This project examines cellular and molecular mechanisms involved in outgrowth of processes from neurons with the ultimate goal of understanding the determinants of neuronal branching patterns and synaptic connections. Current work focuses on the initial formation of axons by dorsal root and sympathetic ganglion neurons grown in vitro. The sequence of changes in neuronal shape and movements of the cytoskeleton during the outgrowth of axons was examined with high resolution videomicroscopy and fluorescence labeling methods. Neurons initially formed multiple filopodia which contained actin but not microtubules or neurofilaments. Formation of axons entailed movement of cytoplasm containing microtubules and neurofilaments from the cell body into filopodia. Neurons grown on substrates coated with a molecule from the extracellular matrix formed multiple axons. However, neurons grown on a synthetic substrate formed axons only if their filopodia contacted an adhesive object such as an adjacent cell or a latex bead coated with adhesive molecules. Paradoxically, beads coated with synthetic substrate molecules triggered conversion of filopodia into axons,indicating that the initiation of axon outgrowth is not dependent on the presence of a specific type of adhesion molecule. Studies with laser scanning reflection microscopy resolved this paradox by showing that neurons adhere more closely to synthetic substrates than to substrates coated with extracellular matrix molecules and that filopodia that contact a cell or coated bead lift off the substrate before they begin to fill with cytoplasm. Movements of beads on the surfaces of neurons were correlated with movements of their cytoplasm. The results of these experiments provide insight into the relationships between adhesion of a neuron to a substrate, movement of cytoplasm within neurons, and formation of neuronal processes.
