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

描述(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产生内源性的分子和电的昼夜节律，共同调节多种生理过程。被打乱的昼夜节律可能会影响一系列人类健康状况，如睡眠和代谢障碍，以及神经精神疾病，如自闭症和抑郁症。要了解生物钟成分的失调如何导致疾病，首先必须了解它们在健康的大脑中是如何工作的。昼夜节律神经生物学中一个关键的悬而未决的问题是，SCN的分子和电节奏如何相互作用，形成一个连贯的起搏器。SCN神经元是一种自主振荡器，每天都有分子转录-翻译反馈环，涉及核心时钟基因PER1/2、Cry1/2、BMal1和Clock。这些神经元可以自发地发射动作电位，更重要的是，可以调节它们的放电频率，以便它们在白天快速放电，在晚上缓慢放电。到目前为止，只有间接证据表明分子反馈环与SCN的电活动有关。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对放电频率的光遗传操作来阐明SCN中基因表达和放电频率节律之间的联系。具体地说，电生理记录将与实时时钟基因表达成像相结合，在一种新型转基因小鼠系(PER1：GFP小鼠)中，荧光是核心时钟基因PER1转录激活的读数，以确定激发速率和PER1启动子活性之间的内在相位关系。这些技术还将用于PER1被敲除或过度表达的动物，以研究PER1本身是否是分子时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 在表达SCN特异性光门离子通道的PER1：GFP小鼠中，将结合使用SCN特异性光遗传操作的激发速率和时钟基因表达的延时共聚焦成像来检测其表达情况。这些技术将被用来确定增加、减少或消除放电频率节律如何改变SCN中的基因表达节律。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，这将有助于我们更好地理解基本生物钟组件的相互作用，这些组件的功能失调会对人类健康造成负面影响。 与公共健康相关：大脑的生物钟-视交叉上核(SCN)-产生昼夜节律，调节一系列生理过程，包括日常/睡眠唤醒周期、体温、摄食行为和内分泌活动。被打乱的昼夜节律有可能影响人类的一系列健康状况，如睡眠和代谢紊乱，以及神经精神疾病，如自闭症和抑郁症。在这里，我使用一种独特的策略，结合电生理学、实时时钟基因表达成像和光遗传学来阐明SCN中固有的基因表达和电活动节律之间的联系，这将提高我们对其失调可能导致疾病的基本生物钟成分的理解。

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. PUBLIC HEALTH RELEVANCE: The brain's biological clock - the suprachiasmatic nucleus (SCN) - generates circadian rhythms that regulate a multitude of physiological processes, including the daily/sleep wake cycle, body temperature, feeding behavior, and endocrine activity. Disrupted circadian rhythms have the potential to influence a large range of human health conditions such as sleep and metabolic disorders and neuropsychiatric illnesses such as autism and depression. Here, I use a unique strategy that combines electrophysiology, real-time clock gene expression imaging, and optogenetics to elucidate the link between the inherent gene expression and electrical activity rhythms in the SCN, which will improve our understanding of essential circadian clock components whose dysregulation may lead to disease.