). Pirch, J.H.; Osterholm, K.C.; Barratt, E.S., and Cohn, R.A.: Marijuana enhancement of shuttle-box performance in rats. Proc. Soc. exp. Biol. Med. 141: 590-592 (1972). Schneider, A.M.: Kapp, B.: Aron, C., and Jarvik, M.E.: Retroactive effects of transcorneal and transpinnatc ECS on step-through latancies of mice and rats. J. comp, physiol. Psychol. 69: 506-509 (1969). Shannon, M.E. and Freid, P.A.: The macro- and microdistribution and polymorphic electroencephalographic effects of A 9 -tetrahydrocannabinol in the rat. Psychopharmacologia 27: 141-156 (1972). Saxby, W.R. and Musty, R.E.: Effects of intravenous trans-1- A 9 -tetrahydrocannabinol on the acquisition of a two-way active avoidance response. Proc. 81st Ann. Convention Amer. Psychol. Ass., vol. 8, pp. 991-992 (1973).
Dr. R.E. Musty, Neuropsychology Laboratory, Department of Psychology, John Dewey Hall, University of Vermont, Burlington, VT 05401 (USA)
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