Post-stress changes in the gut microbiome composition in rats with different levels of nervous system excitability

Alla Shevchenko; Irina Shalaginova; Dmitriy Katserov; Ludmila Matskova; Natalia Shiryaeva; Natalia Dyuzhikova

doi:10.1371/journal.pone.0295709

Peer Review History

Original SubmissionAugust 29, 2023
6 Oct 2023 Decision Letter - Brenda A Wilson, Editor PONE-D-23-27857Post-stress changes in the gut microbiome composition in rats with different levels of nervous system excitabilityPLOS ONE Dear Dr. Shalaginova, 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. In addition to noted major concerns regarding the scientific setup, data presentation and interpretation, both reviewers noted serious concerns regarding the organization, presentation, and clarity of the manuscript. The authors must pay more attention to the quality of the work and presentation in a majorly revised manuscript. Please submit your revised manuscript by Nov 20 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols. We look forward to receiving your revised manuscript. Kind regards, Brenda A Wilson, 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. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match. When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section." 3. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide. Additional Editor Comments: This manuscript barely made it past the initial editorial stage of review. The content, message, data analysis and interpretation, and figures were very poorly prepared, and the manuscript requires major rewriting. [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: No Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: No Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: No Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: No Reviewer #2: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The article needs a major revision and language editing before it could be considered for publication. The manuscript’s content is also rudimentary and structured very poorly. See comments below: Major comments: 1. Introduction: The three main pathways mentioned should also include the HPA axis and the enteric nervous system. 2. Objective: What is the vision behind the experiments performed? What is the conceptual goal and what are the implications of this study that warrants the reader’s attention? 3. Methods: For all software used, please include their respective versions. Even with published data regarding the HT or LT strains, the authors should consider including validation of the strains for clarity of the manuscript. As this is a microbiome study, content of diet should also be mentioned. Preprocessing step is crucial and not mentioned. For reference: a. https://www.nature.com/articles/s41579-018-0029-9 b. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5861821/ c. https://bmcbiol.biomedcentral.com/articles/10.1186/s12915-014-0087-z d. https://academic.oup.com/femsle/article/219/1/87/505487 4. Results: This section requires the most language editing and more elaboration. There is no flow in this section as the authors seemed to describe all the data analyzed. The authors should also include p-values or any other appropriate statistical measures whenever a significant/non-significant change claim is made. Moreover, readers could benefit greatly from a graphical representation of the methodology and analysis to understand what the message of the paper is and how the authors planned to answer the research questions posed. 5. Figures: The quality of all figures provided are very poor. Many of the figures are also attached separately and haphazardly, making the review process very unpleasant. All the barplot figures showcasing the differences in some bacterial phyla/genera could easily be collapsed into 1-2 figures because they carry no meaningful information in deducing changes between the phyla/genera. In truth, the final 2 figures are the only ones carrying any worthwhile information with some statistical analysis done. Moreover, it is very unprofessional for figures to just be screenshots of the software with the data visible. 6. Figure legends: All figure legends are too short and do not help in interpreting the figures. 7. Discussion: Requires more in-depth elaboration and discussion of the results. a. “The high alpha diversity of the gut microbiota” requires a citation. b. The authors mentioned “The genetic characteristics of different rodent strains appear to influence the diversity of the gut microbiota”, yet very little was discussed or mentioned regarding the strain identity of the animals used in the experiments. c. Many of the interpretations derived from the results are coupled with a substantial amount speculations from specific studies that support the authors’ claims, which is not a scientifically-sound practice. 8. Conclusion: From the results and discussion, it is very difficult to reach the same conclusion the authors came to. Minor comments: 1. Many grammatical/language erros, including: a. Abstract: “genetically susceptibility” should be “genetic susceptibility”. b. Introduction: The first sentence seems unnecessary. “HDACS” should be “HDACs”. “At the Pavlov Institute of Physiology of the Russian Academy…” should just be shortened into “A study…” or citing the author’s name. Reviewer #2: The authors compared the changes of gut microbiota after stress in two rat strains that have been inbred to have low (HT) and high excitability (LT) threshold. Previous studies have evaluated their behavior and changes in inflammatory markers. In this study, the authors observed a few differences in the gut microbiota between the two strains in control conditions, such as higher diversity in the HT strain. After stress, both groups showed decrease in relative abundance of Lactobacillus. HT group showed increases in two other species. The authors concluded that the stress resistance in HT strain could be related to its higher gut microbiota diversity, but further research is needed. The authors did not draw any strong conclusions that were not supported by the data and focused more on describing the microbiota changes. The study is well designed, but the results section should contain more details for clarity. Some sentences contain grammar errors that made them hard to understand and should be edited. Comments: 1. The results section can include a bit more details to facilitate the readers. It would be good to indicate the strain, treatment groups, and time points when describing the data in text. For example, it is not clear which time point used for the comparison for results in figure 1. 2. Fig.6 – It is not clear which time point/treatment do the data represent. 3. Fig. 7 – It would be nice to have side-by-side comparison between LT and HT strains for the genus shown, even if one group did not show significant changes. Control group data should also be included to rule out effects not related to stress. 4. Fig. 7 - Are there any changes at the genus level observed only in the LT strain? 5. Fig.3 – The color schemes are a bit hard to read. The authors could consider other color schemes that are more distinguishable between neighboring species. It would also help to make the same species the same color between graphs for comparison. ****** 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: Yes: Andrew Octavian Sasmita 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. https://doi.org/10.1371/journal.pone.0295709.r001
Revision 1
1 Nov 2023 Author Response Thank you for your careful reading of our manuscript and valuable comments. Below are our answers to each of them. Reviewer #1: The article needs a major revision and language editing before it could be considered for publication. The manuscript’s content is also rudimentary and structured very poorly. See comments below: Major comments: 1. Introduction: The three main pathways mentioned should also include the HPA axis and the enteric nervous system. We have added information to the introduction (lines 59-65) 2. Objective: What is the vision behind the experiments performed? What is the conceptual goal and what are the implications of this study that warrants the reader’s attention? The goal of this study is to investigate alterations in the gut microbiota of rat strains with high and low excitability both under normal conditions and following chronic stress exposure. Given that these rat strains exhibit varying susceptibility to post-stress neuroinflammation and behavioral disturbances, understanding the precise taxonomic changes in the microbial community in response to stressors will provide insights into the influence of individual stress susceptibility on the microbiota, with broader implications for personalized medicine and interventions in stress-related disorders. We have added information to the introduction (lines 98-104) 3. Methods: For all software used, please include their respective versions. Even with published data regarding the HT or LT strains, the authors should consider including validation of the strains for clarity of the manuscript. As this is a microbiome study, content of diet should also be mentioned. Preprocessing step is crucial and not mentioned. For reference: a. https://www.nature.com/articles/s41579-018-0029-9 b. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5861821/ c. https://bmcbiol.biomedcentral.com/articles/10.1186/s12915-014-0087-z d. https://academic.oup.com/femsle/article/219/1/87/505487 Thank you for your recommendation and references! In the Materials and Methods section, information regarding excitability thresholds determination has been added. In the Results section, a graph displaying the threshold values for the studied strains is included. We have added information about diet and preprocessing. 4. Results: This section requires the most language editing and more elaboration. There is no flow in this section as the authors seemed to describe all the data analyzed. The authors should also include p-values or any other appropriate statistical measures whenever a significant/non-significant change claim is made. Moreover, readers could benefit greatly from a graphical representation of the methodology and analysis to understand what the message of the paper is and how the authors planned to answer the research questions posed. We have completely rewritten the results section: structured the presentation of the data, provided relevant figures and statistical processing data (p values). 5. Figures: The quality of all figures provided are very poor. Many of the figures are also attached separately and haphazardly, making the review process very unpleasant. All the barplot figures showcasing the differences in some bacterial phyla/genera could easily be collapsed into 1-2 figures because they carry no meaningful information in deducing changes between the phyla/genera. In truth, the final 2 figures are the only ones carrying any worthwhile information with some statistical analysis done. Moreover, it is very unprofessional for figures to just be screenshots of the software with the data visible. All the figures have been revised taking into account the comments. 6. Figure legends: All figure legends are too short and do not help in interpreting the figures. The necessary information, abbreviations, and information about statistical processing have been added to the figure legends. 7. Discussion: Requires more in-depth elaboration and discussion of the results. a. “The high alpha diversity of the gut microbiota” requires a citation. b. The authors mentioned “The genetic characteristics of different rodent strains appear to influence the diversity of the gut microbiota”, yet very little was discussed or mentioned regarding the strain identity of the animals used in the experiments. c. Many of the interpretations derived from the results are coupled with a substantial amount speculations from specific studies that support the authors’ claims, which is not a scientifically-sound practice. We have added material to the discussion in accordance with comments a-c 8. Conclusion: From the results and discussion, it is very difficult to reach the same conclusion the authors came to. We have added information to the conclusion section in accordance with the comments, in addition, the conclusions now are in consistent with new figures and the description of the data. Minor comments: 1. Many grammatical/language erros, including: a. Abstract: “genetically susceptibility” should be “genetic susceptibility”. b. Introduction: The first sentence seems unnecessary. “HDACS” should be “HDACs”. “At the Pavlov Institute of Physiology of the Russian Academy…” should just be shortened into “A study…” or citing the author’s name. Fixed. Reviewer #2: The authors compared the changes of gut microbiota after stress in two rat strains that have been inbred to have low (HT) and high excitability (LT) threshold. Previous studies have evaluated their behavior and changes in inflammatory markers. In this study, the authors observed a few differences in the gut microbiota between the two strains in control conditions, such as higher diversity in the HT strain. After stress, both groups showed decrease in relative abundance of Lactobacillus. HT group showed increases in two other species. The authors concluded that the stress resistance in HT strain could be related to its higher gut microbiota diversity, but further research is needed. The authors did not draw any strong conclusions that were not supported by the data and focused more on describing the microbiota changes. The study is well designed, but the results section should contain more details for clarity. Some sentences contain grammar errors that made them hard to understand and should be edited. Comments: 1. The results section can include a bit more details to facilitate the readers. It would be good to indicate the strain, treatment groups, and time points when describing the data in text. For example, it is not clear which time point used for the comparison for results in figure 1. 2. Fig.6 – It is not clear which time point/treatment do the data represent. 3. Fig. 7 – It would be nice to have side-by-side comparison between LT and HT strains for the genus shown, even if one group did not show significant changes. Control group data should also be included to rule out effects not related to stress. 4. Fig. 7 - Are there any changes at the genus level observed only in the LT strain? 1 – 4: New figures have been made on which time points and comparisons are clear 5. Fig.3 – The color schemes are a bit hard to read. The authors could consider other color schemes that are more distinguishable between neighboring species. It would also help to make the same species the same color between graphs for comparison. A new color scheme was used in Figure 6, which now reflects this data. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0295709.r002
14 Nov 2023 Decision Letter - Brenda A Wilson, Editor PONE-D-23-27857R1Post-stress changes in the gut microbiome composition in rats with different levels of nervous system excitabilityPLOS ONE Dear Dr. Shalaginova, 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. Please submit your revised manuscript by Dec 29 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols. We look forward to receiving your revised manuscript. Kind regards, Brenda A Wilson, Ph.D. Academic Editor PLOS ONE Additional Editor Comments: While most of the concerns of the previous review have been adequately addressed, there still remain some from Reviewer 2 that need to be better addressed and some new concerns regarding the new data. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #2: (No Response) ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #2: Partly ****** 3. Has the statistical analysis been performed appropriately and rigorously? 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 #2: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #2: 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 #2: The figures have been improved. However, with the new data included, I have some concerns. 1. My major concern is the difference between the “control” group time 0 and the “before stress” group. Theoretically these two groups should be similar as both groups are not treated yet. However, there are significant differences in Shannon index (Figure 3A) and levels of g. Prevotella and g. Faecalibacterium (Figure 5). The conclusion that HT strain has more diversity than LT strain is not supported if comparing using the “before stress” groups. Can the authors explain the potential differences? 2. In figure 2, combining data points from HT control group for Chao1 index is questionable as the day 24 is quite different from day 0. ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #2: 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. https://doi.org/10.1371/journal.pone.0295709.r003
Revision 2
18 Nov 2023 Author Response Thank you for your careful reading of our manuscript and valuable comments. Below are our answers to each of them. Reviewer #2: 1. My major concern is the difference between the “control” group time 0 and the “before stress” group. Theoretically these two groups should be similar as both groups are not treated yet. However, there are significant differences in Shannon index (Figure 3A) Fig 3 - The horizontal axis on panel A shows the animals of the control groups at the corresponding time after the end of stress in the experimental groups. The horizontal axis on panel B shows the intact animals of the consolidated control. As we noted (lines 219-220) we did not find any statistically significant effect of stress on alpha diversity indexes in LT and HT rats (paired t-test). 2. and levels of g. Prevotella and g. Faecalibacterium (Figure 5). The conclusion that HT strain has more diversity than LT strain is not supported if comparing using the “before stress” groups. Can the authors explain the potential differences? We noted (lines 245-248), that we found that in one case (Fig 5, g. Prevotella) the control HT rats significantly differed from the experimental group before stress, which makes it incorrect to compare control groups with experimental ones to assess the effect of stress in this case. But in case of g. Faecalibacterium - there were no significant differences (case/control - unpaired t-test). We have explained the potential differences of g. Prevotella (Lines 317-324) 3. In figure 2, combining data points from HT control group for Chao1 index is questionable as the day 24 is quite different from day 0. We specifically checked the absence of significant differences in microbiota diversity indices in rats stool of the experimental group before and at different periods after stress. As the paired t-test shows, there are no significant differences. However, the variability of the data is indeed higher in the HT group both in the case of the Ciao1 index and in the case of the Shannon index. This is also in accordance with the assumption of greater dynamism in the fluctuations of microbiota composition in the stool of high-excitable rats compared with low-excitable ones. Attachments Attachment Submitted filename: Response to Reviewers_V2.docx https://doi.org/10.1371/journal.pone.0295709.r004
27 Nov 2023 Decision Letter - Brenda A Wilson, Editor Post-stress changes in the gut microbiome composition in rats with different levels of nervous system excitability PONE-D-23-27857R2 Dear Dr. Shalaginova, 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, Brenda A Wilson, 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 #2: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? 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 #2: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #2: 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 #2: (No Response) ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #2: No ******** https://doi.org/10.1371/journal.pone.0295709.r005
Formally Accepted
1 Dec 2023 Acceptance Letter - Brenda A Wilson, Editor PONE-D-23-27857R2 Post-stress changes in the gut microbiome composition in rats with different levels of nervous system excitability Dear Dr. Shalaginova: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. 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. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Brenda A Wilson Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0295709.r006

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