NMDA Receptors Enhance the Fidelity of Synaptic Integration

Synthesis

Both reviewers and I agreed that this is a useful and thoughtful study of the impact of NMDARs on synaptic integration. However, we also agreed that you ought to clarify what is novel about your work. There have been many studies of this fundamental subject, and it is important to put your work in the context of the existing literature.

One of the reviewers wrote a particularly detailed and clear critique of the paper (reproduced below). All three of us agreed in whole or in part with the points they made (especially the first one about novelty and context). Please respond to it point by point.

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CRITIQUE BEGINS HERE

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This is an interesting study of basic membrane properties in dendrites of the central nervous system neurons. While dealing with well-known facts and receiving absolutely no help from novel experimental data, relying only on their imagination and cleverness, the authors were able to squeeze out some interesting observations about the behavior of a dendritic branch operating in the environment entirely based on AMPA and NMDA conductances. In this computer simulation study, the author’s best tools are two measures they developed: “CV of synaptic thresholds” and “Effect size (d)", where “d” is derived from “CV of synaptic thresholds” measurements in two subsequent conditions. These two numerical measures (“CV” and “d”) will help them generate enough graphical displays to account for one scientific paper.

There are some straight forward relationships that used to be straight-forward in the past, but are now packaged into a novelty. For example, because AMPA-mediated EPSPs are largest at hyperpolarized potentials (many publications), and NMDA-mediated EPSPs are largest at depolarized potentials (too many publications to cite), it is straightforward that AMPA+NMDA activation will average these extremes over the RMPs (VOLTAGE) and produce a family of EPSPs with smaller amplitude ranges than AMPA-only or NMDA-only.

Another straight forward example follows. Because AMPA-mediated EPSPs are largest when originate in proximal dendrite (tons of published work), and NMDA-mediated EPSPs are largest when originate in distal dendrite (both real data and model data were published), it is straightforward that AMPA+NMDA activation will average these extremes over DISTANCE and produce family of EPSPs with smaller amplitude ranges than AMPA-only or NMDA-only.

Nevertheless, I find certain educational value in the figures and statements of the current manuscript, and I think that some results are worth publishing.

I think that this manuscript should not be published if the authors fail to adequately modify the body of the manuscript, and resolve serious omissions in scientific presentation, plaguing the Results, Figures, Introduction, and Discussion. The manuscript deficiencies are listed below.

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[1]

It is absolutely necessary to resolve the borders between the current study and the previous studies, which have already explained the importance of the AMPA-NMDA synergism, the impact of NMDA receptors on the spatial and voltage aspects of synaptic integration, and the impact of the AMPA-NMDA ratios.

To explain the novelty of their study, these authors wrote: “Here we report that the voltage-dependency and kinetics of NMDA receptors act synergistically to stabilize synaptic integration of excitatory postsynaptic potentials (EPSPs) across spatial and voltage domains.”

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1.1. - Synergistically?

The first main finding of the authors is that AMPA and NMDA receptors act synergistically. I see a major overlap between this “novelty” and previously published facts. It was shown many years ago, in multiple publications, that AMPA receptor activation is essential for the function of NMDA receptors. In this sense, “the news” that AMPA and NMDA receptors act synergistically, is really not a news.

When researchers publish the impact of the AMPA-to-NMDA ratio on EPSP or synaptic integration, this is an example of synergistic action between two receptors.

It is the job of the authors, not the readers, to explicitly state that AMPA-NMDA synergism is well established (cite the original contributors) and then state exactly the author’s contribution to the AMPA-NMDA synergism here that warrants novelty?

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1.2. - Spatial domains?

The second main finding of the authors is that AMPA and NMDA receptors act synergistically to stabilize synaptic integration of excitatory postsynaptic potentials (EPSPs) across spatial domains. I found major overlap between this “novelty” and previously published facts.

[1.2.a] Researchers have already established that NMDA receptors stabilize synaptic integration across spatial domains. In their influential article published in Science, Branco et al (2010) clearly stated: “... the NMDA-dependent mechanism produced strong sensitivity to the direction of synaptic input, even in short pyramidal cell dendrites, making it more general and sensitive to synaptic input, robust against timing jitter, ...”. Pertaining to distributions of synaptic inputs along individual dendritic branches (distribution in space), Branco and colleagues have already shown that NMDA receptors stabilize synaptic integration in the spatial domain.

[1.2.b] Researchers have already established that mechanisms involving NMDA receptors do not require spatially coincident input patterns. EPSP amplification by NMDA receptors occurs when synaptic inputs are either clustered onto individual dendrites, or when they are distributed throughout the dendritic tree (Connelly et al., 2016, Major et al., 2013). This means: “NMDA receptors stabilize synaptic integration across spatial domains, when synaptic inputs are distributed in space onto different dendritic branches of the same neuron”.

[1.2.c] Researchers have already established that “distal inputs are amplified with high gain and integrated over broader time windows”. This allows distal inputs to overcome their electrotonic disadvantage, and become surprisingly more effective than proximal inputs at influencing action potential output” (Branco and Hausser, 2010). This is very similar to the conclusion of the current paper EACH time the current paper deals with synaptic thresholds for action potential output.

[1.2.d] Analysis of the simulations revealed that the synaptic integration gradients can be explained by the interaction between active NMDA conductances and the progressive increase in dendritic input impedance toward the tip of the branch (Branco and Hausser, 2010). This is very similar to the conclusion of the current paper EACH time the current paper mentions that NMDA receptors improve voltage amplitudes in distal dendritic segment.

[1.2.e] Using uniform synaptic NMDA conductance density in the model, the researchers established that the synaptic integration gradients do not require any underlying gradients in the properties of the synapses or in the dendritic distribution of voltage-gated channels. The model could reproduce experimentally observed integration gradients using entirely uniform synaptic parameters and densities of voltage-gated NMDA channels (Branco and Hausser, 2010). This is very similar to uniform densities of voltage-gated NMDA channels in the present study.

[1.2.f] The amplitude of unitary EPSP at the soma of CA1 pyramidal cells is independent of synapse location across the entire range of afferent inputs (Cook and Magee, 2000). This is very similar to the results reported here.

It is the job of the authors, NOT THE JOB of the READERS, to explicitly state that NMDA receptors stabilize synaptic integration across spatial domain (cite the original contributors), and then state exactly the present author’s contribution to the NMDA role in the “space domain” that warrants novelty?

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1.3. - AMPA/NMDA ratio?

[1.3.a] “asynchronous inputs can effectively interact over a wide time window to increase the local membrane depolarization and recruit more NMDAR conductance, thereby producing a broad window for synaptic integration at distal dendrites. This was reproduced with a range of NMDA:AMPA ratios (Figure S1E). This interaction between NMDA and AMPA receptors is sufficient to explain the increased NMDA component of single synapses at distal locations (Branco and Hausser, 2010).

[1.3.b] The impact of AMPA to NMDA receptor ratio on somatic voltage was discussed in Major et al., 2013.

[1.3.c] Desynchronizing the inputs is roughly equivalent to reducing the AMPA component: Time jitter has far less effect on the much slower NMDA component (Major et al., 2013).

It is the job of the authors, NOT THE JOB of the READERS, to explicitly state that AMPA:NMDA ratio was thoroughly explored in the context of synaptic integration (cite the original contributors), and then state exactly the present author’s contribution to this topic that warrants novelty? How is the present author’s AMPA/NMDA ratio conclusion different from the conclusions stated in the published literature studying efficacy of synaptic integration in silico?

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1.4. - Identical outline and similar result

"Figure 1” is conceptually identical, and result-wise similar, to:

Figure 4b, yellow, Magee, 2000.

Figure 1 of Gulledge et al., 2005.

Figure 1F of Major et al., 2013.

This overlap should be mentioned in the paper, and the present author’s novel contribution should be discussed.

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1.5. - NMDA spike width in distal dendrite

"Whereas the increase in local response amplitude with distance was linear when inputs involved AMPA conductances only, incorporation of the NMDA conductance, by itself or in combination with the AMPA conductance, allowed for supralinear distance-dependent increases in response amplitude and width, including long lasting regenerative NMDA spikes, at distal dendritic locations (Figure 2B)”.

Distance-dependent increase in NMDA-mediated response width (duration of NMDA spike) was demonstrated by Gao et al., 2019 (BioRxiv), their figure 5. The authors should state their improvement over Gao et al., 2019.

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[2] - Cardinal error in modeling soma and axon

The authors discuss AMPA and NMDA receptor activation in the context of cortical pyramidal neurons (neocortical pyramidal or hippocampal pyramidal) - 99% of references is on pyramidal neurons. Consequently, they use a model of a hippocampal CA3 pyramidal neuron. However, in the ball-and-stick model (Fig. 2A), and very likely in other figures as well, the authors use a highly inappropriate mechanism for action potential generation, with pronounced fast afterhyperpolarization, characteristic of hippocampal interneurons, not pyramids. Should the authors wish to inform themselves of the natural AP waveforms in the somata of hippocampal pyramids, they should examine Raus Balind et al., 2019.

Anyhow, with super-strong FAST afterhyperpolarization occurring in the axon, soma and proximal dendrites, the authors have unnaturally altered the waveforms of EPSPs in dendrite. For this reason, the computer exercises shown in figures must be revised (repeated) and re-analyzed to include axon mechanisms suitable to pyramidal neurons, producing somatic voltage waveforms that resemble those in pyramidal somata responding to dendritic suprathreshold excitation (Raus Balind et al., 2019).

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[3] - Delete Figure 5.

Figure 5, and all accompanying text, should be deleted from the manuscript. It takes several hours to understand figures 4 and 5.

Figure 5 contributes little to the impact of the paper.

Fig. 5 is too complex and ineligible - the authors are strongly advised to remove it.

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[4] - Simplify Fig. 4

Figure 4 is too dense and ineligible.

The middle panel of 4A should be DELETED. The medium size changes, contributed nothing to the manuscript. Deletion of the medium size panel in 4A will eliminate medium-size lines in 4B, 4C, 4D, to alleviate the clutter.

The strange, tumor-like graph “200 um dendrite (big neuron)", should be deleted from Fig. 4B. It severely violates the logic of presentation, composition of the figure panel, and compromises reader’s understanding of the figure.

The “200 um dendrite (big neuron)” graph insert should be deleted from Fig. 4C.

The “200 um dendrite (big neuron)” graph insert should be deleted from Fig. 4D and 4E,

to alleviate clutter and help important messages come forward.

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[5] - The authors write in a cryptic style, leaving out the important details and explanations

The following text is an example of a highly problematic writing style frequently employed in the Results.

>> “To test whether the impact of dendritic length on synaptic threshold variability was reflective of electrotonic structure of the neuron, we measured synaptic thresholds in a 200-μm dendrite attached to larger neurons having 1-to-5 additional dendrites (600 μm each; Figure 3A). Even as the measured dendrite remained static (at 200 μm), the effect size of combining AMPA and NMDA conductances on synaptic threshold variability was enhanced as neurons became progressively larger, ranging from almost nothing (-0.02 sAMPA-only) in the smallest neuron having no additional dendrites, to very large (-9.65 sAMPA-only) in the largest neuron with five additional dendrites (Figure 3A). Similarly, making our normal ball-and-stick neurons electrotonically “tighter” by removing dendritic spines moderately reduced the impact of synaptic conductance on threshold variability (Figure 3B)”.

The following text is the same text repaired (with some bad English, because the reviewer is not a native speaker).

>> “From experiments shown in Fig. 2, we learned that the synaptic thresholds become more stable (less variable) when synapses incorporated both AMPA and NMDA conductances, as opposed to AMPA-only and NMDA-only synapses. The mean difference in CV (relative to AMPA-only trials) normalized by the standard deviation (s) of the AMPA-only trials, the so called “effect size (d)” (see Methods), here serves as a measure of the improvement in stability. In this respect, if AMPA+NMDA synapses improve the stability of synaptic thresholds over the AMPA-only, or NMDA-only synapses, the effect size “d” becomes negative. Also, “d” is proportional to the magnitude of improvement; a greater negative “d” means greater reduction in synaptic threshold variability caused by the AMPA+NMDA synapse design. To test the impact of neuronal morphology on the observed stabilizing effects of AMPA+NMDA synapse, we measured synaptic thresholds in a 200-μm dendrite attached to neurons having 1-to-5 additional dendrites (600 μm each; Figure 3A). We found that adding dendritic branches to synthetic neurons improved the effect of the AMPA+NMDA synapses on the stability of the synaptic threshold. Even as the measured dendrite remained static (at 200 μm), the effect size (d) was increased as neurons became progressively larger, ranging from almost nothing (d = -0.02 units) in the smallest neuron having no additional dendrites, to very large (d = -9.65 units) in the largest neuron with five additional dendrites (Figure 3A). These data indicate that the stabilizing effect of AMPA+NMDA synapses on synaptic thresholds is even greater in neurons with more elaborate dendritic trees (more branches). Similarly, making our normal ball-and-stick neurons electrotonically “tighter” by removing dendritic spines moderately reduced the impact of synaptic conductance on threshold variability (Figure 3B)”.

The authors should employ similar “repairs” throughout the Results, everywhere. This should be done by adding explanations and helping readers of all levels. In the current form, this manuscript is written for the lab members, who have had multiple prior exposures to these experimental outlines, and who use the same jargon.

George Gopen & Judith Swan (2018) explained my point #[5]: “The fundamental purpose of scientific discourse is not the mere presentation of information and thought, but rather its actual communication. It does not matter how pleased an author might be to have converted all the right data into sentences and paragraphs; it matters only whether a large majority of the reading audience accurately perceives what the author had in mind”.

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[6] - invention of new biophysical units

”... ranging from almost nothing (-0.02 sAMPA-only) in the smallest neuron having no additional dendrites, to very large (-251 9.65 sAMPA-only) in “

Here, the authors should use the syntax “units”. Description of the sAMPA-only unit, and sAMPA-only calculation, should be performed in the Methods (as they have already done).

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[7] - Origin of peak in somatic traces?

In Figure 2B, some of the large somatic voltage waveforms (NMDA-only and NMDA+AMPA) have small (5 ms-wide) peaks on the plateau phase. The dendritic waveforms, which are simultaneously obtained with somatic traces, are without that same peak. In the manuscript, please explain the origin of the small 5-ms wide peak observed in the cell body?

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[8] - Error in Figure 2

"B, Somatic (B1) and dendritic (B2, top) voltage responses for identical subthreshold synaptic barrages at each dendritic location ...”

Semi-transparent black dots above the synaptic currents in Fig. 2-B2 indicate that the temporal structure of inputs was different for AMPA-only versus 2 other conditions. The temporal structure of synaptic barrages were not identical. Therefore, it is impossible for readers to compare three conditions under the same input paradigm.

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[9] - Error in Figure 2

Ten stochastic spatiotemporal patterns of synaptic input (“synaptic barrages,” see Methods) were delivered to progressively more distal 50-μm-spans of the dendrite.

Readers can see multiple superimposed traces in Figure 2B corresponding to different segments of dendrite. The display is complete for “NMDA-only” and “AMPA+NMDA”. The display is incomplete for “AMPA-only", as multiple traces from multiple dendritic segments are missing.

We cannot understand why progressive distances were NOT modelled / displayed for “AMPA-only”? There is no explanation in the manuscript, and no explanation in figure legend.

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[10] - Error in Figure 2

"For simulations involving AMPA only, NMDA-only, or both synaptic conductances, the number of activated synapses within each barrage was iteratively increased until an action potential was initiated”.

Unfortunately, we do not see AP initiation in Figure 2B1 or 2B2. We do not understand this discrepancy between the body of the manuscript and the figure.

"For each pattern of synaptic input, the threshold number of synapses necessary for action potential generation was determined for inputs occurring at different dendritic locations (incremented at 10-μm intervals) and across seven RMPs (-85 to -55 mV)”.

We do not see seven RMPs in Fig. 2B. The entire section of the manuscript devoted to Fig. 2B Results, is one big mistake in scientific communication, with multiple discrepancies between the body of the manuscript and the figure.

"axon (Figure 2A). Ten stochastic spatiotemporal patterns of synaptic input (“synaptic barrages,” see Methods) were delivered to progressively more distal 50-μm-spans of the dendrite. For simulations involving AMPA only, NMDA-only, or both synaptic conductances, the number of activated synapses within each barrage was iteratively increased until an action potential was initiated. For each pattern of synaptic input, the threshold number of synapses necessary for action potential generation was determined for inputs occurring at different dendritic locations (incremented at 10-μm intervals) and across seven RMPs (-85 to -55 mV). As with single EPSPs, identical barrages of synaptic input generated ever-larger local NMDA receptors stabilize synaptic integration, page 8 dendritic depolarization at progressively more distal dendritic locations. Whereas the increase in local response amplitude with distance was linear when inputs involved AMPA conductances only, incorporation of the NMDA conductance, by itself or in combination with the AMPA conductance, allowed for supralinear distance-dependent increases in response amplitude and width, including long lasting regenerative NMDA spikes, at distal dendritic locations (Figure 2B)”.

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[11] - Communication breakdown

In Figure 2D, we learned that the authors have employed sticks of different length, while in Fig. 2ABC we were led to believe that all simulations were performed on the same stick length of 1,000 um.

At what point the authors change their strategy?

When they discuss synaptic inputs delivered at 600 um distance from the cell body in Fig. 2ABC, does that mean that the total length of the stick in that model exercise was 600 um or 1,000 um? The authors need to clarify their procedures.

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[12] I = g (Eq - Vm)

"This shape resulted from distance-dependent voltage attenuation of somatic EPSPs combined with progressively larger synaptic currents at more distal, high-impedance dendritic locations”.

Since the authors promised to deliver IDENTICAL synaptic drives along the length of dendrite at the same voltage, then the synaptic currents cannot possibly be different in repeated trials. Could you please explain your finding that despite identical synaptic conductance (synaptic input), identical equilibrium potential, and identical membrane potential, the synaptic current “I_syn” undergoes changes? Did the authors mean to say: “progressively larger synaptic potentials at more distal location”?

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[13] - in figure 6c?

"while effect sizes for reductions in CV across dendritic location (within a given RMP) ranged from -0.93 to -1.19, primarily because of large variability in the CVs of AMPA-only trials (compare the standard deviations of AMPA-only and AMPA+NMDA trials in Figure 6B)”.

This sentence is perhaps erroneously referencing Figure 6B, when instead it should point to Figure 6C?

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[14] - Figure 7A is AWOL

Figure 7A was never mentioned in the manuscript, and we, the readers, feel uncomfortable. Did we miss something important?

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[15] - I do not see what author see

In ball-and-stick model neurons with short dendrites, when AMPA conductances were small, adding an NMDA conductance did not significantly reduce threshold CVs, as the NMDA conductance became the dominant driver of somatic depolarization, and CVs converged toward those of NMDA-only inputs.

In Fig. 7C, I do not see AMPA+NMDA CVs converging to NMDA-only CVs.

The authors need to either change the figure, or supplement the figure with labeled arrows, or change verbal interpretation, or all three.

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[16] - Physical units of “Fidelity”?

"Thus, the voltage-dependence of the NMDA conductance enhances the fidelity of synaptic integration across a wide range of physiologically relevant AMPA-to-NMDA ratios”.

In their modeling exercises, the authors have produced some numerical values for:

[A] Threshold # of synaptic inputs,

[B] CV of Threshold # of synaptic inputs,

[C] Effect size (d).

Scientific measurements of FIDELITY were not provided. Please define here “Fidelity” in physical terms, or eliminate this term from abstract and discussion.

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[17] - Driving forces are diminished?

"This can reduce synaptic currents in distal dendrites, as greater local depolarization during EPSPs limits synaptic driving forces at the peak of the EPSP”.

” ... conditions in which synaptic driving forces are diminished (see Figures 2 and 8)”.

We do not see any graphical pointers or represents of diminished driving forces in Figure 2.

The authors should provide clean, simple and sufficient explanations on each point they want to make, with figure enhancements (arrows) and auxiliary sentences.

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[18] - Proximal dendrites improve synaptic currents at any given location??

"On the other hand, the increased variability of EPSP-spike coupling when barrages were initiated at different RMPs in proximal dendrites reflects the non-linear amplification of synaptic responses with increased depolarization, leading to a greater range of synaptic currents across voltage at any given location”.

Non-linear amplification of synaptic responses with increased depolarization is not something that happens in proximal dendrites only. I do not understand the significance of “proximal dendrites” in this sentence. “Proximal dendrites improve synaptic currents at any given location”??

What is the meaning of this sentence, and how this sentence improves our understating of the process by which “NMDA receptors stabilize synaptic integration”?

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[19] - NMDA conductances oppose the impact of reduced synaptic driving force

"Similarly, by opposing the impact of reduced synaptic driving force on AMPA-mediated currents, voltage-dependent NMDA conductances can reduce the variability of total synaptic current during temporal integration of clustered excitatory inputs”.

Many believe that NMDA conductances do not oppose the problem with reduced driving force, instead they make it worse.

Could you please explain in the manuscript how: “NMDA conductances oppose the impact of reduced synaptic driving force”?

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CRITIQUE ENDS HERE

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Author Response

Response to Reviewer Comments for eN-NWR-0396-20

We have read and reread the reviewer comments below several times, and tried to interpret them in the most charitable way possible. Below we offer a point-by-point response. Where appropriate, we have modified the manuscript as noted, with changes designated by blue text. For the many comments that we perceive as frivolous, nonconstructive, and/or nonprofessional, we provide detailed rebuttals below.

Comment 1.1: The reviewer seems to be willfully misconstruing our use of the word “synergism”. We use that word only to explain how the voltage-dependance AND slow kinetics of NMDA conductances act synergistically to lower the variability of synaptic spike thresholds (or thresholds for any arbitrary subthreshold voltages) for discrete spatiotemporal patterns of input delivered across multiple locations or voltages. This is a completely independent concept relative to the interaction of AMPA and NMDA conductances at individual synapses, or at multiple synapses within a given dendritic area (both of which have been well described before). We are extraordinarily clear in the text that our results focus solely on the novel and not-necessarily-intuitive finding that the reduction in somatic response variability to spatiotemporal patterns of input generated across all possible dendritic locations and initial membrane potentials depends on interaction of both properties of NMDA receptors (voltage-dependence and slow kinetics), and does not occur when either property is expressed alone. This is “synergy.”

Comment 1.2.a: The reviewer is using a spurious semantic argument to conflate the term “spatial domains”. Branco et al. (2010) did not test “across spatial domains", they looked instead at individual dendritic spans (similar to our 50 um spans) and tested the impact of patterned activation of spines existing in that single domain (e.g., sequential activation of inputs in one direction or the other). They did not attempt to compare identical spatiotemporal patterns of synaptic input delivered to different spatial domains across a dendritic tree, and also did not measure this across a large number of initial membrane potentials. Further, they did not quantify the variability of somatic responses to different, but statistically similar, spatiotemporal patterns of input before and after blockade of NMDA receptors. Thus, their work, while important (and cited in our original and revised manuscripts), does not amount to “overlap” of scientific discovery.

The novelty of our study is that we rigorously test the impact of synaptic conductance on the variability of somatic responses to identical spatiotemporal patterns of input delivered across all possible dendritic locations and across a large variety of initial membrane potentials. This has not previously been done. To our knowledge, the nearest finding to ours is in Larkum et al. (2009), where (as mentioned in our original manuscript) simulations “found the threshold number of synapses necessary for initiating an apical trunk calcium spike to be relatively consistent across tuft locations when NMDA conductances were present, but that thresholds for AMPA-only inputs grew exponentially, and to non-physiological synaptic densities, with distance from the initiation zone in the apical dendrite.”

Comment 1.2.b: Again, the reviewer conflates the term “spatial domain”. Further, EPSP “amplification” is not the point of our paper. Regardless of any amplification (or dampening), we are measuring the somatic fidelity (see below) of input-output functions for spatiotemporal patterns of synaptic input across dendritic domains (50 um spans at 10 um intervals in our tests) and RMP. We recognize the contributions of Connelly et al. (2016) by citing them in the original draft and revised draft of the manuscript, just as we continue to cite the review article by Major et al. (2013). Our novel result, that somatic responses to spatiotemporal patterns of input delivered to different dendritic locations and/or across different initial membrane potentials, lead to less variable somatic responses and EPSP-spike coupling when NMDA and AMPA conductances are co-localized at synapses, does not overlap with the findings of either paper.

Comment 1.2.c: Again, the reviewer seems to be purposefully conflating aspects of synaptic transmission. We make no original claims about NMDA receptors generating larger events at distal locations. This is indeed well known for decades, and we cite many of the fundamental papers that revealed this location-dependent impact of NMDA conductance. As we make abundantly clear to our readers, the novel finding of the present paper is that the co-expression of AMPA and NMDA conductances at synapses greatly reduces the variability of EPSP-spike coupling for spatiotemporal pattens of input delivered to different dendritic domains and/or at different initial membrane potentials.

Comment 1.2.d: We make no novel claim for that finding a relationship between NMDA conductance and local input impedance. This effect is well known, and we cite relevant papers to that effect. The reviewer appears to be working hard to find “overlap” where none exists.

Comment 1.2.e: Again, we make no claims about “discovering” that NMDA currents are larger in high-impedance compartments. We recognize that they do, and cite relevant papers showing as much, even as we note that this particular feature of NMDA conductances is what underlies the very different (and previously unreported) finding that combined synaptic AMPA and NMDA conductances reduce the variability of somatic responses to spatiotemporal patterns of synaptic input delivered at different dendritic locations.

Comment 1.2.f: There is no overlap. Magee and Cook (2000), the paper we believe the reviewer is pointing to, showed that AMPA conductances increase with distance along the apical dendrite of CA1 neurons (and only for CA1 neurons; see for instance, Williams and Stuart, 2002, for an alternative result in layer 5 cortical neurons), and therefore somatic responses to individual synaptic activations tended to be of similar size at the somatic due to increased AMPA receptor conductance at distal synapses having more AMPA receptors. This result is completely unrelated to the findings in our study. To be clear, we show that, for spatiotemporal patterns of input delivered to different dendritic locations and/or across different initial membrane potentials, summed EPSPs and/or EPSP-spike coupling are less variable when uniform NMDA and AMPA conductances are co-localized at synapses. Even as the Magee and Cook result is completely unrelated to any of our findings or simulations, we continue to cite their important work as an example of the many mechanisms by which neurons can reduce somatic variability in synaptic integration.

Comment 1.3a: There is no overlap. We make no claims about “integration windows” or how timing influences NMDA conductances. Perhaps the reviewer is concerned about our results shown in Figure 1, where we show for single EPSPs that NMDA conductances are larger at distal locations. If so, the reviewer is mistaken if he thinks we present Figure 1 as original findings. Figure 1 exists simply to show that our model works as expected given the many decades of study of the interaction of dendritic location (i.e., input impedance) and synaptic conductances. As in our original draft (emphasis ours), we start the section with the sentence, “As expected, given the cable properties of dendrites (Gulledge et al., 2005), the amplitude and kinetics of the resulting EPSPs depended on synaptic conductance type, RMP, and the dendritic location of synaptic input (Figure 1A).”

Comment 1.3.b: Again, there is no overlap. The reviewer seems to willfully conflate the term “AMPA/NMDA ratio”. In the review by Major et al. (2013), they discuss AMPA/NMDA ratios in relation to thresholds necessary to evoke NMDA spikes at individual synapses. We make no claim about AMPA/NMDA ratios in regard to NMDA spike thresholds, but include Figure 7 simply to show that the novel result we find throughout the paper (namely that co-localization of AMPA and NMDA conductances reduce the variability of somatic voltage responses and EPSP-spike coupling in response to spatiotemporal patterns of synaptic input generated at different dendritic locations and/or different initial membrane potentials) is not an artifact of using one particular AMPA:NMDA ratio. This is simply a control experiment to show that the “effect” on somatic response variability is robust across a variety of physiologically relevant AMPA:NMDA ratios.

Comment 1.3.c: Again, there is no overlap. We do not test “time jitter” on synaptic integration of AMPA or NMDA components of inputs. Similarly, Major et al. (2013) do not examine the impact of combined AMPA and NMDA conductances on the variability of somatic responses to spatiotemporal patterns of synaptic input generated at different dendritic locations and/or different initial membrane potentials. Repeatedly throughout the manuscript we clearly communicate the novel and previously unreported result that combining AMPA and NMDA conductances reduces the variability of somatic responses to spatiotemporal patterns of synaptic input generated at different dendritic locations and/or different initial membrane potentials. It is not our responsibility to point out all of the other research papers that have not tested for, and therefore not reported, this finding. While AMPA:NMDA ratios have been measured many times in a variety of systems to study various aspects of synaptic transmission and integration (especially with regard to synaptic plasticity), in no system have AMPA:NMDA ratios been tested in regard to their impact on the variability of somatic responses to spatiotemporal patterns of synaptic input generated at different dendritic locations and/or different initial membrane potentials.

Comment 1.4: As noted above, we included Figure 1 simply to show that our model acts “rationally” and as expected given the well-established knowledge regarding cable properties of dendrites. We do not suggest that these results are “new” or unexpected. To the contrary, we state clearly in the text that they are “as expected”.

Comment 1.5: In our manuscript, we do not claim to have “discovered” that NMDA spikes are larger or longer in duration when occurring in smaller dendrites. This has been known for over two decades, much in advance of the Gao et al. (2020) paper in Journal of Neurophysiology (see for instance the many papers by Jackie Schiller and Matthew Larkum). Regardless, the question is not whether NMDA-mediated responses are larger at distal locations. Our paper tests whether location-dependent increases in NMDA current, combined with the reduced AMPA currents occurring in distal dendrites (due to driving force reductions during the EPSP; see below) reduces the variability of somatic responses to spatiotemporal patterns of input delivered at locations across the dendritic tree and/or across initial membrane potential. This finding is new and previously unreported.

Comment 2: The reviewer again appears to be willfully conflating issues. The action potential waveform has no impact on the results of our study, as we are only measuring the thresholds number of synapses necessary to generate an action potential. Consider the following aspects of our study:

a) We use the occurrence of an action potential only to identify synaptic spike thresholds.

b) Action potentials are not necessary to show this increased fidelity of synaptic integration, as in Figure 6 we used arbitrary somatic “thresholds” far below action potential threshold.

c) Voltage-gated conductances, in the axon or anywhere else, are not necessary for demonstrating the reduced variability of synaptic integration, as Figure 8 is conducted in a purely passive neuron.

Finally, we would point out that we not attempting to simulate “pyramidal neurons”. Our goal is to explore the “synergistic” effect of AMPA and NMDA receptors on somatic response fidelity across a wide range of neuron morphologies.

Comment 3: We disagree. Figure 5 is important as it shows the effect sizes of results in Figure 4. As is evident from Table 2 (which lists effect sizes for results in Figure 4E only), a list of such effect sizes across all dendritic sizes, combinations of conductances for location-specific vs voltage specific responses would be unwieldy. Figure 5 allows one to graphically see how effect sizes for the various conductances are influenced by dendritic location and initial membrane potential.

Comment 4: The middle panel is important in showing that there is a dose dependence to replacing some of the AMPA conductance with a voltage-dependent and/or slow conductance. Thus, there is a real “effect” and not simply large uncorrelated variability from which we happen to choose conductance values that show an “effect”. Further, the results from the neuron with a 200-μm dendrite on a larger neuron is important to show as it demonstrates that the apparent effect of dendrite length on the robustness of NMDA-AMPA interactions is not real, but instead reflects differences in overall electrotonic size of the neurons. By including a larger neuron with a 200-μm dendrite (from Figure 3A) we show that the reduced variability of somatic responses to spatiotemporal patterns of synaptic input generated at different dendritic locations and/or across different initial membrane potentials when AMPA and NMDA receptors are co-expressed occurs even in small (200 μm) dendrites when those dendrites are attached to larger neurons. Not including these data would give the false impression that the presence of NMDA receptors will not enhance the fidelity of EPSP-spike coupling in small dendrites.

Comment 5: While we appreciate the reviewers attempt to help improve our writing, our original text concisely and clearly conveys our results. We see no improvement, and indeed a significant reduction of clarity, in the reviewer’s contribution. We will leave it to the editor to decide whether our writing style is up to the standards of eNeuro.

Comment 6: We are unclear what the reviewer is suggesting. We clearly define the unit of effect size (sAMPA-only) on page 7 (lines 153-164). There is no need to use ambiguous “units” in the text.

Comment 7: As one gets very close to threshold (but remains “just-subthreshold” even at the most excitable dendritic location) there can be significant lift by sodium conductances in the AIS and soma that generates the small peaks of concern to the reviewer. We now explain the origin of these peaks in the Figure 2 legend.

Comment 8]: The reviewer is wrong. The patterns for the different conductances are the same, but each conductance has a different number of “just-subthreshold” synaptic activations within that pattern (based on their different synaptic thresholds for action potential generation). We now provide clear explanation of this on page 9 (lines 218-221) and in the Figure 2 legend (see below).

Comment 9: The reviewer is incorrect. There are no missing data, and all traces for all conductances at all locations (at 20 μm intervals) are shown in Figure 2B.

Comment 10: We apologize for the confusion. We now clarify in the text and figure legend that Figure 2B shows responses to identical subthreshold patterns of synaptic input across all dendritic locations so that the gradual increase in NMDA conductance across location can be visualized. We have added the following text to the Results section on page 9, lines 218-221: “This can best be seen in Figure 2B, which shows somatic and local dendritic responses to identical patterns of subthreshold input (determined for each conductance type as the maximal number of synapses that remained subthreshold across all dendritic locations) in a neuron with an RMP of -70 mV.” We also add the following text to the Figure 2 legend: “While the spatiotemporal pattern is identical for all synaptic conductances, the number of synapses activated is adjusted for each conductance such that the maximal number of synaptic inputs that remain subthreshold across all dendritic locations is delivered. When occurring at locations where they are “just-subthreshold", somatic EPSPs can recruit somatic and axonal voltage-gated sodium conductances, producing the small peaks visible on the largest somatic EPSPs.”

Comment 11: We now clarify on page 8 (line 202) that we tested the impact of combining AMPA and NMDA conductances on the fidelity of EPSP-spike coupling in models with dendrites of various lengths.

Comment 12: The reviewer is wrong. When NMDA receptors are present, synaptic currents (Isyn) increase naturally with distance because the NMDA conductance is voltage dependent. This can clearly be seen in the currents in Figure 2B, but is also extremely well known as the underlying reason why NMDA spikes have lower synaptic thresholds at distal dendritic compartments even when the number of AMPA and NMDA receptors are held constant at synapses across dendritic space. This does not happen with AMPA receptors that lack voltage dependence (also shown in Figure 2B), where synaptic currents actually become smaller due to the collapse in driving force during the EPSP (where Eq-Vm becomes smaller). We feel we have explained these basic physiological principles adequately in the manuscript.

Comment 13: We thank the reviewer for noticing this error. We have now corrected the text to refer to Figure 6C.

Comment 14: We have added a reference to Figure 7A on page 17 (line 455).

Comment 15: The convergence is clear and inescapable. As AMPA conductances are decreased, the CV increases toward the level of NMDA-only inputs (which is depicted as a static horizontal line, as it is independent of AMPA conductance values). Common sense dictates that, as AMPA conductances approach zero, CVs for AMPA+NMDA conductances will converge with the NMDA only conductance (as NMDA conductance is all that remains). It is also clearly evident in Figure 7C as the green symbols curve up toward the NMDA only values as AMPA conductance decreases.

Comment 16: Merriam-Webster defines “Fidelity” as “the degree to which an electronic device (such as a record player, radio, or television) accurately reproduces its effect (such as sound or picture)”. This is analogous to what we are measuring by our CV of synaptic thresholds throughout the paper (i.e., the “fidelity” of EPSP-spike coupling).

Comment 17: We are not testing for, and do not graph, changes in driving force. We expect that the vast majority of readers of eNeuro understand that driving forces are diminished when the membrane potential approaches the synaptic reversal potential, as occurs during EPSPs preferentially in high-impedance dendritic compartments.

Comment 18: We thank the reviewer for this comment. We no longer include the term “at proximal dendrites", and provide on page 19 (lines 523-530) a more elaborate explanation for why the “fast-NMDA” conductance increases synaptic threshold CVs primarily in proximal dendrites.

Comment 19: We believe the reviewer is referencing to the sentence, “Similarly, by opposing the impact of reduced synaptic driving force on AMPA-mediated currents, voltage-dependent NMDA conductances can reduce the variability of total synaptic current during temporal integration of clustered excitatory inputs” (lines 562-565). The sentence is true as written. Reductions in driving force during large-amplitude EPSPs (typical of high-impedance dendritic compartments) act to reduce AMPA currents (synaptic current = driving force * synaptic conductance; see Figure 2B, and notice how the AMPA currents become smaller with distance from the soma). However, because the NMDA conductance is voltage dependent, synapses that also include NMDA receptors have a dynamic conductance that increases with depolarization (i.e., when Vm is closer to Esyn, and driving forces are diminished). This acts to maintain a relatively substantial current even as driving force collapses (see Figure 2B, where AMPA+NMDA currents increase with distance from the soma).