PROJECT SUMMARY/ ABSTRACT Obesity is a growing global health crisis associated with morbidity and mortality, highlighting the need for improved therapeutics. To this end, glucagon-like peptide 1 receptor agonists (GLP1RAs) (e.g., semaglutide), which cause significant weight loss and decreased food intake, are rapidly emerging as the most effective anti- obesity treatments. While GLP1RA appetite suppressing actions are known to involve direct recruitment of central GLP1Rs, which are G-protein coupled receptors (GPCRs), the neural pathways and intracellular signaling mechanisms by which they function are poorly understood. Moreover, despite that these drugs are chronic medications, little is known about the effects of long-term usage on the brain. Here, I seek to investigate the neural mechanisms underlying semaglutide’s effects on appetite across acute and chronic treatment. Previous work and our preliminary data highlight the lateral parabrachial nucleus (lPBN) and area prostrema (AP) as two regions robustly activated by semaglutide. Within the neural circuits that govern appetite, the lPBN is a key satiety promoting node implicated in the control of meal termination and food intake, whereas the AP is a brainstem circumventricular organ located outside of the blood-brain-barrier that is uniquely equipped to sense circulating signals and relay this information to the brain. Importantly, the AP is enriched in GLP1Rs and fluorescently tagged semaglutide accumulates in the AP, but not the PBN, suggesting that the AP is a primary site of semaglutide action and PBN is recruited downstream. In support of this, a recent study showed that Glp1r- expressing AP (APGlp1r) neurons innervate the lPBN and optogenetic stimulation of these APGlp1r projections activate lPBN neurons. Thus, my central hypothesis is that activation of PBN neurons downstream of APGlp1r neurons is a major pathway required for the effects of semaglutide on appetite suppression. This hypothesis will be tested in two specific aims. In Aim 1, I will use in vivo two photon microscopy and acute inhibitory chemogenetic and chronic silencing methods to determine the role and necessity of PBN neurons across acute and chronic semaglutide treatment. In Aim 2, I will first examine the intracellular signaling mechanisms by which semaglutide activates Glp1r-expressing AP neurons (APGlp1r) using ex vivo two-photon brain slice imaging of intracellular cyclic adenosine monophosphate (cAMP) levels and genetic knockout of GPCR-mediated signaling in APGlp1r neurons. Then, I will determine the role of APGlp1r inputs to the PBN across acute and chronic semaglutide treatment using in vivo two-photon imaging of these projections and disruption of GPCR-mediated signaling specifically in in PBN-projecting AP neurons. Execution of the proposed research will facilitate my training and mastery of modern optical imaging using two-photon microscopy, and equip me with knowledge of contemporary tools (e.g. cAMP biosen...