| At synapses of the mammalian central nervous system, release of neurotransmitter occurs at rates transiently as high as 100 Hz, putting extreme demands on the tiny nerve terminals of the brain, typically containing ∼30 functional vesicles. However, the modes of exocytosis and endocytosis that operate at CNS synapses are incompletely understood. Work in this thesis provided the first real-time visualization of a single vesicle fusing at a small central synapse, made possible by highly-intensified CCD imaging of hippocampal synaptic terminals, in which a single vesicle was labelled with the fluorescent membrane marker FM1-43. In a minority of cases, full loss of FM dye was elicited by a single action potential, consistent with classical complete collapse. In most cases, however, action potentials triggered only partial loss of fluorescence, suggesting vesicular retention of membrane marker, consistent with "kiss-and-run" vesicle cycling. An alternative hypothesis, the independent fusion of partially stained vesicles arising from endosomal splitting, could be excluded by observations on the size and timing of successive fusion events. Thus, these experimental evidence support a predominance of kiss-and-run fusion events and rapid vesicular reuse. The results of the single vesicle experiments are most relevant to the readily-releasable pool of vesicles, a subset of the total recycling pool, because of the minimal stimulation protocol used. The properties of the entire vesicle pool were also studied, using novel fluorescent quenching strategies and linear systems theory. They demonstrated that the whole vesicle pool is capable of undergoing kiss-and-run, and that the amount of kiss-and-run on a per fusion basis was greater at low frequencies. Finally, a simple model of the vesicle cycle was used to determine the impact of kiss-and-run on the transfer of information across the synapse. The results indicated that kiss-and-run significantly increased both overall and burst transmission. |
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