Somatic Ca2+ Signaling in CNS Neurons

描述（由申请人提供）： 神经元活动产生细胞钙的动态变化，其在包括基因表达在内的许多细胞功能的调节中起核心作用。最近，我们证明，早期发育小脑浦肯野神经元表达的膜到核体细胞钙信号通路，这是由内源性产生的电活动在一个发展的显着阶段，树突状表达之前。我们的初步研究结果表明，这种信号通路的激活产生胞浆和核钙信号，这些信号导致转录因子的激活和下游细胞蛋白质水平的变化。我们假设，内源性产生的活动，驱动这条通路在未成熟的浦肯野神经元是一个发育相关的信号，该活动的一个作用是诱导基因表达的变化，启动下一个发展阶段，表达功能树突。作为检验这一假设的第一步，我们将应用基因阵列微分析急性分离的浦肯野神经元进行各种刺激范式在相关的发展阶段。这种方法将使我们能够同时分析神经元激活对大量已知有助于神经元功能和发育的神经元转录物的影响。通过使用这些信息结合蛋白质表达分析和功能分析，使用电生理和钙成像技术，我们将能够获得钙调节基因表达在特定发育阶段的影响的理解。我们还将确定膜-核体细胞钙信号通路的分子组成和功能特性，并确定基因阵列微分析确定的蛋白质基因表达所必需的条件。从这些研究的结果将直接相关的浦肯野神经元，这已被很好地表征在形态学水平上的发育程序，并涉及不同的和可识别的发展阶段。然而，这些研究确定的一般原则也将大大有助于我们理解其他CNS神经元类型发育过程中活性依赖性基因表达的作用。

DESCRIPTION (provided by applicant): Neuronal activity produces dynamic changes in cellular calcium that play a central role in the regulation of a number of cellular functions including gene expression. Recently we demonstrated that early developing cerebellar Purkinje neurons express a membrane-to-nucleus somatic calcium signaling pathway that is driven by endogenously generated electrical activity at a developmentally significant stage, just prior to dendritic expression. Our preliminary results show that activation of this signaling pathway produces cytosolic and nuclear calcium signals and that these signals result in activation of transcription factors and changes in the level of downstream cellular proteins. We hypothesize that the endogenously generated activity that drives this pathway in the immature Purkinje neurons is a developmentally relevant signal and that one role for the activity is to induce changes in gene expression necessary for initiation of the next developmental stage, expression of functional dendrites. As a first step toward testing this hypothesis, we will apply gene array microanalysis to acutely isolated Purkinje neurons subjected to various stimulation paradigms at the relevant developmental stage. This approach will enable us to simultaneously analyze the effects of neuronal activation on a large number of neuronal transcripts known to contribute to neuronal function and development. By using this information in combination with analysis of protein expression and functional analysis using electrophysiological and calcium imaging techniques we will be able to gain an understanding of the impact of calcium-regulated gene expression at a specific developmental stage. We will also identify the molecular components and functional properties of the membrane-to-nucleus somatic calcium signaling pathway and determine the conditions that are essential for gene expression of proteins identified by the gene array microanalysis. Results from these studies will be directly relevant to the developmental program of Purkinje neurons, which has been well characterized on a morphological level and involves distinct and identifiable developmental stages. However, the general principles identified by the studies will also contribute significantly to our understanding of the role of activity-dependent gene expression during development in other CNS neuronal types.