Linking Molecular and Electrical Rhythms in the Brain's Biological Clock

描述（由申请人提供）：了解神经系统中基因表达和电活动之间的相互作用是神经科学中的一个基本问题，对突触可塑性和神经发育等领域具有广泛的影响。研究这种相互作用的一个有用的模型系统是大脑的生物钟，视交叉上核（SCN）。SCN产生内源性分子和电昼夜节律，其一起调节多种生理过程。昼夜节律紊乱有可能影响一系列人类健康状况，如睡眠和代谢紊乱以及自闭症和抑郁症等神经精神疾病。要了解生物钟组件的失调如何导致疾病，首先需要了解它们在健康大脑中的工作方式。昼夜神经生物学中一个关键的未解决的问题是SCN的分子和电节律如何相互作用以形成连贯的起搏器。SCN神经元是自主振荡器，其具有涉及核心时钟基因Per 1/2、Cry 1/2、Bmal 1和Clock的日常分子转录-翻译反馈回路。这些神经元可以自发地激发动作电位，重要的是，可以调节它们的放电速率，使它们在白天快速放电，在晚上缓慢放电。到目前为止，只有间接证据将分子反馈回路与SCN电活性联系起来。这项研究计划提出了一种独特的策略，结合电生理学，实时时钟基因表达成像和放电率的光遗传学操纵，以阐明SCN中基因表达和放电率节律之间的联系。具体地说，电生理记录将结合实时时钟基因表达成像在一个新的转基因小鼠系（Per 1：GFP小鼠），其中荧光是一个读出的核心时钟基因Per 1的转录激活，以确定固有的相位之间的关系发射率和Per 1启动子活性。这些技术也将用于Per 1被敲除或过表达的动物，以研究Per 1本身是否是分子钟和电活动节律之间的功能联系。相反，放电频率节律对昼夜节律基因的影响 将通过在表达SCN-特异性光门控离子通道的Per 1：GFP小鼠中使用SCN-特异性光遗传学操作的发射率和时钟基因表达的延时共聚焦成像的组合来检查表达。这些技术将被用来确定如何增加，减少，或消除放电率节律改变基因表达的SCN的节奏。这项研究计划最终将有助于阐明SCN基因表达与电活动节律之间的关系，这将提高我们对基本生物钟组件相互作用的理解，这些组件的功能障碍可能对人类健康产生负面影响。

DESCRIPTION (provided by applicant): Understanding the interaction between gene expression and electrical activity in the nervous system is an essential problem in neuroscience that has broad impacts on such areas as synaptic plasticity and neurodevelopment. A useful model system to study this interaction is the brain's biological clock, the suprachiasmatic nucleus (SCN). The SCN generates endogenous molecular and electrical circadian rhythms that together regulate a multitude of physiological processes. Disrupted circadian rhythms have the potential to influence a range of human health conditions such as sleep and metabolic disorders and neuropsychiatric illnesses such as autism and depression. To understand how the dysregulation of circadian clock components may lead to disease, it is first necessary to understand how they work in a healthy brain. A key unsolved question in circadian neurobiology is how the SCN's molecular and electrical rhythms interact to form a coherent pacemaker. SCN neurons are autonomous oscillators that possess daily molecular transcriptional- translational feedback loops involving the core clock genes Per1/2, Cry1/2, Bmal1, and Clock. These neurons can spontaneously fire action potentials, and, importantly, can modulate their firing rates so that they fire quickly during the day and slowly at night. So far, there has only been indirect evidence connecting the molecular feedback loop and SCN electrical activity. This research plan proposes a unique strategy that combines electrophysiology, real-time clock gene expression imaging, and the optogenetic manipulation of firing rate to elucidate the link between gene expression and firing rate rhythms in the SCN. Specifically, electrophysiological recording will be combined with real-time clock gene expression imaging in a novel transgenic mouse line (Per1:GFP mice) in which fluorescence is a readout of transcriptional activation of the core clock gene Per1 to determine the inherent phase relationship between firing rate and Per1 promoter activity. These techniques will also be used in animals in which Per1 is knocked out or overexpressed to investigate whether Per1 itself is a functional link between the molecular clock and electrical activity rhythms. Conversely, the influence of firing rate rhythms on circadian gene expression will be examined by using a combination of SCN-specific optogenetic manipulation of firing rate and time-lapse confocal imaging of clock gene expression in Per1: GFP mice expressing SCN-specific light-gated ion channels. These techniques will be used to determine how increasing, decreasing, or eliminating firing rate rhythms alter gene expression rhythms in the SCN. This research plan will ultimately help elucidate the relationship between SCN gene expression and electrical activity rhythms, which will improve our understanding of the interplay of essential circadian clock components whose dysfunction can negatively impact human health.