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Constitutive sharing of recycling synaptic vesicles between presynaptic boutons

Nature Neuroscience volume 9, pages 315–321 (2006)Cite this article

Abstract

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.

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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.

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Acknowledgements

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.

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Author notes

  1. Kevin J Darcy and Kevin Staras: These authors contributed equally to this work.

Authors and Affiliations

  1. MRC Laboratory for Molecular Cell Biology and Cell Biology Unit, University College London, Gower Street, London, WC1E 6BT, UK

    Kevin J Darcy, Kevin Staras & Yukiko Goda

  2. Electron Microscopy Facility, MRC Laboratory for Molecular Cell Biology and Cell Biology Unit, University College London, Gower Street, London, WC1E 6BT, UK

    Lucy M Collinson

  3. Department of Pharmacology, University College London, Gower Street, London, WC1E 6BT, UK

    Yukiko Goda

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Correspondence to Yukiko Goda.

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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)

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Darcy, K., Staras, K., Collinson, L. et al. Constitutive sharing of recycling synaptic vesicles between presynaptic boutons. Nat Neurosci 9, 315–321 (2006). https://doi.org/10.1038/nn1640

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