Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

A readily retrievable pool of synaptic vesicles

Abstract

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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

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.

References

  1. 1

    Heuser, J.E. & Reese, T.S. Evidence for recycling of synaptic vesicle membrane during transmitter release at the frog neuromuscular junction. J. Cell Biol. 57, 315–344 (1973).

    CAS  Article  Google Scholar 

  2. 2

    Granseth, B., Odermatt, B., Royle, S.J. & Lagnado, L. Clathrin-mediated endocytosis is the dominant mechanism of vesicle retrieval at hippocampal synapses. Neuron 51, 773–786 (2006).

    CAS  Article  Google Scholar 

  3. 3

    Mueller, V.J., Wienisch, M., Nehring, R.B. & Klingauf, J. Monitoring clathrin-mediated endocytosis during synaptic activity. J. Neurosci. 24, 2004–2012 (2004).

    CAS  Article  Google Scholar 

  4. 4

    Wu, L.G., Ryan, T.A. & Lagnado, L. Modes of vesicle retrieval at ribbon synapses, calyx-type synapses, and small central synapses. J. Neurosci. 27, 11793–11802 (2007).

    CAS  Article  Google Scholar 

  5. 5

    He, L., Wu, X.S., Mohan, R. & Wu, L.G. Two modes of fusion pore opening revealed by cell-attached recordings at a synapse. Nature 444, 102–105 (2006).

    CAS  Article  Google Scholar 

  6. 6

    Zhang, Q., Li, Y. & Tsien, R.W. The dynamic control of kiss-and-run and vesicular reuse probed with single nanoparticles. Science 323, 1448–1453 (2009).

    CAS  Article  Google Scholar 

  7. 7

    Ceccarelli, B., Hurlbut, W.P. & Mauro, A. Turnover of transmitter and synaptic vesicles at the frog neuromuscular junction. J. Cell Biol. 57, 499–524 (1973).

    CAS  Article  Google Scholar 

  8. 8

    Gandhi, S.P. & Stevens, C.F. Three modes of synaptic vesicular recycling revealed by single-vesicle imaging. Nature 423, 607–613 (2003).

    CAS  Article  Google Scholar 

  9. 9

    Koenig, J.H., Yamaoka, K. & Ikeda, K. Omega images at the active zone may be endocytotic rather than exocytotic: implications for the vesicle hypothesis of transmitter release. Proc. Natl. Acad. Sci. USA 95, 12677–12682 (1998).

    CAS  Article  Google Scholar 

  10. 10

    Miesenböck, G., De Angelis, D.A. & Rothman, J.E. Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins. Nature 394, 192–195 (1998).

    Article  Google Scholar 

  11. 11

    Fernández-Alfonso, T., Kwan, R. & Ryan, T.A. Synaptic vesicles interchange their membrane proteins with a large surface reservoir during recycling. Neuron 51, 179–186 (2006).

    Article  Google Scholar 

  12. 12

    Dittman, J.S. & Kaplan, J.M. Factors regulating the abundance and localization of synaptobrevin in the plasma membrane. Proc. Natl. Acad. Sci. USA 103, 11399–11404 (2006).

    CAS  Article  Google Scholar 

  13. 13

    Wienisch, M. & Klingauf, J. Vesicular proteins exocytosed and subsequently retrieved by compensatory endocytosis are nonidentical. Nat. Neurosci. 9, 1019–1027 (2006).

    CAS  Article  Google Scholar 

  14. 14

    Teng, H., Cole, J.C., Roberts, R.L. & Wilkinson, R.S. Endocytic active zones: hot spots for endocytosis in vertebrate neuromuscular terminals. J. Neurosci. 19, 4855–4866 (1999).

    CAS  Article  Google Scholar 

  15. 15

    Teng, H. & Wilkinson, R.S. Clathrin-mediated endocytosis near active zones in snake motor boutons. J. Neurosci. 20, 7986–7993 (2000).

    CAS  Article  Google Scholar 

  16. 16

    Taubenblatt, P., Dedieu, J.C., Gulik-Krzywicki, T. & Morel, N. VAMP (synaptobrevin) is present in the plasma membrane of nerve terminals. J. Cell Sci. 112, 3559–3567 (1999).

    CAS  PubMed  Google Scholar 

  17. 17

    Willig, K.I., Rizzoli, S.O., Westphal, V., Jahn, R. & Hell, S.W. STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis. Nature 440, 935–939 (2006).

    CAS  Article  Google Scholar 

  18. 18

    Miller, T.M. & Heuser, J.E. Endocytosis of synaptic vesicle membrane at the frog neuromuscular junction. J. Cell Biol. 98, 685–698 (1984).

    CAS  Article  Google Scholar 

  19. 19

    Balaji, J. & Ryan, T.A. Single-vesicle imaging reveals that synaptic vesicle exocytosis and endocytosis are coupled by a single stochastic mode. Proc. Natl. Acad. Sci. USA 104, 20576–20581 (2007).

    CAS  Article  Google Scholar 

  20. 20

    Adie, E.J. et al. CypHer 5: a generic approach for measuring the activation and trafficking of G protein-coupled receptors in live cells. Assay Drug Dev. Technol. 1, 251–259 (2003).

    CAS  Article  Google Scholar 

  21. 21

    Martens, H. et al. Unique luminal localization of VGAT-C terminus allows for selective labeling of active cortical GABAergic synapses. J. Neurosci. 28, 13125–13131 (2008).

    CAS  Article  Google Scholar 

  22. 22

    Hüve, J., Wesselmann, R., Kahms, M. & Peters, R. 4Pi microscopy of the nuclear pore complex. Biophys. J. 95, 877–885 (2008).

    Article  Google Scholar 

  23. 23

    Kano, H., Jakobs, S., Nagorni, M. & Hell, S.W. Dual-color 4Pi-confocal microscopy with 3D-resolution in the 100 nm range. Ultramicroscopy 90, 207–213 (2001).

    CAS  Article  Google Scholar 

  24. 24

    Schmidt, R. et al. Spherical nanosized focal spot unravels the interior of cells. Nat. Methods 5, 539–544 (2008).

    CAS  Article  Google Scholar 

  25. 25

    Roos, J. & Kelly, R.B. The endocytic machinery in nerve terminals surrounds sites of exocytosis. Curr. Biol. 9, 1411–1414 (1999).

    CAS  Article  Google Scholar 

  26. 26

    Shupliakov, O. et al. Impaired recycling of synaptic vesicles after acute perturbation of the presynaptic actin cytoskeleton. Proc. Natl. Acad. Sci. USA 99, 14476–14481 (2002).

    CAS  Article  Google Scholar 

  27. 27

    Gundelfinger, E.D., Kessels, M.M. & Qualmann, B. Temporal and spatial coordination of exocytosis and endocytosis. Nat. Rev. Mol. Cell Biol. 4, 127–139 (2003).

    CAS  Article  Google Scholar 

  28. 28

    Sankaranarayanan, S., De Angelis, D., Rothman, J.E. & Ryan, T.A. The use of pHluorins for optical measurements of presynaptic activity. Biophys. J. 79, 2199–2208 (2000).

    CAS  Article  Google Scholar 

  29. 29

    Sankaranarayanan, S. & Ryan, T.A. Real-time measurements of vesicle-SNARE recycling in synapses of the central nervous system. Nat. Cell Biol. 2, 197–204 (2000).

    CAS  Article  Google Scholar 

  30. 30

    Fernández-Alfonso, T. & Ryan, T.A. The kinetics of synaptic vesicle pool depletion at CNS synaptic terminals. Neuron 41, 943–953 (2004).

    Article  Google Scholar 

  31. 31

    Schikorski, T. & Stevens, C.F. Quantitative ultrastructural analysis of hippocampal excitatory synapses. J. Neurosci. 17, 5858–5867 (1997).

    CAS  Article  Google Scholar 

  32. 32

    Murthy, V.N. & Stevens, C.F. Reversal of synaptic vesicle docking at central synapses. Nat. Neurosci. 2, 503–507 (1999).

    CAS  Article  Google Scholar 

  33. 33

    Schikorski, T. & Stevens, C.F. Morphological correlates of functionally defined synaptic vesicle populations. Nat. Neurosci. 4, 391–395 (2001).

    CAS  Article  Google Scholar 

  34. 34

    Tabares, L. et al. Monitoring synaptic function at the neuromuscular junction of a mouse expressing synaptopHluorin. J. Neurosci. 27, 5422–5430 (2007).

    CAS  Article  Google Scholar 

  35. 35

    Takamori, S. et al. Molecular anatomy of a trafficking organelle. Cell 127, 831–846 (2006).

    CAS  Article  Google Scholar 

  36. 36

    Vanden Berghe, P. & Klingauf, J. Synaptic vesicles in rat hippocampal boutons recycle to different pools in a use-dependent fashion. J. Physiol. (Lond.) 572, 707–720 (2006).

    CAS  Article  Google Scholar 

  37. 37

    Staras, K. et al. A vesicle superpool spans multiple presynaptic terminals in hippocampal neurons. Neuron 66, 37–44 (2010).

    CAS  Article  Google Scholar 

  38. 38

    Fredj, N.B. & Burrone, J. A resting pool of vesicles is responsible for spontaneous vesicle fusion at the synapse. Nat. Neurosci. 12, 751–758 (2009).

    Article  Google Scholar 

  39. 39

    Groemer, T.W. & Klingauf, J. Synaptic vesicles recycling spontaneously and during activity belong to the same vesicle pool. Nat. Neurosci. 10, 145–147 (2007).

    CAS  Article  Google Scholar 

  40. 40

    Sara, Y., Virmani, T., Deak, F., Liu, X. & Kavalali, E.T. An isolated pool of vesicles recycles at rest and drives spontaneous neurotransmission. Neuron 45, 563–573 (2005).

    CAS  Article  Google Scholar 

  41. 41

    Hua, Y., Sinha, R., Martineau, M., Kahms, M. & Klingauf, J. A common origin of synaptic vesicles undergoing evoked and spontaneous fusion. Nat. Neurosci. 13, 1451–1453 (2010).

    CAS  Article  Google Scholar 

  42. 42

    Brodin, L. & Shupliakov, O. Giant reticulospinal synapse in lamprey: molecular links between active and periactive zones. Cell Tissue Res. 326, 301–310 (2006).

    Article  Google Scholar 

  43. 43

    Kim, S.H. & Ryan, T.A. Synaptic vesicle recycling at CNS synapses without AP-2. J. Neurosci. 29, 3865–3874 (2009).

    CAS  Article  Google Scholar 

  44. 44

    Glyvuk, N. et al. AP-1/σ1B-adaptin mediates endosomal synaptic vesicle recycling, learning and memory. EMBO J. 29, 1318–1330 (2010).

    CAS  Article  Google Scholar 

  45. 45

    Hosoi, N., Holt, M. & Sakaba, T. Calcium dependence of exo- and endocytotic coupling at a glutamatergic synapse. Neuron 63, 216–229 (2009).

    CAS  Article  Google Scholar 

  46. 46

    Wu, X.S. et al. Ca2+ and calmodulin initiate all forms of endocytosis during depolarization at a nerve terminal. Nat. Neurosci. 12, 1003–1010 (2009).

    CAS  Article  Google Scholar 

  47. 47

    Yao, C.K. et al. A synaptic vesicle-associated Ca2+ channel promotes endocytosis and couples exocytosis to endocytosis. Cell 138, 947–960 (2009).

    CAS  Article  Google Scholar 

  48. 48

    Goslin, K. & Banker, G. Rat hippocampal neurons in low-density culture. in Culturing Nerve Cells 1st edn. (eds. Banker, G. & Goslin, K.) 251–281 (MIT Press, Cambridge, Massachusetts, USA, 1991).

  49. 49

    Staudt, T., Lang, M.C., Medda, R., Engelhardt, J. & Hell, S.W. 2,2′-thiodiethanol: a new water soluble mounting medium for high resolution optical microscopy. Microsc. Res. Tech. 70, 1–9 (2007).

    CAS  Article  Google Scholar 

  50. 50

    Richardson, W.H. Bayesian-based iterative method of image restoration. J. Opt. Soc. Am. 62, 55–59 (1972).

    Article  Google Scholar 

Download references

Acknowledgements

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

Affiliations

Authors

Contributions

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.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4 (PDF 567 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Hua, Y., Sinha, R., Thiel, C. et al. A readily retrievable pool of synaptic vesicles. Nat Neurosci 14, 833–839 (2011). https://doi.org/10.1038/nn.2838

Download citation

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing