Estradiol modulation of pacemaking kisspeptin neurons

 描述(由申请人提供)：这项拟议研究的长期目标是阐明17？雌二醇(E2)调节女性下丘脑Kispeptin(Kiss1)神经元回路的分子和细胞机制。Kiss1神经元是E2的主要靶点，对青春期发育和成年生殖是必不可少的。这些神经元在感觉性腺状态和将性腺状态传递给对调节能量平衡至关重要的神经元方面也很重要，包括下丘脑弓状原阿片黑素皮质素(POMC)和神经肽Y/刺鼠相关肽(NPY/AgRP)神经元。尽管Kiss1神经元很重要，但人们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(RP3V)吻侧脑室周围区(RP3V)的Kiss1神经元表达关键离子通道 以及允许自发的依赖于E2的反弹爆发放电的受体。基于E2依赖的猝发放电特征及其向弓状神经元的投射，我们提出RP3V Kiss1神经元作为中枢起搏神经元，为厌食性POMC神经元提供E2依赖的兴奋性驱动和对厌食性NPY/AgRP神经元提供抑制性驱动。这种Kispeptin输入到POMC和NPY/AgRP神经元，对于雌性排卵周期中雌激素样的能量稳态控制至关重要。在单细胞水平上阐明细胞特有的信号通路和基因表达将有助于开发新的针对中枢神经系统神经元激素作用的策略。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专业知识，以解决以下目标：1)阐明钠通道(NAV)亚单位的组成以及INAP在高与低E2状态下RP3V Kiss1神经元爆发放电的产生中的作用。2)用单细胞RT-PCR和全细胞记录的方法研究Kispeptin对弓状POMC和NPY/AgRP神经元的突触后作用。3)用光发生刺激和全细胞记录的方法研究RP3V Kiss1神经元对弓状POMC和NPY/AgRP神经元的直接突触输入。4)研究体内选择性化学激活或抑制RP3V Kiss1神经元对雌鼠摄食量的影响。总而言之，这些实验将有助于阐明对下丘脑Kiss1神经元的起搏活动至关重要的离子通道，以及这如何转化为下丘脑神经回路中的动作，这些动作不仅对能量稳态至关重要，而且对其他自主功能也至关重要。

 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.