Author Response
eLife assessment
This study assesses homeostatic plasticity mechanisms driven by inhibitory GABAergic synapses in cultured cortical neurons. The authors report that up- or down-regulation of GABAergic synaptic strength, rather than excitatory glutamatergic synaptic strength, is critical for homeostatic regulation of neuronal firing rates. The reviewers noted that the findings are potentially important, but they also raised questions. In particular, the evidence supporting the findings is currently incomplete and demonstration of independent regulation of mEPSCs and mIPSCs is a necessary experiment to support the major claims of the study.
We appreciate the detailed, thoughtful assessment of our paper by the reviewers and editors and will submit a revised version in the future that addresses the reviewers’ comments as detailed below in response to each concern. We will include a more open discussion of alternative possibilities. Further, we will repeat the optogenetic experiments assessing AMPAergic scaling in our mouse cortical cultures in order to demonstrate independent regulation of mEPSCs and mIPSCs as suggested.
Reviewer #1 (Public Review):
In the manuscript titled "GABAergic synaptic scaling is triggered by changes in spiking activity rather than transmitter receptor activation," the authors present an investigation of the role of GABAergic synaptic scaling in the maintenance of spike rates in networks of cultured neurons. Their main findings suggest that GABAergic scaling exhibits features consistent with a key homeostatic mechanism that contributes to the stability of neuronal firing rates. Their data demonstrate that GABAergic scaling is multiplicative and emerges when postsynaptic spike rates are altered. Finally, their data suggest that, in contrast to their prior data on glutamatergic scaling, GABAergic scaling is driven by spike rates. The authors set the paper up as an argument that GABAergic scaling, rather than glutamatergic scaling, serves as the critical homeostatic mechanism for spike rate regulation.
While the paper is ambitious in its rhetorical scope and certainly presents intriguing findings, there are several serious concerns that need to be addressed to substantiate the interpretations of the data. For example, the CTZ data do not support the interpretations and conclusions drawn by the authors. Summarily, the authors argue that GABAergic scaling is measuring spiking (at the time scale of the homeostatic response, which they suggest is a key feature of a homeostat) yet their data in figure 5B show more convincingly that CTZ does not influence spiking levels - only one out of four time points is marginally significant (also, I suspect that the bootstrapping method mentioned in line 454-459 was conducted as a pairwise comparison of distributions. There is no mention of multiple comparisons corrections, and I have to assume that the significance at 3h would disappear with correction).
We certainly understand the criticism here (similar to reviewer 2’s third point). In our resubmission we will do a better job discussing these complications, which we now summarize. First, we are presenting our entire dataset to be as transparent as possible. Unlike most synaptic scaling studies (including our own) that apply drugs to alter activity and assess mPSC amplitude at the final time point, here we are actually showing CTZ’s effect on spiking activity within the culture over time. This is critical because it has informed us of the drug’s true effect on spiking, the variability that is associated with these perturbations, and the ability and timing of the cultured network to homeostatically recover initial levels. This was important because it revealed that the drugs do not always influence activity in the way we assume, and this provides greater context to our results. Second, we are showing all of our data, and presenting it using estimation statistics which go beyond the dichotomy of a simple p value yes or no (Ho J, Tumkaya T, Aryal S, Choi H, Claridge-Chang A. 2019. Moving beyond P values: data analysis with estimation graphics. Nat Methods 16: 565-66). Estimation statistics have become a more standard statistical approach in the last 15 years and is the preferred method for the Society for Neuroscience’s eNeuro Journal. This method shows the effect size and the confidence interval of the distribution. For the 3 hr time point in Fig. 5B the CTZ/ethanol vs. ethanol data points exhibit very little overlap and the effect size demonstrates a near doubling of spike frequency, and the confidence interval shows a clear separation from 0. This was a pairwise comparison as we compared values at each time point after the addition of ethanol or ethanol/CTZ. Third, the plots illustrate an upward trend in spike frequency at 1 and 6 hrs, but that there is also clear variability. It is important to note that while these recordings help us to understand effects on spiking across the cultured network, they cannot directly speak to spiking activity in the principal neurons that we target. This complication along with the variability inherent in these cultures could make simple comparisons difficult to interpret. Regardless, we do see some increase in spiking with CTZ and we clearly see increases in mIPSC amplitude, thus providing some support for the idea that spiking could be a critical player in terms of GABAergic scaling, particularly when put in the context of our other findings. However, it is important to recognize that something other than total spike rate may contribute to GABAergic scaling, such as the pattern of spiking that produces a particular calcium transient, and this will be discussed in the resubmission.
Then, the fact that TTX applied on top of CTZ drives a increase in mIPSC amplitude is interpreted as a conclusive demonstration that GABAergic scaling is sensing spiking. It is inevitable, however, that TTX will also severely reduce AMAP-R activation - a very plausible alternative explanation is that the augmentation of AMPAR activation caused by CTZ is not sufficient to overcome the dramatic impact of TTX. All together, these data do not provide substantial evidence for the conclusion drawn by the authors.
We understand this point when considering the CTZ/TTX experiments by themselves. However, spiking appears to be a more straightforward trigger when the CTZ/TTX results are coupled with the prevention of GABAergic downscaling by optogenetic restoration of spiking in the presence of AMPAR antagonists. Further, an important point here is that our results with TTX vs. TTX + CTZ are different for GABAergic scaling (no difference) and AMPAergic scaling (CTZ diminished upward scaling) suggesting different triggers for the two forms of scaling. We will make this more clear in our resubmission.
Specific points:
- The logic of the basis for the argument is somewhat flawed: A homeostat does not require a multiplicative mechanism, nor does it even need to be synaptic. Membrane excitability is a locus of homeostatic regulation of firing, for example. In addition, synapse-specific modulation can also be homeostatic. The only requirement of the homeostat is that its deployment subserves the stabilization of a biological parameter (e.g., firing rate).
We agree with the reviewer and should not have suggested that this was a necessary requirement for a spike rate hemostat. What we should have said was that historically this definition has been attributed to AMPAergic scaling, which is thought to be a spike rate homeostat. We will correct this in the resubmission.
- Line 63 parenthetically references an important, but contradictory study as a brief "however". Given the tone of the writing, it would be more balanced to give this study at least a full sentence of exposition.
Agreed, we will do this.
- The authors state (line 11) that expression of a hyperpolarizing conductance did not trigger scaling. More recent work ('Homeostatic synaptic scaling establishes the specificity of an associative memory') does this via expression of DREADDs and finds robust scaling.
The purpose of citing this study was to argue that the spike rate homeostat hypothesis doesn’t make sense for AMPAergic scaling based on a study that hyperpolarized an individual cell while leaving the rest of the network unaltered and therefore leaving network activity and neurotransmission largely normal. In this case scaling was not triggered, suggesting reduced spike rate within an individual cell was insufficient to trigger scaling. The study that the reviewer refers to hyperpolarizes a majority of cells in the network and therefore will also alter neurotransmission throughout the network, which does not separate the importance of spiking and receptor activation as in the above-mentioned study. We will make this point more clearly in the resubmission.
- Supplemental figure 1 looks largely linear to me? Out of curiosity, wouldn't you expect the left end to be aberrant because scaling up should theoretically increase the strength of some synapses that would have been previously below threshold for detection?
We agree that the scaling ratio plot is largely linear. To be clear, the linearity of the ratio plot was interesting but our main point here was that this line had a positive slope meaning ratios (CNQX mPSC amplitudes/control mPSC amplitudes) got bigger for the larger CNQX-treated mPSCs. Alternatively, a multiplicative relationship where mPSCs are all increased by a single factor (e.g. 2X) would be a flat line with 0 slope at the multiplicative value (e.g. 2). In terms of the left side of the plot, we do see values that rise abruptly from 1 - this is partially obstructed by the Y axis in this figure and we will adjust this. This left part of the plot is likely due the CNQX-induced increases in mPSC amplitudes of mini’s that were below our detection threshold of 5pA. Therefore, mini’s that were 4pAs could now be 5pAs after CNQX treatment and these are then divided by the smallest control mPSCs which are 5 pAs (ratio of 1). We will try to do a better job describing this in the resubmission.
Given that figure 2B also shows warping at the tail ends of similar distributions, how is this to be interpreted?
The left side of the ratio plot shows evidence consistent with the idea that mIPSCs are dropping into the noise after CNQX treatment (similar to above argument), while most of the distribution suggests mIPSCs are reduced to 50% by CNQX treatment. On the right side of the ratio plot the values appear to mostly increase. We are not sure why this is happening, but it looks like some mIPSCs are not purely multiplicative at 0.5, particularly in TTX. It is also important to point out that this is a relatively small percent of the total population and the biggest mPSCs can vary to a great degree from one cell to the next. We will discuss this in the resubmission.
- The readability of the figures is poor. Some of them have inconsistent boundary boxes, bizarre axes, text that appears skewed as if the figures were quickly thrown together and stretched to fit.
We will address these issues in the resubmission.
- I'm concerned about the optogenetic restoration of activity experiment. Cortical pyramidal neuron mean firing rates are log normally distributed and span multiple orders of magnitude. The stimulation experiments can only address the total firing at a network-level - given than a network level "mean" is meaningless in a lognormal distribution, how are we to think about the effect of this manipulation when it comes to individual neurons homeostatically stabilizing their own activities? In essence, the argument is made at the single-neuron level, but the experiment is conducted with a network-level resolution.
As described above, we do not have the capacity to know what the actual firing rate of a particular neuron was before and after introducing a drug and so we cannot absolutely say that we have restored the original firing rates of neurons. However, there is reason to believe that this is achieved to some extent. Our optogenetic stimulation is only 50-100 ms long activating a subset of neurons. This is sufficient to provide a synaptic barrage that then triggers a full blown network burst where the majority of spikes occur, but this is after the light is off. In other words, the optogenetic light pulse only initiates what becomes a normal network burst that fortunately allows the individual cells to express their relatively normal (pre-drug) activity pattern. In our previous study we show that this is the case for individual units - the spiking of an individual unit during a burst is similar before and after CNQX/optostim (see Figure 4b and Suppl. Fig 4 in Fong et al. 2015 Nat. Comm.). We are not claiming that we have restored spiking to exactly the pre-drug state, but bring it back toward those levels and we see this is associated with a return of the mIPSC amplitude to near control levels. We will include a description of this in the resubmission.
- Line 198-99: multiplicativity is not a requirement of a homeostatic mechanism.
- Line 264-265 - again, neither multiplicativity and synaptic mechanisms are fundamentally any more necessary for a homeostatic locus than anything else that can modulate firing rate in via negative feedback.
Agreed, see above discussion of homeostat requirement. Will adjust these statements in our resubmission.
We were not clear enough here. We actually do mean GABAR. The idea is that CTZ increases network activity and thus increases both AMPAergic and GABAergic transmission. We will clarify this in the resubmission.
- Example: Figure 1A is frustratingly unreadable. The axes on the raster insets are microscopic, the arrows are strangely large, and it seems unnecessary to fill so much realestate with 4 rasters. Only one is necessary to show the concept of a network burst. The effect of time+CNQX on the frequency of burst is shown in B and C.
- Example: Figure 2 appears warped and hastily assembled. Statistical indications are shown within and outside of bounding boxes. Axes are not aligned. Labels are not aligned. Font sizes are not equal on equivalent axes.
We will adjust these issues in the resubmission.
- The discussion should include mention of the limitations and/or constraints of drawing general conclusions from cell culture.
We agree and will adjust the discussion. Also, this is why we cited studies that argue GABAergic neurons have a particularly important role in homeostatic regulation of firing following sensory deprivations in vivo.
- The discussion should include mention of the role of developmental age in the expression of specific mechanisms. It is highly likely that what is studied at ~P14 is specific to early postnatal development.
We will discuss caveats of cortical cultures at DIV 14-20.
It is essential to ensure that the data presented in the paper adequately supports the conclusions drawn. A more cautious approach in interpreting the results may lead to a stronger argument and a more robust understanding of the underlying mechanisms at play.
Agreed.
Reviewer #2 (Public Review):
Synaptic scaling has long been proposed as a homeostatic mechanism for the regulation for the activity of individual neurons and networks. The question of whether homeostasis is controlled by neuronal spiking or by the activation of specific receptor populations in individual synapses has remained open. In a previous work, the Wenner group had shown that upscaling of glutamatergic transmission is triggered by direct blockade of glutamate receptors rather than by the concomitant reduction in firing rate (Nat Comm 2015). In this manuscript they investigate the mechanisms regulating scaling of GABA-mediated responses in cortical cell cultures using whole-cell recordings to detect GABAergic currents and multielectrode arrays to monitor global firing activity, and find that spiking plays a fundamental role in scaling.
Initially, the authors show that chronic blockade (24 h) of glutamatergic transmission by CNQX first reduces spontaneous spiking (at 2 h), but later (24 h) firing grows back towards higher frequencies, suggesting a compensatory mechanism. Then it is shown that either chronic CNQX treatment or TTX cause a reduction in the amplitude of GABAergic mIPSCs. Effects of CNQX on IPSCs are then reverted by replacing spontaneous network firing by chronic optogenetic stimulation of the entire culture, also indicating that GABAergic transmission is homeostatically regulated by global firing. Enhancing glutamatergic transmission with CTZ increases mIPSC amplitude, while addition of TTX in the presence of CTZ causes the opposite effect. Finally, increasing spiking activity using bicuculline also increases mIPSC amplitude, and the authors conclude that spiking activity rather than neurotransmission control homeostatic GABA scaling. The manuscript shows interesting properties in the regulation of global GABAergic transmission and highlight the important role of spiking activity in triggering GABA scaling. However, it is strongly recommended to address some caveats in order to better support the conclusions presented in the manuscript.
Major points:
- The reason why CNQX does not completely eliminate spiking is unclear (Fig. 1). What is the circuit mechanism by which spiking continues, although at lower frequency, in the absence of AMPA-mediated transmission and what the mechanism by which spiking frequency grows back after 24h (still in the absence of AMPA transmission)?
Is it possible that NMDA-mediated transmission takes over and triggers a different type of network plasticity?
The bursting in AMPAR blockade is due to the remaining NMDA receptor mediated transmission. We showed this in our previous study in Suppl. Figure 2 and 6 of Fong et al., 2015 Nat. Comm.. Our ability to optically induce normal looking bursts of spikes was also dependent NMDAR activation. Further, in Dr Fong’s PhD dissertation it was shown that the bursting activity was abolished when AMPA and NMDA receptors were both blocked. There are likely many factors that contribute to the recovery of activity, and certainly one of them is likely to be the weakening of inhibitory GABAergic currents. These points will be discussed in the resubmission.
- A possible activation of NMDARs should be considered. One would think that experiments involving chronic glutamatergic blockade could have been conducted in the presence of NMDAR blockers. Why this was not the case?
Unfortunately, it was not possible to optogenetically restore normal bursting in the presence of NMDAR blockade (even when AMPAergic transmission was intact), as NMDARs appeared to be critical for the optical restoration of the normal duration of the burst (see Suppl. Figure 6 Fong et al., 2015 Nat. Comm). The reviewer raises an excellent point about a possible NMDAR contribution to altered synaptic strength, however. It is likely that NMDAR signaling is reduced in the presence of CNQX since burst frequency was reduced along with AMPAR-mediated depolarizations. We cannot rule out the possibility that NMDAR signaling could contribute to the alterations in GABAergic mIPSCs and will discuss this in the resubmission. However, previous work suggests that 24/48 hour block NMDARs (APV) did not trigger AMPAergic scaling in cortical or hippocampal cultures (see Figure 1 Turrigiano et al., 1998 Nature and Suppl. Figure 4 Sutton et al., 2006 Cell), moreover, our previous study showed that restoring NMDAergic transmission optogentically, at least to some point, had no influence on AMPAergic scaling (Fong et al., 2015, Nat. Comm.). Regardless, we cannot rule out a role for NMDAergic transmission in GABAergic scaling and this discussion will be included in the resubmission.
Also, experiments with global ChR2 stimulation with coincident pre and postsynaptic firing might also activate NMDARs and result in additional effects that should be taken into consideration for the global scaling mechanism.
To be clear, our optical stimulation was turned off before the vast majority of spiking that occurred in the bursts, which played out in a relatively natural manner (see lower panel of Figure 3B optogenetic stimulation – short duration only at onset of burst – we will make this clearer in resubmission). Therefore, we were unlikely to trigger significant synchronous activation that does not normally occur in network bursts.
- Cultures exposed to CTZ to enhance AMPA receptors generated variable results (Fig. 5), somewhat increasing spiking activity in a non-significant manner but, at the same time, strengthening mIPSC amplitude. This result seems to suggest that spiking might be involved in GABAergic scaling, but it does not seem to prove it.Then, addition of TTX that blocked spiking reduced mIPSC amplitude. It was concluded here that the ability of CTZ to enhance GABAergic currents was primarily due to spiking, rather than the increase in AMPA-mediated currents. However, in addition to blocking action potentials, TTX would also prevent activation of AMPARs in the presence of CTZ due to the lack of glutamatergic release. Therefore, under these conditions, an effect of glutamatergic activation on GABAergic scaling cannot be ruled out.
These concerns were very similar to reviewer 1’s first comments. We will address these issues in the resubmission, but to briefly repeat our responses: We are going a step beyond most scaling studies by assessing MEA-wide firing rate, but this still provides an incomplete picture of the particular cells that we target for patch recordings in terms of their firing before and after a drug. Further, we see considerable variability in effect on firing rate from culture to culture, which we will better recognize in the resubmission. Finally, While the CTZ results are not conclusive, taken together with the optogenetic results we think our results are most consistent with idea that GABAergic scaling is a strong candidate as a spike rate homeostat.
- The sample size is not mentioned in any figure. How many cells/culture dishes were used in each condition?
The individual dots represent either individual cells for mIPSC amplitude or individual cultures in MEA experiments. Number of cultures for figures were: Figure 2 – con = 10, TTX = 3, CNQX = 6, Figure 4 – CNQX = 4, con = 10, CNQX/photostim = 6, Figure 5 – ethanol = 3, CTZ = 3, CTZ + TTX =3, Figure 6 – con = 10, bicuculline = 4. We will include the number of cultures for mIPSC amplitude experiments in the figure legends upon resubmission.
- Cortical cultures may typically contain about 5-10% GABAergic interneurons and 90-95 % pyramidal cells. One would think that scaling mechanisms occurring in pyramidal cells and interneurons could be distinct, with different impact on the network. Although for whole-cell recordings the authors selected pyramidal looking cells, which might bias recordings towards excitatory neurons, naked eye selection of recording cells is quite difficult in primary cultures. Some of the variability in mIPSC amplitude values (Fig. 2A for example) might be attributed to the cell type? One could use cultures where interneurons are fluorescently labeled to obtain an accurate representation. The issue of the possible differential effects of scaling in pyramidal cells vs. interneurons and the consequences in the network should be discussed.
We will include this discussion in the resubmission. Briefly, we chose large cells, which will be predominantly glutamatergic neurons as suggested by the reviewer. Ultimately, even among glutamatergic principal cells there may be variability in the response to drug application. All of these issues could contribute to variability and we will expand our description of the variability in our results, including that based on cellular heterogeneity.
Reviewer #3 (Public Review):
This paper concerns whether scaling (or homeostatic synaptic plasticity; HSP) occurs similarly at GABA and Glu synapses and comes to the surprising conclusion that these are regulated separately. This is surprising because these were thought to be co-regulated during HSP and in fact, the major mechanisms thought to underlie downscaling (TTX or CNQX driven), retinoic acid and TNF, have been shown to regulate both GABARs and AMPARs directly. (As a side note, it is unclear that the manipulations used in Josesph and Turrigiano represent HSP, and so might not be relevant). Thus the main result, that GABA HSP is dissociable from Glu HSP, is novel and exciting. This suggests either different mechanisms underlie the two processes, or that under certain conditions, another mechanism is engaged that scales one type of synapse and not the other.
However, strong claims require strong evidence, and the results presented here only address GABA HSP, relying on previous work from this lab on Glu HSP (Fong, et al., 2015). But the previous experiments were done in rat cultures, while these experiments are done in mice and at somewhat different ages (DIV). Even identical culture systems can drift over time (possibly due to changes in the components of B27 or other media and supplements). Therefore it is necessary to demonstrate in the same system the dissociation. To be convincing, they need to show the mEPSCs for Fig 4, clearly showing the dissociation. Doing the same for Fig 5 would be great, but I think Fig 4 is the key.
We understand the concern of the reviewer as we do see significant variability within our cultures and they were plated in different places, by different people, in different species (rat vs mouse). Therefore, in the resubmission to strengthen the conclusions we will repeat our optogenetic studies restoring activity in the presence of AMPAergic blockade in our mouse cortical cultures and measuring AMPA mEPSCs to assess scaling.
The paper also suggests that only receptor function or spiking could control HSP, and therefore if it is not receptor function then it must be spiking. This seems like a false dichotomy; there are of course other options. Details in the data may suggest that spiking is not the (or the only) homeostat, as TTX and CNQX causes identical changes in mIPSC amplitude but have different effects on spiking. Further, in Fig 5, CTZ had a minimal effect on spiking but a large effect on mIPSCs. Similar issues appear in Fig 6, where the induction of increased spiking is highly variable, with many cells showing control levels or lower spiking rates. Yet the synaptic changes are robust, across all cells. Overall, this is not persuasive that spiking is necessarily the homeostat for GABA synapses.
Together our results argue against AMPAR or GABAR activation as a trigger for GABAergic scaling and that this is different than our results for AMPAergic scaling. These points alone are important to recognize. While changes in spiking do not perfectly follow the changes in GABAergic scaling they do always trend in the right direction. As mentioned above, total spiking activity is only one measure of spiking. It is possible that these drugs alter the pattern of spiking that translates into an altered calcium transient that is important for triggering the plasticity. Again, it is important to note that we are going a step beyond most homeostatic plasticity studies that add a drug and simply assume it is having an effect on spiking (e.g. CNQX was initially thought to completely abolish spiking, but clearly does not). Based on the variability that we observe and the nature of our MEA recordings we cannot precisely determine how the total activity or pattern of activity changes with drug application in the specific cells that we target for whole cell recordings. However, we believe our results are more consistent with our proposal that GABAergic scaling is a strong candidate as a spike rate homeostat. Regardless, in the resubmission we will include a broader discussion about these possibilities, and the reality that there could be multiple homeostatic mechanisms that act to recover spiking activity.
The paper also suggests that the timing of the GABA changes coincides with the spiking changes, but while they have the time course of the spiking changes and recovery, they only have the 24h time point for synaptic changes. It is impossible to conclude how the time courses align without more data.
We can only say that by the 24 hour CNQX time point, when overall spiking is recovered, that GABAergic scaling has already occurred. We will state this more clearly in the resubmission.
