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.

Synaptic vesicles transiently dock to refill release sites

Abstract

Synaptic vesicles fuse with the plasma membrane to release neurotransmitter following an action potential, after which new vesicles must ‘dock’ to refill vacated release sites. To capture synaptic vesicle exocytosis at cultured mouse hippocampal synapses, we induced single action potentials by electrical field stimulation, then subjected neurons to high-pressure freezing to examine their morphology by electron microscopy. During synchronous release, multiple vesicles can fuse at a single active zone. Fusions during synchronous release are distributed throughout the active zone, whereas fusions during asynchronous release are biased toward the center of the active zone. After stimulation, the total number of docked vesicles across all synapses decreases by ~40%. Within 14 ms, new vesicles are recruited and fully replenish the docked pool, but this docking is transient and they either undock or fuse within 100 ms. These results demonstrate that the recruitment of synaptic vesicles to release sites is rapid and reversible.

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

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

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

Fig. 1: Zap-and-freeze captures synaptic vesicle fusion.
Fig. 2: Multiple fusion events at single active zones after a single action potential.
Fig. 3: Vesicle fusion during the first 14 ms after an action potential.
Fig. 4: Fusions captured at 5 and 11 ms after an action potential represent synchronous and asynchronous release.
Fig. 5: Transient docking refills the docked vesicle pool within milliseconds.

Data availability

Full data tables underlying the figures are available at https://figshare.com/authors/Shigeki_Watanabe/910686 and in the source data. Raw images and image analysis files are available upon request. Source data are provided with this paper.

Code availability

Custom R, Matlab and Fiji scripts are available at https://github.com/shigekiwatanabe/SynapsEM and are the subject of a manuscript currently in preparation.

References

  1. Heuser, J. E. et al. Synaptic vesicle exocytosis captured by quick freezing and correlated with quantal transmitter release. J. Cell Biol. 81, 275–300 (1979).

    CAS  PubMed  Google Scholar 

  2. Kaeser, P. S. & Regehr, W. G. The readily releasable pool of synaptic vesicles. Curr. Opin. Neurobiol. 43, 63–70 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Imig, C. et al. The morphological and molecular nature of synaptic vesicle priming at presynaptic active zones. Neuron 84, 416–431 (2014).

    CAS  PubMed  Google Scholar 

  5. Hammarlund, M., Palfreyman, M. T., Watanabe, S., Olsen, S. & Jorgensen, E. M. Open syntaxin docks synaptic vesicles. PLoS Biol. 5, 1695–1711 (2007).

    CAS  Google Scholar 

  6. Richmond, J. E., Weimer, R. M. & Jorgensen, E. M. An open form of syntaxin bypasses the requirement for UNC-13 in vesicle priming. Nature 412, 338–341 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Watanabe, S. et al. Ultrafast endocytosis at mouse hippocampal synapses. Nature 504, 242–247 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Watanabe, S. et al. Ultrafast endocytosis at Caenorhabditis elegans neuromuscular junctions. eLife 2013, e00723 (2013).

    Google Scholar 

  9. Chang, S., Trimbuch, T. & Rosenmund, C. Synaptotagmin-1 drives synchronous Ca2+-triggered fusion by C2B-domain-mediated synaptic-vesicle-membrane attachment. Nat. Neurosci. 21, 33–42 (2018).

    CAS  PubMed  Google Scholar 

  10. Ritzau-jost, A. et al. Ultrafast action potentials mediate kilohertz signaling at a central synapse. Neuron 84, 152–163 (2014).

    CAS  PubMed  Google Scholar 

  11. Pyott, S. J. & Rosenmund, C. The effects of temperature on vesicular supply and release in autaptic cultures of rat and mouse hippocampal neurons. J. Physiol. 539, 523–535 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Miki, T. et al. Actin- and myosin-dependent vesicle loading of presynaptic docking sites prior to exocytosis. Neuron 91, 808–823 (2016).

    CAS  PubMed  Google Scholar 

  13. Miki, T., Nakamura, Y., Malagon, G., Neher, E. & Marty, A. Two-component latency distributions indicate two-step vesicular release at simple glutamatergic synapses. Nat. Commun. 9, 3943 (2018).

    PubMed  PubMed Central  Google Scholar 

  14. Neher, E. & Brose, N. Dynamically primed synaptic vesicle states: key to understand synaptic short-term plasticity. Neuron 100, 1283–1291 (2018).

    CAS  PubMed  Google Scholar 

  15. Betz, W. J. & Bewick, G. S. Optical analysis of synaptic vesicle recycling at the frog neuromuscular junction. Science 255, 200–203 (1992).

    CAS  PubMed  Google Scholar 

  16. Dutta, D., Williamson, C. D., Cole, N. B. & Donaldson, J. G. Pitstop 2 is a potent inhibitor of clathrin-independent endocytosis. PLoS ONE 7, e45799 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Von Kleist, L. et al. Role of the clathrin terminal domain in regulating coated pit dynamics revealed by small molecule inhibition. Cell 146, 471–484 (2011).

    Google Scholar 

  18. Watanabe, S. et al. Clathrin regenerates synaptic vesicles from endosomes. Nature 515, 228–233 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Jones, H. C. & Keep, R. F. Brain fluid calcium concentration and response to acute hypercalcaemia during development in the rat. J. Physiol. 402, 579–593 (1988).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Hoppa, M. B., Gouzer, G., Armbruster, M. & Ryan, T. A. Control and plasticity of the presynaptic action potential waveform at small CNS nerve terminals. Neuron 84, 778–789 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Rudolph, S., Tsai, M.-C., von Gersdorff, H. & Wadiche, J. I. The ubiquitous nature of multivesicular release. Trends Neurosci. 38, 428–438 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Sakamoto, H. et al. Synaptic weight set by Munc13-1 supramolecular assemblies. Nat. Neurosci. 21, 41–55 (2018).

    CAS  PubMed  Google Scholar 

  23. Tang, A.-H. et al. A trans-synaptic nanocolumn aligns neurotransmitter release to receptors. Nature 536, 210–214 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Hruska, M., Henderson, N., Le Marchand, S. J., Jafri, H. & Dalva, M. B. Synaptic nanomodules underlie the organization and plasticity of spine synapses. Nat. Neurosci. 21, 671–682 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Dobrunz, L. E., Huang, E. P. & Stevens, C. F. Very short-term plasticity in hippocampal synapses. Proc. Natl Acad. Sci. USA 94, 14843–14847 (1997).

    CAS  PubMed  Google Scholar 

  26. Holderith, N. et al. Release probability of hippocampal glutamatergic terminals scales with the size of the active zone. Nat. Neurosci. 15, 988–997 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Kaeser, P. S. & Regehr, W. G. Molecular mechanisms for synchronous, asynchronous, and spontaneous neurotransmitter release. Annu. Rev. Physiol. 76, 333–363 (2014).

    CAS  PubMed  Google Scholar 

  28. Grauel, M. K. et al. RIM-binding protein 2 regulates release probability by fine-tuning calcium channel localization at murine hippocampal synapses. Proc. Natl Acad. Sci. USA 113, 11615–11620 (2016).

    CAS  PubMed  Google Scholar 

  29. Adler, E., Augustine, J., Duffy, N. & Charlton, P. Alien intracellular calcium chelators attenuate release at the squid giant synapse. J. Neurosci. 11, 1496–1507 (1991).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Chen, C. & Regehr, W. G. Contributions of residual calcium to fast synaptic transmission. J. Neurosci. 19, 6257–6266 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Dittman, J. S. & Regehr, W. G. Calcium dependence and recovery kinetics of presynaptic depression at the climbing fiber to Purkinje cell synapse. J. Neurosci. 18, 6147–6162 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Sakaba, T. & Neher, E. Calmodulin mediates rapid recruitment of fast-releasing synaptic vesicles at a calyx-type synapse. Neuron 32, 1119–1131 (2001).

    CAS  PubMed  Google Scholar 

  33. Redman, S. Quantal analysis of synaptic potentials in neurons of the central nervous system. Physiol. Rev. 70, 165–198 (1990).

    CAS  PubMed  Google Scholar 

  34. Tong, G. & Jahr, C. E. Multivesicular release from excitatory synapses of cultured hippocampal neurons. Neuron 12, 51–59 (1994).

    CAS  PubMed  Google Scholar 

  35. Auger, C., Kondo, S. & Marty, A. Multivesicular release at single functional synaptic sites in cerebellar stellate and basket cells. J. Neurosci. 18, 4532–4547 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. 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  PubMed  Google Scholar 

  37. Abenavoli, A. et al. Multimodal quantal release at individual hippocampal synapses: evidence for no lateral inhibition. J. Neurosci. 22, 6336–6346 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Nakamura, Y. et al. Nanoscale distribution of presynaptic Ca2+ channels and its impact on vesicular release during development. Neuron 85, 145–159 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Rebola, N. et al. Distinct nanoscale calcium channel and synaptic vesicle topographies contribute to the diversity of synaptic function. Neuron 104, 693–710.e9 (2019).

    CAS  PubMed  Google Scholar 

  40. Sabatini, B. L. & Regehr, W. G. Optical measurement of presynaptic calcium currents. Biophys. J. 74, 1549–1563 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Raingo, J. et al. VAMP4 directs synaptic vesicles to a pool that selectively maintains asynchronous neurotransmission. Nat. Neurosci. 15, 738–745 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Turecek, J. & Regehr, X. W. G. Synaptotagmin 7 mediates both facilitation and asynchronous release at granule cell synapses. J. Neurosci. 38, 3240–3251 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Weber, J. P., Toft-Bertelsen, T. L., Mohrmann, R., Delgado-Martinez, I. & Sørensen, J. B. Synaptotagmin-7 is an asynchronous calcium sensor for synaptic transmission in neurons expressing SNAP-23. PLoS ONE 9, e114033 (2014).

    PubMed  PubMed Central  Google Scholar 

  44. Yao, J., Gaffaney, J. D., Kwon, S. E. & Chapman, E. R. Doc2 is a Ca2+ sensor required for asynchronous neurotransmitter release. Cell 147, 666–677 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Hu, Z., Tong, X. J. & Kaplan, J. M. UNC-13L, UNC-13S, and Tomosyn form a protein code for fast and slow neurotransmitter release in Caenorhabditis elegans. eLife 2013, e00967 (2013).

    Google Scholar 

  46. Böhme, M. A. et al. Active zone scaffolds differentially accumulate Unc13 isoforms to tune Ca2+ channel–vesicle coupling. Nat. Neurosci. 19, 1311–1320 (2016).

    PubMed  Google Scholar 

  47. Clustering, C. C. et al. Bruchpilot promotes active zone assembly, Ca2+ channel clustering, and vesicle release. Science 312, 1051–1054 (2006).

    Google Scholar 

  48. Lipstein, N. et al. Dynamic control of synaptic vesicle replenishment and short-term plasticity by Ca2+–calmodulin–Munc13-1 signaling. Neuron 79, 82–96 (2013).

    CAS  PubMed  Google Scholar 

  49. Jackman, S. L., Turecek, J., Belinsky, J. E. & Regehr, W. G. The calcium sensor synaptotagmin 7 is required for synaptic facilitation. Nature 529, 88–91 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Jackman, S. L. & Regehr, W. G. The mechanisms and functions of synaptic facilitation. Neuron 94, 447–464 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Kaech, S. & Banker, G. Culturing hippocampal neurons. Nat. Protoc. 1, 2406–2415 (2006).

    CAS  PubMed  Google Scholar 

  52. Hoopmann, P., Rizzoli, S. O. & Betz, W. J. Imaging synaptic vesicle recycling by staining and destaining vesicles with FM dyes. Cold Spring Harb. Protoc. 7, 77–83 (2012).

    Google Scholar 

  53. Allen, C. & Stevens, C. F. An evaluation of causes for unreliability of synaptic transmission. Proc. Natl Acad. Sci. USA 91, 10380–10383 (1994).

    CAS  PubMed  Google Scholar 

  54. Rosenmund, C., Clements, J. D. & Westbrook, G. L. Nonuniform probability of glutamate release at a hippocampal synapse. Science 262, 754–757 (1993).

    CAS  PubMed  Google Scholar 

  55. Hessler, N. A., Shirke, A. M. & Malinow, R. The probability of transmitter release at a mammalian central synapse. Nature 366, 569–572 (1993).

    CAS  PubMed  Google Scholar 

  56. Jensen, T. P. et al. Multiplex imaging relates quantal glutamate release to presynaptic Ca2+ homeostasis at multiple synapses in situ. Nat. Commun. 10, 1414 (2019).

    PubMed  PubMed Central  Google Scholar 

  57. Watanabe, S. Flash-and-freeze: coordinating optogenetic stimulation with rapid freezing to visualize membrane dynamics at synapses with millisecond resolution. Front. Synaptic Neurosci. 8, 24 (2016).

    PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We are indebted to S. Li, Q. Gan, K. Itoh, D. Lubsanjav, C. Zhang and S. Markert for help with cell culture and freezing and for stimulating discussions. We also thank M. Delanoy and B. Smith for technical assistance with electron microscopy and K. T. DiNapoli for developing R code to randomize images. We thank P. Wurzinger and C. Tomova at Leica for the design and manufacture of the middle plate, M. A. Herman for initial tests of using a capacitor for field stimulation, H. Goldschmidt for help validating the stimulation device using pHluorin imaging, and N. Livingston for help with voltage imaging. We also thank the Marine Biological Laboratory and their neurobiology course for supporting the initial set of experiments (course supported by National Institutes of Health grant R25NS063307). S.W. and this work were supported by start-up funds from the Johns Hopkins University School of Medicine, Johns Hopkins Discovery funds and the National Science Foundation (1727260), and the National Institutes of Health (1DP2 NS111133-01 and 1R01 NS105810-01A1) awarded to S.W. S.W. is an Alfred P. Sloan fellow, a McKnight Foundation Scholar and a Klingenstein and Simons Foundation scholar. E.M.J. is an Investigator of the Howard Hughes Medical Institute. G.F.K. was supported by a grant from the National Institutes of Health to the Biochemistry, Cellular and Molecular Biology program of the Johns Hopkins University School of Medicine (T32 GM007445) and is a National Science Foundation Graduate Research Fellow (2016217537). The EM ICE high-pressure freezer was purchased partly with funds from an equipment grant from the National Institutes of Health (S10RR026445) awarded to S. C. Kuo.

Author information

Authors and Affiliations

Authors

Contributions

M.W.D., S.W. and E.M.J. conceived the zap-and-freeze technique. G.F.K. and S.W. designed the experiments and analyzed the data. S.W., G.F.K. and E.M.J. wrote the manuscript. G.F.K. performed all freezing experiments and single-section electron microscopy sample preparation, imaging and analyses, and FM dye uptake experiments, with technical assistance from S.W., with the exception of the first replicate of the 105-ms time point experiment (which was performed by S.W.) and the 1-s and 10-s time point experiments (which were performed by S.R.). M.C. and G.F.K. performed the serial sectioning 3D reconstruction electron microscopy imaging and analyses. S.W. and M.C. developed the Matlab code for image analyses. K.P.A., E.J.H., K.L., S.R. and T.V. performed pilot zap-and-freeze experiments and electron microscopy sample preparation, imaging and analyses. M.W.D. designed the prototype zap-and-freeze stimulation device. S.W. funded and oversaw the research.

Corresponding author

Correspondence to Shigeki Watanabe.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Neuroscience thanks Erwin Neher and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Schematic of events at the active zone of a hippocampal bouton within the first 15 ms after an action potential.

Vesicles close to the active zone are proposed to transit between docked and undocked states, with the on- and off-rates resulting in a certain number of vesicles docked and ready to fuse at any given time. Synchronous fusion, often of multiple vesicles, begins throughout the active zone within hundreds of microseconds, and the vesicles finish collapsing into the plasma membrane by 11 ms (note that the high local calcium shown only lasts ~ 100 microseconds). Between 5 and 11 ms, residual calcium triggers asynchronous fusion, preferentially toward the center of the active zone. Although shown here as taking place in the same active zone, the degree to which synchronous and asynchronous release may occur at the same active zone after a single action potential is unknown. By 14 ms, the vesicles from the peak of asynchronous fusion, which can continue for tens to hundreds of milliseconds, have fully collapsed into the plasma membrane, and new docked vesicles, which may start to be recruited in less than 10 ms, have fully replaced the vesicles used for fusion. These vesicles then undock or fuse within 100 ms. Whether these new vesicles dock at the same sites vacated by the fused vesicles, and whether newly docked vesicles contributed to synchronous and asynchronous fusion during the first 11 ms, remains to be tested.

Extended Data Fig. 2 Examples of pits in the active zone compared to pits outside the active zone or features not quantified as pits.

a, Examples of pits in the active zone 5 ms after stimulation, indicated by black arrows. b, Examples of features not counted as pits, for instance because the curvature in the presynaptic membrane is mirrored by the postsynaptic membrane, indicated by white arrows. Scale bar: 100 nm c, Full fields-of-view of micrographs from the FM dye experiment described in Fig. 4b. Images from experiments described throughout the manuscript (N = 10 freezes from separate cultures on separate days). Cultured mouse hippocampal neurons were pre-incubated in 30 µM Pitstop 2 in physiological saline (1 mM Ca2+) for 2 min, then either not stimulated or subjected to 10x 1 ms pulses at 20 Hz, at 37 °C in FM 1-43FX, followed by washing and fixation. Note that puncta along the processes became apparent after, suggesting that the zap-and-freeze device induces synaptic vesicle fusion and endocytosis. These images were collected randomly throughout the sapphire disks. Scale bar: 20 microns N = 1 experiment.

Extended Data Fig. 3 Multiple fusion events in serial-sectioning reconstructions of active zones from neurons frozen 5 ms after an action potential.

a, Example transmission electron micrographs from serial sections of active zones from neurons frozen 5 ms after an action potential in 1.2 mM, 2 mM, and 4 mM extracellular Ca2+ (from the same experiments described in Fig. 2). Scale bar: 100 nm. PSD: post-synaptic density. AP: action potential. Arrows indicate “pits” in the active zone (opposite the post-synaptic density), which are presumed to be vesicles fusing with the plasma membrane. Note that pits within the same active zone are often different depths. All data are from two experiments from separate cultures frozen on different days.; experiments in 1.2 mM Ca2+ were performed on separate days from a separate culture from the experiments in 2 mM and 4 mM Ca2+.

Extended Data Fig. 4 Representations of serial-sectioning reconstructions of active zones with exocytic pits.

a, Graphical depictions of serial-sectioned active zones containing exocytic pits, from the experiments described in Fig. 2. b, Sizes of active zones from the data set shown in Fig. 2. Area was calculated as the product of the longest 2-D length of active zone in a 2-D profile from that synapse, the number of sections containing the active zone, and section thickness (that is, as the area of a rectangle). 1.2 mM, no stim, n = 62; 1.2 mM, stim, n = 68; 2 mM, no stim, n = 64; 2 mM, stim, n = 66; 4 mM, no stim, n = 65; 4 mM, stim, n = 64 reconstructed active zones. Error bars indicate median and interquartile range. c, Density of docked vesicles in the active zone, from the experiments shown in Fig. 2. Calculated as the total number of docked vesicles from a given sample x10000, divided by the sum of the total area of active zones from that sample. d, Density of exocytic pits in the active zone, from the experiments shown in Fig. 2. Calculated as the total number of pits from a given sample x10000, divided by the sum of the total area of active zones from that sample. e, Same data shown in b, sorted by whether the active zone contained an exocytic pit or not. See Supplementary Table 1 for full pairwise comparisons and summary statistics.

Source data

Extended Data Fig. 5 Fusion intermediates at multiple time points during the first 14 ms after an action potential.

a-b, Example transmission electron micrographs of synapses from neurons frozen without stimulation or 5, 8, 11, or 14 ms after an action potential (these are other examples from the same experiments shown in Fig. 3). c, Cumulative relative frequency of locations of docked vesicles within the active zone, normalized to the size of the active zone (no stim, n = 447; 5 ms, n = 300; 8 ms, n = 348; 11 ms, n = 188; 14 ms, n = 306 docked vesicles). d, Same data as in c, showing only vesicles from synaptic profiles that contain pits. e, Same data as in c, showing only vesicles from synaptic profiles that contain do not contain pits. f, Size of active zones from data in Fig. 3. Tukey boxplot shown; center: median, lower bound of box: 25th percentile, upper bound of box: 75th percentile, lower whisker: 25th percentile minus 1.5x interquartile range, upper whisker: 75th percentile plus 1.5x interquartile range; dots indicate values outside this range. No stim, n = 274; 5 ms, n = 315; 8 ms, n = 343; 11 ms, n = 192; 14 ms, n = 211; TTX, no stim, n = 121; and TTX, 5 ms, n = 255 synaptic profiles. g, Density of pits in the active zone, from the experiments shown in Fig. 3. Calculated as the total number of pits from a given sample x100, divided by the sum of the total length of active zones from that sample. h, Density of docked vesicles in the active zone, from the experiments shown in Fig. 3. Calculated as the total number of docked vesicles from a given sample x100, divided by the sum of the total length of active zones from that sample. Scale bar: 100 nm. PSD: post-synaptic density. AP: action potential. Arrows indicate “pits” in the active zone (opposite the post-synaptic density), which are presumed to be vesicles fusing with the plasma membrane. All data are from two experiments from separate cultures frozen on different days, except for the data from TTX treatment without stimulation, which are from a single experiment, and data from 5 and 8 ms, which are from three experiments. See Supplementary Table 1 for full pairwise comparisons and summary statistics.

Source data

Extended Data Fig. 6 Chelating residual Ca2+ blocks fusion intermediates at 11 ms but not 5 ms after an action potential.

a-b, Example transmission electron micrographs of synapses from neurons pre-treated with a 0.25% DMSO or b 25 μM EGTA-AM and frozen either without stimulation, 5 ms after stimulation, or 11 ms after stimulation (these are other examples from the same experiments shown in Fig. 4). Scale bar: 100 nm. PSD: post-synaptic density. AP: action potential. Arrows indicate “pits” in the active zone (opposite the post-synaptic density), which are presumed to be vesicles fusing with the plasma membrane. c, Size of active zones from data in Fig. 4. Tukey boxplot shown; center: median, lower bound of box: 25th percentile, upper bound of box: 75th percentile, lower whisker: 25th percentile minus 1.5x interquartile range, upper whisker: 75th percentile plus 1.5x interquartile range; dots indicate values outside this range. See Supplementary Table 1 for full pairwise comparisons and summary statistics. d, Distances of synaptic vesicles from the plasma membrane at the active zone, including both vesicles that were annotated as docked and not docked, from data in Fig. 3. No stim, n = 274; 5 ms, n = 315; 8 ms, n = 343; 11 ms, n = 192; 14 ms, n = 211; TTX, no stim, n = 121; and TTX, 5 ms, n = 255 synaptic profiles. Distances are binned in 2-nm increments, except for “0”, which indicates vesicles ~0 nm from the active zone membrane (“2” indicates vesicles 0.1-2 nm from the membrane, “4” indicates 3-4 nm, etc.). Error bars indicate standard error of the mean. e, Number of pits in the active zone per 100 nm of active zone, from data in Fig. 4. f, Number of docked per 100 nm of active zone, from data in Fig. 4. All data from the experiments described in Fig. 4 are from 4 experiments for no stim and 5 ms time points, 3 experiments for 11 ms, and 2 experiments for 14 ms, from separate cultures frozen on different days (See Supplementary Table 2 for count data from each experiment).

Source data

Supplementary information

Reporting Summary

Supplementary Tables

Full summary statistics and hypothesis testing performed (Supplementary Table 1) and per-replicate mean/median count data (Supplementary Table 2).

Source data

Source Data Fig. 1

Statistical source data for Fig. 1.

Source Data Fig. 2

Statistical source data for Fig. 2.

Source Data Fig. 3

Statistical source data for Figure 3

Source Data Fig. 4

Statistical source data for Fig. 4.

Source Data Fig. 5

Statistical source data for Fig. 5.

Source Data Extended Data Fig. 4

Statistical source data for Extended Data Fig. 4.

Source Data Extended Data Fig. 5

Statistical source data for Extended Data Fig. 5.

Source Data Extended Data Fig. 6

Statistical source data for Extended Data Fig. 6.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kusick, G.F., Chin, M., Raychaudhuri, S. et al. Synaptic vesicles transiently dock to refill release sites. Nat Neurosci 23, 1329–1338 (2020). https://doi.org/10.1038/s41593-020-00716-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41593-020-00716-1

This article is cited by

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