A hallmark of drug addiction is continued drug craving and relapse propensity despite long-term drug abstinence. Drug-induced metaplastic changes in gene expression, protein expression, neuronal morphology, neurochemistry, and electrophysiology are thought to contribute to persistent changes in behavior. Recent studies have identified epigenetic imprinting through histone modifications as a potential regulatory mechanism for development of these metaplastic changes. The proposed studies will address a major knowledge gap in addiction research regarding whether DNA methylation epigenetic imprinting also serves to help maintain drug- induced metaplastic changes, and thereby changes in behavior during drug abstinence. Using our well- characterized cocaine self-administration model with demonstrated abstinence persistent changes in epigenetic imprinting, gene expression, protein expression, and behavior, in Specific Aim #1 we will examine DNA methyltransferase (DNMT) activity and gene promoter DNA methylation patterns in saline self- administering controls, rats self-administering for 10 days on a discrete-trials (DT4) schedule, and rats with 1 and 30 days of drug abstinence from the DT4. Methylation patterns will be examined in >20,000 gene promoters across the genome. In Specific Aim #2, we will use the same animal model and time points to confirm changes in DNA methylation through pyrosequencing and examine mRNA levels for those promoters with differential methylation in an independent set of animals. These studies will not only address the hypothesis that altered DNA methylation occurs with cocaine self-administration but also, whether these DNA methylation changes persist as long during cocaine abstinence as changes in behavior, gene expression, and protein expression. Answering this question will aid in determining whether epigenetic events are only part of the development of persistent neurobiological changes or if they are also needed to maintain persistent neurobiological changes. PUBLIC HEALTH RELEVANCE: One of the hallmarks of drug addiction is continued drug craving and relapse propensity despite long-term drug abstinence. The proposed studies would examine the potential role of DNA methylation in the epigenetic basis of drug craving. The goal of this research is to better understand the neurobiological basis of drug craving and potentially to develop treatments to reduce drug craving/seeking.