9728728 HOLLIDAY There is much evidence indicating that the development of undifferentiated cells into neurons is dependent both upon intrinsic patterns of gene expression as well as on interactions with neighboring cells, including electrical signaling. Early in neuronal differentiation, the elevation of intracellular calcium ions carries much of the electrical signal. Calcium elevation is one of the first steps in the activation of a large variety of cellular signal transduction cascades in all cell types and is therefore very likely to be involved in regulating gene expression and other "experience-dependent" mechanisms that regulate development. There is accumulating evidence that precisely timed changes in the cellular mechanisms that produce transient increases in intracellular calcium may underlie the "critical periods" of neuronal development. This project is designed to determine how the alteration of the level of expression of specific calcium ion regulatory proteins affects transient calcium signaling and subsequent neuronal development. Two calcium regulatory proteins will be separately investigated: a calcium buffering protein (calbindin) and an intracellular calcium induced calcium release channel (CICR). The level of these proteins will be experimentally manipulated by the introduction DNA constructs that either produce additional target protein or specifically reduce endogenous target protein expression. The experiments will be performed in a well- controlled and characterized culture system of rat cerebellar granule neurons. Since intracellular calcium homeostasis is tightly controlled by a variety of mechanisms, alteration of one target may trigger compensatory changes in other regulatory mechanisms. Therefore, the physiological consequences of changing protein expression will be measured with imaging techniques that directly measure intracellular calcium ion concentrations, both at rest and during stimulation. The developmental consequences of changes in target protein expression will be assessed by measuring the acquisition of several developmental markers. The results of these studies will refine our understanding of how calcium transients regulate neuronal development at the single cell level, and develop tools to more definitively test hypotheses about the mechanisms regulating neuronal synaptic organization. The long-range goal of this project is to transiently manipulate calcium regulatory mechanisms during development in vivo to determine whether perturbations of calcium levels, during sensitive defined in vitro, will result in developmental delays or permanent changes in the mature central nervous system.
