Cross-Talk Between Estrogen and Metabolic Hormone Signaling in Arcuate Neurons

项目摘要 拟议研究的长期目标是阐明代谢状态 和17β-雌二醇（E2）调节弓状核（ARC）kisspeptin（Kiss 1）神经元回路，这些神经元回路对 协调女性的能量平衡和生殖。众所周知，E2是致癌的， Kiss 1神经元直接受E2调节，对青春期发育和成年期发育至关重要， 繁殖成功。然而，它们在控制能量稳态中的作用却鲜为人知。我们有 表明ARC Kiss 1神经元直接被瘦素和胰岛素兴奋，表明它们可能是一种神经元。 在控制能量平衡中起重要作用。同时，我们有证据表明谷氨酸是从 ARC Kiss 1神经元和靶向促食欲的阿黑皮素原（POMC）神经元和促食欲的 神经肽Y/刺鼠相关肽（NPY/AgRP）神经元。此外，我们发现谷氨酸可以 通过作用于不同的代谢型谷氨酸组差异调节POMC和NPY/AgRP神经元 受体（mGluRs）。此外，我们发现E2增加囊泡谷氨酸转运体2， （vGluT 2）mRNA在女性ARC Kiss 1神经元，一个指标，提高囊泡谷氨酸包装和 release.我们也有证据表明ARC Kiss 1神经元投射并兴奋AVPV/Pen Kiss 1神经元， 这对诱导GnRH/LH峰很重要。因此，我们认为ARC Kiss 1神经元 整合代谢激素和类固醇信号以调节能量稳态和生殖。 因此，我们提出了新的假设，即ARC Kiss 1神经元的兴奋性在高水平时增加， 雌激素状态，从而释放谷氨酸兴奋POMC神经元和抑制NPY/AgRP神经元， 组I和组II/III的mGluR，其分别减少食物摄入。此外，兴奋性多巴胺能 从ARC Kiss 1神经元到AVPV/Pen Kiss 1神经元的输入构成对GnRH的关键刺激驱动 在GnRH/LH激增的时候。我们的多学科方法结合了一套强大的细胞， 分子和光遗传学工具可实现以下目标：1）阐明ARC Kiss 1神经元中的 E2对Cav 3和HCN离子通道mRNA表达和功能的影响，以及E2对Cav 3和HCN离子通道mRNA表达和功能的影响。 阐明ARC POMC和NPY/AgRP神经元的直接突触输入 在E2处理的雌性中使用光遗传学刺激结合全细胞记录的Kiss 1神经元; 3） 利用光遗传学方法阐明从ARC Kiss 1神经元到AVPV/Pen Kiss 1神经元的直接突触输入， 刺激和全细胞记录在E2处理的女性; 4）阐明高频率的影响， ARC Kiss 1神经元的光遗传学刺激对E2处理的雌性动物的GnRH释放和食物摄入的影响。 因此，阐明E2在下丘脑中作用的电路和信号级联将 提供了一个神经生理学框架，使Kiss 1神经元能够协调生殖与 能源状况。

Project Summary The long range goals of the proposed research are to elucidate the mechanism(s) by which metabolic states and 17β-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.