Recent studies have suggested an association between alcoholism and DNA methylation, a mechanism that can mediate long-lasting changes in gene transcription. Here, we examined the contribution of DNA methylation to the long-term behavioral and molecular changes induced by a history of alcohol dependence. In search of mechanisms underlying persistent rather than acute dependenceinduced neuroadaptations, we studied the role of DNA methylation regulating medial prefrontal cortex (mPFC) gene expression and alcohol-related behaviors in rats 3 weeks into abstinence following alcohol dependence. Postdependent rats showed escalated alcohol intake, which was associated with increased DNA methylation as well as decreased expression of genes encoding synaptic proteins involved in neurotransmitter release in the mPFC. Infusion of the DNA methyltransferase inhibitor RG108 prevented both escalation of alcohol consumption and dependence-induced downregulation of 4 of the 7 transcripts modified in postdependent rats. Specifically, RG108 treatment directly reversed both downregulation of synaptotagmin 2 (Syt2) gene expression and hypermethylation on CpG#5 of its first exon. Lentiviral inhibition of Syt2 expression in the mPFC increased aversion-resistant alcohol drinking, supporting a mechanistic role of Syt2 in compulsive-like behavior. Our findings identified a functional role of DNA methylation in alcohol dependence-like behavioral phenotypes and a candidate gene network that may mediate its effects. Together, these data provide novel evidence for DNA methyltransferases as potential therapeutic targets in alcoholism.

Genetic deletion of the neurokinin 1 receptor (NK1R) has been shown to decrease the reinforcing properties of opioids, but it is unknown whether pharmacological NK1R blockade has the same effect. Here, we examined the effect of L822429, a rat-specific NK1R antagonist, on the reinforcing properties of heroin in rats on short (1 h: ShA) or long (12 h: LgA) access to intravenous heroin selfadministration. ShA produces heroin self-administration rates that are stable over time, whereas LgA leads to an escalation of heroin intake thought to model important dependence-related aspects of addiction. L822429 reduced heroin self-administration and the motivation to consume heroin, measured using a progressive-ratio schedule, in both ShA and LgA rats. L822429 also decreased anxietylike behavior in both groups, measured on the elevated plus maze, but did not affect mechanical hypersensitivity observed in LgA rats. Expression of TacR1 (the gene encoding NK1R) was decreased in reward-and stress-related brain areas both in ShA and LgA rats compared with heroin-naïve rats, but did not differ between the two heroin-experienced groups. In contrast, passive exposure to heroin produced increases in TacR1 expression in the prefrontal cortex and nucleus accumbens. Taken together, these results show that pharmacological NK1R blockade attenuates heroin reinforcement. The observation that animals with ShA and LgA to heroin were similarly affected by L822429 indicates that the SP/NK1R system is not specifically involved in neuroadaptations that underlie escalation resulting from LgA self-administration. Instead, the NK1R antagonist appears to attenuate acute, positively reinforcing properties of heroin and may be useful as an adjunct to relapse prevention in detoxified opioid-dependent subjects.