Figure 4.

Ratio of readily releasable pool (RRP) to recycling pool (RP) for different pool sizes in three different functional fluorescence labeling approaches. (A, C, and E) Representative fluorescence difference images of (A) FM 4-64, (C) synaptopHluorin, and (E) αSyt1-CypHer 5 before and after electrical stimulation (Scale bars, 3 μm). Presynaptic terminals were stimulated with 40 pulses at 20 Hz as a measure for the RRP followed by a twofold stimulation with 900 pulses or a single stimulation with 1200 pulses as a measure for the RP. (B, D, and F) Synapses were grouped according to their recycling pool size (see Materials and Methods). Fluorescence time courses and normalized ratio of RRP to RP of synapses with small and large RP sizes in FM 4-64 (B) (294 synapses, four experiments), synaptopHluorin (D) (494 synapses, six experiments), and αSyt1-CypHer 5 (F) (639 synapses, five experiments) labeling experiments. In synaptopHluorin and αSyt1-CypHer experiments, terminals were stimulated in the presence of folimycin to block reacidification. (Scale bars, 3 μm; two-sample t-test, ^∗p < 0.05, ^∗∗∗p < 0.001). Error bars indicate standard deviation.