Substantial acetylcholine reduction in multiple brain regions of Mecp2-deficient female rats and associated behavioral abnormalities

Hiroyasu Murasawa; Hiroyuki Kobayashi; Jun Imai; Takahiko Nagase; Hitomi Soumiya; Hidefumi Fukumitsu

doi:10.1371/journal.pone.0258830

Peer Review History

Original SubmissionFebruary 18, 2021
20 Apr 2021 Decision Letter - Atsushi Asakura, Editor PONE-D-21-05550 Substantial acetylcholine reduction in multiple brain regions of Mecp2-deficient female rats and associated behavioral abnormalities PLOS ONE Dear Dr. Fukumitsu, 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 Jun 04 2021 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. 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If you did not receive any funding for this study, please state: “The authors received no specific funding for this work.” Please include your amended statements within your cover letter; we will change the online submission form on your behalf. 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: Yes 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: Yes Reviewer #2: No ****** 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 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: Review March 19, 2021 PONE-D-21-05550 Substantial acetylcholine reduction in multiple brain regions of Mecp2-deficient female rats and associated behavioral abnormalities The authors sought to assess the effects of Mecp2 gene deletion on female rats (Mecp2+/−) by examining social behavior, motor function, spatial cognition and other physiological parameters. The study was well-designed and well-constructed. This study represents major advances in the study of animal models of Rett syndrome. The manuscript will likely merit publication with revisions. Financial Disclosure Please include information about the funding for this study. Include a Financial Disclosure. Enter a financial disclosure statement that describes the sources of funding for the work included in this submission. Materials and methods Morris water maze test Line 127 Since the second figure to be mentioned in the text occurs in this line, the figure should be numbered figure 2. Statistical Analysis Are the data normally distributed as demonstrated by the Kolmogorov-Smirnov or Wilks-Shapiro procedures? If the data are not normally distributed, then a nonparametric procedure such as Kruskal-Wallis is appropriate. Results Impaired social behaviors in Mecp2-deficient female rats Page 9 Line 192 Change “disorders” to “disorder” Table 1 Pages 13 to 15 Express the values of each row in a single line by using a smaller font. Legend to figure 7 Line 316 The scale bar appears to be the same length in all panels. What is the magnification indicated by the two scale bars? Relationships between cholinergic and behavioral abnormalities Line 387-408 Include a discussion of the dopaminergic deficits observed as follows: Wong DF, Blue ME, Brašić JR,, Nandi A, Valentine H, Stansfield KH, Rousset O, Bibat G, Yablonski ME, Johnston MV, Gjedde A, Naidu SB. Are dopamine receptor and transporter changes in Rett syndrome reflected in Mecp2-deficient mice? Exp Neurol. 2018; 307: 74-81. https://doi.org/10.1016/ j.expneurol.2018.05.019 PubMed PMID: 29782864. Reference Line 517 Remove the space between “30.” and “Panayotis” Lines 549-550 Reference 41. Include the original title of the article in German as follows: Rett A. Uber ein bisher nicht bekanntes Krankheitsbild einer angeborenen Stoffwechselst örung [On an until now unknown disease of a congenital metabolic disorder]. Krankenschwester. 1966 Sep;19(9):121-2. German. PMID: 5179620. Please add appendix of abbreviations. Define all acronyms before their first use. Supplemental Methods The section in the supplemental methods belong in the body of the text. There are no restrictions on word counts, number of figures, or amount of supporting information. Reviewer #2: In the manuscript “Substantial acetylcholine reduction in multiple brain regions of Mecp2-deficient female rats and associated behavioral abnormalities” The authors assessed the effects of Mecp2 gene deletion on female rats (Mecp2 +/− ) and found severe impairments in social behavior, motor function, and spatial cognition and lower plasma IGF-1 (but not BDNF) and markedly reduced acetylcholine (30%–50%) in multiple brain regions compared to female Mecp2 +/+ rats. This is a purely descriptive study which could be informative but some of the results appears to be disconnected. The correlation of behavioral changes with acetylcholine levels is appears far reaching based on the way the manuscript is organized. Figure 7 images are of poor quality and images of amygdala and hypothalamus should be included. Page 2, Line 25. “…..spatial cognition as well lower plasma” - the word “as” is missing. Page 3, Line 45. “One of most striking findings” - the word “the” is missing Page 9, Line 188. “….and the both” – remove “the” Page 9, Line 193. The author report a decrease in repetitive grooming behavior. Veeraragavan et al. 2016 reported no significant changes in Mecp2-/+ mice, what do you attribute to the divergent results? Page 22, Line 403: “….GABAA (GABAnergic….” – should be “GABAergic” Figure 4 A and B. For consistency capitalize the first word on the y axis. Figure 6 B and C. For consistency capitalize the words on the y axis “goal latency” and “swimming distance”. ****** 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:** James Brasic 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.0258830.r001
Revision 1
16 Jun 2021 Author Response Response to Reviewers: We wish to express our appreciation to the Reviewers for their insightful comments, which have helped us improve our paper substantially. Reply to Reviewer #1 Comments to authors Point 1: Materials and methods Morris water maze test Line 127: Since the second figure to be mentioned in the text occurs in this line, the figure should be numbered figure 2. Answer 1: Thank you for the correction. We have accordingly altered the order of the figure number. Point 2: Materials and methods Statistical Analysis: Are the data normally distributed as demonstrated by the Kolmogorov-Smirnov or Wilks-Shapiro procedures? If the data are not normally distributed, then a nonparametric procedure such as Kruskal-Wallis is appropriate. Answer 2: Thank you for the comment. Because sample size for each experiment was small, it was not appropriate to evaluate the equal distribution of the sample. We therefore changed all our statistical methods to nonparametric procedures, accordingly. Point 3: Results Impaired social behaviors in Mecp2-deficient female rats: Page 9 Line 192 Change “disorders” to “disorder”. Answer 3: Thank you for the correction. We have accordingly changed “disorders” to “disorder.” Point 4: Table 1 Pages 13 to 15 Express the values of each row in a single line by using a smaller font. Answer 4: Thank you for the correction. In accordance, we have corrected the table to express the values of each row in a single line using a smaller font. Point 5: Legend to figure 7 Line 316 The scale bar appears to be the same length in all panels. What is the magnification indicated by the two scale bars? Answer 5: Thank you for the correction. In accordance, we have corrected the scale bars. Point 6: Discussion Relationships between cholinergic and behavioral abnormalities Line 387-408 Include a discussion of the dopaminergic deficits observed as follows: Wong DF, Blue ME, Brašić JR,, Nandi A, Valentine H, Stansfield KH, Rousset O, Bibat G, Yablonski ME, Johnston MV, Gjedde A, Naidu SB. Are dopamine receptor and transporter changes in Rett syndrome reflected in Mecp2-deficient mice? Exp Neurol. 2018; 307: 74-81. https://doi.org/10.1016/ j.expneurol.2018.05.019 PubMed PMID: 29782864. Answer 6: Thank you for the comment and information. In accordance, we have thoroughly corrected the part of the Discussion section concerning the possible involvement of dopaminergic deficits in the behavioral abnormalities of Mecp2+/− rats and cited the article. Point 7: Reference Line 517 Remove the space between “30.” and “Panayotis”　 Answer 7: Thank you for the correction. We have accordingly removed the space between “30.” and “Panayotis.” Point 8: Lines 549-550 Reference 41. Include the original title of the article in German as follows: Rett A. Uber ein bisher nicht bekanntes Krankheitsbild einer angeborenen Stoffwechselst örung [On an until now unknown disease of a congenital metabolic disorder]. Krankenschwester. 1966 Sep;19(9):121-2. German. PMID: 5179620. Answer 8: Thank you for the correction. In accordance, we have corrected the reference to include the original title of the article in German. Point 9: Please add appendix of abbreviations. Define all acronyms before their first use. Answer 9: Thank you for the correction. In accordance, we have added an appendix of abbreviations and defined all acronyms before their first use. Point 10: Supplemental Methods The section in the supplemental methods belong in the body of the text. There are no restrictions on word counts, number of figures, or amount of supporting information. Answer 10: Thank you for the comment. In accordance, we have moved the supplemental methods into the body of the text. Reply to reviewer #2 Comments to authors Point 1: The correlation of behavioral changes with acetylcholine levels is appears far reaching based on the way the manuscript is organized. Answer 1: Thank you for the comment. Considering both reviewers’ comments, we have thoroughly corrected the part of the Discussion section concerning the possible involvement of monoaminergic dysfunctions in the behavioral abnormalities of the Mecp2+/− female rats. Point 2: Figure 7 images are of poor quality and images of amygdala and hypothalamus should be included. Answer 2: Thank you for the comment. In accordance, we have replaced the microscopic photographs and also inserted images of the amygdala and hypothalamus in Figure 7 and Figure 8. Point 3: Page 2, Line 25. “…..spatial cognition as well lower plasma” - the word “as” is missing. Answer 3: Thank you for the correction. In accordance, we have added the word “as.” Point 4: Page 9, Line 193. The author report a decrease in repetitive grooming behavior. Veeraragavan et al. 2016 reported no significant changes in Mecp2-/+ mice, what do you attribute to the divergent results? Answer 4: Thank you for the comments. Concerning self-grooming, we considered that the divergent results would come from the developmental impairment in the cholinergic system. Excessive self-grooming behavior, however, is a typical symptom of autism model mice and has also been observed in Mecp2 null male mice mutants. Therefore, further investigations are necessary to understand the meaning of this behavioral abnormality in the Mecp2-deficient female rat. We have added these limitations to the Discussion section. Point 5: Page 22, Line 403: “….GABAA (GABAnergic….” – should be “GABAergic” Answer 5: Thank you for the correction. In accordance, we have corrected the word to “GABAergic.” Point 6: Figure 4 A and B. For consistency capitalize the first word on the y axis. Answer 6: Thank you for the correction. In accordance, we have capitalized the first word on the y-axis. Point 7: Figure 6 B and C. For consistency capitalize the words on the y axis “goal latency” and “swimming distance”. Answer 7: Thank you for the correction. In accordance, we have capitalized the first word on the y-axis. Attachments Attachment Submitted filename: Response to Reviewer_210602.docx https://doi.org/10.1371/journal.pone.0258830.r002
13 Aug 2021 Decision Letter - Atsushi Asakura, Editor PONE-D-21-05550R1 Substantial acetylcholine reduction in multiple brain regions of Mecp2-deficient female rats and associated behavioral abnormalities PLOS ONE Dear Dr. Fukumitsu, 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 Sep 27 2021 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: http://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, Atsushi Asakura, Ph.D Academic Editor PLOS ONE Journal Requirements: Please 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 rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [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 #1: (No Response) 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 #1: Yes Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: N/A ****** 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 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 #1: No 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 #1: The authors compared and contrasted the neurochemical and behavioral aspects of wild type and Mecp2-deficient female rats utilizing a reasonable experimental design and analysis. They concluded that acetylcholine concentration was associated with behavior. The study was well conducted. The discussion was reasonable. Some readers may be unfamiliar with some technical details so explicitly documenting them will likely facilitate understanding. Some sections may be better worded and organized to help readers comprehend the key points. The manuscript will likely merit publication with revisions. Page 6 Line 131 Change “MWM test” to “Morris water maze (MWM) test” Page 7 Line 157 Change “Friedman’s test” to “Friedman test” Provide a reference for the Friedman test. What statistical program was used? Line 161 Provide a reference for unpaired two-tailed Mann–Whitney U-test. What statistical program was used? Page 11 Lines 245-246 Provide a reference for unpaired two-tailed Mann–Whitney U-test. Line 247 Provide a reference for the Kruskal–Wallis with Dunn’s post hoc multiple comparisons test. Line 248 Provide a reference for the Friedman test. Page 12 Line 269 Provide a reference for the Friedman test. Line 270 Provide a reference for the Kruskal–Wallis with Dunn’s post hoc multiple comparisons test. Line 277 Provide a reference for the Kruskal–Wallis with Dunn’s post hoc multiple comparisons test. Page 13 Lines 292-293 Provide a reference for the Kruskal–Wallis with Dunn’s post hoc multiple comparisons test. Lines 298-299 Provide a reference for unpaired two-tailed Mann–Whitney U-test. Line 308 Change “Fig-2D” to “Fig 2D” Page 15 Line 327 Change “Mecp2-deficient.” to “Mecp2-deficient” Page 16 None of the items utilize . Line 331 Change “p < 0.05 and p < 0.01 vs. female Mecp2+/+” to “p < 0.05 female Mecp2+/- vs. Mecp2+/+” Lines 331-332 Provide a reference for unpaired two-tailed Mann–Whitney U-test. Page 17 Table 2 None of the items utilize . Line 335 Change p < 0.05 vs. female Mecp2+/+ rats” to Non significance female Mecp2+/- vs. Mecp2+/+ rats” Lines 335-336 Provide a reference for unpaired two-tailed Mann–Whitney U-test. Line 339 Change “vs. female Mecp2+/+ rats” to “female Mecp2+/- vs. Mecp2+/+ rats” Provide a reference for unpaired two-tailed Mann–Whitney U-test. Page 19 Line 361 Change p <0.01 vs. female Mecp2+/+ rats” to p <0.01 female Mecp2+/- vs. Mecp2+/+ rats” Line 362 Provide a reference for unpaired two-tailed Mann–Whitney U-test. Line 366 Change “p < 0.01 vs. female Mecp2+/+ rats” to “p < 0.01 female Mecp2+/- vs. Mecp2+/+ rats” Page 20 Line 385 Change “demonstrate” to “demonstrated” Abbreviations Place the abbreviations in alphabetical order. Reviewer #2: The authors have addressed the previous comments. Below are some additional minor comments. Page 4, Line 78. Remove “on so on” Page 7, Line 150. This is the Figure legend for figure 2, not sure why it is in the methods section. Page 12, Line 260-261. “Both the frequency (Fig 4A) and duration 261 (Fig. 4B) of self-grooming behavior were significantly or tended to be reduced in female Mecp2+/−…” However, the graph in figure 4B does not show statistical significance for the duration. The current phrasing is not clear, authors should clarify that frequency was reduced and duration tended to be reduced but not significant. Page 12, Line 283. “Spontaneous locomotor activity was significantly or tended to be lower among female Mecp2+/− rats compared to female Mecp2+/+ rats (Fig 5) during both light and dark phases.” Authors should clarify the specific timepoints of significance. Page 12, Line 285. For consistency lower case w “29 w” Page 13, Line 301. It is unclear why the result section “Impaired spatial learning and memory in Mecp2-deficient female rats” is Figure 2, following Figure 6. Page 14, Line 314. “Surprising, however, biogenic amine and…” – Change to “Surprisingly” Page 14, Line 323. “These results suggest that impaired cholinergic signaling would severely affect the behavioral abnormalities observed...” The word “would” should be changed to “could”. Page 18, Line 344. ” Astrocytes are known to support neuronal function through production and secretion of various extrinsic factors and by the uptake of neurotransmitters, and the number and morphological features are associated with neurodevelopmental abnormalities as well as neurodegeneration and neuroinflammation.” A reference is needed for this sentence. Page 18, Line 355. “Both the neurotrophic factors play important…” – Remove “the” ******* 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:** James Robert Brasic 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. Attachments Attachment Submitted filename: draft review 210728 2204 2147 PONE-D-21-05550R1.pdf Attachment Submitted filename: draft review 210728 2204 2147 PONE-D-21-05550R1.docx https://doi.org/10.1371/journal.pone.0258830.r003
Revision 2
30 Aug 2021 Author Response Response to Reviewers: We wish to express our appreciation to the Reviewers for their insightful comments, which have helped us improve our paper substantially. Reply to Reviewer #1 Point 1: Page 6 Line 131 Change “MWM test” to “Morris water maze (MWM) test”. Answer 1: Thank you for the correction. We have accordingly spelled out “MWM test”. Point 2: Page 7 Line 157 Change “Friedman’s test” to “Friedman test” Provide a reference for the Friedman test. What statistical program was used? Line 161 Provide a reference for unpaired two-tailed Mann–Whitney U-test. What statistical program was used? Answer 2: Thank you for the comment. We have accordingly changed “Friedman’s test” to “Friedman test” Concerning about statistical program, we had used GaphPad Prism (version 9.1.2 for Windows) for all statistical analyses (Friedman test, unpaired two-tailed Mann-Whitney U-test, and Kruskal-Wallis with Dunn’s post hoc multiple comparisons test), according to the software guide. So, we have described it in the revised manuscript. Point 3: Page 13 Line 308 Change “Fig-2D” to “Fig 2D” Answer 3: Thank you for the correction. We have accordingly changed “Fig-2D” to “Fig 2D”. Point 4: Page 15 Line 327 Change “Mecp2-deficient.” to “Mecp2-deficient Answer 4: Thank you for the correction. We have accordingly changed “Mecp2-deficient.” to “Mecp2-deficient”. Point 5: Page 16 Table 1 None of the items utilize *. Line 331 Change “p < 0.05 and *p < 0.01 vs. female Mecp2+/+” to “p < 0.05 female Mecp2+/- vs. Mecp2+/+” Answer 5: Thank you for the correction. In accordance, we have changed “p < 0.05 and p < 0.01 vs. female Mecp2+/+” to “p < 0.05 female Mecp2+/- vs. Mecp2+/+”. Point 6: Page 17 Table 2 None of the items utilize . Line 335 Change p < 0.05 vs. female Mecp2+/+ rats” to Non significance female Mecp2+/- vs. Mecp2+/+ rats”. Line 339 Change “vs. female Mecp2+/+ rats” to “female Mecp2+/- vs. Mecp2+/+ rats” Answer 6: Thank you for the correction. In accordance, we have changed p < 0.05 vs. female Mecp2+/+ rats” to Non significance female Mecp2+/- vs. Mecp2+/+ rats”. We also have changed “vs. female Mecp2+/+ rats” to “female Mecp2+/- vs. Mecp2+/+ rats”. Point 7: Page 19 Line 361 Change p <0.01 vs. female Mecp2+/+ rats” to p <0.01 female Mecp2+/- vs. Mecp2+/+ rats” Line 366 Change “p < 0.01 vs. female Mecp2+/+ rats” to “p < 0.01 female Mecp2+/- vs. Mecp2+/+ rats” Answer 7: Thank you for the correction. In accordance, we have changed p <0.01 vs. female Mecp2+/+ rats” to p <0.01 female Mecp2+/- vs. Mecp2+/+ rats”. We also have changed “p < 0.01 vs. female Mecp2+/+ rats” to “p < 0.01 female Mecp2+/- vs. Mecp2+/+ rats”. Point 8: Page 20 Line 385 Change “demonstrate” to “demonstrated”. Answer 8: Thank you for the correction. We have accordingly changed “demonstrate” to “demonstrated”. Point 9: Abbreviations Place the abbreviations in alphabetical order. Answer 9: Thank you for the correction. In accordance, we have replaced the abbreviations in alphabetical order. Reply to reviewer #2 Comments to authors Point 1: Page 4, Line 78. Remove “on so on”. Answer 1: Thank you for the correction. In accordance, we have removed “on so on”. Point 2: Page 7, Line 150. This is the Figure legend for figure 2, not sure why it is in the methods section. Answer 2: Thank you for the comment. Since we had mentioned the figure in the materials and methods section “Morris water maze (MWM) test”, the reviewer#1 had advised the figure to be numbered figure 2 in the previous comments, according to the Plos One’s instructions. However, since the mismatch between the order of figures and results could confuse readers, we have replaced the Fig 2 after the result section “Impaired spatial learning and memory in Mecp2-deficient female rats” and altered the order of the figure number. Point 3: Page 12, Line 260-261. “Both the frequency (Fig 4A) and duration 261 (Fig. 4B) of self-grooming behavior were significantly or tended to be reduced in female Mecp2+/−…” …… The current phrasing is not clear, authors should clarify that frequency was reduced and duration tended to be reduced but not significant. Answer 3: Thank you for the correction. In accordance, we have revised the sentence to clarify that frequency was reduced and duration tended to be reduced but not significant. Point 4: Page 12, Line 283. “Spontaneous locomotor activity was significantly or tended to be lower among female Mecp2+/− rats compared to female Mecp2+/+ rats (Fig 5) during both light and dark phases.” Authors should clarify the specific timepoints of significance. Answer 4: Thank you for the comments. We have shown the locomotor activities at each 30-min from 16 w or 23 w old rats over 2 consecutive 24-periods as supplemental S1 Fig in the revised manuscript. After statistical analyses (Kruskal–Wallis with Dunn’s post hoc multiple comparisons test), we have found no significant difference between genotypes at any time point. These results indicated that the Mecp2 deficiency is not likely to affect circadian rhythms but locomotor activity itself. So, we have additionally described it in the revised manuscript. Point 5: Page 12, Line 285. For consistency lower case w “29 w” Answer 5: Thank you for the correction. We have accordingly changed “29W” to “29 w”. Point 6: Page 13, Line 301. It is unclear why the result section “Impaired spatial learning and memory in Mecp2-deficient female rats” is Figure 2, following Figure 6. Answer 6: Thank you for the correction. In accordance, we have revised this matter as described in Point 2. Point 7: Page 14 Line 314. “Surprising, however, biogenic amine and…” – Change to “Surprisingly” Line 323. “These results suggest that impaired cholinergic signaling would severely affect the behavioral abnormalities observed...” The word “would” should be changed to “could”. Answer 7: Thank you for the correction. In accordance, we have changed “Surprising” to “Surprisingly”, and “would” to “could”. Point 8: Page 18, Line 344.” Astrocytes are known to support neuronal function....” A reference is needed for this sentence. Answer 7: Thank you for the correction. In accordance, we have added references for the sentence. Point 9: Page 18, Line 355. “Both the neurotrophic factors play important…” – Remove “the”. Answer 9: Thank you for the correction. In accordance, we have removed “the”. Attachments Attachment Submitted filename: Response to Reviewer_210830.docx* https://doi.org/10.1371/journal.pone.0258830.r004
7 Oct 2021 Decision Letter - Atsushi Asakura, Editor Substantial acetylcholine reduction in multiple brain regions of Mecp2-deficient female rats and associated behavioral abnormalities PONE-D-21-05550R2 Dear Dr. Fukumitsu, 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, Atsushi Asakura, Ph.D Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0258830.r005
Formally Accepted
12 Oct 2021 Acceptance Letter - Atsushi Asakura, Editor PONE-D-21-05550R2 Substantial acetylcholine reduction in multiple brain regions of Mecp2-deficient female rats and associated behavioral abnormalities Dear Dr. Fukumitsu: 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 plosone@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. Atsushi Asakura Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0258830.r006

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