Cellular Basis of Circadian Rhythms in Mammals

描述(申请人提供)：哺乳动物视交叉上核(SCN)是日常行为和生理节律所必需的昼夜节律起搏器。在体内和体外，SCN内的多个昼夜节律振荡器彼此同步，以维持近24小时的节律。目前尚不清楚这种节律性是否是特殊的SCN神经元所固有的，也不清楚是什么机制结合了它们的昼夜节律。此外，最近的分子证据表明，其他组织也可以作为昼夜节律振荡器，但其节律性的基础和在行为中的作用尚不清楚。拟议的研究直接利用了长时间记录技术--多电极阵列和基因活动的生物发光报告--以及与昼夜节律有关的基因突变的小鼠和仓鼠的独特特性，直接解决了这些问题。第一个特定的目的是测试假设，即单个SCN神经元是自主的昼夜节律起搏器，以及起搏神经元是SCN神经元的一个小子集。其策略是表征完全分离的SCN神经元的节律能力，然后在固定后，确定它们的神经化学含量。 特殊目的2验证了SCN神经元通过血管活性肠多肽而不是快速突触通讯相互同步的假说。利用特定的拮抗剂、激动剂和基因敲除，这个目标补充了第一个目标，即识别起搏器及其协调活动所需的信号。最近在许多哺乳动物组织中发现了假定的昼夜节律振荡器，这导致了昼夜节律系统是按等级组织的假设。特定的AIMS 3和4将决定主嗅球(OB)的昼夜节律的功能和分子基础，OB是一个模型昼夜节律振荡器，将与SCN进行比较。利用行为学、解剖学和生理学的方法对OB的节律性进行分析，这些目的将为OB中的时钟建立一个活体作用。此外，他们将直接测试这样的假设，即至少有一个参与昼夜节律计时的基因在SCN和OB之间存在差异。这些实验将首次识别大脑两个区域的昼夜节律起搏器，协调其整体节奏的机制，以及它们在行为中扮演的不同角色。

DESCRIPTION (provided by applicant): The mammalian suprachiasmatic nucleus (SCN) is a circadian pacemaker required for daily rhythms in behavior and physiology. In vivo and in vitro, multiple circadian oscillators within the SCN synchronize to each other to sustain near 24-h rhythms. It is presently unclear if this rhythmicity is intrinsic to a specialized population of SCN neurons or what mechanisms couple their circadian rhythms. In addition, recent molecular evidence suggests that other tissues can act as circadian oscillators, but the bases for their rhythmicity and roles in behavior are unknown. The proposed studies directly address these issues by taking advantage of long-duration recording technologies--multielectrode arrays and bioluminescent reporters of gene activity--and the unique properties of mice and hamsters with mutations in genes involved in circadian timekeeping. The first Specific Aim tests the hypotheses that individual SCN neurons are autonomous, circadian pacemakers and that the pacemaking neurons are a small subset of SCN neurons. The strategy is to characterize the rhythmic ability of fully isolated SCN neurons and then, after fixation, their neurochemical content. Specific Aim 2 tests the hypothesis that SCN neurons synchronize to each other via vasoactive intestinal polypeptide and not fast synaptic communication. Using specific antagonists, agonists, and genetic knockouts, this aim complements the first aim in identifying pacemakers and the signals required for their coordinated activity. The recent discoveries of putative circadian oscillators in many mammalian tissues have led to the hypothesis that the circadian system is hierarchically organized. Specific Aims 3 and 4 will determine the function and molecular basis for circadian rhythms in the main olfactory bulb (OB), a model circadian oscillator to be compared to the SCN. Using behavioral, anatomical and physiological assays for rhythmicity in the OB, these aims will establish an in vivo role for the clock in the OB. Furthermore, they will directly test the hypothesis that at least one of the genes involved in circadian timekeeping differ between the SCN and OB. These experiments will, for the first time, identify circadian pacemakers in two brain areas, the mechanisms that coordinate their ensemble rhythms, and the distinct roles they play in behavior.