Dear Dr Bertrand,

Thank you for submitting your manuscript entitled "When Little Means a Lot: Impact of a Brief Early-life Motor Training on Mouse Neuromuscular Development." 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, before we can send your manuscript to reviewers, we need you to complete your submission by providing the metadata that is required for full assessment. To this end, please login to Editorial Manager where you will find the paper in the 'Submissions Needing Revisions' folder on your homepage. Please click 'Revise Submission' from the Action Links and complete all additional questions in the submission questionnaire.

Once your full submission is complete, your paper will undergo a series of checks in preparation for peer review. After your manuscript has passed the checks it will be sent out for review. To provide the metadata for your submission, please Login to Editorial Manager (https://www.editorialmanager.com/pbiology) within two working days, i.e. by Nov 28 2024 11:59PM.

If your manuscript has been previously peer-reviewed at another journal, PLOS Biology is willing to work with those reviews in order to avoid re-starting the process. Submission of the previous reviews is entirely optional and our ability to use them effectively will depend on the willingness of the previous journal to confirm the content of the reports and share the reviewer identities. Please note that we reserve the right to invite additional reviewers if we consider that additional/independent reviewers are needed, although we aim to avoid this as far as possible. In our experience, working with previous reviews does save time.

If you would like us to consider previous reviewer reports, please edit your cover letter to let us know and include the name of the journal where the work was previously considered and the manuscript ID it was given. In addition, please upload a response to the reviews as a 'Prior Peer Review' file type, which should include the reports in full and a point-by-point reply detailing how you have or plan to address the reviewers' concerns.

During the process of completing your manuscript submission, you will be invited to opt-in to posting your pre-review manuscript as a bioRxiv preprint. Visit http://journals.plos.org/plosbiology/s/preprints for full details. If you consent to posting your current manuscript as a preprint, please upload a single Preprint PDF.

Feel free to email us at plosbiology@plos.org if you have any queries relating to your submission.

Kind regards,

Taylor

Taylor Hart, PhD,

Associate Editor

PLOS Biology

thart@plos.org

Dear Dr Bertrand,

Thank you for your patience while your manuscript "When Little Means a Lot: Impact of a Brief Early-life Motor Training on Mouse Neuromuscular Development." was peer-reviewed at PLOS Biology. It 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 would like to invite you to revise the work to thoroughly address the reviewers' reports.

You’ll see that the reviewers think that the study is well executed and find the results of interest. All three reviewers had issues with the level of clarity in the methods, results, and data items, particularly the lack of clarity in how many animals were used in each experiment. R2 and R3 both found the analysis of gene expression changes after training to be of limited insight and suggested a more thorough analysis to justify the inclusion of this data. R3 finds issue with the logical flow throughout the manuscript, while still being quite positive of the paper overall.

We would like to invite a Major Revision of this manuscript before it can be accepted for publication. In particular, the revised manuscript should include additional analysis of the gene expression data as part of a new supplemental item in line with R2's and R3's suggestions, along with videos showing the swimming patterns as indicated by R3. In addition, you should carefully review and modify the data presentation and text to improve the clarity and correct any errors in line with the reviewer's comments.

Given the extent of revision needed, we cannot make a decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is likely to be sent for further evaluation by all or a subset of the reviewers.

We expect to receive your revised manuscript within 3 months. 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 may withdraw it.

**IMPORTANT - SUBMITTING YOUR REVISION**

Your revisions should address the specific points made by each reviewer. Please submit the following files along with your revised manuscript:

1. A 'Response to Reviewers' file - this should detail your responses to the editorial requests, present a point-by-point response to all of the reviewers' comments, and indicate the changes made to the manuscript.

*NOTE: In your point-by-point response to the reviewers, please provide the full context of each review. Do not selectively quote paragraphs or sentences to reply to. The entire set of reviewer comments should be present in full and each specific point should be responded to individually, point by point.

You should also cite any additional relevant literature that has been published since the original submission and mention any additional citations in your response.

2. In addition to a clean copy of the manuscript, please also upload a 'track-changes' version of your manuscript that specifies the edits made. This should be uploaded as a "Revised Article with Changes Highlighted" file type.

*Re-submission Checklist*

When you are ready to resubmit your revised manuscript, please refer to this re-submission checklist: https://plos.io/Biology_Checklist

To submit a revised version of your manuscript, please go to https://www.editorialmanager.com/pbiology/ and log in as an Author. Click the link labelled 'Submissions Needing Revision' where you will find your submission record.

Please make sure to read the following important policies and guidelines while preparing your revision:

*Published Peer Review*

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. Please see here for more details:

https://blogs.plos.org/plos/2019/05/plos-journals-now-open-for-published-peer-review/

*PLOS Data Policy*

Please note that as a condition of publication PLOS' data policy (http://journals.plos.org/plosbiology/s/data-availability) requires that you make available all data used to draw the conclusions arrived at in your manuscript. If you have not already done so, you must include any data used in your manuscript either in appropriate repositories, within the body of the manuscript, or as supporting information (N.B. this includes any numerical values that were used to generate graphs, histograms etc.). For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5

*Blot and Gel Data Policy*

We require the original, uncropped and minimally adjusted images supporting all blot and gel results reported in an article's figures or Supporting Information files. We will require these files before a manuscript can be accepted so please prepare them now, if you have not already uploaded them. Please carefully read our guidelines for how to prepare and upload this data: https://journals.plos.org/plosbiology/s/figures#loc-blot-and-gel-reporting-requirements

*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

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,

Taylor

Taylor Hart, PhD,

Associate Editor

PLOS Biology

thart@plos.org

------------------------------------

REVIEWS:

Reviewer #1: This study explored the influence of increased motor activity in newborn mice during early period of development of spinal cord circuitry and hindlimb muscles. The main conclusion is that a very short motor training performed just after birth is able to induce functional adaptation in the developing

neuromuscular system that could persist several days. The study is impressive in the scope and a number of electrophysiological, morphological and biochemical analyses performed, and the work is very well done. In vitro recordings on spinal cord slices are challenging, and the authors amassed a convincing number of recordings on which they base their conclusions.

The study is implemented with the high degree of rigor and technical excellence. Analyses are carefully performed and presentation is clear in general. The results are definitely interesting and a bit surprising, considering a very short-lasting training sessions. I have no significant criticisms. However, I would advice the Authors to reconsider presentation of all the detailed data in the tables and figures. A number of figures and plots is overwhelming, and some of them repeat the same data already presented in tables 2 and 3. It is a question whether to show all the insignificant data, which are shortly mentioned in the Results, but not discussed further.

The minor points which should be corrected or clarified:

- it is difficult to find a number of mice used in particular groups for analyses, a total number, division into trained and untrained groups; sometimes a number of mice is indicated in the text, but frequently it is not, and this makes more difficult to assess the importance of particular changes among a substantive number of data;

- a number of samples in Table 5 as well as a number of recordings used for violin plots might also be indicated in the plots or in the legends to make them more informative; presentation of a percentage in charts is informative, but should also be supported with a number of neurons/samples (Figs. 1BC, 3C2, 4D, 10C, 11);

- several data in Table 3 and 4 need to be corrected (rheobase is presented in nA? AP rise time in ms? current max in pA?);

- Fig. 3A1: is the scale bars (50ms and 2000pA) real for the presented record? It is stated in methods that "AHP parameters were computed after eliciting a single action potential (AP), with a brief 7 ms, 0.25 nA depolarizing current pulse". Basing o the scale bars the pulse here seems to be 2.5nA and duration at least twice longer, the same doubt concerns the AP and AHP duration. It is difficult for a reader to check such details in each presented chart, but when such a mistake has been found once, I would encourage the Authors to check such and similar details throughout the paper and all figures;

- I have concern about clear outliners visible in some of the violin plots showing distribution of data (especially in Fig. 3A5, Fig. 4 C2 and C3). Are the Authors sure that these measurements were based on reliable recordings, not suffering from an erratic observation or instability of a preparation? Could this be connected to slow-fast differences between MNs, and obviously, slow and fast MNs were not distinguished?

Reviewer #2: In this work, the authors designed a short motor training regimen at P1 and P2 to decode the impact of early training on the development of spinal motor neurons and hindlimb muscles using the combined techniques including behavioral analysis, RNA-seq, electrophysiological recordings, immunostaining, et al. They demonstrated that a short training caused many changes in newborn mice including swimming performances, gene expression, electrophysiological characteristics of spinal motor neurons, muscle development. The study is interesting, but the authors should strengthen the logical explanation of the changes they identified in different aspects.

I have some comments as below:

1. The author should give a brief explanation of the duration time designed in the training paradigm since that 7 sec or 15 sec were very short.

2. In Fig. 1B1�why are the numbers of animals in different test batches different within the same group (untrained or trained)? In the untrained group, the numbers were 64, 66, 61, and 61 at the trial 1, 2, 3, and 4 respectively. In the trained group, they were 110, 99, 103, and 91 at the training 1, 2, 3, and 4 respectively. Why did the authors use such a large number of animals for behavioral screening?

3. In Fig. 1B1 and Fig. 1B2: the changes were strange: At trial 1 of untrained mice, the proportion of mice unable to swim at session 2 increased significantly compared with that at session 1. Does it mean that P1 pups lose swimming ability after a 45-second break?

4. The authors studied training-induced gene expression changes using RNA-seq at P3. The molecules involved in the key signaling pathways should be deeply analyzed combining with the electrophysiological characteristics and morphological changes of training-induced neurons. For example, downregulated DEGs were clustered in neurogenesis, synaptogenesis, developmental NMJ, et al. Which DEGs could support the findings in changes of electrophysiological and morphological characteristics after training? Likewise, upregulated DEGs were clustered in many important biological processes, and these results should be further illustrated and confirmed.

5. At page 21 Line 12-13: "The proportion of MNs expressing LTD, STD, or No Plasticity differed significantly between untrained and trained mice…" What conclusion could be made from this finding? The authors should summarize at the end of this paragraph.

6. The authors described: a significant decrease in the amplitude of sEPSCs, less frequent but larger sIPSP events in trained MNs compared to the untrained MNs. In Fig. 6B2, the frequency of sIPSPs looked higher in the trained MNS (red dots) than the untrained MNs (black dots)? In Fig. 6B3, the amplitude of sIPSPs looked smaller in the trained MNS (red dots) than the untrained MNs (black dots)? Is there any morphological evidence supporting this finding?

7. The authors found a notable increase of 5-HT and NA between P3 and P10 in trained mice, I was wondering if it was possible to present any differences of their fiber density using immunofluorescent staining spinal sections.

Reviewer #3: Review Plos Biology : PBIOLOGY-D-24-03358R1

Title: When Little Means a Lot: Impact of a Brief Early-life Motor Training on Mouse Neuromuscular Development.

Authors : Quilgars et al

Overall comments

The authors compared the impact of an early swim training (5 sessions of 15 sec, twice a day for 2 consecutive days) in neonatal mice pups, compared to untrained pups (2 sessions of 7 sec, twice a day for 2 consecutive days for swimming assessement) on several structural and functional features in the spinal cord and in the interactions between motneurons and muscles. Briefly, they found changes in the acquisition of swimming abilities (acceleration in trained pups), no changes in the intrinsic properties of motoneurons from the lumbar spinal cord except a few aspects of membrane excitability, changes in some features of currents and synaptic plasticity, down- and upregulated genes involved in membrane properties of motoneurons and others structural aspects, changes in some neurotransmitters, and alterations of axonal myelination in the motor regions of the spinal cord. They also found changes in the morphology, phenotype after such an early and short motor training. All the different features studied here are very interesting and correspond to a huge stack of data and work, but sound like a juxtaposition, so they need to be more linked together into an integrated whole study.

The study is very sound and interesting, well-conceived, well-done and well-written but several mistakes and typos are present. The Results part on swim motor training lacks of precision and details that need to be clarified (see description below), otherwise the remaining parts of the manuscript are sound. The manuscript needs more work, precision and clarifications since it really deserves to be published in Plos Biology with a high impact in the field of developmental neuroplasticity. One can deplore the absence of working hypothesis in the Introduction that may link and justify the methods used to test it, and the use of too many abbreviations. English and typos should be corrected.

Abstract

From the Results and Fig 1A, the faster acquisition of swimming was assessed at P1 and P2 in trained and untrained pups; please clarify timing of results.

Methods

The description of the statistics used and their choice should be increased and justified. For example, I don't understand the use of Chi square in many comparisons and make some statistical results unclear, like the numbers of motoneurons expressing STD, LTD or no plasticity. Statistical test description needs to be added in the Results, like the value of Mann-Whitney tests and Spearman…

Results

Part 1: "Motor training accelerated the acquisition of the four-limb motor pattern for swimming."

The Methods, Results, Fig 1 from A to D and the corresponding legend are unclear and hard to follow and decipher, so they need to be clarified as this part is crucial for understanding the whole manuscript.

In Cazalets et al (1990), is written page 127: "During the first 24 hours, only 2/3 of the [rat] pups were swimming, whereas at day 2 they were all able to swim and move around the tank. … As all of them [rat pups] were not able to swim at P0, we did not perform quantitative analysis." The patterns of swimming of mice pups seems critical to be better defined in the present study, compared to rats that served as reference in previous reports (Cazalets et al, 1990) and from the "patterns H0 to H2" in mice pups in Cazalets (2000). Do the patterns of swimming differ between the various strains of mice used in the present study and from "C3H control strain" in Cazalets et al (2000). This is preponderant and crucial for the rest of the study. In addition, the authors use two strains of mice in the present study, the use of each one in the different experiments needs also to be clarified.

How many mice were used in the present study? Why don't the authors show also the numbers of mice for scores 2 to 4? Why the numbers in session 1 but not in session 2? All this part on the "motor training" and "swimming patterns" needs to be clarified from the Methods, Results and corresponding Fig 1 and legend. Why not to add videos of patterns in supplemental data? Supplemental videos may clarify the "non well-defined patterns of swimming" and each pattern of swimming

What is the interest of fig Fig 1C?

The numbers of mice used appears for the body weight comparison. It should appear before in the manuscript, as well as the repartition of the two strains, as previously suggested.

The maximal duration of the righting reflex session should also be described in Methods for ethical consideration since the authors wait for rats to be exhausted in this task (i.e. time to abandon in Fig1D).

The conclusion of this part of the Results may include the difference of training between untrained that performed 7 sec of swimming twice, 2 times a day for 2 consecutive days.

Actually, the part on top of Fig 1A for untrained mice (Trial 1 - Mouse 1 and so on...) is not clear and does not correspond to any description in the legend, Methods or Results. It suggests that each trial was made with different mice. This needs to be clarified.

The difference in swim training between the two groups needs to be clarified and emphasized along the manuscript. I don't understand why the authors used untrained control rats to assess swimming at P1 and P2 (corresponding to a "training") while they did it later from P5 to P14, which may have been enough and leaving untrained control pups really untrained?

Table 1 on Motor Scores. Why is there no score of 1, as it could be 0 for no swim and 1 for swim with 1 limb? Please justify and clarify.

The authors describe the exclusive use of hind limbs around P15, so why do the use of the hind and forelimbs are mixed in scoring from P5 to P12? Actually, the gradation of scores of this scale is not completely clear; please justify.

Part 2: "Motor training induces changes in gene expression in the lateral motor column."

There are discrepancies in the numbers of genes up -and downregulated between text and Fig 2.

The description of the changes in genes is too short and lacks of details to get the insights, only a summary of the results is produced. Maybe more details could be provided in Supplemental data? This raises the question of its interest as it has not been justified previously.

To me, there are also discrepancies between statistics in the text corresponding to the Fig 4C2 and C3. Please clarify.

Last part of Results: "Early motor training impacts the development of postural control during the two first postnatal weeks"

I don' agree with authors on the summary of results: "These data indicate that the postural development of trained pups differs slightly but significantly from that of untrained ones during the first postnatal week and that these differences disappear during the second postnatal week." To me, these results appear stronger than they described and should be emphasized more positively as an improvement and acceleration of the acquisition of sensorimotor abilities, as written in the Abstract.

Discussion

The different parts are very interesting and well-written and suited, but to me, several positive results are underestimated, like the "absence" of changes in the muscle phenotype and others. As previously suggested, all these very interesting results lack of integration and links together to serve a hypothesis on the impact of such an early and short motor training on so many structural, morphological and functional properties for the development and exercise-driven plasticity of the sensorimotor system.

These comments are meant to help the authors to improve the quality of the manuscript, which deserves for me to be published in Plos Biology

Dear Dr Bertrand,

Thank you for your patience while we considered your revised manuscript "When Little Means a Lot: Impact of a Brief Early-life Motor Training on Mouse Neuromuscular Development." for publication as a Research Article at PLOS Biology. This revised version of your manuscript has been evaluated by the PLOS Biology editors, the Academic Editor and the original reviewers.

Based on the reviews, we are likely to accept this manuscript for publication, provided you satisfactorily address the following data and other policy-related requests:

- TITLE: We would like to suggest a different title to increase accessibility for our broad audience: "Brief early-life motor training induces long-lasting changes in behavior and neuromuscular development in mice"

- FINANCIAL DISCLOSURE: 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).

2) Deposition in a publicly available repository. Please also provide the accession code or a reviewer link so that we may view your data before publication.

Regardless of the method selected, please ensure that you provide the individual numerical values that underlie the summary data displayed in the following figure panels as they are essential for readers to assess your analysis and to reproduce it:

Fig 1 B-D

Fig 2 all panels

Fig 3 A2-A6, B2-B3, C1-C2

Fig 4 A2-A3, B, C2

Fig 5 B-D

Fig 6 A2-A3, B2-B3

Fig 7 B-D

Fig 8 B1-B3

Fig 9 B1-B5, C

Fig 10 B1-B3, C1-C3

Fig 11 A1-A3, B1-B4, C

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).

Please also ensure that figure legends in your manuscript include information on where the underlying data can be found, and ensure your supplemental data file/s has a legend.

Please ensure that your Data Statement in the submission system accurately describes where your data can be found.

- DATA AVAILABILITY: For the sequencing data, you should deposit the raw data in a publicly available repository, and mention this in the data availability statement as well as providing a link or DOI.

- CODE POLICY

Per journal policy, if you have generated any custom code during the course of this investigation, please make it available without restrictions. Please ensure that the code is sufficiently well documented and reusable, and that your Data Statement in the Editorial Manager submission system accurately describes where your code can be found.

Please note that we cannot accept sole deposition of code in GitHub, as this could be changed after publication. However, you can archive this version of your publicly available GitHub code to Zenodo. Once you do this, it will generate a DOI number, which you will need to provide in the Data Accessibility Statement (you are welcome to also provide the GitHub access information). See the process for doing this here: https://docs.github.com/en/repositories/archiving-a-github-repository/referencing-and-citing-content

- ORIGINAL GELS: In line with our gel and blot reporting requirements, you should provide uncropped images of all Western blots or other gels.

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:

- a cover letter that should detail your responses to any editorial requests, if applicable, and whether changes have been made to the reference list

- a Response to Reviewers file that provides a detailed response to the reviewers' comments (if applicable, if not applicable please do not delete your existing 'Response to Reviewers' file.)

- a track-changes file indicating any changes that you have made to the manuscript.

NOTE: If Supporting Information files are included with your article, note that these are not copyedited and will be published as they are submitted. Please ensure that these files are legible and of high quality (at least 300 dpi) in an easily accessible file format. For this reason, please be aware that any references listed in an SI file will not be indexed. For more information, see our Supporting Information guidelines:

https://journals.plos.org/plosbiology/s/supporting-information

*Published Peer Review History*

Please note that 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. Please see here for more details:

https://plos.org/published-peer-review-history/

*Press*

Should you, your institution's press office or the journal office choose to press release your paper, please ensure you have opted out of Early Article Posting on the submission form. We ask that you notify us as soon as possible if you or your institution is planning to press release the article.

*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,

Taylor

Taylor Hart, PhD,

Associate Editor

thart@plos.org

PLOS Biology

------------------------------------------------------------------------

Reviewer remarks:

Reviewer #1: I would like to thank the authors for addressing my points in the revised version of the manuscript. The current version is improved and all of my concerns have been considered.

Reviewer #2: The authors carefully addressed my concerns, and I have no further comments.

Reviewer #3 (J-Olivier Coq): Review Plos Biology : PBIOLOGY-D-24-03358R2

Title: When Little Means a Lot: Impact of a Brief Early-life Motor Training on Mouse

Neuromuscular Development.

Authors : Quilgars et al

General comments

The authors have performed most of the changes recommended and have considered and discussed the other changes suggested. The different changes requested by the three reviewers were complementary and the revised version R2 fits to me with the requirements and guidelines for publication in Plos Biology.

Dear Dr Bertrand,

Thank you for your patience while we considered your revised manuscript "Impact of a Brief Early-life Motor Training on Mouse Neuromuscular Development." for publication as a Research Article at PLOS Biology. This revised version of your manuscript has been evaluated by the PLOS Biology editors, and we have noted a few more items to address before your paper can be formally accepted.

----

***Regarding the title, we understand your point and agree that it is best not to say that the changes are 'long-lasting'. The alternative that you have proposed, 'Brief early-life motor training induces changes in behavior and neuromuscular development in mice' is acceptable to us. But we also propose a slight modification 'Brief early-life motor training induces behavioral changes and alters neuromuscular development in mice'.

Whichever version you choose, please change the title in the manuscript file and the meta data to match when you submit the revision.

***Regarding the data, thank you very much for making your sequencing and other source data available. However, there are a few points on this topic still to address.

First, we found that the Research Data Gouv only guarantees maintaining the data for a minimum of 5 years (https://recherche.data.gouv.fr/en/faq). In the interest of maintaining access to your RNAseq data over the long term, we request that you also upload your sequencing data to the GEO database, as this is considered more standard (https://www.ncbi.nlm.nih.gov/geo/).

Second, I have examined the replication data for Figure 1 and quickly noticed what appear to be discrepancies between the plotted data and the corresponding part of the excel file.

- In Fig. 1B in the excel file, the data in the first column sum to 64, which matches the number shown in the figure above the first bar. However, the data in the second column sum to 110, whereas the number above the corresponding bar in the figure is 241. The first 'trained' bar in the figure shows about 36%, which would about match to the raw count of 38 in the file, but only if the total count is indeed 110 - if the count is 241, then there is a substantial mismatch. The same issue appears throughout the figure. It isn't clear to me whether the numbers listed above the histogram bars are wrong, or if the count data in the excel file are wrong, or if the data is displayed incorrectly in the chart.

- For Fig. 1E, there is data for this in the supplemental excel file but there is no panel in the figure with this label. Instead, the second part of Fig. 1D appears to match this data.

I have not comprehensively examined how well the replication data you provided match your figures, so there may be more discrepancies. Please address these issues and also double-check that all data are displayed correctly and that the figures and replication data files match throughout.

***In addition, the figure legends require a bit more information. Please specify in the figure legends where each piece of source data may be found among the different files you uploaded to the repository. It may help to adopt a consistent naming scheme for the source data files, for example "S1_Data.xlsx," which you can then reference in the figure legends parenthetically as ("S1 Data").

Also, some details of the figures need to be explained explicitly in the figure legends. For example, the legends should say what the numbers shown above some of the histogram bars in Fig. 1B represent. The dotted lines shown on the violin plots should also be explained in the legends. This will also make it easier to sort out any other discrepancies.

-----

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:

- a cover letter that should detail your responses to any editorial requests, if applicable, and whether changes have been made to the reference list

- a Response to Reviewers file that provides a detailed response to the reviewers' comments (if applicable, if not applicable please do not delete your existing 'Response to Reviewers' file.)

- a track-changes file indicating any changes that you have made to the manuscript.

NOTE: If Supporting Information files are included with your article, note that these are not copyedited and will be published as they are submitted. Please ensure that these files are legible and of high quality (at least 300 dpi) in an easily accessible file format. For this reason, please be aware that any references listed in an SI file will not be indexed. For more information, see our Supporting Information guidelines:

https://journals.plos.org/plosbiology/s/supporting-information

*Published Peer Review History*

Please note that 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. Please see here for more details:

https://plos.org/published-peer-review-history/

*Press*

Should you, your institution's press office or the journal office choose to press release your paper, please ensure you have opted out of Early Article Posting on the submission form. We ask that you notify us as soon as possible if you or your institution is planning to press release the article.

*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,

Taylor

Taylor Hart, PhD,

Associate Editor

thart@plos.org

PLOS Biology

Dear Dr Bertrand,

Thank you for the submission of your revised Research Article "Brief early-life motor training induces behavioral changes and alters neuromuscular development in mice" for publication in PLOS Biology. On behalf of my colleagues and the Academic Editor, Jacques-Olivier Coq, 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.

We also ask that you take this opportunity to read our Embargo Policy regarding the discussion, promotion and media coverage of work that is yet to be published by PLOS. As your manuscript is not yet published, it is bound by the conditions of our Embargo Policy. Please be aware that this policy is in place both to ensure that any press coverage of your article is fully substantiated and to provide a direct link between such coverage and the published work. For full details of our Embargo Policy, please visit http://www.plos.org/about/media-inquiries/embargo-policy/.

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

Sincerely, 

Taylor Hart, PhD,

Associate Editor

PLOS Biology

thart@plos.org