Dear Ben,

Thank you for submitting your manuscript entitled "Altered auditory feature discrimination in a rat model of Fragile X Syndrome" for consideration as a Research Article by PLOS Biology.

Your manuscript has now been evaluated by the PLOS Biology editorial staff and I am writing to let you know that we would like to send your submission out for external peer review. However, I am not convinced, given the level of mechanistic insight, that the manuscript would work as full Research Article for us. I think it would work better as a Short Report. This would mean to reduce the number of figures to four, but I think this would be possible, so I'd encourage to submit your manuscript as Short Report. You do not need to reduce the number of figures now already. This is something we can work on if we were to invite a revision.

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Kind regards,

Christian

Christian Schnell, PhD

Senior Editor

PLOS Biology

cschnell@plos.org

Dear Dominik,

Thank you for your patience while your manuscript "Altered auditory feature discrimination in a rat model of Fragile X Syndrome" went through peer-review at PLOS Biology. Your manuscript has now been evaluated by the PLOS Biology editors, an Academic Editor with relevant expertise, and by several independent reviewers.

In light of the reviews, which you will find at the end of this email, we are pleased to offer you the opportunity to address the concerns from the reviewers in a revision that we anticipate should not take you very long. We will then assess your revised manuscript and your response to the reviewers' comments with our Academic Editor aiming to avoid further rounds of peer-review, although we might need to consult with the reviewers, depending on the nature of the revisions.

As discussed previously, our Short Reports format allows only four figures. To achieve this, it would probably work best to combine figures 2 and 3, and figures 4 and 5, but we are open to other suggestions, as long as you don't have more than four figures.

In addition to these revisions, 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 shortly.

We expect to receive your revised manuscript within 1 month. Please email us (plosbiology@plos.org) if you have any questions or concerns, or would like to request an extension.

At this stage, your manuscript remains formally under active consideration at our journal; please notify us by email if you do not intend to submit a revision so that we withdraw the manuscript.

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

Sincerely,

Christian

Christian Schnell, PhD

Senior Editor

PLOS Biology

cschnell@plos.org

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REVIEWS:

Reviewer #1: In this timely study, authors perform a thorough analysis of the electrophysiological and behavioral alterations in processing auditory information using rat model of FXS. The study is novel and report impaired auditory frequency resolution in Fmr1 KO rats using behavioral Go/no-Go task. In addition, authors perform in vivo electrophysiological recording and show that the behavioral alterations correlate with neuronal hyperactivity and broader frequency tuning in the auditory cortex but not IC of Fmr1 KO rat. Authors also report that the impaired behavioral and electrophysiological discriminations are observed between spectrally similar tones that are closer in frequency, while no changes in overall tone detection are observed in Fmr1 KO rats. These are very interesting observations that are consistent with findings reported in Fmr1 KO mice and humans with FXS. The studies also suggest that gain enhancement in the cortex may act to preserve signal-to-noise ratios and signal detection threshold at the expense of tuning precision and fine discrimination. The manuscript is well written and the reviewer has no concerns related to the design, presentation, analysis or interpretation of the study. I believe that the manuscript is acceptable for publication in its present form.

Reviewer #2: This manuscript describes a study in male Fmr1 KO rats that used behavioral testing, in vivo electrophysiology, and a computational approach to analyze differences in sound frequency discrimination in KO rats versus WT littermates. The study found that Fmr1 KO rats have a deficit in sound frequency discrimination that is associated with hyperexcitability in the auditory cortex, but not in the IC, and broader frequency tuning curves. They built a decoder to verify that these differences in neuronal responses can account for the observed behavioral effect.

The study seems to be sound and well-performed. The data is presented in a concise way and the manuscript is very well written. I have no major concerns, just a few comments to consider:

1. It would have been really interesting to see to what extent these findings extend to female rats. While I can see that rationale for using only male rats, it would really be valuable to follow up with females.

2. The results parallel some findings in other genetic rodent autism models - maybe worthwhile to briefly discuss?

Minor: Fig. 5D is missing a title for the y-axis.

Reviewer #3: PLOS Biology

Altered auditory feature discrimination in a rat model of Fragile X Syndrome

Gauthier et al. investigate tone discrimination in Fragile X syndrome knockout rats (Fmr1 KO). In their previous study (Auerbach et al, 2021), they demonstrated that these KO rats exhibit auditory hypersensitivity, characterized by faster reaction times in a go/no-go sound detection task. In the current study, they further show that Fmr1 KO rats have impaired tone discrimination abilities. Electrophysiological recordings in anesthetized rats reveal that frequency tuning in the inferior colliculus remains unchanged, whereas the auditory cortex exhibits broader tuning in KO rats. While these findings are interesting, the data analysis lacks some key details that are necessary to interpret the results. These are listed below.

Comments:

In Figure 1, the results from the go/no-go detection task and ABR recordings indicate that KO rats have similar threshold detection across tested frequencies compared to wildtype rats. However, there appears to be a discrepancy between Figures 1C and 1F. Behaviorally, higher frequencies (16, 32 kHz) are detected more easily than lower frequencies (4, 8 kHz), yet ABR data in Figure 1F suggest that low (4 kHz) and high (32 kHz) frequencies have similar thresholds. Could the authors clarify this inconsistency?

Line 331: Fmr1 KO (n = 5 rats, 10 ears) and WT (n = 6 rats, 10 ears).

If there are 6 WT rats, there should be 12 ears. Is this a mistake or some ears were excluded from the analysis for some reason?

The figure legends are missing key information, including:

a) What each error bar represents

b) What each dot in the box plots correspond to.

Figure 4A and 5A panels are missing colorbar values. Should they be spikes/sec?

Line 448: The text refers to "Figure 4E," but this panel does not seem to be included.

Authors state that the neuronal activity was sampled across iso-frequency lamina in the IC and cortical layers in the ACx. Based on Figure 3A, it would be expected that the position of the electrode shank could be determined based on best frequency, but no data (functional or histological) is provided to support this claim.

In addition to my previous comment, in Figure 3A, it seems that recordings were done in multiple subregions of the IC, but this is not clarified in the manuscript. Was the electrophysiological recording performed in the central inferior colliculus, or did it span through multiple subregions? If so, could the authors show that there are no functional differences between these subregions, particularly considering the high variability observed in Figure 3D and F?

In Figure 2C and D, only the false alarm rate is shown. It would be interesting to know if the hit rate also changes when multiple no-go frequencies are introduced. Additionally, since reaction times were extensively studied in a previous publication (tone detection task; Auerbach et al, 2021), could the authors provide the reaction times for the hit trials in the discrimination task as well?

For Figure 2, are KO rats generally unable to discriminate similar tones, or is this effect frequency-dependent? If so, is it consistent across multiple sessions?

In Figure 3, there is a big variability in the data for both wildtype and KO groups within the auditory cortex and inferior colliculus. Is this variability due to between-rat or within-rat differences?

Although the authors report a significant difference between WT and KO groups in the auditory cortex, the distributions of the two groups are highly overlapping. Could the authors discuss whether this significance is meaningful given the overlap?

Figures 4B and 5B lack statistical tests to compare WT and KO groups. What do these results suggest?

In Figure 6D, what do the individual dots represent? What are the two dashed lines?

This figure only shows decoding accuracy results for the auditory cortex, but it would be helpful to see similar analyses for the inferior colliculus. Additionally, to show differences for the auditory cortex in KO rats, a statistical test is needed.

Dear Ben,

Thank you for your patience while we considered your revised manuscript "Altered auditory feature discrimination in a rat model of Fragile X Syndrome" for publication as a Short Reports at PLOS Biology. This revised version of your manuscript has been evaluated by the PLOS Biology editors, the Academic Editor and one of the original reviewers.

Based on the reviews and on our Academic Editor's assessment of your revision, we are likely to accept this manuscript for publication, provided you satisfactorily address the following data and other policy-related requests:

* Please modify the last sentence of the abstract. We think that the current study is too far removed from potential medical applications and want to avoid raising false hopes. We think you can simply delete the second half of the sentence (after the comma).

* Please add the links to the funding agencies in the Financial Disclosure statement in the manuscript details.

* DATA POLICY:

You may be aware of the PLOS Data Policy, which requires that all data be made available without restriction: http://journals.plos.org/plosbiology/s/data-availability. For more information, please also see this editorial: http://dx.doi.org/10.1371/journal.pbio.1001797

Note that we do not require all raw data. Rather, we ask that all individual quantitative observations that underlie the data summarized in the figures and results of your paper be made available in one of the following forms:

1) Supplementary files (e.g., excel). Please ensure that all data files are uploaded as 'Supporting Information' and are invariably referred to (in the manuscript, figure legends, and the Description field when uploading your files) using the following format verbatim: S1 Data, S2 Data, etc. Multiple panels of a single or even several figures can be included as multiple sheets in one excel file that is saved using exactly the following convention: S1_Data.xlsx (using an underscore).

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NOTE: the numerical data provided should include all replicates AND the way in which the plotted mean and errors were derived (it should not present only the mean/average values).

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As you address these items, please take this last chance to 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 cover letter that accompanies your revised manuscript.

In addition to these revisions, 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 shortly.

We expect to receive your revised manuscript within two weeks.

To submit your revision, please go to https://www.editorialmanager.com/pbiology/ and log in as an Author. Click the link labelled 'Submissions Needing Revision' to find your submission record. Your revised submission must include the following:

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*Protocols deposition*

To enhance the reproducibility of your results, we recommend that if applicable 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. 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

Please do not hesitate to contact me should you have any questions.

Sincerely,

Christian

Christian Schnell, PhD

Senior Editor

cschnell@plos.org

PLOS Biology

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Reviewer remarks:

Reviewer #2: I think that the authors have thoroughly addressed the concerns of reviewer #2 and reviewer #3. I have no further comments and congratulate the authors to this study.

Dear Ben,

Thank you for the submission of your revised Short Reports "Altered auditory feature discrimination in a rat model of Fragile X Syndrome" for publication in PLOS Biology. On behalf of my colleagues and the Academic Editor, Jennifer Bizley, I am pleased to say that we can in principle accept your manuscript for publication, provided you address any remaining formatting and reporting issues. These will be detailed in an email you should receive within 2-3 business days from our colleagues in the journal operations team; no action is required from you until then. Please note that we will not be able to formally accept your manuscript and schedule it for publication until you have completed any requested changes.

Please take a minute to log into Editorial Manager at http://www.editorialmanager.com/pbiology/, click the "Update My Information" link at the top of the page, and update your user information to ensure an efficient production process.

PRESS

We frequently collaborate with press offices. If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximise its impact. If the press office is planning to promote your findings, we would be grateful if they could coordinate with biologypress@plos.org. If you have previously opted in to the early version process, we ask that you notify us immediately of any press plans so that we may opt out on your behalf.

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Thank you again for choosing PLOS Biology for publication and supporting Open Access publishing. We look forward to publishing your study. 

Sincerely, 

Christian

Christian Schnell, PhD

Senior Editor

PLOS Biology

cschnell@plos.org