Substance use disorders comprise chronic cycles of drug use, abstinence, and relapse driven by drug-induced transcriptional and circuit adaptations in the brain. Despite consistent evidence that epigenetic chromatin modifications drive these long-term neuronal adaptations, successful druggable treatment targets have not emerged, and further understanding of mechanistic pathways from the epigenome to altered behavior are needed for novel substance use disorder therapies. This proposal examines the link between epigenetic processes, including histone deacetylase (HDAC) function and chromatin accessibility, and mitochondrial morphology and function in the context of nicotine reinforcement learning to bridge this gap. Use of nicotine, the primary psychoactive component of both tobacco and e-cigarettes, alters brain reward pathways, including inputs to spiny projection neurons (SPNs) of the nucleus accumbens (NAc). The subsequent transcriptional and morphological changes in these SPNs support electrophysiological activity driving cue-induced craving and drug- seeking behaviors that make smoking cessation difficult and contribute to nicotine use disorder. Understanding how drugs such as nicotine induce lasting molecular adaptations and alter the functioning of NAc SPNs is critical in untangling the local mechanisms promoting persistent drug use and relapse. SPN mitochondria stand out as an unexplored but likely regulator of NAc SPN adaptations to nicotine, mediating plasticity downstream of epigenetic genome modifications. This proposal uses cutting edge cell-type selective techniques to examine how both nicotine and HDAC inhibitors alter mitochondrial morphology and function, as well as individual SPN gene expression and chromatin accessibility, to provide a new mechanistic understanding of nicotine-reward. Using a mouse model of nicotine intravenous self-administration (IVSA) and drug seeking, I will measure NAc mitochondrial respiratory capacity then will assess dendritic, spine, and mitochondrial morphology in the two main SPN populations in NAc (D1- and D2-dopamine receptor containing SPNs). I will also examine these end points after inhibition of multiple classes of HDACs, determining if nicotine’s effects on mitochondria are related to nicotine’s function as an HDAC inhibitor. Parallel single nuclei RNA and ATAC sequencing will identify molecular adaptations in individual NAc cell types after nicotine IVSA. Finally, after identifying and validating molecular targets regulating transcriptional programs within in SPN cell types, I will disrupt the endogenous mitochondria adaptations to IVSA nicotine with cell-type selective CRISPR activation or interference (CRISPRa/i) manipulations. These studies will inform the relationship between drug-induced epigenetic modifications and downstream mitochondrial functioning, a potential intermediary to morphological and electrophysiological plasticity in response to rewarding drugs. In addition to providing tr...