Dear Dr Glebov,

Thank you for submitting your revised manuscript entitled "Rapid long-range synaptic remodelling in hyperacute ischemic stroke" for consideration as a Research Article by PLOS Biology. I have had a chance to discuss your revised study and your response to reviewers with the Academic Editor and I am writing to let you know that we would like to send your submission back to the original reviewers for another look.

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 Sep 04 2025 11:59PM.

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,

Luke

Lucas Smith, Ph.D.

Senior Editor

PLOS Biology

lsmith@plos.org

Dear Dr Glebov,

Thank you for your patience while we considered your revised manuscript "Rapid long-range synaptic remodelling in hyperacute ischemic stroke" for consideration as a Research Article at PLOS Biology. Your revised study has now been evaluated by the PLOS Biology editors, the Academic Editor and the original reviewers.

As you will see in the comments below, both reviewers 1 and 2 are fully satisfied with the revision. Reviewer 3 also indicates that the paper has been strengthened, but s/he has a few lingering concerns which will need to be carefully addressed. Of note, reviewer 3 has commented that you should clarify how body temperature was maintained during MK801 administration and if body temperature was not carefully controlled during stroke and MK801 administration, this experiment needs to be included. Overall we agree with that assessment.

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 remaining points from the reviewer 3 in a revision that we anticipate should not take you very long. However, if you need to generate new data to address his/her comments, please do let us know and we are happy to provide more time. 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.

In addition to these revisions, you may 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. If you do not receive a separate email within a few days, please assume that checks have been completed, and no additional changes are required.

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.

**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.

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.

*Resubmission Checklist*

When you are ready to resubmit your revised manuscript, please refer to this resubmission 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,

Luke

Lucas Smith, Ph.D.

Senior Editor

PLOS Biology

lsmith@plos.org

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

REVIEWS:

Reviewer #1, Christoph Harms (note, reviewer 1 has signed this review): The authors have increased the transparency of their study. I have no further concerns.

Reviewer #2: The authors succesfully replied to my methodological concerns.

Reviewer #3: Throughout the manuscript the long-distance synaptic changes are referred to as 'enhanced'. This is not appropriate language and not supported by the data as presented. Enhancement has a positive valence associated with it, but none of the changes as reported in the paper can be demonstrated to be enhanced, just changed. A more suitable descriptor should be chosen by the authors because the alteration of synaptic genes, changes in expression of receptors and increased synaptic vesicle cycling could actually be detrimental to brain function and stroke. for example, increased expression of K channels would likely be inhibitory for neuronal function, which would not be considered enhancement.

during the administration of MK801, was the body temperature of the animal carefully controlled? This needs to be described in the methods and is critical because known effects of MK801 include hyperthermia, which can cause broad changes in brain function and has been reported to alter gene expression. Importantly, if body temperature was not carefully controlled during stroke and MK801 administration, this experiment needs to be included.

Fig 1A does not seem to be representative of the data. There is an almost complete loss of BSN labelling (for example) at 2 hrs in OGD, but this is not seen in Fig B

Dear Dr Glebov,

Thank you for your patience while we considered your revised manuscript "Rapid long-range synaptic remodelling in hyperacute ischemic stroke" for publication as a Research Article at PLOS Biology. Your revised study has been evaluated by the PLOS Biology editors and the Academic Editor.

We understand from your responses to reviewer 3, that while you controlled body temperature during the MCAO experiments with a heating pad, that you have not formally measured or controlled the body temperature during the MK801 experiments, as requested in the last round of review. We understand that you instead cite a body of literature showing that MK801's effects on body temperature can be heterogeneous and that they are generally minor. Unfortunately, we do not think that this response has gone far enough in addressing reviewer 3's comment. We would therefore like to invite you to complete one last revision, to address this point with additional data.

After discussing this point with the Academic Editor and with my colleagues on the editorial team, we would not require that you repeat the characterization of all outcomes after MK801 administration. However, at minimum, we think you will need to repeat the MK801 study as previously performed, and measure whether, in your hands and in this context, the drug affects body temperature, or not. Depending on the results of those studies, we think you should then add additional discussion about whether this is a likely confound affecting the results of your experiments, or not.

As an additional note, I actually could not find the details of the MK801 administration studies in your methods section. Please do take a moment to update the methods to include all relevant details about how that experiment was performed, including dosing, method of delivery, timing, etc. Indeed, please double check that the methods section includes the relevant details for all of the experiments conducted in this study.

Given that we think that additional experimental work is needed to address Reviewer 3's concerns, we are offering 3 months for you to complete this next revision. Please note, that 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. This will be the last round of experimental revisions that we will allow.

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.

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,

Luke

Lucas Smith, Ph.D.

Senior Editor

PLOS Biology

lsmith@plos.org

Dear Dr Glebov,

Thank you for your patience while we considered your revised manuscript "Rapid long-range synaptic remodelling in hyperacute ischemic stroke" for publication as a Research Article at PLOS Biology. This revised version of your manuscript has been evaluated by the PLOS Biology editors and the Academic Editor, who is satisfied by the changes made in the most recent revision.

Based on our Academic Editor's assessment of your revision we are likely to accept this manuscript for publication. However, before we can do so, we need you to address a number of data and other policy-related requests in a last short revision. These are detailed below.

IMPORTANT - Please address the following editorial requests:

1) TITLE: We would like to suggest a tweak to your title, to add a verb and a bit more detail about the study. If you agree, we suggest you change your title to:

'Ischemic stroke triggers brain-wide synaptic remodeling within four hours'

2) ABSTRACT: Please note that per journal policy, the model system/species studied should be clearly stated in the abstract of your manuscript.

3) ETHICS STATEMENT: Please update your ethics statement, in your methods section, to include the protocol number of the animal care and use protocol, approved by the Qingdao University IACUC.

4) DATA: Thank you for providing your mass spec and RNA seq data on proteomeXchange and GEO. For the RNA-seq data, please provide me with a reviewer token so I can access this data and make sure it meets our requirements.

5) DATA: Thanks also for providing the other underlying data for your study as a supplemental table. Please add a sentence to every relevant figure legend referencing this file. We also ask that you update your data availability statement to reference this data.

For example, you could change the Data Availability statement to read:

Mass spectrometry proteomics data is available from the ProteomeXchange (https://proteomecentral.proteomexchange.org), dataset identifier PXD058834.

RNAseq data is avialable from GEO (https://www.ncbi.nlm.nih.gov/geo/), accession number GSE283465.

All other relevant data are contained within the manuscript and its supplemental materials.

Other raw data is available from the corresponding Author upon reasonable request

6) CODE: 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

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 may 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. If you do not receive a separate email within a few days, please assume that checks have been completed, and no additional changes are required.

We expect to receive your revised manuscript within three 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,

Luke

Lucas Smith, Ph.D.

Senior Editor

lsmith@plos.org

PLOS Biology

Dear Dr Glebov,

Thank you for the submission of your revised Research Article "Ischemic stroke triggers brain-wide synaptic remodeling within four hours" for publication in PLOS Biology and thank you for addressing our last editorial requests in this revision. On behalf of my colleagues and the Academic Editor, Richard Daneman, 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 NOTE: I saw that you provided an updated data and code availability statement in your manuscript. I have taken the liberty of moving this to the relevant section of our editorial manager system, as that is the version that will be published with your study. I removed the bit about Oe Biotech's analysis pipeline, as that is better left in the methods section, rather than the data availability statement. The current version reads:

Custom code for gene expression analysis has been uploaded to Zenodo (https://zenodo.org/records/17987265). Mass spectrometry proteomics data is available at ProteomeXchange (https://proteomecentral.proteomexchange.org, dataset identifier PXD058834). RNAseq data is available at GEO (https://www.ncbi.nlm.nih.gov/geo/, accession number GSE283465). Numerical data is presented in Table S1. Other raw data is available from the Corresponding Author upon reasonable request.

^Please do take a moment to double check that everything looks good, in our online system, after this change.

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,

Lucas Smith, Ph.D.

Senior Editor

PLOS Biology

lsmith@plos.org