This work was supported by National Institute on Alcohol Abuse and Alcoholism grants awarded to GFK (AA012602) and a National Research Service Award awarded to BMW (AA014723).
Reprint requests: Brendan M. Walker, Molecular and Integrative Neurosciences Department The Scripps Research Institute 10550 N. Torrey Pines Road, SP30-2400 La Jolla, CA 92037; Fax: +1858-784-7405; E-mail: firstname.lastname@example.org
Background: γ-Aminobutyric acid-B (GABAB) receptor agonists have been shown to suppress operant self-administration of ethanol in nondependent rats. However, little work has focused on the effects of GABAB receptor agonists on self-administration of ethanol in dependent animals.
Methods: In the present experiment, the GABAB receptor agonist baclofen was tested for the ability to modulate both fixed- (FR) and progressive-ratio (PR) responding for ethanol in rats while nondependent and subsequently after ethanol dependence induction. Following the acquisition and stabilization of baseline operant ethanol self-administration and after dependence induction, baclofen [0.0, 0.5, 1, 2, and 4 mg/kg, intraperitoneal (IP)] was tested on FR-1 responding for ethanol. The ability of baclofen (2.0 mg/kg) to affect responding under a PR schedule of reinforcement was also evaluated. Dependence was induced in the animals by subjecting them to a 1-month intermittent vapor-exposure period in which animals were exposed to ethanol vapor for 14 h/d. Following the 1-month period, the vapor-exposed animals resumed FR-1 and PR baclofen drug testing (doses as described above) in the operant chambers at a time point corresponding to the animals being 6 hours into withdrawal (i.e., 6 hours after the ethanol vapor had been discontinued for that day).
Results: Baclofen (0.0, 0.5, 1, 2, and 4 mg/kg, IP) dose-dependently decreased ethanol self-administration in both nondependent and dependent rats on a FR schedule of reinforcement. However, the dose of baclofen that significantly reduced responding for ethanol was shifted to the left in the ethanol vapor-exposed animals, indicating an increased sensitivity to baclofen in animals that were chronically exposed to ethanol. When tested using a PR schedule of reinforcement, there was a significant increase in the breakpoint for the vapor-exposed animals (i.e., the animals were willing to work more in a dependent state). Baclofen (2.0 mg/kg, IP) suppressed intake for both nondependent and dependent animals.
Conclusions: Ethanol dependence produced increased self-administration of ethanol as reflected in increased ethanol intake and increased responding on a PR schedule of reinforcement. As baclofen suppressed ethanol self-administration and showed evidence of increased potency in dependent animals, the present experiment suggests that the GABAB receptor could be a potential pharmacotherapeutic target for the treatment of chronic alcoholism.
NUMEROUS LINES OF evidence indicate the involvement of the γ-aminobutyric acid (GABA) neurotransmitter system in the neurobiology of ethanol reinforcement (for a review, see Chester and Cunningham, 2002; Colombo et al., 2004; Davies, 2003; Koob, 2004). γ-Aminobutyric acid is the main inhibitory neurotransmitter in the brain and binds to both ionotropic (GABAA) and metabotropic G-protein receptors (GABAB) to produce its effects. GABAA receptors are located throughout the central nervous system and have long been implicated in alcohol actions. Antagonism of the GABAA receptor has been shown to decrease operant self-administration of ethanol (Hyytia and Koob, 1995; Rassnick et al., 1993a).
In humans, the viability of the GABAB receptor agonist baclofen to reduce the rewarding properties of alcohol and to treat alcohol withdrawal syndrome and alcohol craving has been evaluated using subjective measures in placebo-controlled, double-blind studies (Addolorato et al., 2002a). The results showed that daily dosing with baclofen compared with placebo decreased subjective reports of craving and increased the likelihood of remaining abstinent and the total duration of abstinence from alcohol. Furthermore, if one did drink, baclofen was able to reduce significantly the number of drinks taken. Additionally, patient self-reports identified that baclofen was able to reduce the physiological symptoms of alcohol withdrawal and produced an enhanced sense of well-being (Addolorato et al., 2002b). Later, baclofen was also shown to reduce delirium tremens produced by alcohol withdrawal (Addolorato et al., 2003).
The effects of baclofen in preclinical animal models of ethanol reinforcement, motivation, and withdrawal correspond well with the published clinical research. When evaluating the ability of baclofen to modulate the acute reinforcing effects of ethanol, it was shown that a low dose of a GABAB agonist resulted in a selective suppression of ethanol self-administration (Janak and Gill, 2003). However, if higher doses of baclofen were utilized, a nonselective suppression of both ethanol and sucrose occurred (Anstrom et al., 2003).
An animal model of appetitive behavior that can be used as an index of an animal's motivation to consume a particular drug is extinction responding following stable self-administration of the compound of interest. Indeed, the role of GABAB receptors in extinction responding following ethanol and sucrose self-administration has been evaluated (Colombo et al., 2003). The results identified that baclofen was able to suppress extinction responding for ethanol compared with sucrose responding, which indicated a selective effect of baclofen on the motivation to consume ethanol.
To evaluate the efficacy of baclofen to reduce physiological withdrawal signs and withdrawal-induced anxiogenic behavior produced by chronic ethanol ingestion in rats, File et al. (1991) utilized the social interaction test and elevated-plus maze as animal models of anxiety following baclofen administration during ethanol withdrawal and additionally evaluated withdrawal-induced tremor. Ethanol withdrawal produced tremors and was shown to produce anxiogenic-like behaviors in both the social interaction test (i.e., increased aggression and decreased social interaction) and the elevated-plus maze (i.e., decreased time spent in the open arms of the maze). Baclofen reduced both the physiological tremors and the anxiogenic responses in the social interaction and elevated-plus maze without affecting control rats or producing sedation, identifying the efficacy of baclofen in reducing ethanol withdrawal-induced behaviors.
Although the effects of baclofen have been extensively evaluated using nondependent appetitive and consummatory ethanol self-administration paradigms, as well as dependence-induced measures of physiological withdrawal signs, to date, baclofen has not been tested in ethanol-dependent rats self-administering ethanol during acute withdrawal. Ethanol vapor exposure for 1 month induces dependence that is reflected by not only physical withdrawal signs but also motivationally relevant withdrawal behavior (O'Dell et al., 2004; Roberts et al., 1996, 2000). When measured at 8 hours into ethanol withdrawal (i.e., acute withdrawal), male Wistar rats had increased physiological withdrawal signs and escalated ethanol intake compared with controls. Furthermore, animals exposed to intermittent ethanol vapor exposure showed increased ethanol intake compared with continuous ethanol vapor-exposed animals when tested 2 hours into withdrawal (O'Dell et al., 2004). This increased responsiveness is thought to be a reflection of the organism's response to repetitive cycles of withdrawal.
The previous studies of the effects of baclofen on both the physical and motivational signs of withdrawal suggest that a GABAB agonist may have efficacy in reducing ethanol self-administration in ethanol-dependent animals. To test the hypothesis that ethanol self-administration in dependent animals would be particularly sensitive to a GABAB agonist, the present experiment compared nondependent and dependent ethanol self-administration during acute withdrawal on both continuous and progressive-ratio (PR) schedules of reinforcement.
MATERIALS AND METHODS
Nine male Wistar rats (Charles River Laboratory, Kingston, NY) weighing approximately 200 g upon arrival were communally housed (2–3 per cage) with food and water available ad libitum. The animals were housed within a temperature-controlled (21.5°C) vivarium that was maintained on a 12-hour light/dark cycle (lights on at 08:00 am). Upon their arrival in the vivarium, animals were handled daily over a 1-week period (until the onset of operant conditioning). During the nondependent and dependent pharmacological testing phase, the mean weight of the animals was 528 and 636 g, respectively. The work described herein adheres to the guidelines stipulated in the NIH Guide for the Care and Use of Laboratory Animals and was reviewed and approved by The Scripps Research Institute's Institutional Animal Care and Use Committee.
The operant chambers (Coulbourn Instruments, Allentown, PA) utilized in the present study had 2 retractable levers located 4 cm above a grid floor and 4.5 cm to each side of a 2-well acrylic drinking cup that allowed for up to 2 solutions to be administered upon the pressing of the appropriate lever. Recording of operant responses and subsequent fluid delivery were controlled by custom software running on a PC computer. A lever-press resulted in the activation of a 15 r.p.m Razel syringe pump (Stamford, CT) that delivered 0.1 mL of fluid to the appropriate well over 0.5 seconds. During the 0.5 second pump activation, no responses were recorded. Operant chambers were individually housed in ventilated, sound-attenuated cubicles to minimize environmental disturbances.
(±)Baclofen was purchased from Sigma Chemical Co. (St. Louis, MO) and was soluble in 0.9% physiological saline. Injections were administered via an intraperitoneal (IP) route of administration 30 minutes before operant ethanol self-administration sessions. The animals were weighed immediately before self-administration sessions that occurred on pharmacological test days and administered baclofen on a g/kg basis with the volume of the injections at 1 mL/kg.
The initial operant training was based on an adaptation of Samson's sweetened fading procedure (Samson, 1986) and allowed animals to initially respond for a “SuperSacc” solution (SS) consisting of 3% glucose and 0.125% saccharin. This solution serves as a potent reinforcer and makes it unnecessary to water restrict animals to induce the initial lever-pressing behavior. For 1 week, animals were trained to press on a continuous schedule of reinforcement (FR-1) for SS alone. The animals were then switched to a 2-lever condition with SS+10% EtOH (w/v; 10E) delivered by a response on one of the levers and water delivered by a response on the alternate lever, with the position of the SS+10E lever and water lever being alternated from left to right each session. After 1 week of SS+10E, animals were allowed to self-administer a 0.125% saccharin+10E solution for 4 sessions at which point the animals were switched to a 10E alone solution (see Table 1). Animals were allowed to self-administer 10E and water for approximately 7 weeks to allow for stable self-administration rates before the onset of pharmacological challenges (see Fig. 1). Intraperitoneal vehicle injections were given before the onset of pharmacological testing to protect against the possibility of vehicle effects. All self-administration sessions were for 30 minutes, after which the animals were returned to the vivarium.
Immediately following the completion of the initial dose–response challenge evaluating the effects of baclofen on FR responding (described below), animals were trained to respond for ethanol on a PR schedule of reinforcement on a single lever according to the following progression: 1, 1, 2, 2, 3, 3, 4, 4, 5, 5, 7, 7, 9, 9, 11, 11, 13, 13, 15, 15, 18, 18, 21, 21, 24, 24, etc. As the animals only had previous access to continuous reward self-administration sessions, they were allowed to self-administer ethanol for 4 days on an FR-4 schedule of reinforcement to adjust to a partial reinforcement situation before initiating the PR portion of the experiment. The point at which the animals stopped responding (i.e., no responses within a 30-minute time period) was defined as the breakpoint and reflected the number of reinforcers earned during the session. The PR session length was 180 minutes and 18 sessions were conducted (see Fig. 2) to allow for stable self-administration rates. Once stability had been achieved, an IP vehicle injection was administered to ensure against vehicle effects and pharmacological testing ensued.
Prevapor Baclofen Challenge of FR and PR Self-Administration Responding
Once stable FR-1 responding for ethanol had been established, the effects of baclofen (0.0–4 mg/kg, IP) on FR-1 ethanol self-administration were evaluated according to a Latin square design. Baclofen was administered 30 minutes before the onset of the 30-minute ethanol self-administration session. Following the session, the animals were returned to the vivarium. Following each day of the Latin square, animals were allowed to re-establish baseline ethanol self-administration levels before the drug test day. At the completion of the FR dose–response curve, animals were switched to the PR portion of the experiment.
Following stable PR responding, vehicle or baclofen (2.0 mg/kg, IP) were administered in a counterbalanced order and the effect on ethanol self-administration according to a PR schedule of reinforcement was evaluated over a 180-minute session. The dose of 2.0 mg/kg was chosen based on its efficacy in the FR-1 dose–response curve and administered 30 minutes before the PR self-administration session. Before the second day of drug testing, the animals were allowed to re-establish baseline levels of ethanol self-administration over 3 days. Following each session, the animals were returned to the vivarium and following the completion of the PR drug challenges, the ethanol vapor chamber portion of the experiment was initiated.
Ethanol Vapor Chamber Process
Ethanol vapor exposure has been shown to allow reliably for the titration of blood alcohol levels (BALs) that are sufficient for inducing ethanol dependence (O'Dell et al., 2004; Roberts et al., 1996, 2000). In this paradigm, BALs can be easily titrated by the experimenter to fit established criterion and the animals show normal weight gain and are freely moving (Rogers et al., 1979). Standard rat cages were housed in separate clear plastic chambers that were sealed and ethanol vapor or air was pumped through the chambers. Ethanol vapor was created by dripping 95% ethanol into 2000 mL Erlenmeyer flasks that remained at 50°C due to a warming tray. Air (11 L/min) was passed over the bottom of the flask so that when the ethanol contacted the warm glass and was vaporized, the air carried it into the vapor chamber. Alteration of the ethanol vapor concentration was accomplished by modulating the air flow carrying the vapor into the chamber. Target BALs were 150 to 200 mg% across the 4-week exposure period and were determined by sampling blood collected from the tail (0.25 mL) twice a week and assaying it for ethanol content using the NAD-ADH enzyme spectrophotometric method (Sigma Chemical Co., St. Louis, MO).
In the present experiment, animals were subjected to intermittent vapor exposure (14 hours on/10 hours off) over the course of 4 weeks. Intermittent vapor exposure has been shown to be more effective at inducing dependence (i.e., enhanced ethanol self-administration) when compared with continuous ethanol vapor exposure (O'Dell et al., 2004).
Postvapor Baclofen Challenge of FR and PR Self-Administration Responding
Following the 4-week dependence induction period resulting from ethanol vapor exposure, the animals were tested once at a time point corresponding to 6 hours into withdrawal (i.e., 6 hours after the ethanol vapor was terminated for that day) to confirm differences from prevapor baseline ethanol self-administration and returned to the vapor chambers. Subsequently, a baclofen dose–response curve (0.0–4.0 mg/kg, IP) was conducted according to a Latin square design as described above, except that test days occurred twice weekly to minimize drug carryover effects. On all test days, blood was collected before the ethanol vapor termination for the day to confirm that target BALs were met. Following the test trials, the animals were returned to the vapor chambers. This component of the postvapor testing took 29 days to complete.
Once the baclofen dose–response curve was completed on FR-1 responding for ethanol, animals were allowed to re-establish baseline ethanol self-administration for 180 minutes according to a PR schedule of reinforcement over 3 days. After confirming stable self-administration, the animals were administered baclofen or vehicle (2.0 mg/kg, IP) in a counterbalanced design 30 minutes before the initiation of 180 PR ethanol self-administration sessions. As with the postvapor FR-1 ethanol self-administration and baclofen dose–response challenges, the animals were tested at a time point corresponding to 6 hours of ethanol withdrawal, and testing occurred twice weekly. This component of the postvapor testing required an additional 14 days to complete. Thus, from start to finish, the animals were exposed to intermittent ethanol vapor for 75 days.
The prevapor and postvapor ethanol and water FR-1 responding data, as well as ethanol consumption (g/kg), following baclofen administration were analyzed using a repeated-measures 1-way analysis of variance (ANOVA). Post hoc Least Significant Difference (LSD) tests were conducted if a main effect for dose was found. To further investigate whether there was a dose × vapor treatment interaction, a repeated-measures 2-way ANOVA was computed on the pre and postvapor FR-1 responding and grams per kilogram data to compare the vehicle-treated and 1.0 mg/kg baclofen-treated animals' ethanol responding and intake. To identify possible rate-dependent effects, a median split analysis was conducted on the dependent animal's response rates, which yielded high and low subgroups that were compared using an independent samples t-test. Subsequently, the percent change from baseline following baclofen 1.0 mg/kg administration was compared for the high and low-responding group using an independent samples t-test. Furthermore, a repeated-measures 2-way ANOVA was conducted on the data reflecting the effects of vehicle and baclofen on pre- and postvapor PR breakpoints as well as cumulative responses during the PR sessions.
Figure 3 illustrates the effects of baclofen on nondependent and dependent ethanol and water self-administration, while Fig. 4 displays the effects of baclofen on ethanol consumption (g/kg). Based on previous data from our laboratory (Roberts et al., 1999), these levels of ethanol intake (g/kg) are estimated to result in BALs of approximately 35 mg% and, after extrapolating, 140 mg% for nondependent and dependent animals, respectively. The results of the repeated-measures 1-way ANOVA computed on the nondependent ethanol responding following baclofen administration revealed a main effect for dose [F(4, 32)=9.056, p<0.001]. Post hoc LSD tests revealed that both the 2.0 and 4.0 mg/kg doses were significantly different from vehicle (p<0.05 and 0.01, respectively). Moreover, the ANOVA conducted on the effects of baclofen on ethanol-dependent responding for ethanol indicated a main effect for dose [F(4, 32)=5.865, p=0.001]. Post hoc LSD tests indicated a significant reduction of responding compared with vehicle for doses of 1.0 (p<0.05), 2.0 (p<0.05), and 4.0 mg/kg (p<0.05). No effect of baclofen was seen for either nondependent or dependent water responding. A 1-way ANOVA on ethanol consumption (g/kg) revealed a similar pattern with a significant main effect of baclofen dose for both nondependent [F(4, 32)=9.688, p<0.001] and ethanol-dependent [F(4, 32)=6.795, p<0.001] responding. When a repeated-measures 2-way ANOVA was carried out on the vehicle-treated and 1.0 mg/kg baclofen-treated ethanol responding and consumption (g/kg) data, a significant vapor treatment (nondependent vs dependent) and drug (vehicle vs baclofen) effect was found for both the responding [F(1, 8)=34.869, p<0.001 and F(1, 8)=11.932, p<0.001, respectively] and consumption [F(1, 8)=15.143, p<0.01 and F(1, 8)=18.921, p<0.01, respectively] data. A treatment × drug interaction was found for both the responding [F (1, 8)=7.727, p<0.05] and consumption [F (1, 8)=5.265, p=0.05] data, indicating that baclofen was more potent in ethanol-dependent animals. The median split yielded 2 groups (high and low responders) with mean (±SEM) lever-presses (99.8±7.8 and 53.25±1.43, respectively) that displayed comparable decreases in responding following 1.0 mg/kg baclofen administration (77%±0.1 and 70%±0.11, respectively). This further supports the hypothesis that baclofen acted independently of the response rate in the dependent group. Figures 5 and 6 depict the effect of vehicle and baclofen (2.0 mg/kg) on nondependent and ethanol-dependent PR breakpoints and cumulative PR responses for ethanol. For the PR breakpoint data, a 2-way repeated-measures ANOVA identified a main effect for vapor treatment [F (1, 8)=28.8, p=0.001] and baclofen [F (1, 8)=25.296, p=0.001] and likewise a main effect for vapor treatment [F (1, 8)=48.203, p<0.001] and baclofen [F (1, 8)=5.702, p<0.05] when analyzing the cumulative responses during the PR session. Thus, a 1-month intermittent vapor exposure regimen induced an increased motivational state to consume ethanol, that was suppressed by baclofen. When a 2-way repeated-measures ANOVA was conducted on the baseline cumulative PR responses over time (see Fig. 7), a significant main effect for vapor treatment and time was found [F(1, 8)=25.85, p=0.001 and F(17, 136)=4.76, p<0.001, respectively]. As can been seen in Fig. 7, the vapor-exposed animals worked harder and longer to obtain ethanol. This is supported by the fact that there was a significant vapor × time interaction [F(17, 136)=4.958, p<0.001], which indicated that the vapor treatment affected the groups differentially over time.
The primary purpose of this experiment was to evaluate the effects of GABAB receptor agonist on operant ethanol self-administration during acute withdrawal (i.e., 6 hours into withdrawal) in ethanol-dependent rats. Animals were tested in both nondependent and ethanol-dependent states during acute withdrawal on 2 different schedules of operant ethanol reinforcement following baclofen challenge. Following the establishment of stable baseline ethanol and water self-administration on an FR-1 schedule of reinforcement, the effects of baclofen were tested on ethanol and water self-administration in nondependent and subsequently in ethanol-dependent rats. Ethanol dependence was induced by a 1-month ethanol vapor-exposure regimen and showed that ethanol-dependent animals have a significantly higher level of ethanol self-administration and consumption than when in a nondependent state, confirming the viability of this method for inducing increased ethanol self-administration. Although vapor exposure might be expected to alter alcohol metabolism, the current data set solidly identifies that dependent animals are more motivated to obtain ethanol. Whether this is due to pharmacokinetic or pharmacodynamic changes (or both) in the dependent animals. Additionally, another hypothesis that could explain the increase in responding during the post-vapor phase would be that tolerance to the response-decreasing effects of ethanol occurred. If that were the case, one would expect to observe increases in responding on not only the ethanol lever, but the water lever also. However, water responding remained stable throughout the drug challenges, which suggests that tolerance to the response-depressant effects of ethanol was not contributing to the escalated ethanol intake following vapor exposure. Furthermore, in our vapor-exposure paradigm, the rats had ad libitum food and water during both the vapor-on and vapor-off stages. When the animals were returned to their home cage in the vapor chambers after being tested, they still had a number of hours before the onset of vapor-exposure began and thus would still have had a number of hours for food intake before the onset of the ethanol vapor for that day. Taking this into consideration, it seems unlikely that the rats would be consuming alcohol solely for caloric purposes when a more palatable food reinforcer was available for consumption.
Baclofen (0, 0.5, 1, 2, and 4 mg/kg, IP) dose-dependently attenuated ethanol self-administration and consumption in both nondependent and ethanol-dependent animals during acute withdrawal without affecting water self-administration. When comparing nondependent and ethanol-dependent self-administration responses and ethanol consumption, the dose of baclofen required to attenuate significantly responding and consumption in the nondependent animals was 2.0 mg/kg while the dose that was sufficient to affect ethanol-dependent animals significantly was 1.0 mg/kg, indicating that baclofen was more potent in ethanol-dependent animals. This was confirmed by the significant vapor treatment × baclofen dose interaction that was observed for both self-administration responding (Fig. 3) and consumption (Fig. 4). To ensure that the increased potency established by the treatment × drug interaction was not due to the increased responding seen in the ethanol-dependent group being more susceptible to pharmacologic manipulation simply because it had a higher baseline than the nondependent group (i.e., rate-dependent effects), a median split analysis was utilized to compare the effects of 1.0 mg/kg baclofen on high and low responders during the dependent phase. The median split yielded 2 groups (high and low) with mean (±SEM) lever-presses of 99.8±7.8 and 53.25±1.43, respectively. Baclofen induced a relatively equivalent decrease in percent change from baseline regardless of whether the animals had high or low response rates initially. Thus, baclofen suppressed responding comparably in animals that significantly differed in their responding for ethanol, which supports the finding that baclofen was acting independently of response rate and was more potent in ethanol-dependent animals. The data collected while the animals were in a nondependent state are consistent with the published evidence evaluating the effects of baclofen on operant ethanol self-administration (Anstrom et al., 2003; Janak and Gill, 2003; Stromberg, 2004), in that there was a dose-dependent reduction in ethanol self-administration following baclofen administration.
To confirm that 1 month of ethanol vapor exposure produces a measurable increase in motivation to consume ethanol and that the effects of baclofen were to due to decreased motivation to consume ethanol in both a nondependent and dependent state, the animals were additionally trained to self-administer ethanol according to a PR schedule of reinforcement. This schedule necessitated that the response requirements following a successful reinforcement were increased (i.e., the animals had to work harder for each successive reinforcement). Once stable baseline PR self-administration was achieved, the effect of baclofen was evaluated for the ability to modulate PR responding for ethanol. Accordingly, once the animals were subjected to 1 month of vapor exposure, the effect of baclofen on PR responding was again evaluated. As illustrated in Figs. 5 and 6, the result of a 1-month ethanol-vapor exposure regimen produced a significant increase in both the PR breakpoint (the point at which the animals will no longer lever-press for ethanol) and the cumulative responses during the PR session, supporting the hypothesis that animals in an ethanol-dependent state are willing to work harder for ethanol than when in a nondependent state. This is further supported when taking into account cumulative responses over time. As can be seen in Fig. 7, the vapor-exposed animals worked harder and longer to obtain ethanol. This is supported by the fact that there was a significant vapor × time interaction, which indicated that the vapor treatment affected the nondependent and dependent groups differentially over time. When the effects of baclofen (2.0 mg/kg) were tested on PR responding before and after dependence induction, it was observed that in both nondependent and dependent animals the result was a significant decrease in breakpoints and cumulative responses. This decrease showed that the animals were less motivated to consume ethanol following baclofen challenge and that the data obtained under the FR-1 schedule (see Figs. 3 and 4) are consistent with the hypothesis that the high dose of baclofen is decreasing the reinforcing or rewarding properties of ethanol in both nondependent and ethanol-dependent rats.
γ-Aminobutyric acid-B receptors have been shown to be distributed in brain regions associated with ethanol reinforcement and reward such as the ventral tegmental area (VTA), nucleus accumbens (NAc), and the central nucleus of the amygdala (CeA; Bowery et al., 1987; Cousins et al., 2002; Fadda et al., 2003; Xi and Stein, 1999), which are likely candidates as the site(s) of action for baclofen to modulate ethanol self-administration behavior. This is supported by evidence identifying the ability of GABAB ligands to directly modulate ethanol's effects in the VTA and NAc (Steffensen et al., 2000) and CeA (Roberto et al., 2003) and that baclofen can decrease NAc (Kalivas et al., 1990) and somatodendritic (Klitenick et al., 1992) dopamine release when infused into the VTA. That baclofen was more potent in ethanol-dependent animals (see Figs. 3 and 4) could be explained by baclofen's ability to also decrease both the physiological and anxiogenic withdrawal responses in rats produced by chronic ethanol exposure. The anxiogenic responses observed during ethanol withdrawal have been linked to CeA function (Koob and Le Moal, 2001; Pich et al., 1995; Rassnick et al., 1993b) and recently it has been shown that GABA function is altered in the CeA following chronic ethanol vapor exposure (Roberto et al., 2004). In fact, GABAA receptor modulation in the CeA selectively suppresses ethanol self-administration in ethanol-dependent animals compared with nondependent controls (Roberts et al., 1996). Thus, the CeA could be a putative site of action for the increased potency of baclofen observed in ethanol-dependent animals. It must be noted, however, that the change in sensitivity to baclofen when in a dependent state could also be occurring via alterations in pharmacokinetic mechanisms and should be evaluated in future experiments.
In conclusion, ethanol vapor exposure was an effective method for inducing dependence in rats as confirmed by the differential ethanol intake between nondependent and dependent groups on 2 different operant schedules of reinforcement (i.e., continuous and PR). The activation of GABAB receptors by baclofen dose-dependently reduced both nondependent and ethanol-dependent self-administration of ethanol on a continuous schedule of reinforcement, with an increased sensitivity to baclofen being observed while in an ethanol-dependent state. The decrease in ethanol self-administration that was observed was attributed to the ability of baclofen to decrease the motivation to consume ethanol as demonstrated by the decrease in breakpoints for ethanol in animals that were responding according to a PR schedule of reinforcement. Therefore, a GABAB receptor agonist was effective in suppressing both dependent and nondependent ethanol consumption and could be a viable target for the pharmacotherapeutic treatment of chronic alcohol abuse.
The authors would like to thank Eric Zorrilla, Laura O'Dell, Maury Cole, and Ron Smith for their assistance with this project and Mike Arends for editorial assistance.