Project Summary/Abstract Interoception, the sensing of signals from internal organs, is crucial to achieving homeostasis in unpredictable environments. For instance, appropriate food intake relies on accurate estimation of future energy balance, which in turn relies on gastrointestinal (GI) signals like nutrient content or stomach stretch. A key brain area that assesses visceral state is the lateral parabrachial nucleus (LPBN) in the brainstem. Visceral information is routed to the LPBN by several convergent pathways, including the nucleus of the solitary tract (NTS), which relays signals from vagal afferents to LPBN. Despite this anatomical roadmap, we lack a comprehensive picture of the functional architecture and GI sensory preferences in the LPBN and its NTS inputs. This information is crucial to understanding how representations of body signals associated with fasted, fed, and feeding states are formed, and how they regulate energy balance. To address this gap in knowledge, I propose to assess the functional organization of GI representations and their relation to natural feeding. I will further test if specific NTS cell types mediate the relay of these visceral signals to the LPBN. I will accomplish these aims with a novel method of two-photon imaging in the hindbrain that tracks hundreds of LPBN neurons across days. In Aim 1, I will measure visceral sensory preferences in the LPBN in response to mechanical stimulation and nutrient delivery across regions of the GI tract. These measurements will allow me to determine the spatial organization of LPBN, including testing the presence of a viscerotopic map of internal organs. Next, I will relate these recordings to activity in the LPBN during ingestion, visceral malaise, and across fasted and fed states. I will test the hypothesis that a wave of neural activation in LPBN across many seconds during natural feeding tracks a putative map of the GI tract. In Aim 2, I will determine how specific inputs from the NTS contribute to feeding-related and satiety state-related activity in LPBN. Specifically, I will repeat the stimuli of Aim 1 while recording from calcitonin receptor-expressing (Calcr) neurons of the NTS that have been shown to signal physiological satiety. I will measure when individual axons of these inputs to LPBN (CalcrNTS->PBN) are active, as well as record LPBN responses to the above stimuli while CalcrNTS->PBN axons are silenced. These experiments will demonstrate how a key pathway relays GI state and other satiety information to the LPBN to regulate behavior and interoception. The proposed research will lay the groundwork for understanding how interoceptive signals from the GI tract lead to satiety sensations, influence feeding behaviors, and ultimately regulate energy balance. This knowledge will pave the way for future efforts combining in vivo imaging with spatial transcriptomics to identify specific cell types and subregions within the LPBN as therapeutic targets for obesity an...