The proposed studies will examine the mechanisms by which retinal projection neurons make precise and functional connections with their central targets. The hypothesis that neuronal activity and neurotrophic factors interact synergistically to modulate axon terminal arborization, as well as synapse formation and stabilization will be tested in the live developing brain. Specifically, the roles and interactions between neurotrophins, and pre- and post-synaptic activity during the dynamic elaboration of optic axon terminal arbors and formation of synaptic contracts will be examined in live, anesthetized tadpoles. The Xenopus laevis visual system is a uniquely accessible vertebrate model in which the development of neuronal connections between retinal ganglion cells and their tecta target neurons can be followed over time in the intact embryo. The mechanisms controlling axon growth, arborization, and complexity will be studied in connections with target neurons. Growth and arborization patterns of individual, fluorescently labeled retinal ganglion cell axons will be followed over time using low-light level video microscopy and laser scanning confocal microscopy. The interactions between neurotrophins and pre- and post-synaptic neuronal activity in the dynamics of axon arborization (axon branch addition and withdrawal) and the final complexity of axonal arbors will be examined by perturbing endogenous neurotrophic factor levels and the activity patterns of projection or target neurons. Neurotrophic factor levels will be altered by direct microinjection of neurotrophins, function- blocking antibodies, or control solutions into the tecta of live, anesthetized tadpoles. The contributions of neuronal activity to the dynamics of axon arborization and refinement will be tested by selectively altering pre- or post-synaptic activity by direct microinjection of pharmacological agents into the retina (sodium channel blocks) or rectum (glutamate receptor agonists or antagonists) of the anesthetized tadpole. A combination of in vivo microscopic imaging of individual retinal ganglion cell axon arbors, and targeted expression and in vivo imaging of chimeric, fluorescently labeled synaptic proteins will be used to determine the correlates between axon morphology and synaptic structure. This will provide simultaneous, single cell observation of axon arborization dynamics and refinement, and synapse formation and stabilization. The roles of activity and neurotrophic signals during synapse formation and stabilization will be examined by combining pharmacologic perturbations of neurotrophic factor levels and activity signaling with simultaneous in vivo imaging of axonal arbors and synaptic proteins. The results of these studies will provide valuable insights into fundamental mechanisms of synaptogenesis in the living brain, and will further our understanding of the mechanisms that control the development of regeneration of the visual pathways, that are critically important in the maintenance of normal visual function.
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