PROJECT SUMMARY/ABSTRACT Relapse after abstinence is a key challenge for curbing the ongoing opioid epidemic. One of the common factors for triggering relapse is re-exposure to drug-associated cues. In rats, cue-induced oxycodone seeking progressively increases during abstinence. The Li Lab recently published work demonstrating a critical role of orbitofrontal cortex (OFC) in this incubation. However, molecular mechanisms in OFC underlying this incubation are largely unknown. Moreover, OFC is interconnected with several cortical and subcortical areas. Therefore, the distinct molecular and cellular adaptations in these OFC-associated circuits may underlie the critical role of OFC in oxycodone relapse. Despite recent developments in next-generation sequencing technology, transcriptomic data still tell little about the proteome state due to the complex relationship between transcription and translation, heterogeneous subcellular distributions of proteins (e.g., soma vs. axonal terminal), and other factors. A key limitation to profiling projection-specific proteome has been due to a lack of sufficiently sensitive bioanalytical technologies to quantify the deep proteome in the limited amounts of material available from somas and axonal terminals, particularly using high-resolution mass spectrometry (HRMS), the modern technology of choice for the discovery and/or targeted analysis of the proteome. The Nemes Lab developed ultrahigh- sensitivity HRMS platforms that enabled the quantitative characterization of neuropeptides in distinct brain areas of the mouse and thousands of different proteins in single stem cells, and has recently begun extending these analytical innovations to single-cell-equivalent proteomes and single neurons in the mouse. In this proposal, we aim to profile projection-specific proteome associated with incubation of oxycodone craving by focusing on OFCdorsal striatum (DS) projections. We will profile proteome changes in OFCDS projection neuronal somas in OFC (Aim 1) and in OFCDS projection-specific synaptoneurosomes in DS (Aim 2) during incubation of oxycodone craving. Overall, this proposal will bring the ultrahigh sensitive quantitative HRMS to the field of addiction neuroscience and may provide targets for pharmacological interventions to decrease craving and promote abstinence.