This project investigates the information content of molecules associated with the surfaces over which neuronal growth cones navigate in the normal course of development. The strategy is to employ the neuronal growth cone itself as an assay for the molecular terrain over which it will travel. Research will examine the behavior, structure, free intracellular calcium concentrations, and the activities of the various components involved in calcium homeostasis in these growth cones as they encounter specific substrate molecules. Initial studies will examine the role of particular molecules in the context in which they are normally found by using cryostat sections of denervated muscle. Dissociated neurons will be plated onto these sections, and the behavior, structure, and calcium levels of advancing growth cones will be measured as these growth cones encounter vacated neuromuscular junctions. Then, individual purified molecules (including S-laminin, laminin, fibronectin, L1, J1) will be bound either to culture surfaces, in a patterned fashion, or to microspheres and tested for effects on growth cones. By experimentally provoking encounters between the growth cone and these surface molecules, one can investigate the information content of these individual molecules. Finally, this work will recreate more complex and realistic terrains by a novel experimental approach that employs quantitative combinations of surface associated molecules. Individual molecules will be bound to gold particles. By mixing different gold particles solutions, each containing different molecules, and depositing the solutions onto culture surfaces with defined ratios of specific molecules will be produced. These surfaces will be assayed by encounters with growth cones. This will allow us to examine how the growth cone integrates multiple to dissect the role of particular molecules. Taken together, these sets of data will provide information on a presently unexplored area of great importance in neurogenesis. While we have considerable information about the existence of particular molecules and considerable knowledge about the motility and behavior of growth cone,s little is known about the specifics of interactions between growth cones and surface molecules. This proposal places us in the unique position to make use of the neuronal growth cone as an assay for important molecular features the undoubtedly play major roles in establishing neuronal connectivity.
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