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.

In nerve terminals, synaptic vesicles form large clusters anchored to the presynaptic plasmalemma. Recently, FM1-43 photobleaching experiments carried out a frog motor end-plates demonstrated lack of lateral intermixing of synaptic vesicles within clusters, even during sustained nerve terminal stimulation (Henkel and Betz, 1995; Henkel et al., 1996b). We now have investigated the mobility of synaptic vesicle membranes during the endocytic limb of their exo-endocytic cycle. To this aim, we have carried out photobleaching experiments on nerve terminals of hippocampal neurons prelabeled with CY3-conjugated antibodies directed against lumenal epitopes of synaptotagmin I. This conjugate is taken up specifically by synaptic vesicle membranes during endocytosis and then is recovered in newly formed synaptic vesicles. Using this method, we show that synaptic vesicle membranes intermix after endocytosis. Staurosporine, which at hippocampal synapses partially inhibits unloading of FM1-43, but does not block uptake of antibody probes, prevents this intermixing. Our results indicate that synaptic vesicle docking and/or fusion with the plasmalemma correlate with the release of their membranes from a restraining matrix that hinders their lateral mobility. They suggest that membrane intermediates involved in synaptic vesicle reformation interact with a distinct, highly dynamic cytoskeleton and that newly formed synaptic vesicles are recaptured at random within vesicle clusters. Staurosporine, by inhibiting mobility within the terminal, may favor recapture of new vesicles near sites of endocytosis.

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Development of inhibitory synaptic transmission was studied using a dissociated cell culture from the superior colliculus of neonatal rat. Patch-clamp recordings in the whole-cell configuration were performed to measure evoked (single-cell-activated) inhibitory postsynaptic currents (IPSCs), miniature IPSCs and current responses to maximal concentrations of exogenous gamma-aminobutyric acid (GABA). Over a period of 3 weeks in vitro (DIV3-24), the fraction of synaptically coupled neurons raised from 0% to 76%. Evoked IPSCs were first observed at DIV5. They had an average amplitude of 33.9 pA during the first week (n = 13) and 129.7 pA during the fourth week (n = 48). This increase by a factor of 3.8 represents a significant rise in the efficacy of GABAergic transmission during in vitro development. However, no developmental change has been observed in the average amplitudes of miniature somatic IPSCs. The latter remained at an average level of about 9 pA (symmetrical chloride concentration and a driving force of 68 mV). No increase was found also in whole-cell current densities induced by saturating concentrations of exogenous GABA. Our results suggest that under the given conditions, synapse maturation was primarily the result of presynaptic sprouting. This conclusion is further supported by bouton counts in immunostained collicular cultures, where the number of axosomatic and axodendritic GABAergic contacts per neuron increased from 0.54 and 0.37, respectively, at DIV3, to 13.84 and greater than 23.1, at DIV24. The overall density of GABAergic neurons decreased during this period from about 41,000/cm2 to 15,600 cm2, indicating that a growing number of contacts is formed by a declining number of presynaptic neurons.

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