Synaptic vesicle generation from activity-dependent bulk endosomes requires calcium and calcineurin - PubMed (original) (raw)

SV budding from bulk endosomes requires intracellular calcium. A, Cultures were loaded with HRP (10 mg/ml) for 2 min in the presence of KCl (50 m

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) and washed immediately to remove excess HRP. Cells were then stimulated twice with KCl (50 m

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, 30 s each) to release all available SVs (Immediate Unload) and left to rest for 30 min. Where indicated, cultures were incubated in the presence of low extracellular calcium (Low [Ca2+]e) after the immediate unload, with some solutions supplemented with either BAPTA-AM (300 μ

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) or a DMSO vehicle control. Cultures were fixed after HRP loading (Load), the immediate unload (Unload), or the rest period (Rest), as indicated by arrows. B, Representative electron micrographs of the treatments described above are shown. Scale bar, 500 nm. Black and white arrows indicate HRP-labeled SVs and endosomes, respectively. C, Bar graph displays the mean number of HRP-labeled SVs per nerve terminal ±SEM in either 1.3 m

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extracellular calcium (Normal) or low [Ca2+]e (number of independent experiments: Load, 4; Unload, 3; Rest, 4; Rest-Low [Ca2+]e, 3; Rest-DMSO, 3; Rest-BAPTA, 3; ***p < 0.001, one-way ANOVA). D, E, Frequency distribution of endosome diameter for Load, Unload, and Rest in normal extracellular calcium conditions (D) or Rest for Low [Ca2+]e-, DMSO-, and BAPTA-treated conditions (E). At least 70 HRP-labeled endosomes were used for diameter measurements per independent experiment (**p < 0.01, ***p < 0.001, two-way ANOVA for D and E).