Synaptic vesicle recycling investigated by high-resolution microscopy in a conventional and a sensory synapse

Abstract

Synaptic neurotransmission depends on the action of highly specialized small secretory organelles – synaptic vesicles. At all chemical synapses the synaptic vesicles release in a strictly regulated fashion their neurotransmitter into the synaptic cleft, by fusing with the plasma membrane (exocytosis) at structurally and functionally well organized sites (active zones). The fused vesicles get retrieved (endocytosis), refilled with neurotransmitter and returned to the vesicle cluster near the active zone in order to supply fusion-competent vesicles for further rounds of neurotransmission, thus completing a process termed vesicle recycling. While the molecular key players in the synaptic vesicle cycle have been characterized in detail for conventional synapses, the general mobility of synaptic vesicles is still poorly understood, with only averaged behaviors (over populations of vesicles) known. How could vesicle behavior actually be described when single vesicles are analyzed? The small size of synaptic vesicles (~ 40 nm in diameter) and the dense clustering at the active zone makes it difficult to study their behavior, since conventional imaging techniques are restricted to a resolution of ~200-300 nm by the diffraction limit of light. I investigated here synaptic vesicle mobility throughout the synaptic vesicle cycle, using both conventional and sub-diffraction high-resolution stimulated emission depletion (STED) fluorescence microscopy. I obtained a thorough description of vesicle recycling in hippocampal synapses. Single vesicle tracking revealed that a large resting pool of vesicles has a low mobility, while active, recently endocytosed vesicles exhibit a high-mobility state for a substantial amount of time. They eventually become resting vesicles by integrating into the vesicle cluster (“maturation