Project Summary Given that eating frequency is largely determined by societal norms, a deeper understanding of the neural substrates that control meal size is critical for the development of novel and more effective treatments for obesity. The amount of food consumed over the course of a meal is determined by a competing balance between an early-meal positive feedback process called appetition and a late-meal negative feedback process called satiation. We hypothesize that melanin-concentrating hormone (MCH), an orexigenic neuropeptide produced in the lateral hypothalamic area (LHA) and zona incerta (ZI), is critical in mediating the poorly understood process of appetition. While such a role for MCH has not been directly investigated, this notion is supported by findings revealing that MCH neurons are glucose responsive 1,2 and MCH injections augment food intake by increasing meal size 3. A role for MCH signaling in mediating appetition is further supported by our preliminary data showing that chemogenetic activation of MCH neurons (LHA + ZI populations) potentiates flavor preference learning for a nonnutritive flavor paired with intragastric glucose infusion. MCH is produced in both the LHA and the ZI, which are adjacent regions that are differentiated both functionally and neuroanatomically 4-7. Our preliminary results, using fiber photometry to measure MCH neuron calcium activity in awake behaving animals, reveal that LHA and ZI MCH neurons show distinct calcium activity dynamics during a meal, such that LHA MCH neuron activity increases during active eating relative to interbout intervals, and ZI MCH neuron activity shows the opposite pattern (activity elevated during interbout intervals relative to active eating). These findings introduce the possibility that LHA and ZI MCH neurons subserve distinct but complementary functions in the control of feeding. Based on our preliminary data, here we will evaluate our hypothesis that LHA MCH neurons promote orosensory-mediated appetition, and ZI MCH neurons promote post-oral nutritive-mediated appetition. This hypothesis will be evaluated by separately targeting LHA vs. ZI MCH neuron populations in Aim 1 experiments measuring physiological MCH neuron calcium activity in response to orosensory vs. post-oral nutrient sensing in lean and obese rats, and in Aim 2 functional chemogenetic and caspace-mediated ablation experiments determining the precise oral and post-oral mechanisms through which MCH neuron activation promotes flavor-nutrient learning. In addition to exploring the physiological responses (Aim 1) and functional relevance (Aim 2) of the two MCH neuron populations with regards to appetition and food intake, Aim 3 will combine MCH neuron population-specific pathway tracing, metabolic brain mapping, and single-nucleus RNA sequencing approaches to extensively characterize the anatomical, network, and transcriptional profiles of ZI vs. LHA MCH neurons. Overall, these complementary aims will sig...