Project Summary The long range goals of the proposed research are to elucidate the mechanism(s) by which metabolic states and 17?-estradiol (E2) regulate arcuate nucleus (ARC) kisspeptin (Kiss1) 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 energy homeostasis is less understood. We have shown that the ARC Kiss1 neurons are directly excited by leptin and insulin indicating that they may serve an important role in the control of energy homeostasis. Also, we have evidence that glutamate is released from ARC Kiss1 neurons and targets anorexigenic proopiomelanocortin (POMC) neurons and orexigenic neuropeptide Y/agouti-related peptide (NPY/AgRP) neurons. In addition, we have found that glutamate can differentially regulate POMC and NPY/AgRP neurons by acting on separate groups of metabotropic glutamate receptors (mGluRs). Moreover, we have discovered that E2 increases vesicular glutamate transporter 2 (vGluT2) mRNA in female ARC Kiss1 neurons, an indication of heightened vesicular glutamate packaging and release. We also have evidence that ARC Kiss1 neurons project to and excite AVPV/PeN Kiss1 neurons, which are important for the induction of the GnRH/LH surge. Thus, we believe that ARC Kiss1 neurons integrate metabolic hormone and steroid cues to regulate both energy homeostasis and reproduction. Therefore, we propose the novel hypothesis that the excitability of ARC Kiss1 neurons is increased in high estrogenic states thereby releasing glutamate to excite POMC neurons and inhibit NPY/AgRP neurons via group I and group II/III mGluRs, respectively, which decreases food intake. In addition, excitatory glutamatergic input to AVPV/PeN Kiss1 neurons from ARC Kiss1 neurons constitutes a critical stimulatory drive to GnRH neurons at the time of GnRH/LH surge. Our multidisciplinary approach incorporates a powerful set of cellular, molecular and optogenetic tools to address the following aims: 1) To elucidate in ARC Kiss1 neurons the effects of E2 on the mRNA expression and function of Cav3 and HCN ion channels and the expression of vGluT2 mRNA; 2) to elucidate the direct synaptic input to ARC POMC and NPY/AgRP neurons from ARC Kiss1 neurons using optogenetic stimulation in combination with whole-cell recording in E2-treated females; 3) to elucidate the direct synaptic input to AVPV/PeN Kiss1 neurons from ARC Kiss1 neurons using optogenetic stimulation and whole-cell recording in E2-treated females; 4) to elucidate the effects of high frequency optogenetic stimulation of ARC Kiss1 neurons on GnRH release and on food intake in E2-treated females. Therefore, elucidating the circuits and signaling cascades underlying the actions of E2 in the hypothalamus will provide a neurophysiological framework whereby Kiss1 neurons could coordinate reproduction with changes in energy status.