The principal objective of the proposed research is to identify and characterize some critical cellular mechanisms controlling the growth of optic nerve fibers and the formation of retinotectal synapses, using a combination of morphological, physiological, and biochemical techniques. The retinotectal system provides a very favorable model for studying the dynamics of neurite growth and synaptic development at the cellular and molecular levels, and we plan a set of comparative studies in vivo and in vitro in teleosts (goldfish) and mammals (rats). There are three major goals. The first is to investigate the dynamics of neurite growth in vitro. We have recently discovered that retinal neurite growth cones generate steady currents across their tips, and grow in the direction of an applied field. We plan to examine the effects of local electrical events on cell-substrate adhesion, growth, and the movement and assembly of membrane macromolecules, using internal reflectance and video-enhanced microscopy. Electrogenesis of growing neurites will be monitored with conventional microelectrode recording methods and by the use of a voltage-- sensitive dye. The second goal is to study the development of retinotectal synapses using quantitative EM morphometric and three-dimensional reconstruction methods in order to correlate structural changes and changes in the distribution of membrane macromolecules seen using freeze-fracture methods, with the development of synaptic transmission. The third goal is to establish optimal homogenous culture condtions, by isolating different cell types, and by establishing a continuous cell line of retinal ganglion cells. These studies will involve the development of specific antibodies to different cell types, the production of which would be of great use in the future as tools for cell identification and separation, as molecular probes for membrane macromolecules, and in providing an animal model for autoimmune retinopathies. These studies embrace an area of research (retinal regeneration) targeted by a recent report of the National Advisory Eye Council as being of special interest.
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