Synaptic vesicle dynamics in living cultured hippocampal neurons visualized with CY3-conjugated antibodies directed against the lumenal domain of synaptotagmin

Abstract

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.

Independent Vesicle Pools Underlie Different Modes of Release during Neuronal Development

“…2c), indicating the absence of activity-dependent cycling in axons at 4 DIV. Since contradictory data have been described previously (Kraszewski et al, 1995;Coco et al, 1998;Sabo and McAllister, 2003) and to further confirm our findings, we also used the pHsensitive reporter of synaptic vesicle cycling, synaptophysinpHluorin (SypHy) to measure the levels of evoked and spontaneous release in 4 DIV neurons. SypHy-expressing neurons were incubated in the v-ATPase inhibitor bafilomycin to prevent reacidification of vesicles following exocytosis, thus providing a cumulative measure of release.…”

“…Many previous studies have used high KCl to attempt to label vesicles with either styryl dyes or Syt1 antibodies during neuronal development (Kraszewski et al, 1995;Dai and Peng, 1996;Coco et al, 1998;Zakharenko et al, 1999;Sabo and McAllister, 2003;Tojima et al, 2007). Although it is possible that in some studies, the long exposures to KCl may inadvertently result in labeling of the highly active spontaneous pool, others have clearly shown activitydependent vesicle cycling in young neurons (Dai and Peng, 1996;Zakharenko et al, 1999;Tojima et al, 2007).…”

“…While studies in peripheral and central neuronal culture systems have shown that synapses appear to be capable of both spontaneous and activity-dependent vesicle cycling early in development and possibly before axodendritic contact (Matteoli et al, 1992;Kraszewski et al, 1995;Dai and Peng, 1996;Zakharenko et al, 1999;Sabo and McAllister, 2003;Tojima et al, 2007), there has been no careful examination of the identity of vesicle pools and their mode of mobilization. Previous studies have characterized the functional rearrangements that occur in the pool of vesicles responsible for evoked release during development (Mozhayeva et al, 2002), but do not establish how this relates to spontaneous release nor how contact with the postsynaptic compartment may influence maturation.…”

Postsynaptic GluA1 enables acute retrograde enhancement of presynaptic function to coordinate adaptation to synaptic inactivity

“…We first used the uptake of an antibody that binds to a luminal epitope of synaptotagmin (Syt-Ab) (Fig. 1), which is taken up by synaptic vesicles when they release neurotransmitter (31,32). Excitatory synaptic activity was blocked for 24 h by inhibiting AMPA receptors with 10 μM of NBQX in the culture medium.…”

Synaptotagmin IX Regulates Ca2+-dependent Secretion in PC12 Cells

“…2, D and E). To examine the dynamics of Syt IX molecules during Ca 2ϩ -dependent exocytosis, Abs directed against the luminal domain of Syt IX conjugated to fluorescein and the luminal domain of Syt I conjugated to rhodamine were added to the culture medium (24,35), and the cells were stimulated with a low or high concentration of KCl. Uptake of both the fluorescein-Syt IX Ab and rhodamine-Syt I Ab into neurites and cell body occurred only at depolarizing KCl concentrations (Fig.…”