Hydroxybutyric Acid Intake in Ethanol-preferring sP and -nonpreferring sNP Rats

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Hydroxybutyric Acid Intake in Ethanol-preferring sP and -nonpreferring sNP Rats
  PII S0031-9384(98)00033-X   -Hydroxybutyric Acid Intake in Ethanol-preferring sP and -nonpreferring sNP Rats GIANCARLO COLOMBO, 1 * ROBERTA AGABIO,† GIACOMO DIAZ,‡ MAURO FA`,† CARLA LOBINA,†ROBERTA REALI† AND GIAN LUIGI GESSA† *CNR Center for Neuropharmacology, I-09124 Cagliari, †“Bernard B. Brodie” Department of Neuroscience, Universityof Cagliari, I-09124 Cagliari, and ‡Department of Cytomorphology, University of Cagliari, I-09124 Cagliari, Italy Received 24 July 1997; Accepted 20 January 1998 COLOMBO, G., R. AGABIO, G. DIAZ, M. FA`, C. LOBINA, R. REALI AND G. L. GESSA.    -Hydroxybutyric acid intake inethanol-preferring sP and -nonpreferring sNP rats.  PHYSIOL BEHAV  64 (2) 197–202, 1998.—   -hydroxybutyric acid (GHB) andethanol share several pharmacological similarities, suggesting that GHB may exert ethanol-like effects in the central nervous system.The present study was designed to test whether selectively bred ethanol-preferring rats would, unlike ethanol-nonpreferring ones,self-administer GHB, consistent with their higher preference for ethanol. Male ethanol-naive Sardinian alcohol-preferring (sP) andSardinian alcohol-nonpreferring (sNP) rats were used. In Experiment 1, GHB solution (1% (w/v) in water) was initially offered as thesole fluid available for 14 consecutive days and then presented under the two-bottle, free-choice regimen, one bottle containing waterand the other the GHB solution, for an additional 14 consecutive days. During the free-choice phase, high preference for GHB andintake of pharmacologically relevant daily doses of GHB developed in both rat lines, presumably because the 14-day no-choice periodwould unmask the reinforcing properties of GHB and lead to acquisition of GHB preference also in the supposedly less susceptiblesNP rats. In Experiment 2, the forced GHB drinking phase was reduced to 3 days. Under the subsequent free-choice regimen, dailyGHB preference and intake were initially low in both sP and sNP rats; however, after approximately 10 days, GHB preference andintake in sP rats rose progressively and then stabilized to significantly higher levels than in sNP rats throughout the entire free-choicephase. It is likely that episodic binges of GHB intake occurring during the first 10 days resulted in experiencing the reinforcingproperties of GHB by sP but not sNP rats. The results of the present study suggest that a) sP rats are genetically more sensitive to thereinforcing effects of both ethanol and GHB than sNP rats; and b) disclosure of the higher sensitivity of sP rats to the reinforcing effectsof GHB is a function of the length of the induction procedure. The results are also discussed in terms of differences in GHB receptorscontributing to the predisposition to ethanol preference and avoidance, respectively. © 1998 Elsevier Science Inc.   -Hydroxybutyric acid (GHB) Voluntary oral GHB intake GHB preference Induction procedureSardinian alcohol-preferring (sP) rats Sardinian alcohol-nonpreferring (sNP) rats   -HYDROXYBUTYRIC acid (GHB), an endogenous brain con-stituent likely acting as a neurotransmitter or neuromodulator,produces several neuropharmacological effects in laboratory ani-mals and humans (see (2,26)). The pharmacological profile of GHB shares several relevant similarities with that of ethanol. First,both drugs produce anxiolysis (20,23,31,34), hypothermia (15,25),and anesthesia (24,27) in laboratory animals. Second, GHB andethanol possess similar discriminative stimulus effects (i.e., theanimal correlate of subjective feelings elicited by psychoactivedrugs), as revealed by cross-substitution in a drug discriminationstudy (9). Finally, cross-tolerance to the motor-impairing effects of GHB and ethanol has been reported to occur in rats (8). The closesimilarities between GHB and ethanol constitute the rationale forthe use of GHB in the treatment of alcohol dependence. Indeed, ithas been proposed that GHB reduces ethanol craving and con-sumption (1,17) and also alleviates ethanol withdrawal symptoms(16,32) in alcoholic patients by mimicking the actions of ethanol inthe central nervous system (9,11).A further similarity between GHB and ethanol is constituted bythe abuse liability. Recent studies have demonstrated that GHB a)is self-administered by rodents both orally (5) and intravenously(28); and b) serves as a positive reinforcer in rats tested in aconditioned place preference paradigm (30). Accordingly, clinicalsurveys and reports from U.S. agencies have shown that GHBproduces salient feelings of euphoria, anxiolysis, and relaxation(e.g., (3,12,18)), which are often described as closely resemblingthose of ethanol (18) and lead to the intake of intoxicating doses(1,18).The similarity of the pharmacological effects of GHB andethanol led to the hypothesis that GHB would be self-administeredand preferred by ethanol-preferring rats rather than ethanol-avoid-ing ones. This hypothesis was tested in the present study using ratsfrom the Sardinian alcohol-preferring (sP) and Sardinian alcohol-nonpreferring (sNP) lines, selectively bred for opposite ethanolpreference and consumption in this laboratory (see (4)). Under thestandard two-bottle, free-choice regimen, sP rats consume around 1 To whom requests for reprints should be addressed. E-mail: colomb@unica.it Physiology & Behavior, Vol. 64, No. 2, pp. 197–202, 1998© 1998 Elsevier Science Inc. All rights reserved.Printed in the U.S.A.0031-9384/98 $19.00  .00 197  6 g/kg of ethanol daily and avoid water almost completely; incontrast, sNP rats consume negligible daily amounts of ethanol andgreatly prefer water over the ethanol solution.EXPERIMENT 1A procedure for establishing voluntary oral intake of pharma-cologically relevant doses of GHB in the rat has been recently setup in this laboratory (5). This procedure includes a 14-day periodduring which GHB is the only fluid available, followed by a “GHBversus water” free-choice paradigm. The initial no-choice phasewas necessary because the unpleasant taste of the GHB solutionhampered the rats from discovering the reinforcing properties of the drug (5).Experiment 1 tested GHB preference and consumption in sPand sNP rats under the procedure of the reference study (5). MATERIALS AND METHODS  Animals Male sP [ n    12, 484    11 g (mean initial body weight   SEM)] and sNP [ n  12, 505  14 g (mean initial body weight  SEM)] from the 38th generation and aged 4 months at the start of the study were used. Rats were singly housed in standard plasticcages with wood chip bedding under a 12-h artificial light–dark cycle (lights on at 2000 hours) at a constant temperature of 22  2°C and relative humidity of 60%. Standard laboratory rat chow(MIL Morini, San Polo d’Enza, Italy) was provided ad lib. to therats throughout the experimental period. Rat body weights weremonitored twice per week. Prior to the start of the study, rats wereboth ethanol- and GHB-naive. Procedure Rats were initially forced to drink 1% (w/v) GHB (sodium salt,Laboratorio Farmaceutico C.T., Sanremo, Italy) dissolved in tapwater as the sole drinking fluid available for 14 consecutive days.On the fifteenth day, rats were moved to the free-choice regi-men. Two graduated bottles containing tap water and 1% (w/v)GHB in water, respectively, were continuously offered for 14consecutive days. GHB and water intakes were monitored daily at0800 hours. Bottles were refilled every day with fresh solution andtheir position reversed daily to avoid development of positionhabit.  Data Analyses Data on daily GHB intake (expressed in mg/kg) were analyzedby a two-way (Rat Lines, Days) ANOVA with repeated measureson Days for both experimental phases (forced and voluntary drink-ing).Data on daily water intake (expressed in mL/kg) and GHBpreference (expressed as percentage of GHB solution consumedover total fluid intake) were analyzed by a two-way (Rat Lines,Days) ANOVA with repeated measures on Days for the voluntarydrinking phase. RESULTS AND DISCUSSION Results of the ANOVAs for GHB and water intake as well asGHB preference for both forced and voluntary drinking phases arereported in Table 1.During the 14-day no-choice period, daily GHB intake oc-curred at pharmacologically relevant doses (higher than 600 mg/ kg) in both sP and sNP rats, without significant line differences.Once GHB and water were offered under the free-choice reg-imen, preference for GHB was observed in all rats. However,ANOVA revealed that the only significant effect consisted inperiodic changes in daily GHB and water intakes and GHB pref-erence, which were independent of the rat line. Indeed, as previ-ously observed in Wistar rats (5), all rats alternated periods of highdaily intake of GHB with temporarily self-imposed avoidance of the GHB solution. Average daily GHB intake was not differentbetween sP and sNP rats and ranged between 400 and 600 mg/kg/ day throughout the 14-day period (Fig. 1, top panel). No linedifference in the average daily intake of water (Fig. 1, centerpanel) and preference for GHB (Fig. 1, bottom panel) was found.The results of this experiment a) confirm that GHB can bevoluntarily ingested by the rat at doses that are expected to producesalient psychopharmacological effects and b) extend to sP and sNPrats the ability of the 14-day period of forced GHB drinking toinduce the GHB preference previously observed in Wistar rats (5).No difference in GHB preference and consumption was re-corded between sP and sNP rats during the free-choice phase.Thus, the results of Experiment 1 fail to support the initial predic-tion of a divergence in GHB intake between sP and sNP ratsconsistent with their opposite proclivity to consume ethanol. How-ever, it could not be ruled out at this stage that the procedureemployed to develop GHB preference in the present experimentmight have been inadequate for disclosing possible differences involuntary GHB intake between sP and sNP rats. Indeed, the 14-dayforced GHB drinking might have acted as such a powerful proce-dure for induction of GHB preference and self-administration thatit led to the unmasking of the reinforcing properties of GHB andacquisition of GHB drinking behavior also in the supposedly lesssusceptible sNP rats and, subsequently, to the development of aceiling effect in terms of GHB intake between the two rat lines.EXPERIMENT 2The development of similar GHB preference and consumptionin sP and sNP rats observed in Experiment 1 suggested that ashorter period of forced GHB drinking might differentiate volun-tary GHB consumption between these two rat lines.TABLE 1 TWO-WAY ANOVA FOR GHB AND WATER INTAKE AS WELL ASGHB PREFERENCE IN SARDINIAN ALCOHOL-PREFERRING(sP,  n    12) AND SARDINIAN ALCOHOL-NONPREFERRING(sNP,  n    12) RATS IN EXPERIMENT 1Phase Source Effect df   F p  Level No choiceGHB intake Rat Line 1 3.541 NSDay 13 3.765 0.000014Rat Line  Day 13 1.248 NSFree choiceGHB intake Rat Line 1 0.507 NSDay 13 3.559 0.000035Rat Line  Day 13 1.633 NSWater intake Rat Line 1 1.972 NSDay 13 4.657   0.000001Rat Line  Day 13 0.688 NSGHB preference Rat Line 1 0.090 NSDay 13 5.552   0.000001Rat Line  Day 13 0.540 NSNo-choice phase: GHB [1% (w/v) in water] as the sole drinking fluidavailable for 14 consecutive days; free-choice phase: GHB [1% (w/v) inwater] and tap water under the two-bottle regimen for 14 consecutive days. 198 COLOMBO ET AL.  Thus, in the present experiment, sP and sNP rats wereexposed to forced GHB drinking for only 3 consecutivedays. MATERIALS AND METHODS  Animals Male sP [ n    9, 402    12 g (mean initial body weight   SEM)] and sNP [ n  10, 421  8 g (mean initial body weight  SEM)] from the 39th generation and 3 months old at the start of thestudy were used. Rats were housed under conditions identical tothose in Experiment 1. Rats were ethanol- and GHB-naive. Procedure The procedure was similar to that used in Experiment 1. Theonly exceptions were the duration of the forced drinking period (3days) and the voluntary drinking period (28 days).  Data Analyses Data analyses were performed as indicated in Experiment 1. Inaddition, occurrence of at least 1 day of GHB preference [definedby a daily intake of GHB solution greater than 50% of the totalfluid intake (5)] was evaluated by a Fisher’s exact probability testfor a 2  2 table. RESULTS AND DISCUSSION Results of the ANOVAs for GHB and water intake as well asGHB preference for both forced and voluntary drinking phases arereported in Table 2.During the 3-day no-choice period, sP rats consumed signifi-cantly more GHB than sNP rats (Fig. 2, top panel). Average dailyGHB intake ranged between 800 and 1000 mg/kg in sP rats and700 and 800 mg/kg in sNP rats.Once the GHB solution was presented under the free-choiceregimen with water, the daily mean intake of GHB dropped toapproximately 200 mg/kg in both sP and sNP rats and persisted atthis low level for 10 days (Fig. 2, top panel). Then, quite unex-pectedly, the average daily GHB intake in sP rats increased pro-gressively and subsequently settled at 500–700 mg/kg (Fig. 2, topTABLE 2 TWO-WAY ANOVA FOR GHB AND WATER INTAKE AS WELL ASGHB PREFERENCE IN SARDINIAN ALCOHOL-PREFERRING(sP,  n    9) AND SARDINIAN ALCOHOL-NONPREFERRING(sNP,  n    10) RATS IN EXPERIMENT 2Phase Source Effect df   F p  Level No choiceGHB intake Rat Line 1 4.730 0.044043Day 2 5.274 0.010117Rat Line  Day 2 0.474 NSFree choiceGHB intake Rat Line 1 10.052 0.005590Day 27 2.570 0.000037Rat Line  Day 27 3.063 0.000001Water intake Rat Line 1 0.721 NSDay 27 4.857   0.000001Rat Line  Day 27 4.604   0.000001GHB preference Rat Line 1 6.052 0.024900Day 27 2.922 0.000002Rat Line  Day 27 3.714   0.000001No-choice phase: GHB [1% (w/v) in water] as the sole drinking fluidavailable for 3 consecutive days; free-choice phase: GHB [1% (w/v) inwater] and tap water under the two-bottle regimen for 28 consecutive days.FIG. 1. Daily GHB intake (in mg/kg; top panel), water intake (in mL/kg;center panel), and GHB preference (expressed as percentage of GHB solutionconsumedovertotalfluidintake;bottompanel)inSardinianalcohol-preferring(sP,  n  12; F ) and Sardinian alcohol-nonpreferring (sNP,  n  12; E ) rats inExperiment 1. During the 14-day no-choice phase, GHB (1% (w/v) in water)was presented as the sole drinking fluid available; during the 14-day free-choice phase, GHB (1% (w/v) in water) and tap water were offered under thetwo-bottle regimen. Each point is the mean  SEM of   n  subjects. GHB INTAKE IN ETHANOL-PREFERRING RATS 199  panel). Furthermore, only 1 of the 9 sP rats used in the presentexperiment showed no day of preference for GHB. As previouslymonitored in Experiment 1, a large between-day variability indaily GHB intake occurred in sP rats, with an alternation of daysof high GHB preference and consumption and days of low GHBintake and water preference. In contrast to data from sP rats,average daily GHB intake in sNP rats remained steadily lower than250 mg/kg throughout the 28-day period (Fig. 2, top panel), andonly 3 out of 10 rats displayed at least 1 day of GHB preference(  p  0.01977 with respect to sP rats) during the free-choice phase.Concurrent with the increase in GHB intake, a compensatorydecrease in water consumption occurred in sP rats (Fig. 2, centerpanel). Unfortunately, this phenomenon, well represented in theplot, is not validated by the statistical test because it considers onlythe overall average, disregarding the presence of crossing trends.The pattern of GHB preference closely paralleled that of GHBintake. Initially, the preference ratio was constantly low in both sPand sNP rats. After the first 9–10 days, the mean daily preferenceratio in sP rats increased progressively and then stabilized, aver-aging between 45% and 70% (Fig. 2, bottom panel). In contrast,average daily preference ratio in sNP rats remained steadily lowthroughout the 28-day period.The results of the present experiment indicate that the 3-dayperiod of forced GHB drinking was ineffective in inducing GHBpreference and intake of pharmacologically relevant daily doses of GHB in both sP and sNP rats. Indeed, GHB preference and intakeduring the first 10 days of the subsequent free-choice phase weremoderately low in both rat lines, 2–3 times less than those mon-itored in the corresponding phase after the longer (14 day) induc-tion period (Experiment 1). Thus, although pharmacologicallyrelevant doses of GHB were consumed during the 3-day forceddrinking period in both rat lines, it can be hypothesized that ratswere unable, in such a short time, to experience the reinforcingproperties of GHB and learn consequently that these were second-ary to drinking the GHB solution.However, after this initial period of GHB avoidance, GHBintake in sP rats progressively increased and occurred at dosessimilar to those registered in Experiment 1 in both sP and sNP ratsas well as in Wistar rats in the reference study (5). A likelyexplanation for this result is that episodic binges of GHB intakeoccurring during the first 10 days of the free-choice phase resulted,in these rats, in the perception of the reinforcing properties of GHBand in acquisition of the drinking behavior. In contrast, GHBintake in sNP rats never increased during the free-choice phase of this experiment. It can be speculated that GHB intake in these ratsdid not result in the perception of effects as reinforcing as in sPrats.EXPERIMENT 3Data from Experiment 2 demonstrate that sP rats consumedsignificantly more GHB than sNP rats. However, the oral admin-istration procedure employed in the present study might haveintroduced some potentially confounding taste factor. Indeed, theGHB solution used in this study possesses an unpleasant taste thatprevents naive rats from drinking (5). Experiment 3 was designedto evaluate whether sP and sNP rats may differ in their preferencefor an unpleasant fluid such as a salty solution. To this aim,solutions with different concentrations of sodium chloride wereoffered to sP and sNP rats under a two-bottle, free-choice regimensimilar to that employed in the GHB drinking studies. MATERIALS AND METHODS  Animals Male sP and sNP rats from the 39th generation were used. Ratswere 4 months old and weighed approximately 400–500 g. Ratswere drug- and sodium chloride-naive and were housed underconditions identical to those in Experiments 1 and 2. Procedure Rats were divided into groups of five subjects each and offeredtwo graduated bottles containing tap water and sodium chloride FIG. 2. Daily GHB intake (in mg/kg; top panel), water intake (in mL/kg;center panel), and GHB preference (expressed as percentage of GHBsolution consumed over total fluid intake; bottom panel) in Sardinianalcohol-preferring (sP,  n    9;  F ) and Sardinian alcohol-nonpreferring(sNP,  n  10; E ) rats in Experiment 2. During the 3-day no-choice phase,GHB (1% (w/v) in water) was presented as the sole drinking fluid avail-able; during the 28-day free-choice phase, GHB (1% (w/v) in water) andtap water were offered under the two-bottle regimen. Each point is themean  SEM of   n  subjects. 200 COLOMBO ET AL.  [0.45%, 0.9%, and 1.8% (w/v) (Merck, Darmstadt, Germany)]dissolved in tap water for 24 h.  Data Analyses Comparison of sodium chloride intake (expressed in mL/kg of the solution) between sP and sNP rats was performed by theunpaired, two-tailed Mann–Whitney test. RESULTS AND DISCUSSION As shown in Table 3, virtually identical intakes of the sodiumchloride solution were recorded in sP and sNP rats at each con-centration tested. Thus, taste factors are highly unlikely to accountfor the differential intake of GHB observed in sP and sNP rats(Experiment 2).GENERAL DISCUSSIONThe results of the present study demonstrate that sP rats,selectively bred for high ethanol preference and intake, orallyself-administer larger daily amounts of GHB and display higherpreference for GHB than ethanol-nonpreferring sNP rats; however,disclosure of the differential preference ratio and intake was de-pendent on the experimental procedure. Indeed, presentation of theGHB solution as the sole fluid available for 14 consecutive daysestablished preference for GHB and consumption of pharmacolog-ically relevant daily amounts of GHB in both rat lines (Experiment1), duplicating the results of a previous study from this laboratorywith unselected Wistar rats (5). Once the forced GHB drinkingperiod was restricted to 3 days, GHB preference and intake wereinitially low in both rat lines, indicative of the inability of thisinduction period to establish GHB intake (Experiment 2). How-ever, after the first 10 days of the free-choice regimen, daily GHBintake in sP rats increased to pharmacologically relevant doses,presumably because episodic drinking of the GHB solution re-sulted in the disclosure and experience of the reinforcing proper-ties of GHB. In contrast, daily GHB intake in sNP rats remained atpharmacologically negligible doses throughout the 28 days of thefree-choice phase.The different intakes of GHB monitored in sP and sNP rats arelikely related to a differential perception of GHB reinforcingproperties between the two rat lines, being more salient in sP thanin sNP rats. The contribution of taste factors appears to be mini-mal, since no difference in the intake of sodium chloride solutionswas recorded between sP and sNP rats (Experiment 3). Thus, sPrats appear to possess a genetically based higher sensitivity to thereinforcing properties of GHB than sNP rats.Recently, sP and sNP rats have been differentiated also in termsof sensitivity to the sedative effects of GHB (7), sP rats being moresensitive than sNP rats. Indeed, sP rats displayed a significantlyshorter time to lose the righting reflex and a significantly longersleep time than sNP rats after the intraperitoneal (i.p.) administra-tion of 750 or 1000 mg/kg GHB (7).The brain GHB receptor is the most likely candidate mediatingboth the sedative and reinforcing properties of GHB. Indeed, it hasbeen demonstrated that the GHB receptor antagonist NCS-382dose-dependently a) reversed the sedative and cataleptic effects of GHB in rats (33); b) antagonized the intravenous self-administra-tion of GHB in mice (29); and c) blocked the discriminativestimulus effects (of which the reinforcing effects constitute a part)of GHB in rats trained to discriminate GHB from water (6).Interestingly, a previous observation from this laboratory indicatedthe presence of inherent differences in GHB binding sites incortical membranes from sP and sNP rats, the former showing anincreased affinity (14). Thus, the different sensitivity to GHB of sPand sNP rats (present study; (7)) may be due to genetically deter-mined differences in the GHBergic system. Because these ratshave been primarily selected for opposite ethanol preference, theseresults also raise the possibility of an involvement of the GHBsystem in the predisposition to ethanol preference or avoidance insP and sNP rats.Finally, the results of the present study extend to a further drugand another rat line pair the capability of ethanol-preferring ro-dents to self-administer higher doses of abusive drugs in compar-ison to their ethanol-nonpreferring counterparts. For example, un-der the standard two-bottle, free-choice regimen, selectively bredethanol-accepting AA rats have been reported to consume signif-icantly larger amounts of the opioid agonist etonitazene and co-caine than ethanol-nonaccepting ANA rats (22). Consistently, eth-anol-preferring C57BL/6J mice showed higher intake of andpreference for morphine (13,21) and cocaine (19) than ethanol-nonpreferring DBA/2J mice. These results add support to thehypothesis that common genetic determinants may regulate drug-seeking behavior (see (10)). ACKNOWLEDGEMENTS The authors are grateful to Marinella Boi and M. Elena Vincis foranimal breeding and care and Hugh Sugden for language editing of themanuscript. The present work was partially supported by the LaboratorioFarmaceutico C.T., Sanremo, Italy. REFERENCES 1. Addolorato, G.; Castelli, E.; Stefanini, G. F.; Casella, G.; Caputo, F.;Marsigli, L.; Bernardi, M.; Gasbarrini, G.; GHB Study Group. Anopen multicentric study evaluating 4-hydroxybutyric acid sodium saltin the medium-term treatment of 179 alcohol dependent subjects.Alcohol Alcohol. 31:341–345; 1996.2. Cash, C. D. Gammahydroxybutyrate. An overview of the pros andcons for it being a neurotransmitter and/or a useful therapeutic agent.Neurosci. Biobehav. Rev. 18:291–304; 1994.3. Chin, M. Y.; Kreutzer, R. A.; Dyer, J. E. Acute poisoning from gamma-hydroxybutyrate in California. West. J. Med. 156:380–384; 1992.4. Colombo, G. Ethanol drinking behaviour in Sardinian alcohol-prefer-ring rats. Alcohol Alcohol. 32:443–453; 1997. TABLE 3 INTAKE OF SODIUM CHLORIDE SOLUTION (MEAN (ML/KG)    SEM)IN SARDINIAN ALCOHOL-PREFERRING (sP) AND SARDINIANALCOHOL-NONPREFERRING (sNP) RATS UNDER THETWO-BOTTLE, FREE-CHOICE REGIMEN[Sodium Chloride] (%) sP sNP  p  level* 0.45 63.0  17.1 64.4  17.0 1.00000.90 72.0  19.0 71.4  27.6 1.00001.80 17.5  5.7 16.7  11.2 0.5476Rats ( n  5) were given two graduated bottles containing tap water andsodium chloride [0.45%, 0.9%, and 1.8% (w/v)] dissolved in tap water for24 h.* Unpaired, two-tailed Mann–Whitney test. GHB INTAKE IN ETHANOL-PREFERRING RATS 201
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