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A readily retrievable pool of synaptic vesicles


Although clathrin-mediated endocytosis is thought to be the predominant mechanism of synaptic vesicle recycling, it seems to be too slow for fast recycling. Therefore, it was suggested that a presorted and preassembled pool of synaptic vesicle proteins on the presynaptic membrane might support a first wave of fast clathrin-mediated endocytosis. In this study we monitored the temporal dynamics of such a 'readily retrievable pool' of synaptic vesicle proteins in rat hippocampal neurons using a new type of probe. By applying cypHer5E, a new cyanine dye–based pH-sensitive exogenous marker, coupled to antibodies to luminal domains of synaptic vesicle proteins, we could reliably monitor synaptic vesicle recycling and demonstrate the preferential recruitment of a surface pool of synaptic vesicle proteins upon stimulated endocytosis. By using fluorescence nanoscopy of surface-labeled synaptotagmin 1, we could resolve the spatial distribution of the surface pool at the periactive zone in hippocampal boutons, which represent putative sites of endocytosis.

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Figure 1: Antibodies coupled to cypHer are a reliable tool to measure stimulation-dependent exo-endocytic cycling of endogenous vesicle constituents.
Figure 2: Dose–response curve to analyze the size of the surface pool.
Figure 3: Comparison of vesicle recycling kinetics probed with synaptopHluorin and cypHer-coupled antibodies.
Figure 4: Readily retrievable surface pool of synaptic vesicle constituents.
Figure 5: Repeated stimulation reveals reuse of the RRetP.
Figure 6: Spatial organization of the RRetP.


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We thank M. Pilot and I. Herfort for the preparation of primary cell cultures of hippocampal neurons and E. Neher and R.H. Chow for critical reading of the manuscript. We acknowledge D. Boening and M. Martineau for experimental support and we are grateful to K. Kolmakov and V. Belov (Max Planck Institute for Biophysical Chemistry, Goettingen) for providing us with the new fluorescent dye KK114. We thank G. Miesenböck (Oxford University) for providing superecliptic spH. We would also like to thank M. Hoon and N. Glyvuk for suggestions. This work was supported by grants from the European Science Foundation/Deutsche Forschungsgemeinschaft (DFG) (EUROMEMBRANE programme, EuroSynapse CRP FP-020 to J.K.) as well as from the DFG (Kl 1334/1-1 to C.S.T. and J.K., SFB 944 to J.K., CMPB to A.E. and S.W.H., and SFB755 to A.E. and S.W.H.). Y.H. is supported by a stipend from the Max-Planck Foundation and R. Sinha is supported by a stipend from the International Max Planck Research School in Neurosciences at the University of Goettingen.

Author information




Y.H., R. Sinha and C.S.T. conducted the majority of the experiments. J.H. collected and analyzed the 4Pi microscopy data. IsoSTED microscopy and analysis was performed by R. Schmidt and A.E. in the department of S.W.H. H.M. synthesized the cypHer-conjugated antibodies. J.K. conceptualized the project and together with Y.H. and R. Sinha designed the experiments. R. Sinha and J.K. wrote the paper with the help of Y.H., C.S.T. and R. Schmidt. All authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to Jurgen Klingauf.

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The authors declare no competing financial interests.

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Hua, Y., Sinha, R., Thiel, C. et al. A readily retrievable pool of synaptic vesicles. Nat Neurosci 14, 833–839 (2011).

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