NEURONAL EXCITABILITY IN THE REGULATION OF CIRCADIAN RHYTHMS

描述（由申请人提供）：视交叉上核（SCN）是主要的昼夜节律起搏器，驱动哺乳动物生理和行为的日常节律。SCN神经元利用转录/翻译反馈回路产生电活动的昼夜变化。尽管自1982年以来我们已经知道SCN神经元在白天放电而在夜间沉默，并且大量证据表明阈下K+电导(s)，但临界K+电导(s)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-/-）孔隙形成（¿）亚基的小鼠的运动（轮跑）活动的昼夜节律周期明显短于野生型（WT）小鼠。通过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（目的1）将确定Kv4.2和Kv1.4是否是唯一参与调节SCN兴奋性的IA通道的Kv亚基，并揭示Kv4.2和Kv1.4的联合损失对SCN放电和运动活动节律的影响。目的2的目的是确定缺乏Kv4.2或Kv1.4的SCN神经元中较短的昼夜节律放电周期是否反映了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

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.