The long-term goals of this research are to elucidate the cellular and molecular mechanisms of transmitter release and differentiation of the presynaptic nerve terminal. The present proposal will focus on the active zone, where transmitter release is believed to occur. It has been postulated that Ca 2+ channels are strategically located at active zones. This hypothesis will be tested using w-conotoxin (CTX) as a probe for Ca 2+ channels at the frog neuromuscular junction . In addition, CTX will be used to study the correlation between active zone morphometry and synaptic efficacy as well as the mechanisms of active zone differentiation. (1) Thin-section electron microscopy will be used to reveal the distribution of Ca 2+ channels in the nerve terminal and to examine if Ca 2+ channels are located preferentially at the active zone. (2) Freeze-fracture cytochemical techniques, using CTX tagged with gold particles, will be applied to study whether Ca 2+ channels coincide with the large intramembrane particles seen at the active zone. (3) Quantal contents of identified neuromuscular junctions will be measured with intracellular recording. Active zones of the same junctions will be stained with rhodamine-labeled CTX and visualized with a confocal microscope. The correlation between quantal contents and active zone sizes will be analyzed. (4) Neuromuscular junctions, during reinnervation in the frog and during synaptogenesis in the tadpole, will be double-labeled with rhodamine-CTX and fluorescein alpha bungarotoxin. Whether Ca 2+ channels are initially distributed evenly throughout the nerve terminal and later accumulated at active zones will be investigated. In addition, the temporal and spatial relationship between clusters of Ca 2+ channels and acetylcholine receptors during synaptogenesis will be examined. The proposed research on two of the most important elements at the synapse, active zones and Ca 2+ channels, will provide new insights into the mechanisms on how the synapse works and forms. These results may lead to further understanding on Lambert-Eaton myasthenic syndrome and may provide morphological correlate of learning and memory which are thought to involve changes in synaptic efficacy.
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