After releasing neurotransmitter, synaptic vesicles are retrieved by endocytosis and recycled via fast and slow pathways to be reused for synaptic transmission. To maintain the synaptic efficacy, vesicles must be refilled with neurotransmitter during recycling. However, the refilling speed estimated in isolated or reconstructed vesicles is, thus far, too slow to fill up vesicles within the period of recycling. Here, we re-examined the vesicle refilling rate directly at central glutamatergic synapses in slices, using simultaneous presynaptic and postsynaptic whole-cell recording combined with caged glutamate photolysis. After washing out vesicular glutamate, refilling of vesicles with uncaged glutamate caused a recovery of EPSCs with a time constant of 15 s that varied depending upon temperature, age, and cytosolic Cl(-) concentrations. This time constant is faster than that of the slow recycling pathway (∼30 s) after clathrin-mediated endocytosis but is much too slow to fill up vesicles replenished through fast recycling pathways (<1 s).
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