The long term objective of this proposal is to elucidate the mechanisms involved in transmitter release, and in particular, the role of synaptic vesicles in this process, The Drosophila mutant, shibere, in which vesicle recycling is reversibly blocked at 29C, will be used. By blocking vesicle recycling, it is possible to precisely control the number of vesicles in the synapse by evoking transmitter release at 29C, and gradually depleting the synapse to the desired level. Also, following complete depletion at 29C, a low temperature pulse at 19C allows a limited amount of recycling to occur, the number of vesicles reformed depending on the duration of the pulse. Using these techniques, the contribution of the readily-releasable and reserve vesicle pools to transmitter release will be observed under a variety of conditions. At 29C, depression plots from shibire demonstrate a 2-phase decay, apparently related to readily-releasable and reserve vesicle populations. The two components of the plots will be investigated electrophysiologically to determine the available quanta (n), mobilization characteristics of the reserve population, and the interchangeability between the two populations. Morphological correlates for these two populations will be sought using electron microscopy (EM). We will investigate the possibility that the two components of the plots result from the two vesicle populations, which are reformed by the two recycling pathways (active zone and non-active zone) which have been shown to exist in Drosophila terminals. By giving a short, low temperature pulse, it is possible to create a synapse possessing only the rapidly recycling active zone population. Also, by selectively blocking the active zone recycling pathway with Sr2+ saline, it is possible to create terminals possessing only the non-active zone population. Using these preparations possessing one or the other of the vesicle populations, we will attempt to determine their contributions to evoked and spontaneous release, correlating the findings with the depression plot analyses. Furthermore, by labeling for EM observation one or the other of the two populations, we will observe the interchangeability of the two populations, and under what conditions each comes into play. We will also use immunocytochemical labeling of synaptic vesicle proteins to elucidate the fate of vesicle membrane in the recycling pathway. This research will contribute to our basic understanding of neural processes, and will provide a basis for our understanding of neurological diseases and their treatments.
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