Circadian Clock Cells: Autonomy Persistence and Calcium Dependence

描述（由申请人提供）：哺乳动物生理和行为的日常振荡即使在恒定的环境中也会持续存在，它们的中断会导致时差反应、睡眠障碍和其他疾病，包括情绪障碍。这种“昼夜节律”（约24小时）的节律取决于位于大脑视交叉上核（SCN）中的生物钟。大多数细胞表达“时钟基因”，这是由细胞内时钟组成的转录反馈回路的组成部分，但SCN是主要的起搏器：它可以获得来自视网膜的同步光/暗输入，专门的耦合机制来维持其组成细胞振荡器之间的一致性并增强其稳健性，以及神经元传出投射来同步全身外周组织中的细胞振荡器。然而，最近的工作已经挑战了简单的观点，即SCN神经元都是稳定的、自主的、单细胞转录反馈振荡器。本提案的目的是定义SCN和成纤维细胞生物钟细胞的自主性、持久性和精确性，并探索这些细胞中细胞内转录、电和钙节律的相互依赖性。这将通过机械、药理学和遗传学方法来破坏细胞相互作用和操纵膜电位或细胞内钙。对细胞内生物钟的影响将通过使用光学方法来测量钙和时钟基因转录，以及多电极阵列来监测神经元放电来评估单个细胞。具体来说，我们将测试以下假设：(1)SCN神经元需要来自其他神经元的强直性（而不是节律性）输入来维持节律性；(2)一些SCN神经元的明显非节律性是由于膜超极化而非稳定的非节律亚型的随机事件；(3)细胞需要强直性水平的钙（而不是节律性钙）来维持转录或电节律。这些关于昼夜节律的细胞基础的基本问题的答案对于理解昼夜节律钟如何促进健康和疾病至关重要，并作为新治疗方法的基础。人类大脑中的生物钟记录着一天中的时间，并协调着全身无数的昼夜节律（约24小时）。通过进一步在单细胞水平上描述生物钟的机制，这里提出的实验可能不仅为时差、轮班工作和其他睡眠障碍提供新的治疗方法，而且还为癌症、糖尿病和抑郁症提供了新的治疗方法。

DESCRIPTION (provided by applicant): Daily oscillations in mammalian physiology and behavior persist even in a constant environment, and their disruption leads to jet lag, sleep disorders, and other maladies, including mood disorders. Such "circadian" (ca. 24 hr) rhythms depend on a biological clock located within the brain, in the suprachiasmatic nucleus (SCN). Most cells express "clock genes", components of a transcriptional feedback loop comprising the intracellular clock, but the SCN is the master pacemaker: it has access to synchronizing light/dark input from the retina, specialized coupling mechanisms to maintain coherence among its component cellular oscillators and enhance its robustness, and neuronal efferent projections to synchronize cellular oscillators in peripheral tissues throughout the body. Recent work, however, has challenged the simplistic view that SCN neurons are all stable, autonomous, single cell transcriptional feedback oscillators. The objective of this proposal is to define the autonomy, persistence, and precision of SCN and fibroblast circadian clock cells, and to explore the interdependence of intracellular transcriptional, electrical, and calcium rhythms in these cells. This will be accomplished using mechanical, pharmacologic, and genetic approaches to disrupt cell interactions and manipulate membrane potential or intracellular calcium. Effects on the intracellular circadian clock will be assessed in individual cells by using optical methods to measure calcium and clock gene transcription, and multielectrode arrays to monitor neuronal firing. Specifically, we will test the hypotheses that: (1) SCN neurons require tonic (but not rhythmic) input from other neurons to maintain rhythmicity, (2) apparent non-rhythmicity of some SCN neurons is a stochastic event due to membrane hyperpolarization rather than a reflection of a stable non-rhythmic subtype, and (3) cells require a tonic level of calcium (but not rhythmic calcium) for transcriptional or electrical rhythms. Answers to these fundamental questions about the cellular basis of circadian rhythmicity will be essential for an understanding of how circadian clocks contribute to health and disease, and serve as a basis for novel therapeutic approaches. PUBLIC HEALTH RELEVANCE A biological clock in the human brain keeps track of time of day and orchestrates countless circadian (ca. 24 hr) rhythms throughout the body. By further delineating the mechanisms of this clock at the level of single cells, the experiments proposed here may suggest new therapeutic approaches not only to jet lag, shift work, and other sleep disorders, but also to cancer, diabetes, and depression.