NEURONAL EXCITABILITY IN THE REGULATION OF CIRCADIAN RHYTHMS

描述（由申请人提供）：视交叉上核（SCN）是哺乳动物生理和行为中驱动每日节律的主要昼夜节律起搏器。SCN神经元利用转录/翻译反馈回路来产生电活动的昼夜变化。尽管自1982年以来我们已经知道SCN神经元在白天放电，在晚上沉默，并且有大量证据表明阈下K+电导，但临界K+电导尚未确定。在最近的研究中，我们发现缺乏Kv4.2（Kv4.2-/-）或Kv1.4（Kv1.4-/-）孔形成（<$）亚基的小鼠比野生型（WT）小鼠具有明显更短的运动（车轮运行）活动的昼夜节律周期。使用体外细胞外微电极记录，我们发现，在缺乏Kv4.2或Kv1.4的SCN神经元中，放电的昼夜节律周期同样缩短。这里的初始实验（目的1）将确定Kv4.2和Kv1.4是否是调节SCN兴奋性的IA通道的唯一Kv亚基，并揭示Kv4.2和Kv1.4的组合损失对SCN放电和运动活动节律的影响。目的2的目标是确定缺乏Kv4.2或Kv1.4的SCN神经元中的昼夜节律放电的较短周期是否反映了IA通道在同步中的功能（即，网络特性）或SCN神经元兴奋性的细胞自主调节。这一目标将首次确立 不同SCN细胞类型中的临界K+电导是否不同。该领域长期存在的争论是，基因表达中昼夜节律的产生是否需要膜电位的每日变化。目的3将直接检验Kv4.2和Kv1.4编码的IA通道介导的兴奋性变化也调节基因表达的昼夜节律变化的周期和幅度的假设。最后，观察到SCN神经元放电和自发活动的周期性变化在Kv1.4或Kv4.2的情况下持续存在（尽管周期较短），表明其他K+电导调节SCN神经元膜电位的日常振荡。在目标4中，我们将利用一种新的、基于Taqman的高通量定量RT-PCR方法来同时定量多个K+通道亚基的表达水平，作为昼夜节律时间的函数，并鉴定介导SCN神经元膜电位每日去极化和超极化的阈下K+电导。这些研究将为特定K+电导在调节SCN神经元兴奋性的日常节律中的作用提供根本性的重要新见解。除了指导进一步研究神经元兴奋性，基因表达和行为中的日常节律的分子，细胞和系统机制外，这些见解将转化为理解昼夜节律调节和失调的进展，以及开发新的治疗策略，以使遭受遗传和环境诱导的昼夜节律中断的个体受益。

DESCRIPTION (provided by applicant): The suprachiasmatic nucleus (SCN) is the master circadian pacemaker driving daily rhythms in mammalian physiology and behavior. SCN neurons utilize a transcription/translation feedback loop to generate circadian changes in electrical activity. Although we have known that SCN neurons fire during the day and are silent at night since 1982 and considerable evidence implicates subthreshold K+ conductance(s), the critical K+ conductance(s) have not been identified. In recent studies focused on testing the hypothesis that subthreshold, A-type (IA) voltage-gated K+ (Kv) channels are involved, we found that mice lacking Kv4.2 (Kv4.2-/-) or Kv1.4 (Kv1.4-/-) pore-forming (¿) subunits have markedly shorter circadian periods of locomotor (wheel running) activity than wild-type (WT) mice. Using in vitro extracellular microelectrode recordings, we found that the periods of circadian rhythms in firing are similarly shortened in SCN neurons lacking either Kv4.2 or Kv1.4. Initial experiments here (aim 1) will determine if Kv4.2 and Kv1.4 are the only Kv ¿ subunits contributing to the IA channels that modulate SCN excitability and reveal the effects the combined loss Kv4.2 and Kv1.4 on rhythms in SCN firing and locomotor activity. The goal of aim 2 is to determine if the shorter period of circadian firing in SCN neurons lacking Kv4.2 or Kv1.4 reflects the functioning of IA channels in the synchronization (i.e., network properties) or the cell-autonomous regulation of SCN neuron excitability. This aim will, for the first time, establish whether the critical K+ conductance(s) in different SCN cell types are distinct. A long-standing debate in the field is whether daily changes in membrane potential are required for the generation of circadian rhythms in gene expression. Aim 3 will test directly the hypothesis that Kv4.2- and Kv1.4-encoded IA channel mediated changes in excitability also modulate the period and amplitude of circadian changes in gene expression. Finally, the observation that the cyclic changes in SCN neuron firing and locomotor activity persist (albeit with a shorter period) in the absence of Kv1.4 or Kv4.2 indicates that other K+ conductances regulate the daily oscillations in SCN neuron membrane potentials. In aim 4, we will exploit a novel, high-throughput quantitative Taqman-based RT-PCR based method to quantify the expression levels of multiple K+ channel subunits simultaneously, as a function of circadian time, and to identify the subthreshold K+ conductance(s) that mediates the daily depolarizations and hyperpolarizations in the membrane potentials of SCN neurons. These studies will provide fundamentally important new insights into the roles of specific K+ conductances in regulating/modulating daily rhythms in the excitability of SCN neurons. In addition to guiding further investigations into the molecular, cellular and systemic mechanisms linking daily rhythms in neuronal excitability, gene expression and behavior, these insights will translate to advances in understanding the regulation and dysregulation of circadian rhythms and to the development of novel therapeutic strategies to benefit individuals suffering genetic and environmentally-induced disruptions in circadian rhythms.