Readily releasable vesicles recycle at the active zone of hippocampal synapses

During the synaptic vesicle cycle, synaptic vesicles fuse with the plasma membrane and recycle for repeated exo/endocytic events. By using activity-dependent N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino) styryl) pyridinium dibromide dye uptake combined with fast (<1 s) microwave-assisted fixation followed by photoconversion and ultrastructural 3D analysis, we tracked endocytic vesicles over time, “frame by frame.” The first retrieved synaptic vesicles appeared 4 s after stimulation, and these endocytic vesicles were located just above the active zone. Second, the retrieved vesicles did not show any sign of a protein coat, and coated pits were not detected. Between 10 and 30 s, large labeled vesicles appeared that had up to 5 times the size of an individual synaptic vesicle. Starting at around 20 s, these large labeled vesicles decreased in number in favor of labeled synaptic vesicles, and after 30 s, labeled vesicles redocked at the active zone. The data suggest that readily releasable vesicles are retrieved as noncoated vesicles at the active zone.The mechanisms that govern synaptic vesicle (SV) retrieval have been debated since the discovery of the SV cycle (1, 2). Currently the two original mechanisms via coated vesicles and via “kiss and run” are proposed for mammalian excitatory central synapses (3–6). The proposed clathrin-mediated mechanism that retrieves the membrane via coated vesicles has comparable slow kinetics (7) (15–40 s until the endocytic vesicle separates from the plasma membrane) and a retrieval site outside the active zone (8, 9). First, the SV fully collapses into the release site and diffuses outside the active zone either as an entity or by its parts. At regions outside the active zone, coated pits form that sort SV proteins, and eventually a coated endocytic vesicle pinches off the plasma membrane. It is generally believed that coated vesicles shed their coat and fuse with early endosomes from which SVs bud off that join the SV cluster (8). This endosomal budding step is also believed to be mediated via coated vesicles. In contrast, SV retrieval via kiss and run has faster kinetics (10, 11) (<1 s), and SVs are retrieved at the active zone. During kiss and run, the SV is thought to maintain its identity and SVs are available for redocking and rapid reuse (12–14). Because SVs maintain their identity, fusion steps with potential endosomal compartments after endocytosis are not believed to occur after kiss and run.There is overwhelming evidence that SV retrieval at mammalian central synapses depends on the major coat protein clathrin, but the visualization of coated vesicles shortly after physiological stimulation has only been shown for lower vertebrate synapses (8, 15, 16). Kiss and run, on the other hand, is not generally accepted as a retrieval mechanism at mammalian central synapses. Several fluorescent imaging techniques (e.g., pHluorin-based SV protein chimeras and nanoparticles) recently provided unique insights into kiss and run (6), but many open questions remain. The visualization of a labeled endocytic vesicle at or near the active zone right after a physiological stimulus would provide elegant additional proof for kiss and run. More so, if one could follow such a labeled vesicle until “redocking,” it would greatly facilitate the investigation of the various steps SVs pass through on their way through the SV cycle.Here, we introduce a technique that is based on activity-dependent labeling of SV retrieval with N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino) styryl) pyridinium dibromide (FM1-43) followed by photoconversion and electron microscopic 3D analysis. This technique is combined with fast microwave-assisted fixation, ensuring a high time resolution that allows tracking of endocytic vesicles “frame by frame”—that is, at distinct time points (0, 4, 10, 20, 30, and 40 s) after stimulation.