Minimum Essential Factors Required for Vesicle Mobilization at Hippocampal Synapses

Studies on the mechanisms that underlie the function of small central presynaptic terminals have been hampered by the inaccessibility of these synapses to soluble reagents. Here, we permeabilized hippocampal synapses in culture, manipulated their interior, and monitored the resulting changes in vesicle mobilization with the styryl dye FM2-10. Using this method, we found that 1 μM Ca²⁺ after incubation with GTP or GTP-γ-S could mobilize ∼90% of the total recycling pool, whereas 1 νM Ca²⁺ application after dialysis of permeabilized synapses with GDP-β-S mobilized ∼30% of the recycling vesicles, presumably corresponding to the readily releasable pool. In electron micrographs of permeabilized hippocampal synapses stimulated with 1 μM Ca²⁺, we could detect significant vesicle depletion after preincubation with GTP-γ-S, whereas preincubation with GDP-β-S left the total vesicle pool relatively intact. Taken together, in this system replenishment of the readily releasable pool by the reserve vesicles was strictly GTP dependent. In contrast, vesicle replenishment and release did not require ATP or N-ethylmaleimide-sensitive factor (NSF); however, this process involved formation of new soluble NSF-attachment protein receptor (SNARE) complexes as judged by its sensitivity to tetanus toxin. These results suggest that in hippocampal synapses, vesicle mobilization and replenishment of the readily releasable pool require GTP and Ca²⁺ but do not necessitate ATP-dependent priming and SNARE recycling.