Precise neuronal circuitry underlies function of the nervous system. The vast majority of literature on this topic ascribes unique functions to neuronal growth cones pathfinding and synaptogenesis. Recently, branching along neurites and extension of the new neurite into appropriate regions has been proposed to serve an additional and even a possibly dominant mechanism in neuronal pathfinding. Additionally, we previously demonstrated that cues may be sent from the lead growth cone back along its trailing neurite which lead to defasciculation, enabling their separate search for target areas. These mechanisms enable axons to extend to their appropriate target locations. We developed a system that allows these new mechanisms of axonal guidance to be investigated with a high degree of experimental tractability and promises to allow direct understanding of key, intracellular events underlying this process. The underlying hypothesis of this proposal is that environmental cues induce changes in intracellular messenger systems that initially affect filopodial and lamellipodial changes in the lead growth cone, then subsequently induce changes that underlie the phenomenon of en passant branching described from fixed images of events in vivo. Our first set of investigations will focus on the influence of the immediate neuronal extracellular environment on branching and defasciculation and the second set of experiments will investigate the intracellular signals which induce axonal branching and defasciculation after pioneer growth cone collapse.
Aim number 1) We will investigate how a number of different, specific environmental cues influence the formation and maintenance of branches and defasciculation after growth cone collapse. We will examine target cells or tissue, electrical activity of extending axons, repellent cells or underlying target cells, and brain derived neurotrophic factor applied at either the source or the target.
Aim number 2) We will examine intracellular signals that link growth cone collapse to the induction of branches and defasciculation. In particular, small RhoGTPases, an adapter protein, intracellular calcium and cAMP will be examined. The purpose of these experiments is to better understand what the intracellular events are that carry out the growth cone collapse-induced formation of branches and defasciculation. Together the results will lend valuable insight into fundamental mechanisms that regulate neuronal development.