To sustain neurotransmission, synaptic vesicles and their associated proteins must be recycled locally at synapses. Synaptic vesicles are thought to be regenerated ~20 s after fusion by the assembly of clathrin scaffolds or in ~1 s by the reversal of fusion pores via ‘kiss-and-run’ endocytosis. Here we use optogenetics to stimulate cultured hippocampal neurons with a single stimulus, rapidly freeze them after fixed intervals and examine the ultrastructure using electron microscopy – ‘flash-and-freeze’ electron microscopy. Docked vesicles fuse and collapse into the membrane within 30 ms of the stimulus. Compensatory endocytosis occurs with 50-100 ms at sites flanking the active zone. Invagination is blocked by inhibition of actin polymerization, and scission is blocked by inhibiting dynamin. Because intact synaptic vesicles are not recovered, this form of recycling is not compatible with kiss-and-run endocytosis; moreover it is 200-fold faster than clathrin-mediated endocytosis. It is likely that ‘ultrafast endocytosis’ is specialized to rapidly restore the surface area of the membrane.
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