Figure 3.

Increased spontaneous activity in cpx^−/− boutons drives an increase in the recycling pool of vesicles. A, Preparations passively loaded with FM1–43 dye after different times of dye exposure. Images represent overlay of confocal z-stacks. There is very faint fluorescence at 0.5 min loading, which is increased at 10 min loading, but not increased further at 20 min loading. Scale bar, 5 μm. B, Fluorescence intensity measured over confocal stacks at cpx^−/− preparations. No increase in the fluorescence is observed after 10 min loading, suggesting that the recycling vesicle pool reached its maximal capacity. Data collected from at least 70 boutons (4 larvae) for each data point. C, Overlays of confocal stacks showing WT, syx^3–69, and cpx^−/− preparations loaded passively with FM1–43 for 10 min. Scale bar, 10 μm. D, Florescence intensity of WT, syx^3–69, and cpx^−/− preparations passively loaded for 10 min shows a significantly higher fluorescence for cpx^−/− preparations, and for syx^3–69 boutons, suggesting an increased recycling pool. Data collected from at least 30 boutons (4 larvae) for each genotype. E, Electron micrographs obtained after FM1–43 photoconversion show recycled (black) and nonrecycled (translucent) vesicles in WT and cpx^−/− boutons after 0.5 (top) and 10 (bottom) min passive FM1–43 loading. Scale bar, 200 nm. F, The number of recycled vesicles in WT and cpx^−/− boutons. Vesicles are quantified from electron micrograph obtained using FM1–43 photoconversion at dye loading times and in the absence of stimulation. Data collected from at least 10 boutons (3 larvae) for each data point. *p < 0.05. **p < 0.01. ***p < 0.001.