Alcohol,Vol. 9, pp. 1-7. ©PergamonPress plc, 1991. [hintedin the U.S.A.
0741-8329/92$5.00 + .00
Historical Factors in the Development of ETOH-Conditioned Place Preference D A V I D V. G A U V I N l A N D F R A N K A. H O L L O W A Y
Department of Psychiatry and Behavioral Sciences, University of Oklahoma Health Sciences Center Oklahoma City, OK 73190-3000 Received 1 March 1991; Accepted 1 July 1991 GALrVIN, D. V. AND F. A. HOLLOWAY. Historicalfactors in the development of ETOH-conditionedplace preference. ALCOHOL 9(1) 1-7, 1992.--Three groups of male Sprague-Dawley rats were conditioned with ethanol (ETOH) and water in a Conditioned Place Preference task. To assess the contribution of prior drug/behavioral history on the relative hcdonic valence of ETOH, the three groups differed in the task demands and degree of prior ETOH exposure. One group was a~ainedto self-administer 20% w/v ETOH in a home-cage self-administration task using a "Samson sucrose-fading" procedure prior to place conditioning trials (Group SA/CPP). A second group was initially trained in a two-choice ETOH-Saline drug discrimination (DD) task and subsequently conditioned in the place preference paradigm (Group DD/CPP). The third group of rats had no history of drug exposure and were experimentally naive prior to the place learning task (Group NH]CPP). Group SA/CPP developed a significant conditioned place preference; Group DD/CPP failed to demonstrate either a preference or aversion, whereas the Group NH/CPP demonstrated a significant place aversion. Data suggest that both present and past contingencies contribute to the algebraic summarion of ETOH's hedonic valences. Drug discrimination
Conditioned place preference
Ethanol
ONE of the primary objectives of the present set of experiments was to determine how certain behavioral and/or pharmacological factors influence ETOH's subsequent hedonic effects. To accomplish this aim, we must first consider several theoretical and methodological issues. Preclinical screening to determine the abuse liability of drugs minimally involves the assessment of the discriminative and the reinforcing/rewarding effects of drugs. Stimuli, including drug stimuli, can: a) function as discriminative stimuli that set the occasion of one behavior or another; b) serve as both positive and negative reinforcers, increasing, maintaining, or "consolidating" behaviors that respectively produce or eliminate the reinforcing event; and c) independently, may produce both positive (e.g., reward situations) and negative hedonic (e.g., punishment situations) consequences. The exact role drug stimuli play in a given situation depends both on the behavioral and pharmacological history of the individual and on the current situational constraints under which the drug is being examined. Of special note for the present studies is the lack of a necessary correspondence between positive drug reinforcement and positive hedonic drug effects (see discussion below). One classic drug history factor that clearly can affect subsequent drug effects is that of tolerance. Tolerance is typically defined by a reduced response, with repeated drug exposure, to a given drug close and by a concomitant increase in the dose required to recapture the initial drug effect. There are at least two ways in which tolerance development to ETOH's effects on behavior could interact with subsequent ETOH self-administration (SA): a) tolerance to ETOH's reward/reinforcing effects per se
Self-adminisa'ation
Drug history
could lead to increased ETOH-SA, and b) such tolerance could be mediated by learned behavioral adjustments to ETOH's effects, which in turn allow ETOH's rewarding or positive hedonic stimuli to operate (4). In the latter case, repeated ETOH administration or ingestion could produce tolerance to certain of ETOH's aversive effects, which would allow an individual to be able to consume larger quantities of ETOH (27). Further, self-ingestion of ETOH elicits interoceptive stimulus events, which, when detected (i.e., learned), can be used to assess one's own state of intoxication (2). Thus, with repeated exposure to ETOH, several parallel and/or serial events may occur, any or all of which could enhance ETOH consumption, i.e., a) tolerance may develop to ETOH's reinforcing effects and/or to its positive or negative hedonic effects, and b) the individual may acquire the ability to detect or "experience" the interoceptive discriminative cue produced by ETOH. The preclinical behavioral assay used in animals to model the discriminative or subjective interoceptive effects of drugs in humans is the drug discrimination (DD) paradigm (22). Although ETOH's DD properties in animals have been well characterized (7-9), the influence of such discrimination conditioning as an antecedent to the assessment of ETOH's hedonic effects has not been determined. In order to solve a DD task, an animal must learn to discriminate the difference between relevant drug stimuli and nonrelevant stimuli (12,13). This process of learning about the ETOH discriminative cue may modify ETOH's reinforcing and/or its hedonic effects. The most commonly used preclinical paradigm for the assess-
'Requests for reprints should be addressed to David V. Gauvin, Ph.D., University of Oklahoma Health Sciences Center, Research Building, 306-R, P.O. Box 26901, Oklahoma City, OK 73190-3000.
2
GAUVIN AND HOLLOWAY TABLE 1
Groups
DD/CPP SA/CPP
NH/CPP
Phase 1 Training ETOH vs. SAL Discrimination (n = 12) ETOH Home-Cage Self-Administration (n = 20) None
Phase 2 Training ETOH ConditionedPlace Preference (n = 12) ETOH ConditionedPlace Preference (n = 16) ETOH ConditionedPlace Preference (n = 12)
ment of drug reinforcement properties is the drug self-administration (SA) task. ETOH-SA has been demonstrated using both oral (25) and intravenous (30,31) routes of administration. Although the prior development of physiological tolerance and/or dependence is not required for the self-initiation of ETOH-SA, ETOH-SA can lead to both (14). Further, although ETOH is avidly self-administered, this demonstration does not necessarily confirm that ETOH also has rewarding or positive hedonic properties. It has been amply demonstrated that the same stimulus condition (including drug conditions) can be both positively reinforcing and aversive (i.e., punishing), depending on the task conditions under which it is administered (6, 10, 16, 20) and that, in clinical studies, drugs like ETOH can be reinforcing and aversive at the same time. In the latter case, chronic alcoholics describe their subjective experiences while drinking as not rewarding in the sense of being euphorigenic or relaxing (16,17), but instead are characterized by dysphoria, anxiety, irritability and aggressivity (1, 15, 18, 21). Finally, the behavioral paradigm most often used to assess drug-induced motivational parameters in this and other laboratories is the conditioned place preference (CPP) task (3,29). The repeated pairing of specific exteroceptive stimuli with drug-induced positive or negative hedonic [cf. (32)] interoceptive stimuli may lead to a conditioned preference (approach towards) or aversion (avoidance of) the drug-paired external stimuli when tested undrugged. Although some researchers have used this paradigm to assess the "reinforcing properties of drugs" (29), the functional relationship between behavior and reinforcer delivery (drug delivery and entry into the drug-paired compartment) does not exist in the classic CPP task. Thus, operationally, the "reinforcing effects of drugs" are not explicitly measured. However, another view of this task focuses on the conditioning of hedonically positive or rewarding drug effects (CPP) or conditioning of hedonically negative or aversive drug effects (conditioned place aversion or CPA). Thus, when tested, the undrugged subject will approach or spend more time in the context associated with prior rewarding events and will avoid stimuli associated with prior aversive events. ETOH paired with unique environments has typically produced conditioned place aversions (CPA), independent of route of administration or dose used (1, 24, 26, 28). Typically drug "reinforcement" is operationally defined using operant (lever press) conditioning paradigms, whereas "reward" is operationally defined using "approachavoidance" conditioning. Although some have argued that ETOH's peripheral irritative effects from IP injection may have produced the latter aversion, comparable results with intragastric and IP routes of administration mitigate against this argument. Even when rats were self-administering ETOH within the CPP apparatus, (i.e., functioning as a reinforcer), a CPA was pro-
duced (26). Thus the primary reward properties of ETOH and its ability to establish a secondary reinforcer appear weak at best (1,28). To our knowledge, a systematic analysis of the discriminative stimulus and rewarding properties of ETOH has not been reported in the same animals, using the DD and place learning models. Learning something about the ETOH stimulus and pairing it with food reward in a DD task may influence the acquisition of reward. An analogy can be made between what a human learns during repeated exposure to ETOH and what a rat must learn in a DD task. If the rewarding properties of ETOH are acquired over the course of repeated exposure, it seems possible that a sequence of DD learning and CPP may provide a more direct analysis of the role learning may play in ETOH abuse. The previous reports of CPA from self-administered ETOH (26) are confounded by the "forced"-choice procedure used to elicit self-administration of ETOH. The procedure of ETOH induction used in the Stewart and Grupp (26) study may, in itself, carry negative affective components which would interfere with the development of a CPP. The present study was designed to investigate the relative contribution of prior behavioral/drug history in the development of a CPP induced by ETOH. Using the DD task, we wanted to assess the contributions of allowing rats to learn something about ETOH prior to training a CPP. A second group of rats was first trained to self-administer ETOH in a more "anthropomorphic" model of ETOH drinldng--the Samson sucrose-fading procedure (24). Humans do not acquire their respective drinking patterns from forced-drinking exposures; nor do they usually initiate their drinking with pure ETOH. In contemporary times, the "wine coolers" or mixed drinks are usually used as a "gateway" to drinking large amounts of ETOH. The Samson sucrose-fading procedure mimics this procedure by slowly fading in ETOH to a well-consumed and tolerated sucrose solution, and then slowly fading out the sucrose until rats are consuming high concentrations of ETOH. Importantly, this procedure allows for the ad lib access to both food and water. We hypothesized that learning to consume large quantities of ETOH using this model would allow for the slow development of tolerance to the aversive properties of ETOH, so that subsequent testing for the rewarding properties in the CPP task would produce positive results. We also decided to test the influence of procedural or environmental variables on the development of conditioned place preference in this group. By subdividing the SA/CPP group into two subgroups, we attempted to examine the influence of sequestering the subjects within the compartment that was paired with the self-administration of ETOH. One group was sequestered within the compartment for the full 30-min access session. The other subgroup was allowed free access to all compartments. We added a control group which did not receive prior behavioral demands or exposure to ETOH. METHOD
Subjects Forty-four male Spragne-Dawley rats were purchased from Amitech, Inc. (Omaha, NE) and individually housed in suspended stainless steel cages in a colony room maintained on a 12-h light/dark cycle (lights on at 0630 h). Water was continuously available in the home cages (except for a brief 16-h restricted access in the SA group; see below). The colony room and animal care were administered by an AAALAC-accredited team of technicians from the Department of Comparative Anatomy (University of Oklahoma). Group assignments and descrip-
ETOH-CONDITIONED PLACE LEARNING
tions are depicted in Table 1. Twelve rats were assigned to a "No History" Group and remained drug and experimentally naive prior to CPP training (NH/CPP). Twelve rats were assigned to the DD/CPP group and were subsequently food restricted and trained in a Drug Discrimination Task (see below) prior to CPP training. Twenty rats were assigned to the SA/CPP Group and were subsequently water deprived and trained to self-administer various doses of ETOH prior to CPP training.
3
ing continued over weeks until the final concentration of 20% ETOH was achieved on Day 60. Rats were maintained at 20% ETOH for 60 more days. On Days 121 and 124, rats received a test solution of either 5% or 10% ETOH to assess drinking behaviors. After these tests were completed, each rat was given the 30 min access to 20% ETOH in the CPP apparatus or home cage (dependent upon subgroup assignments, see below).
Conditioned Place Preference Drug Discrimination Twelve rats, reduced to 85% of their free-feeding weights, were used as subjects. Each rat was trained in a two-choice drug discrimination task using 1.5 g/kg ETOH (10% w/v) or saline, administered intraperitoneally (IP). They were trained 6 days per week in 10-min lever press, food-motivated, experimental sessions under an FR-10 schedule of reinforcement in standard operant chambers equipped with two levers, stimulus lamps, house lamp, pellet dispenser, and protruding food trough and enclosed in sound-attenuating cubicles (Lehigh Valley Electronics, Lehigh Valley, PA). The correct lever to earn food reinforcement was cued by the injection administered 15 rain before the experimental session. Experimental contingencies were controlled and monitored by Commodore 64C microcomputer systems interfaced to the operant chambers (American Neuroscience Research Foundation, Yukon, OK). Training continued until each rat emitted less than 20 responses prior to the delivery of the first reinforcer and greater than 90% of the total sessions responses were emitted on the injection-appropriate lever. Each animal had to meet these training criteria for 6 consecutive days (i.e., SAL-ETOHSAL-SAL-ETOH-ETOH) prior to testing. Ten-min reinforced test sessions in which the rat was reinforced for 10 consecutive responses on either lever were alternated with training sessions. If, during a training session, a rat did not meet the training criteria for discriminative performance, further testing was postponed until two consecutive ETOH-SAL training days were achieved at the training criteria. For comparison purposes, a limited number of generalization tests was conducted. Each rat was tested with various doses of: 1) ETOH administered IP and intragastrically (IG) using an 18-g feeding tube (Harvard Bioscience, Inc.); 2) sodium pentobarbital (IP); and 3) caffeine (IP). Once all rats completed drug generalization testing, CPP training commenced.
Self-Administration Twenty rats were trained in a home cage ETOH self-administration procedure using a modified version of the sucrose-fading procedure developed by Samson et al. (22,24). At 1000 h each morning, each rat was given 30 min access to a solution of sucrose and ETOH in a graduated drinking tube temporarily mounted on the home cage. The concentration ratios of the sucrose-ETOH solutions were varied over 60 days by increasing the ETOH concentration from 5% to 20% w/v and reducing, eventually to zero, the sucrose. On Day 1 of the protocol, rats received an initial ETOH preference test for 20% ETOH. Thereafter, access to food and water was ad lib, except during the 30-min access sessions. On Day 2, all rats were deprived of water for 16 h prior to the introduction of a 20% concentration of sucrose. On Day 3, all rats received a solution of 5% ETOH/ 20% sucrose. The volume of solution consumed each day was recorded and the concentration of sucrose was faded down over successive days until rats were freely drinking 5% ETOH. After three consecutive days of drinking 5% ETOH, a solution of 10% ETOH-5% sucrose was administered. This cycle of sucrose fad-
The place preference apparatus consisted of two main "conditioning compartments" (40 × 16 x 24 cm), separated by a small 10-cm alley-way partition and connected to each other by a third compartment (10 x 16x24 cm) in a straight alley-way configuration. The apparatus was constructed of Plexiglas (Cope Plastics, Oklahoma City, OK); the hinged top was clear Plexiglas, and the walls were double-walled clear Plexiglas with sliding black Plexiglas inserts. The central chamber (Compartment 2) had a clear Plexiglas floor, with gray walls. The two conditioning compartments had the following distinguishing stimulus characteristics. In Compartment 1, the walls were decorated with white and black vertical stripes with textured indoor/outdoor carpeting on the floor. In Compartment 3, walls were decorated with white and black horizontal stripes with smooth indoor/outdoor carpeting on the floor. The apparatus was cleaned between rats by removing feces and wiping the walls, ceiling and carpeting with warm soapy water. Sets of removable guillotine doors could be inserted between individual compartments at the beginning of each session. The small partial walls in the alley-ways, separating each of the three compartments, remained in place during the sessions to allow for a clear, detectable, quantitative change of location of the rat from one compartment to the other. The number of compartment entries, time in each compartment, and general activity of the subjects were assessed and recorded by sets of infrared photobeams located near the floors of each compartment and linked by a photobeam controller (DIG-723, Med Associates, Inc., East Fairfield, VT) to a Commodore 64C microcomputer system. The microcomputer system controlled the experimental contingencies and recorded all measures from four sets of conditioning chambers simultaneously (American Neuroscience Research Foundation, Yukon, OK). The first four days of the procedure consisted of a preexposure period with the guillotine doors removed, to allow for free access to all three compartments. Each rat was placed separately into the apparatus for 30 minutes. On the fifth day, a 30-minute "habituation" test session was conducted. The central compartment was sealed off from the two distal compartments by the placement of the guillotine doors. Initially, each rat was sequestered into this small central compartment. Once the computer program was initiated, the guillotine doors were removed which completed a photobeam sensor circuit and started recording time, activity, and chamber data. The rats had free access to all three compartments. The relative time spent in each of the two conditioning comp~rtments was calculated for each rat (total time spent in Compartments 1 and 3). The "preference" score was calculated (the total time spent in the least preferred compartment + total time spent in Compartments 1 plus 3) and expressed as a percentage. This initial "habituation" test session data was used to compare changes in individual preferences/biases for one end compartment over the other. The most nonpreferred compartment was designated as the conditioned stimulus (CS + ) side, which would be paired with ETOH during the following conditioning trials. The alternative compartment was paired with tap water (dependent upon group assignment; see below).
4
GAUVIN AND HOLLOWAY 100 W (J
Z
bJ n~ bd b.. bd n~
0
80 6O
4O
al**
12. 2o
H
T1
T2
FIG. 1. Conditioned place aversion induced by ETOH. Mean (S.E.) preference scores of drug and experimentally naive rats in Group NH/ CPP conditioned with ETOH (2.0 g/kg, IG) in a conditioned place learning assay. The total time of a 30-min session spent in the nonpreferred compartment + total time spent in both conditioning compartments is expressed as a percentage, and is used as a measure of "side preference." Side preference infers a hedonic valence for the environmental cues of the compartment. Initial side preference/bias (H) was used to determine which of two compartments would be paired with ETOH. After double ETOH-water pairings, rats were retested for side preference (T1 and T2). ***p
0741-8329/92$5.00 + .00
Historical Factors in the Development of ETOH-Conditioned Place Preference D A V I D V. G A U V I N l A N D F R A N K A. H O L L O W A Y
Department of Psychiatry and Behavioral Sciences, University of Oklahoma Health Sciences Center Oklahoma City, OK 73190-3000 Received 1 March 1991; Accepted 1 July 1991 GALrVIN, D. V. AND F. A. HOLLOWAY. Historicalfactors in the development of ETOH-conditionedplace preference. ALCOHOL 9(1) 1-7, 1992.--Three groups of male Sprague-Dawley rats were conditioned with ethanol (ETOH) and water in a Conditioned Place Preference task. To assess the contribution of prior drug/behavioral history on the relative hcdonic valence of ETOH, the three groups differed in the task demands and degree of prior ETOH exposure. One group was a~ainedto self-administer 20% w/v ETOH in a home-cage self-administration task using a "Samson sucrose-fading" procedure prior to place conditioning trials (Group SA/CPP). A second group was initially trained in a two-choice ETOH-Saline drug discrimination (DD) task and subsequently conditioned in the place preference paradigm (Group DD/CPP). The third group of rats had no history of drug exposure and were experimentally naive prior to the place learning task (Group NH]CPP). Group SA/CPP developed a significant conditioned place preference; Group DD/CPP failed to demonstrate either a preference or aversion, whereas the Group NH/CPP demonstrated a significant place aversion. Data suggest that both present and past contingencies contribute to the algebraic summarion of ETOH's hedonic valences. Drug discrimination
Conditioned place preference
Ethanol
ONE of the primary objectives of the present set of experiments was to determine how certain behavioral and/or pharmacological factors influence ETOH's subsequent hedonic effects. To accomplish this aim, we must first consider several theoretical and methodological issues. Preclinical screening to determine the abuse liability of drugs minimally involves the assessment of the discriminative and the reinforcing/rewarding effects of drugs. Stimuli, including drug stimuli, can: a) function as discriminative stimuli that set the occasion of one behavior or another; b) serve as both positive and negative reinforcers, increasing, maintaining, or "consolidating" behaviors that respectively produce or eliminate the reinforcing event; and c) independently, may produce both positive (e.g., reward situations) and negative hedonic (e.g., punishment situations) consequences. The exact role drug stimuli play in a given situation depends both on the behavioral and pharmacological history of the individual and on the current situational constraints under which the drug is being examined. Of special note for the present studies is the lack of a necessary correspondence between positive drug reinforcement and positive hedonic drug effects (see discussion below). One classic drug history factor that clearly can affect subsequent drug effects is that of tolerance. Tolerance is typically defined by a reduced response, with repeated drug exposure, to a given drug close and by a concomitant increase in the dose required to recapture the initial drug effect. There are at least two ways in which tolerance development to ETOH's effects on behavior could interact with subsequent ETOH self-administration (SA): a) tolerance to ETOH's reward/reinforcing effects per se
Self-adminisa'ation
Drug history
could lead to increased ETOH-SA, and b) such tolerance could be mediated by learned behavioral adjustments to ETOH's effects, which in turn allow ETOH's rewarding or positive hedonic stimuli to operate (4). In the latter case, repeated ETOH administration or ingestion could produce tolerance to certain of ETOH's aversive effects, which would allow an individual to be able to consume larger quantities of ETOH (27). Further, self-ingestion of ETOH elicits interoceptive stimulus events, which, when detected (i.e., learned), can be used to assess one's own state of intoxication (2). Thus, with repeated exposure to ETOH, several parallel and/or serial events may occur, any or all of which could enhance ETOH consumption, i.e., a) tolerance may develop to ETOH's reinforcing effects and/or to its positive or negative hedonic effects, and b) the individual may acquire the ability to detect or "experience" the interoceptive discriminative cue produced by ETOH. The preclinical behavioral assay used in animals to model the discriminative or subjective interoceptive effects of drugs in humans is the drug discrimination (DD) paradigm (22). Although ETOH's DD properties in animals have been well characterized (7-9), the influence of such discrimination conditioning as an antecedent to the assessment of ETOH's hedonic effects has not been determined. In order to solve a DD task, an animal must learn to discriminate the difference between relevant drug stimuli and nonrelevant stimuli (12,13). This process of learning about the ETOH discriminative cue may modify ETOH's reinforcing and/or its hedonic effects. The most commonly used preclinical paradigm for the assess-
'Requests for reprints should be addressed to David V. Gauvin, Ph.D., University of Oklahoma Health Sciences Center, Research Building, 306-R, P.O. Box 26901, Oklahoma City, OK 73190-3000.
2
GAUVIN AND HOLLOWAY TABLE 1
Groups
DD/CPP SA/CPP
NH/CPP
Phase 1 Training ETOH vs. SAL Discrimination (n = 12) ETOH Home-Cage Self-Administration (n = 20) None
Phase 2 Training ETOH ConditionedPlace Preference (n = 12) ETOH ConditionedPlace Preference (n = 16) ETOH ConditionedPlace Preference (n = 12)
ment of drug reinforcement properties is the drug self-administration (SA) task. ETOH-SA has been demonstrated using both oral (25) and intravenous (30,31) routes of administration. Although the prior development of physiological tolerance and/or dependence is not required for the self-initiation of ETOH-SA, ETOH-SA can lead to both (14). Further, although ETOH is avidly self-administered, this demonstration does not necessarily confirm that ETOH also has rewarding or positive hedonic properties. It has been amply demonstrated that the same stimulus condition (including drug conditions) can be both positively reinforcing and aversive (i.e., punishing), depending on the task conditions under which it is administered (6, 10, 16, 20) and that, in clinical studies, drugs like ETOH can be reinforcing and aversive at the same time. In the latter case, chronic alcoholics describe their subjective experiences while drinking as not rewarding in the sense of being euphorigenic or relaxing (16,17), but instead are characterized by dysphoria, anxiety, irritability and aggressivity (1, 15, 18, 21). Finally, the behavioral paradigm most often used to assess drug-induced motivational parameters in this and other laboratories is the conditioned place preference (CPP) task (3,29). The repeated pairing of specific exteroceptive stimuli with drug-induced positive or negative hedonic [cf. (32)] interoceptive stimuli may lead to a conditioned preference (approach towards) or aversion (avoidance of) the drug-paired external stimuli when tested undrugged. Although some researchers have used this paradigm to assess the "reinforcing properties of drugs" (29), the functional relationship between behavior and reinforcer delivery (drug delivery and entry into the drug-paired compartment) does not exist in the classic CPP task. Thus, operationally, the "reinforcing effects of drugs" are not explicitly measured. However, another view of this task focuses on the conditioning of hedonically positive or rewarding drug effects (CPP) or conditioning of hedonically negative or aversive drug effects (conditioned place aversion or CPA). Thus, when tested, the undrugged subject will approach or spend more time in the context associated with prior rewarding events and will avoid stimuli associated with prior aversive events. ETOH paired with unique environments has typically produced conditioned place aversions (CPA), independent of route of administration or dose used (1, 24, 26, 28). Typically drug "reinforcement" is operationally defined using operant (lever press) conditioning paradigms, whereas "reward" is operationally defined using "approachavoidance" conditioning. Although some have argued that ETOH's peripheral irritative effects from IP injection may have produced the latter aversion, comparable results with intragastric and IP routes of administration mitigate against this argument. Even when rats were self-administering ETOH within the CPP apparatus, (i.e., functioning as a reinforcer), a CPA was pro-
duced (26). Thus the primary reward properties of ETOH and its ability to establish a secondary reinforcer appear weak at best (1,28). To our knowledge, a systematic analysis of the discriminative stimulus and rewarding properties of ETOH has not been reported in the same animals, using the DD and place learning models. Learning something about the ETOH stimulus and pairing it with food reward in a DD task may influence the acquisition of reward. An analogy can be made between what a human learns during repeated exposure to ETOH and what a rat must learn in a DD task. If the rewarding properties of ETOH are acquired over the course of repeated exposure, it seems possible that a sequence of DD learning and CPP may provide a more direct analysis of the role learning may play in ETOH abuse. The previous reports of CPA from self-administered ETOH (26) are confounded by the "forced"-choice procedure used to elicit self-administration of ETOH. The procedure of ETOH induction used in the Stewart and Grupp (26) study may, in itself, carry negative affective components which would interfere with the development of a CPP. The present study was designed to investigate the relative contribution of prior behavioral/drug history in the development of a CPP induced by ETOH. Using the DD task, we wanted to assess the contributions of allowing rats to learn something about ETOH prior to training a CPP. A second group of rats was first trained to self-administer ETOH in a more "anthropomorphic" model of ETOH drinldng--the Samson sucrose-fading procedure (24). Humans do not acquire their respective drinking patterns from forced-drinking exposures; nor do they usually initiate their drinking with pure ETOH. In contemporary times, the "wine coolers" or mixed drinks are usually used as a "gateway" to drinking large amounts of ETOH. The Samson sucrose-fading procedure mimics this procedure by slowly fading in ETOH to a well-consumed and tolerated sucrose solution, and then slowly fading out the sucrose until rats are consuming high concentrations of ETOH. Importantly, this procedure allows for the ad lib access to both food and water. We hypothesized that learning to consume large quantities of ETOH using this model would allow for the slow development of tolerance to the aversive properties of ETOH, so that subsequent testing for the rewarding properties in the CPP task would produce positive results. We also decided to test the influence of procedural or environmental variables on the development of conditioned place preference in this group. By subdividing the SA/CPP group into two subgroups, we attempted to examine the influence of sequestering the subjects within the compartment that was paired with the self-administration of ETOH. One group was sequestered within the compartment for the full 30-min access session. The other subgroup was allowed free access to all compartments. We added a control group which did not receive prior behavioral demands or exposure to ETOH. METHOD
Subjects Forty-four male Spragne-Dawley rats were purchased from Amitech, Inc. (Omaha, NE) and individually housed in suspended stainless steel cages in a colony room maintained on a 12-h light/dark cycle (lights on at 0630 h). Water was continuously available in the home cages (except for a brief 16-h restricted access in the SA group; see below). The colony room and animal care were administered by an AAALAC-accredited team of technicians from the Department of Comparative Anatomy (University of Oklahoma). Group assignments and descrip-
ETOH-CONDITIONED PLACE LEARNING
tions are depicted in Table 1. Twelve rats were assigned to a "No History" Group and remained drug and experimentally naive prior to CPP training (NH/CPP). Twelve rats were assigned to the DD/CPP group and were subsequently food restricted and trained in a Drug Discrimination Task (see below) prior to CPP training. Twenty rats were assigned to the SA/CPP Group and were subsequently water deprived and trained to self-administer various doses of ETOH prior to CPP training.
3
ing continued over weeks until the final concentration of 20% ETOH was achieved on Day 60. Rats were maintained at 20% ETOH for 60 more days. On Days 121 and 124, rats received a test solution of either 5% or 10% ETOH to assess drinking behaviors. After these tests were completed, each rat was given the 30 min access to 20% ETOH in the CPP apparatus or home cage (dependent upon subgroup assignments, see below).
Conditioned Place Preference Drug Discrimination Twelve rats, reduced to 85% of their free-feeding weights, were used as subjects. Each rat was trained in a two-choice drug discrimination task using 1.5 g/kg ETOH (10% w/v) or saline, administered intraperitoneally (IP). They were trained 6 days per week in 10-min lever press, food-motivated, experimental sessions under an FR-10 schedule of reinforcement in standard operant chambers equipped with two levers, stimulus lamps, house lamp, pellet dispenser, and protruding food trough and enclosed in sound-attenuating cubicles (Lehigh Valley Electronics, Lehigh Valley, PA). The correct lever to earn food reinforcement was cued by the injection administered 15 rain before the experimental session. Experimental contingencies were controlled and monitored by Commodore 64C microcomputer systems interfaced to the operant chambers (American Neuroscience Research Foundation, Yukon, OK). Training continued until each rat emitted less than 20 responses prior to the delivery of the first reinforcer and greater than 90% of the total sessions responses were emitted on the injection-appropriate lever. Each animal had to meet these training criteria for 6 consecutive days (i.e., SAL-ETOHSAL-SAL-ETOH-ETOH) prior to testing. Ten-min reinforced test sessions in which the rat was reinforced for 10 consecutive responses on either lever were alternated with training sessions. If, during a training session, a rat did not meet the training criteria for discriminative performance, further testing was postponed until two consecutive ETOH-SAL training days were achieved at the training criteria. For comparison purposes, a limited number of generalization tests was conducted. Each rat was tested with various doses of: 1) ETOH administered IP and intragastrically (IG) using an 18-g feeding tube (Harvard Bioscience, Inc.); 2) sodium pentobarbital (IP); and 3) caffeine (IP). Once all rats completed drug generalization testing, CPP training commenced.
Self-Administration Twenty rats were trained in a home cage ETOH self-administration procedure using a modified version of the sucrose-fading procedure developed by Samson et al. (22,24). At 1000 h each morning, each rat was given 30 min access to a solution of sucrose and ETOH in a graduated drinking tube temporarily mounted on the home cage. The concentration ratios of the sucrose-ETOH solutions were varied over 60 days by increasing the ETOH concentration from 5% to 20% w/v and reducing, eventually to zero, the sucrose. On Day 1 of the protocol, rats received an initial ETOH preference test for 20% ETOH. Thereafter, access to food and water was ad lib, except during the 30-min access sessions. On Day 2, all rats were deprived of water for 16 h prior to the introduction of a 20% concentration of sucrose. On Day 3, all rats received a solution of 5% ETOH/ 20% sucrose. The volume of solution consumed each day was recorded and the concentration of sucrose was faded down over successive days until rats were freely drinking 5% ETOH. After three consecutive days of drinking 5% ETOH, a solution of 10% ETOH-5% sucrose was administered. This cycle of sucrose fad-
The place preference apparatus consisted of two main "conditioning compartments" (40 × 16 x 24 cm), separated by a small 10-cm alley-way partition and connected to each other by a third compartment (10 x 16x24 cm) in a straight alley-way configuration. The apparatus was constructed of Plexiglas (Cope Plastics, Oklahoma City, OK); the hinged top was clear Plexiglas, and the walls were double-walled clear Plexiglas with sliding black Plexiglas inserts. The central chamber (Compartment 2) had a clear Plexiglas floor, with gray walls. The two conditioning compartments had the following distinguishing stimulus characteristics. In Compartment 1, the walls were decorated with white and black vertical stripes with textured indoor/outdoor carpeting on the floor. In Compartment 3, walls were decorated with white and black horizontal stripes with smooth indoor/outdoor carpeting on the floor. The apparatus was cleaned between rats by removing feces and wiping the walls, ceiling and carpeting with warm soapy water. Sets of removable guillotine doors could be inserted between individual compartments at the beginning of each session. The small partial walls in the alley-ways, separating each of the three compartments, remained in place during the sessions to allow for a clear, detectable, quantitative change of location of the rat from one compartment to the other. The number of compartment entries, time in each compartment, and general activity of the subjects were assessed and recorded by sets of infrared photobeams located near the floors of each compartment and linked by a photobeam controller (DIG-723, Med Associates, Inc., East Fairfield, VT) to a Commodore 64C microcomputer system. The microcomputer system controlled the experimental contingencies and recorded all measures from four sets of conditioning chambers simultaneously (American Neuroscience Research Foundation, Yukon, OK). The first four days of the procedure consisted of a preexposure period with the guillotine doors removed, to allow for free access to all three compartments. Each rat was placed separately into the apparatus for 30 minutes. On the fifth day, a 30-minute "habituation" test session was conducted. The central compartment was sealed off from the two distal compartments by the placement of the guillotine doors. Initially, each rat was sequestered into this small central compartment. Once the computer program was initiated, the guillotine doors were removed which completed a photobeam sensor circuit and started recording time, activity, and chamber data. The rats had free access to all three compartments. The relative time spent in each of the two conditioning comp~rtments was calculated for each rat (total time spent in Compartments 1 and 3). The "preference" score was calculated (the total time spent in the least preferred compartment + total time spent in Compartments 1 plus 3) and expressed as a percentage. This initial "habituation" test session data was used to compare changes in individual preferences/biases for one end compartment over the other. The most nonpreferred compartment was designated as the conditioned stimulus (CS + ) side, which would be paired with ETOH during the following conditioning trials. The alternative compartment was paired with tap water (dependent upon group assignment; see below).
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FIG. 1. Conditioned place aversion induced by ETOH. Mean (S.E.) preference scores of drug and experimentally naive rats in Group NH/ CPP conditioned with ETOH (2.0 g/kg, IG) in a conditioned place learning assay. The total time of a 30-min session spent in the nonpreferred compartment + total time spent in both conditioning compartments is expressed as a percentage, and is used as a measure of "side preference." Side preference infers a hedonic valence for the environmental cues of the compartment. Initial side preference/bias (H) was used to determine which of two compartments would be paired with ETOH. After double ETOH-water pairings, rats were retested for side preference (T1 and T2). ***p
