Estimation of the contribution of bulk endocytosis to synaptic vesicle recycling by photoconversion of FM1-43.

<p>Amphibian motor nerve terminals were stimulated at 20 Hz for 10 min and FM1-43 (5 µM) was added 5 min before the end of the stimulation. The preparation was then washed with Ca²⁺-free Ringer's solution, fixed and processed for photoconversion. (<b>A</b>) In unstimulated nerve endings, no apparent photoconversion was detected in the vesicular pool. (<b>B</b>) In stimulated nerve endings, large cisternae surrounded by photoconverted vesicles were observed. (<b>C</b>) Quantification of relative proportion of photoconverted vesicles. Vesicles were classified as recycling (RV; containing photoconverted electron-dense material) or synaptic (SV; non-photoconverted), falling into 3 categories: directly abutting the membrane of cisternae, within 350 nm of the cisternae and further away from the cisternae (color coding as indicated in the figure; n = 7 nerve terminals, 1580 vesicles quantified, 3 NMJ preparations). (<b>D</b>) Large intraterminal bulky structures and enlargement showing a few photolabeled vesicles in close contact with the membrane of the recyclosomes. The arrow indicates a probable nascent recycled vesicle. (<b>E–F</b>) Enlargement showing several photolabeled vesicles (arrowheads) in close contact with the membrane of the cisternae. (<b>G–H</b>) Enlargement showing photolabeled omega-shaped vesicles (arrow) in direct contact with the membrane of the cisternae. Scale bars 500 nm.