Investigation of brainstem neurons in the regulation of gonadotropin secretion

项目总结 这项拟议研究的长期目标是确定生理学上 信号调节促性腺激素的分泌，而促性腺激素最终决定生殖功能。调制方式 黄体生成素(黄体生成素)的分泌决定生育能力并控制性腺类固醇的浓度，性腺类固醇有 对心血管、肌肉骨骼和精神健康有深远影响。促黄体激素分泌脉搏 是由下丘脑弓状核中的神经元组成的，这些神经元含有Kispeptin、神经激肽B和 男性和女性的强啡肽(KNDy神经元)。在女性中，排卵前的黄体生成素高峰是由雌二醇引起的。 并依赖于前腹侧脑室周围区的Kispeptin细胞(AVPVKiss1)。然而， 支配这些Kispeptin细胞群体的高级神经回路仍然是一个重要的 悬而未决的问题。孤束核(NTS)位于脑干内，由 接受丰富的内感和中枢输入和投射的异质神经元群体 大脑。有趣的是，这些神经元与应激时抑制搏动性的促黄体生成素分泌有关， 促排卵前黄体生成素高峰时促黄体生成素分泌增强。为了解决这一明显的问题 悖论，这个K99/R00提议将检验中心假设，不同的神经元亚群在 NTS通过抑制KNDy神经元而抑制搏动性黄体生成素的分泌，并通过 激活AVPVKiss1细胞。在指导阶段，我们将使用病毒介导的细胞激活标记 光学清除组织的技术和光片显微镜以确定相同的神经元是否被激活 在应激和黄体生成素激增期间，以及单细胞RNA测序以确定NTS的亚群 在应激和黄体生成素激增期间被激活的神经元(目标1)。指导阶段将包括关键的 高级神经解剖学和神经成像技术、下一代测序技术、 生物信息学分析，以及过渡到 独立的学术研究岗位。在独立阶段，我建议使用化学发生和细胞- 特定的病毒介导的神经消融技术来确定在 AIM 1对于应激诱导的黄体生成素分泌抑制和KNDy细胞激活(AIM)是充分和必要的 2)或用于排卵前黄体生成素激增和AVPVKiss1细胞激活(目标3)，并确定在大脑中的位置 这些亚群计划(目标2和3)。这些研究将启动我的独立研究计划，并 提供可能影响治疗黄体生成素异常的治疗方法的发展的神经框架 分泌物，包括闭经、不孕症和多囊卵巢综合征。总体而言， 赞助/联合赞助团队对我的科学和专业发展，加上对我的激励 加州大学圣地亚哥分校的学术环境和令人印象深刻的资源将确保我取得 该职业发展提案的目标以及加州大学圣地亚哥分校和美国国立卫生研究院的培训使命。

PROJECT SUMMARY The long-term goal of the proposed research is to determine the neural pathways by which physiological signals regulate gonadotropin secretion, which ultimately determines reproductive function. Modulation of luteinizing hormone (LH) secretion determines fertility and controls gonadal steroid concentrations, which has profound effects on cardiovascular, musculoskeletal, and mental health. Pulses of luteinizing hormone secretion are organized by neurons in the arcuate nucleus of the hypothalamus that contain kisspeptin, neurokinin B and dynorphin (KNDy neurons) in males and females. In females, the preovulatory LH surge is induced by estradiol and is dependent upon kisspeptin cells in the anteroventral periventricular region (AVPVKiss1). However, the higher order neural circuitry that governs these populations of kisspeptin cells remains a significant outstanding question. The nucleus of the solitary tract (NTS) is located in the brainstem and consists of a heterogenous population of neurons that receive rich interoceptive and central inputs and projects widely thought the brain. Interestingly, these neurons are implicated in both the inhibition of pulsatile LH secretion during stress, and the facilitation of enhanced LH secretion during the preovulatory LH surge. To address this apparent paradox, this K99/R00 proposal will test the central hypothesis that distinct subpopulations of neurons in the NTS suppress pulsatile LH secretion via inhibition of KNDy neurons and enhance LH secretion via activation of AVPVKiss1 cells. During the mentored phase, we will employ viral-mediated cell activation labeling techniques and light sheet microscopy of optically cleared tissue to determine if the same neurons are activated during stress and the LH surge, as well as single-cell RNA sequencing to identify the subpopulations of NTS neurons that are activated during stress and the LH surge (Aim 1). The mentored phase will consist of critical training in advanced neuroanatomical and neuroimaging techniques, next generation sequencing technologies, bioinformatic analysis, as well as career development experiences that are necessary for transitioning to an independent academic research position. In the independent phase, I propose to use chemogenetic and cell- specific viral-mediated neural ablation techniques to determine whether subpopulations of neurons identified in Aim 1 are sufficient and necessary for stress-induced suppression of LH secretion and KNDy cell activation (Aim 2) or for the preovulatory LH surge and AVPVKiss1 cell activation (Aim 3) and determine the locations in the brain these subpopulations project (Aims 2 & 3). These studies will launch my independent research program and will provide a neural framework that may influence the development of therapies to treat disorders of altered LH secretion, including amenorrhea, infertility, and polycystic ovary syndrome. Collectively, the commitment of the sponsoring/co-sponsoring team to my scientific and professional development, coupled with the stimulating academic environment and impressive resources at UC San Diego available to me will ensure achievement of the aims of this Career Development proposal and the training mission of UC San Diego and the NIH.