Addiction to cocaine is commonly preceded by experiences with legal or decriminalized drugs, such as alcohol, nicotine, and marijuana. The biological mechanisms by which these gateway drugs contribute to cocaine addiction are only beginning to be understood. We report that in the rat, prior alcohol consumption results in enhanced addiction-like behavior to cocaine, including continued cocaine use despite aversive consequences. Conversely, prior cocaine use has no effect on alcohol preference. Long-term, but not short-term, alcohol consumption promotes proteasome-mediated degradation of the nuclear histone deacetylases HDAC4 and HDAC5 in the nucleus accumbens, a brain region critical for reward-based memory. Decreased nuclear HDAC activity results in global H3 acetylation, creating a permissive environment for cocaine-induced gene expression. We also find that selective degradation of HDAC4 and HDAC5, facilitated by the class II–specific HDAC inhibitor MC1568, enhances compulsive cocaine self-administration. These results parallel our previously reported findings that the gateway drug nicotine enhances the behavioral effects of cocaine via HDAC inhibition. Together, our findings suggest a shared mechanism of action for the gateway drugs alcohol and nicotine, and reveal a novel mechanism by which environmental factors may alter the epigenetic landscape of the reward system to increase vulnerability to cocaine addiction.

( A ) Drug treatment paradigm to study alcohol and cocaine coadministration. Access to voluntary alcohol (10% alcohol, 2 hours per day) and voluntary cocaine (self-administration, 0.8 mg/kg per injection) was restricted to 3 p.m. to 5 p.m. and 11 a.m. to 2 p.m., respectively, to avoid metabolic interaction between the two drugs. ( B ) Alcohol self-administration. Animals in the alcohol-primed group began drinking alcohol on day 1 of the paradigm; animals in the alcohol-concurrent group began drinking on day 11. All groups start cocaine self-administration (0.8 mg/kg per infusion) on day 11. The average alcohol intake during the alcohol-priming period was 1 g/kg during the first 5 days, increasing to 1.2 g/kg during the second 5 days. Alcohol intake was similar in the alcohol-primed versus alcohol-concurrent groups from days 11 to 32 [two-way repeated-measures (RM) analysis of variance (ANOVA): Treatment group: F 1,12 = 1.398, P = 0.26; Treatment day: F 21,252 = 10.71, P < 0.0001; Interaction: F 21,252 = 1.14, P = 0.303; n = 6 to 8 per group]. ( C ) Prior exposure to alcohol does not affect the acquisition of lever pressing on an FR5 schedule of reinforcement. Animals were started on FR1 and gradually increased to FR3 (1 to 2 days) and then to FR5. Animals reached FR5 after 5.9, 6.2, and 6.6 days for water control, alcohol-primed, and alcohol-concurrent groups, respectively (one-way ANOVA: F 2,21 = 0.63, P = 0.5425, not significant; n = 6 to 8 per group). ( D ) Prior alcohol exposure does not enhance lever pressing for cocaine reward (0.8 mg/kg per injection) during the maintenance phase of cocaine self-administration. The analysis showed no main effect for treatment group, treatment day, or an interaction between the two factors (two-way ANOVA: Group: F 2,123 = 1.119, P = 0.3298, not significant; Treatment day: F 6,123 = 1.44, P = 0.20, not significant; Interaction: F 12,123 = 0.16, P = 0.9995, not significant). ( E ) Alcohol preexposure enhances persistence of cocaine seeking during unrewarded time-out sessions, averaged over the last 3 days of the maintenance phase (B) ( one-way ANOVA: F 2,19 = 3.66, P = 0.045; Tukey post hoc: P = 0.047, alcohol primed versus alcohol-naïve; n = 6 to 8 per group). ( F ) Alcohol preexposure enhances motivation for cocaine self-administration in a progressive ratio schedule of reinforcement (one-way ANOVA: F 2,20 = 6.45, P = 0.007; Tukey post hoc analysis: P < 0.01, control versus alcohol-primed; P < 0.01, concurrent versus alcohol-primed; n = 6 to 8 per group). ( G ) Alcohol preexposure enhances compulsive cocaine self-administration. Successive increases of the footshock intensity resulted in a decrease in lever pressing for all groups. Alcohol pretreated animals have significant resistance to footshock (two-way RM ANOVA: Group: F 2,19 = 4.76, P = 0.02; Footshock: F 3,57 = 67.94, P < 0.0001; Footshock × Group interaction: F 6,57 = 1.829, P = 0.1095; n = 6 to 8 per group). The baseline number of cocaine infusions (at 0.0 mA) did not differ between groups (AN, 19.67 ± 2.10; AP, 19.7 ± 2.32; AC, 20.00 ± 2.46). ( H ) Alcohol preexposure does not alter shock-resistant lever pressing for sugar pellets in food-restricted animals (two-way RM ANOVA: Group: F 1,9 = 0.003, P = 0.96, not significant; Footshock: F 3,27 = 131.3, P < 0.0001; Footshock × Group interaction: F 3,27 = 0.64, P = 0.59, not significant; n = 5 to 6 per group). Baseline amount of sugar pellet reward (at 0.0 mA) did not differ between groups (AN, 46.60 ± 3.3; AP, 49.33 ± 2.15). *P < 0.05. Data are means ± SEM.

To study the effects of alcohol on cocaine-related behaviors, we again used a sequential drug administration paradigm, with voluntary access to both alcohol and cocaine ( Fig. 1A ). In this paradigm, daily alcohol use (10% ethanol, 2 hours per day; see Fig. 1A and table S1) precedes the start of daily cocaine self-administration by 10 days. Because most drug users progress to illicit drug use by adding the illicit drug to their preexisting drug regimen (as opposed to switching) ( 4 ), daily alcohol use is continued throughout the duration of the cocaine self-administration paradigm. To avoid acute behavioral, metabolic, or pharmacokinetic interaction between alcohol and cocaine ( 7 – 11 ), we restricted access to alcohol and cocaine to different time periods during the dark (wake) cycle ( Fig. 1A ). We assessed the effect of prior alcohol exposure on three key indices of cocaine addiction observed in humans: (i) persistence of drug seeking in the absence of reward, (ii) motivation for drug use, and (iii) compulsivity. Conversely, we explored the directionality of drug action by reversing the sequence of drug exposure and testing the effect of prior cocaine self-administration on subsequent alcohol preference. Furthermore, to determine whether alcohol creates a permissive epigenetic environment for cocaine-induced gene expression, we used the same alcohol exposure paradigm (10% alcohol, 2 hours per day) to examine the epigenetic effects of daily alcohol use in the nucleus accumbens, a brain region critical for mediating cocaine addiction–like behaviors.

Using a sequential drug administration paradigm that models the stages of drug abuse seen in human populations, we previously found that mice preexposed to nicotine show an enhanced response to the rewarding properties of cocaine ( 5 ). Nicotine primes the brain to cocaine-induced changes by modifying chromatin structure and enhancing cocaine-induced gene expression and long-term synaptic depression in the striatum. The reverse is not observed: Cocaine has no effect on nicotine-induced behavior or gene expression, supporting the hypothesis of a unidirectional progression of drug use ( 5 ). If the different gateway drugs operate through similar biological mechanisms in a rodent model, one would predict that alcohol, a gateway drug, would also potentiate the effects of cocaine. We therefore asked: Does prior alcohol use enhance the behavioral effects of cocaine in a rodent model of addiction and, if so, is the priming process of alcohol mediated by cellular and molecular mechanisms that are similar to those for nicotine? Moreover, is the effect of alcohol on cocaine consumption also unidirectional, as it is with nicotine?

Loss of control over drug use, typified by continued use despite aversive consequences, is a hallmark characteristic of cocaine addiction ( 1 ). The finding that only a small proportion (~21%) of cocaine users progress to compulsive use ( 2 ) highlights the importance of both genetic and environmental factors in conferring vulnerability to cocaine addiction. Prior use of either alcohol or nicotine ranks high among the environmental factors known to influence subsequent use of cocaine. Thus, in 2015, 91% of cocaine users, 18 to 49 years old, had first used alcohol before using cocaine and 5.1% had started both drugs at the same age; 85% had first used nicotine, and 5.8% had started both nicotine and cocaine at the same age ( 3 ). These behavioral regularities in drug involvement have given rise to the gateway hypothesis, which proposes that certain drugs, such as alcohol, nicotine, and marijuana, increase the risk of subsequently using other drugs, such as cocaine ( 4 ). However, the mechanisms by which these initial drug experiences increase vulnerability to cocaine use have only recently begun to be understood ( 5 , 6 ).

RESULTS

Prior alcohol use does not enhance acquisition or amount of cocaine self-administration To distinguish the effects of alcohol preexposure from alcohol coexposure, we compared the outcomes of cocaine self-administration in the alcohol-primed group ( “AP”; Fig. 1A) with two control groups: (i) an alcohol-naïve (AN) group that drank water instead of alcohol and (ii) an alcohol-concurrent (AC) group that started using alcohol and cocaine on the same day. Animals in the AP and AC groups did not differ in their daily alcohol intake patterns (Fig. 1B). Prior exposure to alcohol did not increase the daily intake of cocaine during the acquisition or maintenance phase of the sequential paradigm (fig. S1). Prior exposure to alcohol did not affect the acquisition rate [duration of time required to learn to lever press for cocaine injection on a fixed ratio of 5 (FR5)]. We found that the animals learned to lever press on an FR5 schedule after 5.9, 6.2, and 6.6 days in the alcohol-naïve, alcohol-primed, and alcohol-concurrent groups, respectively (Fig. 1C). Similarly, the groups did not differ in the average number of lever presses for cocaine during a 7-day maintenance phase (Fig. 1D). Overall, our behavioral findings on the effect of prior alcohol use on the early stages of cocaine self-administration (days 11 to 21; Fig. 1A) are consistent with the observations of Fredriksson et al. (12). Using a 12-day cocaine self-administration protocol, they reported no change in acquisition of cocaine self-administration in animals that had an extensive 7-week history of prior alcohol use.

Prior alcohol use enhances cocaine addiction–like behaviors Cocaine addiction is characterized not only by the choice to self-administer cocaine but also by the complex behaviors that define addiction, such as increased motivation for the drug, persistence of drug seeking in the absence of reward, and continued drug use despite negative consequences. To determine whether animals with a history of prior alcohol use have enhanced cocaine addiction–like behaviors, we assayed these key behavioral indices using an approach modeled after Deroche-Gamonet et al. (13) and Belin et al. (14). To examine the persistence of cocaine seeking in the absence of reward, we measured the number of lever presses during “no drug periods” of daily cocaine self-administration (two 15-min intervals during which the levers are available but are not rewarded). Animals in the alcohol-naïve group averaged 18 lever presses during the time-out periods. Animals in the alcohol-primed group had significantly enhanced persistence in comparison to alcohol-naïve animals, averaging 58 lever presses. Animals in alcohol-cocaine concurrent control averaged 38 lever presses during the time-out period but were not significantly different from the alcohol-naïve group (Fig. 1E). We next measured motivation for cocaine self-administration on a progressive ratio schedule of reinforcement, where the animal was required to make increased number of lever presses to earn each subsequent intravenous cocaine reward (0.8 mg/kg per injection), until it reached the breakpoint where it ceased lever pressing (15). We found that alcohol-naïve animals worked for cocaine until an average breakpoint of 310 lever presses, whereas alcohol-primed animals worked for cocaine until they reached an average breakpoint of 563 lever presses. Animals in the alcohol-concurrent group had an average breakpoint of 317 lever presses, similar to the alcohol-naïve group (Fig. 1F). One of the core characteristics of drug addiction in humans is compulsivity, as reflected in continued drug use despite negative consequences (13, 16). Prior studies have found that most rats stop self-administering cocaine when challenged with an aversive footshock 20 days after initiating cocaine self-administration (16). But when rats have had long-term exposure to cocaine (~60 days of daily cocaine self-administration), a subset of animals continue to seek and take cocaine despite an aversive footshock (13, 17). We reasoned that exposure to alcohol before cocaine would reveal a compulsive phenotype that would not otherwise occur in the absence of priming by alcohol. To test for compulsive drug use, we introduced an aversive 2-s foot shock (0.1 to 0.3 mA) to the cocaine self-administration session, with an increase in shock intensity occurring every 40 min. We found that animals in the alcohol-naïve and alcohol-concurrent groups declined similarly, earning 73 and 76% of baseline reward at 0.1 mA, 39 and 31% of baseline reward at 0.2 mA, and 16 and 14% of baseline reward at 0.3 mA. By contrast, animals preexposed to alcohol were significantly more resistant to punishment, earning 90, 68, and 29% of baseline reward at 0.1, 0.2, and 0.3 mA of footshock, respectively (Fig. 1G). Alcohol is a nonspecific and potent neurotoxin, and could decrease sensitivity to footshock by decreasing peripheral pain sensitivity or fear memory. We found, however, that alcohol use did not enhance compulsive lever pressing for sugar pellets in food-restricted animals (Fig. 1H). Thus, the priming effects of alcohol on cocaine compulsivity appear to be mediated by processes that do not generalize to natural rewards. Together, our results indicate that voluntary alcohol use increases vulnerability to cocaine by enhancing persistence of drug seeking, motivation, and continued use despite negative consequences.

Prior cocaine use decreases preference for alcohol To determine whether prior exposure to cocaine enhances alcohol preference, we reversed the order of drug exposure so that cocaine self-administration preceded the start of voluntary alcohol use by 10 days (fig. S2A). Animals in the control group with no prior history of cocaine showed increasing preference for alcohol in a two-bottle free choice paradigm, whereas animals with a prior history of cocaine use actually had decreased alcohol intake and decreased preference for alcohol (fig. S2, B and C). When we analyzed the daily alcohol intake of animals in our sequential alcohol-to-cocaine paradigm (Fig. 1B), we found that animals in the alcohol-primed group had a transient but significant decline in daily alcohol intake during the first 2 days of cocaine self-administration, and after reaching a nadir of alcohol intake, the animals required 4 to 5 days before reaching the prior baseline of daily alcohol intake of 1 to 1.2 g/kg per day (fig. S3). Animals that started alcohol self-administration concurrently with cocaine use also required 4 to 5 days before reaching a daily alcohol intake of 1 to 1.2 g/kg. This is much slower than cocaine-naïve animals that began drinking 1 g/kg by the second day of alcohol use (alcohol-primed group, days 1 to 5). These results suggest that cocaine use may decrease the preference for alcohol even without a priming period. Our findings are consistent with previous reports that prior cocaine use does not enhance alcohol use in rats (18) and that, in mice, prior cocaine use results in decreased alcohol preference (19). Together, our findings are consistent with a unidirectional cross-sensitization as predicted by the gateway hypothesis: from alcohol to cocaine, but not from cocaine to alcohol.

Alcohol use creates a permissive epigenetic environment for cocaine-induced gene expression To gain insight into the molecular mechanisms by which prior alcohol use potentiates the behavioral effects of cocaine self-administration, we examined the epigenetic effects of alcohol use in the nucleus accumbens, a brain region critical for addiction-related learning and memory. In our previous studies with nicotine, we found that nicotine inhibits histone deacetylase (HDAC) activity in the striatum. Inhibition of HDAC activity resulted in global histone acetylation and a permissive epigenetic environment for cocaine-induced gene expression (4). To determine whether alcohol exposure similarly primes the transcriptional response to cocaine, we allowed animals to drink alcohol for 10 daily 2-hour sessions and followed this with a single injection of cocaine (20 mg/kg intraperitoneally) 18 hours after the last alcohol ingestion (Fig. 2A). Accumulation of the ΔFOSB protein in the nucleus accumbens has been found to have a key role in enhancing cocaine self-administration (20), and is induced by cocaine under voluntary (self-administered) conditions as well as experimenter-administered conditions (21). We now asked: Does prior alcohol use enhance the cocaine-induced transcription of this key marker? We carried out quantitative polymerase chain reaction (PCR) using primers specific to the ΔFosB splice variant and found that alcohol preexposure significantly enhanced cocaine-induced expression of ΔFosB in the nucleus accumbens compared to alcohol-naïve animals that also received an injection of cocaine (Fig. 2B). Fig. 2 Alcohol use creates a permissive epigenetic environment for cocaine-induced gene expression. (A) Diagram of sequential drug administration paradigm. Animals drank alcohol in a limited access paradigm [alcohol 10% (v/v); 2 hours per day; average intake, 1.11 ± 0.1 g/kg per day; n = 14] for 10 days. An acute cocaine injection (20 mg/kg intraperitoneally) was given 18 hours after the last alcohol ingestion to avoid pharmacokinetic interaction between alcohol and cocaine. (B) A single cocaine injection causes increased expression of the ΔFosB transcript in alcohol-naïve animals (one-way ANOVA: F 3,20 = 20.15, P < 0.0001; Sidak post hoc: water/saline-injected versus water/cocaine-injected, P < 0.05), whereas alcohol pretreated animals have significantly enhanced cocaine-induced ΔFosB induction (P < 0.05, alcohol/cocaine-injected versus water/cocaine-injected; P < 0.0001, alcohol/cocaine-injected versus water/saline-injected, alcohol/saline-injected; n = 6 to 7 per group). (C) ChIP experiment for acetylated histone H3K27 at the FosB region of interest shows increased acetylation in animals treated with 10% alcohol, 2 hours per day for 10 days (average intake, 1.1 ± 0.1 g/kg per day, n = 5, P < 0.01 versus water control, n = 3 to 5 per group). (D) Immunoblotting experiment for H3K27 acetylation in animals that drank for 10 consecutive days [10% (v/v) alcohol, 2 hours per day; average intake, 0.98 ± 0.12 g/kg per day; n = 5] and euthanized 18 hours after the last alcohol ingestion shows global increase in H3K27 acetylation in the nucleus accumbens (P < 0.05, 0 hours versus water control; n = 4 to 5 per group). P.O., per oral. *P < 0.05, **P < 0.01, ***P < 0.0001. Data are means ± SEM. To determine whether alcohol facilitates acetylation locally at the FosB gene locus, we next performed chromatin immunoprecipitation (ChIP) in lysates of the nucleus accumbens of alcohol-treated animals. A search of the encyclopedia of DNA elements (ENCODE) revealed that acetylation of H3 lysine 27 (H3K27) in the first intronic region of FosB (the gene giving rise to the splice variant ΔFosB) may be a key epigenetic regulatory mark for this gene in several tissues, including brain (fig. S4) (22). We found that alcohol facilitated robust acetylation of H3K27 residues locally at the FosB gene—fourfold above the (water) control group (Fig. 2C). Because the level of global H3 acetylation in the nucleus accumbens has been found to be positively correlated with motivation for cocaine self-administration in rats [although no such effect has been observed for H4 acetylation (23)], we next immunoblotted total histone lysates and found that alcohol also increased global acetylation of H3K27 in the nucleus accumbens (Fig. 2D).

Alcohol use promotes decreased nuclear HDAC activity in the nucleus accumbens To determine whether the local acetylation at the FosB locus and the global acetylation in the nucleus accumbens are due to changes in HDAC activity, we next tested the enzymatic HDAC activity in nuclear lysates of the nucleus accumbens of animals exposed to 10 days of daily alcohol use. Prior studies have found that the effects of alcohol on HDAC activity are dynamic and highly sensitive to both the acute exposure and abstinence stages of alcohol use (24). Therefore, we tested HDAC activity in nucleus accumbens lysates of animals sacrificed at sequential time points after the 10th daily 2-hour alcohol session (on day 10) (Fig. 3A). To determine whether the effects are specific to long-term alcohol exposure, we performed a similar analysis of nuclear HDAC activity in the nucleus accumbens of animals after only 2 days of alcohol exposure (Fig. 3B). Fig. 3 Long-term, but not short-term, alcohol use promotes progressive decrease of nuclear HDAC activity following alcohol cessation. (A) Diagram of 10-day voluntary drinking protocol. Alcohol-treated animals were sacrificed at sequential time points after the last alcohol ingestion on day 10: 0 (end of alcohol drinking session), 12, 18, and 22 hours after the last alcohol ingestion (average intake, 0.95 ± 0.05 g/kg per day; n = 20). (B) Short-term (2-day) alcohol exposure paradigm (average alcohol intake, 1.07 ± 0.16 g/kg per day; n = 20). (C) Ten-day exposure to alcohol causes a decrease in nuclear HDAC activity in the nucleus accumbens (one-way ANOVA: F 4,19 = 3.669, P = 0.0224; Sidak post hoc: P = 0.026, water versus alcohol 22 hours after treatment). (D) Two-day exposure to alcohol does not result in decreased nuclear HDAC activity (one-way ANOVA: F 4,18 = 0.794, P = 0.5442). (E) HDAC activity decreases progressively following cessation of alcohol treatment in animals treated with alcohol for 10 days (two-way ANOVA: Time after treatment cessation: F 3,32 = 3.165, P = 0.038; Treatment group: F 1,32 = 17.18, P = 0.0002; Interaction: F 3,32 = 4.715, P = 0.0078; Sidak post hoc: no significant change at 0 and 12 hours, P = 0.035 at 18 hours, and P = 0.0003 at 22 hours; n = 4 to 5 per group). (F) Immunoblotting experiment for H3K27 acetylation shows enhancement of global H3K27 acetylation at 12, 18, and 22 hours after the last alcohol ingestion (one-way ANOVA: F 4,16 = 5.45, P = 0.0058; Sidak post hoc: P = 0.0033, P = 0.006, P = 0.010 at 12, 18, and 22 hours, respectively, versus water control group; n = 4 to 5 per group). (G) Two-day exposure to alcohol does not result in enhanced H3K27 acetylation in the nucleus accumbens (one-way ANOVA: F 4,14 = 0.3981, P = 0.8067; n = 4 to 5 per group). *P < 0.05, **P < 0.01. Data are means ± SEM. Ten days, but not 2 days, of alcohol use resulted in a significant decrease in HDAC activity in comparison to water control (Fig. 3, C and D). Ten days of alcohol use caused a progressive decrease in HDAC activity after the end of the alcohol drinking session (Fig. 3E). We observed no significant change in HDAC activity at the time of acute alcohol exposure (“time 0,” during which blood alcohol levels were 193 ± 43 μg/ml; see table S1) and a significant decrease in HDAC activity at time points during which no serum alcohol was detectable (18 and 22 hours; Fig. 3E and table S1). In line with this decrease in HDAC activity, we also observed an increase in global H3K27 acetylation in the nucleus accumbens, starting at 12 hours after the last alcohol intake (Fig. 3F). Two days of alcohol use did not have a significant effect on H3K27 acetylation in the nucleus accumbens (Fig. 3G). The finding that short-term alcohol use does not affect HDAC activity or histone acetylation suggests that HDACs behave quite differently under short-term versus long-term exposure conditions, fostering a permissive epigenetic environment only after long-term exposure. Together with the findings observed in our earlier studies with nicotine, the present findings suggest that HDAC inhibition, global acetylation in the nucleus accumbens, and specific hyperacetylation of key genes in the nucleus accumbens may be general mechanisms of action for gateway drugs (5).

Alcohol use decreases nuclear HDAC4 and HDAC5 levels in the nucleus accumbens We next asked: Which specific HDACs are involved in alcohol-induced acetylation, and how are these inhibited by alcohol? Eleven HDACs have been identified in mammals. An extensive body of literature has demonstrated key roles of these enzymes in integrating a diverse array of molecular responses to stimulants such as cocaine (25, 26). We focused on the class IIa HDACs HDAC4 and HDAC5 because they have been previously implicated in regulating the behavioral effects of cocaine, including cocaine self-administration (23, 27, 28). The class II HDACs shuttle between the cytoplasm and nucleus in an activity-dependent manner (29, 30), and are heavily regulated by posttranslational modifications such as phosphorylation, carbonylation, SUMOylation, and ubiquitination (31). Although the class II HDACs carry a mutation in their catalytic domains that render them inactive as deacetylases (32, 33), genetic and pharmacological knockdown of class II HDACs causes decreased HDAC activity (34) and global histone hyperacetylation and enhanced neuronal plasticity (35–37). Class II HDACs mediate histone acetylation by forming a multiprotein co-repressor complex that includes the class I HDAC, HDAC3 (38). HDAC3 is a potent deacetylase. Focal deletion of HDAC3 has been found to enhance long-term memory (39) and to facilitate conditioned place preference to cocaine (40). The catalytically inactive C terminus of HDAC4 is crucial for the recruitment and deacetylase activity of the multiprotein complex, which forms only in the nucleus of the cell. Thus, class II HDACs may function as activity-dependent scaffolds that target the co-repressor complex to specific promoters (38). HDAC4 has been found to be critical for activity-dependent regulation of H3K27 deacetylation in specific gene promoter regions such as BDNF (brain-derived neurotrophic factor) (41). We first asked: Does either HDAC4 or HDAC5 interact with the FosB gene locus? To address this question, we performed ChIP experiments in the rat striatum with antibodies against HDAC4 and HDAC5 and found that HDAC4, but not HDAC5, interacted with the FosB locus (fig. S5A). This finding is consistent with that of Wang et al. (23), who reported that overexpression of HDAC4 in the nucleus accumbens resulted in a decreased expression of FosB. Having found that HDAC4 is present at the FosB gene locus, we next examined the specific regulation of HDAC4 by asking whether alcohol regulates acetylation at the FosB promoter by altering the subcellular localization of HDAC4 in the nucleus accumbens. Another class IIa HDAC, HDAC5, limits cocaine reward by shuttling from the cytoplasm to the nucleus following acute exposure to cocaine (28). Although purified class II HDACs have weak deacetylase activity (32), Fischle et al. (38) found that, in vivo, nuclear HDAC4 (but not cytoplasmic HDAC4) exists in an enzymatically active multiprotein complex. To explore the effects of alcohol use on subcellular localization of HDAC4, we performed immunohistochemistry on striatal slices of animals exposed to alcohol for 10 days (fig. S5, B and C). We found that an acute exposure to alcohol (time 0) did not result in a significant change in HDAC4 subcellular localization compared to control animals. The HDAC4 stain completely and precisely overlaid the nuclear stain in 81% of nucleus accumbens cells in both the water control group and the acutely exposed alcohol group (time 0). By contrast, 18 hours after the last alcohol exposure on day 10 (when blood alcohol was not detectable; table S1), a significantly lower number of cells had HDAC4-positive nuclei, suggesting that HDAC4 accumulated less in the nucleus during the alcohol abstinence interval (fig. S5, B and C). We next immunoblotted for HDAC4 in nuclear and cytoplasmic fractions of nucleus accumbens cells from animals sacrificed at four successive time points after the last alcohol ingestion (0, 12, 18, and 22 hours). Similar to immunohistochemical stains, we found that acute alcohol exposure (time 0) did not lead to a change in the nuclear accumulation of HDAC4, whereas the lack of alcohol resulted in a significant decrease in the nuclear accumulation of HDAC4 at 18 and 22 hours after the last alcohol ingestion (Fig. 4A). In parallel, we also performed immunoblotting studies of HDAC5 following a 10-day alcohol exposure, which revealed a similar decrease in nuclear accumulation 18 hours after the last alcohol ingestion (fig. S6B). Although HDAC4 may be a specific regulator of FosB, it is possible that other HDACs may be involved and may account for the changes that we observed in nuclear HDAC activity and global histone acetylation. Fig. 4 Alcohol use promotes degradation of nuclear HDAC4 and HDAC5 in the nucleus accumbens. (A) Immunoblotting experiment for HDAC4 in the nuclear lysates of nucleus accumbens cells shows that alcohol cessation following 10 days of alcohol use (Fig. 3A) is associated with significantly decreased levels of HDAC4 in the nucleus accumbens 18 hours after the last alcohol ingestion (one-way ANOVA: F 4,17 = 4.37, P = 0.0130; Sidak post hoc: P < 0.05, alcohol/18-hour cessation versus water control; n = 4 to 5 per group). (B) Alcohol use is not associated with changes in HDAC4 levels in the cytoplasmic fractions of nucleus accumbens cells (one-way ANOVA: F 4,20 = 0.5071, P = 0.7310; n = 4 to 5 per group). (C) Quantitative real-time PCR (qRT-PCR) analysis for HDAC4 mRNA in nucleus accumbens lysates after 10 days of alcohol use shows no change in HDAC4 mRNA 18 hours after the last alcohol ingestion (P > 0.05, alcohol pretreated versus water control; n = 4 to 5 per group). (D) Intra-nucleus accumbens delivery of the proteasomal inhibitor lactacystin rescues the decrease in HDAC activity observed following alcohol cessation (one-way ANOVA: F 2,13 = 7.97, P = 0.0055; Sidak post hoc: P < 0.01, water versus alcohol; P < 0.05, alcohol versus alcohol + lactacystin; n = 6 to 8 per group). (E) Intra-nucleus accumbens delivery of the proteasomal inhibitor lactacystin rescues the decrease in nuclear HDAC4 observed following alcohol cessation (one-way ANOVA: F 2,13 = 7.97, P = 0.0055; Sidak post hoc: P < 0.01, water versus alcohol; P < 0.05, alcohol versus alcohol + lactacystin; n = 6 to 8 per group). (F) Alcohol treatment and lactacystin infusion had no significant effects on HDAC4 levels in the cytoplasm (one-way ANOVA: F 4,20 = 0.507, P = 0.7310; n = 6 to 8 per group). *P < 0.05, **P < 0.01. Data are means ± SEM.

Decrease of nuclear HDAC4 is facilitated by proteasome-mediated degradation Surprisingly, we found that alcohol-induced decrease of nuclear HDAC4 accumulation was not paralleled by a comparable increased accumulation in the cytoplasmic compartment, as might be expected for an enzyme that shuttles between the nucleus and the cytoplasm (Fig. 4, A and B). In addition, quantitative analysis of HDAC4 mRNA revealed no change at the level of transcription in response to alcohol exposure (Fig. 4C). These findings suggested that the observed decrease in nuclear HDAC accumulation might be due to the degradation in the nucleus, not to shuttling to the cytoplasm. Studies by Potthoff et al. (42) have demonstrated that the activity-dependent differentiation of mature skeletal muscle is mediated by selective proteasomal degradation of class IIa HDACs in the nucleus of muscle fibers. To test for this possibility, we placed in-dwelling cannulae into the nucleus accumbens of rats bilaterally and allowed the animals to drink either alcohol or water for 10 days. Twelve hours after the last exposure to alcohol, we injected a covalent inhibitor of the proteasome in one hemisphere [one-time injection of 200 μM lactacystin, 0.4 μl into the nucleus accumbens, as described by Massaly et al. (43)] and injected dimethyl sulfoxide (DMSO) vehicle in the contralateral, control hemisphere. Lactacystin rescued the decrease in nuclear HDAC activity (Fig. 4D) and kept nuclear HDAC4 and HDAC5 protein levels at control levels, with no change in the vehicle-treated contralateral hemisphere (Fig. 4E and fig. S6B). Lactacystin infusion did not affect HDAC4 or HDAC5 accumulation in the cytoplasm, suggesting that the degradation of these HDACs is occurring in the nuclear compartment (Fig. 4F and fig. S6C). Proteasome-mediated degradation of HDAC4 in the nuclear compartment has been found to mediate growth factor–induced cell motility (44) and activity-dependent striatal muscle differentiation (42). Our findings therefore suggest that, in addition to the well-described phosphorylation-dependent shuttling of the class II HDAC (28), other posttranslational modifications, such as SUMOylation (45) and ubiquitinization (44), may have key mechanistic roles in the way in which class II HDACs regulate vulnerability to drug addiction. Moreover, these data suggest that the class II HDACs, HDAC4 and HDAC5 may act as nodal regulators, integrating environmental stimuli (alcohol use) with behavioral response (potentiated reward-based learning, resulting in addiction-like behavior).