REGULATION OF NEURITE OUTGROWTH AND CONNECTIVITY

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The overall objective of this proposal is to define the intracellular mechanisms that regulate neuronal growth and connectivity.The experiments focus on movements of growth cones at the elongating tips of nerve fibers to reveal mechanisms that govern their function. These structures contain the motile machinery and decision making apparatus involved in neurite elongation, path finding, and synaptogenesis. The hypothesis that will be tested is that alterations in growth cone movements are mediated by changes in internal Ca. The experiments exploit the large identified neurons of the snail Helisoma in culture. These neurons develop large growth cones whose structure and movements can be studied quantitatively. Moreover, identified neurons provide an opportunity to reveal common, as well as unique, regenerative properties based upon study of individual neurons. The first Specific Aim addresses how Ca regulates growth cone movement. Progress over the last four years has established multiple stimuli that have a broad range of effects on growth cones. The effects of graded alterations in growth cone movements caused by these stimuli on internal Ca levels will be tested by imaging growth cones loaded with fura-2. The second Specific Aim focuses on the role of Ca in axonal regeneration using a newly developed preparation that makes it possible to study dynamic events associated with extension of neurites from an axon stump. This preparation duplicates in culture the regeneration that normally occurs in vivo after an axon is damaged. The importance of Ca in allowing conditioning factors to induce outgrowth from the axon stump will be examined. Specific Aim 3 will investigate how changes in the growth cone cytoskeleton are related to changes in growth cone movement.The structure and motility of growth cones depends on the organization of cytoskeletal components.Computer enhanced DIC and fluorescence microscopy will be used to reveal how dynamic changes in the cytoskeleton regulate growth cone structure and function. The hypothesis that changes in internal Ca influence the cytoskeleton and thereby alter growth cone structure and movement will be tested using multiple stimuli that have been identified as growth regulators.