Project Summary Alcohol misuse and addiction is a growing and increasingly destructive socioeconomic national and global crisis. There is a critical need for novel therapeutic interventions that, rather than managing symptoms, reverse alcohol-induced neurophysiological changes. One effect that alcohol, and myriad other addicitive drugs, produce in the brain, is the disruption of long-term synaptic depression (LTD), particularly the presynaptic form of LTD that occurs at corticostriatal synapses. The striatum is associated with compulsive and habitual behaviors, including drug addiction. Drug-induced disruption of LTD in dorsolateral striatum (DLS) is associated with increased habitual behavior whereas LTD restoration restores drug-disrupted goal-directed behavior. We have found that alcohol disrupts striatal presynaptic mu opioid receptor (MOR)-mediated LTD that, in the DLS, occurs exclusively at anterior insular cortex inputs (AIC-DLS synapses). MOR-short-term depression at thalamic striatal inputs are unaffected by alcohol exposure. Identifying the molecular changes that occur presynaptically following alcohol exposure at AIC-DLS synapses, but not at thalamostriatal synapses, may reveal new targets for pharmacological interventions in alcohol use disorder and addiction. The specific problem though is that identifying presynaptic-specific changes in protein-protein interactions using pharmacological and genetic tools is laborious, inefficient, and relies on a priori knowledge of signaling pathways, likely missing key molecular players that could be affected. Based on our preliminary data, our central hypothesis is that in vivo alcohol exposure alters PKA-dependent presynaptic protein organization that is required for MOR-mediated LTD induction. We will capitalize on the combined physiological and proteomic expertise of the investigators to develop a novel biochemical methodology that is able to selectively determine the precise ex vivo and in vivo changes that occur in the protein interactome precisely within AIC synaptic terminals (but do not occur within thalamostriatal terminals) in DLS during the induction of MOR-mediated LTD and following in vivo exposure to alcohol. The comparison between these alcohol-sensitive AIC-DLS synapses and the alcohol-insensitive thalamic inputs will allow us to determine what makes some synapses susceptible to alcohol’s deleterious effects and others resistant. This methodology employs mouse brain slice electrophysiology, in vivo alcohol exposure, and viral vector-driven expression of proteins that allow for proximity labeling within presynaptic terminals with subsequent proteomic analyses. Using this new tool we will open the “black box” of presynaptic proteome changes that we predict will uncover previously unidentified synaptic plasticity protein-protein interactions that are disrupted by alcohol and other addictive drugs. Furthermore, we aim to identify novel protein interactions that may prove to be therape...