SUMMARY: Opioid use disorder and opioid overdose death rates in the United States reached unprecedented levels during the COVID-19 pandemic. Understanding how endogenous opioid activity affects natural reward seeking is crucial to understanding the neuropharmacological basis of opioid use disorder. Previous research implicates the mesolimbic dopamine pathway, which refers to dopamine neurons projecting from the Ventral Tegmental Area (VTA) to the Nucleus Accumbens (NAc), in reward and addiction. Recent studies show that gamma-aminobutyric acid (GABA) containing neurons in the VTA (VTAGABA) provide local inhibition of dopamine neurons that synapse onto the NAc, thus playing a role in regulating reward behaviors. Importantly, these VTAGABA neurons contain a variety of G-protein coupled receptors, specifically the µ-opioid receptor (MOR). However, the exact role of these receptors and their signaling play in VTAGABA neurons and consequent regulation of natural reward is unknown. The source of endogenous opioid neuropeptide onto these MORs, and how these impacts signaling and activity has not been described. The central hypothesis of this proposal is that µ-opioid receptor signaling on VTAGABA neurons is regulated by afferent endogenous opioid peptides, resulting in disinhibition of VTAGABA neuron excitability. This results in control of dopamine neuron activity, and ultimately the expression of natural reward-seeking. This proposal directly addresses NIDA's Priority Scientific Area 1 that aims to further understand the molecular, neuropharmacological and circuit changes induced by drug use. Aim 1 will isolate the role of endogenous µ-opioid peptides in the VTA on dopamine signaling and natural reward- seeking. Aim1A uses ex vivo two-photon imaging and neuropharmacology approaches to visualize VTAGABA and VTA DA dynamics. Aim1B will investigate the effects of MOR ablation in the VTA on dopamine activity and behavior. Aim 2 will isolate the source and dynamics of endogenous µ-opioids in the VTA during natural reward behavior. In Aim 2A, I will use viral tracing techniques to anatomically visualize inputs from the lateral hypothalamus (LH) to the VTA. Aim 2B will test the effects of endogenous µ-opioid signaling on VTAGABA and DA activity in the NAc during reward seeking behaviors. In Aim 2C, I will use molecular approaches including CRISPR gene-editing and a recently developed µ-opioid biosensor (µMASS1) to understand the spatiotemporal aspects of MOR signaling in VTAGABA neurons during reward seeking. This career development training and series of experiments will provide insight into the role of endogenous MOR signaling on dopaminergic activity and natural reward-seeking. For this proposal, I will train in slice electrophysiology, two-photon slice imaging, gene-editing, molecular neuropharmacology, and behavioral approaches to understand how specific neuropeptides regulate reward circuits. This F31 proposal will greatly advance my career development...