The phenomenon of synaptic vesicle clustering as the prefusion state in the model system of exocytosis.

Our findings concern to the synaptic vesicle interactions that were reconstructed in the cell-free system and are thought to represent the different states of exocytosis pathway. The combination of different technical approaches allowed to study the features of aggregation and calcium-dependent homotypic fusion of synaptic vesicles. Electron microscopy observations of synaptic vesicle fraction purified from the rat brain showed the appearance of large particles formed by aggregated synaptic vesicles in the presence of the nerve terminal cytosolic proteins only. This data were confirmed by dynamic light scattering measurements indicating an importance of the cytosolic proteins for the formation of synaptic vesicle clusters. The scanning confocal microscopy and imaginative exploitation of fluorescence probe R18 allowed to distinguish the process of synaptic vesicle clustering from the synaptic vesicle fusion. The stimulating effect of antiepileptic drug, ethosuximide and sodium valproate on the formation of synaptic vesicle aggregates has been revealed. Experiments with the removal of cholesterol showed that such modification of synaptic vesicle membranes did not change the ability of synaptic vesicles to form the clusters, reducing their Ca2+-triggered membrane fusion. Thus, our data have shown that aggregated state of synaptic vesicles represent an intermediate stage of the fusion pathway, where aggregation of synaptic vesicles is preceded by Ca2+-dependent membrane fusion.