MECHANISM OF NERVE GROWTH AND NAVIGATION

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Understanding the mechanism of axonal pathfinding is a central issue in developmental neurobiology. The work outlined in this proposal is focused on the molecular and cellular mechanisms underlying the growth and navigation of nerve processes. The experimental system l will use combines in vitro study of Xenopus neurons with state-of-the-art imaging molecular manipulation techniques in the laboratory of Dr. Mu-ming Poo. The main question addressed in this proposal is how second messengers and cytoskeletaI proteins are involved in directing growth cone motility. There are two specific aims of this proposal. In AIM 1, I will determine the role of Ca2+ and cAMP as second messengers in regulating the rate and direction of neurite extension under different environmental conditions. This will be assayed by measuring [Ca2+]i at regular time intervals using the standard fura-2 ratio imaging method and measuring the rate of neurite extension by recording bright-field images using time-lapse video recording. Whether cytoplasmic Ca2+ or cAMP gradient is sufficient in inducing growth cone turning will be tested by using focal UV-activated release of Ca2+ and cAMP from caged compounds at the growth cone. Whether Ca2+ and cAMP-dependent pathways interact with each other will also be examined. In AIM 2, I will determine the early cellular events associated with the growth cone turning induced by three different chemotropic gradients (glutamate, forskolin, and NT-3). This will be accomplished by analyzing the filopodia activity, microtubule/microfilament polymerization/depolymerization and distribution of gelsolin, an actin binding protein, using combined DIC and fluorescence imaging methods. Completion of these two aims will provide insight into molecular events that precede growth cone turning. These results will both advance our understanding of neurodevelopment as well as provide conceptual framework for designing means to direct growth of regenerating axons after nerve injury.