Estrogen Modulation of Bursting Activity in GnRH neurons

雌激素对 GnRH 神经元爆发活性的调节

• 批准号: 7805411
• 负责人: Oline Karin Rønnekleiv
• 金额: $ 33.35万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-01-01 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7805411
• 关键词: Agonist; Animal Model; Applications Grants; Back; Cell model; Cell physiology; Cells; Complex; Contraceptive methods; Coupled; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Estradiol; Estrogen Receptors; Estrogens; Exhibits; Feedback; Feeds; Female; Fertility; G Protein-Coupled Receptor 54; GTP-Binding Proteins; Gene Expression; Glutamates; Hypogonadism; Hypothalamic structure; Infertility; Ion Channel; KISS1R gene; Knowledge; Lead; Ligands; Luteinizing Hormone; Measurement; Mediating; Membrane; Metabolic; Molds; Molecular; Mutation; Neurons; Neuropeptides; Neurosecretion; Neurotransmitters; Opioid; Opioid Receptor; Ovarian; Ovulation; Patients; Phase; Phosphorylation; Pituitary Gland; Play; Regulation; Research; Reverse Transcriptase Polymerase Chain Reaction; Reverse Transcription; Role; Sexual Development; Signal Pathway; Signal Transduction; Signaling Molecule; Steroids; Synapses; T-Type Calcium Channels; Techniques; Time; Whole-Cell Recordings; Work; attenuation; computerized data processing; density; experience; feeding; gamma-Aminobutyric Acid; kisspeptin; mRNA Expression; mouse model; neuronal excitability; novel; patch clamp; postsynaptic; presynaptic; public health relevance; receptor; receptor coupling; reproductive; research study; response

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.

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，该机制控制GNRH神经分泌和生育能力。这些神经元位于下丘脑中，构成了垂体黄体激素（LH）分泌和女性排卵的最后一步。重要的是，E2分别通过阴性和阳性反馈分别抑制和刺激GNRH神经分泌。广泛的研究表明，这些E2作用很复杂，涉及多个神经递质和代谢因素。但是，我们对通过调节GNRH神经元的细胞和分子机制有限，因此对生育能力的控制不完全了解。最近的证据表明，E2通过雌激素受体（ER）或新型膜ER（MER）以及通过ER和MER突触前直接作用于GnRH神经元上。我们已经确定，由E2调节的内部矫正K+（KIR）电导在介导GnRH细胞兴奋性中起主要作用，并且可能与GNRH分泌的负反馈有关。此外，我们发现生殖必要的神经肽亲肽素通过抑制KIR通道和非选择性阳离子（TRPC样）通道的激活来使GNRH神经元去极化。 KIR的抑制和TRPC通道的激活是重要机制，在GNRH激增时，Kisspeptin消除了抑制性驱动并将GNRH神经元去极化。在此提案中，我们寻求进一步探讨E2使用全细胞贴片夹和单细胞逆转录PCR方法的GNRH神经元兴奋性的机制，以及我们具有丰富经验的技术。我们将重点介绍E2调节关键兴奋性输入（例如Kisspeptin，谷氨酸）和抑制性输入（例如阿片类药物，GABA）的机制。我们的具体目的将研究重要因素，这是对GNRH兴奋性理解的关键：（1）阐明E2增加GNRH神经元中KIR通道活性的信号传导级联； （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Kisspeptin-GPR54动作的E2调节和由Kisspeptin激活的细胞信号传导级联反应，从而增加了GNRH神经元中TRPC通道活性； （4）使用MER配体STX阐明了GNRH神经元中T型钙通道的E2调节。可以预见，这些研究的结果将有助于理解控制GNRH神经元兴奋性的E2的细胞作用，这对于脉冲神经分泌至关重要，最终是女性的排卵。公共卫生相关性：我们的研究重点是了解女性生殖周期的负反馈阶段和正面反馈阶段，通过雌激素调节GnRH神经元的细胞机制。功能性GNRH神经元对于该物种的存活至关重要，并且对这些重要神经元受调节的细胞和分子机制知之甚少。我们精心制作了一组仔细的实验​​，以阐明与雌激素介导的抑制作用（负反馈）和GNRH神经元的激发（正反馈）有关的信号传导途径和通道。可以预见的是，提出的实验的结果将有助于塑造GNRH神经元爆发的细胞模型，搏动性神经分泌以及最终在女性中排卵。