The severing of retinal ganglion cell axons in man leads to permanent blindness. In the goldfish, however, such injury initiates a remarkable regenerative process during which axons of retinal ganglion cells grow back into the optic tectum with the formation of new synapses and the recovery of sight. During this regeneration, ganglion cells show marked increases in new protein biosynthesis while glia of the optic tract proliferate. I propose a Target Regulation Hypothesis which suggests that retinal ganglion cells are stimulated in part by a contact with the synaptic target site and in part by an association with """"""""activated"""""""" glia. I will use biochemical and morphological markers in order to characterize the signals which stimulate ganglion cells and to investigate what influence activated glia may have upon regenerating neurons. A population of retinal ganglion cells will be obtained by microdissection. Seven different surgical preparations will be employed in an effort to uncover putative influences upon these cells or upon glia of the optic tract. The response of isolated retinal ganglion cells during regeneration will be monitored by examining biosynthetically labeled proteins using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), by quantitating lectin binding to plasma membranes, by observing changes in cell surface antigenic sites, by monitoring axonal growth in vitro, and by testing the specificity of cell aggregation. The biologic response of specific glial cell populations will be monitored by cell counts using immuno-histochemical markers, by quantitating incorporation of radiolabeled precursors using electron microscopic autoradiography, by measuring the activity of the myelin membrane enzyme 2':3'-cyclic nucleotide, 3'-phosphohydrolase, and by analyzing specific biosynthetically labeled proteins using SDS-PAGE. Application of these techniques to tissues recovered from the various surgical preparations will allow identification of cell populations that help to regulate growth of retinal ganglion cells. Conventional biochemical methods will be used to isolate soluble and membrane-associated factors that stimulate ganglion cells in vitro. In this way I hope to further our understanding of the molecular basis for a regeneration of the visual systems that leads to recovery of sight.
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