To sustain neurotransmission, synaptic vesicles and their associated proteins must be recycled locally at synapses. Synaptic vesicles are thought to be regenerated approximately 20 s after fusion by the assembly of clathrin scaffolds or in approximately 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 within 50 to 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 restore the surface area of the membrane rapidly. Sustained neurotransmission requires recycling of synaptic vesicles, but the proposed mechanisms have been controversial; here a ‘flash-and-freeze’ method for electron microscopy reveals a new ultrafast form of endocytosis that is actin- and dynamin-dependent and occurs within 100 milliseconds of stimulation. Sustained neurotransmission requires recycling of synaptic vesicles but the proposed mechanisms — clathrin-mediated endocytosis and 'kiss-and-run' reversal of fusion — have been controversial. Now Erik Jorgensen and colleagues, using ultrafast, 'flash-and-freeze' electron microscopy, have identified a previously unknown actin- and dynamin-dependent mechanism of endocytosis, occurring within 100 milliseconds of stimulation in mouse hippocampal neurons. This is 200-times faster than the clathrin-mediated process, and morphological characteristics rule out the 'kiss-and-run' model. This work suggests that rapid internalization of membrane from the surface is the first step in endocytosis.

Ultrafast endocytosis at mouse hippocampal synapses

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