Genetically diverse mice exhibit divergent domain-specific, sex-dependent behavioral outcomes following exposure to early life stress

Jennifer Nguyen; Kevin Shimizu; Varvara Zlotnik; Matthew N. Nguyen; Sandra Del Toro; Michael T. Nguyen; Janet Ronquillo; Lindsay R. Halladay

doi:10.1371/journal.pone.0331457

Peer Review History

Original SubmissionMay 20, 2025
20 May 2025 Author Response Transfer Alert This paper was transferred from another journal. As a result, its full editorial history (including decision letters, peer reviews and author responses) may not be present. https://doi.org/10.1371/journal.pone.0331457.r001
24 Jun 2025 Decision Letter - Alexandra Kavushansky, Editor PONE-D-25-27338Genetic diversity shapes behavioral outcomes and reveals sex differences in mice exposed to early life stressPLOS ONE Dear Dr. Halladay, 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 Aug 08 2025 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. 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Currently, your Funding Statement reads as follows: L.R.H., The Jackson Laboratory Diversity Outbred Pilot Grant Program https://www.jax.org/ L.R.H., National Institute of Mental Health (NIMH) award R15MH127514 https://www.nimh.nih.gov/ Funders did not play a role in the study design, data collection, analysis, decision to publish, or preparation of the manuscript. Please include your amended statements within your cover letter; we will change the online submission form on your behalf. 3. Please update your submission to use the PLOS LaTeX template. The template and more information on our requirements for LaTeX submissions can be found at http://journals.plos.org/plosone/s/latex. 4. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: 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: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes ******** 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 manuscript entitled ‘Genetic diversity shapes behavioral outcomes and reveals sex differences in mice exposed to early life stress’ by Nguyen et al. described the effect of maternal separation with early weaning (MSEW) on behaviors in the Diversity Outbred (DO) mouse strain. A series of behavioral battery tests showed the sex difference in anxiety and learning/memory. The experimental designs are well organized, and the results are clear. However, the manuscript contains several points that require improvement. Major comments 1. The title is misleading. Four behavioral assays were performed in the current study: two-period social interaction, 3-D radial arm maze, fear conditioning, and operant conditioning. However, the DO strain was compared with C57BL/6J only in the two-period social interaction test. Although the authors mentioned their previous reports using B6 mice in references 4 and 5 regarding social interaction, those studies employed different experimental conditions and cannot be directly compared to the results in the present study. Therefore, it is hard to claim that ‘Genetic diversity shapes behavioral outcomes and reveals sex differences…’. ‘Diversity Outbred mouse strain showed sex differences in behavioral outcomes caused by the exposure to early life stress’ seems more appropriate. The Discussion and conclusion should also be revised carefully. 2. The manuscript should be organized carefully. For example, Statistical analysis (Page 11, lines 251-254) must be included in the Materials & Methods section, not the Results section. Many figure legends are redundant and contain sentences to be included in the Materials & Methods or Results sections. Overall, it is quite hard to conclude that genetic diversity affects behavioral outcomes and reveals sex differences in mice exposed to the MSEW. The manuscript should be carefully revised. Minor comments 1. Are the error bars indicated as SD or SEM? 2. Symbols of significance between groups (, , or ) are confusing in Fig 2, 3, and 5. Different symbols should be used like Fig. 4 ****** 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: Hitoshi Inada ******** [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.0331457.r002
Revision 1
8 Aug 2025 Author Response Response to Editor Comments: We have updated our formatting to adhere to PLOS ONE’s style requirements. We have removed funding information from the Acknowledgment section. The Funding Statement (online submission) is accurate as is. We have included a caption for our supporting information. Response to Reviewers' comments: Reviewer #1: The manuscript entitled ‘Genetic diversity shapes behavioral outcomes and reveals sex differences in mice exposed to early life stress’ by Nguyen et al. described the effect of maternal separation with early weaning (MSEW) on behaviors in the Diversity Outbred (DO) mouse strain. A series of behavioral battery tests showed the sex difference in anxiety and learning/memory. The experimental designs are well organized, and the results are clear. However, the manuscript contains several points that require improvement. Thank you for the positive feedback regarding experimental organization and clear results, and we appreciate your feedback related to improving the manuscript. Major comments 1. The title is misleading. Four behavioral assays were performed in the current study: two-period social interaction, 3-D radial arm maze, fear conditioning, and operant conditioning. However, the DO strain was compared with C57BL/6J only in the two-period social interaction test. Although the authors mentioned their previous reports using B6 mice in references 4 and 5 regarding social interaction, those studies employed different experimental conditions and cannot be directly compared to the results in the present study. Therefore, it is hard to claim that ‘Genetic diversity shapes behavioral outcomes and reveals sex differences…’. ‘Diversity Outbred mouse strain showed sex differences in behavioral outcomes caused by the exposure to early life stress’ seems more appropriate. The Discussion and conclusion should also be revised carefully. Thank you for this feedback. As requested, we have modified our title to avoid wording that alludes to a comparison between DO and isogenic mice. The updated title mentions only genetically diverse mice and summarizes the main findings relevant to the genetically diverse mice. It now reads: Genetically diverse mice exhibit divergent domain-specific, sex-dependent behavioral outcomes following exposure to early life stress We appreciate the point related to our citing previous work (Refs 4 and 5), and we acknowledge that the social behavior testing apparatus we used here, a t-maze, is not physically identical to the apparatus we used in our past social behavior studies, which was more of an open field design. Nonetheless, the 2pSI is similarly intended to test both social motivation and social novelty preference, and in each case (Refs 4 and 5, as well as data presented here) social motivation (novel conspecific vs general novelty) was affected by MSEW. But because the tasks were indeed not identical, regarding the Introduction text that refers to 4 and 5, we removed the word “similar,” but retain the idea that each of these studies evidence an alteration to social motivation following exposure to MSEW. 2. The manuscript should be organized carefully. For example, Statistical analysis (Page 11, lines 251-254) must be included in the Materials & Methods section, not the Results section. Many figure legends are redundant and contain sentences to be included in the Materials & Methods or Results sections. We appreciate this feedback. We have moved the Statistical Analysis section to Materials and Methods. Regarding the figure legends, after careful review, we removed text that may be overly redundant, but kept text that would enable a reader to better understand the figure without having to unnecessarily refer back to the methods / results. We feel that the legends are now more succinct while still containing information pertinent to the main findings of each figure. Overall, it is quite hard to conclude that genetic diversity affects behavioral outcomes and reveals sex differences in mice exposed to the MSEW. The manuscript should be carefully revised. We have carefully considered the statements in our manuscript and have made several changes throughout. For example, in the Discussion subsection “Cued fear learning and expression”, we made the following revision to remove (strikethrough) and replace text (underlined): These results emphasize that genetic background may interact with environmental stressors and learning conditions to shape fear responses, emphasizing the value of using genetically diverse animals alongside traditional inbred strains but future studies would be warranted to determine this since for this set of experiments, we did not directly compare DO to inbred mice. Similarly in the subsection “Operant reward-seeking”, we added the following underlined statement: This sex-specific discrepancy may again be attributed to the genetic heterogeneity of DO mice compared to other strains used in prior work, but additional studies are necessary to more thoroughly examine this possibility. Minor comments 1. Are the error bars indicated as SD or SEM? Error bars depict SEM. We now specify that in the figure captions. 2. Symbols of significance between groups (, , or **) are confusing in Fig 2, 3, and 5. Different symbols should be used like Fig. 4 Thank you for this feedback. We have updated Figures 2 and 3 with additional symbols that denote different comparisons, as well as the captions for figures 2, 3, and 5 to clearly define the comparisons being highlighted in each graph. https://doi.org/10.1371/journal.pone.0331457.r003
17 Aug 2025 Decision Letter - Alexandra Kavushansky, Editor Genetically diverse mice exhibit divergent domain-specific, sex-dependent behavioral outcomes following exposure to early life stress PONE-D-25-27338R1 Dear Dr. Halladay, 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 will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. For questions related to billing, please contact billing support . 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, Alexandra Kavushansky, PhD Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: (No Response) ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy . Reviewer #1: Yes: Hitoshi Inada ******** https://doi.org/10.1371/journal.pone.0331457.r004
Formally Accepted
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