PONE-D-24-10034Depolarization of mouse DRG neurons by GABA does not translate into acute pain or hyperalgesia in healthy human volunteersPLOS ONE

Dear Dr. Schmelz,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. It will be essential for you to respond to all of the reviewers' comments, but it is particularly important that you can successfully address: reviewer one's concerns about clarity/statistics and reviewer two's concern that the lack of effect of GABA could be due to the possibility that the chloride reversal potential is not above threshold for an AP (a significant consideration of this idea must be developed in the discussion).

Please submit your revised manuscript by Jun 16 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Peter Wenner

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at 

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and 

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Thank you for stating the following financial disclosure: 

"The study was supported by the German Research Foundation (Deutsche Forschungsgemeinschaft, https://www.dfg.de/en/research-funding/funding-opportunities), grant project 397846571 (RR) and FOR 2690 (MS, RR) and SFB 1158 (RC, MS)."

Please state what role the funders took in the study.  If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." 

If this statement is not correct you must amend it as needed. 

Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf.

3. In the online submission form, you indicated that [The data underlying the results presented in the study are available from the authors upon request.]. 

All PLOS journals now require all data underlying the findings described in their manuscript to be freely available to other researchers, either 1. In a public repository, 2. Within the manuscript itself, or 3. Uploaded as supplementary information.

This policy applies to all data except where public deposition would breach compliance with the protocol approved by your research ethics board. If your data cannot be made publicly available for ethical or legal reasons (e.g., public availability would compromise patient privacy), please explain your reasons on resubmission and your exemption request will be escalated for approval. 

4. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.

5. We note that S1 and S2 Figures in your submission contain copyrighted images. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For more information, see our copyright guidelines: http://journals.plos.org/plosone/s/licenses-and-copyright.

We require you to either (1) present written permission from the copyright holder to publish these figures specifically under the CC BY 4.0 license, or (2) remove the figures from your submission:

a. You may seek permission from the original copyright holder of S1 and S2 Figures to publish the content specifically under the CC BY 4.0 license. 

We recommend that you contact the original copyright holder with the Content Permission Form (http://journals.plos.org/plosone/s/file?id=7c09/content-permission-form.pdf) and the following text:

“I request permission for the open-access journal PLOS ONE to publish XXX under the Creative Commons Attribution License (CCAL) CC BY 4.0 (http://creativecommons.org/licenses/by/4.0/). Please be aware that this license allows unrestricted use and distribution, even commercially, by third parties. Please reply and provide explicit written permission to publish XXX under a CC BY license and complete the attached form.”

Please upload the completed Content Permission Form or other proof of granted permissions as an "Other" file with your submission. 

In the figure caption of the copyrighted figure, please include the following text: “Reprinted from [ref] under a CC BY license, with permission from [name of publisher], original copyright [original copyright year].”

b. If you are unable to obtain permission from the original copyright holder to publish these figures under the CC BY 4.0 license or if the copyright holder’s requirements are incompatible with the CC BY 4.0 license, please either i) remove the figure or ii) supply a replacement figure that complies with the CC BY 4.0 license. Please check copyright information on all replacement figures and update the figure caption with source information. If applicable, please specify in the figure caption text when a figure is similar but not identical to the original image and is therefore for illustrative purposes only.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The paper by Sohns et al. investigated the effects of GABA in the periphery, on rodent neurons and on humans. The work on rodent neurons slightly extends existing knowledge, the human experiments represent the novelty of the paper.

Major:

- The experiments were not preregistered as a trial, and therefore the protocol and number of subjects not laid down in a public database before the investigation. Nevertheless, the experiments are approved by an ethics committee, which requires to provide hypotheses, endpoints, and a power calculations. None of this is provided in the manuscript. This should be explicitly mentioned for every question, and clearly distinguish preset and adequately powered hypotheses and those which are exploratory and might have a less favorable type I and II error.

This includes the experimental timeline, which is not easy to understand. E.g. who of the subjects in Fig. S2 proceeded from B to C, and who from B to D. A flowchart might help making this more accessible

- “The inability of GABA to generate action potentials in DRG neurons is also consistent with the lack of sensations accompanying intradermal GABA observed here and reported previously (27)”. Although the pain directly induced by GABA might be minimal, and this might explain why modulation of evoked please was investigated: Please provide data on the pain caused by GABA vs. control injection, in particularly given the failure of the only published human study cited (citation 27) to do so.

Minor:

- Figure 3a: In case the SD is negative, the data are probably skewed. The authors need to decide whether non-parametric visualization is more suitable.

- Line 187: ‘to less than 10% of the initial fluorescence ‘should rather be to less than 10% increase above the initial …

- Independent repetitions of cellular experiments should be mentioned, in addition to the cell count. E.g. in line 358, assuming 100 cells in a field of view and a 20% response rate to GABA, 19 cells might well be a single run of the experiment, which I would consider highly questionable.

- Rundown of GABA responses with butemetanide cannot be judged without an identical protocol without this substance.

- Line 277: Why where experiments single and not double blinded?

- Many values are provided with a precision which is probably not justified by the measurement, and a digit less might be the better

- Fig 1B. Is the concentration response constructed only from protocols like in Fig 1A, which has, despite tachyphylaxis in other panels, starts with a high concentration

- Fig 1I. Legend mentions 145 mM KCl, which is probably 45 mM as mentioned in the methods

- Fig 1J) A Venn diagram seems more suited to visualize the populations than a bar chart

- Fig 2 C,D,E: Is a scale up to 12 reasonable for a measure in the range 0-10?

- Fig 3B: Why did half of the skin blood flow increase in the furosemide pretreated spots occur before the iontophoresis?

Reviewer #2: This manuscript addresses the question of whether modulation of cutaneous terminals of primary somatosensory neurons by GABA alters human responses to stimuli that evoke mild to moderate pain or itch. This is a question of potential clinical importance as peripheral GABA signaling might provide opportunities to treat pain and itch caused by peripheral insults. Using dissociated mouse DRG neurons and Ca-imaging, the authors replicate previous studies indicating that GABA depolarizes DRG neuron somata sufficiently to increase intracellular Ca2+ that depends upon a high concentration of intracellular Cl- maintained by NKCC1 pump activity, and that GABA sensitivity correlates with TTX insensitivity (the sensory neurons more likely to be nociceptors or pruriceptors). They then describe various experiments with human volunteers to determine whether excitatory responses to GABA injected into the skin alter neurogenic inflammation, pain, or itch (apparently for the first time, as no references to previous studies involving cutaneous injection of GABA are cited). The most important finding is that GABA injection failed to produce pain or itch, and that injection of neither GABA nor furosemide (which inhibits NKCC1 to reduce intracellular Cl- concentration and hyperpolarize the Cl- equilibrium potential, ECl) altered the volunteers’ ratings of pain or itch induced by other cutaneous stimuli. While these are noteworthy findings and the methods are solid, I have concerns about how the experimental design and results are interpreted, which should be considered when revising the manuscript.

1. The manuscript stresses the “contrast” and “surprising” difference between the observations of GABA-evoked Ca2+ transients in dissociated mouse DRG neurons and the lack of GABA effects upon injection into human skin. Ignored completely are well-established principles about how a ligand such as GABA can both depolarize a neuron if membrane potential is below the reversal potential for Cl- and produce a net inhibitory effect via a decrease in input resistance (Rin) and shunting of excitatory currents. In nociceptive and pruriceptive DRG neurons, ECl is often depolarized relative to resting membrane potential (RMP) but hyperpolarized relative to AP threshold. For example, if a dissociated soma or peripheral process has an RMP of -65 mV, ECl of -40 mV, and an AP threshold of -35 mV (all of which have been reported for DRG neuron somata), opening of Cl- channels by themselves will not be able to depolarize RMP to the AP threshold. In addition, the increase in Cl- conductance will reduce Rin, decreasing the amplitude of sensory generator potentials and spontaneous depolarizing fluctuations of RMP, and the Cl-mediated depolarization that approaches (but cannot exceed) ECl will inactivate voltage-gated Na+ channels. Thus, there is no reason for surprise at the lack of human GABA-evoked pain and itch when extrapolating from the observations of GABA-evoked Ca2+ transients in dissociated DRG neuron somata.

2. The fact that the Ca2+ transients are relatively small suggests that the GABA-evoked depolarizations are not large enough to activate a substantial fraction of voltage-gated Ca channels and thus that these depolarizations usually fail to exceed AP threshold. A major limitation of the manuscript is that no electrophysiological measurements were made, so none of these potentials (RMP, ECl, AP threshold, GABA-evoked depolarization relative to AP threshold) are actually known. Of course, these values are not yet obtainable from the cutaneous terminals of rodent or human DRG neurons, but the inhibitory principles are clear and should be discussed as part of the experimental design and interpretation of the results.

3. Similarly, the results do not really suggest that “pharmacological intervention of GABAAR signaling in human skin is not … promising.” This is because the study is mainly designed to test interventions that target depolarizing effects of GABA. The work of Gamper, Du and colleagues has provided strong evidence that, at least in the DRG, even when depolarizing a nociceptor, GABA has strong inhibitory effects. In principle, the same mechanisms could operate on conduction through peripheral branch points and on the effectiveness of generator potentials in terminals.

4. This study did not examine conditions that might optimize inhibitory effects of GABA in cutaneous receptive fields. The closest it came to testing this idea was in the experiments of Fig.2D,E, examining the effects of furosemide pretreatment and GABA injection on pain evoked by electrical stimulation. There was a lack of apparent analgesic effects from GABA, but this might be because the electrical test stimuli produced only modest pain that was further attenuated by the injection volume effect seen in all the human experiments in this manuscript. Furthermore, the effectiveness of peripheral injections of GABA may be limited by desensitization of GABA receptors, and by potentially rapid uptake and degradation of injected GABA within the skin.

5. Because no pro-algesic effects of GABA were found in this study (Figures 2, 3), the furosemide experiments (expected to reduce the depolarizing effects of GABA) provide very limited information. However, furosemide should enhance the inhibitory effects of GABA, so the cited study reporting itch reduction by furosemide (line736) supports the possibility that enhancement of GABA effects might be therapeutically useful. Again, it would have been useful also to explicitly test whether peripheral GABA injection can have inhibitory effects on pain or itch, and perhaps to test the effects of blocking transport of Cl- out of the cells (in addition to their use of furosemide to block transport of Cl- into the cells).

MINOR COMMENTS

Line 53 – Furosemide should reduce the outwardly directed Cl- gradient, but brief applications of GABA should mainly reveal the effects of this gradient reduction, not by itself cause “depletion” of the gradient.

Line 141 – poly-L-lysine

Line 191 – what is the duration of each GABA application?

Line 352 – why show a weak effect of Ni2+ (n=47, p=0.037) rather than the strong effect mentioned from the highly specific T-type VGCC blocker TTA-P2?

Line 360 – the important effect is inhibiting transport of Cl- out of the cell to change the Cl- gradient, not the small current mediated by NKCC1 transporters.

Lines 414-418 – the responses should be higher in the neurons with a lower, not higher, TTX sensitivity, if I understand this experiment correctly. Also, which traces are being referred to?

Line 436 – electrically evoked calcium signals (not “electrical calcium signals”)

Line 704 – a reference is needed for this statement about mast cell degranulation

Line 729 – the observation that human DRG neurons have a more hyperpolarized ECl than rodent DRG neurons should be mentioned (Zhang et al., PMID: 26415765).

**********

6. 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: No

Reviewer #2: Yes: Edgar T. Walters

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment 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. Registration is free. 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 PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PONE-D-24-10034R1Depolarization of mouse DRG neurons by GABA does not translate into acute pain or hyperalgesia in healthy human volunteersPLOS ONE

Dear Dr. Schmelz,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

The authors have responded well to the reviewers' comments and the manuscript is much improved. However, the authors will need to make minor adjustments in the text of the results that refer to Figure 3C-E (3E is not referred to, and 3D appears to be referred to as 3C, there is no downward arrow that was referred to in the text). In addition there are minor errors in spelling in the body of the text. Please adjust these minor changed and resubmit the manuscript.

Please submit your revised manuscript by Aug 16 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Peter Wenner

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: (No Response)

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: (No Response)

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: (No Response)

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: (No Response)

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: (No Response)

**********

7. 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 #2: Yes: Edgar T. Walters

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment 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. Registration is free. 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 PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Depolarization of mouse DRG neurons by GABA does not translate into acute pain or hyperalgesia in healthy human volunteers

PONE-D-24-10034R2

Dear Dr. Schmelz,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Peter Wenner

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: