NEURONAL EXCITABILITY IN THE REGULATION OF CIRCADIAN RHYTHMS

描述(申请人提供)：视交叉上核(SCN)是控制哺乳动物生理和行为日常节律的主要昼夜节律起搏器。SCN神经元利用转录/翻译反馈环来产生电活动的昼夜变化。自1982年以来，虽然我们已经知道SCN神经元白天放电，夜间沉默，并且有相当多的证据表明阈值下K电导(S)，但临界K电导(S)还没有被确定。在最近的研究中，我们发现缺乏Kv4.2(Kv4.2-/-)或Kv1.4(Kv1.4-/-)孔形成亚基的小鼠的运动(轮跑)活动的昼夜周期明显短于野生型(WT)小鼠。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。这里的初步实验(目标1)将确定Kv4.2和Kv1.4是否是调节SCN兴奋性的IA通道中仅有的Kv？亚基，并揭示Kv4.2和Kv1.4的联合丢失对SCN放电和运动活动节律的影响。目标2的目的是确定缺乏Kv4.2或Kv1.4的SCN神经元较短的昼夜节律放电周期是否反映了IA通道在同步过程中的功能(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 不同类型SCN细胞的临界K电导(S)是否存在差异。该领域的一个长期争论是，在基因表达中是否需要膜电位的每日变化来产生昼夜节律。目的3将直接验证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昼夜变化的周期和幅度的假设。最后，观察到在没有Kv1.4或Kv4.2的情况下，SCN神经元放电和运动活动的周期性变化仍然存在(尽管周期较短)，这表明其他K电导调节SCN神经元膜电位的每日振荡。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，作为昼夜时间的函数，并确定调节SCN神经元膜电位每日去极化和超极化的亚阈值K电导(S)。这些研究将对特定钾电导在调节/调节SCN神经元兴奋性的日常节律中的作用提供重要的新见解。除了指导对神经元兴奋性、基因表达和行为中日常节律联系的分子、细胞和系统机制的进一步研究外，这些见解还将转化为在理解昼夜节律的调节和失调以及开发新的治疗策略方面的进展，以造福于遭受遗传和环境诱导的昼夜节律紊乱的个体。

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.