Project Summary The current opioid crisis is a major public health problem, and it persists despite the availability of Food and Drug Administration–approved treatments (methadone, buprenorphine, and naltrexone). A critical challenge is the occurrence of lapses or relapses in treated patients, especially given that relapses carry a risk of overdose. Therefore, there is an unmet need to advance novel alternative interventions that may overcome limitations of currently available treatment options for opioid use disorders (OUDs). The reason for the limited success of treatments for OUDs are complex and multifactorial. One potential limitation could be attributed to classical animal model of OUDs which rarely incorporate social factors. We recently developed an operant rat model of choice between heroin and social interaction and showed that: (1) rats strongly prefer operant social interaction over heroin, and (2) social choice-induced abstinence (voluntary abstinence) prevents heroin seeking over time (in a model of drug craving). The genetic and circuit mechanisms mediating the protective effect of social reward on heroin craving are unknown. The overarching goal of this proposal is to study the genomic profiling contributing to the buffering effect of social interaction on heroin craving. We will focus on the central nucleus of the amygdala (CeA) and its projections based on our findings on the role of CeA in incubation of psychostimulant craving after social choice-induced voluntary abstinence and previous studies on its role in incubation of drug craving after forced abstinence across drug classes. In Aim 1, we will generate single- nucleus RNA-seq from CeA and its projections (using anterograde transynaptic viruses) in rats during the incubation’s tests after either social-based voluntary or forced abstinence. Additionally. we will interpret the transcriptomic data within detailed ontologies of transcriptomic cell types in each brain region from the BRAIN Initiative Cell Census Network, as well as with snRNA-seq of post-mortem amygdala and insula of human donors with opioid use disorder from the Single-Cell Opioid Responses in the Context of HIV (SCORCH) consortium. In Aim 2, we will use retrograde adeno-associated viruses to label CeA projection neuron nuclei and integrate the transcriptomic data with complementary transcriptomic datasets from large consortia. In Aim 3, we will combine our translationally relevant social choice animal model with advanced CRISPRa and CRISPRi AAV tools to manipulate transcription of key hub genes in either CeA or projection regions to demonstrate their causal role on social-based protection of heroin craving. Our proposal will provide new insights into genomic profiling and its mechanisms underlying the protective effect of social reward on heroin craving in a novel social-based rat model. These results will improve our mechanistic understanding of the role of social interaction in OUDs.