The nascent technology of electron microscope tomography (EMT), which for the first time provides nanometer scale spatial resolution in tissue sections, offers a powerful approach to increasing our understanding of the structural basis of synaptic transmission. EMT studies have begun on active zones, the sites in presynaptic cells where synaptic vesicles dock at and fuse with the plasma membrane to release their neurotransmitter into the synaptic cleft. At a model synapse, the frog's neuromuscular junction (NMJ), qualitative studies exposed the macromolecular composition of protein aggregates known as active zone material (AZM). The organization and associations of its components indicate that the AZM helps dock synaptic vesicles and anchor plasma membrane channels and other proteins that regulate vesicle fusion. EMT also exposed filaments in the lumen of synaptic vesicles and connections of cytoskeleton to AZM, which are also likely to play a role in synaptic transmission. The research proposed here will extend EMT analysis of active zones at the frog's NMJ by implementing quantitative methods for characterizing structures and relationships already identified and by systematically exposing additional ones. It will also apply EMT analysis to active zones at other synapses, where the gross organization differs from that at the frog's NMJ.
The specific aims are to: 1) Generate a quantitative architectural map of the AZM at the frog's NMJ. 2) Determine the spatial relationship of docked synaptic vesicles to the AZM and presynaptic membrane at the frogs' NMJ. 3) Define the associations of cytoskeletal filaments with AZM at the frog's NMJ. 4) Characterize the lumenal filaments in synaptic vesicles at the frog's NMJ. 5) Compare the structure of the AZM at the mouse's NMJ and at neuron-to-neuron synapses with that of the AZM at the frog's NMJ. The quantitative and systematic structural characterization of constituents of the active zone made accessible by EMT is essential for generating and testing hypotheses as to their chemical nature and function in synaptic transmission. At a broader level, a comprehensive knowledge of the mechanisms involved in synaptic transmission is requisite to understanding factors that regulate synapse development and maintenance, bring about disease and influence regeneration.
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