PROJECT SUMMARY A major barrier in the field of opioid research is our limited understanding of the organization of the opioid system in the brain: we still do not know which cell types express each opioid receptor (mu, delta, kappa, nociceptin) to mediate the effects of endogenous and exogenous opioids, nor what other molecules are present in these cells. Without this knowledge, understanding how opioids alter activity in circuits to produce behavioral effects remains elusive. This gap in knowledge prevents the identification of molecular targets to potentiate opioid analgesia and mitigate the deleterious effects of opioid use disorder (OUD), including addiction and respiratory depression. To fill this gap in knowledge, we propose to leverage the uniquely massive single-cell RNA sequencing (scRNA-seq) database generated by the Allen Institute for Brain Science (>3.4 million cells throughout the entire mouse brain) as part of the BRAIN Initiative Cell Census Network (BICCN) effort. Using this database, we will establish a comprehensive catalog of all the cell types that express each opioid receptor and peptide throughout the brain, as well as the co-expression of gene networks that mediate or regulate opioid actions, including other G protein-coupled receptors and cellular effectors of opioid receptors (Aim 1). Further, we will use our well- established high-throughput single-cell RNA-seq pipelines to characterize the cell-type-specific molecular adaptations that occur during chronic opioid exposure, withdrawal, and abstinence, using a clinically relevant model of post-surgical pain for which opioids are typically prescribed (Aim 2). Finally, we will leverage this novel knowledge to dissect the mechanisms of action of opioids by performing advanced circuit mapping and in vivo functional imaging studies of cell types expressing opioid receptors in the prefrontal cortex (PFC), a region critical to both opioid analgesia and addiction (Aim 3). Our transformative work will be the first to combine several highly innovative technologies at the molecular, circuit, and neural ensemble levels, including high-throughput scRNA-seq, new viral strains with improved transsynaptic transfer and decreased toxicity for circuit mapping, the crystal skull and miniscope- microprism optical approaches for in vivo wide-field imaging of brain state and for recording dynamics of molecularly defined neuron types in freely moving mice undergoing opioid analgesia and addiction paradigms. We have an extraordinary interdisciplinary team of investigators with highly complementary expertise in the neurobiology of opioids and the distribution and function of their receptors in pain and addiction circuits, the molecular and anatomical brain architecture, large-scale cell type characterization and circuit mapping, and highly innovative brain imaging methods as applied to the study of neural circuits. Overall, this research aims to generate an exceptional resource for the opioid...