Estrogen Modulation of Bursting Activity in GnRH neurons

描述(申请人提供)：本项目的总体目标是确定卵巢类固醇17-雌二醇(E2)调节促性腺激素释放激素神经元兴奋性的机制，从而控制促性腺激素释放激素神经分泌和生育。这些神经元位于下丘脑，构成了调节女性垂体黄体生成素分泌和排卵的最后一步。重要的是，E2分别通过负反馈和正反馈交替抑制和刺激GnRH神经分泌。广泛的研究表明，这些E2的作用是复杂的，涉及多种神经递质和代谢因素。然而，我们对GnRH神经元调控的细胞和分子机制的了解有限，因此对生育控制的了解还不完全。最近的证据表明，E2通过雌激素受体(ER)或一种新的膜ER(Mer)直接作用于GnRH神经元，并通过ER和mer作用于突触前。我们已经证实，受E2调节的内向整流钾(Kir)电导在调节GnRH细胞兴奋性方面发挥着重要作用，并可能参与GnRH分泌的负反馈。此外，我们还发现，生殖必需神经肽Kispeptin通过抑制KIR通道和激活非选择性阳离子通道(TRPC样通道)来去极化GnRH神经元。KIR的抑制和TRPC通道的激活是Kispeptin在GnRH峰时取消抑制驱动和去极化GnRH神经元的重要机制。在这项建议中，我们试图利用我们拥有丰富经验的全细胞膜片钳和单细胞逆转录聚合酶链式反应技术，进一步探索E2调控GnRH神经元兴奋性的机制。我们将集中于阐明E2调节关键的兴奋性输入(例如Kispeptin、谷氨酸)和抑制性输入(例如阿片类物质、GABA)的机制。我们的具体目标将探讨理解GnRH兴奋性的关键因素：(1)阐明E2增加GnRH神经元KIR通道活性的信号级联；(2)阐明-阿片受体激动剂对GnRH神经元突触前和突触后的影响；(3)阐明E2对Kisspeptin-Gpr54作用的调节以及Kisspeptin激活的增加GnRH神经元TRPC通道活性的细胞信号级联；以及(4)利用配体和STX来阐明GnRH神经元T型钙通道对E2的调节。可以预见，这些研究的结果将有助于理解E2控制GnRH神经元兴奋性的细胞作用，而GnRH神经元兴奋性是女性脉动性神经分泌和最终排卵的关键。

DESCRIPTION (provided by applicant): The overall objectives of this project are to ascertain the mechanisms by which the ovarian steroid 17 - estradiol (E2) regulates GnRH neuronal excitability, which controls GnRH neurosecretion and fertility. These neurons are located in the hypothalamus and constitute the final step in the regulation of pituitary luteinizing hormone (LH) secretion and ovulation in females. Importantly, E2 feeds back to alternately inhibit and stimulate GnRH neurosecretion, via negative and positive feedback, respectively. Extensive studies have demonstrated that these E2 actions are complex and involve multiple neurotransmitters and metabolic factors. However, we have limited knowledge about the cellular and molecular mechanisms by which GnRH neurons are regulated, and therefore, incomplete understanding of the control of fertility. Recent evidence suggests that E2 acts directly on GnRH neurons through estrogen receptor (ER) or a novel membrane ER (mER), as well as presynaptically through ER and mER. We have identified that inwardly rectifying K+ (Kir) conductances that are regulated by E2 play a major role in mediating GnRH cellular excitability and may be involved in negative feedback on GnRH secretion. In addition, we have discovered that the reproductively essential neuropeptide kisspeptin depolarizes GnRH neurons through inhibition of Kir channels and activation of nonselective cationic (TRPC-like) channels. The inhibition of Kir and activation of TRPC channels are important mechanisms by which kisspeptin abrogates inhibitory drive and depolarizes GnRH neurons at the time of the GnRH surge. In this proposal, we seek to further explore the mechanisms by which E2 governs GnRH neuronal excitability using whole-cell patch clamp and single cell reverse transcription PCR approaches, techniques with which we have extensive experience. We will focus on elucidating the mechanisms by which E2 modulates critical excitatory input (e.g. kisspeptin, glutamate) and inhibitory input (e.g. opioids, GABA). Our Specific Aims will examine important factors key to the understanding of GnRH excitability: (1) elucidate the signaling cascade by which E2 increases Kir channel activity in GnRH neurons; (2) elucidate the pre- and postsynaptic effects of - opioid receptor agonists on GnRH neurons; (3) elucidate E2 modulation of kisspeptin-GPR54 actions and the cellular signaling cascades activated by kisspeptin that increase TRPC channel activity in GnRH neurons; and (4) elucidate the E2 regulation of T-type calcium channels in GnRH neurons using the mER ligand STX. It is envisioned that the results from these studies will help in understanding the cellular actions of E2 that govern GnRH neuronal excitability, which is critical for pulsatile neurosecretion and ultimately ovulation in the female.