A role for hypothalamic astrocytes in neural circuits controlling reproduction

项目摘要 星形胶质细胞是一种神经胶质细胞亚型，对许多中枢神经系统功能至关重要。失调 已知星形胶质细胞功能的缺失会导致多种神经和神经肌肉疾病。有趣的是， 下丘脑星形胶质细胞的破坏具有损害生育能力的能力。大约五分之一的夫妇 美国人患有某种类型的不育症，因此阐明了生殖的完整回路， 具有本质意义视前区和下丘脑中的促性腺激素释放激素（GnRH）神经元形成了 中央控制生育的最终共同途径。脉冲式GnRH分泌引起促黄体生成素释放 垂体前叶分泌的促卵泡激素（LH）和促卵泡激素（FSH），激活性腺功能 包括类固醇生成。一个传入网络可能控制着GnRH的脉动分泌，包括类固醇和激素。 弓状核中的敏感kisspeptin神经元（KNDy神经元）和胶质细胞。虽然GnRH脉冲释放 是男性和女性生殖的共同点，女性表现出独特的过程，包括生殖周期 和排卵，从而改变神经胶质细胞的相互作用重要的是，星形胶质细胞表现出形态可塑性， 神经胶质细胞对下丘脑神经元鞘化的水平在周期的不同阶段发生变化，表明神经胶质细胞对下丘脑神经元鞘化的作用可能与神经元鞘化有关。 星形胶质细胞的调节随周期阶段而变化。星形胶质细胞介质如前列腺素E2（PGE2） 还可以调节GnRH神经元的活性，PGE2是及时性成熟和成年所必需的。 啮齿动物的繁殖为了研究星形胶质细胞调节生殖神经回路的背景， 将使用电生理学、钙成像和神经胶质细胞培养的组合。目标1将研究是否有因素 包括性别、周期阶段和一天中的时间影响星形胶质细胞调节GnRH和/或KNDy的能力 神经元放电率通过转导的Gq信号受体体外活化星形胶质细胞 hM3D（Gq）将用于诱导细胞内钙瞬变，其代表内源性Gq诱导的 星形胶质细胞的活动，同时监测所识别的神经元的活动。在目标2中， 进行原代星形胶质细胞培养，然后进行上清液的高效液相色谱， 阐明下丘脑星形胶质细胞的胶质递质谱。为了赞美这一点，内源性Gq- 将在用GFAP-GCaMP6f转导的神经胶质培养物和脑切片中评估偶联受体谱 通过用候选Gq配体处理。这些结果将为我随后的实验提供信息， 内源性Gq偶联受体将在下丘脑星形胶质细胞和胶质递质中被敲低 GnRH神经元中的受体，为这些神经胶质神经信号的必要性提供了测试， 在体内繁殖。完成拟议的工作将阐明星形胶质细胞在神经内分泌系统中的作用。 控制生育。阐明星形胶质细胞与GnRH来回通信的机制 KNDy神经元将为确定治疗不孕症的潜在治疗靶点铺平道路。

PROJECT SUMMARY Astrocytes are a glial subtype that are essential for numerous central nervous system functions. Dysregulation of astrocyte function is known to cause a variety of neurological and neuromuscular disorders. Interestingly, disruption of hypothalamic astrocytes has the capability to compromise fertility. Roughly one in five couples in the USA suffer from some type of infertility, thus elucidating the complete circuits underlying reproduction is essential. Gonadotropin-releasing hormone (GnRH) neurons in the preoptic area and hypothalamus form the final common pathway for the central control of fertility. Pulsatile GnRH secretion causes release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from the anterior pituitary, activating gonadal functions including steroidogenesis. An afferent network likely governs pulsatile GnRH secretion, including both steroid- sensitive kisspeptin neurons in the arcuate nucleus (KNDy neurons) and glia. Although pulsatile GnRH release is common to male and female reproduction, females exhibit unique processes including reproductive cycles and ovulation that could alter neural-glial interactions. Critically, astrocytes exhibit morphological plasticity and the levels of hypothalamic neuron ensheathment by glia change across cycle stages, pointing to potential astrocytic regulation that varies with respect to cycle stage. Astrocytic mediators like prostaglandin E2 (PGE2) can also regulate GnRH neuron activity, and PGE2 is required for timely sexual maturation and adult reproduction in rodents. To investigate the contexts in which astrocytes regulate reproductive neural circuits, a combination of electrophysiology, calcium imaging, and glial cell cultures will be used. Aim 1 will study if factors including sex, cycle stage, and time of day influence the ability of astrocytes to regulate GnRH and/or KNDy neuron firing rates. In vitro chemogenetic activation of astrocytes via the transduced Gq signaling receptor hM3D(Gq) will be employed to induce intracellular calcium transients that typify endogenous Gq-induced astrocyte activity, while monitoring the activity of identified neurons. In Aim 2, chemogenetic activation of primary astrocyte cultures followed by high-performance liquid chromatography of supernatants will be done to elucidate the gliotransmitter profiles of hypothalamic astrocytes. To compliment this, the endogenous Gq- coupled receptor profiles will be assessed in glial cultures and brain slices transduced with GFAP-GCaMP6f via treatment with candidate Gq ligands. These results will inform my subsequent experiments in which an endogenous Gq-coupled receptor will be knocked down in hypothalamic astrocytes and a gliotransmitter receptor in GnRH neurons, providing a test for the necessity of these components of glial-neural signaling for reproduction in vivo. Completion of the proposed work will clarify roles of astrocytes in the neuroendocrine control of fertility. Elucidating the mechanisms by which astrocytes communicate back-and-forth with GnRH and KNDy neurons will pave the way to identifying potential therapeutic targets to treat types of infertility.