Kisspeptin Synaptic Input to POMC Neurons

描述(申请人提供)：这项拟议研究的长期目标是通过调节下丘脑厌食症神经元控制食物摄入量和能量消耗，了解Kispeptin在调节能量平衡中的作用。瘦素和胰岛素等外周代谢信号的中枢信号异常已导致能量平衡紊乱，如代谢综合征和厌食症-恶病质。前阿片黑素皮质素原(POMC)神经元在能量平衡中起关键作用，至少部分地调节这两种代谢激素的中枢信号。此外，已发现Kispeptin神经元表达瘦素和胰岛素。 受体，并且给药直接刺激POMC神经元。因此，Kispeptin神经元可能通过它们对POMC神经元的突触输入，在能量稳态的控制中起着不可或缺的作用。这一建议的中心假设是，弓状Kispeptin神经元，也被称为KNDy(Kispeptin/Neurokinin B/Dynorphin)神经元，通过激活规范的瞬时受体电位(TRPC)通道，直接兴奋弓状核中的POMC细胞，这种兴奋在 禁食状态。为了解决这一假设，我设计了两个具体的目标，其中包括将转基因和光遗传工具与分子生物学(目标1)和电生理学(目标2)创造性地结合起来。在目标1中，我将通过分散和采集POMC-EGFP雄性小鼠POMC神经元池进行定量PCR分析，阐明喂养和食物限制POMC-EGFP雄性小鼠POMC神经元中的Kispeptin信号成分。我们的实验室在这项技术的开发和实施方面走在了前列，这为我们提供了一个非常强大的工具来表征神经元中的信号分子(例如，受体和离子通道)。在目标2中，我将利用光遗传学的创新工具选择性地光激活KNDy神经元，同时记录弓状核POMC神经元，来探讨KNDy神经元与弓状核POMC神经元之间的功能联系。通过使用选择性拮抗剂，我将确定Kispeptin诱导的POMC神经元的激活是否通过TRPC通道发生，就像我们已经证明的那样，Kispeptin诱导的下丘脑其他神经元(即GnRH神经元)的激活。总之，对KNDy和POMC神经元之间TH功能连接的基因表达和细胞通路的研究将有助于确定治疗干预的细胞靶点，以对抗与能量稳态相关的疾病。

DESCRIPTION (provided by applicant): The long-term goal of the proposed research is to understand the role of kisspeptin in the regulation of energy homeostasis through its modulation of hypothalamic anorexigenic neurons that control food intake and energy expenditure. Abnormalities in the central signaling of peripheral metabolic cues such as leptin and insulin have led to disorders in energy homeostasis such as the metabolic syndrome and anorexia-cachexia. Pro- opiomelanocortin (POMC) neurons play a critical role in energy homeostasis and mediate, at least in part, the central signaling of these two metabolic hormones. In addition, kisspeptin neurons have been shown to express leptin and insulin receptors, and kisspeptin administration directly stimulates POMC neurons. Therefore, kisspeptin neurons may play an integral role in the control of energy homeostasis through their synaptic input to POMC neurons. The central hypothesis of this proposal is that arcuate kisspeptin neurons, also known as KNDy (kisspeptin/neurokinin B/dynorphin) neurons, directly excite POMC cells in the arcuate nucleus through activation of canonical transient receptor potential (TRPC) channels, and this excitation is reduced in the fasted state. To address this hypothesis, I have designed two specific aims, which include a creative combination of transgenic and optogenetic tools in conjunction with molecular biology (aim 1) and electrophysiology (aim 2). In aim 1, I will elucidate the kisspeptin signalng components in POMC neurons of fed and food restricted POMC-EGFP male mice by dispersing and harvesting pools of POMC neurons for quantitative PCR analysis. Our lab has been in the forefront in the development and implementation of this technique, which gives us an extremely powerful tool to characterize signaling molecules (e.g., receptors and ion channels) in neurons. In aim 2, I will interrogate the functional connection between KNDy neurons and POMC neurons in the arcuate nucleus by using the innovative tool of optogenetics to selectively photoactivate KNDy neurons while simultaneously recording from arcuate POMC neurons. By using selective antagonists I will determine if the kisspeptin-induced activation of POMC neurons occurs via TRPC channels, as we have shown for kisspeptin-induced activation of other hypothalamic neurons (i.e., GnRH neurons). Together, the investigation of the gene expression and cellular pathways specific to th functional connectivity between KNDy and POMC neurons will help identify cellular targets for therapeutic interventions to counteract disorders associated with energy homeostasis.