This grant proposes to use newly developed laser-activated micro mass analysis (LAMMA), tissue culture, and patch-clamp techniques to study the physiology of vertebrate photoreceptors and other retinal neurons. LAMMA and intracellular recording will be used to provide more detailed measurements of rod Ca influx and efflux from rods in darkness, to examine the mechanism of light-dependent release of Ca from rods, to measure the time course of Ca re-uptake into rods during dark adaptation and its relationship to photoreceptor sensitivity, and to provide basic information about Ca content and Ca movements in cones. Patch-clamp techniques will be used to characterize outer segment and inner segment membrane channels in intact, isolated rods and to attempt to perfuse the rods internally with various nucleotides and light-activated proteins or enzymes. A combination of histochemical and tissue culture techniques will be used to grow simple goldfish horizontal and ganglion cells in culture and to identify the various morphological classes of cells and the distribution of their post-synaptic receptors. Patch-clamp techniques will be used to investigate the mechanism of the sensitivity of horizontal cells to putative synaptic transmitters, to characterize the membrane conductances of ganglion cells, and to identify and investigate the sensitivities of ganglion cells to classical transmitter substances and peptides. These experiments should contribute basic information about the mechanisms and excitation and adaptation in vertebrate photoreceptors and about the physiology and pharmacology of vertebrate retina.
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