Author Response
Thank you for your thorough critique and thoughtful suggestions for improving our manuscript, "Homeostatic Synaptic Plasticity of Miniature Excitatory Postsynaptic Currents in Mouse Cortical Cultures Requires Neuronal Rab3A.” The reviewers’ detailed comments suggest that showing multiple types of graphs to demonstrate the presence of divergent scaling of mEPSC amplitudes in cultures from Rab3A wild type, and its disruption in cultures from Rab3A knockout mice, had the unintended consequence of obscuring the major results of our study. Furthermore, our proposal that the difference in characteristics of scaling of GluA2 receptor expression compared to that of mEPSC amplitudes, based on the ratio plots, indicated that a mechanism other than postsynaptic receptors likely contributes to the homeostatic increase in mEPSC amplitude was not convincing to the reviewers. Reviewers 2 and 3 point out these results might be explained by differences in the limitations and artifacts of the two very distinct techniques, electrophysiology and fluorescence imaging. In the revision we will acknowledge that a greater variability in the signal, or, more issues with signal over noise, might be present in imaging experiments compared to electrophysiology. This could explain the lack of identical effects on GluA2 receptors compared to mEPSC amplitudes in the matched experiments, but we maintain it is also possible that a greater variability in GluA2 responses is biologically meaningful. Further, an issue with the accuracy of imaging experiments to report the true receptor effects would also call into question the conclusion that receptors always increase after activity blockade. Finally, the graphs illustrating the detailed characteristics of scaling with rank order and ratio plots required pooling multiple samples per cell, which precludes application of standard statistical methods to determine whether effects or differences reach statistical significance. Therefore, we will remove the cumulative distribution functions, rank order plots, and ratio plots, and show only analyses that involve a single sample per cell. This major change will simplify and clarify the main findings, that homeostatic plasticity of both mEPSC amplitude and GluA2 receptor expression in mouse cortical cultures involves the synaptic vesicle protein Rab3A operating in neurons rather than astrocytes. We will focus our comparison between mEPSC amplitudes and receptors in the same cultures to differences between the magnitude of effects on the mean or median, and will make clear that overall, our data can be explained by two possibilities: 1) the presynaptic vesicle protein is acting via regulation of postsynaptic receptors alone, or, it is regulating both postsynaptic receptors and another contributor to mEPSC amplitude, possibly amount of transmitter released by a single vesicle. Either way, it is very surprising that this presynaptic protein is involved in postsynaptic changes, so our results represent a novel contribution to the field of homeostatic plasticity. In sum, the changes we propose should go a long way towards addressing the majority of the reviewers’ major critiques.
A related issue raised by the reviewers was that the model describing potential presynaptic mechanisms of Rab3A in homeostatic plasticity was not supported by direct evidence (Figure 10). We meant the model to introduce the possibility of a presynaptic contribution to mEPSC amplitude and to stimulate future research, but clearly did not communicate its speculative nature, neither in the Figure legend nor in our discussion of potential mechanisms. In the revision, we will restrict the model to the direct findings in this study. Additionally, we will state where appropriate, that while previous findings at the mouse NMJ are consistent with a presynaptic role for Rab3A (Wang et al., 2011), in the current study there is no direct evidence for this idea in cortical cultures other than the quantitative differences in the fold increases in mEPSC amplitudes and GluA2 receptors which were assayed in the same cultures.
We will submit a revised version addressing each of the reviewer’s concerns and suggestions as described above and below; these major modifications will greatly improve the readability of the manuscript and clarify the main results.
Reviewer #1
Koesters and colleagues investigated the role of the presynaptic small GTPase Rab3A in homeostatic scaling of miniature synaptic transmission in primary mouse cortical cultures using electrophysiology and immunohistochemistry. The major finding is that TTX incubation for 48 hours does not induce an increase in the amplitude of excitatory synaptic miniature events in neuronal cultures derived from Rab3A KO and Rab3A Earlybird mutant mice. NASPM application had comparable effects on mEPSC amplitude in control and after TTX, implying that Ca2+-permeable glutamate receptors are unlikely modulated during synaptic scaling. Immunohistochemical analysis revealed an increase in GluA2 puncta size and intensity in wild type, but not Rab3A KO cultures. Finally, they provide evidence that loss of Rab3A in neurons, but not astrocytes, blocks homeostatic scaling. Based on these data, the authors propose a model in which presynaptic Rab3A is required for homeostatic scaling of synaptic transmission through GluA2-dependent and independent mechanisms.
While the title of the manuscript is mostly supported by data of solid quality, many conclusions, as well as the final model, cannot be derived from the results presented. Importantly, the results do not indicate that Rab3A modulates quantal size on both sides of the synapse. Moreover, several analysis approaches seem inappropriate.
The following points should be addressed:
- The model shown in Figure 10 is not supported by the data. The authors neither provide evidence for two different functional states of Rab3A being involved in mEPSC amplitude modulation, nor for a change in glutamate content of vesicles. Furthermore, the data do not fully support the conclusion of a presynaptic role for Rab3A in homeostatic scaling.
We will revise the model, removing presynaptic mechanisms for Rab3A and restricting it to the direct findings in this study.
- The analysis of mEPSC data using quantile sampling followed by ratio calculation is not meaningful under the tested experimental conditions because of the following reasons:
(i) The analysis implicitly assumes that all events have been detected. The prominent mEPSC frequency increase after TTX suggests that this is not the case, i.e., many (small) mEPSCs are likely missed under control conditions.
We explicitly addressed the potential contribution of missed mEPSCs that are below threshold in (Hanes et al., 2020). We found that even simulating a threshold of 7 pA, applied to data artificially modified by uniformly multiplying the control data set, did not generate a ratio plot with the increasing ratio over 75% of the data that we observe in the experimental data. Overall, the findings from simulating a threshold and a uniform multiplicative factor illustrate that the threshold issue does not cause major changes to the data. Furthermore, in cultures from Rab3A+/+ mice from the Rab3AEbd/+ colony, the mEPSC amplitudes were significantly smaller than those recorded in cultures from Rab3A+/+ mice from the Rab3A+/- colony (lines 327-329, 11 pa vs 13 pA), indicating that if there were smaller mEPSCs occurring in the Rab3A+/+ data set, we would have detected them. Although for these reasons we feel it is unlikely our ratio plot analysis is invalid, to clarify the result that homeostatic plasticity of mEPSC amplitude requires functioning Rab3A, we will remove the ratio plots.
(ii) The analysis is used to conclude how events of a certain size are altered by TTX treatment. However, this analysis compares the smallest mEPSCs of the TTX condition with the smallest control mEPSCs, but this is not a pre-post experimental design. Variation between cells and between coverslips will markedly affect the results and lead to misleading interpretations.
The rank order plot is a well-established plot to examine the mathematical transformation caused by homeostatic plasticity, first used in (Turrigiano et al., 1998). We included it here to facilitate comparison of our findings with previous results. We introduced the ratio plot in (Hanes et al., 2020), finding it shows more clearly differences occurring in the range of small mEPSC values. The reviewer is correct in that we are assuming the smallest mEPSCs before treatment should be matched with the smallest mEPSCs after treatment. It is almost impossible to do a pre-post experimental design for mEPSCs. Even when applying a treatment, for example acute perfusion with a receptor antagonist, to a single cell and recording mEPSCs before and after the treatment, it is not a true pre-post design at the level of mEPSC amplitudes, which come from many different inputs. The power of the method is that different characteristic mathematical transformations for different experimental conditions (e.g., genotype or activity protocol) support the idea that those conditions either involve different mechanisms or have altered the mechanism. Such differences might be missed by only comparing means or medians. However, we found no evidence that loss of Rab3A or expression of the Rab3A Earlybird mutant altered the mathematical transformation due to homeostatic plasticity, other than to reduce its magnitude across all amplitudes. Therefore, including these complex analyses is not adding anything to the finding that Rab3A plays a role in homeostatic plasticity of mEPSC amplitudes and they will be removed in the revision.
(iii) The ratio (TTX/control) vs. control plots seem to suffer from a division by small value artifact (see Figure 6F).
The reviewer is referring to findings on the ratio plot for receptor cluster area. Because the large ratios for the smallest control areas occur in the cultures prepared from wild type mice, and to a much lower extent in cultures prepared from Rab3A knockout mice, we think there is a biologically relevant increase in the TTX/CON ratio, since an artifact due to division by small values should be present in both data sets. However, we cannot rule out that the differences in ratio plot behavior between receptors and mEPSC amplitudes result from the different limitations in detection of receptor clusters vs. the limits of detection of mEPSCs, so we will remove the ratio plots and focus on comparison of means or medians.
Correspondingly, ratio-analysis differs considerably for different control conditions (Fig. 1Giii, Fig. 2Giii, Fig. 6C, Fig. 9A).
The reviewer is correct to point out that the ratio plot shows differences across control conditions (note, these differences are not obvious with the more standard rank order plot). The magnitude of the 50th percentile ratio differs across control conditions, and behaviors of the largest mEPSCs also differ, with some ratios going down at the highest control amplitudes (1Giii, 6C), and others continuing to increase with increasing control amplitude (2Giii, 9A). They all share the divergent increasing ratio from smallest mEPSC amplitude to around the 20 pA level. We attribute the differences in magnitude to the differences in experimental conditions: 1Giii is Rab3A+/+ from the +/+ colony; 1Giii is Rab3A+/+ from the Ebd/+ colony; 6C is a set of Rab3A+/+ cultures assayed several years after the set in 1Giii; 9A is a different culture condition altogether, with neurons being plated onto an already formed bed of astrocytes. Effects on the largest mEPSCs are likely attributable to the small number and high variability of amplitudes in this range. Since the variability in the very sensitive ratio plot have taken away from the main findings of homeostatic plasticity being disrupted in the absence of functioning Rab3A in neurons, we will remove the rank-order and the ratio plots from the manuscript.
- As noted by the authors in a previous publication (Hanes et al. 2020), statistical analysis of CDFs suffers from ninflation. In addition, the quantile sampling method chosen violates an important assumption of the K-S test. Indeed, pvalues for these comparisons are typically several orders of magnitude smaller. Given that the statistical N most likely corresponds to the number of cultures (see, e.g., https://doi.org/10.1371/journal.pbio.2005282), CDF comparisons are not informative and should thus not be used to draw conclusions from the data. The plots can be informative, though.
As the reviewer acknowledges, we were very careful in (Hanes et al., 2020) to state that the p values could not be used to determine significance in the KS test of cumulative distributions for pooled data because the KS test assumes a single sample per cell. We also suggested in that study that the p values could be used in a comparative way for looking at data sets with similar (inflated) n values to say something about bigger or smaller differences. We failed to reiterate those caveats here. In reviewing the article “What is N” by (Lazic et al., 2018) (which we very much appreciate being shown by the reviewer), we agree that in the current study where we are attempting to show how the effect of homeostatic plasticity is or is not altered by loss of Rab3A function, it is imperative that we be able to make conclusions about statistical significance. The pooling approach is essential for having some sense of the mEPSC amplitude distributions, but that is not necessary for looking at the effect of Rab3A. Therefore, we will remove all analyses that involve pooling of multiple mEPSC amplitudes per cell.
- How does recoding noise and the mEPSC amplitude threshold affect "divergent scaling"?
We addressed this in our 2020 paper (Hanes et al., 2020) where we showed that the experimental homeostatic increase in mEPSC amplitude cannot be simulated with uniform, multiplicative synaptic scaling whether we included or excluded distortion caused by a detection threshold.
- What is the justification for the line fits of the ratio data/how was the fit range chosen?
We are assuming the reviewer is referring to the line fits for the rank-order data. If so, the fit range is the entire range of the data. This issue will be addressed by the removal of the rank-order plots from the manuscript.
- TTX application induces a significant increase in mEPSC amplitude in Rab3A-/- mice in two out of three data sets (Figs. 1 and 9). Hence, the major conclusion that Rab3A is required for homeostatic scaling is only partially supported by the data.
Based on the p-values for comparison of means with a Kruskal-Wallis test, we would argue that TTX application does not show a significant increase in mEPSC amplitude in Rab3A-/- neurons (Figure 1 p-value = .318; Figure 9 p-value = .125) when comparing to untreated control mEPSC amplitude means. It is only when we use the KS test and the inflated n’s that we get a barely significant results, p = 0.042. Based on the Lazic article (Lazic et al., 2018), we would now conclude that we cannot use the KS p value in that analysis. We have tried to be clear that the effect of TTX application on mEPSC amplitude in Rab3A-/- neurons is not completely abolished, but rather is dramatically reduced, which we acknowledge in the manuscript (line 279). This issue will be addressed by removal of CDFs from the manuscript.
- Line 289: A comparison of p-values between conditions does not allow any meaningful conclusions.
Although we feel that comparison of magnitude of effects can be stated in a qualitative way for similar sized pooled data sets with larger or smaller p-values, we agree that statistical significance has no meaning. This issue will be addressed by removing the CDF plots from the manuscript.
- There is a significant increase in baseline mEPSC amplitude in Rab3AEbd/Ebd (15 pA) vs. Rab3Aebd/+ (11 pA) cultures, but not in Rab3A-/- (13.6 pA) vs. Rab3A+/- (13.9 pA). Although the nature of scaling was different between Rab3AEbd/Ebd vs. Rab3AEbd/+, and Rab3AEbd/Ebd with vs. without TTX, the question arises whether the increase in mEPSC amplitude in Rab3AEbd/Ebd is Rab3A dependent. Could a Rab3A independent mechanism occlude scaling?
We have acknowledged in the manuscript that one explanation for a failure to exhibit homeostatic plasticity in the cultures from Rab3A Earlybird mutant mice is that the already large basal amplitude occludes any further increase (line 366). In the revision we will make sure the occlusion possibility is highlighted, but we will also discuss other proteins that have been implicated in homeostatic plasticity that have caused an increase in mEPSC amplitude and/or AMPA receptors at baseline, for example, Arc/Arg3.1 KO (Shepherd et al., 2006; Beique et al., 2011); Homer KO (Hu et al., 2010) and inhibition of mir-186-5p (Silva et al., 2019).
- Figure 4: NASPM appears to have a stronger effect on mEPSC frequency in the TTX condition vs. control (-40% vs. 15%). A larger sample size might be necessary to draw definitive conclusions on the contribution of Ca2+-permeable AMPARs.
We will acknowledge that Ca2+-permeable AMPARs could be contributing to the frequency increase following activity blockade and will also include analyses of frequency throughout the manuscript.
- The authors discuss previous papers showing changes in VGLUT1 intensity. Was VGLUT intensity altered in the stainings presented in the manuscript?
We will perform analyses VGLUT1 intensity and include them in the manuscript.
- The change in GluA2 area or fluorescence intensity upon TTX treatment in controls is modest. How does the GluA2 integral change?
The changes in GluA2 integrals look exactly like the changes in cluster size and were not included to simplify the results. But with the removal of the CDFs, rank order, and ratio plots, we can easily include integral measurements. What we did not observe was an additive effect with intensity and size such that the effects on integral were of greater magnitude or statistical significance than either alone. We will include the integral plots in the revised manuscript.
- The quantitative comparison between physiology and microscopy data is problematic. The authors report a mismatch in ratio values between the smallest mEPSC amplitudes and smallest GluA2 receptor cluster sizes (l. 464; Figure 8). Is this comparison affected by the fluorescence intensity threshold?
What was the rationale for a threshold of 400 a.u. or 450 a.u.?
We have acquired AOIs of receptor clusters at multiple threshold levels, and can examine whether the results are altered when using a low, medium or high threshold level.
How does this threshold compare to the mEPSC threshold of 3 pA?
The issue with values being below threshold in untreated cultures has been the concern in interpreting effects on mEPSC amplitudes, specifically, whether this mismatch contributes to divergent scaling. A problem of values being below a toohighly set threshold in the control and becoming detectable after the homeostatic plasticity produces a lower ratio than expected, because now there are values in the treated condition that were not present in the control condition. Instead, for GluA2 receptor cluster size, we observed higher TTX/CON ratios at the low end of the data set. So, based on this, the thresholds chosen for imaging are not having the same effect, if that is what is being asked. This issue will be addressed by removing ratio plots.
The conclusion that an increase in AMPAR levels is not fully responsible for the observed mEPSC increase is mainly based on the rank-order analysis of GluA2 intensity, yielding a slope of ~0.9. There are several points to consider here: (i) GluA2 fluorescence intensity did increase on average, as did GluA2 cluster size. (ii) The increase in GluA2 cluster size is very similar to the increase in mEPSC amplitude (each approx. 18-20%). (iii) Are there any reports that fluorescence intensity values are linearly reporting mEPSC amplitudes (in this system)?
We agree that our data show GluA2 receptors increase as based on cluster size, and did not mean to imply otherwise. Our conclusion that there is another contributor to mEPSC amplitude other than receptors is based on two main findings, 1) that the ratio plots for mEPSC amplitudes and receptor cluster size have distinctively different behaviors, and 2) that there are differences in either magnitude or direction of the TTX effect across 6 matched cultures, 3 from WT animals and 3 from TTX animals (see more explanation of this point below, in response to Reviewer 3). To our knowledge, no one has reported homeostatic plasticity effects on a culture by culture basis, and no one has compared imaging results and physiological results for the same cultures. We will remove the ratio plots and the conclusions based on the differences in behavior for mEPSC amplitudes and receptor cluster size. We will acknowledge in the revision that the differences in magnitude and direction across the 6 matched cultures could be due to the differences in limitations and artifacts of imaging fluorescent antibody staining vs. the limitations and artifacts of detecting mEPSCs electrophysiologically. However, we will continue to state that our results could also be due to the possibility that mEPSC amplitude is not changing in lockstep with receptor levels in every situation. To support this proposal, we will discuss those articles that include both measurements, and point out where mEPSC amplitude measurements and receptor levels match and where they do not.
Antibody labelling efficiency, and false negatives of mEPSC recordings may influence the results. The latter was already noted by the authors.
We will add the caveat that antibody labeling efficiency can vary between coverslips. Although we prepared single solutions that were applied to all coverslips in an experiment, this was not possible for the primary antibody to GluA2, which was added to live cultures in individual wells.(iv) It is not entirely clear if their imaging experiments will sample from all synapses. We will add to Materials and Methods that we sample from all the synapses that could be detected by the researcher on the primary dendrite of the pyramidal cell.
Other AMPAR subtypes than GluA2 could contribute, as could kainate or NMDA receptors.
This is true, other AMPARs (GluA3 and/or GluA4) could be contributing, but we only looked at the receptors well established to be contributing to homeostatic plasticity (GluA1 and GluA2). We will acknowledge the possible contribution of other AMPARs in the revised manuscript.
Furthermore, the statement "complete lack of correspondence of TTX/CON ratios" is not supported by the data presented (l. 515ff). First, under the assumption that no scaling occurs in Rab3A-/- , the TTX/CON ratios show a 20-30% change, which indicates the variation of this readout. Second, the two examples shown in Figure 8 for Rab3A+/+ are actually quite similar (culture #1 and #2), particularly when ignoring the leftmost section of the data, which is heavily affected by the raw values approaching zero.
We will remove the ratio plots from the manuscript and the arguments about differences between GluA2 receptors and mEPSC amplitudes that were based on them. However, we maintain that we have demonstrated a lack of consistent effect for GluA2 receptors and mEPSCs in the matched culture experiments. Yes, the readout of homeostatic plasticity in ratio plots for mEPSCs in the Rab3AKO reach over 1.1 in Figure 1, and as high a 1.2 in the cultures where Rab3AKO neurons were plated on Rab3AWT glia (Figure 9). Our point is that if we had measured GluA2 receptor responses to TTX in those same experiments, the ratios should have been above 1. However, in the experiments in which we measured both mEPSCs and GluA2 receptors, the ratios do not match. In culture #1, the ratio for mEPSCs was at 1 for more than 50% of the data, but for GluA2 receptors, was below 1 for more than 50% of the data. In culture #3, the ratio for mEPSCs was below 1 for more than 50% of the data, but for GluA2 receptors was close to 1.2 for 50% of the data. Only for culture #2 do the ratios appear to match. In the revised manuscript, the evidence that GluA2 receptors and mEPSCs are not changing in parallel will be based on the behavior of means or medians in untreated vs TTXtreated cultures, rather than ratio plots. It could be argued that we need a greater number of matched experiments to make conclusions, but the whole point of a matched experiment is that it should always show the same result—we are no longer dealing with the variability in the homeostatic plasticity itself. We will add a statement that the only three explanations left for the failure of mEPSC amplitudes and GluA2 receptors to change in parallel are 1) a true mismatch, 2) a sampling issue, or 3) technical artifacts that occur in one culture and not another.
- Figure 7A: TTX CDF was shifted to smaller mEPSC amplitude values in Rab3A-/- cultures. How can this be explained?
Figure 7A depicts the pooled data that are shown separately for 3 cultures in Figure 8. We observed mEPSC amplitudes being smaller after TTX treatment in some range of the data for all three Rab3AKO cultures, suggesting that this may be a biological result rather than random variation around no change (which would be a ratio of 1). However, this effect is not significant at the level of means, nor in the KS test (which has the issue of inflated n in any case), so we did not highlight this point. This issue will be addressed by the removal of the CDF plots from the manuscript.
Reviewer #2
Technical concerns:
- The culture condition is questionable. The authors saw no NMDAR current present during spontaneous recordings, which is worrisome since NMDARs should be active in cultures with normal network activity (Watt et al., 2000; Sutton et al., 2006).
The (Watt et al., 2000) study recorded mEPSCs in 0 Mg2+ (Figure 1). The (Sutton et al., 2006) study also shows an average mEPSC waveform (Figure 1D) that was recorded from in 0 Mg2+. Our extracellular recording solution contains Mg2+ (1.3 mM) so we likely are not observing NMDA-mediated currents because they are blocked with Mg2+ when strong depolarizations are prevented with TTX in the recording solution. We will add the idea that the NMDA currents are blocked by Mg2+ to Material and Methods.
It is important to ensure there is enough spiking activity before doing any activity manipulation.
We agree that it would be best if network spiking activity were monitored alongside mEPSC recordings, for example by culturing on multi-electrode arrays. Data from these measurements might explain culture to culture variability in homeostatic responses. To our knowledge, most other studies investigating homeostatic plasticity do not monitor network spiking activity in the same cultures that assay mEPSC amplitudes. This is something that the field should move towards. We will add the caveat that activity was not directly measured to the manuscript.
Similarly, it is also unknown whether spiking activity is normal in Rab3A KO/Ebd neurons.
Since we did not measure spiking activity, we cannot address whether the disruption in homeostatic plasticity in cultures prepared from Rab3A KO and Rab3AEbd/Ebd mutant mice is due to an alteration in network activity. If activity were already low in cultures prepared from these genetically altered mice, we would expect mEPSC amplitudes to be increased, compared to those measured in cultures from WT animals. That is not the case in cultures from Rab3A KO mice, so it is unlikely that network activity is reduced. However, mEPSC amplitudes are increased in Rab3AEbd/Ebd cultures, leaving open this possibility. It would have to be a defect unique to neurons in culture, since the Rab3AEbd/Ebd mouse appears normal in every way, suggesting action potential activity is occurring in the brains of these animals in vivo. We will add the possibility that activity is altered in the cultures from Rab3AKO and Rab3AEbd/Ebd to the manuscript.
- Selection of mEPSC events is not conducted in an unbiased manner. Manually selecting events is insufficient for cumulative distribution analysis, where small biases could skew the entire distribution. Since the authors claim their ratio plot is a better method to detect the uniformity of scaling than the well-established rank-order plot, it is important to use an unbiased population to substantiate this claim.
MiniAnalysis (a standard program used for mEPSC event detection and analysis) selects many false positives with the automated feature (due to the very small sizes of events that are close to the noise level) so manual re-evaluation of the automated process is necessary to eliminate false positives. As soon as there is a manual step, bias is introduced. Interestingly, a manual reevaluation step was applied in a recent study that describes their process as ‘unbiased” (Wu et al., 2020). The alternative is to apply a very large threshold, reducing or eliminating false positives. However, this has the effect of biasing the data towards large events. In sum, we do not believe it is currently possible to perform a completely unbiased detection process. We feel that it is important to include as many small events as possible to reduce the problem of having events in the TTX experimental group that were not matched by events in the control experimental group, for the rank order and ratio plots, so setting the threshold low and manually detecting events accomplishes this. We will add to the Materials and Methods section that the person selecting events did not have information on whether the record was from an untreated or a TTX-treated cell at the time of selection. All of these issues, the potential for skewing the CDFs, and bias potentially interfering in the true rank order and ratio relationships, are addressed by removal of the CDFs, ratio and rank-order plots from the manuscript.
- Immunohistochemistry data analysis is problematic. The authors only labeled dendrites without doing cell-fills to look at morphology, so it is questionable how they differentiate branches from pyramidal neurons and interneurons. Since glutamatergic synapses on these two types of neuron scale in the opposite directions, it is crucial to show that only pyramidal neurons are included for analysis.
MAP2, in addition to labeling dendrites, also labels the cell body, and we used the cell structure revealed by MAP2 staining to select pyramidal-shaped neurons. The selection of the primary dendrite of a pyramidal neuron was stated in lines 239-240 in Materials and Methods and lines 1094 in the figure legend, but we had not explicitly stated how we knew it was a pyramidal neuron. We will include a low power picture of each of the selected pyramidal neurons in the revision.
Conceptual concerns:
The only novel finding here is the implicated role for Rab3A in synaptic scaling, but insights into mechanisms behind this observation are lacking. The author claims that Rab3A likely regulates scaling from the presynaptic side, yet there is no direct evidence from data presented. In its current form, this study's contribution to the field is very limited.
We acknowledge that a presynaptic mechanism is involved in the regulation of homeostatic plasticity by Rab3A is not supported by direct evidence in cortical cultures in this study. But we disagree that the study’s contribution is very limited.
The revised manuscript will emphasize that there are only two possible mechanisms by which Rab3A is acting in homeostatic plasticity. Either this presynaptic vesicle protein is regulating postsynaptic receptors (an extremely surprising result for which we do have direct evidence), or, it is regulating quantal size from both sides of the synapse (supported by direct evidence from our previous study at the mouse neuromuscular junction in vivo, where receptors are not being upregulated during homeostatic plasticity, and, by indirect evidence in the current study, that receptors and mEPSCs are not being identically regulated in the same cultures). Furthermore, the first idea that follows from the effect of Rab3A on receptors is that it would be regulating release of factors from astrocytes, since this is a mechanism that has been shown to be involved in homeostatic plasticity, and we clearly disprove this hypothesis.
- Their major argument for this is that homeostatic effects on mEPSC amplitudes and GluA2 cluster sizes do not match. This is inconsistent with reports from multiple labs showing that upscaling of mEPSC amplitude and GluA2 accumulation occur side by side during scaling (Ibata et al., 2008; Pozo et al., 2012; Tan et al., 2015; Silva et al., 2019).
We agree with the reviewer that many studies show an increase in receptors and mEPSC amplitudes after activity blockade. This is why we were very surprised in our initial experiments to find that there was not a consistent robust increase in receptors in our cultures. At that point we were only imaging, and we assumed that it was homeostatic plasticity that was not always robust. We decided it was essential to measure mEPSC amplitudes and image receptors in the same cultures. We expected to observe larger and smaller effects on mEPSC amplitudes from culture to culture that were paralleled by larger and smaller effects on receptors, but this is not what happened. We have gone back to the literature to look more closely at whether variability across cultures has ever been shown for mEPSC amplitudes, receptors, or both. In a survey of 14 studies, none report results culture by culture. To our knowledge, we are the first to report this variability in the receptor response, and the lack of correlation between mEPSC amplitudes and receptor responses, in the same cultures. That said, for the 4 examples provided by the reviewer, only 1 reports evidence relevant to our study that receptors and mEPSC amplitudes ‘occur side by side,’ which is the (Ibata et al., 2008) study. Here, 24 hr of TTX treatment of rat cortical cultures causes synaptically localized GluA2 receptors in confocal imaging, and mEPSC amplitudes, to both increase to around 130%. The (Pozo et al., 2012) study is not a study of activity blockade but of the effects of overexpressing beta-integrins in rat hippocampal cultures, and this causes both GluA2 receptors and mEPSC amplitudes to increase, but the GluA2 level is not restricted to synaptic sites, and, is expressed as the surface fraction (surface receptor/total receptor—total receptor being surface intensity plus internalized intensity) which increases from 0.5 to 0.55, or to 110%, while mEPSC amplitude increases to ~180%. The (Tan et al., 2015) study only provides Western blot data to show an increase of receptors to 125% in mouse cortical cultures in response to 48 hr TTX, with mEPSC amplitudes increased to ~140%, but the Western blot technique measures synaptic and nonsynaptic receptors on excitatory and inhibitory neurons, as well as receptors on astrocytes. Finally, in (Silva et al., 2019), the culture conditions for the imaging data and the mEPSC amplitude data are markedly different, with ‘low-density’ Banker cultures being used for the former, and ‘high-density’ cultures used for the latter, and the protocol to induce activity blockade is different from ours (noncompetitive AMPA and NMDA blockers); synaptic GluA2 receptors are increased to ~280% and mEPSC amplitudes to ~170%. In the revision we will carefully summarize the previous evidence for receptors and mEPSC amplitude responses to activity blockade. Since it is known that different protocols trigger different molecular mechanisms, for example, TTX + APV triggers a homeostatic plasticity that can be completely reversed by acute application of blockers of Ca-permeable receptors, whereas TTX alone triggers a plasticity that is insensitive to these blockers (Sutton et al., 2006), Figure 4E; (Soden and Chen, 2010); Figure 4A), we will keep our discussion restricted to studies using TTX alone for at least 24 hr. We will acknowledge that our finding that GluA2 receptors and mEPSC amplitudes are not varying in lockstep from culture to culture suggests there is another contributor to mEPSC amplitude, but that we cannot rule out it is due to a greater variability in signal, or more issues with signal over noise, in imaging experiments compared to electrophysiology experiments.
Studies surveyed about reporting results by culture:
(Ju et al., 2004; Stellwagen et al., 2005; Shepherd et al., 2006; Sutton et al., 2006; Cingolani and Goda, 2008; Hou et al., 2008; Ibata et al., 2008; Chang et al., 2010; Hu et al., 2010; Jakawich et al., 2010; Beique et al., 2011; Tatavarty et al., 2013; Diering et al., 2014; Sanderson et al., 2018)
Further, because the acquisition and quantification methods for mEPSC recordings and immunohistochemistry imaging are entirely different (each with its own limitations in signal detection), it is not convincing that the lack of proportional changes must signify a presynaptic component.
We agree with the reviewer that there is no way to compare absolute levels from one type of experimental technique to another, but whatever differences in technical issues there are for the two techniques, they should cause systemic errors and should not contribute to the differences between experiments. Most of the issues with imaging come down to variability in the intensity of fluorescence from experiment to experiment, since the antibody solutions are made anew each time, as is the fixation solution. In addition, the confocal microscope function can vary over time and give brighter or dimmer images. But those kinds of artifacts are addressed by using the same solutions on control and TTX-treated coverslips, and imaging control and TTX-treated coverslips in the same single 2-3 hour imaging session, so that whatever issues there are, they cannot contribute to the TTX effect itself. Therefore when we compare the TTX effect (TTX measurements compared to untreated measurements) from culture to culture and find that in one WT culture there was no increase in receptors but there was in mEPSC amplitude, it is difficult to explain how a limitation specific to the antibody imaging technique could produce such a result. Similarly, when we get the opposite result, that in one KO culture, receptors increased but mEPSC amplitudes did not, it is unclear how limitations in signal detection would produce such a result in one culture but not another. The one exception to this is that the primary GluA2 antibody has to be added individually to each coverslip before returning the dishes to the incubator in order to avoid the disruption to live cells that a complete removal of media would have had. The only remaining ‘artifact’ that could explain the results would be a greater variability in the imaging experiments due to limitations in the signal or the signal to noise ratio. In the revision we will report additional characteristics of imaging experiments, such as average intensity for each coverslip, and for each experiment, to address whether variability in fluorescence levels could explain the variability in TTX effects we observe. We will include the possibility that the mismatches in GluA2 receptors and mEPSCs could be caused by greater variability in the imaging experiments.
- The authors also speculate in the discussion that presynaptic Rab3A could be interacting with retrograde BDNF signaling to regulate postsynaptic AMPARs. Without data showing Rab3A-dependent presynaptic changes after TTX treatment, this argument is not compelling. In this retrograde pathway, BDNF is synthesized in and released from dendrites (Jakawich et al., 2010; Thapliyal et al., 2022), and it is entirely possible for postsynaptic Rab3A to interfere with this process cell-autonomously.
In the revision, the model will focus on the direct findings of the manuscript and tone down the speculation about BDNF signaling, but in the Discussion we will add the possibility that a Rab3A-BDNF interaction could occur either presynaptically or postsynaptically. Interestingly, these articles suggest the postsynaptic BDNF is affecting presynaptic function, namely mEPSC frequency. It is conceivable it could presynaptically affect the vesicle’s release of transmitter.
- The authors propose that a change in AMPAR subunit composition from GluA2-containing ones to GluA1 homomers may account for the distinct changes in mEPSC amplitudes and GluA2 clusters. However, their data from the Naspm wash-in experiments clearly show that GluA1 homomer contributions have not changed before and after TTX treatment.
Our apologies to the reviewer that we were not clear on this point. In lines 396 to 400 we were describing the significant effects that NASPM had on mEPSC frequency on both untreated and TTX-treated cells, despite having only modest, and not quite significant effects on mEPSC amplitude. We conclude from these results that there are synaptic sites that have only GluA1 homomers, and the mEPSCs from these sites are blocked 100% by NASPM. There may be an increase in such GluA1-only synapses after activity blockade, but nevertheless, these events do not contribute to the amplitude increase. So we did not mean to suggest that there is a shift from Glua2 containing to GluA1 containing receptors that leads to the amplitude increase and fully agree with the reviewer that the GluA1 homomer contributions to amplitude have not changed before and after TTX. We will clarify the difference between the contribution of GluA1 homomers to amplitude and frequency in the revised manuscript.
Reviewer #3
Summary: The authors clearly demonstrate the Rab3A plays a role in HSP at excitatory synapses, with substantially less plasticity occurring in the Rab3A KO neurons. There is also no apparent HSP in the Earlybird Rab3A mutation, although baseline synaptic strength seems already elevated. In this context, it is unclear if the plasticity is absent or just occluded by a ceiling effect due the synapses already being strengthened. The authors do appropriately discuss both options. There are also differences in genetic background between the Rab3A KO and Earlybird mutants that could also impact the results, which are also noted. The authors have solid data showing that Rab3A is unlikely to be active in astrocytes, Finally, they attempt to study the linkage between synaptic strength during HSP and AMPA receptor trafficking, and conclude that trafficking is largely not responsible for the changes in synaptic strength.
Strengths: This work adds another player into the mechanisms underlying an important form of synaptic plasticity. The plasticity is only reduced, suggesting Rab3A is only partially required and perhaps multiple mechanisms contribute. The authors speculate about some possible novel mechanisms.
Weaknesses: However, the rather strong conclusions on the dissociation of AMPAR trafficking and synaptic response are made from somewhat weaker data. The key issue is the GluA2 immunostaining in comparison with the mESPC recordings. Their imaging method involves only assessing puncta clearly associated with a MAP2 labeled dendrite. This is a small subset of synapses, judging from the sample micrographs (Fig 5). To my knowledge, this is a new and unvalidated approach that could represent a particular subset of synapses not representative of the synapses contributing to the mEPSC change. (they are also sampling different neurons for the two measurements; an additional unknown detail is how far from the cell body were the analyzed dendrites for immunostaining. While the authors acknowledge that a sampling issue could explain the data, they still use this data to draw strong conclusions about the lack of AMPAR trafficking contribution to the mEPSC amplitude change. This apparent difference may be a methodological issue rather than a biological one, and at this point it is impossible to differentiate these. It will unfortunately be difficult to validate their approach. Perhaps if they were to drive NMDA-dependent LTD or chemLTP, and show alignment of the imaging and ephys, that would help. More helpful would be recordings and imaging from the same neurons but this is challenging. Sampling from identified synapses would of course be ideal, perhaps from 2P uncaging combined with SEP-labeled AMPARs, but this is more challenging still. But without data to validate the method, it seems unwarranted to make such strong conclusions such as that AMPAR trafficking does not underlie the increase in mEPSC amplitude, given the previous data supporting such a model.
We chose the primary dendrite to ensure we were not assaying dendrites from inhibitory neurons or on axons, but we will add in the revision that it is a limitation of our methods that we are not sampling all the synapses for each neuron. The majority of previous studies that establish that receptors are increased side by side with mEPSCs did not measure receptors and mEPSCs in the same cells, nor even in the same cultures. There is a recent study which employs dual recordings, transfection of GluA2 and VGlut1 constructs, and infusion of dyes to highlight cell morphology (Letellier et al., 2019), so in principle an experiment could be done in which synaptic GluA2 sites are imaged in a cell in which the mEPSCs are also measured. It would be difficult to make these measurements in the same cells before and after TTX treatment, since there is a high likelihood of damaging the cell upon electrode withdrawal and with the imaging process itself. In theory, only a few such experiments would be necessary to establish whether receptors and mEPSC amplitudes are varying in lockstep, and we will consider this for a future study. As stated in response to conceptual concern #1 in Reviewer 2’s comments, we will review the literature on previous studies’ demonstrations of increases in receptors and mEPSC amplitudes following activity blockade in more detail, including how the synaptic sites to be imaged were chosen, to address whether our selection of sites touching the primary dendrite is unvalidated.
A sample from 3 articles:
(Ibata et al., 2008), only information is that ‘distal dendrites’ were examined. The authors do not use a dendritic label. (Jakawich et al., 2010), ‘neurons with pyramidal-like morphology were selected for imaging,’ and ‘principal dendrite of each neuron was linearized’—but how these were identified is not clear, since MAP2 or other cellular labels are not described.
(Silva et al., 2019), ‘dendrites with similar thickness and appearance were randomly selected using MAP2 staining,’ which suggests synaptic sites with GluA2 and VGLUT1 were selected on the basis of being close to or touching the MAP2 positive dendrite, although this is not stated explicitly.
We can perform length measurements on the dendrites imaged and report this information in the revision, but the primary dendrite is the closest dendrite to the cell body.
We have addressed the potential contribution of technical artifacts arising from the two distinct methods of measurement, imaging and electrophysiology, in our response to conceptual concern #1 of Reviewer 2.
Other questions arise from the NASPM experiments, used to justify looking at GluA2 (and not GluA1) in the immunostaining. First, there is a frequency effect that is quite unclear in origin. One would expect NASPM to merely block some fraction of the post-synaptic current, and not affect pre-synaptic release or block whole synapses. It is also unclear why the authors argue this proves that the NASPM was at an effective concentration (lines 399-400).
We observed a clear effect of NASPM reducing mEPSC frequency. We will state more clearly that we infer from the loss of mEPSCs after NASPM that such mEPSCs were from synaptic sites that had only GluA1 homomers, and acknowledge that this is an interpretation. We will also clarify that if our inference is correct, it would indicate that the dose of NASPM we used was 100% effective at blocking GluA1 homomers. The alternative explanation would be a presynaptic effect of NASPM, which has never been reported, to our knowledge.
Further, the amplitude data show a strong trend towards smaller amplitude. The p value for both control and TTX neurons was 0.08 - it is very difficult to argue that there is no effect. And the decrease is larger in the TTX neurons. Considering the strong claims for a pre-synaptic and the use of this data to justify only looking at GluA2 by immunostaining, these data do not offer much support of the conclusions. Between the sampling issues and perhaps looking at the wrong GluA subunit, it seems premature to argue that trafficking is not a contributor to the mEPSC amplitude change, especially given the substantial support for that hypothesis. Further, even if trafficking is not the major contributor, there could be shifts in conductance (perhaps due to regulation of auxiliary subunits) that does not necessitate a pre-synaptic locus. While the authors are free to hypothesize such a mechanism, it would be prudent to acknowledge other options and explanations.
We did not mean to suggest that there is no effect of NASPM on mEPSC amplitude. We will clarify that our data indicate that there is no effect of NASPM on the TTX effect on mEPSC amplitude. We agree with the reviewer that the effect of NASPM on frequency is of larger magnitude after TTX treatment, although the p value is larger than that for untreated cells, likely due to greater variability. We interpret this to mean that TTX treatment increases the proportion of synapses that have only GluA1 homomers. Nevertheless, the increase in GluA1 homomer sites does not appear to contribute to the overall increase in amplitude following TTX treatment, and we wanted to find the mechanism of the amplitude increase. That is why we focused on GluA2 receptors. We will acknowledge the limitation of basing our conclusions on only GluA2 receptors in the revision, as well as the possibility that there is a change in conductance. As stated in our response to Reviewer 2, we do not mean to state that GluA2 receptors do not go up after activity blockade, we find that this is the case. We are proposing an additional mechanism contributing to mEPSC amplitude to explain the different responses for GluA2 receptors vs. mEPSC amplitudes in some of the 6 matched experiments (3 WT and 3 KO).
The frequency data are missing from the paper, with the exception of the NASPM dataset. The mEPSC frequencies should be reported for all experiments, particularly given that Rab3A is generally viewed as a pre-synaptic protein regulating release. Also, in the NASPM experiments, the average frequency is much higher in the TTX treated cultures. Is this statistically above control values?
We will report frequency measurements for all experiments shown. Following TTX treatment, frequency variability increases enormously, with cells having as high as > 10 mEPSCs per second, and other TTX-treated cells with frequencies as low as < 1 mEPSC per second, so the TTX effect on frequency, and whether this effect is present or not in Rab3A KO and Rab3AEbd/Ebd is not completely clear, which is why we did not include those results previously.
Unaddressed issues that would greatly increase the impact of the paper:
- Is Rab3A acting pre-synaptically, post-synaptically or both? The authors provide good evidence that Rab3A is acting within neurons and not astrocytes. But where it is acting (pre or post) would aid substantially in understanding its role (and particularly the hypothesized and somewhat novel idea that the amount of glutamate released per vesicle is altered in HSP). They could use sparse knock-down of Rab3A, or simply mix cultures from KO and WT mice (with appropriate tags/labels). The general view in the field has been that HSP is regulated post-synaptically via regulation of AMPAR trafficking, and considerable evidence supports this view. The more support for their suggestion of a pre-synaptic site of control, the better.
We agree with the reviewer that this is the most important question to answer next. The approach suggested by the reviewer would be to record from Rab3A KO neurons in a culture where the majority of its inputs are Rab3A positive. If the TTX effect is absent from these cells, it would strongly indicate that postsynaptic Rab3A is required for homeostatic plasticity. There are not currently transgenic mice expressing GFP forms of Rab3A, so we would have to create one, or, transiently transfect Rab3A-GFP into Rab3AKO neurons. Given that under our experimental conditions, we require a very high density of neurons to observe the increase in mEPSC amplitude, it would be difficult to get the ratio of Rab3A-expressing neurons high enough using transfection to be sure that a given postsynaptic cell lacking Rab3A had a normal number of Rab3A-positive inputs and almost no Rab3A-negative inputs. It may be that the opposite experiment is more doable—an isolated Rab3A-positive neuron in a sea of Rab3A-negative neurons, which could be accomplished with a very low transfection efficiency. Another approach would be to use the fast off rate antagonist gamma-DGG, which is more effective against low glutamate concentrations than high glutamate concentrations (see (Liu et al., 1999; Wu et al., 2007). If gamma-DGG were less effective at reducing mEPSC amplitude in TTX-treated cells, compared to untreated cells, it would support the hypothesis that activity blockade leads to an increase in the amount of transmitter per vesicle fusion event. Further, if the change in gamma-DGG sensitivity after activity blockade were disrupted in cultures from Rab3A KO cells, it would support a presynaptic role for Rab3A in homeostatic plasticity of mEPSC amplitude. We have begun these experiments but are finding the surprising result that within a single recording, small mEPSCs and large mEPSCs appear to be differentially sensitive to gamma-DGG. To confirm that this is a biological characteristic, rather than an issue with the detection threshold, we will be repeating our experiments with a slow off rate antagonist that has same effect regardless of transmitter concentration. The complexity of these results precludes including them in the current manuscript.
- Rab3A is also found at inhibitory synapses. It would be very informative to know if HSP at inhibitory synapses is similarly affected. This is particularly relevant as at inhibitory synapses, one expects a removal of GABARs and/or a decrease of GABA-packaging in vesicles (ie the opposite of whatever is happening at excitatory synapses). If both processes are regulated by Rab3A, this might suggest a role for this protein more upstream in the signaling; an effect only at excitatory synapses would argue for a more specific role just at these synapses.
The next question, after it is determined where Rab3A is acting, is whether it is required for other forms of homeostatic plasticity. This includes plasticity of GABA mIPSCs on pyramidal neurons, but also mEPSCs on inhibitory neurons, and, the downscaling of mEPSCs (and upscaling of mIPSCs) when activity is increased, by bicuculline for example. We will add a statement about future experiments examining other forms of plasticity to the discussion, and include examples where a molecular mechanism has mediated multiple forms, and those that have been shown to be very specific.
Beique JC, Na Y, Kuhl D, Worley PF, Huganir RL (2011) Arc-dependent synapse-specific homeostatic plasticity. Proc Natl Acad Sci U S A 108:816-821.
Chang MC, Park JM, Pelkey KA, Grabenstatter HL, Xu D, Linden DJ, Sutula TP, McBain CJ, Worley PF (2010) Narp regulates homeostatic scaling of excitatory synapses on parvalbumin-expressing interneurons. Nat Neurosci 13:1090-1097.
Cingolani LA, Goda Y (2008) Differential involvement of beta3 integrin in pre- and postsynaptic forms of adaptation to chronic activity deprivation. Neuron Glia Biol 4:179-187.
Diering GH, Gustina AS, Huganir RL (2014) PKA-GluA1 coupling via AKAP5 controls AMPA receptor phosphorylation and cell-surface targeting during bidirectional homeostatic plasticity. Neuron 84:790-805.
Hanes AL, Koesters AG, Fong MF, Altimimi HF, Stellwagen D, Wenner P, Engisch KL (2020) Divergent Synaptic Scaling of Miniature EPSCs following Activity Blockade in Dissociated Neuronal Cultures. J Neurosci 40:4090-4102.
Hou Q, Zhang D, Jarzylo L, Huganir RL, Man HY (2008) Homeostatic regulation of AMPA receptor expression at single hippocampal synapses. Proc Natl Acad Sci U S A 105:775-780.
Hu JH, Park JM, Park S, Xiao B, Dehoff MH, Kim S, Hayashi T, Schwarz MK, Huganir RL, Seeburg PH, Linden DJ, Worley PF (2010) Homeostatic scaling requires group I mGluR activation mediated by Homer1a. Neuron 68:1128-1142.
Ibata K, Sun Q, Turrigiano GG (2008) Rapid synaptic scaling induced by changes in postsynaptic firing. Neuron 57:819826.
Jakawich SK, Nasser HB, Strong MJ, McCartney AJ, Perez AS, Rakesh N, Carruthers CJ, Sutton MA (2010) Local presynaptic activity gates homeostatic changes in presynaptic function driven by dendritic BDNF synthesis. Neuron 68:1143-1158.
Ju W, Morishita W, Tsui J, Gaietta G, Deerinck TJ, Adams SR, Garner CC, Tsien RY, Ellisman MH, Malenka RC (2004) Activity-dependent regulation of dendritic synthesis and trafficking of AMPA receptors. Nat Neurosci 7:244-253.
Lazic SE, Clarke-Williams CJ, Munafo MR (2018) What exactly is 'N' in cell culture and animal experiments? PLoS Biol 16:e2005282.
Liu G, Choi S, Tsien RW (1999) Variability of neurotransmitter concentration and nonsaturation of postsynaptic AMPA receptors at synapses in hippocampal cultures and slices. Neuron 22:395-409.
Pozo K, Cingolani LA, Bassani S, Laurent F, Passafaro M, Goda Y (2012) beta3 integrin interacts directly with GluA2 AMPA receptor subunit and regulates AMPA receptor expression in hippocampal neurons. Proc Natl Acad Sci U S A 109:1323-1328.
Sanderson JL, Scott JD, Dell'Acqua ML (2018) Control of Homeostatic Synaptic Plasticity by AKAP-Anchored Kinase and Phosphatase Regulation of Ca(2+)-Permeable AMPA Receptors. J Neurosci 38:2863-2876.
Shepherd JD, Rumbaugh G, Wu J, Chowdhury S, Plath N, Kuhl D, Huganir RL, Worley PF (2006) Arc/Arg3.1 mediates homeostatic synaptic scaling of AMPA receptors. Neuron 52:475-484.
Silva MM, Rodrigues B, Fernandes J, Santos SD, Carreto L, Santos MAS, Pinheiro P, Carvalho AL (2019) MicroRNA186-5p controls GluA2 surface expression and synaptic scaling in hippocampal neurons. Proc Natl Acad Sci U S A 116:5727-5736.
Soden ME, Chen L (2010) Fragile X protein FMRP is required for homeostatic plasticity and regulation of synaptic strength by retinoic acid. J Neurosci 30:16910-16921.
Stellwagen D, Beattie EC, Seo JY, Malenka RC (2005) Differential regulation of AMPA receptor and GABA receptor trafficking by tumor necrosis factor-alpha. J Neurosci 25:3219-3228.
Sutton MA, Ito HT, Cressy P, Kempf C, Woo JC, Schuman EM (2006) Miniature neurotransmission stabilizes synaptic function via tonic suppression of local dendritic protein synthesis. Cell 125:785-799.
Tan HL, Queenan BN, Huganir RL (2015) GRIP1 is required for homeostatic regulation of AMPAR trafficking. Proc Natl Acad Sci U S A 112:10026-10031.
Tatavarty V, Sun Q, Turrigiano GG (2013) How to scale down postsynaptic strength. J Neurosci 33:13179-13189.
Turrigiano GG, Leslie KR, Desai NS, Rutherford LC, Nelson SB (1998) Activity-dependent scaling of quantal amplitude in neocortical neurons. Nature 391:892-896.
Wang X, Wang Q, Yang S, Bucan M, Rich MM, Engisch KL (2011) Impaired activity-dependent plasticity of quantal amplitude at the neuromuscular junction of Rab3A deletion and Rab3A earlybird mutant mice. J Neurosci 31:3580-3588.
Watt AJ, van Rossum MC, MacLeod KM, Nelson SB, Turrigiano GG (2000) Activity coregulates quantal AMPA and NMDA currents at neocortical synapses. Neuron 26:659-670.
Wu XS, Xue L, Mohan R, Paradiso K, Gillis KD, Wu LG (2007) The origin of quantal size variation: vesicular glutamate concentration plays a significant role. J Neurosci 27:3046-3056.
Wu YK, Hengen KB, Turrigiano GG, Gjorgjieva J (2020) Homeostatic mechanisms regulate distinct aspects of cortical circuit dynamics. Proc Natl Acad Sci U S A 117:24514-24525.
