Figure 4.

Dynamin I GTPase activity is required for replenishment of the reserve pool by bulk endosome‐derived synaptic vesicles. (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. Where indicated cultures were incubated with dynasore (DynS, 80 µM) immediately after this wash step. The readily releasable pool (RRP) and reserve pool (RP) were sequentially unloaded using 60 action potentials (30 Hz) followed by three trains of 400 action potentials (40 Hz, Immediate Unload). The same unloading stimuli were delivered after a 30 min rest period (Second Unload). (b) Representative traces in arbitrary fluorescence units are shown for the unloading of FM1‐43 in cells without (Black) or with (Green) DynS. Bars indicate the period of stimulation. (c) Mean RP size normalized to the total immediate recycling pool is plotted for both immediate and second unloads ± SEM (Ctrl n = 4, DynS n = 4, from 2 culture preparations **p < 0.01, one‐way anova).