Project Summary/Abstract Despite considerable efforts to address and reverse the obesity epidemic, obesity remains highly prevalent in the U.S. The brain plays an important role in regulating body weight, both through modulating energy expenditure and driving feeding behavior, and thus understanding how the brain regulates energy balance is critical for developing effective obesity treatments. Melanin-concentrating hormone (MCH) is a neuropeptide that acts in the brain to promote weight gain by increasing food intake and reducing energy expenditure. Due to its dual actions on both aspects of the energy balance equation, the MCH system is a promising target for obesity pharmacotherapies. Currently, the mechanisms and neural sites of action by which MCH modulates energy balance are largely unknown. The overarching goal of this proposal is to identify neural sites of action and mechanisms by which MCH promotes hyperphagia and susceptibility to diet induced obesity. Specific Aim 1A and 1B utilizes two novel virogenetic RNA interference approaches to promote a loss of function to the MCH system in the paraventricular hypothalamus (PVH). Animals are then challenged with a palatable high fat, high sugar, Western cafeteria diet in order to determine whether the PVH MCH system plays a role in the development of diet induced obesity. Experiments are performed in male and female rats, with the potential for further investigation into sex differences should sexually dimorphic effects be observed. Specific Aim 1C investigates a novel molecular mechanism by which MCH may alter feeding effects, namely by modulating the length of neuronal primary cilia and/or gene expression of the ciliary signaling protein adenylyl cyclase 3 (AC3). Specific Aim 2A investigates the function of zona incerta (ZI) MCH neurons, a population whose behavioral function has not been studied in isolation with regards to feeding behavior. Based on our unpublished preliminary data and recent published work, we propose that ZI MCH neurons increase binge-eating behavior. We will examine this question using our MCH neuron-specific chemogenetic DREADDs approach. We further plan to determine the downstream anatomical outputs of ZI MCH neurons, which will enable us to employ our dual virus chemogenetic approach in future studies in order to better understand the function of the multi-order circuitry of this unique population of MCH neurons. These experiments build on the experiments from the applicant's K01 proposal and are specifically designed to produce necessary data for the applicants R01 application. The studies outlined herein will additionally contribute significantly to understanding the role of the MCH signaling in regulating energy balance, which is an important step toward effectively developing obesity pharmacotherapies based on this system.