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.

  • Article
  • Published:

Fast resupply of synaptic vesicles requires synaptotagmin-3

Abstract

Sustained neuronal activity demands a rapid resupply of synaptic vesicles to maintain reliable synaptic transmission. Such vesicle replenishment is accelerated by submicromolar presynaptic Ca2+ signals by an as-yet unidentified high-affinity Ca2+ sensor1,2. Here we identify synaptotagmin-3 (SYT3)3,4 as that presynaptic high-affinity Ca2+ sensor, which drives vesicle replenishment and short-term synaptic plasticity. Synapses in Syt3 knockout mice exhibited enhanced short-term depression, and recovery from depression was slower and insensitive to presynaptic residual Ca2+. During sustained neuronal firing, SYT3 accelerated vesicle replenishment and increased the size of the readily releasable pool. SYT3 also mediated short-term facilitation under conditions of low release probability and promoted synaptic enhancement together with another high-affinity synaptotagmin, SYT7 (ref. 5). Biophysical modelling predicted that SYT3 mediates both replenishment and facilitation by promoting the transition of loosely docked vesicles to tightly docked, primed states. Our results reveal a crucial role for presynaptic SYT3 in the maintenance of reliable high-frequency synaptic transmission. Moreover, multiple forms of short-term plasticity may converge on a mechanism of reversible, Ca2+-dependent vesicle docking.

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: SYT3 is localized to presynaptic terminals.
Fig. 2: SYT3 accelerates vesicle resupply at the calyx of Held.
Fig. 3: Recovery from depression is accelerated by Ca2+ binding to presynaptic SYT3.
Fig. 4: SYT3 accelerates recovery from depression in cerebellar climbing fibres.

Similar content being viewed by others

Data availability

 Source data are provided with this paper.

Code availability

Custom-written codes used for analysis and simulations are available upon request.

References

  1. Wang, L. Y. & Kaczmarek, L. K. High-frequency firing helps replenish the readily releasable pool of synaptic vesicles. Nature 394, 384–388 (1998).

    Article  ADS  CAS  PubMed  Google Scholar 

  2. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Hilbush, B. S. & Morgan, J. I. A third synaptotagmin gene, Syt3, in the mouse. Proc. Natl Acad. Sci. USA 91, 8195–8199 (1994).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  4. Sugita, S., Shin, O.-H., Han, W., Lao, Y. & Südhof, T. C. Synaptotagmins form a hierarchy of exocytotic Ca2+ sensors with distinct Ca2+ affinities. EMBO J. 21, 270–280 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. 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).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  6. Xu, J., Mashimo, T. & Südhof, T. C. Synaptotagmin-1, -2, and -9: Ca2+ sensors for fast release that specify distinct presynaptic properties in subsets of neurons. Neuron 54, 567–581 (2007).

    Article  CAS  PubMed  Google Scholar 

  7. Zucker, R. S. & Regehr, W. G. Short-term synaptic plasticity. Annu. Rev. Physiol. 64, 355–405 (2002).

    Article  CAS  PubMed  Google Scholar 

  8. Sutton, R. B., Ernst, J. A. & Brunger, A. T. Crystal structure of the cytosolic C2a-C2b domains of synaptotagmin III. J. Cell Biol. 147, 589–598 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Bhalla, A., Chicka, M. C. & Chapman, E. R. Analysis of the synaptotagmin family during reconstituted membrane fusion. J. Biol. Chem. 283, 21799–21807 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Gao, Z., Reavey-Cantwell, J., Young, R. A., Jegier, P. & Wolf, B. A. Synaptotagmin III/VII isoforms mediate Ca2+-induced insulin secretion in pancreatic islet β-cells. J. Biol. Chem. 275, 36079–36085 (2000).

    Article  CAS  PubMed  Google Scholar 

  11. Awasthi, A. et al. Synaptotagmin-3 drives AMPA receptor endocytosis, depression of synapse strength, and forgetting. Science 363, eaav1483 (2019).

    Article  CAS  PubMed  Google Scholar 

  12. Butz, S., Fernandez-Chacon, R., Schmitz, F., Jahn, R. & Sudhof, T. C. The subcellular localizations of atypical synaptotagmins III and VI. Synaptotagmin III is enriched in synapses and synaptic plasma membranes but not in synaptic vesicles. J. Biol. Chem. 274, 18290–18296 (1999).

    Article  CAS  PubMed  Google Scholar 

  13. Xiao, L. et al. Developmental expression of synaptotagmin isoforms in single calyx of Held-generating neurons. Mol. Cell. Neurosci. 44, 374–385 (2010).

    Article  CAS  PubMed  Google Scholar 

  14. Whittaker, V. P. Thirty years of synaptosome research. J. Neurocyt. 22, 735–742 (1993).

    Article  CAS  Google Scholar 

  15. Phillips, G. R. et al. The presynaptic particle web: ultrastructure, composition, dissolution, and reconstitution. Neuron 32, 63–77 (2001).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  17. Chen, Z., Cooper, B., Kalla, S., Varoqueaux, F. & Young, S. M. Jr. The Munc13 proteins differentially regulate readily releasable pool dynamics and calcium-dependent recovery at a central synapse. J. Neurosci. 33, 8336–8351 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Thanawala, M. S. & Regehr, W. G. Presynaptic calcium influx controls neurotransmitter release in part by regulating the effective size of the readily releasable pool. J. Neurosci. 33, 4625–4633 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  19. Elmqvist, D. & Quastel, D. M. A quantitative study of end-plate potentials in isolated human muscle. J. Physiol. 178, 505–529 (1965).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Thanawala, M. S. & Regehr, W. G. Determining synaptic parameters using high-frequency activation. J. Neurosci. Methods 264, 136–152 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Kusick, G. F. et al. Synaptic vesicles transiently dock to refill release sites. Nat. Neurosci. 23, 1329–1338 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Silva, M., Tran, V. & Marty, A. Calcium-dependent docking of synaptic vesicles. Trends Neurosci. 44, 579–592 (2021).

    Article  CAS  PubMed  Google Scholar 

  25. Hui, E. et al. Three distinct kinetic groupings of the synaptotagmin family: candidate sensors for rapid and delayed exocytosis. Proc. Natl Acad. Sci. USA 102, 5210–5214 (2005).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  26. Helmchen, F., Borst, J. G. & Sakmann, B. Calcium dynamics associated with a single action potential in a CNS presynaptic terminal. Biophys. J. 72, 1458–1471 (1997).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  27. Wang, L. Y., Neher, E. & Taschenberger, H. Synaptic vesicles in mature calyx of Held synapses sense higher nanodomain calcium concentrations during action potential-evoked glutamate release. J. Neurosci. 28, 14450–14458 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Schneggenburger, R., Meyer, A. C. & Neher, E. Released fraction and total size of a pool of immediately available transmitter quanta at a calyx synapse. Neuron 23, 399–409 (1999).

    Article  CAS  PubMed  Google Scholar 

  29. Lipstein, N. et al. Munc13-1 is a Ca2+-phospholipid-dependent vesicle priming hub that shapes synaptic short-term plasticity and enables sustained neurotransmission. Neuron 109, 3980–4000 e3987 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Padmanarayana, M. et al. A unique C2 domain at the C terminus of Munc13 promotes synaptic vesicle priming. Proc. Natl Acad. Sci. USA 118, e2016276118 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Camacho, M. et al. Control of neurotransmitter release by two distinct membrane-binding faces of the Munc13-1 C1C2B region. eLife 10, e72030 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Lein, E. S. et al. Genome-wide atlas of gene expression in the adult mouse brain. Nature 445, 168–176 (2007).

    Article  ADS  CAS  PubMed  Google Scholar 

  33. Foster, K. A. & Regehr, W. G. Variance-mean analysis in the presence of a rapid antagonist indicates vesicle depletion underlies depression at the climbing fiber synapse. Neuron 43, 119–131 (2004).

    Article  CAS  PubMed  Google Scholar 

  34. Eshra, A., Schmidt, H., Eilers, J. & Hallermann, S. Calcium dependence of neurotransmitter release at a high fidelity synapse. eLife 10, e70408 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Muller, M., Felmy, F., Schwaller, B. & Schneggenburger, R. Parvalbumin is a mobile presynaptic Ca2+ buffer in the calyx of Held that accelerates the decay of Ca2+ and short-term facilitation. J. Neurosci. 27, 2261–2271 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Foster, K. A., Kreitzer, A. C. & Regehr, W. G. Interaction of postsynaptic receptor saturation with presynaptic mechanisms produces a reliable synapse. Neuron 36, 1115–1126 (2002).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Luo, F. & Südhof, T. C. Synaptotagmin-7-mediated asynchronous release boosts high-fidelity synchronous transmission at a central synapse. Neuron 94, 826–839.e3 (2017).

    Article  CAS  PubMed  Google Scholar 

  39. Weyrer, C., Turecek, J., Harrison, B. & Regehr, W. G. Introduction of synaptotagmin 7 promotes facilitation at the climbing fiber to Purkinje cell synapse. Cell Rep. 36, 109719 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Dittman, J. S., Kreitzer, A. C. & Regehr, W. G. Interplay between facilitation, depression, and residual calcium at three presynaptic terminals. J. Neurosci. 20, 1374–1385 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Turecek, J., Jackman, S. L. & Regehr, W. G. Synaptotagmin 7 confers frequency invariance onto specialized depressing synapses. Nature 551, 503–506 (2017).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  43. Wen, H. et al. Distinct roles for two synaptotagmin isoforms in synchronous and asynchronous transmitter release at zebrafish neuromuscular junction. Proc. Natl Acad. Sci. USA 107, 13906–13911 (2010).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  44. Malagon, G., Miki, T., Tran, V., Gomez, L. C. & Marty, A. Incomplete vesicular docking limits synaptic strength under high release probability conditions. eLife 9, e52137 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Atluri, P. P. & Regehr, W. G. Determinants of the time course of facilitation at the granule cell to Purkinje cell synapse. J. Neurosci. 16, 5661–5671 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Regehr, W. G. Short-term presynaptic plasticity. Cold Spring Harb. Perspect. Biol. 4, a005702 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  47. Saunders, A. et al. Molecular diversity and specializations among the cells of the adult mouse brain. Cell 174, 1015–1030.e16 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Dimassi, S. et al. A subset of genomic alterations detected in rolandic epilepsies contains candidate or known epilepsy genes including GRIN2A and PRRT2. Epilepsia 55, 370–378 (2014).

    Article  CAS  PubMed  Google Scholar 

  49. Sato, D. et al. SHANK1 deletions in males with autism spectrum disorder. Am. J.f Hum. Genet. 90, 879–887 (2012).

    Article  CAS  Google Scholar 

  50. Chakrabarti, S. et al. Impaired membrane resealing and autoimmune myositis in synaptotagmin VII-deficient mice. J. Cell Biol. 162, 543–549 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Pilpel, N., Landeck, N., Klugmann, M., Seeburg, P. H. & Schwarz, M. K. Rapid, reproducible transduction of select forebrain regions by targeted recombinant virus injection into the neonatal mouse brain. J. Neurosci. Methods 182, 55–63 (2009).

    Article  PubMed  Google Scholar 

  52. Wadiche, J. I. & Jahr, C. E. Multivesicular release at climbing fiber–Purkinje cell synapses. Neuron 32, 301–313 (2001).

    Article  CAS  PubMed  Google Scholar 

  53. Taschenberger, H., Leao, R. M., Rowland, K. C., Spirou, G. A. & von Gersdorff, H. Optimizing synaptic architecture and efficiency for high-frequency transmission. Neuron 36, 1127–1143 (2002).

    Article  CAS  PubMed  Google Scholar 

  54. Meyer, A. C., Neher, E. & Schneggenburger, R. Estimation of quantal size and number of functional active zones at the calyx of Held synapse by nonstationary EPSC variance analysis. J. Neurosci. 21, 7889–7900 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Hallermann, S. et al. Bassoon speeds vesicle reloading at a central excitatory synapse. Neuron 68, 710–723 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Ritzau-Jost, A. et al. Apparent calcium dependence of vesicle recruitment. J. Physiol. 596, 4693–4707 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Fekete, A. et al. Underpinning heterogeneity in synaptic transmission by presynaptic ensembles of distinct morphological modules. Nat. Commun. 10, 826 (2019).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  58. Schneggenburger, R. & Neher, E. Intracellular calcium dependence of transmitter release rates at a fast central synapse. Nature 406, 889–893 (2000).

    Article  ADS  CAS  PubMed  Google Scholar 

  59. 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).

    Article  CAS  PubMed  Google Scholar 

  60. Guan, Z., Quinones-Frias, M. C., Akbergenova, Y. & Littleton, J. T. Drosophila synaptotagmin 7 negatively regulates synaptic vesicle release and replenishment in a dosage-dependent manner. eLife 9, e55443 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  61. Shin, O. H., Maximov, A., Lim, B. K., Rizo, J. & Sudhof, T. C. Unexpected Ca2+-binding properties of synaptotagmin 9. Proc. Natl Acad. Sci. USA 101, 2554–2559 (2004).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  62. Shin, O.-H. et al. Munc13 C2B domain is an activity-dependent Ca2+ regulator of synaptic exocytosis. Nat. Struct. Mol. Biol. 17, 280–288 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank C. Dean for providing Syt3 KO mice and virus plasmids; the OHSU Molecular Virology Core for packaging AAV vectors; K. Wright, K. Monk, S. Kaech Petrie and the staff at the Advanced Light Microscopy Core for imaging assistance; G. Westbrook, H. von Gersdorff, L. Trussell, M. Freeman and P. Brehm and members of the Jackman Lab for comments on the manuscript; J. Vazquez for illustrations; P. Kaeser and myriad colleagues from Vollum for reagents, technical assistance and discussions. This work was supported by the Whitehall Foundation (S.L.J), the Medical Research Foundation (S.L.J) and the NIH Imaging Core Facility (P30NS061800).

Author information

Authors and Affiliations

Authors

Contributions

D.J.W., A.S. and S.L.J. conceived and designed the study. D.J.W. performed electrophysiological recordings and analyses. S.A.K., K.J.-N. and S.L.J. performed histology and imaging. D.J.W. and E.S. performed biophysical modelling and data fitting. D.J.W. and S.L.J. wrote the manuscript with input from all authors.

Corresponding author

Correspondence to Skyler L. Jackman.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature thanks Lu-Yang Wang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

Additional information

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

Extended data figures and tables

Extended Data Fig. 1 Immunolabeling for SYT3 in WT and Syt3 KO animals.

a-b) Representative fluorescent images showing immunolabeling for SYT3 and VGLUT1 in the brainstem (a) and cerebellum (b) of one WT and one Syt3 KO animal. Representative images were acquired using tissue from a single animal of each genotype, but similar results were obtained using tissue from >5 animals of each genotype while optimizing immunohistochemistry.

Extended Data Fig. 2 Controls for spectral bleed-through and secondary antibody specificity.

a) Representative fluorescent images of calyces of Held from a WT mouse, stained using primary antibodies against SYT3, VGLUT1, and Bassoon, followed by secondaries for all primaries. b-d) Representative images of calyces, where secondaries were applied after tissue was treated with only one primary antibody against either SYT3 (b), VGLUT1 (c), or Bassoon (d). Aside from the omission of primary antibodies, all slices were imaged using the same microscope settings and processed identically. Representative images were acquired using tissue from a single WT mouse, but similar results were obtained from 2 or more animals while optimizing immunohistochemistry.

Extended Data Fig. 3 Localization of active zone proteins using structured-illumination microscopy.

a) Representative fluorescence images of a calyx of Held from a WT mouse, immunolabeld using primary antibodies against Bassoon, followed by multiple secondaries with different fluorophores. To ensure proper SIM microscope channel alignment, XY displacement between channels was determined, and this shift was used to perform post hoc alignment of images for localization in Fig. 1. b) Inset from a showing overlap of channels following post hoc channel alignment. Active zone fluorescence profiles were assessed using a 100 nm-wide rectangle drawn through the center of Bassoon-labeled puncta, extending from the presynaptic terminal to the postsynaptic cell. c) Averaged fluorescence profiles of Bassoon puncta imaged in 3 channels, showing the full-width at half maximum (FWHM) of gaussian fits to each channel, and the relative offset of fits to fluorescent peaks. (N = 100 puncta from 4 calyces). The increase in FWHM with fluorophore wavelength is not expected to affect the localization analyses presented in Fig. 1. d) Representative calyx of Held from a WT mouse, immunolabelled for SYT3 and PSD-95 (for analysis in Fig. 1c). Data are mean ± s.e.m. Error bands are obscured by the mean. SIM images used for fluorescence profiles were acquired using tissue from one WT animal, but similar results were obtained from 2 or more animals of each genotype while optimizing immunohistochemistry.

Source data

Extended Data Fig. 4 Basal synaptic properties are unchanged at the calyx of Held in Syt3 KOs.

a) Representative spontaneous excitatory postsynaptic currents (sEPSCs) recorded from MNTB neurons in WT (black) and Syt3 KO animals (magenta). b) Averaged sEPSC waveforms. c) Rise (P = 0.87) and decay time constants (P = 0.11) of sEPSCs. d) Average (P = 0.41) and cumulative distribution (P = 0.51) of sEPSC amplitudes. e) Average (P = 0.78) and cumulative distribution (P = 0.86) of sEPSC frequency. f) Representative EPSCs elicited by afferent fibre stimulation of the calyx of Held. g) Average rise (P = 0.79) and decay (P = 0.91) time constants, and amplitudes (P = 0.82) of EPSCs. h) Schematic showing presynaptic Ca2+-current (ICa) recordings at the calyx of Held. i) ICa elicited by 1 ms depolarizations from −80 mV to 0 mV at 100 Hz. j) Average amplitudes of initial ICa (P = 0.5). k) Normalized ICa evoked by 20 depolarization steps at 100 Hz. Data are mean ± s.e.m. Number of experiments are shown in Extended Data Table 1. Significances were tested using Kruskal-Wallis tests (c,g (rise time and EPSC amplitude)) and two-tailed Student’s t-tests (d, g (τ), j and k). Cumulative distributions in d and e were tested using Kolmogorov-Smirnov tests.

Source data

Extended Data Fig. 5 The readily releasable pool of vesicles is decreased in Syt3 KOs.

a) Averaged cumulative amplitudes of EPSCs elicited by 200 Hz stimulation at the calyx of Held in WT (black) and Syt3 KO (magenta) animals. Back-extrapolation from EPSC81-10028 corrected for vesicle replenishment early in the train18 was used to estimate the RRPTrain (P = 0.02). b) Amplitudes of the first 40 EPSCs at 200 Hz stimulation plotted against cumulative release. Linear forward-extrapolation of the first 4 EPSCs was used to estimate the RRPEQ19 (P = 0.006). c) Normalized amplitudes of the first 20 EPSCs at 200 Hz stimulation. τDecay was calculated with a single exponential fit. RRPDecay was estimated from τDecay20 (P = 0.03). Data are mean ± s.e.m. Number of experiments are shown in Extended Data Table 1. Statistical significances are shown as *: P < 0.05, **: P < 0.01. Significances were evaluated using Kruskal-Wallis tests.

Source data

Extended Data Fig. 6 Modeling the role of SYT3 in vesicle trafficking at the calyx of Held.

a) Illustrated model of vesicle docking and fusion based on previous studies55,56 (see Methods). An infinite pool of reserve vesicles reversibly transitions to a fusion-incompetent loosely docked state. Loosely docked vesicles reversibly transition to a fusion-competent tightly docked state, and release with a fixed probability (p). For the conceptual basis of this model, see21. b) Best fit of the model (magenta) to Syt3 KO data (light magenta) for EPSC trains (left), steady-state amplitudes for frequencies from 1-200 Hz (center), and recovery from depression after 100 EPSCs at 200 Hz (right). c) Illustration of a model where SYT3 reduces vesicle depletion by permanently lowering p, as has been suggested for other synaptotagmin isoforms60. d) Best fit of the model shown in c (black) to data (gray) for WT calyces of Held. e) A model where SYT3 increases p immediately after each action potential, after which p decreases exponentially back to its baseline value. f) Best fit of the model shown in e to WT data. g) A model where SYT3 transiently increases k1. After each action potential k1 increases by a fixed amount, after which k1 decreases exponentially back to its baseline value. h) Best fit of the model shown in g to WT data. i) A model where SYT3 transiently increases k2. After each action potential k2 increases by a fixed amount, after which k2 decreases exponentially back to its baseline value. j) Best fit of the model shown in i to WT data. Note that this model produced the lowest χ2 values among all models tested, supporting a scenario where SYT3 accelerates the transition of vesicles to a tightly docked state. Data were reproduced from Fig. 2. Data are mean ± s.e.m. Number of experiments are shown in Extended Data Table 1.

Source data

Extended Data Fig. 7 Biophysical models of docking and fusion based on Ca2+ binding.

a) Ca2+-dependent model of vesicle docking and fusion. The transition from loose to tight docking was accelerated by SYT3 binding to residual calcium ([Ca2+]res). Tightly docked vesicles fused via 5 cooperative binding steps58 that were driven by local calcium ([Ca2+]local) (see Methods). b) Best fit of the model (dark lines) to WT (grey) and KO (light magenta) data. c) Simulated Ca2+-dependent membrane binding by different SYT isoforms driven by an action potential evoked increase in [Ca2+]res. The fraction of active SYT isoforms was governed by their reported Ca2+ affinity and binding kinetics 4,25,61. χ2 values show best fits to WT data for models where each SYT isoform increased docking rates. d) Modified version of the model in a where an additional [Ca2+]local-dependent mechanism was introduced to increase the docking rate (klocal). Because Munc13-1 has been shown to accelerate vesicle priming at multiple synaspes30,31, including the calyx of Held29,59, we modeled this additional mechanism using the Ca2+-binding properties reported for Munc13-162. e) Best fit of the model in e to WT and KO data. The improved model fit to Syt3 KO data supports a role for additional mechanisms such as Munc13-1, in promoting the transition of vesicles to tight docking. Data were reproduced from Fig. 2. Data are mean ± s.e.m. Number of experiments are shown in Extended Data Table 1.

Source data

Extended Data Fig. 8 SYT3 does not affect quantal parameters or Ca2+-dependence of release from cerebellar climbing fibres.

a) Superimposed recordings of 10 climbing fibre EPSCs during 0.1 Hz stimulation in varying [Ca2+]e. b) Average EPSC amplitudes at varying [Ca2+]e concentrations. c) Average variance of EPSCs plotted against mean EPSC amplitude across varying [Ca2+]e concentrations. Parabola fits were used to estimate quantal parameters54 (q: P = 0.76; N: P = 0.38; p: P = 0.78). Data are mean ± s.e.m. Number of experiments are shown in Extended Data Table 1. Statistical significances were evaluated using two-tailed Student’s t-tests (b,c (EPSC variance, q, and p)) and Kruskal-Wallis tests (c(N)).

Source data

Extended Data Fig. 9 Recovery from depression is slowed by loss of Syt3 at cerebellar mossy fibre synapses.

a) Schematic of cerebellar mossy fibre recordings. EPSCs were recorded from voltage-clamped granule cells (GC) while mossy fibres (MF) were activated by electrical stimulation in the white matter 50-100 μm from the cell. b) Representative EPSCs elicited by 100 stimuli at 200 Hz, followed by stimuli at varying intervals to probe recovery from depression (ΔtRecovery). c-d) Time course of recovery of EPSCs in the first 0.5 s after trains (c, linear scale) and 10 s (d, logarithmic scale). e) Weighted time constants of biexponential recovery (τw) (P = 0.01). Data are mean ± s.e.m. Number of experiments are shown in Extended Data Table 1. Statistical significances are shown as *: P < 0.05. Significances were evaluated using two-tailed Student’s t-tests.

Source data

Extended Data Fig. 10 Recovery from depression is not affected in Syt7 KOs at calyx of Held or climbing fibre synapses.

a) Representative EPSCs elicited by 100 stimuli at 200 Hz, followed by stimuli at varying intervals to probe recovery from depression (ΔtRecovery) at the calyx of Held in WT (black) and Syt7 KO (red) synapses. b) First second of recovery of EPSCs after 200 Hz stimulation, fit with biexponential curves. c) Full time-course of recovery of EPSCs after 200 Hz stimulation. d) Weighted time constant of biexponential recovery (τw) for both genotypes (P = 0.40). e) Superimposed recordings of EPSCs evoked by pairs of stimuli of climbing fibre synapses with varying ΔtRecovery. f-g) Time-course of recovery from depression in linear (f) and logarithmic scale (g). h) Weighted time constant of biexponential recovery (τw) for both genotypes (P = 0.14). WT data were reproduced from Figs. 2 & 4. Data are mean ± s.e.m. Number of experiments are shown in Extended Data Table 1. Significances were evaluated using Kruskal-Wallis tests (d) and two-tailed Student’s t-tests (h). Critical significance thresholds were post hoc Šidák corrected.

Source data

Extended Data Fig. 11 SYT3 drives facilitation at depressing synapses in low extracellular Ca2+.

a) Superimposed recordings of EPSCs evoked by pairs of stimuli at the calyx of Held at varying intervals (Δt) in 0.6 mM [Ca2+]e. b) Average paired-pulse ratios at the calyx of Held in 0.6 mM [Ca2+]e fit exponentially. c) Average paired-pulse ratios at Δt = 3 ms and time constant of exponential fits to data in b (P = 7.5 * 10−5). d) Representative EPSCs at the calyx of Held evoked by stimulation at 200 Hz. e) Average normalized EPSCs during 200 Hz stimulation. f) Normalized amplitude of the last 3 EPSCs (EPSC8-10) during 200 Hz stimulation (P = 3.3 * 10−5). g) Superimposed recordings of EPSCs evoked by pairs of stimuli at the calyx of Held at varying Δt in 0.3 mM [Ca2+]e. h) Paired-pulse ratios in climbing fibres in 0.3 mM [Ca2+]e fit exponentially. i) Average paired-pulse ratios at Δt = 10 ms and time constant of exponential fits to data in h (P = 5.5 * 10−7). j) Representative EPSCs evoked by climbing fibre stimulation at 50 Hz. k) Average normalized amplitudes of climbing fibre EPSCs during 50 Hz stimulation. l) Normalized amplitude of the last 3 EPSCs (EPSC8-10) during 50 Hz stimulation (P = 0.02). Data are mean ± s.e.m. Number of experiments are shown in Extended Data Table 1. Statistical significances are shown as *: P < 0.05, **: P < 0.01, ***: P < 0.001. Significances were evaluated using Kruskal-Wallis tests (c) and two-tailed Student’s t-tests (b,e,f,h,i,l).

Source data

Extended Data Fig. 12 SYT3 drives facilitation at calyx of Held at 34 °C in low extracellular Ca2+.

a) Superimposed recordings of EPSCs evoked by pairs of stimuli at the calyx of Held at varying intervals (Δt) in 0.6 mM [Ca2+]e. b) Average paired-pulse ratios at the calyx of Held in 0.6 mM [Ca2+]e fit exponentially. c) Average paired-pulse ratios at Δt = 2 ms and time constant of exponential fits to data in b (P = 0.001). d) Representative EPSCs at the calyx of Held evoked by stimulation at 200 Hz. e) Average normalized EPSCs during 200 Hz stimulation. f) Normalized amplitude of the last 3 EPSCs (EPSC8-10) during 200 Hz stimulation (P = 0.004). g) Best fit to WT and KO data of the biophysical model of SYT3-dependent vesicle docking (Extended Data Fig. 7d). Data are mean ± s.e.m. Number of experiments are shown in Extended Data Table 1. Statistical significances are shown as **: P < 0.01. Significances were evaluated using two-tailed Student’s t-tests (b, e) and Kruskal-Wallis tests (c, f).

Source data

Extended Data Fig. 13 SYT3 contributes to facilitation at cerebellar parallel fibre synapses.

a) Schematic of cerebellar parallel fibre recordings. EPSCs were recorded from voltage-clamped Purkinje cells (PC) while parallel fibres were activated by electrical stimulation in the molecular layer. b) Superimposed recordings of EPSCs evoked by pairs of stimuli at the at varying intervals (Δt) in WT (black), Syt3 KO (magenta) and Syt7 KO (red) synapses. c) Average paired-pulse ratios fit exponentially. d) Average paired-pulse ratios at Δt = 5 ms and time constant of exponential fits to data in b. e) Representative EPSCs evoked by 20 stimuli at 50 Hz. f) Average normalized EPSCs during 50 Hz stimulation. Magenta and red bars indicate significant differences between WT and Syt3 KO, or WT and Syt7 KO, respectively. Gray bar indicates significant differences between Syt3 KO and Syt7 KO. g) Normalized amplitude of the last 5 EPSCs (EPSC16-20) during 50 Hz stimulation (WT vs. Syt3 KO: P = 0.0002; WT vs. Syt7 KO: P = 0.009; Syt3 KO vs. Syt7 KO: P = 0.007). Data are mean ± s.e.m. Number of experiments are shown in Extended Data Table 1. Statistical significances are shown as *: P < 0.05, **: P < 0.01, ***: P < 0.001. Significances were evaluated using one way ANOVA followed by two-tailed Student’s t-tests. Critical significance thresholds were post hoc Šidák corrected.

Source data

Extended Data Table 1 Number of recordings from WT and Syt3 KO animals
Extended Data Table 2 Parameters for biophysical models

Supplementary information

Supplementary Fig. 1

Uncropped images of western blots used in Fig. 1h.

Reporting Summary

Peer Review File

Source data

Rights and permissions

Springer Nature or its licensor holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Weingarten, D.J., Shrestha, A., Juda-Nelson, K. et al. Fast resupply of synaptic vesicles requires synaptotagmin-3. Nature 611, 320–325 (2022). https://doi.org/10.1038/s41586-022-05337-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41586-022-05337-1

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

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