The long term goal of this project is to determine the extracellular cues and the intracellular signals involved in axonal growth and synaptogenesis. Here we propose to examine in vivo the growth and differentiation of optic nerve axons in the frog Xenopus laevis. Recently developed techniques permit direct microscopic examination of individual optic nerve fibers as they grow into and branch to form terminal arbors within their primary target, the optic tectum. The ability to observe identified axons in vivo offers the opportunity to experimentally dissect the signals that guide axonal growth in a biologically relevant setting. The growth and branching of individual optic nerve fibers will be followed using low light level video microscopy and laser scanning confocal microscopy; in the same axons fluorescent imaging techniques will be used to assay the levels of the intracellular messengers Ca2+ and cAMP. Any correlations between growth events and changes in second messenger levels will be further explored by experimentally perturbing the second messenger levels or metabolism. In a second series of experiments, we will examine the effects of defined growth factors on the growth and arborization of individual optic nerve fibers. BDNF is expected to be the relevant growth factor; we will confirm this by altering the levels of BDNF in the optic tectum (by adding exogenous BDNF, by overexpressing it or by inhabiting its synthesis). Parallel studies will alter the expression of the receptor. Transfection with another growth factor receptor will be used to validate any positive effects.
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