At most synapses in the brain, short-term plasticity dynamically modulates synaptic strength. Rapid frequency-dependent changes in synaptic strength have key roles in sensory adaptation, gain control and many other neural computations1,2. However, some auditory, vestibular and cerebellar synapses maintain constant strength over a wide range of firing frequencies3,4,5, and as a result efficiently encode firing rates. Despite its apparent simplicity, frequency-invariant transmission is difficult to achieve because of inherent synaptic nonlinearities6. Here we study frequency-invariant transmission at synapses from Purkinje cells to deep cerebellar nuclei and at vestibular synapses in mice. Prolonged activation of these synapses leads to initial depression, which is followed by steady-state responses that are frequency invariant for their physiological activity range. We find that synaptotagmin 7 (Syt7), a calcium sensor for short-term facilitation7, is present at both synapses. It was unclear why a sensor for facilitation would be present at these and other depressing synapses. We find that at Purkinje cell and vestibular synapses, Syt7 supports facilitation that is normally masked by depression, which can be revealed in wild-type mice but is absent in Syt7 knockout mice. In wild-type mice, facilitation increases with firing frequency and counteracts depression to produce frequency-invariant transmission. In Syt7-knockout mice, Purkinje cell and vestibular synapses exhibit conventional use-dependent depression, weakening to a greater extent as the firing frequency is increased. Presynaptic rescue of Syt7 expression restores both facilitation and frequency-invariant transmission. Our results identify a function for Syt7 at synapses that exhibit overall depression, and demonstrate that facilitation has an unexpected and important function in producing frequency-invariant transmission.
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.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Abbott, L. F., Varela, J. A., Sen, K. & Nelson, S. B. Synaptic depression and cortical gain control. Science 275, 220–224 (1997)
Abbott, L. F. & Regehr, W. G. Synaptic computation. Nature 431, 796–803 (2004)
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)
Bagnall, M. W., McElvain, L. E., Faulstich, M. & du Lac, S. Frequency-independent synaptic transmission supports a linear vestibular behavior. Neuron 60, 343–352 (2008)
Turecek, J., Jackman, S. L. & Regehr, W. G. Synaptic specializations support frequency-independent Purkinje cell output from the cerebellar cortex. Cell Reports 17, 3256–3268 (2016)
Zucker, R. S. & Regehr, W. G. Short-term synaptic plasticity. Annu. Rev. Physiol. 64, 355–405 (2002)
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)
Cook, D. L., Schwindt, P. C., Grande, L. A. & Spain, W. J. Synaptic depression in the localization of sound. Nature 421, 66–70 (2003)
Galarreta, M. & Hestrin, S. Frequency-dependent synaptic depression and the balance of excitation and inhibition in the neocortex. Nat. Neurosci. 1, 587–594 (1998)
Brenowitz, S., David, J. & Trussell, L. Enhancement of synaptic efficacy by presynaptic GABA(B) receptors. Neuron 20, 135–141 (1998)
McElvain, L. E., Faulstich, M., Jeanne, J. M., Moore, J. D. & du Lac, S. Implementation of linear sensory signaling via multiple coordinated mechanisms at central vestibular nerve synapses. Neuron 85, 1132–1144 (2015)
Zhou, H. et al. Cerebellar modules operate at different frequencies. eLife 3, e02536 (2014)
Liu, H. et al. Synaptotagmin 7 functions as a Ca2+-sensor for synaptic vesicle replenishment. eLife 3, e01524 (2014)
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)
Luo, F. & Sudhof, T. C. Synaptotagmin-7-mediated asynchronous release boosts high-fidelity synchronous transmission at a central synapse. Neuron 94, 826–839, (2017)
Müller, M., Goutman, J. D., Kochubey, O. & Schneggenburger, R. Interaction between facilitation and depression at a large CNS synapse reveals mechanisms of short-term plasticity. J. Neurosci. 30, 2007–2016 (2010)
Wu, D. et al. Postsynaptic synaptotagmins mediate AMPA receptor exocytosis during LTP. Nature 544, 316–321 (2017)
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)
Borst, J. G. The low synaptic release probability in vivo. Trends Neurosci. 33, 259–266 (2010)
Arenz, A., Silver, R. A., Schaefer, A. T. & Margrie, T. W. The contribution of single synapses to sensory representation in vivo. Science 321, 977–980 (2008)
Kuenzel, T., Borst, J. G. & van der Heijden, M. Factors controlling the input-output relationship of spherical bushy cells in the gerbil cochlear nucleus. J. Neurosci. 31, 4260–4273 (2011)
Sugita, S. et al. Synaptotagmin VII as a plasma membrane Ca2+ sensor in exocytosis. Neuron 30, 459–473 (2001)
Bakken, T. E. et al. A comprehensive transcriptional map of primate brain development. Nature 535, 367–375 (2016)
Lein, E. S. et al. Genome-wide atlas of gene expression in the adult mouse brain. Nature 445, 168–176 (2007)
Jackman, S. L. & Regehr, W. G. The mechanisms and functions of synaptic facilitation. Neuron 94, 447–464 (2017)
Brandt, D. S., Coffman, M. D., Falke, J. J. & Knight, J. D. Hydrophobic contributions to the membrane docking of synaptotagmin 7 C2A domain: mechanistic contrast between isoforms 1 and 7. Biochemistry 51, 7654–7664 (2012)
Robinson, D. A. The use of control systems analysis in the neurophysiology of eye movements. Annu. Rev. Neurosci. 4, 463–503 (1981)
Sullivan, W. E. & Konishi, M. Segregation of stimulus phase and intensity coding in the cochlear nucleus of the barn owl. J. Neurosci. 4, 1787–1799 (1984)
Chakrabarti, S. et al. Impaired membrane resealing and autoimmune myositis in synaptotagmin VII-deficient mice. J. Cell Biol. 162, 543–549 (2003)
Telgkamp, P., Padgett, D. E., Ledoux, V. A., Woolley, C. S. & Raman, I. M. Maintenance of high-frequency transmission at Purkinje to cerebellar nuclear synapses by spillover from boutons with multiple release sites. Neuron 41, 113–126 (2004)
Sakaba, T. & Neher, E. Calmodulin mediates rapid recruitment of fast-releasing synaptic vesicles at a calyx-type synapse. Neuron 32, 1119–1131 (2001)
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)
Stevens, C. F. & Wesseling, J. F. Activity-dependent modulation of the rate at which synaptic vesicles become available to undergo exocytosis. Neuron 21, 415–424 (1998)
Wang, L. Y. & Kaczmarek, L. K. High-frequency firing helps replenish the readily releasable pool of synaptic vesicles. Nature 394, 384–388 (1998)
Yang, H. & Xu-Friedman, M. A. Impact of synaptic depression on spike timing at the endbulb of Held. J. Neurophysiol. 102, 1699–1710 (2009)
McElvain, L. E., Bagnall, M. W., Sakatos, A. & du Lac, S. Bidirectional plasticity gated by hyperpolarization controls the gain of postsynaptic firing responses at central vestibular nerve synapses. Neuron 68, 763–775 (2010)
Telgkamp, P. & Raman, I. M. Depression of inhibitory synaptic transmission between Purkinje cells and neurons of the cerebellar nuclei. J. Neurosci. 22, 8447–8457 (2002)
Atluri, P. P. & Regehr, W. G. Delayed release of neurotransmitter from cerebellar granule cells. J. Neurosci. 18, 8214–8227 (1998)
Hefft, S. & Jonas, P. Asynchronous GABA release generates long-lasting inhibition at a hippocampal interneuron-principal neuron synapse. Nat. Neurosci. 8, 1319–1328 (2005)
Lu, H. W. & Trussell, L. O. Spontaneous activity defines effective convergence ratios in an inhibitory circuit. J. Neurosci. 36, 3268–3280 (2016)
Trommershäuser, J., Schneggenburger, R., Zippelius, A. & Neher, E. Heterogeneous presynaptic release probabilities: functional relevance for short-term plasticity. Biophys. J. 84, 1563–1579 (2003)
Thanawala, M. S. & Regehr, W. G. Determining synaptic parameters using high-frequency activation. J. Neurosci. Methods 264, 136–152 (2016)
We thank P. Kaeser and the Regehr laboratory for comments on the manuscript. This work was supported by grants from the NIH (R01NS032405 and R35NS097284) and a Nancy Lurie Marks grant 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.
Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
Short-term plasticity at synapses can be tuned to perform different computations. a, Many synapses in the brain undergo short-term depression that is use-dependent: elevations in presynaptic firing rate (top) result in more profound depression of postsynaptic currents (EPSCs, middle). The charge transfer that ultimately drives postsynaptic firing increases rapidly with presynaptic firing, but short-term depression of the synapse reduces charge transfer back to a similar steady state. Thus, short-term depression is suited to convey temporal information about changes in presynaptic firing. b, Some synapses maintain constant strength across firing frequencies (top, middle). Charge transfer at these synapses can therefore reliably reflect the absolute rate of presynaptic firing. c, Typical depressing synapses can be well-approximated by intrinsic synaptic properties alone. Synapses have a limited number of vesicles in the readily releasable pool (RRP). High-frequency presynaptic activity depletes the RRP until it can be replenished at some rate (R). Less recovery occurs as the interval between stimuli is reduced, thereby leading to an increase in depletion during high-frequency stimulation. d–f, Other synaptic features must operate in order to generate frequency-invariant synapses, and several possibilities have been proposed. d, One way of making transmission frequency-invariant would be to balance depletion with facilitation3,5 (Extended Data Fig. 9). Activating a mechanism of short-term facilitation with high-frequency stimulation increases PR, releasing more vesicles from the RRP. The increase in release results in steady-state transmission that is consistent across the physiological firing range. e, Another way of generating a frequency-invariant synapse would be to have a rapid, calcium-dependent increase in R31,32,33,34,35. When the presynaptic frequency is elevated, presynaptic calcium and the rate of replenishment are increased to maintain the same RRP size regardless of activation frequency. However, this cannot explain the frequency invariance of PC and vestibular synapses, where recovery from depression is insensitive to stimulation frequency in juvenile animals5,36. f, In this proposed model36, each release site can hold two vesicles, but only one can be released per stimulus (green, releasable; orange, non-releasable). Vesicle replenishment to each release site is very rapid, and release is limited by a decrease in PR. Decreases in PR are independent of stimulation frequency. The high replenishment rate and additional vesicle per release site results in very little vesicle depletion, and responses are instead shaped by decreases in PR that are constant across frequencies. Another possibility is that a synapse could have very low PR to limit vesicle depletion at each release site, but still maintain synaptic strength by having a very large number of release sites (not shown). However, such a mechanism would require an extremely large number of release sites, and is inconsistent with the depression present at PC and vestibular synapses. Finally, postsynaptic mechanisms of short-term plasticity, such as receptor saturation and desensitization, could also contribute to frequency-invariant transmission (not shown). These mechanisms are unlikely to contribute to frequency invariance at PC and vestibular synapses because frequency-independent transmission is unaltered when saturation and desensitization are minimized by competitive low-affinity postsynaptic receptor antagonists4,5.
To determine whether Syt7 was present at PC synapses in the DCN, we compared immunohistochemical labelling in mice expressing synaptophysin–TdTomato (Syp–TdT) specifically in PCs (PCP2–Cre × Syp–TdT). a, Syp–TdT could be observed in boutons surrounding neurons in the DCN (upper left). Immunolabelling of calbindin could be seen both in PC boutons labelled with Syp–TdT, and throughout the length of PC axons traversing the DCN (upper middle). Syt2 was also highly expressed in PC boutons, with most Syt2 puncta co-localizing with Syp–TdT (upper right). We found that the vast majority of inhibitory synapses labelled by VGAT were Syp–TdT positive, suggesting that most inhibitory input to the DCN arises from PCs (lower middle). Syt7 was also prominent in PC boutons labelled by Syp–TdT, but with less punctate expression compared to Syt2 or VGAT. Scale bar, 15 μm. b, The expression of Syt2 was stable across development, and showed no prominent differences in intensity between wild-type and Syt7 knockout mice. Scale bar, 20 μm.
Extended Data Figure 3 Alterations in short-term depression at PC to DCN synapses in Syt7 knockout mice are consistent with the predicted consequences of eliminating facilitation.
High-frequency stimulation of the PC–DCN synapse leads to depressing IPSCs that can be approximated by the equation IPSC = IPSCSS + (1 − IPSCSS)e−S/λ, where S is the stimulus number, IPSCSS is the steady-state IPSC amplitude, and λ is the exponential decay constant. a, Average normalized IPSC amplitude for 10- and 100-Hz trains and fits in P13–15 wild-type (left) and Syt7 knockout (middle) mice. λ is plotted as a function of stimulation frequency (right). b, As in a, but for P21–32 mice. c, As in a, but for P80–110 mice. According to a model of the PC–DCN synapse, both depression and facilitation are present, but depletion of the readily releasable pool dominates and leads to depression during high-frequency stimulation5. In wild-type mice, λ is prolonged in a frequency-dependent manner, and it has been hypothesized that this arises from short-term facilitation that is more prominent as the stimulus frequency is increased. In young animals, when Syt7 expression is low (Fig. 1c), λ is weakly modulated by stimulation frequency. The prolongation of λ is more prominent in juveniles and adults. In the absence of Syt7, λ is not frequency dependent at any age. These observations are consistent with an age-dependent increase in Syt7 expression in wild-type mice leading to an age-dependent increase in facilitation, which in turn leads to age-dependent increases in the frequency dependence of λ. Data are mean ± s.e.m. Number of experiments shown in Extended Data Table 1.
Extended Data Figure 4 The frequency dependence of synaptic strength is consistent for different cells for a given age and genotype.
a, The steady-state IPSC amplitude as a function of frequency for each cell analysed (thin lines) and averages (markers) for each genotype and age. Normalized steady-state IPSC amplitudes across frequencies are plotted for wild-type (left) and Syt7 knockout mice (right) at P13–15, P21–32, and P80–110. b, Ratio of the steady-state IPSC amplitudes at 100 Hz divided by steady-state amplitudes at 10 Hz is summarized for different ages of wild-type and Syt7 knockout mice, and in P21–32 mice in the presence of the low-affinity GABAA receptor antagonist TPMPA (2 mM, far right). Data from young animals are consistent with previous reports5,30,37. **P < 0.01, unpaired two-tailed Student’s t-test. Data are mean ± s.e.m. Number of experiments shown in Extended Data Table 1.
Extended Data Figure 5 Altered recovery from depression in Syt7 knockout mice cannot account for the loss of frequency invariance at the PC–DCN synapse in Syt7 knockout mice at P21–32.
It has been shown previously that the loss of Syt7 in hippocampal cultures slows recovery from depression13. If recovery from depression were slower in Syt7 knockouts, it could partially explain the reduction of sustained transmission at high frequencies. We therefore examined recovery from depression at PC–DCN synapses. One hundred stimuli at 100 Hz were followed by a single stimulus after an interval. This was repeated for many trials and a range of time intervals. Experiments were performed in wild-type (a) and Syt7 knockout (b) mice. a, PC–DCN synapses recovered slowly with a single exponential of 7.7 s (left). b, In Syt7 knockout mice, recovery could not be approximated by a single exponential but was well approximated by a double exponential with time constants of 280 ms and 5.1 s. These findings indicate that a slowed recovery from depression does not occur in Syt7 knockout mice and thus does not contribute to the reduced steady-state responses in Syt7 knockout mice. The rapid time constant of recovery from depression that is apparent in Syt7 knockout mice is consistent with the prediction of a model of the PC–DCN synapse in which the decay of facilitation obscures this rapid phase of recovery in wild-type mice5 (Extended Data Fig. 9). The role of Syt7 in recovery from depression at the PC–DCN synapse differs from that at cultured hippocampal synapses, where calcium-dependent recovery from depression relies on Syt7, and recovery from depression is slowed in the absence of Syt713. Data are mean ± s.e.m. Number of experiments shown in Extended Data Table 1.
a, PCs fire at high rates spontaneously in vivo. We therefore examined whether the PC–DCN synapse could sustain transmission for prolonged high-frequency activation. a, Example of prolonged stimulation of PC axons (500 stimuli, 100 Hz), average of four trials (top), and average across cells (bottom) and fit with the equation IPSC = IPSCSS + (1 − IPSCSS)e−S/λ as in Extended Data Fig. 3. Vertical scale bar, 1 nA. b–g, At some synapses, asynchronous release becomes more prominent with prolonged high-frequency stimulation14,15,38,39. At these synapses, fast synchronous release can be seen riding on top of a slowly decaying current that is, in part, mediated by asynchronous release. We therefore investigated whether asynchronous release contributes to transmission during trains at the PC–DCN synapse. b, Example trains of 100 stimuli at 100 Hz from a wild-type (black) and Syt7 knockout (red) mouse. Responses evoked by the entire train are plotted (top) and regions within the dashed boxes are shown on an expanded scale (bottom). We measured the average decay time constant of IPSCs during the train (average IPSC number 50–99, τIPSC), and for the last IPSC in the train (τTrain). IPSCs were well fit by a single exponential decay with similar values of τ. This indicates that asynchronous release is not prominent at this synapse. Vertical scale bars, 0.5 nA; dashed line indicates baseline before the train. c, Average decay time for IPSCs during the train, and for the last IPSC in the train to decay back to baseline measured before the onset of stimulation. No significant differences were found between wild-typeτIPSC and wild-type τtrain (P = 0.36), Syt7 knockout τIPSC and Syt7 knockout τtrain (P = 0.71), wild-type τIPSC and Syt7 knockout τIPSC (P = 0.23), or wild-type τtrain and Syt7 knockout τtrain (P = 0.48), unpaired two-tailed student’s t-test. d, Charge transfer was measured in two different ways to isolate different components of release. The average incremental IPSC amplitude was multiplied by the stimulation rate (left), or traces were integrated and multiplied by stimulation rate (right). The average charge transfer as a function of stimulation frequency, calculated either by IPSC amplitude, or by integration, for young (e, P12–15), juvenile (f, P21–32), and adult (g, P80–110) wild-type and Syt7 knockout mice is shown. Data are mean ± s.e.m. Number of experiments shown in Extended Data Table 1.
Single PC axons provide powerful inhibition on their postsynaptic targets in the DCN, and can be identified by their all-or-nothing nature. If the loss of Syt7 caused elevations in PR, it would be expected to increase the strength of unitary fibre inputs. Experiments were done using minimal stimulation to determine the amplitudes of single PC–DCN inputs. a, The stimulus was adjusted so that, at a constant intensity, synaptic inputs were activated approximately half the time in a stochastic manner. This is shown for 100 superimposed traces (left), along with average of failures (thick grey) and average of successes (thick black), for the amplitude histogram for the events recorded in that cell (middle), and for the IPSC amplitude as a function of trial number (bottom). Vertical scale bar, 0.5 nA. b, Amplitude histogram of single-fibre conductances for wild-type (black) and Syt7 knockout mice (red). c, Cumulative amplitude histograms of single-fibre conductances. No significant difference was found between wild-type (black) and Syt7 knockout mice (red). P = 0.43, Kolmogorov–Smirnov test. Number of experiments shown in Extended Data Table 1.
a, The steady-state EPSC size as a function of stimulation frequency for each cell analysed (thin lines) and averages (markers) for wildtype (left, black) and Syt7 knockouts (red, right). b, Ratio of the steady-state EPSC amplitudes at 100 Hz divided by steady-state amplitudes at 10 Hz is summarized for vestibular synapses in wildtype and Syt7 knockouts. **P < 0.01, unpaired two-tailed Student’s t-test. Data are mean ± s.e.m. Number of experiments shown in Extended Data Table 1.
Extended Data Figure 9 A model of the PC–DCN synapse indicates that eliminating facilitation can explain the loss of frequency-invariant transmission and other synaptic changes observed in Syt7 knockout mice.
We extended a previously described model of PC–DCN synapses to explore transmission in Syt7 knockout mice5. The model consists of two pools of vesicles. Pool 1 vesicles have a high release probability and are replenished slowly. Pool 2 vesicles have a low initial release probability that increases with facilitation (in wild-type animals only), and replenish rapidly. The existence of multiple vesicle pools has been proposed at several types of synapses40,41,42. The model was constrained by many experiments and in the simplest configuration could account for all experimental observations5. The model is compared to data from wild-type and Syt7 knockout mice (a1–d1). The contribution of each pool and its properties (IPSC size a2–d2, release probability PR a3–d3, and readily releasable pool size RRP a4–d4) are also shown for each experiment. a, During high-frequency stimulation in wild-type mice (a1–2, black), Pool 1 primarily contributes to depression seen during the onset of stimulation, but is strongly depleted during sustained firing (a2, dashed grey). Pool 2 facilitates and maintains release at steady-state (a2–3, solid grey). We modelled Syt7 knockout mice by removing facilitation (a3, red). When facilitation was eliminated, steady-state transmission was reduced during high-frequency stimulation because fewer vesicles were released from Pool 2 (a4, light red). b, In the model of wild-type synapses, the magnitude of facilitation increased with stimulation frequency (b3, grey), allowing more of the RRP of Pool 2 (RRP2) to be released at high frequencies, resulting in similar IPSC amplitude across frequencies (b1–2, black). When facilitation was removed (b3, red), transmission was no longer frequency invariant, because fewer vesicles were released at high frequencies (b1,4, red). c, When the frequency of stimulation was stepped from 10 to 100 Hz, a transient enhancement was observed in wild-type mice (c1, black markers). In the model, this transient enhancement is mediated by facilitation of Pool 2 (c3, grey). Facilitation is weakly activated by 10 Hz stimulation, but increases when stepping to 100 Hz stimulation. As more vesicles are released, RRP2 is partially depleted and the IPSC amplitude depresses, ultimately reaching steady-state levels that are similar to those reached during 10 Hz stimulation. When facilitation is removed, no transient enhancement occurs, and the IPSC amplitude simply depresses (c1–2, red). d, The model is also able to explain recovery from depression. In wild-type mice, a single slow recovery is observed (d1, black markers) because the decay of facilitation obscures the rapid recovery of RRP2 (d3–4, τFdeact). When facilitation is removed, this rapid component of recovery is unmasked (d1–2, red). The known role of Syt7 in facilitation, and the fact that the many alterations in synaptic responses in Syt7 knockout animals are explained by eliminating facilitation in this model, support the importance of Syt7-mediated facilitation in frequency-invariant transmission.
About this article
Cite this article
Turecek, J., Jackman, S. & Regehr, W. Synaptotagmin 7 confers frequency invariance onto specialized depressing synapses. Nature 551, 503–506 (2017). https://doi.org/10.1038/nature24474
Current Opinion in Neurobiology (2020)
Loss of Doc2b does not influence transmission at Purkinje cell to deep nuclei synapses under physiological conditions
The Synaptic Vesicle Priming Protein CAPS-1 Shapes the Adaptation of Sensory Evoked Responses in Mouse Visual Cortex
Cell Reports (2020)