This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Synaptic vesicles undergo exo-endocytosis in response to action potentials, and underlie synaptic transmission. One of the unknown parameters is the number of synaptic vesicles released by a single action potential. The central dogma has been that only a single vesicle is released at a single active zone by a single action potential, based on the pioneering and extensive work by Bernard Katz in frog neuromuscular junctions. In the mammalian central nervous system, a wide range of hypotheses have been proposed, with the number ranging from 1 to 5. But the research has been hampered by technical problems. Electrophysiology provides temporal and amplitude information of synaptic responses, but it is not clear how many nerve terminals are stimulated for the recorded synaptic responses. Recently developed optical imaging methods provide similar responses with additional spatial information at the light microscopic level, but there still remains an uncertainty about how many active zones are present in the stimulated nerve terminal. We are addressing this question by stimulating all nerve terminals in a cultured rat hippocampal neuron using a single action potential, then directly counting the released synaptic vesicles by electron tomography. Exo-endocytosed synaptic vesicles are visualized by a fluorescent marker, FM1-43, and by changing the fluorescence into electron-dense product with a photoconversion technique that we have established. This project not only will provide the number of released vesicles, but also allows us to count the number of active zones in individual nerve terminals. The combination of physiological and ultrastructural analyses surpasses any of the published methods so far, and is expected to reveal critical information about synaptic transmission in the CNS.
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