Project Summary The misuse and abuse of prescription pain relievers, such as oxycodone, contributed to the unprecedented opioid epidemic in the United States. Despite substantial knowledge of the pharmacokinetic and behavioral effects of oxycodone in various animal models, only a small number of candidate genes and neuroanatomical systems affected by opioids have been studied. Recent technological advances in the field of single cell genomics are promising avenues for the unbiased discovery and characterization of brain cell types that respond to opioids. In the parent R01 grant, we leverage a multi-omics methodology (Single Cell Multiome ATAC + Gene Expression) to map the transcriptome and epigenome from the same cell across thousands of cells in brain regions relevant to the effects of opioid exposure. To this aim, we will use a rat model of extended access to oxycodone intravenous self-administration that recapitulates several neuroadaptations also observed in humans with opioid use disorders (OUD). This approach provides an exceptional opportunity to systematically explore the cellular diversity of the opioid system and, at the same time, the causative mechanisms that regulate cellular states based on the associations between epigenetic changes and the expression of target genes in individual cells. We will integrate this multi-omics methodology with rigorous computational approaches to explore the cellular organization of the opioid system in multiple brain regions and different stages of OUD progression. In this supplement request, we are extending the studies funded by the parent grant to focus on glucocorticoid receptor (GR)--regulated enhancers. Specifically, we will generate GR binding profiles by CUT&RUN and calculate the enrichment in specific cell types by integrating the single-cell omics datasets produced by the parental R01.