Neurobiology of the Circadian Clock

项目摘要 神经科学中的一个基本问题是基因表达的变化如何转化为神经系统的变化。 神经生理学，并最终转化为行为的变化。大脑的24小时计时机制，或者 生物钟是一个独特的适合于研究神经可塑性和基因-行为的系统， 问题.神经网络产生和驱动生理和行为的昼夜节律， 位于下丘脑的视交叉上核（SCN）内。SCN神经元表现出内源性 自发尖峰频率的昼夜节律，即使是孤立的单细胞，在这些神经元内， 一个确定的“时钟基因”网络，形成自动调节转录/翻译反馈环（TTFL） 产生接近24小时的节律。 关于SCN夹带和起搏器的机制，我们的知识存在关键空白 可塑性是由它引起的。与节律生成不同，实时动态SCN分子和神经网络 在夹带期间的响应以前是不可观察的，因此关于 在这些水平上夹带的实际动态拓扑。我们已经开发并建立了新的方法 使得能够使用一种方法直接观察SCN分子和神经网络夹带拓扑， 它将时钟神经元电活动的ChrimsonR光遗传学操纵与PER 2：：LUC真实的- 生物钟基因激活的时间报告（体外循环计时和夹带，EXCITE）。EXCITE提供 对隔离SCN的重复输入刺激的精确定时、持续时间和强度，并跟踪SCN时钟 在离体3-5周内以高时间和空间分辨率观察分子节律。其中的孤立SCN 系统惊人地概括了完整动物昼夜节律钟夹带的典型特征，包括 具有周期后效、周期匹配和系统相角差的类光相位响应， 偏离24小时的刺激，以及对具有最小可耐受夜间的光周期的差异诱导。 我们将使用EXCITE来检查-（1）SCN夹带的分子和神经网络拓扑， 可塑性，（2）夹带的SCN神经网络拓扑结构，和（3）光周期的分子机制。 诱导SCN网络可塑性。这些目标的成功实现将为SCN提供新的见解 夹带和可塑性-SCN分子和神经网络如何被光输入修改， 行为可塑性定义SCN编码光历史和光周期的机制将 为操纵SCN神经和转录网络以改善昼夜节律紊乱开辟了道路。

PROJECT SUMMARY A fundamental question in neuroscience is how changes in gene expression are translated into changes in neuronal physiology, and ultimately into changes in behavior. The brain’s 24-hour timing mechanism, or biological clock, is a system that is uniquely suited to the study of neural plasticity and the genes-to-behavior problem. The neural network that generates and drives circadian rhythms in physiology and behavior is located within the suprachiasmatic nuclei (SCN) of the hypothalamus. SCN neurons exhibit endogenous circadian rhythms in spontaneous spike frequency, even as single cells in isolation, and within these neurons is a defined network of “clock genes” that forms autoregulatory transcription/translation feedback loops (TTFLs) to generate near 24-hour rhythms. There are key gaps in our knowledge regarding the mechanisms of SCN entrainment and the pacemaker plasticity it induces. Unlike rhythms generation, real-time dynamic SCN molecular and neural network responses during entrainment have been previously un-observable, and thus knowledge is limited regarding the actual dynamic topologies of entrainment at those levels. We have developed and instituted novel methods enabling direct observation of SCN molecular and neural network entrainment topologies using an approach that combines ChrimsonR optogenetic manipulation of clock neuron electrical activity with PER2::LUC real- time reporting of clock gene activation (EX vivo CIrcadian Timing and Entrainment, EXCITE). EXCITE provides precise timing, duration and intensity of recurring input stimulation to the isolated SCN, and tracks SCN clock molecular rhythms at high temporal and spatial resolution for 3-5 weeks ex vivo. The isolated SCN in this system strikingly recapitulates canonical features of circadian clock entrainment in intact animals, including light-like phase responses with period after-effects, period matching and systematic phase angle differences to stimuli that deviate from 24 hours, and differential entrainment to photoperiods with a minimum tolerable night. We will use EXCITE to examine - (1) Molecular and Neural Network Topologies for SCN Entrainment and Plasticity, (2) SCN Neural Network Topology of Entrainment, and (3) Molecular Mechanisms of Photoperiod- Induced SCN Network Plasticity. Successful completion of these aims will provide novel insight into SCN entrainment and plasticity - how the SCN molecular and neural networks are modified by light input to result in behavioral plasticity. Defining the mechanisms by which the SCN encodes light history and photoperiod will open the way for manipulation of SCN neural and transcriptional networks to ameliorate circadian disorders.