PROJECT SUMMARY Growing evidence suggests that agouti-related protein (AgRP) neurons expressed in the hypothalamic arcuate nucleus play an important role in health and disease. In response to hormonal, nutrient, and neural-related input, these neurons, when activated potently stimulate feeding and engage a broad array of other metabolic and behavioral functions. Our recent findings suggest that rather than being a homogeneous population, AgRP neurons are a heterogeneous population that contain functionally distinct subsets of AgRP neurons that conduct distinct biological functions. Specifically, using a single-cell transcriptomics approach with RNAScope, our Co-PI, Dr. Tune H. Pers, University of Copenhagen, has shown that multiple subsets of AgRP neurons exist, and these exhibit differential responses to various physiological stimuli such as leptin and glucose. This work supports our recent findings which shows that whereas fasting activates the majority of AgRP neurons, acute cold-exposure only activates a small subset. Moreover, our findings further show that AgRP neurons are required for cold-induced increases in energy intake, but not energy expenditure, suggesting that those AgRP neurons involved in feeding are distinct from those involved in energy expenditure. We therefore hypothesize that in response to different afferent input, distinct subpopulations of AgRP neurons project to separate brain regions to mediate their biological effects. Proposed aims seek: 1) to identify and transcriptionally characterize distinct subsets of AgRP neurons and 2) to map the projections and responsiveness of transcriptionally distinct AgRP neuron subsets. This project leverages the complementary expertise and resources of the Morton and Pers laboratories to create synergy that is essential to the success of this project. Dr. Morton has extensive experience examining the role of the brain in the regulation of energy- and glucose homeostasis, while Dr. Pers has expertise in transcriptomics and computation techniques. Thus, to accomplish this, we will use a combination of mouse genetic, viral and immunohistochemical techniques along with single-cell RNA sequencing (RNA-seq) strategies and bioinformatic analysis. Together, this work will fundamentally advance our knowledge and understanding of the AgRP neurocircuits that regulate feeding, relative to other biological functions, and create opportunities to develop novel strategies for the treatment of obesity.