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Journal of Neuroscience, Vol 15, 4328-4342, Copyright © 1995 by Society for Neuroscience
Synaptic vesicle dynamics in living cultured hippocampal neurons visualized with CY3-conjugated antibodies directed against the lumenal domain of synaptotagmin
K Kraszewski, O Mundigl, L Daniell, C Verderio, M Matteoli and P De Camilli
Department of Cell Biology, Yale University School Medicine, New Haven, Connecticut 06510, USA.
Antibodies directed against the lumenal domain of synaptotagmin I
conjugated to CY3 (CY3-Syt1-Abs) and video microscopy were used to study
the dynamics of synaptic vesicles in cultured hippocampal neurons. When
applied to cultures after synapse formation, CY3-Syt1-Abs produced a strong
labeling of presynaptic vesicle clusters which was markedly increased by
membrane depolarization. The increase of the rate of CY3-Syt1-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 isolation, CY3-Syt1-Abs, in
combination with electron microscopy immunocytochemistry, revealed the
presence of synaptic vesicle clusters which move in bulk in anterograde and
retrograde direction. Clusters are present both in the axon shaft and in
filopodia but not in the filopodia of the growth cone. Both presynaptic
vesicle clusters and clusters present in isolated axons were disrupted by
okadaic acid as previously shown for synaptic vesicle clusters at the frog
neuromuscular junction. These findings indicate that synaptic vesicle
aggregation may occur independently of cell-cell interaction, but that, in
the absence of a synaptic contact, vesicle clusters are not stably anchored
to a given region of the cell surface. Labeling of synaptic vesicles in
immature isolated neurons was found to be depolarization and Ca2+
dependent, demonstrating that Ca(2+)-regulated exocytosis is an intrinsic
characteristic of synaptic vesicles irrespective of their localization at a
synapse.
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