Cellular and molecular bases for rhythmic GnRH release

生殖健康是全面健康的窗口。15%到20%的夫妇难以怀孕;失败 因此，生殖系统的疾病影响了相当多的人口。除了生育能力，性类固醇还会改变发育 和许多系统的功能，例如，骨骼、中枢神经系统和心血管系统。 两种脊椎动物的生育能力都需要间歇性释放促性腺激素释放激素（GnRH） 这些脉冲的频率变化对女性生殖周期至关重要。这个目标 建议是增加我们对GnRH间歇性释放产生的基本理解， 这是理解和调节生育率的关键环节。拟议的工作将定义GnRH的内在特性 神经元，局部网络的相互作用，以及胶质细胞和假设的驱动神经元位于弓状核的作用 共表达kisspeptin、神经激肽B和强啡肽的下丘脑核（KNDy神经元）。我们 工作假设是内在和网络机制在局部回路中相互作用，产生最终的 GnRH神经分泌系统的间歇性输出。我们将使用最先进的 电生理学、成像和化学发生学方法结合GnRH释放的局部测量 在转基因小鼠中，通过荧光报告蛋白的表达鉴定特定的神经元。我们 我还将扩展我们的研究与数学建模，以产生假设和返回这些假设 送去实验室检测我们有两个具体目标。在目标1中，我们将研究自分泌和旁分泌是如何 促性腺激素释放激素神经元和神经胶质细胞的因子改变动作电位、细胞内钙和 GnRH的释放，以及男性和女性的类固醇反馈如何改变这些关系。我们将 应用这些知识来研究GnRH神经元如何协调产生脉冲。我们假设 来自这些电路元件（GnRH神经元、传入神经元和神经胶质）的信号是产生脉冲所必需的。我们 将使用药理学和化学遗传学的方法来研究这些元素的具体作用， 建立GnRH网络。在目标2中，我们将描述GnRH的内在特性和突触输入 两种性别的完整小鼠与去势小鼠中的神经元。我们将利用这些数据， 表征孤立的参数，并直接研究突触传递如何与内在的相互作用 GnRH神经元改变动作电位产生的特性。动态夹持是一种方法， 互动要研究。这是通过对这些电导进行仔细建模来实现的，然后可以 在记录期间添加到单元和/或从单元减去。这通过迭代交互在真实的时间中发生 与细胞的膜电位，使电压依赖的电导变化得到精确控制， 并记录电导对膜电位和细胞放电的影响。通过检查 从这两个角度产生脉冲，一个综合的图片将出现，增加了我们的理解， 这种现象。所有必要的动物模型和方法都已到位，以完成这些研究。

Reproductive health is a window to overall health. Fifteen to 20% of couples have difficulty conceiving; failures of the reproductive system thus affect a substantial population. Beyond fertility, sex steroids alter development and function of many systems, for example, bone, the central nervous system and the cardiovascular system. Episodic release of gonadotropin-releasing hormone (GnRH) is required for fertility in vertebrates of both sexes, and shifts in frequency of these pulses are essential for female reproductive cycles. The goal of this proposal is to increase our fundamental understanding of the generation of episodic release of GnRH as a critical link to understanding and modulating fertility. The proposed work will define intrinsic properties of GnRH neurons, local network interactions, and roles of glia and postulated driver neurons located in the arcuate nucleus of the hypothalamus that coexpress kisspeptin, neurokinin B and dynorphin (KNDy neurons). Our working hypothesis is that intrinsic and network mechanisms interact in local circuits to produce the final episodic output of the GnRH neurosecretory system. We will study this system using state-of-the-art electrophysiological, imaging and chemogenetic approaches combined with local measures of GnRH release in transgenic mice in which specific neurons are identified by expression of fluorescent reporter proteins. We will also extend our studies with mathematical modeling to generate hypotheses and return these hypotheses to the lab for testing. We have two specific aims. In Aim 1, we will examine how autocrine and paracrine factors from GnRH neurons and glia alter the relationship among action potentials, intracellular calcium and GnRH release, as well as how steroid feedback in males and females modifies these relationships. We will apply this knowledge to study how GnRH neurons are coordinated to produce pulses. We hypothesize factors from these circuit elements (GnRH neurons, afferent neurons, and glia) are needed for pulse generation. We will use pharmacologic and chemogenetic approaches to study the specific roles of these elements in organizing the GnRH network. In Aim 2, we will characterize intrinsic properties and synaptic input to GnRH neurons in intact vs. castrate mice of both sexes. We will use these data to move beyond independent characterization of isolated parameters and directly study how synaptic transmission interacts with intrinsic properties of GnRH neurons to alter action potential generation. Dynamic clamp is a method that allows these interactions to be studied. This is accomplished by careful modeling of these conductances, which can then be added to and/or subtracted from cells during recording. This occurs in real time through iterative interaction with the cell’s membrane potential, so that voltage-dependent changes in conductance are precisely controlled, and effects of the conductances on the membrane potential and firing of the cell are recorded. By examining pulse generation from these two angles, an integrated picture will emerge that adds to our understanding of this phenomenon. All necessary animal models and methods are in place to complete these studies.