PONE-D-21-16756

The effect of salicylate on the auditory cortex of guinea pigs

PLOS ONE

Dear Dr. Ochi,

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.

Both reviewers raised concerns about data analysis including single unit sorting, statistical distributions and statistical analysis.  Please also address concerns that the distinction between tinnitus and hearing loss has not been addressed.  Tinnitus can occur in the absence of hearing loss and hearing loss can occur without tinnitus.  Please also address the limitations of using salicylate as a tinnitus inducer.

Please submit your revised manuscript by September 30, 2021. 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: http://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,

Susan E Shore, Ph.D.

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 funders had no role in study design, data collection and analysis, decision to

publish, or preparation of the manuscript.'

At this time, please address the following queries:

1. Please clarify the sources of funding (financial or material support) for your study. List the grants or organizations that supported your study, including funding received from your institution.
2. State what role the funders took in the study. If the funders had no role in your study, please state: “The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.”
3. If any authors received a salary from any of your funders, please state which authors and which funders.
4. If you did not receive any funding for this study, please state: “The authors received no specific funding for this work.”

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

3. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information.

Additional Editor Comments (if provided):

[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: Partly

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: No

**********

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: Yes

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: In “The effect of salicylate on the auditory cortex of guinea pigs”, Kenmochi et al. recorded unit activity in the primacy AC and DC from anesthetized guinea pigs before and after applying salicylate intraperitoneally. They observed a drop in spontaneous activity in primary AC, and a rise in spontaneous activity in DC. Both areas showed an increase in spiking threshold. The authors discuss these findings in the context of tinnitus mechanisms and previous similar work in the cat.

Overall, the work presented appears to be carefully done, but the data presented are very limited in scope and thus this work represents an incremental advance in our understanding of the impact of salicylate on the AC.

There are several suggestions that I have for improving the manuscript.

Major:

1. There is very little analysis provided. After recording from 300+ neurons in the AC, the authors only provide information on thresholds and spontaneous activity. It would be very useful, both from the perspective of comparison to previous work and to better understand the currently-presented findings, if the authors provided additional analyses. For example, the authors measured many units simultaneously, but provide no information about correlations between neurons (though reference previous work about correlations between neurons). There is no information about the impact of CF, of frequency tuning, of depth or any other parameter, in their analysis. The absence of these types of analyses diminish the impact of this manuscript.

2. There is no mention of the impact of the salicylate exposure to the peripheral auditory system/ABRs, etc. Was any analysis of the peripheral system done?

3. Figure 1: Several voltage excursions exceed threshold. Which ones were counted as spikes? Where is the signal taken from that is expanded on the right?

4. Figure 2: Please show histograms of the CFs in the two areas. Seeing their mean/IQRs is not as helpful as just seeing the distributions

5. Figure 3: Please label the y-axis as “Tone threshold (dB)” and please separate AC from DC so that the reader can see the time trends more clearly. Please label significant changes as appropriate.

6. Figure 4: Please label the y-axis as “Spontaneous activity (Hz)” and please separate AC from DC so that the reader can see the time trends more clearly. Please label significant changes as appropriate.

7. The authors make the statement on P15, line 215-216 that the animals were likely to have tinnitus. There is no basis for this statement. Please remove.

Minor:

1. Title and abstract should indicate that salicylate was systemically administered

2. L18 should be “salicylate as a tinnitus-inducing…”

3. L39 should be “regardless of where salicylate acts…”

4. L42 should be “among all single units…”

5. L49 should be “is more scarce…”

6. L55 “unsuitable” is probably not the right word because it implies that this is not an appropriate structure/species to record from. Consider “difficult”, “challenging” or “suboptimal”

7. L141 should be “levels were recorded.”

8. L143 – was only the p-p amplitude recorded? What about spike width or other features? Usually there is an additional dimension that is used for clustering.

9. L250 should be “In conclusion, intraperitoneal salicylate…”

Reviewer #2: In this study, the authors measured the effects of salicylate treatment on the response threshold and spontaneous firing activity in the primary auditory cortex (AI) and dorsocaudal (DC) field of the auditory cortex in guinea pigs. They report that the threshold was increase by salicylate for both AI and DC. Spontaneous activity was suppressed in AI and elevated in DC by salicylate. This study is an extension of their previously published work in which they examine the salicylate effects in Cats. The study was generally well carried out. The findings are interesting and important in understanding salicylate-induced auditory pathologies such as hearing loss and tinnitus. However, there are several points that need to be clarified.

1. The result section and the figure legends are too short and not very clear.

2. The single unit separation was not described in sufficient detail. Was it entirely based on the amplitude of the spikes? Were the same single units tracked over the entire experiment, before and after salicylate application? Were they not confirmed with spike shape?

3. If the 160 and 156 single units were tracked over the four recording periods, the Friedman test (nonparametric version of repeated measure ANOVA) should be used to test the effects of salicylate. It seems that the current statistical tests did not correct for multiple comparison.

4. The graphs plotted top and bottom 10% points as outliers. Are the outliers included in the statistical analysis?

5. The authors used guinea pigs weighing between 355 and 830 g (mean ± 1SEM = 575 ± 61 g). Why are they so different in weight? Is there any age difference? If so, is salicylate effect dependent on the age.

6. In the discussion, the authors argue that threshold increase is evidence of tinnitus. That does not make much sense. The findings that hearing loss happens after tinnitus in humans taking salicylate indicate that hearing loss and tinnitus are two separate effects of salicylate, not causally related.

**********

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: No

[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.

The effect of systemic administration of salicylate on the auditory cortex of guinea pigs

PONE-D-21-16756R1

Dear Dr. Ochi,

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 for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, 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,

Susan E Shore, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

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 #1: All comments have been addressed

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 #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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 #1: Yes

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 #1: Yes

Reviewer #2: Yes

**********

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 #1: The added frequency analysis and edits have improved the paper.

Please be sure to label the y-axis in Figure 3

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 #1: No

Reviewer #2: No