Stress reduces an animal’s drive for seeking natural rewards but increases drug-seeking behavior. In humans, anxiety disorders are also highly comorbid with substance-use disorder, and therefore a better understanding of the underlying circuitry that connect these behaviors is necessary for the development of better treatment strategies. Examining the role neuropeptide S (NPS), a neuropeptide shown to regulate anxiety-related behavior through activation of the Gq-coupled protein receptor (NPSR1), plays in seeking social and natural rewards could provide an interesting link between the body’s anxiety response with drug-seeking behaviors. The peri locus coeruleus (periLC) produces NPS and sends projections to the orbitofrontal cortex (OFC), which contains dense expression of NPSR1. Using a combination of retrograde tracing paired with in situ hybridization, as well as whole-cell patch clamp electrophysiology, I found that the OFCNPSR1 neurons receive input from the periLC, and that periLCNPS neurons project to the OFC. Therefore, I hypothesize that periLCNPS neurons drive natural reward- seeking behavior through activation of OFCNPSR1 neurons. In order to dissect the periLCNPS-OFCNPSR1 projection’s role in reward-seeking behavior, during my training period, I will learn to conduct novel in vivo calcium imaging of network and single-cell activity, neuropharmacological and molecular manipulations. The first aim of this research proposal is to determine the endogenous neuronal activity of periLCNPS during natural reward- seeking behaviors. Previous research indicates that LC phasic activity is associated with reward stimuli, using fiber photometry, I will measure the endogenous soma and terminal network activity of the periLCNPS neurons during reward-related behaviors such as classical conditioning and social interaction tasks. Aim 1 will establish how periLCNPS neurons behave in vivo to neutral and appetitive stimuli. The second aim of this research proposal will investigate whether periLCNPS-OFCNPSR1 projections are necessary and sufficient for the modulation of natural reward-seeking. I plan to use two-photon calcium imaging in a behaving mouse to investigate OFCNPSR1 single-cell activity during delivery of appetitive stimuli with and without periLC terminal activation using an excitatory opsin expressed in periLC excitatory terminals in the OFC. Finally, I will examine the sufficiency and necessity of the periLCNPS-OFCNPSR1 projection in reward-seeking behaviors. To examine the sufficiency, I will utilize in vivo optogenetic strategies to photoactivate the periLC-NPS terminals in the OFC during classical conditioning and social interaction tasks. To test the projection’s necessity, I will use a conditional, viral-based Crispr-Cas9 system to inactivate Nps or Npsr1 genes in the region of interest. These mice will then undergo classical conditioning and social interaction assays. Here we will test the hypothesis that periLCNPS neurons enhance natura...