β-Endorphin administration interferes with the acquisition and initial maintenance of ethanol preference in the rat

Physiology & Behavior, Vol. 45, pp. 87-92. Copyright©Pergamon Press plc, 1989. Printed in the U.S.A.

0031-9384/89 $3.00 + .00

/3-Endorphin Administration Interferes With the Acquisition and Initial Maintenance of Ethanol Preference in the Rat CARMEN

S A N D I , 1 JOSI~ B O R R E L L

AND CARMEN

GUAZA 2

Department o f Psychobiology, Cajal Institute, CSIC, Veldzquez 144, 28006 Madrid, Spain R e c e i v e d 22 A p r i l 1988 SANDI, C., J. BORRELL AND C. GUAZA. fl-Endorphin administration interferes with the acquisition and initial maintenance of ethanol preference in the rat. PHYSIOL BEHAV 45(1) 87-92, 1989.--Attention has been focused on the possibility of an interaction between the endorphinergic system and ethanol intake. In the present study, the effects of subcutaneous (SC) administration of/3-endorphin (/3-E) (0.25, 1 and 5 ~.g/kg) and/or naloxone (NX) (1 or 2.5 mg/kg) on ethanol preference (EP) have been investigated in rats. Under our procedural conditions, rats developed ethanol preference (EP) by a forced ethanol drinking session (conditioning session). Preconditioning administration of fl-E (1 p.g/kg) reduced later EP. When fl-E was administered postconditioning, the opioid affects ethanol preference depending on the dose: both 0.25/~g/kg and 5/xg/kg reduced EP, but the dose of 1/zg/kg did not alter it. Administration of/3-E (1 and 5/.tg/kg) before the first testing session attenuated EP. NX antagonized the effects of fl-E on EP in the three experimental procedures used, indicating that/x-opioid receptors might be involved in the/3-E-induced reductions on EP. Our results provide further evidences for a/3-endorphinergic system involvement on the mechanisms leading to consumption of ethanol. /3-Endorphin

Naloxone

Ethanol preference

Rat

A line of evidence supporting the idea of a particular involvement of opioid peptides on ethanol intake is derived mainly from assessments of the effects of morphine and naloxone on ethanol consumption. Morphine has been reported to have dose-related opposite effects: low doses increase (12,13) and high doses decrease ethanol intake (10,33). Opioid antagonists, such as naltrexone or naloxone, reduce ethanol intake (1, 5, 12, 21, 29, 30). Although not all rats avoid all concentrations of alcohol (34), naive rats normally consume little alcohol in a spontaneous choice situation (6). Forced consumption of low ethanol concentrations seems to be one way of increasing a rat's subsequent preference (12). We have previously shown (8) that ethanol solution (2.5%) is avoided by naive male Wistar rats in favor of water. However, when these animals are forced to drink such ethanol solution as their only source of fluid prior to having a free choice, they reverse Jheir original aversion and consume more ethanol solution than water across the following days. It has been suggested that the ability of ethanol to reinforce ethanol intake might be related to an incremented activity at the dopaminergic mesolimbic system (7) or through interactions with endogenous opioids (25). There is considerable evidence supporting the idea that endorphins can induce motivational effects (2, 7, 28). The ability of opioid effects to serve as positively reinforcing, as well as aversive stimuli, is supported by results from different behavioral procedures (38); for example, using taste and

place conditioning procedures, preference/aversion conditioning have been reported for different/~-opioid agonists (22,24). Although the idea of opioid-alcohol interactions has been largely assessed, their common mechanisms still remain unexplained (26). It is noteworthy that some of the factors leading to increased ethanol intake (food and water deprivation, continued exposure to ethanol, etc.) (3,12) have also been shown to cause changes in endogenous opioids (4, 27, 31). The aim of the present study was to investigate further whether or not/3-endorphin can influence the acquisition and initial maintenance of preference for ethanol. METHOD

Subjects The subjects were 443 adult male Wistar rats (CIB, Spain) weighing 200--250 g at the beginning of the experiments. They were housed in group cages (4--5 animals per group) and maintained under a light-dark schedule (lights on between 7:00 and 19:00 hr) and temperature (22°C) controlled conditions. Food and water were freely available in the home cages.

Procedure After one week of adaptation to the drinking bottles, rats were weighed, individually housed and deprived of water for 72 hr. The duration of deprivation was deemed necessary to assure that all rats drank immediately when fluid was again

1Supported by a FPI Predoctoral Fellowship from the MEC. 2Requests for reprints should be addressed to C. Guaza, Department of Psychobiology, Cajal Institute, Velazquez 144, 28006 Madrid, Spain.

87

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FIG. 1. (A) Effect of/3-endorphin administration 15 minutes before the conditioning session on ethanol preference throughout the 3 consecutive retention testing days. Number of animals: saline=27; fl-end (0.25 g.g/kg)=16; /3-end (1 /~g/kg)=28; fl-end (5 tzg/kg)=18. Ap<0.001 vs. saline (3 retention days). (B) Effect of administration of/3-endorphin and/or naloxone 15 minutes before the conditioning session on ethanol preference in the first retention testing day. Number of animals: l0 rats per group, ep<0.05 and &p<0.005 vs. saline; ©p<0.05 vs./3-E; Ap<0.001 vs. NX. Results show ethanol preference scores: mean-+standard deviation.

made available; it resulted in a mean weight loss of less than 13%. Subsequently, on the 5 consecutive days of the experimental procedure, the animals were maintained on a 24 hr water-deprivation schedule with fluids only available during the 15-rain period of the drinking sessions. A two-bottle choice procedure was used in order to evaluate ethanol preference over water. Our previous experiments (8) showed that rats forced to drink a weak ethanol solution (2.5%) develop a stable baseline on alcohol preference in consecutive days, while rats with concentrations of 4% or 7% failed to develop ethanol preference in subsequent testing. Thus, the forced alcohol session (2.5%) is termed a "conditioning session"--in terms of operant behavior--since rats without this session do not develop a preference for alcohol. In summary, the experiments started with one day of habituation when the animals were allowed access to tap water in the two bottles. The following day (conditioning day) the two bottles were filled with a 2.5% ethanol solution. On the 3 subsequent days (retention days) the animals were presented with a two-bottle choice between 2.5% ethanol solution and tap water.

Treatments Animals were treated with/3-endorphin and/or naloxone according to one of the three procedures: Preconditioning. Rats were injected 15 min before the ethanol exposure (f'wst ethanol presentation). Postconditioning. Rats were injected immediately after the forced ethanol exposure. Preretention. Injections were made 15 min before the first retention day (choice test).

Drugs /3-Endorphin (Bachem, Inc. Torrance, CA) and/or naloxone hydrochloride (Dupont Pharmaceuticals, Switzerland) were used throughout the e x p e ~ n t s . The drugs (at the designated doses) were dissolved in physiolngical saline to obtain a constant injection volume of 0.5 ml. Control animals received the same volume of sah'ne as ptaeebo.

Statistics Fluid intake measures were corrected for body weight (ml/100 g body weight). Ethanol preference is expressed by the index: EP = [Ethanol intake/(Ethanol + Water intake)] x 100. Data were statistically analyzed using one or two way analyses of variance. For statistical analysis, the preference scores for each subject were subjected to aresin transformation in order to satisfy the assumptions of the analysis of variance before carrying out ANOVA, This transformation is used to adjust for the lack of normality of p e r c e n ~ or proportional data (37). Posteriori Tukey's multiple e ~ son tests were carried out when A N O V A ' s revealed S ~ i f i cant effects. RESULTS

Experiment 1: Preconditioning Administration The effects offl-E administration (0.25, 1 and 5/~g/i~) on ethanol preference are shown in Fig. IA. A N O V A of the data showed a significant treatment effect, F(3,225)=6.21, p<0.001, due to the attenuation in ethanol preference by the dose of 1/zg/kg offl-E, which is maintained throughout the 3 consecutive retention days.

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FIG. 2. (A) Effect of/3-endorphin administration immediately after the conditioning session on later ethanol preference throughout 3 consecutive retention testing days. Number of animals: saline=33;/3-end (0.25/zg/kg)=24; B-end (1 ~.g/kg)= 16; B-end (5 p.g/kg)= 14. lip<0.05 vs. saline (3 retention days). Op<0.05 and Ap<0.001 vs. saline (first retention day). (B) Effect of administration of/3-endorphin and /3-endorphin plus naloxone, immediately after the first ethanol exposure, on ethanol preference in the first retention day. Number of animals: saline=33; B-end (0.25 /~g/kg)=24; /3-end (0.25 /~g/kg) + NX (2.5 mg/kg)= 18;/3-end (5/.Lg/kg)=14;/3-end (0.25 p.g/kg) + NX (2.5 mg/kg)= 15. Op<0.05 and Ap<0.001 vs. saline; Op<0.05 vs. corresponding/3-E group. Results show ethanol preference scores; mean+-standard deviation.

The possibility that the observed effect of #-E could be mediated by interactions with opioid receptors was assessed by the concomitant administration of/3-E (1/zg/kg) and NX (2.5 mg/kg) (Fig. 1B). A N O V A of the data revealed a significant treatment effect, F(3,36)=7.76, p<0.001, fl-E significantly reduced ethanol preference. EP was also reduced when NX was administered alone. However, the simultaneous administration of/3-E and NX significantly reversed the effect o f the opioid agonist. Preconditioning administration of/3-E, at all doses tested, did not modify ethanol fluid consumption at the conditioning session. The effects of preconditioning administration of/3-E (1 /zg/kg) and/or NX (2.5 mg/kg) on ethanol drinking are shown in Table 1. A N O V A of the data showed a significant treatment effect, F(3,36)=31.36, p<0.001. ~-E did not alter ethanol intake, whereas the antagonist NX significantly reduced ethanol intake. However, the simultaneous administration o f / 3 - E and NX reversed the reduction on ethanol consumption induced by the antagonist.

Experiment 2: Postconditioning Administration Effects o f / 3 - E administration (0.25, 1 and 5 /zg/kg) on ethanol preference are shown in Fig. 2A. A N O V A of the three retention days yielded a significant effect, F(3,249)=7.46, p<0.001, for the factor associated with/3-E treatment, due to a persistent reduction on EP with the dose of 0.25 /zg/kg. It is interesting to note that A N O V A of ethanol preference scores at the first retention day revealed a

TABLE 1 FLUID CONSUMPTION (ml/100g BODY) IN THE CONDITIONING SESSION BY RATS SUBJECTED TO/3-E AND/OR NX PRECONDITIONING ADMINISTRATION Total Intake (Ethanol) Saline /3-E 1/zg/kg NX 2.5 mg/kg fl-E + NX

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significant treatment effect, F(3,83)=7.86, p<0.001: the lower dose of/3-E reduced EP, whereas the medium dose had no effect and the higher dose attenuated EP. The effects of fl-E (0.25, and 5/zg/kg) and fl-E plus NX (2.5 mg/kg) on ethanol preference are shown in Fig. 2B. A N O V A of the data revealed a significant effect, F(4,99)= 6.28, p<0.001, of the treatments. The effect on EP of both doses of fl-E were reversed by administration of NX. In a previous study (29) it was found that postconditioning administration of NX failed to modify EP.

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FIG. 3. (A) Effect of/3-endorphin administration 15 minutes before the first retention test on ethanol preference in the same and consecutive retention days. Number of animals: saline= 19; /3-end (0.25/xg/kg)= 19; fl-end (1/zg/kg)=20; fl-end (5/zg/kg)=20. &p <0.005 vs. saline (3 retention days). (B) Effect of administration of fl-endorphin and/or naloxone 15 minutes prior to the first retention test on ethanol preference in the same day. Number of animals: saline=28;/3-end (5 /zg/kg)=29; NX (1 mg/kg)= 10;/3-end (5/.tg/kg) + NX (1 mg/kg)= 10; NX (2.5 mg/kg)= t9; B-end (5 /xg/kg) + NX (2.5 mg/kg)=19. Ap<0.001 vs. saline; Op<0.05 and ~p<0.005 vs. /3-E. Results show ethanol preference scores: mean--standard deviation.

Experiment 3: Preretention Administration The effects of/3-E administration (0.25, 1 and 5/zg/kg) on ethanol preference are shown in Fig. 3A. A N O V A of the data showed a significant treatment effect, F(3,222)=5.72, p<0.001. This effect was due to a reduction, throughout the 2 earlier days on EP induced by the administration o f 1 and 5 /zg/kg of/3-E. The effects of fl-E (5/zg/kg), NX (1 and 2.5 mg/kg) and /3-E plus NX on ethanol preference are shown in Fig. 3B. A N O V A of the data showed a significant treatment effect, F(5,110) = 10.60, p <0.001. Administration of 1 mg/kg of NX failed to modify EP, whereas a dose of 2.5 mg/kg significantly reduced EP. However, both doses of NX significantly antagonized the reduction on EP induced by the opioid peptide. DISCUSSION

The results of the present study show that under the schedule followed (one conditioning session in which alcohol is offered as the only source of drinking, followed by a free two-bottle choice test), rats acquired preference for a weak (2.5%) ethanol solution. This preference is maintained for at least three consecutive days. It has been suggested that learning and memory processes, likely associated to the reinforcing properties of ethanol, seems to .play an important role in the free consumption of the drug (6,34). Learning and memory may be related to neurohumoral changes that persist into the posttraining period (16). In particular, the brain/3-E system seems to be activated by the simple exposure o f rats to a

novel situation as well as during training (16,32). In addition, endogenous opioids seem to be also involved in the reinforcing mechanisms of ethanol intake (39). Therefore, it would appear that the acquisition and maintenance o f preference to ethanol could be influenced by alterations on the endogenous opioid systems. As in a previous paper (30), here again NX injected immediately prior to the drinking sessions attenuated EP. Since preconditioning administration o f NX reduced significantly ethanol intake in the conditioning session, the amount of ethanol consumed by the rats might not be enough to induce the ethanol reinforcing effects likely associated to the acquisition of preference to the drug. The fact that postconditioning administration of NX failed to affect later EP s e e m s to indicate the absence o f aversive effects from NX. Preretention administration of NX diminished EP, a fact consistent with previous works in which N X decreased intake of preferred solutions (19). It can be suggested that the attenuated intake of ethanol induced by the prior administration of NX could be due to a blockade of the reinforcement properties o f alcohol and/or a shift in the gustatory or olfactory threshold o f the animals for alcohol, as it has been previously shown for alcohol (5, 20, 36) and other flavors (18). When rats were injected with/3-E prior to the first exposure to ethanol their endogenous endorphinergic s y s t e m is expected to be activated. In the present study, only the dose of 1/xg/kg of the opioid induced reductions in e fl~mol preference. It has been postulated that individual d ~ e r e n c e s in production of endogenous opioid peptides may be related to individual differences in the propensity to drink alcoholic

fl-ENDORPHIN AND E T H A N O L P R E F E R E N C E

91

beverages (12). Thus, each /3-E dose-treated group could drink ethanol depending on their endorphinergic status. Drug administration shortly before the conditioning session may influence both acquisition and consolidation of newly acquired information (9). Assuming that learning and memory processes may be involved in the acquisition and maintenance of taste preferences, it can be suggested that preconditioning administration of fl-E could interfere with mechanisms involved in the storage of the information acquired during the conditioning session. Posttraining treatment (i.e., injection of a drug following the presentation of a flavored solution) is the procedure more generally used to establish conditioned taste preference/aversion (CTP/CTA). The CTA paradigm involves the classical conditioning of a taste-conditioned stimulus (CS) paired with an aversive unconditioned stimulus (UCS). Postconditioning administration of/3-E affected EP depending on the dose used: 0.25/xg/kg reduced EP, 1/xg/kg did not reliably alter EP and 5 /xg/kg attenuated EP. It seems unlikely that the reduction in PE induced by administration of the lower dose of/3-E could be the result o f a CTA to ethanol induced by the drug since the dose of 1 /xg/kg was ineffective. However we cannot dismiss the possibility that the reduction of EP caused by administration of the higher dose of fl-E could be due to aversive properties of the opioid and consequently through the induction of a CTA to ethanol. Using repeated pairings of/x-agonists with neutral flavours, it has been shown that taste preferences can be induced by administration of low doses of opioids whereas at higher doses taste preferences are absent or replaced by taste aversion (23). However, in that study, at variance with the schedule followed in our work, the ability of opioids to serve as a UCS might be linked to the repeated drug-flavour pairings. The effects of agents administered shortly after learning have been interpreted in terms of effects upon consolidation of memory (9). A common factor for several putative modulators of memory is its nonlinear dose-response effects. It is interesting to note that in the present study postconditioning administration of the lowest and highest dose of/3-E reduced ethanol preference, whereas the medium dose was ineffective. As in our study, nonlinear dose-response effects have been described for the effects of endogenous opioids on learning and memory processes (14). Taking into consideration that fl-E may cause retrograde amnesia (16), it can be postulated that under our experimental conditions, administration of the opioid immediately after the conditioning ses-

sion, might attenuate the development of preference to ethanol by interfering with memory storage, as it has been postulated for other behavioural paradigms. Administration of 1 and 5/xg/kg of/3-E shortly before the first testing session attenuated ethanol preference. Thus, fl-E was able to interfere with mechanisms involved in the consumption of ethanol when preference has just previously been acquired. Hubbell et al. (12) reported that morphine enhances ethanol intake; however, in their study rats had a prolonged history of taking the alcoholic beverage prior to experimental procedures. Nevertheless, in agreement with the results of the present study, reductions in ethanol consumption after preadministration of morphine (35) or opioid peptides, like met-enkephalin (11), have been previously reported. As suggested for preconditioning administration of /3-E, a different activation of endogenous opioid systems may modulate avidity for ethanol at the In'st retention testing day. Even more, preretention administration of/3-E could interfere with mechanisms making the information stored at the conditioning session available for retrieval in the retention session, as suggested for other tasks involving learning and memory (17). The fact that reductions of preference remained through subsequent retention days extend the possibility that/3-E, interfering with the mechanisms leading to drinking more ethanol than water at the first choice day, modify later propensity to drink ethanol in rats. NX antagonized the effects of/3-E on EP in the three experimental procedures used. Since fl-E shows high affinity for the/x-subtype receptor, the blockage of/3-E-induced decreases on EP with NX supports the idea that this type of receptors might be involved in the effect of the opioid. The present findings provide stronger evidence for the idea that endorphins may be involved in the reinforcing mechanisms which lead to consumption of ethanol; in particular, in ethanol preference behaviour in which learning and memory processes display an important role. Indeed, a justdeveloped ethanol preference behaviour seems to be highly sensitive to alterations in the activity of the endogenous /3-endorphinergic system.

ACKNOWLEDGEMENTS The authors express their appreciation to C. Bail6n and P. Baranda for their expert technical assistance. This work has been supported by CAICYT grants.

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