Estradiol modulation of pacemaking kisspeptin neurons

 描述（由申请人提供）：拟议研究的长期目标是阐明17 β-雌二醇（E2）调节女性下丘脑kisspeptin（Kiss 1）神经元回路的分子和细胞机制。Kiss 1神经元是E2的主要靶点，对青春期发育和成年生殖至关重要。这些神经元对于感知和将性腺状态传递给对调节能量平衡至关重要的神经元也很重要，包括下丘脑弓状前阿黑皮素（POMC）和神经肽Y/刺鼠相关肽（NPY/AgRP）神经元。尽管Kiss 1神经元很重要，但对这些细胞的生物物理特性和分子特征知之甚少， 以及它们运作的神经回路。我们发现第三脑室头端室周区（RP 3V）的Kiss 1神经元表达关键离子通道 以及允许自发性E2依赖性反弹爆发放电的受体。基于E2依赖的爆发放电特征及其对弓状神经元的投射，我们提出了一个新的假说，即RP 3VKiss 1神经元作为中枢起搏神经元，为促食欲POMC神经元提供E2依赖的兴奋驱动，并为促食欲NPY/AgRP神经元提供抑制驱动。这种kisspeptin输入POMC和NPY/AgRP神经元是至关重要的雌激素控制的能量稳态在女性排卵周期。阐明细胞特异性信号通路和基因表达在单细胞水平将有助于开发新的策略，在中枢神经系统神经元靶向激素的行动。我们的多学科方法结合了一套独特的细胞，分子，光遗传学和化学遗传学工具，以及我们在分子生物学，电生理学，组织化学和整个动物生理学方面的综合专业知识，以解决以下目标：1）阐明钠通道（Nav）亚基组成和INaP在高与低E2状态下RP 3V Kiss 1神经元中产生爆发放电的贡献。2)采用单细胞RT-PCR和全细胞记录技术研究kisspeptin对弓状POMC和NPY/AgRP神经元的突触后作用。3)利用光遗传学刺激和全细胞记录技术阐明RP 3V Kiss 1神经元对弓状POMC和NPY/AgRP神经元的直接突触输入。4)分别研究选择性化学发生激活或抑制RP 3V Kiss 1神经元对低E2和高E2雌性动物摄食量的体内影响。总的来说，这些实验将允许阐明的离子通道的重要性的起搏器活动在下丘脑Kiss 1神经元，以及如何将其转化为行动内的下丘脑神经回路，不仅是至关重要的能量稳态，但其他自主功能。

 DESCRIPTION (provided by applicant): The long-range goals of the proposed research are to elucidate the molecular and cellular mechanisms by which 17ß-estradiol (E2) regulates hypothalamic kisspeptin (Kiss1) neuronal circuits in females. Kiss1 neurons are a major target of E2 and are essential for pubertal development and adult reproduction. These neurons are also important for sensing and relaying gonadal status to neurons vital for regulation of energy balance, including the hypothalamic arcuate proopiomelanocortin (POMC) and neuropeptide Y/agouti-related peptide (NPY/AgRP) neurons. In spite of the importance of Kiss1 neurons, relatively little is known about the biophysical properties and molecular signature of these cells, as well as the neural circuits by which they operate. We have discovered that the Kiss1 neurons in the rostral periventricular area of the third ventricle (RP3V) express the critical ion channels and receptors that permit spontaneous E2-dependent rebound burst firing. Based on the E2-dependent burst firing characteristics and their projections to arcuate neurons, we propose the novel hypothesis that the RP3V Kiss1 neurons serve as the central pacemaker neurons that provide E2-dependent excitatory drive to the anorexigenic POMC neurons and inhibitory drive to the orexigenic NPY/AgRP neurons. This kisspeptin input to POMC and NPY/AgRP neurons is vital for the estrogenic control of energy homeostasis during the ovulatory cycle in females. Elucidating the cell-specific signaling pathways and gene expression at the single cell level will help in developing new strategies for targeting hormone actions in CNS neurons. Our multidisciplinary approach incorporates a unique set of cellular, molecular, optogenetic and chemogenetic tools and our combined expertise in molecular biology, electrophysiology, histochemistry and whole animal physiology to address the following aims: 1) To elucidate the sodium channel (Nav) subunit composition and the contribution of INaP to the generation of burst firing in RP3V Kiss1 neurons in high- versus low-E2 states. 2) To elucidate the postsynaptic actions of kisspeptin on arcuate POMC and NPY/AgRP neurons using single cell RT-PCR and whole-cell recording. 3) To elucidate the direct synaptic input to arcuate POMC and NPY/AgRP neurons from RP3V Kiss1 neurons using optogenetic stimulation and whole-cell recording. 4) To examine the in vivo effects of selective chemogenetic activation or inhibition of RP3V Kiss1 neurons on food intake in low- versus high-E2 females, respectively. Collectively, these experiments will allow for the elucidation of the ion channels important for the pacemaker activity in hypothalamic Kiss1 neurons, and how this translates into actions within hypothalamic neurocircuits that are vital for not only energy homeostasis but other autonomic functions.