PROJECT SUMMARY/ABSTRACT Opioid use, dependence, and addiction have dramatically increased to epidemic proportions in recent years, leading to substantial financial and societal health burdens, as well as an increasing number of overdoses. To combat this epidemic, it is imperative that we understand the neurobiological underpinnings that lead to opioid use disorder. We must identify disrupted neuron subtypes in the brain in opioid use disorders and dysregulated molecules within these neurons that underlie cellular, circuit, and ultimately behavioral adaptations. Use of rat drug self-administration (SA) and relapse assays, which are considered the best available animal models of addiction, will allow a more complete understanding of the molecular mechanisms underlying the genomic, epigenetic, and transcriptional-induced cellular plasticity that drives the long-lasting drug seeking and propensity for heroin relapse. We will perform genome-wide transcriptome and open-chromatin profiling of ventral pallidum (VP) projection neuron subtypes in rat heroin SA, both acutely following drug cessation and after prolonged periods of drug abstinence. Here, we focus on the VP as a critical node in the brain’s reward circuit. Our studies will profile VP neurons that project to the nucleus accumbens, ventral tegmental area, medial dorsal thalamus, and lateral habenula. We will then integrate the transcriptomic and epigenomic data with complementary transcriptomic and epigenomic datasets, including multimodal data from the BRAIN Initiative describing cell type diversity in the VP and its output circuits. We will reconstruct cell type-specific gene co-expression and open chromatin networks and identify hub genes predicted to have central roles in immediate and prolonged abstinence from heroin, which could underlie subsequent relapse behavior. This collection of datasets and models will be made available through a biologist-friendly web portal based on our BRAIN Initiative-funded Neuroscience Multi-Omic Analytics platform. Using the data generated we will develop rat gene loci-specific CRISPR epigenomic targeting tools to determine the functional significance of key hub genes that are regulated in VP projection neuron subtypes. To achieve this goal, we will employ rat models of relapse in combination with advanced CRISPRa and CRISPRi AAV tools to enhance or reduce transcription of key hub genes during heroin SA or abstinence from heroin SA followed by cue-induced reinstatement. The studies proposed in this grant application will, for the first time, identify the distinct heroin-induced gene network adaptations occurring temporally in a cell-type-specific manner within a novel neurobiological circuit.