Readily releasable vesicles recycle at the active zone of hippocampal synapses

Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 03/2014; 111(14). DOI: 10.1073/pnas.1321541111
Source: PubMed


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.

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    • "Fast microwave - assisted aldehyde fixation and photoconversion of endocytic vesicles labeled with FM1 – 43 dyes revealed that , after release , 50% of the RRP vesicles are retrieved close to the AZ within 4 s after stimulation in a clathrin - independent manner . Within 30 s , the rest of the RRP is endocytosed through large intermediate vesicles , and a small proportion of the pool rejoins the RRP ( Schikorski , 2014 ) . As discussed above , following ultrafast endocytosis SVs are reformed from endosomal compartments in a clathrin - dependent manner . "
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    ABSTRACT: Synapses are the fundamental functional units of neural circuits, and their dysregulation has been implicated in diverse neurological disorders. At presynaptic terminals, neurotransmitter-filled synaptic vesicles are released in response to calcium influx through voltage-gated calcium channels activated by the arrival of an action potential. Decades of electrophysiological, biochemical, and genetic studies have contributed to a growing understanding of presynaptic biology. Imaging studies are yielding new insights into how synapses are organized to carry out their critical functions. The development of techniques for rapid immobilization and preservation of neuronal tissues for electron microscopy has led to a new renaissance in ultrastructural imaging that is rapidly advancing our understanding of synapse structure and function.
    Frontiers in Cellular Neuroscience 05/2015; 9(196). DOI:10.3389/fncel.2015.00196 · 4.29 Impact Factor
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    • "SV Reformation from Endosomes via Clathrin/AP-2 individual rate constants for membrane retrieval and acidification (k CIE , k CME ), but differ with respect to the stoichiometry of membrane retrieval: the size of clathrin-coated pits (Heuser and Reese, 1973) and vesicles (Maycox et al., 1992) at nerve terminals indicates that CME retrieves single vesicles. By contrast, CIE may occur via large invaginations that internalize multiple vesicle equivalents (Schikorski, 2014; Watanabe et al., 2013) and, thus, is cooperative (Figure 2K; Figures S3G–S3I). Fitting the mathematical model to the experimental data obtained from pHluorin imaging allowed us to accurately reproduce stimulus-dependent changes in fluorescence decay as well as the different fractions of during-stimulus endocytosis elicited by 5 Hz or 40 Hz stimulation trains (Figures 2I and 2J). "
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    ABSTRACT: Neurotransmission depends on presynaptic membrane retrieval and local reformation of synaptic vesicles (SVs) at nerve terminals. The mechanisms involved in these processes are highly controversial with evidence being presented for SV membranes being retrieved exclusively via clathrin-mediated endocytosis (CME) from the plasma membrane or via ultrafast endocytosis independent of clathrin. Here we show that clathrin and its major adaptor protein 2 (AP-2) in addition to the plasma membrane operate at internal endosome-like vacuoles to regenerate SVs but are not essential for membrane retrieval. Depletion of clathrin or conditional knockout of AP-2 result in defects in SV reformation and an accumulation of endosome-like vacuoles generated by clathrin-independent endocytosis (CIE) via dynamin 1/3 and endophilin. These results together with theoretical modeling provide a conceptual framework for how synapses capitalize on clathrin-independent membrane retrieval and clathrin/AP-2-mediated SV reformation from endosome-like vacuoles to maintain excitability over a broad range of stimulation frequencies.
    Neuron 06/2014; 82(5):981-988. DOI:10.1016/j.neuron.2014.05.007 · 15.05 Impact Factor
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    • "Loss of dgit in this model does not affect membrane retrieval per se, but selectively impairs slow, presumably clathrin-(Heerssen et al., 2008) and stoned B-mediated reformation of acidified SVs. We thus favor a hypothetical scenario in which the dGIT-stoned B complex in conjunction with other factors regulates SV reformation from internalized endosomal structures in order to replenish SVs close to the AZ (as recently observed at mammalian synapses; Schikorski, 2014). Further studies are required to put this model to the test. "
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    ABSTRACT: Neurotransmission involves the exo-endocytic cycling of synaptic vesicles (SVs) within nerve terminals. Exocytosis is facilitated by a cytomatrix assembled at the active zone (AZ). The precise spatial and functional relationship between exocytic fusion of SVs at AZ membranes and endocytic SV retrieval is unknown. Here, we identify the scaffold G protein coupled receptor kinase 2 interacting (GIT) protein as a component of the AZ-associated cytomatrix and as a regulator of SV endocytosis. GIT1 and its D. melanogaster ortholog, dGIT, are shown to directly associate with the endocytic adaptor stonin 2/stoned B. In Drosophila dgit mutants, stoned B and synaptotagmin levels are reduced and stoned B is partially mislocalized. Moreover, dgit mutants show morphological and functional defects in SV recycling. These data establish a presynaptic role for GIT in SV recycling and suggest a connection between the AZ cytomatrix and the endocytic machinery.
    Cell Reports 05/2014; 7(5). DOI:10.1016/j.celrep.2014.04.051 · 8.36 Impact Factor
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