Suppression of reward-seeking behaviors is critical for survival, yet chronic opioid use and relapse commonly occur despite known social, economic, and health consequences among Veterans suffering from opioid use disorder (OUD). Thus, defining how opioid use influences the neuronal systems responsible for suppressing reward-motivated behaviors is critical for understanding the etiology of OUD. Despite this knowledge, no studies to date have tracked activity in single neurons from initial opioid use to dependence, limiting our understanding of how brain circuits responsible for suppressing maladaptive behavioral actions are modulated to unleash excessive opioid seeking and consumption. My lab has overcome this issue by developing a novel mouse behavioral assay that allows longitudinal tracking of activity in precisely defined neuronal circuit elements across heroin self-administration, extinction, and reinstatement. Using this paradigm, I will test the hypothesis that a thalamoaccumbal to parvalbumin interneuron circuit is weakened by opioid use to causally disinhibit opioid self-administration and relapse to opioid seeking. Recent work in my laboratory, as well as the work of others, reveals that neurons in the posterior paraventricular nucleus of the thalamus (pPVT) provide a critical ‘brake’ that suppresses inappropriate reward- motivated behaviors through the activation of downstream nucleus accumbens (NAc) parvalbumin interneurons (PV-INs). Despite the demonstrated role of this pPVT→NAcPV-IN circuit for suppression of reward seeking; the activity, physiology, and function of this circuit for controlling opioid-seeking behaviors remains unclear. Based on extensive preliminary findings, here I will test the overarching hypothesis that heroin self-administration reduces pPVT→NAcPV-IN circuit activity and connectivity, causally disinhibiting heroin use and relapse to heroin seeking. I will test independent components of this hypothesis through the completion of 3 independent Aims. In Aim 1, I will use two-photon calcium imaging to measure the activity dynamics of pPVT→NAc axons and downstream NAc PV-INs across heroin self-administration, extinction, and reinstatement. In Aim 2, I will use slice patch-clamp electrophysiology to characterize synaptic adaptations within pPVT→NAcPV-IN circuits caused by heroin self-administration. Finally, in Aim 3 I will use optogenetic and chemogenetic strategies to determine the necessity and sufficiency of pPVT→NAcPV-IN circuit dynamics for heroin self-administration and relapse to heroin seeking. Overall, I investigate how a principle, but understudied, behavioral suppression circuit is modified by opioid use to unleash maladaptive opioid-seeking behaviors. Our preliminary findings suggest that this project will be highly impactful for the field and could inform circuit-based therapeutics that normalize neuronal activity for treatment of Veterans suffering from OUD.