This project uses structural methods to study neurons grown in vitro with the goal of understanding the molecular mechanisms involved in neurite outgrowth and pathfinding. One initiative focuses on the initial outgrowth of neurites from neuronal cell bodies. Neurite formation by isolated peripheral ganglion neurons from chick embryos was examined by time-lapse microscopy with conventional and laser scanning microscopes. Differential interference contrast optics were used to visualize movements of neuronal cytoplasm, as well as movements of small beads attached to the surface membrane, and interference reflection optics were used to monitor the concomitant pattern of adhesion to the substrate (polyornithine or laminin). Related changes in the distributions of specific components of the cytoskeleton were determined by immunofluorescence labeling methods. Neurons grown in normal medium were compared with neurons grown in medium containing drugs that disrupt microtubules or actin filaments. The results provide a comprehensive picture of the cytoskeletal movements and substrate interations that lead to the initiation of neurite outgrowth and suggest a plausible model of the underlying molecular mechanisms. Experiments designed to test this model are in progress. A second initiative examines the adhesive interactions of growth cones with substrates that support their growth in vivo. Retinal axon growth cones growing on substrates consisting of different, naturally-occurring adhesion molecules (laminin, merosin, N-cadherin, or L1) were visualized with time-lapse interference reflection microscopy to determine the distance between the growth cone and the substrate. Growth cones on all substrates formed transient areas of close apposition to the substrate, but the sizes, distributions and lifetimes of these contacts differed. These differences help to explain why growth cones migrate more rapidly on some substrates than others.
