Synaptotagmin-7 is required for synchronous but not asynchronous facilitation of glutamate release at cortical boutons

  1. Dimitris Kotzadimitriou
  2. Helen Langley
  3. Eleanor McGowan
  4. Philipe R. F. Mendonca
  5. Erica Tagliatti
  6. Yulia Timofeeva
  7. Shyam S. Krishnakumar
  8. Kirill E. Volynski

  • Curated by eLife

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    eLife Assessment

    This important work substantially advances our understanding of the role of synaptotagmin-7 (Syt7) in short-term plasticity at cortical glutamatergic synapses. The evidence supporting the conclusions is convincing, with rigorous and elegant quantal-level iGluSnFR imaging and failure-based analyses at single boutons. The work will be of broad interest to synaptic physiologists and molecular biologists.

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Abstract

Short-term synaptic plasticity and the kinetics of neurotransmitter release vary widely across synapses, but population measurements obscure the mechanisms that generate this diversity. While the Ca 2+ sensor Synaptotagmin-7 (Syt7) has been implicated in facilitation, vesicle replenishment and asynchronous vesicle exocytosis, its precise contributions to these processes remain debated. We used quantal-resolution imaging to measure synchronous and asynchronous glutamate release at individual cortical boutons in wild type and Syt7 -/- neurons. Stratifying boutons by release efficacy and applying failure-based analysis to isolate trials where the first action potential evoked no release allowed us to separate facilitation from vesicle depletion. Syt7 deletion selectively eliminated activity-dependent facilitation of synchronous release but left facilitation of asynchronous release intact, although its overall magnitude was reduced. We further show that synchronous and asynchronous events arise from functionally distinct vesicle populations. These findings demonstrate that activity-dependent facilitation of synchronous and asynchronous exocytosis are mechanistically separable, enabling synapses to independently tune distinct temporal components of neurotransmission.

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  1. eLife

    eLife Assessment

    This important work substantially advances our understanding of the role of synaptotagmin-7 (Syt7) in short-term plasticity at cortical glutamatergic synapses. The evidence supporting the conclusions is convincing, with rigorous and elegant quantal-level iGluSnFR imaging and failure-based analyses at single boutons. The work will be of broad interest to synaptic physiologists and molecular biologists.

  2. eLife

    Reviewer #1 (Public review):

    Kotzadimitriou et al. investigate how synaptotagmin-7 (syt7) contributes to short-term plasticity at cortical glutamatergic synapses. Using quantal-level iGluSnFR imaging and failure-based analyses at single boutons, the authors distinguish between synchronous and asynchronous glutamate release across boutons with differing baseline efficacy. They show that knocking out syt7 abolishes facilitation of synchronous release while leaving asynchronous facilitation largely intact, although reduced in magnitude. Furthermore, they argue that synchronous and asynchronous events arise from functionally distinct vesicle pools. The manuscript concludes that syt7 is essential for the facilitation of synchronous release, while other calcium sensors govern asynchronous release.

    Strengths:

    (1) The use of iGluSnFR provides a robust readout of single-synapse activity. Unlike traditional ephys methods that average the activity of thousands of synapses (which may mask the facilitation of low Pr synapses), the authors employ quantal imaging to analyze thousands of individual boutons and stratify them by efficacy. The representative images and traces in Figure 1 are of high quality, and the quantal analysis demonstrating multiple quantal peaks aligns well with previously published work (Mendonca et al., 2022; Wang et al., 2022).

    (2) The failure-based analysis is thoughtfully implemented. By isolating trials in which no release occurred, the authors effectively separate facilitation from depletion, strengthening their central argument that syt7 is required for facilitation independent of vesicle depletion.

    (3) The proposed model (depicted in Figure 7) is interesting and may reconcile the contradictory roles attributed to syt7, as described by others in the field. Specifically, the authors provide data to address syt7's potential function in facilitation, asynchronous release, and replenishment. However, to further support their model, which argues that "multiple Ca2+ sensors have both unique and overlapping roles in regulating synaptic plasticity," additional experiments are needed (see point 2 below).

    Weaknesses:

    (1) While the authors use cultures from syt7 knockout mice (and wild-type controls), there are no acute rescue experiments (e.g., syt7 viral transduction in KO cultures) or checks for compensatory changes in other proteins. Previous studies (Bacaj et al., 2013; Jackman et al., 2016) have utilized viral rescues to confirm specificity. Without such experiments, it remains theoretically possible that the chronic loss of syt7 leads to downregulation of another protein essential for facilitation. At a minimum, the authors should perform rescue experiments for at least some of their findings. Additionally, western blots for syt1 and syt7 should be conducted to confirm that their knockout is specific to syt7.

    (2) The manuscript acknowledges the possible roles of Doc2a and syt3 but fails to address them experimentally. Recent work (Wu et al., 2024; Weingarten et al., 2024) has identified Doc2a as the primary sensor for asynchronous release. Even if its expression in cortical cultures remains unconfirmed (as claimed by the authors), they should, at the very least, perform Western blots for Doc2a and syt3 in both wild-type (to determine basal expression levels) and syt7 knockout cultures. Without analyzing the levels of these proteins, the mechanism/model behind the "remaining" asynchronous release remains speculative. Is it possible that these other calcium sensors are upregulated in their syt7 KO cultures and could instead explain their results?

  3. eLife

    Reviewer #2 (Public review):

    Summary:

    In this elegant study, the authors employ live iGluSnFR-based imaging of glutamate release from cortical boutons to dissect the distinct roles of the Ca²⁺ sensor synaptotagmin-7 (Syt7) in synaptic transmission. Although multiple functions have been attributed to Syt7 over the years, the field remains conflicted. The authors argue that one major obstacle for resolving some of these discrepancies lies in a fundamental limitation of electrophysiological recordings, which aggregate signals across all synapses to yield averaged readouts, dominated by strong, high-release-probability synapses. By using a live glutamate imaging approach combined with sensitive detection of action potential-evoked activity across different stimulation regimes, and a dedicated analysis pipeline, the authors confirm a role for Syt7 in facilitating synchronous release and in regulating the magnitude of asynchronous release. In contrast, they find no evidence that Syt7 contributes to the facilitation of asynchronous release, do not find evidence for a role for Syt7 in synaptic vesicle replenishment during AP trains, and provide evidence suggesting that the maintenance of facilitation by Syt7 may occur independently of vesicle depletion.

    Strengths:

    This study offers a fresh perspective on a debated issue, using a new experimental approach that the authors previously explored in the context of Synaptotagmin 1 (Mendonca et al. 2022). The authors record the response to a series of pair-pulse stimulations, followed by an AP train. By carefully quantifying individual events and by sorting events based on their efficacy, the authors extract quantitative information that they assign to different properties of synaptic function. They also devised an interesting approach for monitoring aspects of facilitation, in which they isolate PPR events where the first response did not elicit detectable release (thus regarding the release in response to the second AP as facilitating), and compare them with successful events. Together, the authors provide semi-quantitative descriptions of synchronous and asynchronous release during single, paired, and AP trains, yielding a weighted estimate of Syt7's contribution to distinct features of synaptic vesicle release that are independent of postsynaptic readouts. A major strength of the study is the confirmation of two principal proposed functions of Syt7: facilitation of synchronous release and regulation of the magnitude of asynchronous release.

    Weaknesses:

    The experimental approach presented here is elegant and well-executed. However, a principal limitation lies in translating electrophysiological terminology to imaging-based measurements. For instance, interpreting signals persisting beyond 10 ms as a proxy for asynchronous release relies on assumptions that would be good to experimentally justify. Could such signals arise from iGluSnFR saturation, or be affected by desensitization?. Moreover, the quantification of asynchronous release is based on very small signals that represent only a fraction of the already small synchronous release component, raising concerns about signal-to-noise limitations. A key issue is that failures to evoke glutamate release may arise from AP failures, such that the second response in a PPR does not necessarily represent facilitation. Given that many of the findings largely confirm existing literature, the study might have benefited from a different framing, for example, as an additional validation of the correspondence between electrophysiological measures and the authors' imaging-based readouts. Another point concerns the analysis of synaptic vesicle replenishment following depletion, which would ideally be addressed using alternative stimulation protocols, such as quantifying the response/success rate to single APs at varying time points after a train. Although the authors are appropriately cautious in their conclusions (e.g., with respect to Figure 5b), this limitation remains. Finally, the use of heterogeneous cortical neuronal cultures is likely to introduce substantial variability, as the authors themselves acknowledge, which may arise from the co-expression of multiple Ca²⁺ sensors across diverse cell types.

    In summary, the authors were able to confirm previously-described changes in neurotransmission properties upon the loss of Syt7 using live imaging of glutamate release at the level of single boutons. They also present preliminary evidence for the interdependence of Syt7 function, synaptic vesicle replenishment, and the facilitation of asynchronous release, although these results will need to be substantiated in future studies using alternative stimulation protocols and complementary methodologies. Taken together with the group's prior work on synaptotagmin-1, this study illustrates that live imaging of glutamate release offers an alternative approach that recapitulates some elements detectable via electrophysiological analysis, while possibly revealing new insights into the function of synaptic proteins. As a whole, taking a live imaging approach may be a broadly accessible way forward to analyze synaptic function. The potential of studying synaptic proteins in diverse cell types that are difficult to access with patch-clamp electrophysiology is particularly compelling.

  4. eLife

    Reviewer #3 (Public review):

    In this manuscript, the authors examine the role of Syt7 in the plasticity of synchronous and asynchronous release in cultured neurons. The experimental approach is the imaging of SF-iGluSnFR.A184V expressed in cultured neurons while delivering stimulation through whole-cell patch clamping of single neurons in the culture. In this manner, they could examine the optical signature of glutamate release in single presynaptic terminals, while separating the release events into synchronous (<10ms) and asynchronous (>10ms) while delivering both paired pulses or trains of stimuli (at 20 Hz, 50 ms between stimuli).

    This manuscript employs techniques previously reported by the research group in their Mendoca et al., Nat Comms 2022 paper. This paper uses this approach to specifically examine the role of Syt7. The use of iGluSnFR in this manner provides significant rigor to the paper. The most significant weakness is that some of the events the authors discuss in this manuscript are rare, and the strength of the conclusions regarding those is somewhat unclear.

    The main novel contribution of this manuscript is that single-bouton high-frequency imaging allowed them to examine paired-pulse plasticity in boutons that had not released neurotransmitter during the first pulse (failure-based analysis), thus separating between the effects of vesicle depletion and facilitation of the release machinery. This approach also allowed them to segregate their observations according to bouton-specific release efficacy. Both examinations are unavailable when performing cell-level analysis of neurotransmitter release, as is reported by most electrophysiological approaches.

    The authors conclude that Syt7 contributes specifically to facilitation of synchronous release, not asynchronous release, while reducing the magnitude of the asynchronous component. Finally, the authors suggest segregation of synchronous and asynchronous release, either by differential use of calcium sensors or spatial segregation of the vesicles contributing to both modes of release.

    This report contributes significantly to the discussion of the control of synaptic plasticity by different molecular players. It is not the first to examine Syt7, but its contribution to the examination of this protein is significant.

    I find this report to be well executed and reasoned. In my opinion, the authors could improve the manuscript by clarifying the description of several methodological and experimental sections. Furthermore, in my opinion, some of the conclusions are overstated.

    The authors state: "Because boutons along a single axon originate from the same presynaptic neuron, they are expected to share broadly similar molecular components of the vesicular release machinery and experience comparable presynaptic action potential waveforms." The authors should address the idea that presynaptic terminals from the same neuron on different postsynaptic targets can differ in the molecular components, as well as in the presynaptic side. There is ample evidence for differences between synapses onto glutamatergic and GABAergic neurons of the same neuron.

    The authors used 4ms-long frames, but the stimuli are delivered at 20Hz (50ms apart). Therefore, in paired pulse stimulation, isn't there going to be a difference between the first and second stimuli regarding the timing of the frames relative to the stimulus? Isn't the deconvolution sensitive to such an offset?

    A 10ms threshold for defining synchronous vs. asynchronous release full in-between frames. Doesn't this increase the chance of assigning borderline events to the wrong category?

    On page 11 of the conclusion, the authors state that "Our data indicate that in our conditions during paired-pulse protocol Syt7 primarily enhances release probability rather than increasing the RRP size." While I understand the reasoning behind this statement, it should be toned down. The authors did not directly address the RRP size.

    In failure-based analysis, the number of failure events in high-efficiency boutons is expected to be low. How does this affect the conclusions of the authors concerning the effects of Syt7 deletion on facilitation in high-efficiency boutons?
    SourceData.xlsx was not available to me, as far as I could tell.

    How can the conclusions of the authors on the differential molecular composition of vesicles contributing to synchronous and asynchronous release be related to the reported effect of strontium on the nature of release? (see 10.1523/JNEUROSCI.20-12-04414.2000)

    Is this the first use of failure-based analysis? If not, the authors should cite precedents. In 10.1016/s0896-6273(00)80338-4, failure of release during the 1st AP was presented, with facilitation during the 2nd, although no formal analysis was performed.

  5. Version published to 10.64898/2025.12.19.695358 on bioRxiv