| The formation of precise neuronal connections during development is initiated by correct pathfinding of the tip of the growing axon, the growth cone, in response to attractive and repulsive guidance cues that are either diffusible or substrate-bound. By establishing a reproducible gradient of diffusible guidance cues, I have quantitatively examined the molecular and cellular mechanisms underlying the turning response of growth cones of cultured Xenopus spinal neurons.; Using molecular and pharmacological approaches, I found that many diffusible guidance cues share same downstream signaling mechanisms and can be divided into two groups: Group I factor-induced turning responses are cAMP- and calcium-dependent, and require co-activation of PLC-γ and PI3K pathways; Group II factor-induced turning responses are cGMP-dependent. Furthermore, the growth cone response to a particular guidance cue depends on the internal state of the neuron, which, in turn, is under influence of other coincident signals. I found that brief periods of electrical stimulation resulted in a marked alteration in the growth cone turning response in gradients of Group I factors. The effect appears to be mediated by an elevation of both calcium and cAMP. Interestingly, the repulsion induced by inhibitory molecules associated with myelin of the central nervous system (CNS), is converted to attraction by electrical stimulation or elevating cytoplasmic levels of cAMP. Thus, manipulations to increase cytoplasmic cAMP levels, such as intermittent electrical stimulation, may provide a means to promote nerve regeneration after CNS injury.; Analogous to chemotaxis of microorganisms, I found that the neuronal growth cone exhibits adaptation in its chemotactic migration, undergoing consecutive phases of desensitization and resensitization in the presence of increasing concentrations of a guidance cue. The desensitization is accompanied by a reduction of calcium signaling induced by the guidance cue, whereas the resensitization requires MAPK activation and local protein synthesis. Such adaptive behavior allows the growth cone to readjust its sensitivity over a wide range of concentrations of guidance cues and is an essential feature for long-range chemotaxis. |
