Project Summary and Abstract Substance use disorder (SUD) is a debilitating disorder that has a societal cost of more than 700 million dollars per year in the US alone. Despite the prevalence and significant global financial burden of SUD, the pathophysiology of the disorder is poorly understood, and current treatments have limited efficacy. At the core of SUD is a dysregulation in reward learning and appetitive motivation. These effects are mediated by drug- induced alterations in dopamine transmission in the ventral tegmental area (VTA) to nucleus accumbens (NAc) projection pathway, which has been causally linked to these behavioral phenotypes. Further, animal models of SUD show dysregulation of VTA to NAc excitability and accumbal dopamine release at terminals, further supporting the critical role that this system plays in the expression of dysregulated drug and non-drug associated behaviors. Within this VTA to NAc pathway, dopamine is released from multiple cellular compartments – including somatodendritic and terminals - that are regulated by different molecular, circuit-based, and receptor- based mechanisms. However, while work has focused on ex vivo measurements of dopamine release at terminals, relatively little is known about the roles of release in other compartments within this pathway, such as somatodendritic release within the VTA and how this relates to the execution of motivated behaviors in a basal state. The goal of this proposal is to understand the compartment-specific experience-dependent plasticity that underlies appetitive motivation and reward learning in vivo and then define the molecular mechanisms for this plasticity. To this end, we will use a combination of cutting-edge techniques, including in vivo fiber photometry combined with fluorescent dopamine sensors during operant behavior to image dopamine transients within the VTA and NAc longitudinally over time. We will then use ex vivo fast scan cyclic voltammetry combined with optogenetic stimulation and pharmacology in somatodendritic and terminal compartments to define the receptor- based mechanisms by which this dopaminergic plasticity occurs. By defining these molecular targets that underlie reward and motivation, these experiments will help to identify targets that may be driving learning- dependent plasticity mechanisms within the mesolimbic pathway. As such, the results of these experiments have broad implications for identifying and understanding the potential mechanisms of dopamine dysregulation in SUD and may provide therapeutic avenues in order to reverse deficits in these processes.