BEHAVIORAL BIOLOGY 24, 533-538 (1978)
BRIEF REPORT Self-administration of Diazepam by the Rat NANCY Y . WALTON AND J. A . DEUTSCH
Department of Psychology, C-009, University of California, San Diego, La Jolla, California 92093 Male Charles River rats were offered diazepam either orally or by counterinjection over a 6-day period, with drug concentrations increasing every other day. Although diazepam intoxication was observed at all drug concentrations and with increasing severity as the drug concentration was increased, no decrease in intake ensued. This apparent lack of conditioned aversion formation was not due to diazepam-induced amnesia, since rats poisoned with lithium chloride after diazepam consumption showed no attenuation of conditioned aversion when compared to rats poisoned after drinking flavored water.
Diazepam is a very widely used drug in the United States, but its behavioral pharmacology has not been studied intensively in the rat. This may, in part, be due to the rat's well-documented unwillingness to selfadminister psychoactive compounds (Berger, 1972; Berman and Cannon, 1974; Cappell and LeBlanc, 1975; Cappell et al., 1973; Eckardt, 1975, 1976; Elsmore and Fletcher, 1972; Lester et al., 1970), a factor which greatly limits the design of experiments investigating such drugs. Amit et al. (1973) reported that rats show conditioned aversion to diazepam in concentrations as low as 0.06 mg/ml. However, since they reported neither the method used to obtain this finding nor any statistical analysis of their data, the information is difficult to evaluate. Randall and Kappell (1973) report on the effects of diazepam on a variety of behavioral tasks, but apparently did not attempt to investigate self-administration. These experiments were undertaken to discover if rats could be induced to self-administer diazepam, and the conditions under which such self administration could be obtained. Our initial experiment was designed to encourage self-administration by offering diazepam after a 23bhr water deprivation period. In order to assess the gustatory aversiveness of diazepam, we allowed one group to drink the diazepam suspension orally, while another group self-administered through a chronic intragastric fistula. 533 0091-6773/78/0244-0533502.00/0 Copyright© 1978by AcademicPress, Inc. All rightsof reproductionin any formreserved.
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Subjects were 20 naive, male, Charles River Sprague-Dawley rats, weighing 400 to 600 g at the time of surgery. Animals were housed singly in Plexiglas cages, 18 x 28 cm and 38 cm high, with sawdust-covered floors. Food was available ad libitum throughout the experiment in the home cages. Surgical implantation of intragastric fistulas was performed under sodium pentobarbital anesthesia (50 mg/kg of body weight) using the procedure described by Deutsch and Walton (1977). Rats were assigned at random to oral intake or untasted intake groups, and the experiment began 7 days after surgery. Diazepam intake was tested in counterinjection test cages, as described by Deutsch and Hardy (1976). The floor of each test cage was fitted with a U-tube, one end of which was inside the cage and the other end outside. The U-tube was connected to a pumping mechanism which refilled the tube as the rat drank and simultaneously pumped a volume of fluid equal to that drunk into the rat's intragastric fistula. The oral intake group had diazepam suspensions available to drink from the U-tube, with vanilla flavored water counterinjected into the fistula, while the untasted intake group had vanilla flavored water available to drink in the U-tube with diazepam counterinjection. The flavored water was composed of 1% Schilling vanilla flavoring (v/v) in tap water. Diazepam suspensions were composed of crushed tablets (Valium, Hoffman-La Roche) mixed in 3% ethanol [95% ethanol (v/v) in tap water]. This mixture was allowed to stand overnight and was then shaken and filtered with fine wire mesh to remove solid particles of inert binder ingredients. Diazepam suspensions have been shown to retain a uniform drug concentration for at least 5 days (Newton, 1976), and our suspensions were always shaken prior to use, and containers were refilled each day. Drug concentrations were not tested, but observed behavioral effects corresponded appropriately to the drug concentrations as prepared, and we have since replicated these effects with diazepam suspensions prepared from an injectable Valium preparation (thus eliminating the need to filter the suspension). All rats were water deprived, and testing was begun 23 hr later. Tests were begun each day at 2200 hr. Rats were placed in test cages for 10 min each day, returned to their home cages, and given 10-min access to water in the home cages 30 min after the end of the test session. Effective diazepam concentrations used in testing increased every other day, from 0% on Days 1 and 2 to 0.25 mg/ml on Days 3 and 4, 0.50 mg/ml on Days 5 and 6, and 1 mg/ml on Days 7 and 8. (The actual diazepam suspension concentration for the counterinjection group was twice that of the effective concentration, since effective concentration reflects equal simultaneous intake of diazepam suspension and flavored water.) Volume of diazepam consumed by each rat during each test session was recorded. Figure 1 presents the fluid intake volume plus the drug dosage self-
FIG. I. Mean dosage of diazepam self-administered by both groups over days (upper graph). Mean fluid intake of both groups of rats over days (lower graph). Solid lines connect points from oral intake group, while broken lines connect points from counterinjection group. Diazepam concentration was 0 mg/ml on Days 1 and 2, 0.25 mg/ml on Days 3 and 4, 0.50 mg/ml on Days 5 and 6, and 1 mg/ml on Days 7 and 8. administered by both groups over the 8 days of testing. Each of these measures was analyzed in a two-way A N O V A , with repeated measures over days. This analysis confirmed what is evident on visual inspection of the figure: Volume of fluid intake was invariant over days for both groups, with differences between groups also nonsignificant. This constant intake produced a significant increase in quantity of drug self-administered as the drug concentration was increased, F (2, 36) = 191.66, P < 0.001. These results are in sharp contrast to the rat's behavior when alcohol is offered at varying concentrations. Wallgren and Barry (1970) have demonstrated that the typical decrease in volume of alcohol consumed as the alcohol concentration increases actually represents a careful metering of drug dosage by the animals with the gram per kilogram dose of alcohol remain-
WALTON AND DEUTSCH
ing remarkably constant across all concentrations. The presence of diazepam, on the other hand, was apparently completely ignored by the rats in the present experiment, with no aversion being shown to either the taste of the drug or its postingestional consequences. It has been suggested that diazepam may cause amnesia in the rat (Soubrie et al., 1976), thus leaving the rat with no memory of the drug's taste, the aftereffects associated with drug ingestion, or both. Experiment 2 was conducted to test the ability of rats to form conditioned aversions to the taste of diazepam, when a known aversive agent, lithium chloride, was used to ensure that an aversive state would follow the ingestion of diazepam. Rats used in the first experiment again served as subjects. Half of the animals from each of the groups of Experiment 1 were assigned to each of two groups. The first group drank a diazepam suspension, followed by an intraperitoneal (ip) injection of lithium chloride, while the second group drank a flavored water solution, followed by injection of lithium chloride. The procedure for both groups was identical. They were tested in cages identical to the home cages, except that no food was present and they were fitted with a graduated buret drinking tube. All animals were tested once a day at 2200 hr. Rats were water deprived but food was available ad libitum in the home cages. After being placed on deprivation the animals were run for 3 days with water available in the 10-rain test session, with water again available for 10 min in the home cages 30 min after the completion of the test session. On the fourth day one group of rats was given a 1 mg/ml diazepam suspension to drink during the test session, while the other group was given a 1% chocolate flavored water solution [(v/v) Schilling chocolate flavoring in tap water] to drink during the test session. Immediately after the test session all animals were injected ip with 20 ml/kg of body weight of 0.075 M lithium chloride solution and returned to their home cages, where they had 10-min access to water 30 min later. On the following day the rats were again given either diazepam or flavored water to drink. The volume of diazepam or flavored water drunk before and after the lithium chloride injections was recorded for each animal. A two-way analysis of variance with repeated measures over one factor (consumption before and after lithium chloride poisoning) was used to evaluate the data. The analysis revealed a significant effect due to lithium chloride poisoning, with all rats drinking less after poisoning than they had before, F (1, 18) = 383.827, P < 0.001. There were no significant effects due to the fluid paired with poisoning (diazepam or chocolate) nor to the interaction. Mean volume drunk before poisoning was 17.75 ml of chocolate flavored water and 15.01 ml of diazepam. Mean volume consumed after poisoning fell to 1.02 ml of chocolate and 1.22 ml of diazepam.
Symptoms of intoxication, including gross ataxia, loss of grasp reflex, and lack of normal orienting response to loud noises, were observed at all drug concentrations in both experiments, with overall severity increasing as drug concentration increased. Severity of such symptoms varied markedly among individual rats at lower concentrations, but all rats showed symptoms of intoxication when drinking the 1 mg/ml diazepam suspension. The aversion formed to a psychoactive compound such as ethanol has been attributed to the state of intoxication induced by drug intake (Eckardt, 1975; Lester et al., 1970). The lack of any diminution of volume of diazepam consumed during Experiment 1 was, therefore, rather startling. The second experiment demonstrates clearly that this lack of diminution cannot be ascribed to amnesia, since in this experiment the rats showed a clear-cut conditioned aversion after drinking volumes of diazepam comparable to those consumed in the first experiment. Diazepam appears to be a drug which is not unpalatable and which does not produce a strong conditioned aversion, under the conditions of our experiment, even when given in doses large enough to produce obvious intoxication. This property may make diazepam a good compound for an animal model of environmental factors leading to drug abuse. Much work remains to be done to explicate the reasons why diazepam does not produce a strong conditioned aversion, while other psychoactive compounds tested have produced such aversions in the rat. REFERENCES Amit, Z., Corcoran, M. E., Charness, M. E., and Shizgal, P. (1973). Intake of diazepam and hashish by alcohol preferring rats deprived of alcohol. Physiol. Behav. 10, 523-527. Berger, B. D. (1972). Conditioning of food aversions by injections of psychoactive drugs. J. Comp. Physiol. Psychol. 81, 21-26. Berman, R. F., and Cannon, D. S. (1974). The effect of prior ethanol experience on ethanol-induced saccharin aversions. Physiol. Behav. 12, 1041-1044. Cappeli, H., and LeBlanc, A. E. (1975). Conditioned aversion by ampthetamines: Rates of acquisition and loss of the attenuating effects of prior exposure. Psychopharmacologia 43, 157-162. Cappetl, H., LeBlanc, A. E., and Endrenyi, L. (1973). Aversive conditioning by psychoactive drugs: Effects of morphine, alcohol, and chlordiazepoxide. Psychopharmacologia 29, 239-246. Deutsch, J. A., and Hardy, W. T. (1976). Ethanol tolerance in the rat measured by the untasted intake of alcohol. Behav. Biol. 17, 379-389. Deutsch, J. A., and Walton, N. Y. (1977). A rat alcoholism model in a free choice situation. Behav. Biol. 19, 349-360. Eckardt, M. J. (1975). Conditioned taste aversions produced by the oral ingestion of ethanol in the rat. Physiol. Psychol. 3, 317-321. Eckardt, M. J. (1976). Alcohol-induced conditioned taste aversions in rats: Effect of concentration and prior exposure to alcohol. J. Stud. Alc. 37, 334-346. Elsmore, T. F., and Fletcher, G. V. (1972). Delta 9 tetrahydrocannabinol: Aversive effects in rats at high doses. Science 175, 911-912.
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Lester, D., Nachman, M., and LeMagnen, J. (1970). Aversive conditioning by ethanol in the rat. Quart. J. Stud. Alc. 31, 578-586. Newton, D. W. (1976). Limitations of compounding diazepam suspensions from tablets. Amer. J. Hosp. Pharmacol. 33, 450-452. Randall, L. O., and Kappell, B. (1973). Pharmacological activity of some benzodiazepines and their metabolities. In S. Garattini, E. Mussini, and L. O. Randall (Eds.), "The Benzodiazepines." New York: Raven Press. Soubrie, P., Simon, P., and Boissier, J. R. (1976). An amnesic effect of benzodiazepines in rats? Experientia 32, 359-360. Wallgren, H., and Barry III, H. (1970). "Actions of Alcohol." Amsterdam: Elsevier.