Dear Dr Raimondo,

Thank you very much for submitting your manuscript 'Chloride dynamics alter the input-output properties of neurons' for review by PLOS Computational Biology. Your manuscript has been fully evaluated by the PLOS Computational Biology editorial team and in this case also by independent peer reviewers. The reviewers appreciated the attention to an important problem, but raised some substantial concerns about the manuscript as it currently stands. While your manuscript cannot be accepted in its present form, we are willing to consider a revised version in which the issues raised by the reviewers have been adequately addressed. We cannot, of course, promise publication at that time.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

Your revisions should address the specific points made by each reviewer. In particular, make sure to address the points raised by reviewer #3 regarding originality of your model and novelty of your findings.

Please return the revised version within the next 60 days. If you anticipate any delay in its return, we ask that you let us know the expected resubmission date by email at ploscompbiol@plos.org. Revised manuscripts received beyond 60 days may require evaluation and peer review similar to that applied to newly submitted manuscripts.

In addition, when you are ready to resubmit, please be prepared to provide the following:

(1) A detailed list of your responses to the review comments and the changes you have made in the manuscript. We require a file of this nature before your manuscript is passed back to the editors.

(2) A copy of your manuscript with the changes highlighted (encouraged). We encourage authors, if possible to show clearly where changes have been made to their manuscript e.g. by highlighting text.

(3) A striking still image to accompany your article (optional). If the image is judged to be suitable by the editors, it may be featured on our website and might be chosen as the issue image for that month. These square, high-quality images should be accompanied by a short caption. Please note as well that there should be no copyright restrictions on the use of the image, so that it can be published under the Open-Access license and be subject only to appropriate attribution.

Before you resubmit your manuscript, please consult our Submission Checklist to ensure your manuscript is formatted correctly for PLOS Computational Biology: http://www.ploscompbiol.org/static/checklist.action. Some key points to remember are:

- Figures uploaded separately as TIFF or EPS files (if you wish, your figures may remain in your main manuscript file in addition).

- Supporting Information uploaded as separate files, titled Dataset, Figure, Table, Text, Protocol, Audio, or Video.

- Funding information in the 'Financial Disclosure' box in the online system.

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see here. 

We are sorry that we cannot be more positive about your manuscript at this stage, but if you have any concerns or questions, please do not hesitate to contact us.

Sincerely,

Hugues Berry

Associate Editor

PLOS Computational Biology

Kim Blackwell

Deputy Editor

PLOS Computational Biology

A link appears below if there are any accompanying review attachments. If you believe any reviews to be missing, please contact ploscompbiol@plos.org immediately:

[LINK]

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: The paper combines experimental work and computer simulations to investigate the impact of fluctuations in Cl- concentration on neural signalling which is an important topic. I congratulate the authors for an interesting an well written paper. I like the concept of Cl- index. I however had the following questions/comments

1) The simulations last for 1 second. If I understand correctly, intracellular Cl concentration starts at a low level and then accumulates throughout the simulations. For the results to be relevant, Cl concentration must have reached some kind of steady-state or mean steady state level by the end of the simulations. How do they authors ensure that Cl concentration has converged during the 1s timespan of the simulation? Since dynamics of ionic concentrations are typically very slow, it is not clear to me a priori that 1 second is enough? I suggest the authors increase the duration of the simulations or at least provide a convincing argument for the convergence of Cl concentration.

2)Cl concentration is taken as dynamical while other ionic concentrations are taken as constant. While in some cases this can be a good approximation, this assumption is ultimatey inconsistent with assumptions of constant osmolority and the almost electroneutrality of the intracellular space. Given the importance of intracellular potassium in particular, I suggest the author consider intracellular K+ as dynamically evolvoing in their model. A reasonable guess is that the sum of intracellular Cl- and K+ concentrations would be roughly constant. This would impact KCC2 tranport by making the impact of Cl increase on the rate of transport less important. Maybe this could be done just for a few simulations at least to estimate how large would the impact of fluctuating potassium concentration would be.

3) The authors adjust parameters so the mean firing rate is 5 Hz when each of the synapse receives input at 5 Hz. This seems reasonable, and seems to come from a steady-state assumption such as every neuron in a network firing at 5Hz. However this is not satisfactorily justified or referenced in the paper.

4) In the discussion, the author refer to epilepsy. While the design of the paper is good is it stands, the impact of the paper could be increased if relvance of the results with respect to a particular pathology or physiological scenario would be more fleshed out.

Reviewer #2: In their computational study, the authors studied how chloride dynamics affects distal and proximal inhibition and how it modulates subtractive or divisive changes of the input-output function of neurons, which are driven by concurrent balanced excitatory synaptic drive. They found that spatial variations in chloride concentration can emerge despite uniform expression of chloride pumps. In addition, they observed that whereas chloride accumulation in dendrites diminishes the ability of distal inhibition to trigger subtractive shifts in the input-output curve, the ability of proximal inhibition to trigger divisive changes of the input-output curve remained largely unaffected by dynamical chloride. The methods are well described and the results are appropriately interpreted.

Major points:

* The authors show convincingly that concurrent inhibitory and excitatory synaptic drive may lead to spatial variations in chloride despite uniform expression of chloride pumps. However, experimental data show that the expression of chloride pumps may not be uniform leading to a dendro-somato-axonal gradient in EGABA with chloride concentration becoming progressively higher toward soma (Gulyas et al. Eur J Neurosci 2001, Szabadics et al. Science 2005, Khirug et al. J Neurosci 2008). The authors should explore such scenario in their model – e.g. by making the KCC2 pump strength spatially inhomogeneous with a higher expression in dendrites/soma and low/zero expression in the axon. Can enhanced KCC2 pump strength in dendrites counteract the inhibition-compromising effects of dendritic chloride accumulation?

* Line 155: “Combined with mechanisms to describe Cl- influx via both tonic and synaptic activation of GABAARs…” In Methods, the authors describe the synaptic models of phasic GABA-A responses. Did they include also tonic GABA-A receptors/conductances in their models? I could not find the description of them. They probably mean tonic chloride currents via leak chloride channels. This has to be clearly distinguished and described in the paper.

* Line 582-583: The authors report that they modelled excitatory synapses as voltage-dependent NMDA conductances. Were AMPA synapses not included? If not, why? Mixed AMPA/NMDA synaptic models would be biologically more realistic than pure NMDA inputs. Recent compartmental modelling of chloride concentration changes used stochastic AMPA/GABA-A inputs (Lombardi et al. Front Cell Neurosci 2018, Lombardi et al. IMJS 2019). Are the results of the current paper in line with this modelling or are there some important differences when NMDA synapses are included?

* Lombardi et al. IMJS 2019 showed that membrane resistance (determined by gpas) critically influences the amount of chloride changes. Is that the case in the authors’ model? Do their conclusion hold for a wide range of gpas values? (Modeling in the paper was performed only for a selected input resistance of 350 Mohm.)

* Prescott, Sejnowski and De Koninck (Mol Pain 2006) showed that inhibitory regulation of firing can be compromised by decreasing hyperpolarization and/or shunting. The authors should discuss their results in the light of this paper/ these findings.

* Figure 4 illustrates subtractive and divisive effects of distal and proximal inhibition, respectively. Subtractive inhibition should result in a rightward shift of the IO curve without affecting its slope whereas divisive inhibition should alter the slope of the IO curve. However, in the figure it seems that distal inhibition affects the slope, whereas proximal inhibition shifts the curve and only minimally affects the slope. Can authors comment on this?

Minor points:

* Figure 5: Replace “Excitatatory” by “Excitatory” (twice).

* Line 265: Add “blue”

Reviewer #3: Major concerns:

The study examines the effect of dynamic changes in [Cl]i on neuronal input-output relationship. Although there are some interesting aspects in this work, much of the results are intuitive and lack significant insight to be gained from a modeling study. For example, it is expected that the effect of inhibition on input-output function is weaker when inhibition is inter-mixed with excitation. Similarly, if [Cl]i is dynamically changing, any time delay in these changes could minimize the effects on the input-output relationship when excitation/inhibition are colocalized (Fig 4B). Most importantly, the model does not seem to reproduce the divisive effects of proximal inhibition on input-output relationship (Fig. 4D). This limits the confidence in the model itself. Model innovation is minimal. Overall, the results are not significant to improve our understanding of inhibitory effects on input-output processing in neurons.

Minor comments:

Lines 85: “inputs can perform a divisive operation by changing the slope (gain) of the input-output function, or”

What is the direction of this “change” – be specific.

Lines 97 – 99: “Intracellular Cl- concentration ([Cl-]i), and hence EGABA, can differ between

subcellular neuronal compartments, which has been suggested to result from spatial differences in

the function or expression of cation-chloride cotransporters in the neuronal membrane (10–12).”

Clarify the above statement.

Lines 110 – 113: “Whilst the vast majority of theoretical models of inhibitory signalling and neuronal computation assume static values for EGABA, previous studies have addressed the biophysical underpinnings of Cl homeostasis and ionic plasticity in neurons (20,22), the effects of neuronal morphologies on Cl accumulation (19,21,23), and how dynamic Cl- affects neural coding (24).”

Please summarize briefly what these studies suggest on Cl- dynamics rather than simply stating that Cl- dynamics has been explored.

Lines 135 – 141: Please clarify the rational for using organotypic slices as a suitable preparation to measure Cl-extrusion. These sentences are too long and should be restructured for readability.

Lines 177 – 180: The sentence begins with “Extensive evidence….”, but there are no relevant citations provided. Clarifying what type of methods and preparations revealed differential [Cl]i in those experimental studies would also be pertinent.

Line 224: It may be better to call “synaptic structure” as “synaptic configuration” here and elsewhere.

Lines 235 – 237: This sentence is too long and cryptic. Please explain clearly.

Lines 243 – 244: “predicted” must be replaced with “speculated”

Lines 202 – 222: Some of the descriptions provided here in the Figure legend (e.g., balanced input) can go in the main manuscript. Figure legends must be very brief only describing the graphic components of the figure.

It would be useful to provide a figure panel showing the time series of [Cl]i in the model in response to balanced input with different synaptic configurations.

**********

Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Computational Biology data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Nicolas Doyon

Reviewer #2: No

Reviewer #3: No

Dear Dr Raimondo,

Thank you very much for submitting your manuscript "Chloride dynamics alter the input-output properties of neurons" for consideration at PLOS Computational Biology. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. 

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Hugues Berry

Associate Editor

PLOS Computational Biology

Kim Blackwell

Deputy Editor

PLOS Computational Biology

***********************

A link appears below if there are any accompanying review attachments. If you believe any reviews to be missing, please contact ploscompbiol@plos.org immediately:

[LINK]

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: I feel the authors have satisfactorily answered the comments of the reviewers and that the current version of the manuscript is worthy of publication as it is.

A few minor comments:

1) At several places, when the authors write Cl- and K+, the - and + are not in exponent format. Please verify.

2) I'm still uneasy with p values such as 10^(-140). Is it different from 0 then? Is it within some numerical uncertainty? Or if the p-value is so small, can we say that something is certain? Also, just above the discussion sectio, -140 should be in exponent

3) Page 19, line 560, what are ' mature and stable Cl- homeostasis mechanisms'?

4) line 579, is it mOsm or mOsM?

5) Line 606, could the authors specify what is the meaning of (\\xi=1)?

6) Figure 1 c, the units are written as (1/mMs) which can be misleading. Would it possible to write mM^-1 s^-1 or (1/(mM\\cdot s))?

Reviewer #2: The authors have addressed all my concerns. Minor points:

* A new important experimental study on dendritic inhibition has recently been published - the authors might want to cite it: https://www.nature.com/articles/s41467-019-13533-3

* The authors performed new simulations with manipulations of ionic leak conductances and the associated input resistance (Fig. 6 & S1A). However, they don't mention the rationale or the motivation for it - namely previous results of Lombardi et al. https://www.ncbi.nlm.nih.gov/pubmed/30897846 For a better understanding of the background for these simulations, this could be mentioned at the beginning of the relevant results section.

* L. 464 - replace "stength" by "strength"

Reviewer #3: The authors have made significant changes to the manuscript since last review which has greatly improved the manuscript and satisfactorily clarified much of the reviewer concerns. However, there are a few minor points to be addressed as noted below:

1) Ref Fig 2 and other related simulations. Please provide the stdev of the input firing rates and note whether it differs for different mean input firing rates (e.g.., mean rate of 5 Hz versus 20 Hz).

2) Line 414: As per the illustration in Fig 6A inset, the authors have to spell out x50 as the relative excitatory conductance at half-maximal firing rate in the results section. Currently it seems like they call the half-maximal firing rate as x50 which is not the case.

3) Figure 3, panels A and B: Please change Y-axis label to Firing Rate (Hz).

4) Line 513: Previous work such as Venugopal S, J Neurophysiol, 2011 has to be cited.

Line 189 Remove "well" or provide multiple references

Line 206 Begin sentence with "Note that multiple candidate..."

Line 207 Remove extra period

**********

Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Computational Biology data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

Reviewer #2: None

Reviewer #3: Yes

**********

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Nicolas Doyon

Reviewer #2: No

Reviewer #3: No

Figure Files:

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example in PLOS Biology see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, PLOS recommends that you deposit laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see http://journals.plos.org/ploscompbiol/s/submission-guidelines#loc-materials-and-methods

Dear Dr Raimondo,

We are pleased to inform you that your manuscript 'Chloride dynamics alter the input-output properties of neurons' has been provisionally accepted for publication in PLOS Computational Biology.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Computational Biology. 

Best regards,

Hugues Berry

Associate Editor

PLOS Computational Biology

Kim Blackwell

Deputy Editor

PLOS Computational Biology

***********************************************************