Intracellular free Ca2+ concentrations determine several important functions in retinal neurons including neuronal development and differentiation, synaptic signaling, neurotransmitter release, gene expression and neurodegeneration. The application's broad, long-term objectives are to understand the contribution of intracellular Ca2+ channels (ICCs) to the function of retinal neurons and to identify the regulation of these proteins as potential targets for the treatment of neurodegeneration in the retina. The central hypothesis of the present application is that ICCs determine (a) physiological and pathophysiological processes in retinal neurons, and (b) functional properties of retinal neurons through their differential distribution and their interaction with regulatory factors.
The specific aims for testing this hypothesis in neurons of the mammalian retina are: 1) to determine the subcellular localization of ICCs and of their signaling partners in the retina; 2) to analyze the biophysical and pharmacological characteristics of ICCs and their regulatory interactions with other proteins in the retina; 3) to identify and measure the contribution of ICCs to intracellular Ca2+ signaling of retinal neurons.
In Aim 1, experiments will identify the structure and functional organization of intracellular Ca2+ signaling mechanisms with respect to glutamatergic synapses in the retina. ICCs and functionally associated proteins will be localized using immunocytochemistry, confocal laser scanning and electron microscopy.
In Aim 2, the mechanisms of action and regulation of ICCs will be determined with single channel electrophysiology. The contribution of ICCs to intracellular Ca2+ signaling in glutamatergic retinal neurons will be measured by experiments in Aim 3 using imaging of intracellular Ca2+ concentrations, and confocal laser scanning microscopy.
Both Aim 2 and 3 will lay the groundwork for a possible pharmacological modulation of ICCs and the control of intracellular Ca2+ concentration that are essential for the development of treatments of neurodegenerative processes in the retina such as glaucoma, ischemia and photoreceptor degeneration.
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