The differential outgrowth of axons and dendrites results in the development of neuronal polarity, which is essential for the functional connectivity of the nervous system. At a basic level, axonal outgrowth is characterized by rapid rates of elongation and growth cone exploration of the environment (104b), while dendritic growth is orders of magnitude slower. In addition, patterns of branching and the control of process diameter differ dramatically between axons and dendrites (34). There is abundant evidence that microtubules (MTs) are involved in these processes (17, 39, 60, 70, 100, 108). They provide structural support (56), are required for axonal elongation (64, 77, 116), and direct the rapid transport of organelles throughout the processes (100, 107). Despite the acknowledged importance of MTs, their exact role in the growth of axons and dendrites, their mechanism of assembly and transport, and role in growth cone functions are still unresolved. The objective of this proposal will be to determine how MTs contribute to the differential outgrowth of axons and dendrites. Three approaches will be used: 1.) Evaluate the dynamic properties of MTs in axons and dendrites of mature and immature neurons. 2.) Determine the mechanism of MT transport in axons and dendrites, and evaluate changes with maturation 3.) Examine and compare MT behavior in axonal and dendritic growth cones. Hippocampal neuronal cultures, which have well differentiated axonal and dendritic domains and identifiable axonal and dendritic growth cones, will be used for all studies. Microinjection of labeled tubulin will be used to look for rates and sites of tubulin incorporation, sites of nucleation, and MT behavior in growth cones. Transport of MTs will be assessed by injection of caged-fluorescein-labeled tubulin, followed by photoactivation. These issues are significant both for normal neuronal function and for the response of neurons to damage or disease. The development and maintenance of structurally and functionally distinct axonal and dendritic processes underlies nearly every aspect of normal neuronal function. And the properties of the axonal and dendritic cytoskeleton in part determine the responses of axons and dendrites to neuronal injury and their capacity for structural reorganization.
