To elucidate mechanisms underlying axon outgrowth in the mammalian CNS, morphological and biochemical studies will be carried out in the corticospinal and callosal pathways that develop postnatally from the hamster sensorimotor cortex. The first goal is to understand the role of axon-target interactions in the survival of axotomized CNS neurons. Axotomy-induced retrograde cellular responses of dye-labeled corticospinal neurons will be studied in vivo and in vitro when target innervation by growing corticospinal axons is experimentally prevented or delayed. What role does innervation of spinal targets play in survival of corticospinal neurons after axotomy? The second goal is to understand how the motile exploratory tips of growing axons, growth cones, interact with their environments during axon guidance. Dissociated cortical neurons growing on tissue sections of hamster cerebral cortex will provide a means to observe CNS growth cone motility on a relevant CNS substrate. High-resolution video microscopy (VEC-DIC) will be used to analyze changes in gowth cone morphologies and behaviors in response to cell surface cues in different regions of CNS tissue of different ages. In a second in vitro preparation, the behavior of fluorescently labeled growth cones in the corpus callosum will be observed directly as growth cones extend in real time within a living slice of the newbom hamster sensorimotor cortex. Laser confocal microscopy will permit an analysis of changes in growth cone morphologies at specific decision regions in the callosal pathway when growth cones must alter their direction of movement. The third goal is to determine the role in axon outgrowth of a novel, developmentally regulated neuron-specific 33 kDa protein. This protein, designated GRP-33 for its growth-relatedness, will be further characterized by determining with gel electrophoresis its mode of axonal transport in the developing corticospinal pathway. Subcellular fractionation will be used to localize GRP-33 to specific subcellular structures and thereby provide insight into its function. The intracellular localization of GRP-33 in growth cones of cultured neurons with EM immunocytochemistry will address its role in neurite extension, and a light microscopic immunocytochemical study of changes in GRP-33 distribution in the developing hamster CNS will shed light on the expression of this protein in relationship to development of different CNS pathways. An elucidation of mechanisms related to axon outgrowth, i.e., the function of growth-related proteins, the behaviors of growth cones during axon guidance decisions, and the role of target innervation in neuron survival after injury are directed toward the goal of promoting regeneration of the injured mammalian CNS.
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