Figure 1 Spatial distribution and relation between recycling pool size ΔFtot and electrically evoked FM1-43 release half-decay time constant t50. (A) (Left) Difference-image of FM 1-43 labeled neurons in a typical experiment. Presynaptic terminals were maximally loaded with 1200 pulses at 40 Hz. A test pulse of 600 pulses at 30 Hz was applied after washing and synapses were completely destained by two subsequent stimulation cycles with 900 pulses at 30 Hz. (Right) The fluorescence half-decay time constants from the left image during stimulation were calculated to generate a time constant image. Synapses with large recycling pool size colocalize with areas of high t50 values (red arrows; scale bars 11 μm). (B) Synapse fluorescence intensity histogram (bold dotted blue line) and correlation between recycling pool size ΔFtot and half-decay time constants t50 (Spearman's ρ = 0.23 ± 0.07, p < 0.05), derived from individual synapses (black circles, 3149 synapses, five experiments). The mean value of ΔFtot (2620 a.u.) is indicated by the red dotted line. (C) Synapses were grouped in bins and averaged. t50 values were normal distributed affirmed by one-sample Kolmogorov-Smirnov test in each group and significant different (one-way ANOVA: F6,31 = 17.23; p < 0.001). The half-decay time t50 increases with increasing ΔFtot with a strong effect size (Cohen's d = 1.10 ± 0.18). (D) Synapses with large recycling pool (RP) sizes released their dye content relatively slower than synapses with small recycling pool sizes (two-sample t-test, ∗∗∗p < 0.001; 315 synapses). All error bars indicate standard error of the mean.

Figure 2 Disuse hypersensitivity leads to higher release time constants. (A) Histograms for preparations incubated with 500 nM TTX for three days (1526 synapses, three experiments) and control preparations (1748 synapses, three experiments), respectively. Disuse hypersensitivity increased recycling pool size by 10.5%. (B) Corresponding t50 increased by 20% (two-sample t-test, p < 0.001). Error bars indicate standard deviation.

Figure 3 Absolute and relative fluorescence release in recycling pool size groups (8481 synapses, nine experiments). Fluorescence profiles were binned according to recycling pool size and were averaged. A triangular legend symbol indicates the smallest (tip of the triangle) and largest (base of the triangle) recycling pool size. Fluorescence profiles of the different recycling pool size bins in absolute (A) and relative values (B).