Calcium Signaling in Suprachiasmatic Nucleus Neurons

描述（由申请人提供）：光通过改变两个时钟基因Per1和Per2的表达来携带位于视交叉上核（SCN）神经元中的昼夜节律钟，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由光敏视网膜神经节细胞组成，这些细胞含有与SCN神经元突触的色素黑视素。RHT输入到SCN启动细胞内信号级联，最终导致改变的时钟基因的表达，但具体的途径仍然知之甚少。在一个模型中，RHT输入激活电压依赖性Ca2+通道，触发Ca2+从细胞内储存释放，可能通过一氧化氮信号传导介导。然而，这些发现还没有直接与生物钟基因表达的改变联系起来。在本提案中，我们将利用一种新的动物模型，使其有可能研究活动依赖性诱导Per1在个别SCN神经元。本研究的总体目标是了解视交叉上核神经元调控光夹带的信号转导通路。基于在目前的资助期间获得的数据，我们假设，Ca2+进入SCN神经元主要通过L型电压依赖性Ca2+通道在兴奋性谷氨酸能或兴奋性GABA能突触传递触发的动作电位放电诱导Per1基因表达。四个特定目的将研究在昼夜节律的不同部分SCN神经元中Ca 2+和Per1基因表达的调节。我们将使用一种创新的钙离子成像和单细胞电生理记录方法的组合，应用于从转基因小鼠（Per1：金星）制备的SCN神经元，表达由Per1启动子驱动的荧光蛋白金星。这些方法将使我们能够监测Ca2+浓度和膜电位的变化，同时记录Per1在单个SCN神经元中的表达。通过这项研究，我们希望确定光夹带途径的组成步骤，更普遍地说，以提供更好地理解调节基因表达的活性依赖性变化的机制。

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.