Figure 4.

The replenishment of the recycling pool by bulk endosome-derived SVs is independent of activity. A, Cultures were loaded with FM1-43 (10 μm) using a train of 800 action potentials (80 Hz) and washed for 2 min to remove excess dye. The recycling pool was then released using three trains of 400 action potentials (40 Hz, Immediate Unload). The same unloading stimulus was delivered after a 30 min rest period (Second Unload). Where indicated, two additional trains of 400 action potentials (40 Hz) were delivered at 10 min intervals (Stim. Unloads). B, Representative traces in arbitrary fluorescence units are shown for the unloading of FM1-43 in cells without (Rest) or with (Active) additional stimulations during the 30 min rest period. Bars indicate the period of stimulation. C, Mean SV recycling pool sizes after 30 min rest normalized to total immediate recycling pool are shown. The accumulated amount of dye unload during the additional unloading steps was also plotted (gray bar). Number of independent experiments: Rest, 3; Active, 4; all ±SEM, two-tailed Student's t test.