Calcium Signaling in Suprachiasmatic Nucleus Neurons

描述(申请人提供)：光通过改变两个时钟基因PER1和PER2的表达来改变位于视交叉上核(SCN)神经元的生物钟，从而确保生理过程在一天中的适当时间发生。光信息通过视网膜下丘脑束(RHT)传递到SCN，RHT由含有与SCN神经元突触的光色素黑素的光敏性视网膜神经节细胞组成。RHT进入SCN启动了细胞内的信号级联，最终导致时钟基因表达的改变，但具体的途径仍然知之甚少。在一个模型中，RHT输入激活电压依赖的钙通道，触发细胞内钙释放，可能是通过一氧化氮信号调节的。然而，到目前为止，这些发现还没有直接与时钟基因的表达变化联系在一起。在本提案中，我们将利用一种新的动物模型，该模型使研究单个SCN神经元中PER1的活性依赖诱导成为可能。我们研究的总体目标是了解调节视交叉上核神经元光携带的信号转导通路。根据本研究期间所获得的数据，我们推测在兴奋性谷氨酸或兴奋性GABA能突触传递所触发的动作电位放电过程中，Ca~(2+)主要通过L类电压依赖性钙通道进入SCN神经元，诱导PER1基因表达。四个特定的目标将研究在昼夜节律的不同时间段SCN神经元中钙和PER1基因表达的调节。我们将使用钙成像和单细胞电生理记录方法的创新组合，应用于从表达由PER1启动子驱动的荧光蛋白Venus的转基因小鼠(PER1：Venus)制备的SCN神经元。这些方法将使我们能够监测钙离子浓度和膜电位的变化，同时记录单个SCN神经元中PER1的表达。通过这项研究，我们希望确定光携带途径的组成步骤，更广泛地说，为更好地理解调控活性依赖的基因表达变化的机制。

DESCRIPTION (provided by applicant): Light entrains the circadian clock located in suprachiasmatic nucleus (SCN) neurons by altering the expression of two clock genes, Per1 and Per2, thus ensuring that physiological processes occur at the appropriate time of day. Light information is transmitted to the SCN via the retinohypothalamic tract (RHT), composed of light-sensitive retinal ganglion cells containing the photopigment melanopsin that synapse on SCN neurons. RHT input to the SCN initiates an intracellular signaling cascade that ultimately leads to altered expression of clock genes, but the specific pathways remain poorly understood. In one model, RHT input activates voltage-dependent Ca2+ channels, triggering release of Ca2+ from intracellular stores, possibly mediated through nitric oxide signaling. However, these findings as yet have not been tied directly to altered clock gene expression. In the present proposal, we will take advantage of a new animal model that makes it possible to study activity-dependent induction of Per1 in individual SCN neurons. The overall goal of our research is to understand the signal transduction pathways regulating photic entrainment of suprachiasmatic nucleus neurons. Based on the data obtained during the current funding period, we hypothesize that Ca2+ entering SCN neurons primarily through L-type voltage-dependent Ca2+ channels during action potential firing triggered by excitatory glutamatergic or excitatory GABAergic synaptic transmission induces Per1 gene expression. Four Specific Aims will study the regulation of Ca2+ and Per1 gene expression in SCN neurons during different portions of the circadian day. We will use an innovative combination of Ca2+ imaging and single cell electrophysiological recording methods applied to SCN neurons prepared from transgenic mice (Per1:Venus) expressing the fluorescent protein Venus driven by the Per1 promoter. These methods will allow us to monitor changes in Ca2+ concentration and membrane potential while simultaneously recording Per1 expression in individual SCN neurons. Through this research, we expect to identify the component steps of the light entrainment pathway, and more generally, to provide a better understanding of the mechanisms regulating activity- dependent changes in gene expression.