545 Gen. Physiol. Biophys. (2013), 32, 545–558 doi: 10.4149/gpb_2013037 Te phenomenon of synaptic vesicle clustering as the prefusion state in the model system of exocytosis Vitaliy P. Gumenyuk 1 , Alexander Yu. Chunikhin 2 , Nina H. Himmelreich 1 and Irene O. Trikash 1 1 Department of Neurochemistry, Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, 9 Leontovich str., 01601, Kiev, Ukraine 2 Laboratory of Optical Methods Investigations, Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, 9 Leontovich str., 01601, Kiev, Ukraine Abstract. 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. Te 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. Tis data were confirmed by dynamic light scattering measurements indicating an importance of the cytosolic proteins for the formation of synaptic vesicle clusters. Te scanning confocal microscopy and imaginative exploita- tion of fluorescence probe R18 allowed to distinguish the process of synaptic vesicle clustering from the synaptic vesicle fusion. Te 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, while reducing their Ca 2+ -triggered membrane fusion. Tus, 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 Ca 2+ -dependent membrane fusion. Key words: Synaptic vesicle aggregation — Membrane fusion — Cell-free system — Cholesterol — Antiepileptic drugs Correspondence to: Irene O. Trikash, Department of Neuro- chemistry, Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, 9 Leontovich str., 01601, Kiev, Ukraine E-mail: trikash@biochem.kiev.ua Introduction Docking is considered to be the necessary intermediate step that occurs before the synaptic vesicles gained the compe- tence to fuse and release the neurotransmitters. Recently, only a fraction of docked synaptic vesicles was shown to fuse with plasmalemma upon Ca 2+ -influx (Wadel et al. 2007). Te electron tomography studies revealed that synaptic vesicles are linked to each other and organized in clusters or tight aggregations (Fernandez-Busnadiego et al. 2010). In order to complete this, the synaptic vesicles have a distinct set of proteins comprised including the vesicular SNARE protein synaptobrevin/VAMP-2, synaptophysin and the calcium- and phospholipid-binding protein synaptotagmin 1 (Sudhof 2004). It was suggested that MUNC18-1 that bind to the neuronal SNARE-complex (Dulubova et al. 2007) by form- ing a complex with syntaxin, takes place in neurotransmit- ter release through the mediation of docking and fusion of synaptic vesicles with presynaptic membranes (de Wit et al. 2006; Verhage and Sorensen 2008). Moreover, there has been a model proposed where several presynaptic proteins selec- tively localized in synaptic vesicle clusters form a complex proteinaceous net that restrains their mobility (Shupliakov 2009). Te majority of vesicles in the nerve terminal support neurotransmitter release indirectly being cross-linked to each other by acting as a molecular buffer. Disrupting the clusters by application of black widow spider venom resulted