PROJECT SUMMARY/ABSTRACT – PROJECT 4 The objective of Project 4 is to characterize the transcriptional and epigenetic mechanisms of opioid use disorder (OUD) with a focus on dorsal striatum (DS) and nucleus accumbens (NAc), two key brain reward regions. In all prior molecular studies of the human OUD brain, we analyzed isolated neurons vs. non-neuronal cells, which displayed major differences in opioid action. We are now advancing this effort by performing multiomic analyses at the single-cell level, using single nucleus RNAseq (snRNAseq) and snATACseq. Since genetic tags are not possible in humans, such single-cell approaches offer the only available means of studying individual cell populations (e.g., D1 vs. D2 medium spiny neurons) in the human brain. We are performing equivalent single-cell studies on DS and NAc of rats that self-administer (SA) an opioid drug across multiple phases of drug intake including relapse. These temporal assessments are instrumental in guiding analysis of human tissue. Our proposed experiments incorporate not only heroin, which our group has studied extensively, but also fentanyl given its current dominance in OUDs and the virtual complete lack of information about neurobiological aspects of fentanyl use in humans. Overlaying the human and rat snRNAseq and snATACseq data with sequencing datasets on bulk DS and NAc or on sorted neurons from these regions, as well as with sequencing datasets available from the PPG’s work on mouse SA models, provides insight into the most important mechanisms underlying abnormal gene expression associated with OUDs. With this approach, our research has identified PRC2 (polycomb repressive complex 2) as one of the strongest-implicated mediators of molecular pathology in DS and NAc across the three species. Based on these deductions, we are now examining the causal role of PRC2 in opioid-induced behavioral and transcriptional abnormalities by use of viral and genetic mouse tools already validated in PPG laboratories. We are also studying PRC2’s role in opioid-induced regulation of the dendritic morphology of striatal D1 and D2 neurons. Given the prominent development of PRC2 inhibitors for the treatment of specific cancers, characterization of PRC2 contributions to OUDs offers a translational opportunity to advance novel therapies for drug addiction.