MOLECULAR PHYSIOLOGY OF CIRCADIAN PACEMAKING

许多关键的日常节律是内源性的，由内部主起搏器或生物钟驱动，自由运行周期约为一天（即昼夜节律）。哺乳动物下丘脑的视交叉上核（SCN）是主生物钟的明显位置。最近的研究表明：(1)产生神经生理昼夜节律的能力存在于单个SCN神经元中；(2)在这些神经元中产生节律的基本机制涉及生物钟基因之间的转录-翻译负反馈回路。昼夜神经生物学的一个关键挑战是定义细胞内分子钟与其神经生理输入和输出之间的联系。为此，我们创建了含有昼夜节律报告结构的转基因小鼠，该结构在mPer1启动子的控制下表达可降解形式的GFP。我们将利用双标记免疫细胞化学、膜片钳电生理学、基因表达谱和多电极阵列记录技术，将活体SCN中per1启动子激活的细胞动力学可视化结合起来，以(i)识别光夹带转导的SCN神经元以及在相位重置过程中发生的生理和分子事件；（ii）在SCN切片中测量单个神经元的节律，以提供起搏器结构的新视角；（iii）识别有节律的SCN神经元的特定群体，并检查分子时钟环路输出到细胞膜的机制。这些目标的成功完成将阐明基因调控与SCN昼夜节律功能之间的关键联系。这将增加我们对正常生物钟功能的基本机制的理解，以及我们对生物钟紊乱的疾病过程的理解。

Many critical daily rhythms are endogenous, driven by an internal master pacemaker or biological clock, with a free-running period of about a day (i.e. circadian). The suprachiasmatic nuclei (SCN) of the hypothalamus are the apparent site of the master biological clock in mammals. Recent research has revealed that (i) the ability to generate neurophysiological circadian rhythms resides in single SCN neurons, and (ii) the fundamental mechanisms for rhythms generation within these neurons involve transcriptional-translational negative feedback loops among circadian clock genes. A critical challenge for circadian neurobiology is to define the links between the intracellular molecular clock with its neurophysiological inputs and outputs. Toward this end we have created transgenic mice incorporating a circadian reporter construct which expresses a degradable form GFP under the control of the mPer1 promoter. We will combine our ability to visualize the cellular dynamics of per1 promoter activation in the living SCN with double label immunocytochemistry, patch clamp electrophysiology, gene expression profiling and multielectrode array recording to (i) identify light- entrainment-transducing SCN neurons and the physiological and molecular events which occur in them during the phase-resetting process, (ii) measure individual neuronal rhythms in SCN slices en masse to provide a novel view of pacemaker structure, (iii) identify specific populations of rhythmic SCN neurons and examine the mechanisms by which the molecular clock loop is output to the cell membrane. Successful completion of these aims will elucidate critical links between gene regulation and circadian function of the SCN. This will increase our understanding of the fundamental mechanisms underlying normal biological clock function and our understanding of disease processes in which the clock is deranged.