Estrogen Modulation of Bursting Activity in GnRH neurons

描述（由申请人提供）：该项目的总体目标是确定卵巢类固醇17 -雌二醇（E2）调节GnRH神经元兴奋性的机制，从而控制GnRH神经分泌和生育能力。这些神经元位于下丘脑，构成了调节垂体促黄体激素（LH）分泌和女性排卵的最后一步。重要的是，E2反馈分别通过负反馈和正反馈交替抑制和刺激GnRH神经分泌。大量研究表明，E2的作用是复杂的，涉及多种神经递质和代谢因素。然而，我们对调节GnRH神经元的细胞和分子机制的了解有限，因此，对生育控制的理解不完整。最近的证据表明，E2通过雌激素受体（ER）或新型膜ER （mER）直接作用于GnRH神经元，并通过ER和mER在突触前作用。我们已经发现E2调节的内校正K+ (Kir)电导在介导GnRH细胞兴奋性中起主要作用，并可能参与GnRH分泌的负反馈。此外，我们发现生殖必需的神经肽kisspeptin通过抑制Kir通道和激活非选择性阳离子（trpc样）通道使GnRH神经元去极化。Kir的抑制和TRPC通道的激活是kisspeptin在GnRH激增时消除抑制驱动并使GnRH神经元去极化的重要机制。在这项提议中，我们寻求进一步探索E2控制GnRH神经元兴奋性的机制，使用全细胞膜片钳和单细胞逆转录PCR方法，我们拥有丰富的经验。我们将重点阐明E2调节关键兴奋性输入（如kisspeptin，谷氨酸）和抑制性输入（如阿片类药物，GABA）的机制。我们的具体目标将研究理解GnRH兴奋性的关键因素：(1)阐明E2增加GnRH神经元中Kir通道活性的信号级联；(2)阐明-阿片受体激动剂对GnRH神经元的突触前和突触后作用；(3)阐明E2调控kisspeptin- gpr54的作用以及kisspeptin激活的细胞信号级联反应，从而增加GnRH神经元中TRPC通道的活性；(4)利用mER配体STX阐明E2对GnRH神经元中t型钙通道的调控。据设想，这些研究的结果将有助于理解E2的细胞作用，控制GnRH神经元的兴奋性，这对女性脉动性神经分泌和最终排卵至关重要。公共卫生相关性：我们的研究重点是了解GnRH神经元在女性生殖周期的负反馈和正反馈阶段受雌激素调节的细胞机制。GnRH神经元的功能对物种的生存至关重要，但人们对这些重要神经元的细胞和分子机制知之甚少。我们精心设计了一套实验来阐明参与雌激素介导的GnRH神经元的抑制（负反馈）和兴奋（正反馈）的信号通路和通道。据设想，从提出的实验结果将有助于塑造细胞模型的爆发发射GnRH神经元，脉动神经分泌，并最终在女性排卵。

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. PUBLIC HEALTH RELEVANCE: The focus of our research is to understand the cellular mechanisms by which GnRH neurons are regulated by estrogen during the negative and positive feedback phases of the female reproductive cycle. Functioning GnRH neurons are essential for survival of the species, and very little is known about the cellular and molecular mechanism by which these vital neurons are regulated. We have crafted a careful set of experiments to elucidate the signaling pathways and channels that are involved in estrogen mediated inhibition (negative feedback) and excitation (positive feedback) of GnRH neurons. It is envisioned that the results from the proposed experiments will help mold a cellular model of burst firing of GnRH neurons, pulsatile neurosecretion, and ultimately ovulation in the female.