Function and Organization of Gonadotropin-Releasing Hormone Neuronal Circuitry

 描述(申请人提供)：多囊卵巢综合征(PCOS)是无排卵性不孕的最常见原因，其特征是月经周期不规律和雄激素升高。调节下游生殖功能的中枢神经系统通过促性腺激素释放激素(GnRH)神经元进行交流。在正常月经周期中，促性腺激素释放激素脉冲频率的变化向脑下垂体发出信号，以释放调节卵泡成熟和卵巢类固醇产生的激素。激素反馈是通过GnRH神经元的传入网络传递给GnRH神经元的，然而该网络的电路和活动尚不清楚。在大多数患有多囊卵巢综合征的成年女性中，这种神经系统持续过度活跃，导致排卵失败和高雄激素产生。这种增加的神经内分泌活动何时开始尚不清楚，然而，发育过程中的神经元过度活动可能会导致成年后网络连接和功能的改变。在发育期间暴露于升高的雄激素会导致许多物种的表型类似于多囊卵巢综合征。我们的假设是，GnRH神经元在青春期前的活动对于吸引适当的突触输入是关键的，而产前的雄激素释放(PNA)改变了这种活动 以及突触发生，从而影响成人GnRH神经元回路的连接和功能。为了验证这一假设，我们将研究PNA如何改变GnRH神经元网络的成人组织，以及青春期前发育期间GnRH神经元的传入输入的功能。在目标1中，我们将验证PNA增加青春期前GnRH神经元的GABA能和谷氨酸能突触后电流(PSCs)的频率和幅度的假设，并且这是通过增加突触连接密度来实现的。将进行PSCs的全细胞电生理记录，以检测这些输入在青春期前发育期间的自发功能。诱发活动将被用来识别已经建立但尚未激活的突触连接。在目标2中，我们将检验这一假设，即发育过程中暴露于高雄激素会导致成年女性GnRH神经元突触前网络组织发生变化。我们将利用一种针对表达Cre重组酶的GnRH神经元的病毒示踪剂来绘制GnRH神经元网络的网络图，并结合免疫荧光和/或原位杂交来识别神经元表型。这将使我们能够确定GnRH神经元传入的数量、位置和识别，并检查这种连接是否因发育过程中暴露于雄激素而改变。这两个目标都将促进我们对典型GnRH神经元发育的了解，超越对单个神经元的研究，以理解网络相互作用，具有解决长期存在的关于下丘脑调节生育的问题的巨大潜力。它们还将进一步加深我们对PNA如何改变GnRH神经元网络的理解。这个项目的广泛含义和知识深度是我作为一名内科科学家接受培训的基本要素。

 DESCRIPTION (provided by applicant): Polycystic ovary syndrome (PCOS) is the most common cause of anovulatory infertility and is characterized by irregular menstrual cycles and elevated androgens. The central neuronal system that regulates downstream reproductive function communicates through gonadotropin-releasing hormone (GnRH) neurons. Shifts in GnRH pulse frequency during the normal menstrual cycle signal the pituitary to release hormones that regulate follicular maturation and ovarian steroid production. Hormonal feedback is conveyed to GnRH neurons through their afferent network, however the circuitry and activity of this network is not well understood. In most adult women with PCOS this neuronal system is persistently overactive leading to failure of ovulation and high androgen production. When this increased neuroendocrine activity begins is not known, however neuronal hyperactivity during development could result in altered network connectivity and function in adulthood. Exposure to elevated androgens during development results in phenotypes that are similar to PCOS in many species. Our hypothesis is that GnRH neuron activity during the prepubertal period is critical for attracting appropriate synaptic inputs and that prenatal androgenization (PNA) alters this activity and synaptogenesis and thus the adult connectivity and function of the GnRH neuronal circuitry. To test this hypothesis we will study how PNA alters the adult organization of the GnRH neuronal network and the function of afferent inputs to GnRH neurons during prepubertal development. In Aim 1 we will test the hypothesis that PNA increases the frequency and amplitude of GABAergic and glutamatergic postsynaptic currents (PSCs) to prepubertal GnRH neurons and that this occurs through an increased density of synaptic connections. Whole-cell electrophysiological recordings of PSCs will be made to examine the spontaneous function of these inputs during prepubertal development. Evoked activity will be used to identify synaptic connections that have been established but are not yet active. In Aim 2 we will test the hypothesis that developmental exposure to elevated androgens results in altered adult organization of the presynaptic network to GnRH neurons in females. We will utilize a viral tracer targeted to Cre recombinase-expressing GnRH neurons to map the circuitry of the GnRH neuronal network in combination with immunofluorescence and/or in situ hybridization to identify neuron phenotype. This will allow us to characterize the number, location and identify of GnRH neuron afferents and to examine if this connectivity is altered by developmental exposure to androgens. Both Aims will further our knowledge of typical GnRH neuron development beyond the study of individual neurons to comprehension of network interactions with great potential to address long-standing questions about hypothalamic regulation of fertility. They will also further our understanding of how PNA alters the GnRH neuronal network. The broad implications and intellectual depth of this project are essential elements for my training as a physician-scientist.