? 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. ? ?
