Skip to main content

Thank you for visiting 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.

Constitutive sharing of recycling synaptic vesicles between presynaptic boutons


The synaptic vesicle cycle is vital for sustained neurotransmitter release. It has been assumed that functional synaptic vesicles are replenished autonomously at individual presynaptic terminals. Here we tested this assumption by using FM dyes in combination with fluorescence recovery after photobleaching and correlative light and electron microscopy in cultured rat hippocampal neurons. After photobleaching, synapses acquired recently recycled FM dye–labeled vesicles originating from nonphotobleached synapses by a process requiring dynamic actin turnover. The imported vesicles entered the functional pool at their host synapses, as revealed by the exocytic release of the dye upon stimulation. FM1-43 photoconversion and ultrastructural analysis confirmed the incorporation of imported vesicles into the presynaptic terminal, where they mixed with the native vesicle pools. Our results demonstrate that synaptic vesicle recycling is not confined to individual presynaptic terminals as is widely believed; rather, a substantial proportion of recycling vesicles are shared constitutively between boutons.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type



Prices may be subject to local taxes which are calculated during checkout

Figure 1: Mature synapses incorporate nonlocally recycled vesicles into their functional pool.
Figure 2: Visualizing fluorescently labeled and photobleached vesicles by CLEM.
Figure 3: Ultrastructural analysis of vesicle incorporation.
Figure 4: Spatial distribution of newly incorporated vesicles within boutons.
Figure 5: CLEM shows the departure of vesicle clusters from FM1-43–labeled synapses.


  1. Murthy, V.N. & De Camilli, P. Cell biology of the presynaptic terminal. Annu. Rev. Neurosci. 26, 701–728 (2003).

    Article  CAS  Google Scholar 

  2. Sudhof, T.C. The synaptic vesicle cycle. Annu. Rev. Neurosci. 27, 509–547 (2004).

    Article  Google Scholar 

  3. 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).

    Article  CAS  Google Scholar 

  4. 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).

    Article  CAS  Google Scholar 

  5. Ahmari, S.E., Buchanan, J. & Smith, S.J. Assembly of presynaptic active zones from cytoplasmic transport packets. Nat. Neurosci. 3, 445–451 (2000).

    Article  CAS  Google Scholar 

  6. Hopf, F.W., Waters, J., Mehta, S. & Smith, S.J. Stability and plasticity of developing synapses in hippocampal neuronal cultures. J. Neurosci. 22, 775–781 (2002).

    Article  CAS  Google Scholar 

  7. Krueger, S.R., Kolar, A. & Fitzsimonds, R.M. The presynaptic release apparatus is functional in the absence of dendritic contact and highly mobile within isolated axons. Neuron 40, 945–957 (2003).

    Article  CAS  Google Scholar 

  8. Matteoli, M., Coco, S., Schenk, U. & Verderio, C. Vesicle turnover in developing neurons: how to build a presynaptic terminal. Trends Cell Biol. 14, 133–140 (2004).

    Article  CAS  Google Scholar 

  9. Ahmari, S.E. & Smith, S.J. Knowing a nascent synapse when you see it. Neuron 34, 333–336 (2002).

    Article  CAS  Google Scholar 

  10. Ziv, N.E. & Garner, C.C. Cellular and molecular mechanisms of presynaptic assembly. Nat. Rev. Neurosci. 5, 385–399 (2004).

    Article  CAS  Google Scholar 

  11. Murthy, V.N., Sejnowski, T.J. & Stevens, C.F. Heterogeneous release properties of visualized individual hippocampal synapses. Neuron 18, 599–612 (1997).

    Article  CAS  Google Scholar 

  12. Bonhoeffer, T., Staiger, V. & Aertsen, A. Synaptic plasticity in rat hippocampal slice cultures: local “Hebbian” conjunction of pre- and postsynaptic stimulation leads to distributed synaptic enhancement. Proc. Natl. Acad. Sci. USA 86, 8113–8117 (1989).

    Article  CAS  Google Scholar 

  13. Cochilla, A.J., Angleson, J.K. & Betz, W.J. Monitoring secretory membrane with FM1–43 fluorescence. Annu. Rev. Neurosci. 22, 1–10 (1999).

    Article  CAS  Google Scholar 

  14. Ryan, T.A. et al. The kinetics of synaptic vesicle recycling measured at single presynaptic boutons. Neuron 11, 713–724 (1993).

    Article  CAS  Google Scholar 

  15. Klingauf, J., Kavalali, E.T. & Tsien, R.W. Kinetics and regulation of fast endocytosis at hippocampal synapses. Nature 394, 581–585 (1998).

    Article  CAS  Google Scholar 

  16. Aravanis, A.M., Pyle, J.L. & Tsien, R.W. Single synaptic vesicles fusing transiently and successively without loss of identity. Nature 423, 643–647 (2003).

    Article  CAS  Google Scholar 

  17. Hiruma, H., Katakura, T., Takahashi, S., Ichikawa, T. & Kawakami, T. Glutamate and amyloid beta-protein rapidly inhibit fast axonal transport in cultured rat hippocampal neurons by different mechanisms. J. Neurosci. 23, 8967–8977 (2003).

    Article  CAS  Google Scholar 

  18. Bridgman, P.C. Myosin-dependent transport in neurons. J. Neurobiol. 58, 164–174 (2004).

    Article  CAS  Google Scholar 

  19. Pennuto, M., Dunlap, D., Contestabile, A., Benfenati, F. & Valtorta, F. Fluorescence resonance energy transfer detection of synaptophysin I and vesicle-associated membrane protein 2 interactions during exocytosis from single live synapses. Mol. Biol. Cell 13, 2706–2717 (2002).

    Article  CAS  Google Scholar 

  20. Tanaka, H. et al. Molecular modification of N-cadherin in response to synaptic activity. Neuron 25, 93–107 (2000).

    Article  CAS  Google Scholar 

  21. Li, Z. & Murthy, V.N. Visualizing postendocytic traffic of synaptic vesicles at hippocampal synapses. Neuron 31, 593–605 (2001).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  23. Henkel, A.W., Lubke, J. & Betz, W.J. FM1–43 dye ultrastructural localization in and release from frog motor nerve terminals. Proc. Natl. Acad. Sci. USA 93, 1918–1923 (1996).

    Article  CAS  Google Scholar 

  24. Harata, N., Ryan, T.A., Smith, S.J., Buchanan, J. & Tsien, R.W. Visualizing recycling synaptic vesicles in hippocampal neurons by FM 1–43 photoconversion. Proc. Natl. Acad. Sci. USA 98, 12748–12753 (2001).

    Article  CAS  Google Scholar 

  25. Micheva, K.D. & Smith, S.J. Strong effects of subphysiological temperature on the function and plasticity of mammalian presynaptic terminals. J. Neurosci. 25, 7481–7488 (2005).

    Article  CAS  Google Scholar 

  26. Rizzoli, S.O. & Betz, W.J. The structural organization of the readily releasable pool of synaptic vesicles. Science 303, 2037–2039 (2004).

    Article  CAS  Google Scholar 

  27. Kraszewski, K., Daniell, L., Mundigl, O. & De Camilli, P. Mobility of synaptic vesicles in nerve endings monitored by recovery from photobleaching of synaptic vesicle-associated fluorescence. J. Neurosci. 16, 5905–5913 (1996).

    Article  CAS  Google Scholar 

  28. Henkel, A.W., Simpson, L.L., Ridge, R.M. & Betz, W.J. Synaptic vesicle movements monitored by fluorescence recovery after photobleaching in nerve terminals stained with FM1–43. J. Neurosci. 16, 3960–3967 (1996).

    Article  CAS  Google Scholar 

  29. Jordan, R., Lemke, E.A. & Klingauf, J. Visualization of synaptic vesicle movement in intact synaptic boutons using fluorescence fluctuation spectroscopy. Biophys. J. 89, 2091–2102 (2005).

    Article  CAS  Google Scholar 

  30. Shtrahman, M., Yeung, C., Nauen, D.W., Bi, G.Q. & Wu, X.L. Probing vesicle dynamics in single hippocampal synapses. Biophys. J. 89, 3615–3627 (2005).

    Article  CAS  Google Scholar 

  31. Shepherd, G.M. & Harris, K.M. Three-dimensional structure and composition of CA3 → CA1 axons in rat hippocampal slices: implications for presynaptic connectivity and compartmentalization. J. Neurosci. 18, 8300–8310 (1998).

    Article  CAS  Google Scholar 

  32. Matteoli, M., Takei, K., Perin, M.S., Sudhof, T.C. & De Camilli, P. Exo-endocytotic recycling of synaptic vesicles in developing processes of cultured hippocampal neurons. J. Cell Biol. 117, 849–861 (1992).

    Article  CAS  Google Scholar 

  33. Zhai, R.G. et al. Assembling the presynaptic active zone: a characterization of an active one precursor vesicle. Neuron 29, 131–143 (2001).

    Article  CAS  Google Scholar 

  34. Kraszewski, K. et al. Synaptic vesicle dynamics in living cultured hippocampal neurons visualized with CY3-conjugated antibodies directed against the lumenal domain of synaptotagmin. J. Neurosci. 15, 4328–4342 (1995).

    Article  CAS  Google Scholar 

  35. Ma, L., Zablow, L., Kandel, E.R. & Siegelbaum, S.A. Cyclic AMP induces functional presynaptic boutons in hippocampal CA3–CA1 neuronal cultures. Nat. Neurosci. 2, 24–30 (1999).

    Article  CAS  Google Scholar 

  36. Antonova, I. et al. Rapid increase in clusters of presynaptic proteins at onset of long-lasting potentiation. Science 294, 1547–1550 (2001).

    Article  CAS  Google Scholar 

  37. Zakharenko, S.S., Zablow, L. & Siegelbaum, S.A. Visualization of changes in presynaptic function during long-term synaptic plasticity. Nat. Neurosci. 4, 711–717 (2001).

    Article  CAS  Google Scholar 

  38. Colicos, M.A., Collins, B.E., Sailor, M.J. & Goda, Y. Remodeling of synaptic actin induced by photoconductive stimulation. Cell 107, 605–616 (2001).

    Article  CAS  Google Scholar 

  39. Morales, M., Colicos, M.A. & Goda, Y. Actin-dependent regulation of neurotransmitter release at central synapses. Neuron 27, 539–550 (2000).

    Article  CAS  Google Scholar 

  40. Xia, Z., Dudek, H., Miranti, C.K. & Greenberg, M.E. Calcium influx via the NMDA receptor induces immediate early gene transcription by a MAP kinase/ERK-dependent mechanism. J. Neurosci. 16, 5425–5436 (1996).

    Article  CAS  Google Scholar 

  41. Cottrell, J.R., Borok, E., Horvath, T.L. & Nedivi, E. CPG2: a brain- and synapse-specific protein that regulates the endocytosis of glutamate receptors. Neuron 44, 677–690 (2004).

    CAS  Google Scholar 

  42. Stinchcombe, J.C., Nomoto, H., Cutler, D.F. & Hopkins, C.R. Anterograde and retrograde traffic between the rough endoplasmic reticulum and the Golgi complex. J. Cell Biol. 131, 1387–1401 (1995).

    Article  CAS  Google Scholar 

Download references


We thank M. Raff, C. Stevens and C. Dillon for comments on the manuscript, O. Shupliakov for helpful advice on electron microscopy methodologies and analysis and G. Kemenes for advice on statistics. We also thank L. Yu for technical assistance, T. Branco for the GluR1-labeling protocol, A. Ferrari for the SypI-EGFP construct, Y. Hayashi for the EGFP-GluR2 construct and members of the Goda lab and E. Koo for helpful discussions. This work was supported by the Medical Research Council, the US National Institutes of Health and the National Alliance for Research on Schizophrenia and Depression in association with the Sidney Baer Trust.

Author information

Authors and Affiliations


Corresponding author

Correspondence to Yukiko Goda.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Time-lapse imaging of inter-synaptic vesicle transport. (PDF 2322 kb)

Supplementary Fig. 2

Photobleaching was not detrimental to synaptic vesicle turnover. (PDF 2468 kb)

Supplementary Fig. 3

Kymograph plots of FM FRAP (PDF 2747 kb)

Supplementary Fig. 4

FM4-64 fluorescence at synapses neighboring photobleached boutons is not influenced by the FRAP protocol. (PDF 1967 kb)

Supplementary Fig. 5

Characterizing the two phases of fluorescence recovery at photobleached synapses. (PDF 1774 kb)

Supplementary Fig. 6

Example FRAP sequence from experiments characterizing the release competence of recovered fluorescence. (PDF 1120 kb)

Supplementary Fig. 7

FM loading protocol does not influence vesicle incorporation. (PDF 1773 kb)

Supplementary Video 1

Inter-synaptic movement of FM4-64 labeled vesicles. (MOV 162 kb)

Supplementary Video 2

Recovery of FM4-64 fluorescence at photobleached synapses. (MOV 259 kb)

Supplementary Video 3

Departure of FM1-43 fluorescent packets from synapses. (MOV 715 kb)

Supplementary Video 4

Departure of FM1-43 fluorescent packets from synapses. [This region was subsequently examined in EM, see Figure 5.] (MOV 187 kb)

Supplementary Methods (PDF 10 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Darcy, K., Staras, K., Collinson, L. et al. Constitutive sharing of recycling synaptic vesicles between presynaptic boutons. Nat Neurosci 9, 315–321 (2006).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


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