Project Summary Obesity is currently affecting nearly 40% of adults in the U.S. and clearly a major health problem. Energy intake exceeding the homeostatic need is an essential component for the development of obesity. While the hypothalamus plays a key role in regulating energy homeostasis, brain imaging studies in humans consistently found neural activities in the extrahypothalamic region being highly correlated with the consumption of energy-dense food and obesity. The most profound associations are located in reward- or salience-related brain structures. It is therefore imperative to understand how these higher brain regions contribute to the homeostatic regulation of food intake. Our long-term goal is to understand how higher cortical structures exert top-down control of food intake and homeostatic regulation. During the K01 period, enabled by whole-brain CLARITY and lightsheet imaging, we conducted multiple brain-wide screenings to search for extrahypothalamic circuits recruited by fasting. A previously uncharacterized posterior insular cortex to basolateral amygdala projection (pINS-BLA) was unbiasedly identified as one of the most active projections recruited by overnight fasting based on immediate early gene expression. The insular cortex, a key site for integrating internal and external sensory information and encoding valances, is also one of the most prominent brain regions found to be associated with food reward and obesity across numerous human imaging studies. In this proposal, we will test the hypothesis that the activity of this novel pINS-BLA projection encodes top-down hunger signal and therefore positively regulates food intake. We will pursue the following two specific aims: (1) Employ fiber photometry to track and quantify circuit dynamics of the pINS-BLA projection in relation to fasting and re-feeding. (2) Use optogenetics to determine the causal significance of the pINS-BLA projection in food consumption. The studies proposed here will build upon the unique brain-wide screening capacity and the original discovery of an insular cortex to amygdala projection, both achieved during the K01, to unmask a new top-down mechanism of feeding regulation. The completion of this proposal will greatly expand the existing hypothalamic-centered understanding of homeostatic control. This knowledge will provide insight into how higher brain functions are altered during obesity and offer a novel perspective of targeting obesity, both of which will lay the groundwork for our subsequent R01 application.