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This principle imposes a large challenge in maintaining synaptic transmission during repetitive firing, because the small recycling pool may limit the number of available vesicles for release and nerve terminals would have to distinguish the recycling pool from the reserve pool and keep reserve pool vesicles from being used. By recording the presynaptic capacitance changes and the postsynaptic EPSC at rat calyx of Held synapses in the absence or presence of transmitter glutamate in nerve terminals, we developed a new method to count functional recycling vesicles. We found that essentially all vesicles in calyces participated in recycling, challenging the small-recycling-pool principle established by FM dye labeling. 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