OPERANT RESPONDING FOR ETHANOL IN RATS WITH A LONG-TERM HISTORY OF FREE-CHOICE ETHANOL DRINKING

Eliza Koros1, Wojciech Kostowski1,2 and Przemyslaw Bienkowski1,3,*

1 Department of Pharmacology and Physiology of the Nervous System, Institute of Psychiatry and Neurology, Al. Sobieskiego 1/9, PL-02957 Warsaw,
2 Department of Experimental and Clinical Pharmacology, Warsaw Medical University, ul. Krakowskie Przedmiescie 26/28, PL-00527, Warsaw and
3 Clinical Psychopharmacology Laboratory, National Centre of Sport Medicine, ul. Wawelska 5, PL-02034 Warsaw, Poland

Received 17 March 1999; in revised form 7 June 1999; accepted 21 June 1999


    ABSTRACT
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 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 METHOD
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Wistar rats were allowed to drink ethanol in a two-bottle (water vs 2–8% v/v ethanol) and then in a three-bottle choice paradigm (water vs 8% ethanol vs 16% ethanol, v/v). After 7 months of free access to alcohol, the subjects were trained to respond for 8% ethanol in an operant procedure. No relationship was found between prior alcohol drinking and lever pressing for ethanol.


    INTRODUCTION
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 METHOD
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Non-operant choice procedures are widely used for screening rats towards high (or low) home-cage ethanol preference. For example, a standard two-bottle choice procedure (water vs 10% ethanol) has been successfully employed for selection of rat lines with genetically determined high or low alcohol preference (Sinclair et al., 1989; Files et al., 1997Go). Although non-operant choice procedures enable assessment of consumatory behaviours (ethanol-taking), preparatory behaviours (ethanol-seeking) may not be directly determined in these procedures.

In operant paradigms, a small amount of ethanol is available upon completion of each response requirement (e.g. lever press). Importantly, operant procedures enable examination of both ethanol-seeking and ethanol-taking behaviour (Markou et al., 1993Go; Piasecki et al., 1998Go). The most popular version of the operant ethanol self-administration paradigm was developed by Samson (1986). In Samson's sucrose-fading procedure, rats are first trained to respond for small amounts of sucrose in daily 30-min sessions. Then, over the next 2 to 3 weeks, sucrose is gradually eliminated and ethanol concentration increased to the required value (Samson, 1986Go; Files et al., 1997Go; Piasecki et al., 1998Go). Finally, the above paradigm allows assessment of ethanol-taking in food- and water-sated animals. Persistence of lever pressing in extinction, i.e. in the absence of ethanol reinforcement, may be treated as a simple measure of ethanol seeking (Markou et al., 1993Go; Piasecki et al., 1998Go).

Little is known about the relationship between free-choice ethanol consumption and ethanol-reinforced behaviour. It is not known, for example, whether long-term ethanol drinking might lead to any tolerance or sensitization to the reinforcing properties of ethanol. Thus, the reasons for the present study were twofold. First, we aimed to investigate whether a 7-month history of voluntary ethanol drinking might alter lever pressing for ethanol either in the presence (ethanol-taking) or absence (ethanol-seeking) of ethanol reinforcement. Second, we looked for possible correlations between individual home-cage ethanol intakes and ethanol-reinforced behaviour.


    METHOD
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 METHOD
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Subjects
Male Wistar rats (250–300 g at the beginning of the study) served as subjects. The animals were obtained from a breeder (HZL, Warsaw, Poland) 2 weeks before the start of the investigation. The rats were kept under standard laboratory conditions at 22 ± 1°C, 60% relative humidity, and a 12 h light/ 12 h dark cycle (lights on at 07:00). After 2 weeks of acclimatization, the subjects were transferred to individual wire cages (20 x 25 x 28 cm; L x W x H) with graduated drinking tubes mounted at the front. Standard lab chow (Bacutil, Poland) was always freely available. Tap water was generally available ad libitum, except as noted below. Treatment of the rats in the present study was in full accordance with the respective Polish and European regulations and was approved by our institution's Ethics Committee.

Long-term free-choice ethanol drinking
The subjects were assigned randomly to one of two experimental groups, i.e. to an ethanol-drinking group (n = 26 rats) or to a water-drinking control group (n = 19 rats). To minimize the aversive effects of ethanol, the animals in the ethanol group were first exposed (22 days) to increasing concentrations of alcohol (2–8%, v/v) and tap water in a two-bottle choice situation. For the next 28 days, these animals were presented with two different ethanol solutions (8 and 16%, v/v) and tap water in a three-bottle choice test. All solutions were presented in the drinking tubes which were rotated daily to prevent position preference. After 50 days of continuous access to alcohol and water, the rats were deprived of alcohol for 5 days. During the deprivation period, water was available in all three bottles. After the 5-day deprivation period, both alcohol solutions were presented again along with water for the next 28 days (Koros et al., 1998Go, 1999Go). The ethanol group completed six 28-day three-bottle ethanol drinking cycles. Accordingly, this group had a choice between ethanol and water for 218 days (~7 months). The mean ethanol intake in the three-bottle choice situation was relatively stable and ranged from 3.5 to 4.5 g/kg/day.

The alcohol deprivation effect (ADE) was first described by Sinclair and Senter (1968) as a transient increase in ethanol intake after a period of forced abstinence. The 5-day deprivation episodes were introduced to monitor possible development of ADE. The detailed pattern of ethanol-drinking behaviour and the development of ADE in the above procedure has been recently described by Koros et al. (1999).

Depending on the phase of the procedure, the control group had free access to two or three tubes of tap water. Two weeks after the end of the last three-bottle ethanol-drinking cycle, an operant procedure was started (see below).

Operant responding for ethanol
Responding for ethanol was tested in eight standard operant conditioning chambers (Coulbourn, Allentown, PA, USA). The chambers (see Piasecki et al., 1998Go for details) consisted of modular test cages enclosed within sound-attenuating cubicles with fans for ventilation and background white noise. A white house light was centred near the top of the front of the cage. The start of any experimental session was signalled by turning the house light on. The cage was also equipped with two response levers, separated by a liquid delivery system (the liquid dipper). Only one lever (‘active’ lever) activated the liquid dipper. Presses on the ‘inactive’ lever were recorded, but not reinforced. The liquid delivery system presented different solutions (sucrose, sucrose + ethanol or ethanol; see below) in a 0.1-ml portion for 5 s. The availability of reinforcer was signalled by an audible click and a small white light (4 W) located inside the liquid dipper hole. Programming of all sessions as well as data recording made use of the L2T2 Software package (Coulbourn) running on an IBM-PC compatible.

The rats were trained to respond for 8% (v/v) ethanol according to Samson's sucrose-fading procedure (Samson, 1986Go) with some minor modifications (Piasecki et al., 1998Go). The animals were deprived of water for 22 h/day during the first 4 days of training and trained to lever press for 10% sucrose (w/v) solution on a fixed ratio 1 (FR1) schedule of reinforcement. As soon as lever pressing was established, water started to be freely available in the home cages. All training sessions were 30 min long. Only one session was given each day (6 to 7 sessions/week). Starting on day 5, the animals received 2% ethanol–10% sucrose. Then, over the next 10–14 days, the ethanol concentration was gradually increased from 2% to 8% and sucrose concentration decreased from 10% to 0%. The rats were allowed to lever press for 8% ethanol in the next 30 sessions (stabilization period). Only the animals that systematically emitted >=20 responses/ 30 min were considered to be initiated to lever press for ethanol.

After the stabilization period was completed, the rats were tested in extinction sessions. In the single 30-min extinction session, the liquid delivery system was off and responding had no consequences.


    RESULTS
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 METHOD
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Ethanol-reinforced behaviour in rats with prior ethanol-drinking history
Approximately 40% of all subjects were successfully initiated to lever press for ethanol with no difference between the animals with prior ethanol history and the water-drinking control group (Table 1Go). The initiated animals from both groups showed comparable numbers of responses and consumed similar amounts of absolute alcohol in the last three sessions of the stabilization period (Table 1Go).


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Table 1. Effects of long-term history of free-choice ethanol drinking on parameters of ethanol-reinforced behaviour in the operant procedure
 
When the liquid dipper was inactive and ethanol reinforcement absent (extinction), lever pressing substantially decreased in both groups (‘Extinction’; Fig. 1Go). However, on the next day (‘Re-exposure’), when the liquid dipper was active again, responding reached baseline levels. These observations were confirmed by a two-way ANOVA (Drinking History x Session Type) which indicated a significant effect of Session Type, F(2,32) = 28.13, P < 0.0001. In contrast, the ANOVA did not reveal any significant differences between the ethanol-drinking and the water-drinking subjects (Fs < 1.88, Ps > 0.15).



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Fig. 1. Operant responding at baseline, extinction, and re-exposure in rats with or without a previous history of ethanol consumption.

Values are means (± SEM) of the numbers of responses/30 min in an ethanol self-administration session performed either before (Baseline) or after (Re-exposure) a single extinction session (Extinction). {square}, operant responding in rats without a prior history of ethanol drinking (n = 8 rats); {blacksquare}, operant responding in rats with a prior long-term history of ethanol drinking (n = 10 rats), *P < 0.05 vs the respective Baseline and Re-exposure session.

 
The mean number of ‘inactive’ lever presses in the operant experiments was negligible (<1.25 presses/30 min) and did not differ between the experimental and control groups (data not shown).

Correlations between individual ethanol intakes in a three-bottle choice situation and ethanol-reinforced behaviour
Only the rats (n = 10) from the ethanol-drinking group which were successfully initiated in the operant procedure were used in a correlational analysis. Total ethanol intake in the sixth 28-day cycle of the three-bottle choice test did not predict operant behaviour in the ethanol self-administration or extinction sessions (rs < 0.47, Ps > 0.15; regression analysis).

Total ethanol intake in the sixth cycle of the three-bottle test was 4.66 ± 0.54 g/kg/day for the initiated rats and 3.85 ± 0.47 g/kg/day for the 16 non-initiated rats (P > 0.25; Student's t-test).


    DISCUSSION
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 METHOD
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
The animals used in the present study were first allowed to drink ethanol in the long-term, free-choice procedure. The pattern of ethanol consumption in this paradigm has been recently described in detail by Koros et al. (1998, 1999). The present study demonstrated that neither initiation nor maintenance of operant responding for ethanol was substantially affected by prior history of home-cage ethanol drinking. Similarly, ethanol-seeking behaviour (lever pressing in extinction) remained unaffected in ethanol-experienced subjects. Thus, it seems that long-term ethanol consumption in our procedure does not lead to any persistent adaptations that would be critical for initiation, maintenance or extinction of ethanol-reinforced behaviour.

The correlational analysis indicated that individual ethanol intakes in the three-bottle choice situation did not predict operant responding for ethanol. Our findings agree with several previous observations. In a series of experiments reviewed by George and Ritz (1993), no consistent relationship between free- choice ethanol preference and ethanol-reinforced behaviour was found across a number of rodent genotypes, including genetically selected alcohol preferring and non-preferring rats. Importantly, another initiation procedure, i.e. the post-prandial drinking technique, was employed to facilitate responding for ethanol in these latter studies (George, 1990Go; George and Ritz, 1993Go; Ritz et al., 1994Go). Samson et al. (1989) have reported successful initiation of ethanol-reinforced behaviour in alcohol non-preferring rats tested in the secondary conditioning procedure.

The obvious limitation of the present study is that only one strain of rats was used. It is possible that prior history of ethanol drinking would facilitate lever pressing for ethanol in other outbred strains. The possibility also exists that other schedules of ethanol pre-exposure leading to higher ethanol intakes would influence subsequent lever pressing behaviour. Considering the parameters of the operant procedure, it can be hypothesized that any differences between ethanol-experienced and ethanol-naive subjects would become apparent if higher ethanol concentrations were used. Recently, Files et al. (1997) have shown that alcohol-preferring AA rats consumed more ethanol in the operant procedure than their alcohol-avoiding ANA counterparts only when higher ethanol concentrations (>15%) were introduced. Last but not least, access to ethanol in the operant paradigm was limited to 30 min/day. Possibly, a 24-h operant paradigm (e.g. Hölter et al., 1997Go) would be more appropriate for rats drinking ethanol in the 24-h choice procedure.

Taken together, the results of the present study suggest that: (1) long-term, free-choice ethanol drinking alters neither ethanol-taking nor ethanol-seeking behaviour in the operant procedure; (2) ethanol drinking in the operant and non-operant paradigms may be regulated by differing neural mechanisms.


    ACKNOWLEDGEMENTS
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 METHOD
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
This study was supported in part by IPiN (grant #53/99), PARPA (grant Alc-1/99), and the State Committee for Scientific Research (grant 4PO5A 00916).


    FOOTNOTES
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 ABSTRACT
 INTRODUCTION
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* Author to whom correspondence should be addressed at: Department of Pharmacology and Physiology of the Nervous System, Institute of Psychiatry and Neurology, ul. Sobieskiego 1/9, PL-02957 Warsaw, Poland. Back


    REFERENCES
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 INTRODUCTION
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 ACKNOWLEDGEMENTS
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