PROJECT SUMMARY Research demonstrates that substance-use disorder, including opiate use disorder, affects one's decision making and reward processing resulting in impaired goal-directed behaviors. However, there remains a lack of understanding of the neurobiology that underlies persistent reward-seeking despite value changes that would normally alter goal-directed behavior. The neuropeptide S (NPS) system, made up of the peptide and its receptor (NPSR1), drives reward-seeking behaviors, however the underlying mechanism is not understood. We generated both NPS-Cre and NPSR1-Cre driver mouse lines for accessing and examining the key circuit components of NPS/NPSR1-mediated behaviors. I isolated a population of NPS-containing cells adjacent to the locus coeruleus (LC) and found that these neurons project to the orbitofrontal cortex (OFC), a region with dense expression of NPSR1. I found that these neurons respond to delivery of reward-predictive cues as well as consumption, but to a lesser extent. I then preliminarily tested the effects of oral fentanyl self-administration on this population and uncovered a bidirectional response to delivery of the conditioned stimulus (enhanced activity), and fentanyl reward (quiescence). In this proposal, I first aim to determine the dynamics of OFCNPSR1 neuronal ensembles during natural- and drug-seeking behaviors (Aim 1) using two-photon microscopy. This aim seeks to further investigate OFCNPSR1 activity during self-administration of fentanyl reward as well as how devaluation of either a natural or drug reward is modulated by OFCNPSR1 activity. My second aim is to examine how hindbrain NPS neuron activity and neuropeptide release impact OFC encoding during reward-seeking behaviors (Aim 2). This aim serves to close this gap in knowledge by using multi-site fiber photometry to simultaneously image hindbrain NPS, and OFC neuron activity during reward-seeking behaviors. Finally, in the R00 portion of this grant, I will expand the scope of this study by determining the sufficiency of the OFCNPSR1-Basolateral Amygdala (BLA) projection in driving drug-seeking behaviors (Aim 3). I found that OFCNPSR1 neurons are connected with the BLA. In addition, the BLA expresses NPSR1, however, the source of endogenous NPS to the BLA is unknown as well as any modulation the OFCNPSR1 neurons may provide to this region. This aim serves to uncover the source of BLA NPS as well as investigate the role of BLANPSR1 neurons in reward-seeking behavior and how this activity may be affected by OFCNPSR1 projections. I will utilize a series of complimentary cutting-edge neuroscience techniques to dissect the role of the OFCNPSR1 neurons in drug- seeking behavior. The central goal is to aid in future investigation of whether NPS signaling represents an important avenue for altering drug-seeking behavior. This proposal provides substantial new training in SLM, in vivo calcium imaging of network and single-cell activity, neuropharmacological and m...