Optical measurement of glutamate release robustly reports short-term plasticity at a fast central synapse

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1. Version published to 10.3389/fnmol.2024.1351280

   Feb 28, 2024
2. 

   Review Commons

   May 1, 2023

   Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

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   Reviewer #1

   __Summary __

   The authors used a novel imaging technique to monitor glutamate release and correlated these measurements with gold standard electrophysiological measurements. The genetically encoded glutamate reporter, iGluSnFR, was expressed in mouse spiral ganglion neurons using the approach described in Ozcete and Moser (2021, EMBO J). The iGluSnFR signals and the postsynaptic currents were measured at the endbulb of Held synapse. A small effect of the expression of iGluSnFR on the mEPSC kinetics was found (but see comment 1). Furthermore, deconvolution of the iGluSnFR signals was performed enabling the …

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   Reply to the reviewers

   Detailed Answer to the Reviewers

   Reviewer #1

   __Summary __

   The authors used a novel imaging technique to monitor glutamate release and correlated these measurements with gold standard electrophysiological measurements. The genetically encoded glutamate reporter, iGluSnFR, was expressed in mouse spiral ganglion neurons using the approach described in Ozcete and Moser (2021, EMBO J). The iGluSnFR signals and the postsynaptic currents were measured at the endbulb of Held synapse. A small effect of the expression of iGluSnFR on the mEPSC kinetics was found (but see comment 1). Furthermore, deconvolution of the iGluSnFR signals was performed enabling the comparison of some presynaptic properties assessed with either iGluSnFR or electrophysiology.

   We thank the reviewer for her/his appreciation of our work and for the comments that have helped/will help us further improve our manuscript.

   __Major comments __

   1. The central finding of the study is the prolonged decay time constant of the mEPSC. The difference is small but astonishingly significant (0.172 {plus minus} 0.002 and 0.158 {plus minus} 0.001, P=0.003). The SEM is about 100 times smaller than the measured time constant. This is biologically not plausible. Therefore, I am skeptical about the statistical significance of the results.

   We appreciate the feedback of the reviewer. We agree that our presentation of the data was easy to misunderstand and we changed it (see below). We modeled the statistical relationship of kinetic parameters with a mixed effects model (as described in methods). Since the presentation of regression parameters for this kind of data is not very usual in synaptic neuroscience (nor very informative in this study), we instead opted to report SEM and a p-value derived from the fit of the linear mixed effects model. For the SEM, there is no clear way to take into account the clustered nature of the data, so we calculated the SEM over all observations. Since the SEM is proportional to 1/sqrt(n) and the number of recorded mEPSCs is very large, this does indeed yield a very small SEM. We agree that reporting the SEM over all observations is unusual and leads to misunderstandings in this case. Now, we instead report the re-calculated the mean / SEM for all parameters over the median values per cell. We changed the presentation of the data also for the other values presented in the MS in all tables and the relevant parts of the main texts.. We note that the summary statistics do not directly influence the further statistical modeling.

   1. Analysis of the size of RRP with electrophysiology and iGluSnFR is potentially interesting but iGluSnFR recordings could not resolve the spontaneous fusion of single vesicles. Therefore, it is not possible to estimate RRP with these iGluSnFR recordings. This limitation of the approach should be emphasised more clearly.

   Yes, we think the inability to resolve single vesicles is one of the major limitations of the study and we note this in the introduction and in the relevant section of the discussion. We agree that it should be clear in the relevant section that we are not able to measure RRP size without resolving single vesicle release and modified the wording of the relevant results section to reflect this better (line 267, 497). We still believe that the cumulative release analysis is potentially interesting to researchers in the field, as RRP size is not the only parameter that can be estimated in this way. In particular, an estimation of the release probability in resting conditions is possible by dividing the amplitude of the first response (i.e. response to a single stimulus) by the RRP estimate even without knowing the exact number of vesicles that comprise either.

   1. The control conditions (no surgery/no virus injection) are not the correct conditions for comparison with the experimental conditions (surgery/virus injection and sensor expression). The control group should be operated and injected with saline or ideally with a virus expressing GFP at the extracellular membrane. The authors addressed this issue by citing their previous work (Özcete and Moser, 2021). However, I am not convinced that surgery does not induce subtle changes that could explain the small differences in mEPSCs.

   This is an excellent point that should be addressed in further research. A slowed decay would be consistent with the idea that iGluSnFR affects glutamatergic transmission by buffering glutamate, but we cannot rule out subtle changes due to the postnatal surgery or AAV-mediated transgene expression. In response to the reviewer’s comment, we modified the text to reflect the possibility of surgery and / or other parts of the expression system being responsible for the changes. We also discuss further control experiments (line 408). Finally, we believe that our comparison is still relevant for researchers using iGluSnFR in the system, as they will be asking if introducing a measurement system affects the underlying quantity.

   __Minor comments __

   The supplementary figures are not listed in the order in which they appear in the main text.

   We now list the supplementary figures in the order in which they appear in the main text.

   Figure 2B and 3 are not referenced in the main text.

   We now reference the figures in the text.

   The PPR in Figure 3 shows a PPR that cannot be evaluated because of the unusual plot with lines that are too thick.

   We updated Fig.3 and chose a more straight forward way to display the PPRs.

   Line 105: "...while simultaneously monitoring currents in postsynaptic cells". This sentence is not correct given that the EPSCs have not been shown yet at this point of the manuscript.

   We removed this part of the sentence.

   Line 110: "SV and are not cause are cause by spontaneous action potentials...". The sentence does not make sense.

   We corrected the sentence.

   Line 168-9: "...we did not find significant differences in amplitude and kinetics...". According to Table 2 (2mM Ca2+ condition), both Imax and Q appear to be almost twice as high in iGluSnFR as in control (2.05 {plus minus} 0.06 and 1.34 {plus minus} 0.03, respectively; P=0.241). Is this not a significant difference?

   The difference was not significant. The misunderstanding likely stems from the same problem in the presentation of the values as for the mEPSCs. We replaced the SEMs with the SEMs of the cell median to avert this.

   Table 4. 2mM Ca2+ condition. The Rrefill parameter is about an order of magnitude smaller in the iGluSnFR-expressing group. Is this correct or just a typo?

   Thank you for spotting this: it was an error with regards to unit conversion. The value for the control group was off by a factor of 10. We corrected this mistake.

   Referees cross-commenting

   I also agree with the comments of the other reviewers.

   Significance

   General assessment

   This topic is currently of interest because iGluSnFR techniques are widely used. However, the study is preliminary. The scientific progress in terms of quantity and quality is limited. For example, Figs. 1 and 5 show only images and traces with little scientific significance.

   Advance

   The main advance of the study is the implementation of the deconvolution of the iGluSnFR signal and the comparison of the back extrapolation with the first stimulus (Fig. 6). This comparison was similar between electrophysiology and iGluSnFR when deconvolution of the iGluSnFR data was performed. These data therefore argue against saturation of iGluSnFR, as expected from a large number of previous analyses of iGluSnFR.

   There is little methodological improvements compared with the group's previous study (Ozcete and Moser, 2021 EMBO J). In this earlier study, a different synapse was analyzed but the same iGluSnFR was injected into the scala tympani of the right ear through the round window in the same way as in this study. Surprisingly, the authors do not refer to Ozcete and Moser (2021) in the relevant methods section.

   Thank you for spotting this omission. We now cite Özçete and Moser (2021) in the appropriate place in the methods section as well.

   Reviewer #2

   Summary

   In the manuscript 'Optical measurement of glutamate release robustly reports short-term plasticity at a fast central synapse' the authors present a careful analysis of whether direct measurements of transmitter release using the genetically-encoded indicator iGluSnFR, are suitable for assessing changes in transmitter release at the spiral ganglion neuron end bulbs of Held in the mouse cochlear nucleus. What sets this study apart from other studies, which have demonstrated the utility of iGluSnFR measurements, is the use of a camera-based fluorescence readout as opposed to confocal or 2P microscopy methods and that it is performed in the cochlear nucleus.

   The primary methodology is the comparison of electrophysiological measurements of excitatory postsynaptic currents from bushy cells with fluorescence changes in the end bulbs of iGluSnFR expressing auditory nerve fibers with and without stimulation of the auditory nerve fibers. The experiments are technically demanding and introducing genetically encoded indicators in neurons of the cochlea is no small accomplishment. An important observation is that mEPSCs are slightly modified (prolonged) due to expression of iGluSnFR in the presynaptic end bulbs. This is perhaps not surprising as iGluSnFR binds glutamate and may act as a buffer to reduce the peak and slightly prolong the increase in cleft glutamate concentration after release from synaptic vesicles. To my knowledge, others have not reported iGluSnFR effects on mEPSCs. Perhaps earlier studies have not checked as carefully, alternatively previous studies had a too-low fraction of presynaptic terminals expressing iGluSnFR (or less expression of iGluSnFR) to detect a change in EPSC parameters, or this is a synapse-specific phenomenon. However, the authors demonstrate that EPSCs evoked by electrical stimulation of the auditory nerve fibers are unaffected by expression of iGluSnFR in the presynaptic neurons. Further findings are that the determined decay time constant is substantially longer than at other synapses (~16 ms at hippocampal synapses, Dürst et al., 2018). Synaptic depression was robustly reported by iGluSnFR at this synapse, but determination of single quantal events and thus quantal analysis was not really possible at this synapse using iGluSnFR in conjunction with the imaging and analysis techniques presented. The manuscript is carefully written and presented.

   We thank the reviewer for her/his appreciation of our work and for the comments that have helped/will help us further improve our manuscript.

   Major points

   1. The ROIs are selected to be 'outer bounds' of the glutamate spread from the synapses being studied. My concern is that these generously-sized ROIs include signal from many iGluSnFR molecules which are distal to the release sites and thus will be reached only slowly by low concentrations of glutamate or be contributing only noise and no changes in fluorescence. I suggest the temporal resolution could be improved by restricting the analysis of fluorescence changes to fewer pixels within the ROIs with the fastest rising/highest amplitude responses.

   Thanks for this helpful comment: The data in our data set should be well-suited to perform this analysis in addition to the presented analysis and so we added this new analysis to the Revision Plan.

   1. The observation that despite a 2 fold increase in eEPSCs when changing from 2 mM to 4 mM extracellular calcium there is no change in iGluSnFR peak is curious as pointed out by the authors but not really discussed.

   We don’t currently have an obvious explanation but consider saturation of the iGluSnFR peak response likely to contribute. In response to the comment of the reviewer, we have added the analysis of integrated iGluSnFR data, which we previously found to be more robust toward saturation than the peak, to the revision plan. We plan to add the relevant discussion along with the new analysis.

   Are the traces presented in Figure 5 examples from the same recording?

   Traces in fig. 5 are grand averages (wording modified for clarity). Unfortunately, it was not possible to routinely measure iGluSnFR responses from the same cell in 2mM Ca2+ and 4 mM Ca2+ as the time needed for the protocols was rather long which influenced cell stability and imaging conditions would deteriorate during the exchange of the bath solution. We think it is not quite possible to directly compare the absolute iGluSnFR responses at different extracellular Ca2+ levels.

   Assuming the examples are from one cell I first assumed the lack of change of peak was saturation of iGluSnFR but the larger fluorescence change with 100 Hz stimulation suggests otherwise. How many endbulbs are contacting one BC? Do you capture iGluSnFR responses from only one or several? In the previous point I suggested that restricting analysis to the soonest reacting pixels might improve temporal resolution but in the case of detecting the peaks with higher and normal calcium, these fastest reacting signals are probably also more likely to be saturated with glutamate.

   The eEPSCs elicited by this stimulation paradigm are monosynaptic (see methods / electrophysiology section), but there might be other iGluSnFR expressing endbulbs on the same bushy cell. Since we reduce the current just enough such that any further reduction leads to a complete failure to elicit an EPSC, we believe these additional endbulbs are not releasing glutamate. We cannot, however, exclude the possibility that iGluSnFR on neighboring structures captures any potential spillover glutamate.

   Minor points

   • mEPSCs are usually recorded in tetrodotoxin, I didn't find any mention in methods/results

   In this system, sEPSCs are not affected by TTX (Oleskevich and Walmsley, 2002) and thus usually recorded without adding TTX. We discuss this more explicitly and added a clarification to reflect this assumption (on line 111).

   • the large numbers of abbreviations make it difficult in places to follow the manuscript please at least define them again in the figure legends (e.g. BC, AVCN in figure 1, Q, FWHM in figure 2 etc.)

   We went over the manuscript again and removed some abbreviations or redefined them in captions.

   • it is a bit unusual to report results of a Wilcoxon test and at the same time mean and SEM instead of medians, if different tests were used then it is important to indicate this where the p values are given or make the sentence in the methods more definitive

   We agree that the initial presentation of the data was ambiguous. We changed the presentation to reflect this (see also answer to reviewer 1).

   • the liquid junction potential is reported as 12 mV, pretty sure it should be -12 mV (unless the QX-314 or some other of the more exotic ingredients in the extracellular solution is having a dramatic effect on the LJP).

   We follow the usual conventions of P. H. Barry, Methods in Enzymology, Vol. 171, p. 678, as described in E. Neher, Methods in Enzymology, Vol. 207, p. 123, in which the LJP is defined as the potential of the bath solution with respect to the pipette solution. We subtracted this positive potential (+12mV) in the end to obtain the membrane potential which therefore was more hyperpolarized than the nominal potential.

   I wonder if one of the faster/lower affinity iGluSnFR variants would be better suited for studying this synapse.

   We agree with the reviewer that future studies should explore the potential of faster/lower affinity iGluSnFR variants for studying the endbulb synapse. The reasons why we employed the original version include: i) sharing the same mice for studies of cochlear ribbon synapses (Özcete & Moser, 2021) and cochlear nucleus synapses (this MS) for the sake of reducing animal experiments, ii) good signal to background facilitating our first study establishing the recording in brainstem slice, iii) less signal to background and shorter signals with the new variants (as found in preliminary recordings from cochlear ribbon synapses) that would make the endbulb recordings more challenging. We have added the following statement to discussion. “Future imaging studies of glutamate release at calyceal synapses should explore the potential of new iGluSnFR variants with lower affinity that provide more rapid signal decay. This will ideally go along with imaging at higher framerate and might require stronger intensities of the excitation light to boost the fluorescence signal.” on line 430.

   The paper would benefit from a careful reading to shorten the text and to check for clarity. For instance page 15 line 436 I don't understand how 'the results can reduce the likelihood of biologically relevant changes'. I think the authors meant something different

   Thank you for spotting this. We reworded the sentence (now on line 399): "The data on hand suggests that this is not the case. Firstly, even if a larger sample size may uncover more subtle effects neurotransmission of evoked events, our measurements suggest a small effect size. Secondly, even as we did find changes in mEPSC, it is probable that the biological significance is limited"

   • page 5 'width' is misspelled

   Fixed.

   • page 18 'strychnine' is misspelled

   Fixed.

   • on many of the figures is text that it much too small

   We went through the manuscript and increased the text size in the figures, where appropriate.

   __Referees cross-commenting __

   I agree with all the comments of the other reviewers - both raise the point that there should be a 'control' AAV injected for comparison of the mEPSCs which I missed but is of course quite important. See https://pubmed.ncbi.nlm.nih.gov/24872574/ for a study of AAV serotype-dependent effects on presynaptic release.

   We now added a section on other possible factors influencing the results, citing the study above.

   Significance

   The main audience for this paper will be fairly specialized. Researchers interested in properties of presynaptic release and some specialists in synaptic transmission in the auditory system will be the main readers/citers of this work.

   The work is an important technical/methodological report. It highlights an important effect of expressing iGluSnFR and also demonstrates that the effect is overall not very problematic. Additional problems using iGluSnFR are also indicated.

   I am an electrophysiologist, studying synaptic transmission and plasticity with experience using a wide range of optogenetic tools

   Reviewer #3

   __Summary __

   In the present manuscript, the authors explore the information that can be obtained using optical measurement of glutamate release with iGluSnFR on synaptic dynamics in the endbulb of Held.

   They virally express iGluSnFR in presynaptic terminals, patch the postsynaptic cells and combine high-frame-rate optical recordings with electrophysiological measurements. Their first finding is that mEPCSs are prolonged when presynaptic cells express the glutamate indicator, which they interpret as buffering of extracellular glutamate by the indicator. Next, they repeated the experiment, this time with stimulating evoked EPSCs. In contrast to the previously observed effects, iGluSnFR did not affect the time course or the amplitude of the evoked EPSCs. The authors then asked whether iGluSnFR signals can be used to study synaptic dynamics, specifically, synaptic depression. In these experiments, the authors observed a change in the paired-pulse ratio with ISI of 10ms, but not longer intervals. They analyzed presynaptic release and did not find statistically significant differences.

   Can iGluSnFR signals be used for the analysis of synaptic release? When stimulated at a low frequency of 10Hz (allowing the fluorescence to return close to baseline levels in between pulses), iGluSnFR dynamics were somewhat comparable to postsynaptic signals. At higher frequencies, the slow time course of the indicator prevented the identification of individual responses and the resulting fluorescence had a very different shape. To resolve this problem, the authors used deconvolution analysis (fig 6). This analysis revealed a linear relationship between the optical readout and the patch-clamp data.

   I find the manuscript to be clearly written, the findings are well presented and discussed and are novel and of substantial interest to neuroscientists in the field. I do have a number of questions about experiments and analysis that may have an effect on the conclusions of this work.

   We thank the reviewer for her/his appreciation of our work and for the comments that have helped/will help us further improve our manuscript.

   1. In experiments comparing the effects of iGluSnFR expression on release dynamics (figure 1-4), the authors compare infected presynaptic cells to control (uninfected). The assumption is that synaptic buffering by iGluSnFR may affect glutamate diffusion in the synaptic cleft. However, it is possible that viral infection itself changes presynaptic properties. The authors should compare release from cells infected with GFP or a comparable indicator.

   We agree that this is an important control experiment to be done in the future and that causal attribution is not in the scope of this study. A slowed decay would be consistent with the idea that iGluSnFR affects glutamatergic transmission by buffering glutamate, but we cannot rule out subtle changes due to the postnatal surgery or AAV-mediated transgene expression. In response to the reviewer’s comment, we modified the text to reflect the possibility of surgery and / or other parts of the expression system being responsible for the changes. We now also discuss further control experiments (line 408). Finally, we believe that our comparison is still relevant for researchers using iGluSnFR in the system, as they will be asking if introducing a measurement system affects the underlying quantity.

   1. Analysis of pool parameters presented in table 4 indicates almost doubling of RRP size with iGLuSnFR with 2 mM Ca++. While not significant, this result may indicate a real effect that may have been missed due to low power (N=3 and 7 for these experiments). I do not believe the authors did a power analysis in this study. How was the number of experiments determined? I would suggest increasing the number of experiments to avoid type II errors.

   We thank the reviewer for this critical comment. Indeed, we would also have liked to have a greater statistical power for these experiments, but had to face the situation that the establishing the method required more animals than expected and the animal license did not offer further animals for the analysis. Moreover, we note that the obtained RRP size estimates were generally lower compared to previous estimates of our lab for the endbulb synapse (e.g. Butola et al., 2021: ~20 nA for 2 mM Ca2+ in Fig. 5). This can partially be attributed to the use of cyclothiazide in previous studies, which we avoided given reports of presynaptic effects of cyclothiazide. As the series resistances of the included recordings were below 8 MOhm (mean series resistances: 2mM Ca, injected: 5.58 MOhm; 2mM Ca, control: 5.93 MOhm; 4mM Ca, injected: 5.75 MOhm; 4mM Ca, control: 6.0 MOhm) and series resistance compensation was set to 80% we do not expect clamp-quality to contribute to the smaller estimates in the present data set.

   We have now added a statement noting the preliminary nature of these results and indicated that further experiments will be required to more certainly conclude on potential effects of iGluSnFR or the manipulation on endbulb transmission: “Our preliminary train stimulation analysis of vesicle pool dynamics in the presence and absence of AAV-mediated iGluSnFR expression in SGNs has not revealed significant differences between the two conditions. Further experiments, potentially involving faster versions of iGluSnFR and employing trains of different stimulation rates for model based analysis of vesicle pool dynamics (Neher and Taschenberger, 2021) will help to assess the value and impact of iGluSnFR in the analysis of transmission at calyceal synapses.” on line 381.

   1. The deconvolution analysis assumes an instantaneous rise time. Yet previous work (Armbruster et al., 2020) that took into account diffusion, suggested potentially slower rise time dynamics. More importantly, the deconvolved waveforms do not match the shapes of the EPSCs (figures 5 and supp 6-2).What is the aim of the deconvolution? It was not clear from the text, but I assume it shows iGluSnFR binding to glutamate - in which case the slow waveforms are indicative of extrasynaptic iGluSnFR activation.

   The deconvolution analysis was mainly used to recover the average responses to stimuli in the train without contamination by previous responses (see also Taschenberger et al. 2016, their figure 6).

   We did also try to use the average singular response instead of the exponential fit as a kernel for the (Wiener) deconvolution analysis, which more closely resembled the observed (fast) rise. Unfortunately, this led to markedly worse results, likely because of the noise levels in the measurements. We believe that it would be beneficial to model the rise of the signal more precisely if glutamate imaging data is acquired at higher framerates.

   The broad wave forms may be due to extrasynaptic binding of glutamate, but we also note that each frame corresponds to ~10ms and there is only ~10 data points between stimuli, so the responses are unlikely to be as sharp as eEPSCs.

   However, I suppose that the more interesting question is whether iGluSnFR could be deconvolved to reveal the underlying release events, similar to how calcium signals can be used to inform about single action potentials.

   We agree that it would be particularly interesting to use a "mini iGluSnFR" signal to deconvolve the resulting traces. Unfortunately, we failed to detect iGluSnFR signals reporting individual release events at this time, preventing this kind of analysis.

   1. I suggest referencing and discussing (Aggarwal et al., 2022; Srivastava et al., 2022) . These highly relevant papers analyzed iGluSnFR to probe synaptic release.

   References:

   Aggarwal, A., Liu, R., Chen, Y., Ralowicz, A. J., Bergerson, S. J., Tomaska, F., Hanson, T. L., Hasseman, J. P., Reep, D., Tsegaye, G., Yao, P., Ji, X., Kloos, M., Walpita, D., Patel, R., Mohr, M. A., Tilberg, P. W., Mohar, B., Team, T. G. P., . . . Podgorski, K. (2022). Glutamate indicators with improved activation kinetics and localization for imaging synaptic transmission. bioRxiv, 2022.2002.2013.480251. https://doi.org/10.1101/2022.02.13.480251

   Armbruster, M., Dulla, C. G., & Diamond, J. S. (2020). Effects of fluorescent glutamate indicators on neurotransmitter diffusion and uptake. Elife, 9. https://doi.org/10.7554/eLife.54441

   Srivastava, P., de Rosenroll, G., Matsumoto, A., Michaels, T., Turple, Z., Jain, V., Sethuramanujam, S., Murphy-Baum, B. L., Yonehara, K., & Awatramani, G. B. (2022). Spatiotemporal properties of glutamate input support direction selectivity in the dendrites of retinal starburst amacrine cells. Elife, 11. https://doi.org/10.7554/eLife.81533

   We thank the reviewer for the suggestions. Some of these studies were not available when we first drafted the manuscript. We now added a section discussing these studies starting on line 466:

   Optimizing the imaging technique may reduce noise level, while the development of improved GEGIs could improve the signal to a level, at which spontaneous release events can be identified reliably in the cochlear nucleus. In retinal slices, where quantal events have been reliably observed with two-photon imaging, temporal deconvolution was successfully employed to estimate release rates from iGluSnFR signal (Srivastava et al., 2022; James et al., 2019). Subcellular targeting of iGluSnFR variants to the postsynaptic membrane may reduce measurement errors introduced by contributing extrasynaptic iGluSnFR signal and improve spatial resolution of glutamate imaging data(Hao et al., 2023; Aggarwal et al., 2022).

   Referees cross-commenting

   I also agree with the comments made by other reviewers!

   Significance

   Overall, this study addresses an important problem in basic neuroscience research. With the developing reliance on optical measurement of neuronal function, it is important to understand the impact of the indicators on physiological function and the limitations of the technique. The study is well-executed and will be informative to neuroscientists performing optical glutamate recording to study single-cell and circuit function in and beyond the auditory system.

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   Referee #3

   Evidence, reproducibility and clarity

   In the present manuscript, the authors explore the information that can be obtained using optical measurement of glutamate release with iGluSnFR on synaptic dynamics in the endbulb of Held.

   They virally express iGluSnFR in presynaptic terminals, patch the postsynaptic cells and combine high-frame-rate optical recordings with electrophysiological measurements. Their first finding is that mEPCSs are prolonged when presynaptic cells express the glutamate indicator, which they interpret as buffering of extracellular glutamate by the indicator. Next, they repeated the experiment, this time with stimulating evoked EPSCs. In contrast to … More

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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   Referee #3

   Evidence, reproducibility and clarity

   In the present manuscript, the authors explore the information that can be obtained using optical measurement of glutamate release with iGluSnFR on synaptic dynamics in the endbulb of Held.

   They virally express iGluSnFR in presynaptic terminals, patch the postsynaptic cells and combine high-frame-rate optical recordings with electrophysiological measurements. Their first finding is that mEPCSs are prolonged when presynaptic cells express the glutamate indicator, which they interpret as buffering of extracellular glutamate by the indicator. Next, they repeated the experiment, this time with stimulating evoked EPSCs. In contrast to the previously observed effects, iGluSnFR did not affect the time course or the amplitude of the evoked EPSCs. The authors then asked whether iGluSnFR signals can be used to study synaptic dynamics, specifically, synaptic depression. In these experiments, the authors observed a change in the paired-pulse ratio with ISI of 10ms, but not longer intervals. They analyzed presynaptic release and did not find statistically significant differences. Can iGluSnFR signals be used for the analysis of synaptic release? When stimulated at a low frequency of 10Hz (allowing the fluorescence to return close to baseline levels in between pulses), iGluSnFR dynamics were somewhat comparable to postsynaptic signals. At higher frequencies, the slow time course of the indicator prevented the identification of individual responses and the resulting fluorescence had a very different shape. To resolve this problem, the authors used deconvolution analysis (fig 6). This analysis revealed a linear relationship between the optical readout and the patch-clamp data.

   I find the manuscript to be clearly written, the findings are well presented and discussed and are novel and of substantial interest to neuroscientists in the field. I do have a number of questions about experiments and analysis that may have an effect on the conclusions of this work.

   1. In experiments comparing the effects of iGluSnFR expression on release dynamics (figure 1-4), the authors compare infected presynaptic cells to control (uninfected). The assumption is that synaptic buffering by iGluSnFR may affect glutamate diffusion in the synaptic cleft. However, it is possible that viral infection itself changes presynaptic properties. The authors should compare release from cells infected with GFP or a comparable indicator.
   2. Analysis of pool parameters presented in table 4 indicates almost doubling of RRP size with iGLuSnFR with 2 mM Ca++. While not significant, this result may indicate a real effect that may have been missed due to low power (N=3 and 7 for these experiments). I do not believe the authors did a power analysis in this study. How was the number of experiments determined? I would suggest increasing the number of experiments to avoid type II errors.
   3. The deconvolution analysis assumes an instantaneous rise time. Yet previous work (Armbruster et al., 2020) that took into account diffusion, suggested potentially slower rise time dynamics. More importantly, the deconvolved waveforms do not match the shapes of the EPSCs (figures 5 and supp 6-2).What is the aim of the deconvolution? It was not clear from the text, but I assume it shows iGluSnFR binding to glutamate - in which case the slow waveforms are indicative of extrasynaptic iGluSnFR activation. However, I suppose that the more interesting question is whether iGluSnFR could be deconvolved to reveal the underlying release events, similar to how calcium signals can be used to inform about single action potentials.
   4. I suggest referencing and discussing (Aggarwal et al., 2022; Srivastava et al., 2022) . These highly relevant papers analyzed iGluSnFR to probe synaptic release.

   References:

   Aggarwal, A., Liu, R., Chen, Y., Ralowicz, A. J., Bergerson, S. J., Tomaska, F., Hanson, T. L., Hasseman, J. P., Reep, D., Tsegaye, G., Yao, P., Ji, X., Kloos, M., Walpita, D., Patel, R., Mohr, M. A., Tilberg, P. W., Mohar, B., Team, T. G. P., . . . Podgorski, K. (2022). Glutamate indicators with improved activation kinetics and localization for imaging synaptic transmission. bioRxiv, 2022.2002.2013.480251. https://doi.org/10.1101/2022.02.13.480251

   Armbruster, M., Dulla, C. G., & Diamond, J. S. (2020). Effects of fluorescent glutamate indicators on neurotransmitter diffusion and uptake. Elife, 9. https://doi.org/10.7554/eLife.54441

   Srivastava, P., de Rosenroll, G., Matsumoto, A., Michaels, T., Turple, Z., Jain, V., Sethuramanujam, S., Murphy-Baum, B. L., Yonehara, K., & Awatramani, G. B. (2022). Spatiotemporal properties of glutamate input support direction selectivity in the dendrites of retinal starburst amacrine cells. Elife, 11. https://doi.org/10.7554/eLife.81533

   Referees cross-commenting

   I also agree with the comments made by other reviewers!

   Significance

   Overall, this study addresses an important problem in basic neuroscience research. With the developing reliance on optical measurement of neuronal function, it is important to understand the impact of the indicators on physiological function and the limitations of the technique. The study is well-executed and will be informative to neuroscientists performing optical glutamate recording to study single-cell and circuit function in and beyond the auditory system.

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   Read the original source
4. 

   Review Commons

   May 1, 2023

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   Referee #2

   Evidence, reproducibility and clarity

   In the manuscript 'Optical measurement of glutamate release robustly reports short-term plasticity at a fast central synapse' the authors present a careful analysis of whether direct measurements of transmitter release using the genetically-encoded indicator iGluSnFR, are suitable for assessing changes in transmitter release at the spiral ganglion neuron end bulbs of Held in the mouse cochlear nucleus. What sets this study apart from other studies, which have demonstrated the utility of iGluSnFR measurements, is the use of a camera-based fluorescence readout as opposed to confocal or 2P microscopy methods and that it is performed in … More

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   Referee #2

   Evidence, reproducibility and clarity

   In the manuscript 'Optical measurement of glutamate release robustly reports short-term plasticity at a fast central synapse' the authors present a careful analysis of whether direct measurements of transmitter release using the genetically-encoded indicator iGluSnFR, are suitable for assessing changes in transmitter release at the spiral ganglion neuron end bulbs of Held in the mouse cochlear nucleus. What sets this study apart from other studies, which have demonstrated the utility of iGluSnFR measurements, is the use of a camera-based fluorescence readout as opposed to confocal or 2P microscopy methods and that it is performed in the cochlear nucleus.

   The primary methodology is the comparison of electrophysiological measurements of excitatory postsynaptic currents from bushy cells with fluorescence changes in the end bulbs of iGluSnFR expressing auditory nerve fibers with and without stimulation of the auditory nerve fibers. The experiments are technically demanding and introducing genetically encoded indicators in neurons of the cochlea is no small accomplishment. An important observation is that mEPSCs are slightly modified (prolonged) due to expression of iGluSnFR in the presynaptic end bulbs. This is perhaps not surprising as iGluSnFR binds glutamate and may act as a buffer to reduce the peak and slightly prolong the increase in cleft glutamate concentration after release from synaptic vesicles. To my knowledge, others have not reported iGluSnFR effects on mEPSCs. Perhaps earlier studies have not checked as carefully, alternatively previous studies had a too-low fraction of presynaptic terminals expressing iGluSnFR (or less expression of iGluSnFR) to detect a change in EPSC parameters, or this is a synapse-specific phenomenon. However, the authors demonstrate that EPSCs evoked by electrical stimulation of the auditory nerve fibers are unaffected by expression of iGluSnFR in the presynaptic neurons. Further findings are that the determined decay time constant is substantially longer than at other synapses (~16 ms at hippocampal synapses, Dürst et al., 2018). Synaptic depression was robustly reported by iGluSnFR at this synapse, but determination of single quantal events and thus quantal analysis was not really possible at this synapse using iGluSnFR in conjunction with the imaging and analysis techniques presented. The manuscript is carefully written and presented.

   Major points:

   1. The ROIs are selected to be 'outer bounds' of the glutamate spread from the synapses being studied. My concern is that these generously-sized ROIs include signal from many iGluSnFR molecules which are distal to the release sites and thus will be reached only slowly by low concentrations of glutamate or be contributing only noise and no changes in fluorescence. I suggest the temporal resolution could be improved by restricting the analysis of fluorescence changes to fewer pixels within the ROIs with the fastest rising/highest amplitude responses.
   2. The observation that despite a 2 fold increase in eEPSCs when changing from 2 mM to 4 mM extracellular calcium there is no change in iGluSnFR peak is curious as pointed out by the authors but not really discussed. Are the traces presented in Figure 5 examples from the same recording? Assuming the examples are from one cell I first assumed the lack of change of peak was saturation of iGluSnFR but the larger fluorescence change with 100 Hz stimulation suggests otherwise. How many endbulbs are contacting one BC? Do you capture iGluSnFR responses from only one or several? In the previous point I suggested that restricting analysis to the soonest reacting pixels might improve temporal resolution but in the case of detecting the peaks with higher and normal calcium, these fastest reacting signals are probably also more likely to be saturated with glutamate.

   Minor points:

   • mEPSCs are usually recorded in tetrodotoxin, I didn't find any mention in methods/results
   • the large numbers of abbreviations make it difficult in places to follow the manuscript please at least define them again in the figure legends (e.g. BC, AVCN in figure 1, Q, FWHM in figure 2 etc.)
   • it is a bit unusual to report results of a Wilcoxon test and at the same time mean and SEM instead of medians, if different tests were used then it is important to indicate this where the p values are given or make the sentence in the methods more definitive
   • the liquid junction potential is reported as 12 mV, pretty sure it should be -12 mV (unless the QX-314 or some other of the more exotic ingredients in the extracellular solution is having a dramatic effect on the LJP). I wonder if one of the faster/lower affinity iGluSnFR variants would be better suited for studying this synapse. The paper would benefit from a careful reading to shorten the text and to check for clarity. For instance page 15 line 436 I don't understand how 'the results can reduce the likelihood of biologically relevant changes'. I think the authors meant something different
   • page 5 'width' is misspelled
   • page 18 'strychnine' is misspelled
   • on many of the figures is text that it much too small

   Referees cross-commenting

   I agree with all the comments of the other reviewers - both raise the point that there should be a 'control' AAV injected for comparison of the mEPSCs which I missed but is of course quite important. See https://pubmed.ncbi.nlm.nih.gov/24872574/ for a study of AAV serotype-dependent effects on presynaptic release.

   Significance

   The main audience for this paper will be fairly specialized. Researchers interested in properties of presynaptic release and some specialists in synaptic transmission in the auditory system will be the main readers/citers of this work.

   The work is an important technical/methodological report. It highlights an important effect of expressing iGluSnFR and also demonstrates that the effect is overall not very problematic. Additional problems using iGluSnFR are also indicated.

   I am an electrophysiologist, studying synaptic transmission and plasticity with experience using a wide range of optogenetic tools

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5. 

   Review Commons

   May 1, 2023

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   Referee #1

   Evidence, reproducibility and clarity

   Summary

   The authors used a novel imaging technique to monitor glutamate release and correlated these measurements with gold standard electrophysiological measurements. The genetically encoded glutamate reporter, iGluSnFR, was expressed in mouse spiral ganglion neurons using the approach described in Ozcete and Moser (2021, EMBO J). The iGluSnFR signals and the postsynaptic currents were measured at the endbulb of Held synapse. A small effect of the expression of iGluSnFR on the mEPSC kinetics was found (but see comment 1). Furthermore, deconvolution of the iGluSnFR signals was performed enabling the comparison of some presynaptic … More

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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   ---

   Referee #1

   Evidence, reproducibility and clarity

   Summary

   The authors used a novel imaging technique to monitor glutamate release and correlated these measurements with gold standard electrophysiological measurements. The genetically encoded glutamate reporter, iGluSnFR, was expressed in mouse spiral ganglion neurons using the approach described in Ozcete and Moser (2021, EMBO J). The iGluSnFR signals and the postsynaptic currents were measured at the endbulb of Held synapse. A small effect of the expression of iGluSnFR on the mEPSC kinetics was found (but see comment 1). Furthermore, deconvolution of the iGluSnFR signals was performed enabling the comparison of some presynaptic properties assessed with either iGluSnFR or electrophysiology.

   Major comments

   1. The central finding of the study is the prolonged decay time constant of the mEPSC. The difference is small but astonishingly significant (0.172 {plus minus} 0.002 and 0.158 {plus minus} 0.001, P=0.003). The SEM is about 100 times smaller than the measured time constant. This is biologically not plausible. Therefore, I am skeptical about the statistical significance of the results.
   2. Analysis of the size of RRP with electrophysiology and iGluSnFR is potentially interesting but iGluSnFR recordings could not resolve the spontaneous fusion of single vesicles. Therefore, it is not possible to estimate RRP with these iGluSnFR recordings. This limitation of the approach should be emphasised more clearly.
   3. The control conditions (no surgery/no virus injection) are not the correct conditions for comparison with the experimental conditions (surgery/virus injection and sensor expression). The control group should be operated and injected with saline or ideally with a virus expressing GFP at the extracellular membrane. The authors addressed this issue by citing their previous work (Özcete and Moser, 2021). However, I am not convinced that surgery does not induce subtle changes that could explain the small differences in mEPSCs.

   Minor comments

   The supplementary figures are not listed in the order in which they appear in the main text.

   Figure 2B and 3 are not referenced in the main text.

   The PPR in Figure 3 shows a PPR that cannot be evaluated because of the unusual plot with lines that are too thick.

   Line 105: "...while simultaneously monitoring currents in postsynaptic cells". This sentence is not correct given that the EPSCs have not been shown yet at this point of the manuscript.

   Line 110: "SV and are not cause are cause by spontaneous action potentials...". The sentence does not make sense.

   Line 168-9: "...we did not find significant differences in amplitude and kinetics...". According to Table 2 (2mM Ca2+ condition), both Imax and Q appear to be almost twice as high in iGluSnFR as in control (2.05 {plus minus} 0.06 and 1.34 {plus minus} 0.03, respectively; P=0.241). Is this not a significant difference?

   Table 4. 2mM Ca2+ condition. The Rrefill parameter is about an order of magnitude smaller in the iGluSnFR-expressing group. Is this correct or just a typo?

   Referees cross-commenting

   I also agree with the comments of the other reviewers.

   Significance

   General assessment This topic is currently of interest because iGluSnFR techniques are widely used. However, the study is preliminary. The scientific progress in terms of quantity and quality is limited. For example, Figs. 1 and 5 show only images and traces with little scientific significance.

   Advance The main advance of the study is the implementation of the deconvolution of the iGluSnFR signal and the comparison of the back extrapolation with the first stimulus (Fig. 6). This comparison was similar between electrophysiology and iGluSnFR when deconvolution of the iGluSnFR data was performed. These data therefore argue against saturation of iGluSnFR, as expected from a large number of previous analyses of iGluSnFR.

   There is little methodological improvements compared with the group's previous study (Ozcete and Moser, 2021 EMBO J). In this earlier study, a different synapse was analyzed but the same iGluSnFR was injected into the scala tympani of the right ear through the round window in the same way as in this study. Surprisingly, the authors do not refer to Ozcete and Moser (2021) in the relevant methods section.

   Audience „basic research"

   My field of expertise imaging and electrophysiology; basic research

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6. 

   Version published to 10.1101/2022.11.27.518035v3 on bioRxiv

   May 1, 2023
7. 

   Version published to 10.1101/2022.11.27.518035v2 on bioRxiv

   Feb 13, 2023
8. 

   Version published to 10.1101/2022.11.27.518035v1 on bioRxiv

   Nov 27, 2022