In vivo synaptic recovery following optogenetic hyperstimulation.

Local recycling of synaptic vesicles (SVs) allows neurons to sustain transmitter release. Extreme activity (e.g., during seizure) may exhaust synaptic transmission and, in vitro, induces bulk endocytosis to recover SV membrane and proteins; how this occurs in animals is unknown. Following optogenetic hyperstimulation of Caenorhabditis elegans motoneurons, we analyzed synaptic recovery by time-resolved behavioral, electrophysiological, and ultrastructural assays. Recovery of docked SVs and of evoked-release amplitudes (indicating readily-releasable pool refilling) occurred within ∼8-20 s (τ = 9.2 s and τ = 11.9 s), whereas locomotion recovered only after ∼60 s (τ = 20 s). During ∼11-s stimulation, 50- to 200-nm noncoated vesicles ("100nm vesicles") formed, which disappeared ∼8 s poststimulation, likely representing endocytic intermediates from which SVs may regenerate. In endophilin, synaptojanin, and dynamin mutants, affecting endocytosis and vesicle scission, resolving 100nm vesicles was delayed (>20 s). In dynamin mutants, 100nm vesicles were abundant and persistent, sometimes continuous with the plasma membrane; incomplete budding of smaller vesicles from 100nm vesicles further implicates dynamin in regenerating SVs from bulk-endocytosed vesicles. Synaptic recovery after exhaustive activity is slow, and different time scales of recovery at ultrastructural, physiological, and behavioral levels indicate multiple contributing processes. Similar processes may jointly account for slow recovery from acute seizures also in higher animals.