This proposed research will study the interactions of axonal growth cones and their synaptic target neurons, in order to address the cellular mechanisms that direct and accompany synaptogenesis. Recent evidence from our laboratory suggests that a reciprocal relationship exists between afferent axon and target cell regulation of the growth of afferents by target cells, and the regulation of target cell differentiation by afferents. This relationship is the theme of this proposal. We will use the developing mouse cerebellum and analyze intact brain and tissue culture models, by static and dynamic microscopy. To address how the growth of afferents is influenced by target cells, we will examine the coordination of timing of afferent ingrowth with target cell availability in vivo. After dye labeling afferent axons, the cell contacts of mossy fibers will be analyzed in two situations where mossy fibers arrive before granule cells are present: vestibular mossy fiber ingrowth at E15, and in the neurological mutant meander tail, in which granule cells are missing in the anterior cerebellum. Second, the patterns of climbing fiber growth and growth cone behavior during their exuberant growth onto multiple Purkinje cells, and during focused arborization on individual Purkinje cells will be studied in slices of cerebellum, using time-lapse video microscopy. Third, in an in vitro model system based on purified Purkinje cells, the neural specificity of axon-target interactions will be investigated by analyzing the regulation of neurite growth and branching patterns of appropriate climbing and parallel fiber afferents and inappropriate afferents, with static and real time microscopy. Fourth, with the same tissue culture model, the cellular events associated with Purkinje and granule cell synaptogenesis, including afferent growth cone behavior, formation of synaptic junctions, and expression of synapse-associated proteins, will be examined. The regulation of survival and differentiation of Purkinje cells by afferent-target and other cell-cell interactions will be investigated, by co-culturing purified Purkinje cells with neurons and nonneuronal cells intrinsic and afferent to the cerebellum as well as other brain cell types. The extent to which these interactions regulate the construction of synaptic machinery in the target cell will also be examined. These studies will delineate temporal and spatial features of axon-target contacts, and the key cellular interactions that regulate afferent axonal growth and survival and differentiation of targets. The fundamental information obtained from the analysis of intact brain, coupled with the insight into cellular mechanisms obtained from in vitro experiments, will be essential to interpreting new molecular approaches to neuronal development.
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