The proposed studies address basic mechanisms of selective synapse formation during central nervous system (CNS) development. They focus on the formation of a single identified synapse in the rat hippocampus, the en passant synapse from mossy fiber to CA3 pyramidal cell. Two complementary experimental systems from neonatal rat will be used: (1) a hippocampal slice, and (2) dentate- hippocampal cell co-cultures. The experiments will employ confocal time-lapse analysis of cytoarchitectural changes, functional imaging measurements and electrophysiology, supplemented by retrospective cytologies to identify and characterize nascent synapses. Three major questions will be pursued: 1. What motions of the prospective synaptic partners -- one mossy fiber and one dendrite -- bring about the initial synaptogenic contact? What is the role of the dendritic filopodium in synaptogenesis? Do afferent axons induce the extension of filopodia? Must synaptogenic contacts occur at specialized regions such as leading growth cones or the tips of axonal or dendritic filopodia? How are axon growth and synapse formation events orchestrated to form the periodic en passant synaptic varicosities? 2. How does the initial contact site mature into a definitive synapse? A new fluorescence method for imaging synaptic vesicle recycling, electrophysiology, Ca imaging and retrospective immunohistochemistry and electron microscopy will be used to analyze the sequence of changes in molecular architecture and functional status that intervene between the initial contact and the fully differentiated synapse. 3. Are initial contact or maturation processes influenced by electrical activity? After baseline analyses of the developmental processes defined in the preceding paragraphs, experiments will be carried out to detect effects of electrical activity of each of those processes. By providing new information about the basis of cellular and molecular mechanisms of CNS synapse formation, these studies may offer new insights into developmental disorders such as neurological birth defects, learning deficits, and mental retardation, and suggest therapies to combat the neural deficits produced by stroke, CNS trauma and degenerative diseases such as Parkinson's and Alzheimer's. In addition, since abnormal mossy fiber sprouting is implicated in several seizure disorders, the proposed research could produce results immediately applicable to epilepsy.
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