Project Summary: Cannabinoids, both endogenous and exogenous, exert a profound impact on the hypothalamic control of biological processes such as reproduction and energy homeostasis. The overall goal of this proposal is to gain a better understanding of the cannabinoid regulation of energy homeostasis, and the cellular mechanism(s) by which they affect the excitability of anorexigenic proopiomelanocortin (POMC) neurons in the arcuate nucleus (ARC) of the hypothalamus to alter these actions. The fundamental hypothesis is that endocannabinoids modulate energy balance in a sex- and diet-dependent fashion that can be differentially influenced by gonadal steroid hormones. They do so, in large part, via retrograde inhibition of excitatory glutamatergic neurotransmission in POMC neurons in a manner that is sexually differentiated, and dependent on dietary composition and the gonadal steroid milieu. Endocannabinoid regulation of energy homeostasis and synaptic transmission onto POMC neurons will be studied using a transgenic, Nr5a1-cre animal model, in which either channelrhodpsin-2 or a modified Gq-coupled muscarinic M3 receptor is delivered via a viral vector construct into the hypothalamic ventromedial nucleus (VMN) and integrated into neurons expressing steroidogenic factor- 1 (SF-1; encoded by the Nr5a1 gene) for in vivo and in vitro optogenetic experimentation. Females are ovariectomized and males orchidectomized one week prior to experimentation, respectively, and treated with estradiol, testosterone, or their respective vehicles according to well-established protocols. In Specific Aim 1, I will evaluate the mechanisms through which long-term exposure to a Westernized high-fat diet differentially enhances endocannabinoid sensitivity in males and females by assessing how it influences the capability of endogenous cannabinoids to retrogradely inhibit light- and chemically- evoked glutamatergic neurotransmission at VMN SF-1/ARC POMC synapses. In Specific Aim 2, the objective is to determine whether these high-fat diet-induced alterations in endocannabinoid sensitivity effectively translate into changes in energy balance. Specifically, I will evaluate whether high-fat diet exposure differentially attenuates the capability of opto- and chemogenetically stimulated VMN SF-1 neurons to suppress energy intake and enhance energy expenditure, and whether these adaptations are due to alterations in the expression/activity of the energy-sensing signaling molecules like phosphatidylinositol-3-kinase and AMP-activated protein kinase. This proposal describes an integrated approach to study sex differences in, and gonadal steroid hormonal influences on, the cannabinoid regulation of energy balance. Further, it will elucidate the relevant cellular mechanisms and neuroanatomical substrates that differentially predispose males and females to dysregulated endocannabinoid signaling and food consumption under conditions of diet-induced obesity/insulin resistance.