It has been known for more than 70 years that synaptic strength is dynamically regulated in a use-dependent manner1. At synapses with a low initial release probability, closely spaced presynaptic action potentials can result in facilitation, a short-term form of enhancement in which each subsequent action potential evokes greater neurotransmitter release2. Facilitation can enhance neurotransmitter release considerably and can profoundly influence information transfer across synapses3, but the underlying mechanism remains a mystery. One proposed mechanism is that a specialized calcium sensor for facilitation transiently increases the probability of release2,4, and this sensor is distinct from the fast sensors that mediate rapid neurotransmitter release. Yet such a sensor has never been identified, and its very existence has been disputed5,6. Here we show that synaptotagmin 7 (Syt7) is a calcium sensor that is required for facilitation at several central synapses. In Syt7-knockout mice, facilitation is eliminated even though the initial probability of release and the presynaptic residual calcium signals are unaltered. Expression of wild-type Syt7 in presynaptic neurons restored facilitation, whereas expression of a mutated Syt7 with a calcium-insensitive C2A domain did not. By revealing the role of Syt7 in synaptic facilitation, these results resolve a longstanding debate about a widespread form of short-term plasticity, and will enable future studies that may lead to a deeper understanding of the functional importance of facilitation.
This is a preview of subscription content, access via your institution
Open Access articles citing this article.
Cancer Cell International Open Access 20 December 2021
Scientific Reports Open Access 31 May 2021
Orphanet Journal of Rare Diseases Open Access 13 May 2021
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Get time limited or full article access on ReadCube.
All prices are NET prices.
Feng, T. P. Studies on the neuromuscular junction. XVIII. The local potentials around n-m junctions induced by single and multiple volleys. Chin. J. Physiol. 15, 367–404 (1940)
Zucker, R. S. & Regehr, W. G. Short-term synaptic plasticity. Annu. Rev. Physiol. 64, 355–405 (2002)
Abbott, L. F. & Regehr, W. G. Synaptic computation. Nature 431, 796–803 (2004)
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)
Bertram, R., Sherman, A. & Stanley, E. F. Single-domain/bound calcium hypothesis of transmitter release and facilitation. J. Neurophysiol. 75, 1919–1931 (1996)
Felmy, F., Neher, E. & Schneggenburger, R. Probing the intracellular calcium sensitivity of transmitter release during synaptic facilitation. Neuron 37, 801–811 (2003)
Südhof, T. C. A molecular machine for neurotransmitter release: synaptotagmin and beyond. Nature Med. 19, 1227–1231 (2013)
Matveev, V., Zucker, R. S. & Sherman, A. Facilitation through buffer saturation: Constraints on endogenous buffering properties. Biophys. J. 86, 2691–2709 (2004)
Blatow, M., Caputi, A., Burnashev, N., Monyer, H. & Rozov, A. Ca2+ buffer saturation underlies paired pulse facilitation in calbindin-D28k-containing terminals. Neuron 38, 79–88 (2003)
Mochida, S., Few, A. P., Scheuer, T. & Catterall, W. A. Regulation of presynaptic CaV2.1 channels by Ca2+ sensor proteins mediates short-term synaptic plasticity. Neuron 57, 210–216 (2008)
Sippy, T., Cruz-Martin, A., Jeromin, A. & Schweizer, F. E. Acute changes in short-term plasticity at synapses with elevated levels of neuronal calcium sensor-1. Nature Neurosci. 6, 1031–1038 (2003)
Tsujimoto, T., Jeromin, A., Saitoh, N., Roder, J. C. & Takahashi, T. Neuronal calcium sensor 1 and activity-dependent facilitation of P/Q-type calcium currents at presynaptic nerve terminals. Science 295, 2276–2279 (2002)
Müller, M., Felmy, F. & Schneggenburger, R. A limited contribution of Ca2+ current facilitation to paired-pulse facilitation of transmitter release at the rat calyx of Held. J. Physiol. (Lond.) 586, 5503–5520 (2008)
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)
Li, C. et al. Ca2+ -dependent and -independent activities of neural and non-neural synaptotagmins. Nature 375, 594–599 (1995)
Sugita, S. et al. Synaptotagmin VII as a plasma membrane Ca2+ sensor in exocytosis. Neuron 30, 459–473 (2001)
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)
Bacaj, T. et al. Synaptotagmin-1 and synaptotagmin-7 trigger synchronous and asynchronous phases of neurotransmitter release. Neuron 80, 947–959 (2013)
Liu, H. et al. Synaptotagmin 7 functions as a Ca2+ -sensor for synaptic vesicle replenishment. eLife 3, e01524 (2014)
Deschênes, M. & Hu, B. Electrophysiology and pharmacology of the corticothalamic input to lateral thalamic nuclei: an intracellular study in the cat. Eur. J. Neurosci. 2, 140–152 (1990)
Lømo, T. Potentiation of monosynaptic EPSPs in the perforant path-dentate granule cell synapse. Exp. Brain Res. 12, 46–63 (1971)
Geiger, J. R. & Jonas, P. Dynamic control of presynaptic Ca2+ inflow by fast-inactivating K+ channels in hippocampal mossy fiber boutons. Neuron 28, 927–939 (2000)
Geppert, M. et al. Synaptotagmin I: a major Ca2+ sensor for transmitter release at a central synapse. Cell 79, 717–727 (1994)
Dingledine, R. & Somjen, G. Calcium dependence of synaptic transmission in the hippocampal slice. Brain Res. 207, 218–222 (1981)
Manabe, T. & Nicoll, R. A. Long-term potentiation: evidence against an increase in transmitter release probability in the CA1 region of the hippocampus. Science 265, 1888–1892 (1994)
Gustavsson, N. et al. Impaired insulin secretion and glucose intolerance in synaptotagmin-7 null mutant mice. Proc. Natl Acad. Sci. USA 105, 3992–3997 (2008)
Martinez, I. et al. Synaptotagmin VII regulates Ca2+-dependent exocytosis of lysosomes in fibroblasts. J. Cell Biol. 148, 1141–1150 (2000)
Klyachko, V. A. & Stevens, C. F. Excitatory and feed-forward inhibitory hippocampal synapses work synergistically as an adaptive filter of natural spike trains. PLoS Biol. 4, e207 (2006)
MacLeod, K. M., Horiuchi, T. K. & Carr, C. E. A role for short-term synaptic facilitation and depression in the processing of intensity information in the auditory brain stem. J. Neurophysiol. 97, 2863–2874 (2007)
Mongillo, G., Barak, O. & Tsodyks, M. Synaptic theory of working memory. Science 319, 1543–1546 (2008)
Chakrabarti, S. et al. Impaired membrane resealing and autoimmune myositis in synaptotagmin VII-deficient mice. J. Cell Biol. 162, 543–549 (2003)
Kim, J. H. et al. High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice. PLoS ONE 6, e18556 (2011)
Zolotukhin, S. et al. Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield. Gene Ther. 6, 973–985 (1999)
Jackman, S. L., Beneduce, B. M., Drew, I. R. & Regehr, W. G. Achieving high-frequency optical control of synaptic transmission. J. Neurosci. 34, 7704–7714 (2014)
Kamiya, H., Shinozaki, H. & Yamamoto, C. Activation of metabotropic glutamate receptor type 2/3 suppresses transmission at rat hippocampal mossy fibre synapses. J. Physiol. (Lond.) 493, 447–455 (1996)
Zhao, M., Hollingworth, S. & Baylor, S. M. Properties of tri- and tetracarboxylate Ca2+ indicators in frog skeletal muscle fibers. Biophys. J. 70, 896–916 (1996)
Kreitzer, A. C. & Regehr, W. G. Modulation of transmission during trains at a cerebellar synapse. J. Neurosci. 20, 1348–1357 (2000)
Brenowitz, S. D. & Regehr, W. G. Calcium dependence of retrograde inhibition by endocannabinoids at synapses onto Purkinje cells. J. Neurosci. 23, 6373–6384 (2003)
Grynkiewicz, G., Poenie, M. & Tsien, R. Y. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J. Biol. Chem. 260, 3440–3450 (1985)
Sabatini, B. L. & Regehr, W. G. Detecting changes in calcium influx which contribute to synaptic modulation in mammalian brain slice. Neuropharmacology 34, 1453–1467 (1995)
Maravall, M., Mainen, Z. F., Sabatini, B. L. & Svoboda, K. Estimating intracellular calcium concentrations and buffering without wavelength ratioing. Biophys. J. 78, 2655–2667 (2000)
Sabatini, B. L. & Svoboda, K. Analysis of calcium channels in single spines using optical fluctuation analysis. Nature 408, 589–593 (2000)
Regehr, W. G. Short-term presynaptic plasticity. Cold Spring Harb. Perspect. Biol. 4, a005702 (2012)
Kaeser, P. S. & Regehr, W. G. Molecular mechanisms for synchronous, asynchronous, and spontaneous neurotransmitter release. Annu. Rev. Physiol. 76, 333–363 (2014)
Kamiya, H. & Zucker, R. S. Residual Ca2+ and short-term synaptic plasticity. Nature 371, 603–606 (1994)
Celio, M. R. Calbindin D-28k and parvalbumin in the rat nervous system. Neuroscience 35, 375–475 (1990)
We thank P. Kaeser and L. Bickford for help with producing AAVs, B. Sabatini and J. Levasseur for help with plasmids, K. Ennis, M. Ocana and the Neurobiology Imaging Center for help with immunohistochemistry, B. Sabatini, P. Kaeser, D. Fioravante, C. Hull and L. Glickfeld for comments on the manuscript. This work was supported by grants from the National Institutes of Health (NIH; NS032405) and Nancy Lurie Marks Foundation to W.G.R., the Vision Core and NINDS P30 Core Center grant (NS072030) to the Neurobiology Imaging Center at Harvard Medical School, and a Nancy Lurie Marks Fellowship to S.L.J.
The authors declare no competing financial interests.
Extended data figures and tables
a–d, It is established that calcium has an important role in synaptic facilitation, and several mechanisms have been proposed that involve different aspects of calcium signalling2. Here we discuss the calcium signals that evoke rapid vesicle fusion, and also those thought to be involved in facilitation (a), and three mechanisms of facilitation are presented schematically43 (b–d). a, To understand the mechanisms that have been proposed to account for facilitation, it is important to appreciate different aspects of presynaptic calcium signalling. Calcium signals are complex, but can be approximated by two components. An action potential opens calcium channels for less than a millisecond, and near open channels the calcium levels reach tens of micromolar. Release sites near calcium channels experience high local calcium levels (Calocal) that are highly dependent on the distance from open calcium channels. Calocal can be reduced by high concentrations of fast calcium buffers that rapidly bind calcium. In addition, there is a residual calcium signal (Cares) that results from calcium equilibrating within presynaptic terminals, before calcium is gradually removed over tens to hundreds of milliseconds. The amplitude of Cares (and also total influx of Ca2+, Cainflux) is determined by all of the calcium channels that open, not only those that produce Calocal that drives release, and after initial equilibration Cares is roughly uniform throughout the presynaptic bouton. It is generally accepted that fast synaptic transmission is produced by calcium binding to Syt1, Syt2 or Syt9, which have low-affinity binding sites, fast kinetics, and require the binding of multiple calcium ions7,44. The time course of release follows the time course of calcium channel opening, but with a brief delay (<1 ms). Cares after a single stimulus is much smaller than Calocal. Typical fluorescence-based approaches to measure calcium readily detect Cares, but are insensitive to Calocal, which is too localized and short-lived to measure. Note the y axis is logarithmic to show both Calocal and Cares in a, but not in b–d. b, For one mechanism of facilitation, a fast calcium buffer is present in presynaptic terminals that binds calcium and reduces Calocal. Stimulation twice in rapid succession results in the same calcium influx for both stimuli. If there is no fast presynaptic buffer, the amplitudes of Calocal and the EPSCs are the same for both stimuli (red traces). If a fast high-affinity buffer is present (black traces), it reduces the initial Calocal and reduces the amplitude of the initial EPSC, but if enough calcium enters and binds to the buffer, it reduces its ability to buffer calcium. As a result, the second stimulus produces larger Calocal than the first, and the EPSC is facilitated. c, A second possible mechanism is that more calcium enters for the second stimulus, and as a result there is more neurotransmitter release. This could arise from a spike broadening, or from the modulation of calcium channels. It is possible that influx through all calcium channels in the presynaptic terminal would be increased, in which case both Cares and Calocal would be increased. It is also possible that the only calcium channels that are modulated are the subset that produce Calocal that triggers release, in which case Cares would not be significantly increased. d, Finally, it is possible that there is a specialized calcium sensor that produces facilitation that is distinct from Syt1 (refs 2, 4, 45). Previous studies have shown that such a sensor would need to be sensitive to Cares based on the observation that facilitation is altered at some synapses by manipulations that affect Cares without affecting Calocal. According to this scheme, release is mediated by Syt1 but calcium binding to a second sensor would increase p. The sensor is sufficiently slow that it does not influence release evoked by the first stimulus, but it is able to influence release evoked by a second stimulus.
a–d, Fluorescent images of immunostaining for vGlut1 (top) and syt7 (bottom) in slices from wild-type and Syt7-knockout animals, showing the stratum radiatum (SR) of hippocampal CA1 region (a), the ventral thalamus (b), mossy fibres (MF) in hippocampal CA3 (c), and the lateral and medial performant paths (LPP and MPP) in the outer molecular layer of the dentate gyrus (d). Notably, Syt7 expression in wild-type animals was higher in the LPP, where synapses exhibit facilitation, than in the MPP, where synapses exhibit depression. Scale bar, 50 μm. The presence of Syt7 labelling in regions containing CA3–CA1 synapses, layer 6 to thalamus synapses, mossy fibres synapses and LPP–granule-cell synapses that are also colabelled with antibodies to the presynaptic marker for glutamatergic synapses vGlut1, suggests that Syt7 is located presynaptically at these synapses. It is, however, difficult to obtain sufficient resolution with confocal microscopy in brain slices to unambiguously establish that Syt7 is located presynaptically at these synapses. Importantly, the Allen Brain atlas (http://www.brain-map.org) suggests that the presynaptic cells for these synapses contain messenger RNA for Syt7. Lastly, immunoelectron microscopy revealed selective staining of presynaptic boutons in the CA1 region of the hippocampus16.
Extended Data Figure 3 Immunohistochemistry of Syt7 and calbindin expression at mossy fibre synapses.
Fluorescent images of immunostaining for calbindin-D28k, which predominantly labels mossy fibres in the CA3 region of the hippocampus9,46 (top) and Syt7 (bottom) in slices from wild-type and Syt7-knockout animals. Colocalization of Syt7 and calbindin staining in wild-type animals provides further support for the expression of Syt7 in mossy fibre terminals. Scale bar, 20 μm.
Average normalized synaptic responses evoked by extracellular stimulation with trains at frequencies from 5 to 50 Hz at four synapses in slices from wild-type and Syt7-knockout animals. Enhancement during trains was eliminated for all synapses other than mossy fibre synapses, where significant enhancement was present by the fifth stimulus for 5 Hz and 10 Hz, the third stimulus for 20 Hz, and the sixth stimulus for 50 Hz (compared to 1 by a Wilcoxon signed rank test, P < 0.05). This indicates that another form of synaptic enhancement gradually builds during repetitive activation and is consistent with a specialized form of synaptic enhancement that has been described at mossy fibre synapses in which spike broadening gradually builds during repetitive activation and leads to increased calcium influx. The numbers of experiments are shown in Extended Data Table 1.
a, Representative spontaneous EPSCs (sEPSCs) recorded from voltage-clamped hippocampal CA1 cells in wild-type (black) and knockout (red) animals. Vertical scale bars, 20 pA. b, Representative sEPSCs, averaged from >50 events recorded in wild-type and knockout animals. Vertical scale bars, 10 pA. c, d, Average sEPSC amplitude (c) and frequency (d) in wild-type (n = 16) and Syt7-knockout animals (n = 18).
Extended Data Figure 6 MK801 blockade of NMDAR-mediated EPSCs reveals similar initial release probability in wild-type and knockout synapses.
a, Representative NMDAR-EPSCs recorded in wild-type and knockout animals before the application of MK801 (average of 10 traces) and after stimulation in the presence of MK801 (average response of fifteenth to twentieth stimuli). Vertical scale bars, 100 pA. b, Average NMDAR-EPSCs recorded in the presence of MK801, normalized to the first stimulus. c, Half-decay times of NMDAR-EPSC amplitudes. *P < 0.05, one-way ANOVA with Tukey’s post-hoc test. Data represent mean ± s.e.m. The number of experiments is shown in Extended Data Table 2.
Extended Data Figure 7 Effect of virally expressed Syt7 wild-type and Syt7(C2A*) in wild-type animals.
a, b, Top, AAV was injected into the hippocampal CA3 region in wild-type animals to express ChR2 and either wild-type Syt7 (a) or Syt7(C2A*). Bottom, representative EPSCs and average paired-pulse ratios for responses evoked electrically and optically in wild-type slices with AAV-driven expression of wild-type Syt7 (electrical, n = 12; optical, n = 13) (a) and Syt7(C2A*) (electrical, n = 5; optical, n = 13) (b). Vertical scale bars, 100 pA.
Extended Data Figure 8 Evidence suggests that Syt7 does not produce facilitation by acting as a local calcium buffer at the CA3–CA1 synapse.
This graph illustrates the general relationship between PPR and external calcium for synapses in which buffer saturation produces facilitation (green) and for facilitation observed at the CA3–CA1 synapse and many other synapses (black)9. It has been shown previously that the for buffer saturation mechanism (Extended Data Fig. 1b) the amplitude of facilitation is reduced when Cainflux is reduced by lowering external calcium9. This can be understood by considering that this form of facilitation is thought to require sufficient Cainflux to saturate the endogenous buffer, and thereby reduce its ability to buffer calcium for subsequent stimuli. If Cainflux is low, then there is insufficient calcium entry to bind very much of the endogenous buffer, and little facilitation would result. In addition, as shown in Extended Data Fig. 1, for a calcium buffer to produce facilitation it would need to buffer calcium sufficiently that it would reduce initial p. We have shown, however, that p is unaltered in Syt7 knockouts. This is perhaps not surprising in light of the fact that Syt7 is thought to be located on the plasma membrane, and in cases where this type of facilitation has been observed it is associated with high concentrations of a fast cytosolic buffer9.
About this article
Cite this article
Jackman, S., Turecek, J., Belinsky, J. et al. The calcium sensor synaptotagmin 7 is required for synaptic facilitation. Nature 529, 88–91 (2016). https://doi.org/10.1038/nature16507
This article is cited by
Orphanet Journal of Rare Diseases (2021)
Cancer Cell International (2021)
Subcellular patch-clamp techniques for single-bouton stimulation and simultaneous pre- and postsynaptic recording at cortical synapses
Nature Protocols (2021)
Synaptotagmin 7 switches short-term synaptic plasticity from depression to facilitation by suppressing synaptic transmission
Scientific Reports (2021)
Scientific Reports (2021)