PROJECT SUMMARY Opioid use disorder (OUD) is a major epidemic within the United States, and this issue is exacerbated by the highly addictive nature of opioids. When presented with drug-associated environmental cues, patients suffering from OUD are more likely relapse to compulsive opioid seeking and taking, even with the risk of significant negative consequences. Despite knowing this, how opioid-associated cues engage brain reward circuits for the control of opioid-seeking behaviors is unclear. Having a better understanding of how cues affect brain reward circuits to provoke drug seeking could provide better insight into novel therapeutics for the treatment of OUD. One particular brain region known to be engaged by reward-associated cues is the paraventricular nucleus of the thalamus (PVT). The PVT is architecturally organized between areas that are associated with conditioned reward-seeking behaviors, and has been demonstrated to play roles in appetitive motivation, feeding, memory, and natural reward or drug-seeking behaviors. In addition to these inputs, the PVT itself has dense opioid receptor (OR) expression, which is known to modulate PVT activity. Furthermore, my data reveals that opioid receptors are specifically expressed on PVT neurons that project to the nucleus accumbens (PVTàNAc), a pathway that provides a critical “brake” for reward seeking. Despite this knowledge, the influence of opioid use and the presentation of opioid-associated cues on PVTàNAc activity is unknown. Furthermore, the function of this pathway for opioid seeking is unknown. Here I propose the central hypothesis that pPVTàNAc neurons are inhibited during opioid use and by the presentation of opioid-associated cues, and that this inhibition is required for opioid seeking. These data are supported by my extensive preliminary datasets, wherein I show that PVTàNAc neurons are inhibited during the presentation of heroin predictive cues, whereas optogenetically mimicking this inhibition in extinction conditions drives immediate and voracious goal-directed heroin seeking. Using innovative techniques developed in our laboratory, I will test my hypothesis with two experiments. In Aim 1, I will use a novel head-restrained heroin self-administration protocol that allows simultaneous in vivo two-photon calcium imaging to determine the precise activity dynamics of pPVTàNAc neurons during acquisition, extinction, and reinstatement phases of self-administration. In Aim 2, I will use behavioral optogenetics to determine the function of pPVTàNAc neurons for cue-induced reinstatement of heroin seeking. Together, these experiments will identify the activity dynamics and function of precisely-defined PVT output neurons for heroin seeking. This information will provide an overall better understanding of OUD for the development of novel therapeutics.