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的机制(S) 17β-雌二醇(E_2)调节弓状核(Arc)-Kispeptin(Kiss1)神经元回路，这些神经元回路对 协调女性体内的能量平衡和生殖。众所周知，雌二醇是厌食性的，而且 由E2直接调节的Kiss1神经元对青春期发育和成人是必不可少的 繁衍成功。然而，它们在能量动态平衡控制中的作用却鲜为人知。我们有 结果表明，ARC Kiss1神经元可被瘦素和胰岛素直接兴奋，这表明它们可能为 在控制能量动态平衡方面起着重要作用。此外，我们有证据表明谷氨酸是从 ARC Kiss1神经元和靶点厌氧性前阿片黑素皮质素(POMC)神经元和食欲性 神经肽Y/刺鼠相关肽(NPY/AgRP)神经元。此外，我们还发现谷氨酸可以 作用于不同代谢性谷氨酸的POMC和NPY/AgRP神经元的差异调节 受体(MGluRs)。此外，我们还发现，E2增加了囊泡型谷氨酸转运蛋白2 (VGluT2)mRNA在雌性ARC Kiss1神经元中的表达，这是囊泡性谷氨酸包装和 放手。我们也有证据表明ARC Kiss1神经元投射并兴奋AVPV/PEN Kiss1神经元， 它们在GnRH/LH峰的诱导中起重要作用。因此，我们认为ARC Kiss1神经元 整合代谢激素和类固醇信号，调节能量平衡和生殖。 因此，我们提出了一个新的假设，即ARC Kiss1神经元的兴奋性在HIGH中增加 雌激素通过释放谷氨酸兴奋POMC神经元和抑制NPY/AgRP神经元 I组和II/III组的mGluRs分别减少了食物的摄入量。此外，兴奋性谷氨酸能 ARC Kiss1神经元对AVPV/PEN Kiss1神经元的输入构成对GnRH的关键刺激驱动 促性腺激素释放激素/促黄体生成素峰时的神经元。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，以解决以下目标：1)阐明在ARC Kiss1神经元 雌二醇对Cav3和HCN离子通道基因表达及功能的影响 VGluT2 mRNA；2)阐明ARC POMC和NPY/AgRP神经元的直接突触输入 用光遗传刺激结合全细胞记录的雌鼠Kiss1神经元； ARC-Kiss1神经元向AVPV/PEN-Kiss1神经元的直接突触传入 雌激素组雌鼠的刺激和全细胞记录；4)阐明高频的影响 ARC Kiss1神经元的光遗传刺激对雌鼠GnRH释放和食物摄入量的影响。 因此，阐明下丘脑中E2作用的电路和信号级联将 提供一个神经生理学框架，使Kiss1神经元能够协调生殖与 能量状态。

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.