Synaptic vesicles can be released at extremely high rates, which places an extraordinary demand on the recycling machinery. Previous ultrastructural studies of vesicle recycling were conducted in dissected preparations using an intense stimulation to maximize the probability of release. Here, a single light stimulus was applied to motor neurons in intact Caenorhabditis elegans nematodes expressing channelrhodopsin, and the animals rapidly frozen. We found that docked vesicles fuse along a broad active zone in response to a single stimulus, and are replenished with a time constant of about 2 s. Endocytosis occurs within 50 ms adjacent to the dense projection and after 1 s adjacent to adherens junctions. These studies suggest that synaptic vesicle endocytosis may occur on a millisecond time scale following a single physiological stimulus in the intact nervous system and is unlikely to conform to current models of endocytosis.
Neurons communicate with one another at junctions called synapses. When an electrical signal travels along a neuron and arrives at a synapse, vesicles filled with small neurotransmitter molecules fuse with the cell membrane and release the neurotransmitter. These chemicals rapidly bind to receptors on the downstream neuron that induce an electrical response in that cell.
Vesicles can be consumed at prodigious rates, up to 500 a second, so the cell must recover the membrane rapidly and regenerate more vesicles filled with neurotransmitter. Experiments in the 1970s and 1980s suggested that when vesicles empty their contents into the synapse, they fuse completely with the membrane and are lost. To recover the membrane, the cell forms ‘pits’, by means of a coat protein called clathrin, which then bud off into the cell as new vesicles. It takes roughly 15–20 s for vesicles to be recycled in this way. By contrast, synapses with very high firing rates are thought to recycle vesicles through a faster process known as ‘kiss and run’, in which vesicles are not fully integrated into the membrane, but instead fuse transiently with it to form a reversible pore within about a second.
However, these studies triggered vesicle release using conditions that are unlikely to occur naturally inside cells. Now, Watanabe et al. have used optogenetics to study vesicle recycling in response to single stimuli at the synapse between neurons and muscles in an intact living animal, the nematode C. elegans. The worms had been genetically modified to express a light-sensitive ion channel called channelrhodopsin in their motor neurons. Watanabe et al. used a single pulse of light to stimulate vesicle release, and then rapidly froze the worms before studying their synapses with electron microscopy.
They found that vesicle recycling occurred at the edges of the synapse or at a specialized structure in the middle of the synapse. Vesicle recycling took less than 50 ms—much faster than anything previously observed. This ultrafast recycling is unlikely to occur via ‘kiss and run’ since recycling occurred at sites lateral to the sites of fusion and because the recycled vesicles were larger than the originals, implying that they had not simply re-formed after a brief fusion event.
By using physiologically relevant stimuli in an intact animal, Watanabe et al. reveal that vesicles can be recycled at synapses much more rapidly than previously thought, suggesting that our current models of this process may need to be reassessed.