THE MOTIVATIONAL PROCESSES that control the

Transcription

1 GLASNER, OVERMIER AND BALLEINE 53 The Role of Pavlovian Cues in Alcohol Seeking in Dependent and Nondependent Rats* SUZETTE V. GLASNER, PH.D., J. BRUCE OVERMIER, PH.D., AND BERNARD W. BALLEINE, PH.D. Department of Psychiatry, Cedars Sinai Medical Center, Thalians E-142, 8730 Alden Drive, Los Angeles, California ABSTRACT. Objective: Recent treatment approaches to substance use disorders have focused on reducing drug use by modifying drug-seeking behaviors in response to drug-associated cues. Understanding the effect of alcohol-related stimuli on alcohol-seeking responses is therefore of interest in the study of alcoholism. The present study examined the impact of ethanol- (ETOH) associated cues on selective ETOH-seeking behavior, using a Pavlovian-instrumental transfer design in groups of alcohol-dependent and nondependent rats. Method: Rats (N = 24) received Pavlovian conditioning in which each of two stimuli, a tone and white noise, was paired alternately with a 10% sweetened ETOH solution and a polycose-quinine solution. The rats were trained to perform two instrumental actions, with one action earning access to the sweetened ETOH and the other to the polycose-quinine. After training, half of the animals were made ETOH-dependent by intragastric administration of 36 g/kg of ETOH over 4 days, whereas the remainder received intragastric administration of an isocaloric polycose solution. On the following day, subjects were given a choice extinction test in which they were free to choose between both actions with no outcomes being delivered. During this test, the ETOH- and polycose-associated Pavlovian cues were presented to assess performance of the two instrumental actions both in the presence and absence of these stimuli. Results: Pavlovian cues associated with both the ETOH or the polycose exerted a nonspecific excitatory influence on reward-seeking behavior in both nondependent and alcohol-dependent rats. Conclusions: Responses through which rats gain access to ETOH appear to be subject to the general excitatory influence of the general motivational arousal induced by reward-related cues. It appears the rats performance did not depend on encoding the specific consequences of their actions and thus was not affected by the selective retrieval or priming of those consequences in memory. (J. Stud. Alcohol 66: 53-61, 2005) THE MOTIVATIONAL PROCESSES that control the intake of alcohol and other drugs of abuse have recently received increasing attention in theory and research that assess the factors determining drug-seeking behavior (e.g., Humans: LaBerg and Ellertson, 1987; Reich et al., 2004 and Animals: Koob and Weiss, 1992; Robinson and Berridge, 2001). An issue that has received relatively less examination, however, is the relationship between the motivational processes that control the pursuit of nondrug rewards and those that determine alcohol-seeking. Although several reviews have suggested the motivational mechanisms that control drug-seeking behavior and those that control the pursuit of nondrug rewards share commonalities (e.g., Newlin, 1992; Kelley and Berridge, 2002), this issue has yet to be systematically and empirically explored. It is well established in studies of rats that reward-related cues powerfully facilitate the performance of deliberated actions such as lever pressing when the actions provide Received: June 21, Revision September 1, *Preparation of this article was supported by an individual National Research Service Award to Suzette V. Glasner from the National Institute on Alcoholism and Alcohol Abuse (AA ). Correspondence may be sent to Suzette V. Glasner at the above address, or via at: suzette.glasner@cshs.org. J. Bruce Overmier is with the Department of Psychology, University of Minnesota, Minneapolis, MN. Bernard Balleine is with the Department of Psychology and the Brain Research Institute, University of California, Los Angeles, Los Angeles, CA. access to rewarding events an effect referred to as Pavlovian-instrumental transfer (Balleine, 1994; Colwill and Motzkin, 1994; Colwill and Rescorla, 1988; Kruse et al., 1983). In these experiments, rats are first given Pavlovian conditioning in which a neutral stimulus (CS) is paired with a rewarding event (US), after which they are trained in an instrumental conditioning procedure to press a lever for a particular reward, for example. The influence of the Pavlovian CS on lever press performance is then assessed in a final extinction test. The Pavlovian CS typically elevates lever pressing in this test as a result of two quite distinct processes: (1) a stimulus-induced increase in general appetitive arousal that acts to elevate the performance of all actions indiscriminately (e.g., Balleine 1994) or (2) a specific priming or retrieval process specific to actions that gain access to the specific reward predicted by the CS (e.g., Colwill and Rescorla, 1988; Kruse et al., 1983). Whether the general or the specific process predominates appears to depend not only on the specificity of the incentive factors that control reward-related responses in any situation but also on the specificity with which the reward associated with an action has been encoded. Thus, a CS associated with access to sugar solution will generally elevate all actions when rats are thirsty but will have a more selective effect on the performance of actions trained with sugar if the rats are trained to discriminate action from other actions on the basis of their consequences. 53

2 54 JOURNAL OF STUDIES ON ALCOHOL / JANUARY 2005 Several lines of evidence suggest that the incentive effects of Pavlovian cues may be involved in initiating and maintaining the performance of actions that gain access to ethanol (ETOH). For example, detoxified alcohol-dependent individuals given a priming dose of ETOH report higher subjective craving ratings and perform instrumental actions for access to ETOH at a significantly higher rate when presented with ETOH-associated Pavlovian cues than do individuals who are not presented with such cues (Ludwig et al., 1974). In rats, there has been no direct assessment of the effect of ETOH-related cues on the performance of ETOH-related actions. Krank (1989), however, reported that the intragastric (i.g.) infusion of ETOH enhanced consumption of sweetened ETOH but only in the presence of ETOH-associated cues. It has also been established that, when given adequate pre-exposure to ETOH, rats will prefer contexts in which they have received ETOH over other contexts (Bozarth, 1990). These results are consistent with the claim that the incentive processes recruited by Pavlovian signals for ETOH can contribute to ETOHseeking behavior, although whether changes in ETOH-induced preferences of place and ETOH consumption reflect processes common to alcohol-seeking is clearly open to debate (e.g., Dickinson and Balleine, 2002). In short, the precise nature of the processes that control alcohol-seeking in rats has only recently begun to be characterized. In an important study in this area, Dickinson et al. (2002) reported that ETOH-seeking responses in nondependent rats were impervious to the effects of a posttraining reduction in the value of the ETOH induced by taste aversion learning. Dickinson et al. (2002) interpreted this result as suggesting that ETOH-seeking responses are not maintained by the incentive properties of the ETOH but by its ability to serve as a reinforcer of responses that result in ETOH delivery. From this perspective, at least in rats, ETOH seeking is a habitual response that is not directly mediated by its relationship to the representation or the incentive value of its consequences. The findings of Dickinson et al. may have important implications for understanding the addictive properties of ETOH by demonstrating its unique propensity to establish automatic, habitual behavior patterns a position that has much in common with Tiffany s (1990) argument that substance-seeking and use are partially mediated by automatic action schemata triggered in the presence of drug-associated cues. Taken together, these claims are of interest in the study of incentive effects of Pavlovian cues on ETOH seeking because they indicate that the behavioral effects of these stimuli may not depend upon their relationship specifically to ETOH but to reward processes in general. The aim of the present experiment was therefore to characterize the effects of Pavlovian signaling processes on ETOH-seeking behavior in ETOH-dependent and nondependent rats. ETOH and polycose drinking were first induced in the home cage, after which rats were trained in a Pavlovian conditioning paradigm in which each of two stimuli, a tone (T) and white noise (N), was paired alternately with ETOH delivery and polycose delivery. All rats were subsequently trained to perform two instrumental actions, with one action earning access to a 10% sweetened ETOH solution and the other to a polycose-quinine solution. Following this training, half of the animals were made ETOH dependent by i.g. administration of ETOH, and the remainder received i.g. administration of an isocaloric polycose solution. On the day following the completion of this treatment, all subjects were given a response choice test in extinction during which no outcomes were delivered. In this test, the ETOHand polycose-associated cues were presented, interspersed with periods in which no conditioned stimuli were present, to assess performance of the two instrumental actions in the presence and in the absence of these stimuli. Consistent with prior work (Kruse et al., 1983), we predicted that, during the extinction test, the action trained with the outcome that is consistent with the Pavlovian signal should be selectively elevated, relative to the other action. If, however, ETOH-seeking performance is controlled by a habit mechanism as suggested by Dickinson et al. (2002), it may be anticipated that the ETOH-seeking response will not be selectively impacted by ETOH-related cues that ETOH-related cues may not exert a selective influence on reward-related actions but rather act to generate a more general motivational effect, particularly in dependent rats. If this is the case then we should expect the ETOH-related cues to generate a general transfer effect and elevate both actions equally in this situation. Subjects and apparatus Method The animal subjects were 24 experimentally naïve male Long-Evans rats, weighing on average 250 g at the start of the experiment. The rats were housed individually under a 12:12 light-dark cycle (lights on at 0600 hours) and were handled daily for 1 week prior to the initiation of instrumental training. Training and testing took place in four Med Associates (East Fairfield, VT) operant chambers measuring cm, and all chambers were housed within sound and light resistant shells. A ventilation fan in each of the chambers provided a background noise level of 55 db. Each chamber was equipped with a liquid dipper that, when operated, delivered 0.10 ml of a sweetened 10% ETOH solution or a quinine-adulterated polycose solution through a dipper cup into a recessed magazine in the chamber. The chambers contained one lever located 8 cm to the right of the magazine and a second lever located 8 cm symmetrically to the left of the magazine. Throughout the period of instrumental training, the animals were trained to

3 GLASNER, OVERMIER AND BALLEINE 55 press either the left or right lever in a series of separate sessions in which only one lever was available to them in a given session, the other lever being removed from the chamber. One lever delivered only the ETOH solution; the other delivered only the polycose solution. The left and right levers were introduced concurrently only during the choice extinction sessions. A house light mounted top-center on the wall opposite the magazine provided illumination, and a microcomputer equipped with MED-PC program located in the adjacent room controlled the equipment and recorded the lever presses. The sweetened 10% ETOH solution and quinine-adulterated polycose solution served as the instrumental outcomes. As established in a pilot procedure for induction of equivalent ETOH and polycose consumption described elsewhere (see Glasner, unpublished manuscript), the polycose solution contained 300 µg/ml of quinine and was isocaloric to the sweetened 10% ETOH solution. The animal subjects were assigned to either the ETOH-dependent (DEP) or to the nondependent (NON) group, following completion of instrumental and Pavlovian training, as described below. Procedure Induction of ETOH and polycose drinking. To induce reliable consumption of an ETOH-saccharin solution (10% w/v and 0.15% w/v, respectively), all animals were deprived of food and water for 1 day before they were exposed to a sweetened 1% (w/v) ETOH solution in a calibrated glass drinking tube with a metal spout. This tube, when presented, always held 100 ml of solution and could be fastened to the front of the animal s home cage. On the day following this initial 24-hour exposure to the sweetened 1% ETOH solution, the animals were returned to free water access and maintained on a 23-hour food deprivation schedule, with 1 hour daily access to their maintenance diet for the duration of the experiment. The animals were given one further day of exposure to the sweetened 1% (w/ v) ETOH solution with concurrent access to 100 ml of tap water before the ETOH concentration was gradually increased to 10% (w/v), with saccharin concentration held constant at 0.15% (w/v). This drinking induction stage was conducted over a period of 12 days, with 2 days of exposure to each of six solutions of ETOH containing 1%, 2%, 4%, 6%, 8% and 10% ETOH concentration, respectively. Throughout the period of concurrent exposure to ETOH solution and tap water in the home cage, the drinking tube containing ETOH was placed on the right-hand front side of the cage for half of the animals, with the water placed on the left-hand front side of the cage. The remaining animals received the opposite positioning of ETOH- and water-containing drinking tubes. To control for side preferences, the position of these tubes was switched daily for all subjects. Water and ETOH tubes were removed for 1 hour daily to measure intake and replace solutions. On the final 2 days of the ETOH exposure phase, all animals were given concurrent access to the polycose-quinine solution in the home cage. This solution was presented in the exact same manner as the ETOH solution with 23 hours of exposure per day. Positioning of the ETOH and polycose solutions on the home cages was again counterbalanced and switched daily. All subjects received 1 hour of water access on each of the 2 days of concurrent ETOH and polycose access, after which they were returned to free water access. General training procedures. Magazine training (MT) sessions were conducted over 4 days and followed by lever training. The animals were fed daily after the training sessions. As in the drinking induction stage, animals had free access to tap water while in the home cage throughout the training phase of the experiment. Each training session started with illumination of the house light and ended when the light was extinguished. Session duration, unless otherwise specified, was 30 minutes or until 35 ETOH outcomes were earned, whichever came first. MT. Following the ETOH and polycose drinking induction phase, all animals were given four sessions of MT, with the ETOH and polycose solutions presented as the reinforcers on alternate days. For half of the animals, the ETOH solution was presented on the first and third sessions, with the polycose solution presented on the second and fourth sessions. The remaining animals received the reverse order of presentation of the two outcomes during MT. Training was conducted on a random time 60-second schedule in the operant chambers with the levers removed. All subjects were gradually returned to free feeding status over the final 2 days of MT, and were maintained on free feeding throughout instrumental training and Pavlovian conditioning to mitigate any caloric-based preference for ETOH or polycose that may have emerged during the drinking induction stage (cf., Dickinson et al., 2002). Instrumental training. All animals were trained to lever press for either the sweetened ETOH or the polycose-quinine solutions on alternate days. In each session the respective inactive lever was removed from the chambers so that only one lever was present during any given session. For half of the animals, the right lever delivered the ETOH solution, and the left lever delivered the polycose solution; the remaining animals received the opposite action-outcome assignments. Animals were first trained to lever press for the sweetened 10% ETOH solution or the polycose-quinine solution on a continuous reinforcement (CRF) schedule. Once the animals earned 35 dipper deliveries of the ETOH or polycose solutions on three consecutive CRF training sessions, the response requirement for access to the solutions was increased, using random ratio (RR) schedules of reinforcement. The animals were first shifted to an RR-2

4 56 JOURNAL OF STUDIES ON ALCOHOL / JANUARY 2005 schedule (i.e., each response delivered the ETOH or polycose outcome with a probability of 0.5). Once the subjects earned 35 outcomes on each of three consecutive RR- 2 training sessions, the schedule was changed to RR-5 (i.e., each response delivered the ETOH or polycose outcome with a probability of 0.2). The animals were likewise maintained on this schedule until they earned 35 outcomes on each of three consecutive sessions. Pavlovian conditioning. On the day following the final instrumental training session, all animals were given Pavlovian conditioning in the operant chambers with the levers removed. For this conditioning, two stimuli, a tone and a white noise, served as the CSs and were paired with either the sweetened 10% ETOH or the polycose-quinine solution. Each of the stimuli was 80 db in intensity against a background noise level of 55 db. For half of the animals, the tone was paired with delivery of the ETOH solution, whereas the white noise was paired with delivery of the polycose solution. The remaining animals received the opposite pairings. The tone and white noise were presented in separate daily conditioning sessions over a 7-day period, with a total of seven conditioning sessions on each stimulus. Each conditioning session was 32 minutes long with four stimulus presentations interspersed with periods during which no stimuli were present. The length of each CS presentation was 2 minutes. The length of the intertrial intervals (i.e., off-stimulus periods) varied but were 3 minutes long on average. During each CS presentation, the instrumental outcome was delivered as a Pavlovian US on an RT-30-second schedule through the dipper cup in the magazine. Head entries to the magazine during each of the CS presentations, as well as during a pretrial interval of equal duration, were recorded. On the day following the final Pavlovian conditioning sessions, all animals were given one final session of instrumental training on each of the right and left levers on the RR-5 training schedule. Half of the animals were given training on the polycose-delivering lever before receiving a session on the ETOH-delivering lever; the remaining half received the reverse order of lever training prior to the initiation of the dependence induction stage. Following this training, 14 of the animals were randomly assigned to the DEP group; the remaining 10 animals were assigned to the NON group. Induction of ETOH dependence and assessment of ETOH withdrawal. On the basis of pilot observations for establishing physiological ETOH dependence in rats (see Glasner, 2002), ETOH was administered to DEP subjects by i.g. intubation at intervals of 8 hours across a 4-day period, with a resulting total of 12 doses. NON subjects received i.g. intubation of an isocaloric solution of polycose dissolved in tap water at the same intervals. The first ETOH dose administered to group DEP was 5 g/kg of a 45% (v/v) ETOH-water solution, the second and third dosages consisted of 2 g/kg of an 18% (v/v) ETOH-water solution, and all subsequent dosages were 3 g/kg of a 27% (v/v) ETOHwater solution. Using these various concentrations of ETOH among DEP subjects and corresponding concentrations of the isocaloric polycose solution among NON subjects, the volume of all doses throughout the 4-day intubation period was kept equivalent within each animal subject. Intubation tubes were coated with Vaseline to prevent dehydration of throat and mouth mucosa. All animals were deprived of food 24 hours prior to the initiation of the intubation stage and were given 1-hour access to their maintenance diet daily throughout the 4-day intubation period. The subjects continued to have free water access during this stage and were returned to free feeding status following the final intubation dose. Prior to each intubation, each animal was observed for failure to meet dosage criteria. Based on previously established behavioral signs of ETOH overdose (Majchrowicz, 1975), animal subjects were not administered their scheduled dosage of ETOH if they evidenced either (1) loss of righting reflex, defined by an inability of the animal to right itself when placed on its back, or (2) coma, defined by the absence of the blinking reflex and/or spontaneous breathing. Omission of scheduled ETOH dosages was rare and constituted less than 5% of all doses. Assessment of ETOH withdrawal. The effectiveness of the ETOH intubation procedure in producing ETOH dependence was inferred on the basis of an assessment of ETOH withdrawal signs 12 hours after administration of the last intubation dose. The withdrawal reaction was assessed in all animal subjects using a withdrawal score comprising the sum of the scores of three individual items (see Roberts et al., 1996). Each item was scored on a subjective three-level scale, with a score of 0 representing undetectable, 1 representing moderately severe and 2 representing severe. On the basis of a previously reported procedure for the behavioral assessment of ETOH withdrawal (Macey et al., 1996; Roberts et al., 1996), the items comprising the withdrawal severity score were (1) the ventromedial distal limb flexion response, measured by observing retraction of the lower legs of the rat toward the body upon being lifted by the scruff of the neck; (2) tail rigidity, measured by observing elevation of the tail above the floor and arching toward the back; and (3) ataxia, indicated by the presence of a broad-based, irregular rigid gait, by which movement appeared slow and deliberate. A total withdrawal severity score, ranging from 0 to 6, was calculated for each animal subject by summing the scores of the three individual items used in the behavioral assessment. Pavlovian instrumental transfer test. Testing was conducted immediately following the behavioral withdrawal assessment. All subjects were placed in the operant chambers with the right and left levers present concurrently for the choice extinction test. During this test, the ETOH and

5 GLASNER, OVERMIER AND BALLEINE 57 polycose-associated cues were each presented four times, interspersed with equal periods during which no stimulus (Ø) was presented. The test was 34 minutes long, and the first 2 minutes were an adaptation period in which the levers were available but no stimuli were presented. The adaptation period was followed by 16 two-minute bins comprising the on- and off-stimulus trials. All subjects received a total of eight stimulus trials during this test (four tone trials and four noise trials intermixed with eight Ø trials in the following order: T, Ø, N, Ø, N, Ø, T, Ø, N, Ø, T, Ø, T,Ø, N, Ø). Results Of the 24 animal subjects included in the study, one expired during the dependence induction stage, apparently as the result of ETOH overdose. Two animals failed to acquire lever pressing for both the ETOH and polycose solutions during the instrumental training stage and were excluded from the study. Of the remaining 21 animals, those assigned to the DEP group (n = 12) received a mean total dosage of 34 g/kg during the dependence induction stage, taking into account adjustments for failure to meet dosage criteria. Instrumental training Figure 1 shows the mean rate of lever pressing on the final day of instrumental training for the ETOH and polycose outcomes, respectively, separated by group DEP vs NON). As is clear from this figure, the groups did not differ in their respective rates of lever pressing for the ETOH and polycose outcomes. Correspondingly, a two-way analysis of variance revealed no effect of group (F < 1) or effect of reinforcer type (F < 1) or interaction between group and reinforcer type (F < 1). Pavlovian conditioning To determine whether the subjects learned the relationship between the Pavlovian stimuli and the ETOH and polycose presentations, the number of magazine entries during the Pavlovian stimuli was compared with the number of entries made during a comparable prestimulus period using the ratio of A/B, where A = the number of head entries during the CS, and B = the number of head entries during the pre-cs period of equal duration (2 minutes). Preliminary analysis indicated there was no effect of stimulus type (tone vs noise; F < 1) or of group (F = 1.97, 1/19 df, p >.05), and the data were therefore collapsed across these factors. The ratio of head entries to the CS relative to the pre-cs increased from 2.3 to 4.6 from the first day of conditioning to the last day of conditioning, showing that the tone and noise CS s gained associative strength as signals for the two reinforcers (t = -5.37, 20 df, p <.001). Observational withdrawal assessment Figure 2 shows the ETOH withdrawal severity scores resulting from the behavioral withdrawal assessment among DEP and NON rats just prior to the choice extinction test. As indicated by this figure, the DEP rats scored higher on the withdrawal severity index than the NON rats (F = 9.76, 1/19 df, p <.01). FIGURE 1. Mean (SE) responses per minute for ETOH and polycose at baseline as a function of reinforcer type and of to-be-assigned group (DEP = dependent group; NON = nondependent group) FIGURE 2. Mean (SE) total subjective withdrawal severity score prior to the Pavlovian-to-instrumental transfer test as a function of group (DEP = dependent group; NON = nondependent group)

6 58 JOURNAL OF STUDIES ON ALCOHOL / JANUARY 2005 Pavlovian-to-instrumental transfer test To assess the effects of the ETOH- and polycose-associated Pavlovian cues on instrumental performance of the responses previously trained to access each of these outcomes, the number of lever presses during the off-stimulus trials was compared to the number of lever presses performed during each of the stimulus trials in the choice extinction test. These comparisons are plotted in Figure 3 separately for the DEP and NON groups on the basis of performance on each of the two levers that delivered the ETOH or polycose solutions, respectively, during previous training. For each lever, instrumental performance is identified by stimulus. Performance of the action in the presence of the Pavlovian signal associated during conditioning with the same US that the action delivered during instru- FIGURE 3. Mean (SE) responses on the ETOH-trained (left panels) and polycose-trained (right panels) levers during the Pavlovian-to-instrumental transfer test in the presence and in the absence of the Pavlovian stimuli (DEP = dependent group; NON = nondependent group; OFF = instrumental performance during off-stimulus [i.e., baseline] periods with no Pavlovian stimuli presented; SAME = instrumental performance during the stimulus that was paired with the same outcome as that earned in training on that lever; DIFF = instrumental performance during the stimulus that was paired with a different outcome than that earned in training on that lever)

7 GLASNER, OVERMIER AND BALLEINE 59 mental training (SAME) is thus distinguished from performance of the action in the presence of the Pavlovian signal associated with a US different from the US delivered by the action during instrumental training (DIFF). In each of these plots, performance in the presence of each SAME and DIFF stimuli is compared to the rate of performance during the off-stimulus (i.e., baseline) periods (OFF), during which no Pavlovian stimuli were presented. The rate of performance during the OFF periods was determined separately by lever (ETOH vs polycose) by averaging the total performance during the OFF periods that followed each of the SAME and DIFF trials, respectively. As is clear from this figure, positive transfer emerged among both NON and DEP rats, each of which performed more lever presses during the stimulus trials than during the baseline periods, with relatively greater lever pressing overall observed among NON than among DEP rats. To normalize the distribution of responding observed during the choice extinction test, the individual animal subjects numbers of responses displayed in Figure 3 were transformed to their respective square roots for the statistical analysis. The transformed values are presented within each of the bars plotted in Figure 3. Analysis of variance revealed an overall effect of group (DEP vs NON) that approached significance (F = 3.99, 1/19 df, p <.07) and a highly significant effect of stimulus (OFF vs SAME vs DIFF) (F = 15.15, 2/38 df, p <.01). There was, however, no effect of lever (ETOH vs polycose), and none of the interactions among any of the factors reached significance (all F s < 1). To assess the selectivity of the observed positive transfer effect to instrumental performance in the presence of the SAME stimulus, the transformed data were collapsed across lever and group. Using a t test, we compared the mean total lever press performance in the presence of the SAME stimulus with the mean total lever press in the presence of the DIFF stimulus. This analysis revealed no difference between instrumental performance in the presence of the SAME and DIFF stimuli (t = 0.67, 20 df, p >.05), indicating that the Pavlovian-to-instrumental transfer effect was not selective overall. Having established that there was no selective transfer effect, we performed further analysis to confirm the presence of a general, nonselective positive transfer effect. The transformed data were collapsed across lever, group and stimulus trial (SAME vs DIFF), and the mean lever press performance during the stimulus trials was compared to the mean lever press performance during the OFF periods. A t test on these data confirmed the presence of a strong and positive general transfer effect (t = 10.1, 20 df, p <.01). Following our a priori interests, we next assessed whether the magnitude of the increments in responding in the presence of the two types of Pavlovian stimuli relative to the OFF periods differed as a function of ETOH dependency. Because there were differences between the DEP and NON groups in overall responding during the extinction test, this assessment was conducted using a ratio score that accounted for differential rates of responding during the OFF periods. This score, therefore, was computed separately by lever and stimulus trial to assess the magnitude of the transfer effect in the presence of the SAME and DIFF stimuli, using the ratio A-B/A+B, where A = lever press performance during the stimulus trial and B = lever press performance during the OFF trial, determined separately by lever as described earlier. Analyses of variance on these ratios, conducted separately by stimulus trial (SAME vs DIFF) and lever (ETOH vs polycose), revealed no significant differences between the DEP and NON groups in the magnitude of the transfer effect (all F s < 1). Discussion The results of our study suggest that alcohol-seeking in rats was generally facilitated by reward-related cues, whether or not those cues were associated with ETOH (i.e., the excitatory effect of Pavlovian cues on lever press responses rewarded by ETOH was similar whether those cues were predictive of polycose or of ETOH). In this study, therefore, the Pavlovian appetitive stimuli appeared to act to increase the general appetitive motivation of the rats (cf., Rescorla and Solomon, 1967), irrespective of whether those cues were associated with drug or nondrug rewards. Furthermore, this general motivating impact of Pavlovian cues was observed consistently among both ETOH-dependent and nondependent animals. The finding that ETOH-associated cues do not exert an outcome-specific motivational effect on ETOH seeking in DEP subjects is inconsistent with the current clinical conceptualization of the role of ETOH-associated cues in ETOH dependence and relapse. Current treatment approaches generally assume that stimuli paired with drug rewards have unique directive properties; consequently, these treatment approaches generally focus on eliminating drug-seeking behaviors in response to drug-associated cues (see Monti et al., 1999). The pattern of findings observed among DEP animals suggests, however, that instead of increasing the control by alcohol-specific cues, the shift from the nondependent to the dependent state may result in relatively enhanced control of drug seeking by reward-related stimuli in general. As is consistent with aspects of Tiffany s (1990) analysis of drug addiction, a theoretical implication of the nonselective excitatory effect of ETOH-associated cues is that ETOH seeking is habitual and is mediated by automatic action schemata triggered in the presence of cues associated with the drug use environment. On this view, any appetitive cue associated with the drug- or ETOH-seeking (or taking) environment could act to generate a general increase in ETOH seeking. This assumption is consistent with the observed increase in ETOH seeking in the presence

8 60 JOURNAL OF STUDIES ON ALCOHOL / JANUARY 2005 of both the ETOH- and the polycose-associated Pavlovian cues. Our findings are also consistent with the suggestion by Dickinson et al. (2002) that, unlike nondrug rewards, ETOH has response reinforcing properties that allow it to support and maintain drug-seeking behavior in a manner that does not depend on encoding a specific action-drug contingency. The present results extend those of Dickinson et al. (2002) in demonstrating that this habit mechanism may control ETOH seeking in both ETOH-dependent and nondependent rats. It is possible, however, that direct experience with ETOH while in an ETOH-dependent state is necessary before ETOH-related cues can have any selective effect on ETOHassociated instrumental actions. This suggestion is consistent with incentive learning theory (see Dickinson and Balleine, 1994), which posits that changes in the value of a reward (i.e., ETOH) after a shift in primary motivational state (i.e., from intoxication to withdrawal) are learned through consummatory experience, with the incentive when the rat is in the new motivational state. This account contrasts with the claim that ETOH seeking is habitual and assumes instead that control by the incentive value of the ETOH, or by cues that predict ETOH, is low in nondependent animals (and, at least initially, also in dependent animals) but that this control increases with successive consummatory experience with ETOH in ETOH withdrawal state. This suggestion is consistent, in fact, with studies of cue-induced reinstatement of ETOH seeking in animal subjects (see Bienkowski et al., 2000) and is consistent, furthermore, with the findings of the study cited earlier performed by Ludwig et al. (1974) in human alcoholics. As described earlier, in that study, recently detoxified human alcoholic subjects (i.e., subjects who have probably experienced multiple episodes of physiological dependency) were given a priming dose of ETOH before instrumental button-pressing for access to ETOH was assessed, either in the presence or the absence of Pavlovian ETOH-associated cues. Although this study found that the ETOH-associated cues had a profound excitatory impact on the button-pressing response, all of the subjects in this experiment were given an opportunity to drink a small quantity of ETOH prior to this test. Hence, it is quite possible that in dependent rats ETOH priming interacts with the excitatory effect of ETOH-associated cues to generate a selective excitatory effect on ETOH-seeking behavior. Investigation of the differential effects of ETOH dependence induction on cueelicited reward-seeking relative to reward consumption is therefore a promising area for future research. Several limitations to the above interpretations warrant comment. First, the observed nonselective effect of ETOHassociated cues on ETOH seeking in the withdrawal state may reflect a failure of the DEP subjects to discriminate between the ETOH- and polycose-associated cues; we have no evidence that the rats encoded the specific sensory features of the US s as a component of the CS-US associations acquired during Pavlovian training. Alternatively, the rats may have had difficulty discriminating their corresponding ETOH- and polycose-delivering instrumental actions during the extinction test. Recent work has shown impaired discrimination performance after posttraining, low-dose ETOH administration among adolescent but not adult rats (Land and Spear, 2004), and chronic ETOH intake is known to induce neurotoxicity and to result in cognitive impairments (e.g., Fadda and Rossetti, 1998; Ratti et al., 1999). Increased neurodegeneration and associated impairments in learning and memory have been observed in animals when large amounts of ETOH in the brain are followed by periods of abstinence under a dosing procedure similar to that employed in the present study (Crews et al., 2001; Obernier et al., 2002; Penland et al., 2001). Other recent work has shown impaired acquisition of associative learning in a fear conditioning paradigm among adult animals that had undergone repeated alcohol withdrawal (Stephens et al., 2001), and selective deficits in Pavlovian-to-instrumental transfer in an appetitive conditioning paradigm have been reported among adult animals subjected to a single withdrawal episode (Ripley et al., 2004). The nonselective transfer effect observed among DEP subjects may thus reflect a posttraining retrieval deficit resulting from ETOH withdrawal and related alterations in brain functioning. In summary, although there is considerable evidence that instrumental performance for nondrug rewards is at least partially controlled by a process that encodes the actionoutcome contingency (cf., Dickinson and Balleine, 1994), the results of the present study suggest that ETOH-seeking in both nondependent and recently dependent rats is not so controlled and may be habitual. It is possible that this finding was mediated by known neurotoxic effects of ETOH and a consequent deficit in learning and discrimination. Nevertheless, investigation of the conditions that alter responsivity to incentive manipulations (e.g., multiple dependency cycles, consummatory experience with ETOH during ETOH withdrawal) will offer an empirical basis for delineating the influence of habit learning on the clinical course of alcoholism. Acknowledgments The authors thank Eric Klinger and Marilyn Carroll for helpful discussions and comments on the issues surrounding this research project. 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