SYNAPTIC VESICLE DYNAMICS IN LIVING CULTURED HIPPOCAMPAL-NEURONS VISUALIZED WITH CY3-CONJUGATED ANTIBODIES DIRECTED AGAINST THE LUMENAL DOMAIN OF SYNAPTOTAGMIN

Antibodies directed against the lumenal domain of synaptotagmin I conj ugated to CY3 (CY3-Syt(1)-Abs) and video microscopy were used to study the dynamics of synaptic vesicles in cultured hippocampal neurons, Wh en applied to cultures after synapse formation, CY3-Syt(1)-Abs produce d a strong labeling of presynaptic vesicle clusters which was markedly increased by membrane depolarization, The increase of the rate of CYS -Syt(1)-Ab uptake in a high K+ medium was maximal during the first few minutes but persisted for as long as 60 min, In axons developing in i solation, CY3-Syt(1)-Abs, in combination with electron microscopy immu nocytochemistry, revealed the presence of synaptic vesicle clusters wh ich move in bulk in anterograde and retrograde direction, Clusters are present both in the axon shaft and in filopodia but not in the filopo dia of the growth cone, Both presynaptic vesicle clusters and clusters present in isolated axons were disrupted by okadaic acid as previousl y shown for synaptic vesicle cluster's at the frog neuromuscular junct ion, These findings indicate that synaptic vesicle aggregation may occ ur independently of cell-cell interaction, but that, in the absence of a synaptic contact, vesicle clusters are not stably anchored to a giv en region of the cell surface, Labeling of synaptic vesicles in immatu re isolated neurons was found to be depolarization and Ca2+ dependent, demonstrating that Ca2+-regulated exocytosis is an intrinsic characte ristic of synaptic vesicles irrespective of their localization at a sy napse.