The principal objective of the proposed research is to identify and characterize some critical cellular mechamisms controlling the growth of optic nerve fibers and the formation of retinotectal synapses. There are four major goals. The first is to investigate the role of calcium in growth of retinal ganglion cells. We have recently discovered that retinal neurite growth cones generate steady Ca2+ currents across their tips, and grow in the direction of an applied field. We plan to measure calcium dynamics in the growth cones of retinal ganglion cells of goldfish, frogs, and rates in culture using a calcium sensitive dye and a new fast digital imaging camera. Using vibrating probe and patch clamp recordings to examine macroscopic and microscopic ion currents, we will determin whether variations in intraterminal calcium levels affect the conductrances of other ion channels; and, using video-enhanced microscopy, we will determine whether this variation is associated with morphological changes in the growth cone and with changes in the rate of growth. The second goal is to examine the effect of electric fields on the growth of optic nerve fibers. We plan to determine the effects of fields on the rate of growth of retinal ganglion cell axons, and to correlate this with changes in the rate of axoplasmic transport and with changes in the calcium dynamics of growth cones. The third goal is to examine synaptic interactions between retinal ganglion cell terminals and tectal interneurons. We will employ voltage sensitive dyes to determine detailed synaptic interactions between developing and regenerating retinal ganglion cells and interneurons in the tectum, including the isthmo-tectal input, which appears to modulate retinotectal synaptogenesis. We will use a new method to examine changes in the expression of specific proteins that occur during retinoctectal synaptogenesis in single isolated tectal neurons. These studies should provide detailed insights into molecular mechamisms that determine how and where retinal neurites grow, and how specific connections form between different cells in the visual system. This information is of paramount importance in understanding the normal development and function of the visual system, as well as its response to injury.
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