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Home , Alcohol dependence, Calcium cyanamide, Cyanamide, Disulfiram

Japan. J. Pharmacol. 35, 123-128 (1984) 123

Selective Suppression of Schedule-Induced Drinking by Antialcoholic Drugs in Rats

Hisashi KURIBARA, Akihiro HIGASHIDA* and Sakutaro TADOKORO Divisionfor BehaviorAnalysis, Behavior Research Institute, GunmaUniversity School of ,3-39-22 Showa-machi,Maebashi 371, Japan Accepted February29, 1984

Abstract•\Effects of and , antialcoholic drugs, on schedule-induced ethanol drinking as well as on schedule-controlled response (lever-pressing) under a fixed interval 1 min schedule of food were investigated in Wistar strain rats. When ethanol solution was available, the schedule- induced ethanol drinking decreased depending on the ethanol concentration (2-8%). However, the dose of ethanol intake during the 1 hr experimental session was at maximum (2.8 g/kg) when 4% ethanol solution was available. Thereafter, 4% ethanol solution was used in the experiment for studying the effects of disulfiram and on the schedule-induced ethanol drinking. Disulfiram

(100-200 mg/kg, p.o.), pretreated at 1 hr before the start of the experiment, tended to suppress schedule-induced drinking. However, the same treatment of calcium cyanamide (5-10 mg/kg, p.o.) did not produce a marked change in it. In contrast, disulfiram (100 and 200 mg/kg) and calcium cyanamide (5 and 10 mg/ kg) markedly suppressed schedule-induced ethanol drinking without eliciting a marked change in schedule-controlled response. The present results suggest that both disulfiram and calcium cyanamide selectively suppress ethanol drinking in rats.

Disulfiram and calcium cyanamide have relatively few reports (14-16) which have been commonly used as antialcoholic drugs. examined effects of these drugs on ethanol- It has been well known that these drugs drinking in rodents. In these experiments, inhibit and ac- ethanol preferring animals were selectively cumulate in the body after used for the experimental subjects. taking alcoholic beverages, producing Previously, Falk (17) reported that rats aversive effects such as warmth, flush, voluntarily drank a relatively large amount throbbing headache, , , of water under an intermittent food delivery drowsiness and in humans, i.e., schedule. This behavior has been called the disulfiram-ethanol or cyanamide-ethanol "schedule -induced drinking" or "adjunctive reaction (1). drinking". Thereafter, many researchers (18- In preclinical and clinical evaluation of the 25) applied the schedule-induced drinking combined effects of disulfiram or calcium to induce voluntary drinking of ethanol cyanamide with ethanol, blood pressure, solution in rats and to make rats physically heart rate and respiration rate as well as dependent on ethanol. However, there have ethanol and acetaldehyde levels in the blood been no reports on the effect of antialcoholic and/or the brain have been used as the drugs on schedule-induced ethanol drinking. indicators (2-13). However, there are In this experiment, we investigated changes in schedule-induced ethanol drink- * Present address: Tsumura Research Institute for ing developed under a fixed interval 1 min , 3586 Yoshiwara, Ami-machi, (F1 I) schedule of food reinforcement after Inashiki-gun, Ibaragi 300-11, Japan administration of disulfiram and calcium 124 H. Kuribara, A. Higashida & S. Tadokoro cyanamide. In addition, the effects of these method for measurement were previously drugs on the schedule-controlled response reported by us (26). Experimental sessions (lever-pressing for food reinforcement) were consisted of 1 hr per day and were held every also examined. day except Sunday. After 15 sessions of conditioning using the routine procedure Materials and Methods (27, 28), drug testing was started. The Animals: The experimental animals used experiment was carried out between 9:00- were 10 adult male rats of the Wistar strain 17 :00. which had been provided by the Institute of Drugs: The drugs used were ethanol Experimental Animal Research, Gunma (Kanto Chemical Co., Tokyo), disulfiram University School of Medicine. They were (Antabuse "D" Tablet; Tokyo Tanabe Pharm. housed in groups of 3-4 in stainless steel Co., Tokyo) and calcium cyanamide wire mesh cages of 45 (D) •~25 (W) •~20 (H) (Cyanamide Solution; Yoshitomi Pharm. Co., cm, and they were allowed to freely take Osaka). Ethanol was diluted by tap water and solid diet (MF: Oriental Yeast Co., Tokyo) was voluntarily drunk by the rats during the and tap water until the start of the experiment. experimental sessions. Hereafter, the sessions The breeding room was artificially illuminated in which water and ethanol solution were by fluorescent lamps on a 12 hr light-dark available are defined as the water session and schedule (lights on at 6:00 and off at 18:00), the ethanol session, respectively. Disulfiram and the room temperature was regulated to was suspended in a 0.5% carboxymethyl 23•}2•Ž. The rats were employed for the cellulose Na suspension, and calcium experiments when they were 10 weeks of age cyanamide was diluted by distilled water. and weighed 300-350 g. Prior to the start They were orally given 1 hr before the start of conditioning in the Fl 1 min food reinforce- of the experiment. Each volume administered ment situation, they were reduced to 80-85% was fixed at 1 ml/kg body weight. Ethanol of their free feeding body weight by food sessions were held once per 2 weeks, and deprivation. Daily food intake was limited to disulfiram or calcium cyanamide was admin- 10-15 g/rat/day throughout the experimental istered once per week. Therefore, effects of period. However, water was still freely these drugs on rats' behaviors in water and available. ethanol sessions were monitored every other Schedule: Each food-deprived rat was week. Carboxymethyl cellulose Na sus- trained to press a lever in an operant chamber pension or distilled water was given as the of 20 (D) •~ 25 (W) •~ 20 (H) cm for food control administration. pellets of 90 mg each as the reinforcement. Statistical analysis: The data obtained The spherical food pellet was specially made were statistically analyzed by Student's t- and was hard enough to prevent pulveri- test. When P values were equal to or less zation at delivery. On the 1st session of the than 0.05, there was considered to be a conditioning, each lever-pressing was re- significant difference. inforced (continuous reinforcement schedule: Results CR F). On the 2nd session and thereafter , the food reinforcement schedule was changed After 15 sessions of conditioning, all the from CR F to Fl 1 min . Thus, every lever- rats exibited a stable schedule-controlled pressing which was emitted 1 min after the response which was characterized with an previous food delivery was reinforced. The accelerating pattern of the response rate other lever-pressings did not affect the food during the Fl. Eight out of 10 rats exhibited reinforcement schedule, but were recorded . typical schedule-induced water drinking and During the conditioning, water was freely drank 15-50 ml during a 1 hr session. available in the operant chamber through a However, the other 2 rats failed to drink drinking spout (SE TV-15: O'hara & Co . Ltd., more than 10 ml of water. These rats were Tokyo), and amount of water intake was excluded from further drug testing. measured by a drinkometer (LA-1: O'hara & Figure 1 shows changes in the schedule- Co. Ltd.). The principle of the device and the controlled response and schedule-induced Antialcoholic Drugs and Ethanol Drinking 125 ethanol drinking by means of the overall shown in this figure. In the 8% ethanol response rate and the amount of fluid intake, session, the overall response rate increased respectively, during 2, 4, 6 and 8% ethanol significantly as compared with that in the sessions. Doses of ethanol intake are also water session. The decrease in the amount

Fig. 1. Changes in schedule-controlled response and schedule-induced drinking, expressed in terms of response rate (left panel) and amount of fluid intake (middle panel), respectively, under a fixed interval 1 min schedule of food reinforcement in rats during the 2, 4, 6 and 8% ethanol session. Doses of ethanol intake (right panel) are also shown in this figure. The rat's behaviors were observed for 1 hr. *P<0.05, **P<0 .01 : Significant difference as compared with the value in the water session (Student's t-test).

Fig. 2. Effects of disulfiram (100 and 200 mg/kg, p.o.) on schedule-controlled response (left panel) and schedule-induced water or ethanol drinking (right panel) under a fixed interval 1 min schedule of food reinforcement in rats. Disulfiram was pretreated at 1 hr before start of the experiment, and the rat's behaviors were observed for 1 hr. Open and hatched columns indicate the results in the water session and the 4% ethanol session, respectively. +PG0.05: Significant difference as compared with the value after administration of carboxymethyl cellulose Na solution (dose=0) in the water session. **P<0.01: Significant difference as compared with the value after administration of carboxymethyl cellulose Na suspension (dose=0) in the 4% ethanol session. 126 H. Kuribara, A. Higashida & S. Tadokoro

Fig. 3. Effects of calcium cyanamide (5 and 10 mg/kg, p.o.) on schedule-controlled response (left panel) and schedule-induced water or ethanol drinking (right panel) under a fixed interval 1 min schedule of food reinforcement in rats. Calcium cyanamide was pretreated at 1 hr before start of the experiment, and the rat's behaviors were observed for 1 hr. The data are expressed in the same way as in Fig. 2, except that distilled water (dose=0) was administered in the control sessions. of fluid intake was dependent on the ethanol was more marked than that observed in the concentration. However, the dose of ethanol water sessions. intake was at maximum (2.8 g/kg) in the Figure 3 shows the effects of calcium 4% ethanol session. Therefore, this ethanol cyanamide (5 and 10 mg/kg, p.o.), pre- concentration was used in the experiment treated at 1 hr before the start of the experi- for studying effects of disulfiram and calcium ment, on the schedule-controlled response cyanamide on schedule-induced ethanol and schedule-induced water and ethanol drinking. drinking. The data are presented in the same Figure 2 shows the effects of disulfiram way as in Fig. 2. In the water sessions, (100 and 200 mg/kg, p.o.), pretreated at 1 hr calcium cyanamide did not produce a marked before the start of the experiment, on the change in either the schedule-controlled schedule-controlled response and schedule- response or schedule-induced water drinking. induced water and ethanol drinking by However, calcium cyanamide significantly means of the overall response rate and the suppressed schedule-induced ethanol drink- amount of fluid intake, respectively, during ing in a dose-dependent manner without the 1 hr session. In the water sessions, eliciting a marked change in the schedule- disulfiram tended to suppress the schedule- controlled response in the ethanol sessions. controlled response, but the change was not significantly different as compared with Discussion the value in the water session after adminis- The present experiment demonstrated that tration of carboxymethyl cellulose Na. under the Fl 1 minschedule of food reinforce- Disulfiram (200 mg/kg) significantly de- ment, rats voluntarily drank ethanol solutions creased the amount of water intake. In at concentrations of more than 4%, which contrast, disulfiram (100 and 200 mg/kg) rats generally refuse to drink under normal did not induce a marked change in the breeding conditions (29, 30). The dose of schedule-controlled response in the ethanol ethanol intake during the 1 hr sessions sessions, but produced a significant decrease attained a maximum (2.8 g/kg) in the 4% in the amount of ethanol intake. The change ethanol session. These results are almost Antialcoholic Drugs and Ethanol Drinking 127 identical with those reported by many strong as that of disulfiram. Clinically, the researchers (18-25). daily doses of disulfiram and calcium The schedule-controlled response in- cyanamide for treatment of alcoholic patients creased in the 8% ethanol sessions. However, are reported to be 250-500 mg and 50-100 this behavioral change is unlikely to be mg, respectively (1 ). The results obtained attributable to a pharmacological effect of from our experiment are fairly consistent with ethanol. This is because although the dose the clinical data. However, it can not be of ethanol intake was higher in the 4 and 6% explained only by the present experiment why ethanol sessions than in the 8% ethanol no marked change in the schedule-controlled sessions, no marked change in the schedule- response was produced even though marked controlled response was found in these suppression of schedule-induced ethanol sessions. We have observed that 2-3 g/kg drinking was induced after administration of p.o. of ethanol, given to rats immediately disulfiram and calcium cyanamide. A further before the start of the session, scarcely study is required to elucidate the effect of induced changes in either the schedule- these drugs on the schedule-controlled controlled response or schedule-induced response in ethanol sessions. water drinking (H. Kuribara et al., un- For studying the combined effect of published data). In addition, Cook and disulfiram or calcium cyanamide with Singer (31) reported a marked increase in ethanol, physical signs or acetaldehyde the response rate after artificial suppression levels in the blood and/or the brain have been of the schedule-induced water drinking by usually measured as the indicators (2-13). taking off the drinking spout. These results We did not examine them in the present suggest that the marked increase in the experiment. However, it can be concluded schedule-controlled response observed in the that schedule-induced ethanol drinking in 8% ethanol session is attributable to sup- rats is applicable to behavioral studies on the pression of the schedule-induced ethanol interaction between ethanol and antialcoholic drinking due to nonspecific aversive effects drugs. This is not only because rats voluntarily of ethanol such as taste, etc. drink relatively large dose of ethanol within Disulfiram (200 mg/kg) tended to suppress a short period under the experimental both the schedule-controlled response and situation, but also because the ethanol schedule-induced water drinking. These drinking can be used as the indicator of the results are probably due to a effect effect of antialcoholic drugs. of this drug (1). In contrast, no marked change in either the schedule-controlled References response or schedule-induced water drinking 1 Ritchie, J.M.: The aliphatic alcohols. 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