Recent studies indicate that within individual synapses spontaneous and evoked release processes are segregated and regulated independently. In the hippocampus, earlier electrophysiological recordings suggested that spontaneous and evoked glutamate release can activate separate groups of postsynaptic NMDA receptors with limited overlap. However, it is still unclear how this separation of NMDA receptors is distributed across individual synapses. In a previous paper (Reese and Kavalali, 2015) we showed that NMDA receptor mediated spontaneous transmission signals to the postsynaptic protein translation machinery through Ca2+-induced Ca2+ release. Here, we show that in rat hippocampal neurons although spontaneous and evoked glutamate release driven NMDA receptor mediated Ca2+ transients often occur at the same synapse, these two signals do not show significant correlation or cross talk.https://doi.org/10.7554/eLife.21170.001
The vast majority of neuronal subtypes in the mammalian central nervous system exhibit both action potential (AP) synchronized evoked neurotransmitter release and stochastic, spontaneous neurotransmitter release (Kavalali, 2015). Previous studies by our group and others have shown that spontaneous neurotransmission is critical in the homeostatic regulation of synaptic strength and this regulation may have implications for the efficacy of rapidly acting antidepressants (Nosyreva et al., 2013; Gideons et al., 2014; Sutton et al., 2006). In particular, our recent work has shown that the Ca2+ dependence of these effects is driven by NMDA receptor mediated Ca2+ signals that are amplified via Ca2+-induced Ca2+ release via ryanodine receptor mediated coupling to postsynaptic Ca2+ stores (Reese and Kavalali, 2015). These earlier results highlight a novel mechanism by which spontaneous release can trigger large Ca2+ transients — and in turn activates postsynaptic signaling — but it remains unclear if these transients are generated at the same synapses that participate in evoked release.
An early investigation into the possibility that evoked and spontaneous neurotransmission may be separated spatially found that glutamate from evoked neurotransmitter release fell upon a subset of NMDA receptors distinct from those activated by spontaneous neurotransmission (Atasoy et al., 2008). However, these results lack the ability to distinguish quantal events at the single synapse level and do not show the extent to which evoked and spontaneous release processes are co-localized. More recent experiments in the Drosophila neuromuscular junction have come to mixed conclusions about the propensity of a single active zone to participate in both evoked and spontaneous transmission; finding either considerable overlap, but with a population of spontaneous only active zones or limited overlap with a negative correlation between the two modes of transmission (Melom et al., 2013; Peled et al., 2014). To date, no such measurement has been made in mammalian synapses. Here, we utilize the genetically encoded Ca2+ sensor GCaMP6f-PSD95 to image neurotransmission at individual synaptic regions to measure the amount of transmission mode overlap in hippocampal neurons.
In order to detect Ca2+ signals in the postsynaptic compartment, we utilized the postsynaptically targeted indicator GCaMP6f-PSD95. Dissociated hippocampal cultures DIV 14–16 were transfected using Lipofectamine 3000 (Thermo Fisher, Waltham MA) to produce sparse cell labeling (<1% efficiency). The indicator was expressed in the postsynaptic compartment where its clustering produced punctate signals consistent with a postsynaptic localization (Figure 1A,B). ROIs (regions of interest) were placed systematically over individual fluorescent puncta so as to minimize the chance of an ROI receiving signals from multiple postsynaptic fluorescence signals. Fluorescence traces were generated from ROIs and peaks corresponding to synaptic activity were counted such that one ROI correlates to one synapse (Figure 1A, see Materials and methods). To determine if the observed transients were in fact NMDA receptor mediated currents, we recorded cells for 4 min in Tyrode’s solution containing 1 µM TTX and then TTX + 50 µM AP5. The addition of AP5 produced a 98.3% reduction in detected events over 4 min which is consistent with the recorded signals originating from postsynaptic NMDA currents (Figure 1C–D).
When subjected to field stimulation, individual puncta responded in a sparse, synchronous manner consistent with the evoked release of synaptic vesicles. To allow recording of both evoked and spontaneous responses, cells were perfused with Tyrode’s solution containing 5 µM NBQX, 5 µM muscimol and 20 µM ryanodine. In this system, the AMPA receptor antagonist NBQX and the GABAa agonist muscimol function to prevent spontaneous action potentials and reverberatory network activity during stimulation by shunting excitation in the dendrite and soma but not the axon (Figure 2—figure supplement 1A,B) (Sperk et al., 1997). Ryanodine was included to block efflux of Ca2+ from the endoplasmic reticulum which we have previously shown to contribute to synaptic Ca2+ measurements (Reese and Kavalali, 2015).
Under these conditions we detected spontaneous and evoked response rates consistent with earlier estimates of evoked and spontaneous release probabilities at single synapses (Murthy et al., 1997; Leitz and Kavalali, 2011, 2014). To assess if spontaneous response rate and evoked response probability (reported here as Rp due to the postsynaptic nature of our measurement and so as not to be confused with presynaptic Pr) may be functionally correlated, cells were recorded for 8 min during which single action potentials were stimulated every 30 s for a total of 15 action potentials (see example Video 1, Figure 2A). Signal peaks detected within 1 s of a stimulus were considered to be evoked, and all peaks falling out of the 1 s window were considered to be spontaneous in origin (see Methods). When signal peak times are plotted, synchronous evoked activity across multiple ROIs is apparent on a background of spontaneous activity (Figure 2A).
Plotting the spontaneous response rate (Figure 2B) against the evoked response rate for each synapse reveals no linear correlation between the two parameters (Figure 2C). A fit line describes a trend towards lower spontaneous response rates in high evoked Rp ROIs, but with very weak correlation. Interestingly, there is a large population of ROIs (~20%) that exhibit spontaneous but not evoked responses during the recording period. These ROIs may represent synapses that exclusively release vesicles spontaneously, or have a very low evoked Rp (Figure 2C, along y axis). The presence of these ROIs suggests that a population of synapses may exist which exclusively signal spontaneously rather than by action potential driven chemical neurotransmission. The distribution of observed frequencies for spontaneous responses shows clustering around a mean rate of 0.78 events / ROI / minute (Figure 2B). Evoked responses show a more even distribution with a mean Rp of 0.34 (Figure 2D). In addition, we found that neither evoked Rp or spontaneous response rate vary with respect to their distance to the soma (data not shown). Further analysis of this data was performed in which the coefficient of variance was calculated for the event amplitudes recorded at each ROI. These calculations reveal no correlation between spontaneous or evoked response rate and the variance in amplitudes that would be indicative of multiple synaptic inputs per ROI (Figure 2—figure supplement 1C,D).
Previous studies have shown that the distribution of spontaneous synaptic responses in time is not consistent with the events being independent at the population or single-synapse level and thus do not follow the Poisson distribution (Abenavoli et al., 2002; Melom et al., 2013). In our data, neither the Poisson distribution matching the measured average (λ = 6.2, p<<0.001, excluding zeros, not shown) or the theoretical best fit Poisson distribution (λ = 4.3, p<<0.001, excluding zeros) adequately describe the measured distribution as compared by the χ2 test (Figure 2E). We therefore conclude that the occurrence of spontaneous transmission events is a non-Poisson process and not truly random at the single synapse level.
To investigate whether evoked and spontaneously released glutamate activate common or separate pools of NMDARs within a single synapse, we utilized the use-dependent NMDAR blocker MK-801(Huettner and Bean, 1988). Cells were given 10 single stimuli, 10 s apart to establish a baseline Rp before washing in 10 µM MK-801 for 10 min. After treatment and while MK-801 was still in the bath solution, cells were again stimulated 10 times, 10 s apart to measure Rp after treatment (Figure 3A, bottom trace). To account for photobleaching and cellular run-down during this long recording period, control experiments were performed without MK-801 (Figure 3A, top). During the 10 min of stimulation-free MK-801 treatment, spontaneous response rate decreased by 89% which is consistent with MK-801 induced block of NMDARs that respond to spontaneous glutamate release (Figure 3A,B) (p=0.010 comparing first and last minute of MK-801 with student’s paired t-test). In contrast, there was no significant decrease in spontaneous response rate in the control experiments when comparing the first and last minute (p=0.20, student’s paired t-test) (Figure 3B).
In control cells, of the ROIs that responded one or more times during the first stimulation, spontaneous responses were observed in 75% of those ROIs and 58% responded to stimulation after the 10 min control perfusion. In the MK-801 treated cells, of the ROIs that responded during the first stimulation, 66% responded spontaneously and 56% responded after treatment (Figure 3C, blue bars). We expect that if MK-801 application at rest blocked a large number of both spontaneous and evoked release responding NMDA receptors, there would be a steep decrease in the number of synapses that respond to evoked stimulus after MK-801 treatment. However, the ability of ROIs to respond one or more times to evoked stimulus after MK-801 treatment is unaffected compared to control (Figure 3C, p=0.31 via one-way ANOVA). The application of MK-801 did not affect the number of spontaneously active ROIs (Figure 3C), though it did significantly decrease the rate of detected events at those ROIs (Figure 3B). While the vast majority of ROIs exhibit both spontaneous and evoked responses (Figure 2C, spontaneous bars), the inability of MK-801 to halt evoked responses after spontaneous block suggests a separation of the two modes of transmission within the same ROI.
During stimulation there was a decrease in amplitude with every subsequent response in the MK-801 treated cells and no single ROI responded more than eight times with MK-801 in the bath solution (Figure 3D). This decrease in amplitude is accompanied by a decrease in Rp after MK-801 treatment that preferentially affects high responding ROIs which is consistent with its usage-dependent mechanism of action (Figure 3E). Channels that open during the spontaneous-only period of MK-801 treatment will be blocked and unavailable to participate in the second round of evoked responses if they were to bind glutamate.
Comparing the spontaneous event rate measured before the addition of MK-801 against the percent reduction in evoked Rp after MK-801 treatment shows little correlation between the two parameters (Figure 4A). If NMDARs were equally likely to receive neurotransmitter from a spontaneous vesicle fusion as an evoked vesicle fusion, we would expect that the ROIs with the highest rate of spontaneous activity would exhibit the largest amount of MK-801 induced blockade when measuring evoked responses. However, we find that the rate of spontaneous activity is a poor predictor of the amount of MK-801-induced decrease in evoked responses. In contrast, comparing the initial Rp against the MK-801 induced change reveals that ROIs with the highest initial Rp see the largest decrease in Rp after treatment (Figure 4B). These findings are consistent with the blockade of NMDA receptors responding to stimulation during MK-801 treatment being due to evoked glutamate release alone, with little contribution from spontaneous neurotransmission. In other words, the plot shown in Figure 4B validates the classical assumption on the nature of MK-801 block at the single synapse level ‘higher the Rp (a proxy for release probability), larger the percentage of block by MK-801’. However, this increase in block – while proportional to initial Rp – is not correlated to the amount of spontaneous events seen prior to MK-801 application (Figure 4A). This result indicates that a classical measure of release probability does not show a correlation between spontaneous response rate and evoked response rate at the single synapse level. Taken together, these results indicate that the majority of hippocampal synapses participate in both evoked and spontaneous neurotransmission while a small subpopulation (~20%) may only transmit spontaneous signals. The occurrence of spontaneous signals appears to be non-random which may indicate some form of presynaptic regulation (e.g. Bal et al., 2013). Finally, the dynamics of NMDA receptor blockade with MK-801 illustrate that in synapses that release glutamate via both modes of transmission, the postsynaptic NMDA receptors activated are largely unique to one mode or the other with limited overlap within the synapse. This result implies a nanoscale segregation of sites for spontaneous versus evoked neurotransmission within individual synapses (Atasoy et al., 2008; Tang et al., 2016).
Prepared as described previously in (Reese and Kavalali, 2015).
Tyrode’s solution and equipment are the same as described previously. 8 hr after transfection, 40x images were collected at five frames per second with 33% illumination intensity and a neutral density 0.6 filter in the light path. In order to maximize NMDA receptor currents, cells were imaged in Mg2+ free solution (Jahr and Stevens, 1990). Stimulation was given with a 3 mm spaced parallel bipolar electrode using 30 mA of current.
Beginning at the first stimulation for each experiment, the image stack was divided into 30 s segments (Figure 2) or 10 s segments (Figures 3 and 4). From each segment, an average intensity projection was created and subtracted from a maximum intensity projection. This manipulation highlights puncta and eliminates background. Using this image, 2.5 µm diameter circular ROI’s were placed manually over isolated puncta that did not overlap. 50 ROIs were selected in each experiment in the order that they appear, using as many segments as necessary. ROIs found to overlap multiple puncta in the following projections were relocated to measure only a single punctum. Fluorescence traces were obtained from each ROI and raw fluorescence values converted to a running ∆F/Fo using a 5 s baseline. A point was identified as a peak were identified if its ∆F/Fo was greater than 0.1, its slope calculated in 400 ms steps was greater than 687 units / sec and the peak value was greater than two standard deviations greater than the signal from the previous 2 s. Based on peak time, events that fell within a 1 s window after a stimulation were considered to be evoked and all other events were considered to be spontaneous. Relative to the presynaptic voltage deflection, the postsynaptic Ca2+ signal has a longer rise time and the 1 s window safely includes all evoked events. As the average spontaneous event rate is less than one event per minute, the chance of incorrectly marking a spontaneous event as an evoked event remains relatively low.
All statistical tests were performed using Graphpad Prism 6.01.
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Spontaneous and evoked release are independently regulated at individual active zonesJournal of Neuroscience 33:17253–17263.https://doi.org/10.1523/JNEUROSCI.3334-13.2013
Acute suppression of spontaneous neurotransmission drives synaptic potentiationJournal of Neuroscience 33:6990–7002.https://doi.org/10.1523/JNEUROSCI.4998-12.2013
Indira M RamanReviewing Editor; Northwestern University, United States
In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.
[Editors’ note: a previous version of this study was rejected after peer review, but the authors submitted for reconsideration. The first decision letter after peer review is shown below.]
Thank you for submitting your work entitled "Single synapse evaluation of the postsynaptic NMDA receptors targeted by evoked and spontaneous neurotransmission" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Eve Marder as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Ling-Gang Wu (Reviewer #1).
Our decision has been reached after consultation among the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife.
All the reviewers found the subject matter to be interesting and important, but all converged on a common set of concerns that not only would require substantial further experimentation to address, but which also call into question the central claim of the manuscript. Because it is unlikely that such fundamental matters can be addressed within a few weeks, as is expected for a Research Advance, we cannot consider the present manuscript further. The specific issues, which are laid out fully in the reviews, are as follows:
1) The study hinges on distinguishing spontaneous and evoked release within a single bouton, but there is insufficient evidence that the level of resolution of the techniques used is sufficient to support this claim adequately. More evidence is needed.
2) A number of quantitative factors reduce confidence in the present results, including a) apparently large differences in the mini rate compared to previous studies (from the same group), b) the properties of the indicator used, c) the low statistical power, d) possible rundown, e) analysis methods that give contradictory results on the same data, and f) analysis methods that may not represent accurate measurements of the desired quantities. All these matters would require addressing, several of which would demand additional experiments.
If you choose to undertake the extensive revisions along the lines suggested by the reviewers, we are willing to consider a new, full-length manuscript on this topic.
In the last decade, the authors have discovered that spontaneous and evoked release are regulated separately. In the present work, the author further refines the finding by showing that at the single synapse level, spontaneous and evoked release are regulated separately by releasing to separate NMDA receptors. The finding is of significance in our understanding of synaptic strength, synaptic plasticity and homeostasis.
1) The study implies that spontaneous and evoked release are separated anatomically even within a single bouton. However, this is not supported by electron microscopic evidence. They identified single calcium transient event in postsynaptic compartment by 2.5 μm diameter ROI (in method), which is bigger than a single spine. It is hard to say "single events" in case of ROI is bigger than single spine due to records in several dendritic spines. Stronger evidence would be needed to clarify this point.
2) In Figure 2A, they showed average of spontaneous response was 0.79 events/ROI/min, which is much larger than the value (~0.1 events/ROI/min) shown in a previous study from the same lab (Reese and Kavalali, 1F, GCaMP5K-PSD-95, Mg free condition). This large difference needs to be reconciled.
3) In the previous study, they used Fluo-4 which showed better results than GCaMP5K-PSD95 for detecting spontaneous calcium transient in 1.25 mM Mg, which is close to physiological condition. I wonder if the same conclusion would hold in the more physiological solution (e.g., use Fluo-4 in 1.25 mM Mg).
4) Usually, the frequency of spontaneous release is very low, less than 1 Hz, and has big variation. Therefore, it is necessary to collect enough number of examples for analysis. The cell number and culture number are in generally rather low, often in the range of 2-4. I would suggest to increase the cell number and culture number substantially to consolidate the conclusion. The authors did not show the number of experiment in Figure 3B (with large error bar). That may weaken the conclusion of Figure 3.
5) Figure 3C, control, the ROIs responding to the 2nd 10AP are much less than that to the first 10AP. Does this means a significant system rundown?
In this study, Reese and Kavalali use fluorescent calcium sensors to measure the activity of NMDA receptors in response to glutamate release. Spatially resolving these calcium signals allowed the authors to examine the likelihood that a given synaptic site 'transmits' via spontaneous and/or AP-evoked release. This is an interesting question, and it was addressed by combining the use of the activity-dependent NMDA receptor antagonist, MK-801, to illustrate any overlap between NMDA receptor activity that was driven by spontaneous versus evoked release. From these experiments the authors conclude that while many synapses participate in both evoked and spontaneous release, some synapses seem to preferentially participate in one form of release or the other.
Again, the topic of this study is of great interest, but there are a number of significant concerns that must be addressed, as detailed below.
1) Being able to clearly distinguish between NMDA-calcium signals that arise from spontaneous release versus AP driven release is paramount for the conclusions drawn in this study. As such, the authors need to justify the effectiveness of using NBQX and muscimol to preventing spontaneous APs, either through additional experiments or via very clear citations.
2) On a similar note, the use of NBQX and muscimol must also be shown to not interfere with the triggering or propagation of induced action potentials. This concern is exacerbated by the use of a bipolar electrode which is typically used to provide focused stimulation, so there is a concern that APs are not be driven throughout field of view. In order to claim that some release sites only function spontaneously and not by evoked APs, it must be demonstrated that the stimulation protocol was sufficient to always cause APs. Correlations between spontaneous and evoked rates of release may be confounded by failures to evoke APs particularly if an 'AP failure rate' is unevenly distributed in space relative to the simulating electrode.
3) The study would be much more informative if some discussion of the anatomical correlative of an ROI was provided in the main text. For example, does an ROI contain a single input or multiple inputs? And how does this parameter affect the conclusions of the study?
4) In regards to the MK-801 experiments, it was difficult to understand the various ways the data were analyzed and why certain methods were selectively applied in some cases but not in others. For example, the text for Figure 3A states that the spontaneous response rate was reduced by 87%. However, Figure 3C appears to contradict this claim, as no difference between MK801 and control conditions was apparent. Apparently, by using different methods of analysis for the same experiment, both a significant 87% reduction (vs. no change in controls) and a non-significant 25% reduction (vs 24% in controls) were observed regarding spontaneous response release in the presence of MK-801. This is particularly worrisome given a similar problem seems occur for evoked responses: Figure 3C shows no difference in ROI responders between control and MK-801 treated neurons while Figure 4D shows a nearly 50% reduction in the signal intensity for the first response after MK-801 treatment (compared to controls). This might suggest that a good portion of the individual NMDA receptors participating in evoked release, and not the entire post-synaptic NMDA pool, were indeed blocked by MK-801 during spontaneous release; these findings seem to directly contradict the primary conclusion for the figure. At the very least, a clear, persuasive argument needs to be made as to why different methods of analysis are appropriate; also, a clear, convincing explanation for their apparently different conclusions needs to be provided.
5) It is difficult to understand the main conclusions of Figure 3E through 4A and B because it is not explicitly stated how Rp was measured. If the response probability of a given ROI is measured over the entire post-treatment stimulation protocol (as in Figure 2), then stimulating in the presence of MK-801 would lower the 'Rp' causing it to decrease over the course of measuring it (and as stated, eventually lowering it to 0). Because the act of measuring significantly changes Rp, it is unclear what that number means or what it is actually measuring. The significant confound from these evoked effects (illustrated by Figure 4B) makes it hard to draw or justify conclusions from comparisons with spontaneous release (as attempted in 4A). If the goal here is to examine the ability of spontaneous release to reduce 'Rp', then the analysis should only compare the first stimulation,post-treatment, or somehow otherwise remove the confounding influence that stimulation in MK-801 would have on measuring Rp.
In this manuscript the authors report an investigation of the overlap of spontaneous and evoked transmitter release in individual synapses. The topic is controversial and its investigation therefore clearly important. The authors provide impressive imaging data showing that most synapses show both, spontaneous and stimulated events 22% of synapses show only spontaneous events. The authors claim that there is little correlation between spontaneous and evoked release, which is not so clearly explained.
While the data appears to be quite interesting, some clarification is needed:
An important question is the definition of a synapse. The Materials and methods section states that a 2.5 µm diameter ROI was placed over fluorescent puncta. How certain is it that there is only 1 synapse in such an area and that it does not include 2 synapses. This is important for the main points of the manuscript.
The authors claim that the ability to respond to evoked stimulus after MK-801..is unaffected compared to control. Figure 3C suggests an increased fraction o ROIs responding although clearly not significant. However, absence of significance is not significance of absence. On the other hand Figure 3E indicates decreased release probability. This seems contradictory and should be explained.
Related to this point: Figure 4 and the related text are not very clear.
Figure 3 legend and related text are inconsistent and confusing legend say Figure 3A top is with MK-801 but this is probably wrong. It would also help to increase the separation between top and bottom panel of Figure 3A to make clear to which trace the MK-801 bar belongs. The bar is inconsistent with the legend, which says it starts 4 min after the action potentials were evoked but it seems less than 2 min in the graph.
The term "evoked release probability" needs to be defined and probably a different term should be used because release probability is usually reserved for the probability that a vesicle of the releasable pool is released. Does it mean the probability that a synapse produces a detectable signal in response to an AP? Is that signal always from a single vesicle or could it be from multiple vesicles? The authors need to provide a clear definition.
[Editors’ note: what now follows is the decision letter after the authors submitted for further consideration.]
Thank you for submitting your article "Single synapse evaluation of the postsynaptic NMDA receptors targeted by evoked and spontaneous neurotransmission" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Eve Marder as the Senior Editor. The following individual involved in review of your submission has agreed to reveal his identity: Ling-Gang Wu (Reviewer #2).
The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.
Previous work from this lab has provided evidence that spontaneous release of neurotransmitter and evoked release of neurotransmitter are regulated separately. The present work further shows that this separation can extend to the single synapse level. By using fluorescent calcium sensors to measure the activity of NMDA receptors with high spatial resolution, the authors tested whether a given synaptic site "transmits" via spontaneous and/or AP-evoked release. The results suggest that while many synapses participate in both evoked and spontaneous release, some synapses seem to preferentially participate in one form of release or the other, such that spontaneous and evoked release events act at separate NMDA receptors. The finding is of significance to the understanding of synaptic strength, synaptic plasticity and homeostasis.
While there was considerable enthusiasm for the manuscript and the response to previous comments, the three reviewers were largely in agreement about issues that still require addressing. Two major points emerged. 1) All reviewers found that the rebuttal letter was well written and convincing, but all agreed that the manuscript itself had not been sufficiently edited to ensure that the important information provided in the response to reviewers would be available to readers of the manuscript. Please incorporate the figures and information from the rebuttal either directly into the manuscript main text or add them as supplementary figures. 2) Specific concerns remained about Figures 3 and 4 regarding self-consistency and interpretability.
The combined reviews with key points from the consultation session are included below to facilitate your revision.
Combined reviewers' points regarding essential revision 1:
– The rebuttal letter is, overall, very good. But, the text of the manuscript does not appear to have been changed in any significant way to reflect the issues that were raised, and largely addressed, in the reviews and response to the reviews. Specifically, all three referees asked for detailed discussion of the "ROI" – a key question was: are these single synapses or do they contain multiple synapses? This issue is addressed in the rebuttal but not in the manuscript. Our concerns about the stimulation protocols and recording conditions were also addressed in the rebuttal but not in the revised manuscript. This is an interesting study, but the issues raised do need to be addressed in the revised text of the manuscript.
– The manuscript is now much clearer but the some of the figures in the rebuttal with detailed description should be part of the manuscript. Some could be supplementary information. Author response image 4, lower figure may need detailed description and analysis to make the point. What is the vertical scale in these graphs?
Combined reviewers' points regarding essential revision 1:
– In addition, Figure 3 and 4 still remain confusing – we asked for clarification but clarification was not provided: we still do not know what the bars, labeled "spontaneous" in Figure 3C, or how they relate to the graph show in in Figure 3B.
– The clarification of Figures 3 and 4 is essential. While Figure 3C suggests no effect of MK-801 on spontaneous responses, Figure 3B does. I can somehow imagine what this means but it needs to be spelled out clearly in the text.
– The ∆F/F0 values in Figure 3D seem almost an order of magnitude smaller than in the example traces of panel A. Is the scale bar in A wrong or the values in D or do the traces show unrepresentative signal amplitudes?
– I still do not understand the arguments surrounding Figure 4B. If MK-801 were to block all evoked responses entirely, all data points would lie on the line from x,y = 0,0 to 1,-1 simply because the maximum -∆Rp equals Rp before. The figure shows a rather linear slope of -0.8 suggesting 80% inhibition of Rp whatever it was before MK-801 was added and I do not recognize any relation between blocking efficiency with Rp. ∆Rp may therefore also not be a good choice as analyzed parameter in panel A. Maybe the relative change ∆Rp/Rp(before) might be more instructive.
[Editors' note: further revisions were requested prior to acceptance, as described below.]
Thank you for resubmitting your work entitled "Single synapse evaluation of the postsynaptic NMDA receptors targeted by evoked and spontaneous neurotransmission" for further consideration at eLife. Your revised article has been favorably evaluated by Eve Marder (Senior editor), a Reviewing editor, and three reviewers.
The manuscript is greatly improved but there are some minor points that must be addressed before acceptance, as outlined below:
There remains some confusion about Figure 4B. One reviewer points out that the fact that the data fall on a line suggest that MK801 blocks about 80% of the channels regardless of initial Rp; the percent block necessarily decreases as Rp decreases because there are fewer channels activated that can be blocked. Another reviewer suggests that this is actually the point: MK801 blocking efficiency may be constant at ~80% but the fact that it is predictable from evoked Rp but not spontaneous rate supports the separation of the groups. Since the reviewers had such difficulty working this out, a clarification in the text would be helpful. Also, Figure 4B is not called in the text. Please add a reference to this plot at the appropriate place in the manuscript. Finally, a minor question remains regarding the relationship between the amplitudes of the example signal in Figure 3A and the mean summary data in Figure 3D (see reviewer comment below). Please indicate whether the traces shown unusually large or whether there is an error in the scale bar.
Additional comments from the reviewers are included below so that you can see the nature of the confusion of the reviewers.
The response to the following point is not clear to me:
"I still do not understand the arguments surrounding Figure 4B. If MK-801 were to block all evoked responses entirely, all data points would lie on the line from x,y = 0,0 to 1,-1 simply because the maximum -ΔRp equals Rp before. The figure shows a rather linear slope of -0.8 suggesting 80% inhibition of Rp whatever it was before MK-801 was added and I do not recognize any relation between blocking efficiency with Rp. ΔRp may therefore also not be a good choice as analyzed parameter in panel A. Maybe the relative change ΔRp/Rp(before) might be more instructive."
While the vertical scale of Figure 4B was changed by the authors, the data points are still the same and give in my opinion a misleading impression. The graph does not show ∆Rp/Rp(before). Such a graph will show points scattered around a constant value of ~0.8 for all Rp before values, indicating that the blocking efficiency is independent of Rp. I do not understand what Figure 4B tells us.
The review stated:
"The ΔF/F0 values in Figure 3D seem almost an order of magnitude smaller than in the example traces of panel A. Is the scale bar in A wrong or the values in D or do the traces show unrepresentative signal amplitudes?"
In the rebuttal the authors write:
"The example trace in Figure 1C was chosen for aesthetic reasons and is not a good representative of the average amplitudes. A new representative trace has been chosen that closely matches the average amplitude of 0.67 dF/F for that experiment."
However, the discrepancy the review pointed out is not between Figure 3D and Figure 1C but between panels A and D of Figure 3 because panel A presumably shows representative recorded data that led to the quantification shown in in panel D.https://doi.org/10.7554/eLife.21170.012
- Austin L Reese
- Ege T Kavalali
- Ege T Kavalali
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Animal experimentation: Animal experimentation: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All of the animals were handled according to approved institutional animal care and use committee (IACUC) protocols of the UT Southwestern Medical Center (APN# 0866-06-05-1).
- Indira M Raman, Reviewing Editor, Northwestern University, United States
© 2016, Reese et al.
This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.