Project Summary The long range goals of our research program has been to elucidate the mechanism(s) by which metabolic states and 17β-estradiol (E2) regulate arcuate nucleus kisspeptin (Kiss1ARH) neuronal circuits that are critical for coordinating energy homeostasis and reproduction in females. It is well known that E2 is anorexigenic, and that Kiss1 neurons, which are directly regulated by E2, are essential for pubertal development and adult reproductive success. However, their role in the control of other homeostatic functions is just emerging. Earlier, we found that Kiss1ARH neurons are depolarized/excited by leptin and insulin via canonical transient receptor potential (TRPC) 5 channel signaling and proposed that they may serve as an important hub in the control of energy homeostasis. Recently, we found that high frequency optogenetic stimulation of Kiss1ARH neurons releases glutamate to excite the anorexigenic proopiomelanocortin (POMC) neurons but inhibit the orexigenic neuropeptide Y/agouti-related peptide (AgRP) neurons. E2 increases vesicular glutamate transporter 2 (Vglut2) mRNA expression and glutamate release from female Kiss1ARH neurons to augment the POMC excitation and AgRP inhibition. Also, Kiss1ARH neurons project to and excite AVPV/PeN Kiss1 neurons via glutamate, which drives the GnRH and LH surges. Thus, Kiss1ARH neurons appear to integrate metabolic hormone and gonadal steroid signaling to regulate both energy homeostasis and reproduction via multiple neurotransmitters. Key excitatory cationic channels are upregulated by E2 leading to increased excitability and glutamatergic synaptic transmission, whereas peptide expression and transmission are attenuated by the classical estrogen receptor (ER) signaling pathways. Recently, we have found that the selective membrane estrogen receptor (GqmER) agonist STX increases the excitability of Kiss1ARH neurons without downregulating the peptide expression. Therefore, we hypothesize that estrogenic signaling in Kiss1ARH neurons is important for increasing Kiss1ARH neuronal excitability and maintenance of homeostatic functions critical for reproductive success. Our multidisciplinary approach incorporates a powerful set of cellular, molecular, genetic and optogenetic tools, and our combined expertise in molecular biology, electrophysiology, and whole animal physiology to address the following aims: (1) to measure the estrogenic-mediated increase in excitability of Kiss1ARH neurons using GCaMP6 and Voltron recordings; (2) to elucidate the estrogenic modulation of the synaptic input from Kiss1ARH to hypothalamic paraventricular nucleus neurons using optogenetic stimulation and its effects on food intake; and (3) to elucidate the estrogenic modulation of synaptic input from Kiss1ARH neurons to hypothalamic dorsomedial nucleus neurons and its effects on energy expenditure. Elucidating the circuits and signaling cascades underlying the actions of E2 and the selective GqmER agonist STX will prov...