The proposed research would investigate the maturation of growing axons, using mouse cerebellum as a model system, and would address three related issues: 1) the changes in form and cytology during the transition of growth cone to mature synaptic arbor; 2) to what extent the growing tip and mature synaptic bouton reflect the origin of the axon versus the interaction of the growing tip with its microenvironment; (3) to what extent the strategies taken by growing axons in formation of synaptic connection include error, and if so, what rearrangements they make to form proper connections. Once the steps in the formation of specific connections are delineated, the molecular basis for such specificity can in the future be explored. The form of immature axons and growing tips will be characterized during outgrowth towards target structures, during the period axons wait and during the formation of synaptic connections. Axons will be labeled with horseradish peroxidase (HRP) by injection into brain slices, followed by light and electron microscopic analysis. The three dimensional forms of axons and their growing tips will be correlated with their cytology, synaptic patterns and interactions with surrounding cells. The growing tips on axons during outgrowth will be contrasted with those during periods of waiting and synaptogenesis. The relations of waiting axons with target cells will be determined with HRP labeling and specific antisera to synapses and Purkinje cells. Climbing and mossy fibers will be identified by injection of their specific axon pathways or nucleus of origin, and the parameters of the rearrangement of transient connections of supernumerary climbing fiber branches and of axons with both climbing and mossy fiber features will be documented. The effect of deletion of the source of climbing fibers on the maturation of mossy fibers will be assessed. The interaction of axonal growing tips with target cells will be studied by co-culturing cerebellar afferents with purified granule or Purkinje cells.
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