Cross-frequency coupling between slow harmonics via the real brainstem oscillators: An in vivo animal study

Yoshinori Kawai

doi:10.1371/journal.pone.0289657

Peer Review History

Original SubmissionFebruary 24, 2023
25 May 2023 Decision Letter - Gennady S. Cymbalyuk, Editor PONE-D-23-04711Cross-Frequency Coupling between Slow Harmonics via the Real Brainstem Oscillators: An in vivo Animal StudyPLOS ONE Dear Dr. Kawai, 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, revise introduction to emphasize the the lack of previous research in the area. The sample size (number of rats) needs to be stated. Determining the statistical validity of the findings is difficult without this sample size stated in the materials and methods. The reference section needs revision to ensure consistency in reference formatting. Edit the figure legends for clarity and coherence with the main text (ex. lines 213-220). Minor points 1. Abstract. Too long Abstract. First part, before "In this article …", includes sentences that belong to Introduction. 2. Line 127 and other epoch durations. Please, provide the number of points of the epochs with the durations of 10, 20, and 100 s that were analyzed. 3. Line 167. What is the meaning of vertical bars in Panels Ba-Be of Fig. 2? 4. Line 182. 100 sec epoch is not longer than the previously mentioned 1000 sec epoch. Please correct wording. Please also introduce PZT, NTS, and LP in Fig. 3 in the first sentence, such as “Figure 3 shows … and so on”. 5. Line 188. I assume Figs. A1 and B1 stand for Fig. 3A1 and 3B1? 6. Line 190. There are no panels A2, A3, A4, B2, B3, and B4 in Fig. 2. Are those for Fig. 3? 7. Line 192. Should "patterns in quality" be substituted with "qualitative patterns" 8. Line 194. What do arrows mean in panels A3 and A4 in Fig. 3? 9. Line 213. Standard deviation for Fig. 4Bb seems incorrect. It is of the order of 1. 10. Line 354. Here and further throughout the text: Fig. S1. 11. Lines 393-395. Please edit for clarity. 12. Line 400. The author can use either FFT or spell it out, but not both. 13. Lines 558-560. I am not clear about the meaning of the last sentence: It looks like coherence disappear, but panels do not show that signals disappear. 14. Line 613. Should you remove "two"? 15. Line 618. (d) and (q) must be removed. Please submit your revised manuscript by Jul 09 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, Gennady S. Cymbalyuk, 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. 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For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability. Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized. Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access. We will update your Data Availability statement to reflect the information you provide in your cover letter. 4. 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. 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 Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: I Don't Know 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: 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 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 introduction is too brief and relies heavily on the researcher’s own work. Additional references should be included to support the lack of previous research in the area. These could be reviews or articles that mention this gap in the literature. The sample size (number of rats) needs to be stated. Determining the statistical validity of the findings is difficult without this sample size stated in the materials and methods. The reference section needs revision due to inconsistent formatting. First, there are variations in the listing of the journal’s full name versus abbreviations (ex. 2 vs 4). Second, there are variations in the capitalization of the article name (ex. 4 vs 5). Third, the doi is not always listed even if it is available (ex. 25 vs. 26). The article should be edited to ensure consistency in reference formatting. Several pieces of the paper could be included in the figure legends and vice versa (ex. lines 213-220). Reviewer #2: In this paper, the authors investigated brainstem rhythms of the rat and their interactions. One of the rhythms seems to govern respiratory function (delta range) and another rhythm drives heart beats (theta range). It was demonstrated that these rhythms were synchronized in many cases of the brainstem activity by creation of temporary neural circuits. The author found a synchronization between central rhythmic and peripheral cardiorespiratory activities. In general, the paper is well-written, the results seem solid, and the experimental data supports the conclusions of the paper. However, some sentences in the paper are not clearly written. Basically, writing style must be improved. However, I have only minor points that need to be corrected. Minor points (lines are numbered as a sequence from the beginning of the paper) 1. Abstract. Too long Abstract. First part, before "In this article …", includes sentences that belong to Introduction. 2. Line 127 and other epoch durations. I would suggest to provide the number of points of the epochs with the durations of 10, 20, and 100 s that were analyzed. Those are important for the evaluation of the errors in Fast Fourier Transforms. 3. Line 167. What is the meaning of vertical bars in Panels Ba-Be of Fig. 2? 4. Line 182. 100 sec epoch is not longer than the previously mentioned 1000 sec epoch. Please correct wording. Please also introduce PZT, NTS, and LP in Fig. 3 in the first sentence, such as “Figure 3 shows … and so on”. I think, the reader must observe what is shown in the figure, which is quite complicated. 5. Line 188. I assume Figs. A1 and B1 stand for Fig. 3A1 and 3B1? 6. Line 190. There are no panels A2, A3, A4, B2, B3, and B4 in Fig. 2. I assume those are for Fig. 3? 7. Line 192. I think "patterns in quality" must be "qualitative patterns" 8. Line 194. What arrows mean in panels A3 and A4 in Fig. 3? 9. Line 213. Standard deviation for Fig. 4Bb seems incorrect. It is of the order of 1. 10. Line 354. Here and further throughout the text: Fig. S1. 11. Lines 393-395. Bad sentence. Please write more clear sentence or two. 12. Line 400. The author can use either FFT or spell it out, but not both. FFT has been defined above. 13. Lines 558-560. I am not clear about the meaning of the last sentence: It looks like coherence disappear, but panels do not show that signals disappear. 14. Line 613. I would remove "two". 15. Line 618. (d) and (q) must be removed. ****** 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: No 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.0289657.r001
Revision 1
29 May 2023 Author Response 29 May, 2023 Editorial Office PLOS ONE Journal Dear Editorial Office: I wish to submit a revised version of an Original Research Article (PONE-D-23-04711) for publication in PLOS ONE, titled “Cross-Frequency Coupling between Slow Harmonics via the Real Brainstem Oscillators: An in vivo Animal Study.” I appreciate the Reviewers’ careful and encouraging suggestions for revision of this article. In that case, the following revisions or rebuttals (in red in below) were made as our response to comments from the Reviewers. Reviewer #1: The introduction is too brief and relies heavily on the researcher’s own work. Additional references should be included to support the lack of previous research in the area. These could be reviews or articles that mention this gap in the literature. This is a very important suggestion. Surprisingly, many physiologists of respiration have seemed to believe still that respiratory rhythmic activity is originated in specific neuronal groups in the brainstem as a pace-maker, not neuronal networks, while cardiac one is in the heart, in contrast to my claim that both rhythms of respiration and heart could be generated in the brainstem in a cooperative manner. Few of them have referred to any relationship with cardiac rhythm. Autonomic nervous system physiologists, on the other hand, have suggested that the cardiac rhythm may be associated with the brainstem neurons, however, few have mentioned any relationship with respiratory rhythm. I suspect that clinical cardiologists must recognize the interrelation of cardiorespiratory rhythm but could not yet describe this rhythm relation analytically. Therefore, as far as my knowledge adequate literature concerning cardiorespiratory cooperative activity in terms of rhythmic correlation could not be found for bridging the gap pointed out by this Reviewer. However, most recent references (5&6) concerning respiration networks were added newly with a sentence of “Furthermore, it has been generally stressed that cardiorespiratory rhythms be generated based on pacemaker-like activity originating from particular brainstem neurons, rather than network activity.” in the Introduction of the revised manuscript. (L.49-52) According to a suggestion of “Please, revise introduction to emphasize the lack of previous research in the area” raised in the mail letter, sentences of “Furthermore, it has been generally stressed that cardiorespiratory rhythms be generated based on pacemaker-like activity originating from particular brainstem neurons, rather than network activity. However, the body-brain interrelationship of rhythmicity….” were incorporated with additional two references (5&6) in the revised manuscript. (LL.49-52) The sample size (number of rats) needs to be stated. Determining the statistical validity of the findings is difficult without this sample size stated in the materials and methods. According to the suggestions, number of rats were added in the revised version. (LL.98-99) The reference section needs revision due to inconsistent formatting. First, there are variations in the listing of the journal’s full name versus abbreviations (ex. 2 vs 4). Second, there are variations in the capitalization of the article name (ex. 4 vs 5). Third, the doi is not always listed even if it is available (ex. 25 vs. 26). The article should be edited to ensure consistency in reference formatting. The reference list was generated using EndNote with PubMed. However, as pointed out by the Reviewer (#1), the format of listing seems to be not consistent. I have reflected the consistency of the bibliography list notation in the revised version as far as I have noticed, as pointed out by this Reviewer. (LL.685-800) Several pieces of the paper could be included in the figure legends and vice versa (ex. lines 213-220). According to the suggestion, that part was changed. (LL.556-564) Reviewer #2: In this paper, the authors investigated brainstem rhythms of the rat and their interactions. One of the rhythms seems to govern respiratory function (delta range) and another rhythm drives heart beats (theta range). It was demonstrated that these rhythms were synchronized in many cases of the brainstem activity by creation of temporary neural circuits. The author found a synchronization between central rhythmic and peripheral cardiorespiratory activities. In general, the paper is well-written, the results seem solid, and the experimental data supports the conclusions of the paper. However, some sentences in the paper are not clearly written. Basically, writing style must be improved. However, I have only minor points that need to be corrected. Minor points (lines are numbered as a sequence from the beginning of the paper) 1.Abstract. Too long Abstract. First part, before "In this article …", includes sentences that belong to Introduction. According to the suggestion, a long sentence in the Abstract, “In this article, by focusing on the physiology and anatomy of a certain rodent brainstem region, the nucleus of the tractus solitarius, we demonstrated the characteristics of harmonic brain waves and their interactions, with a probable anatomical configuration of the responsible oscillator circuits.”, was moved to in the Introduction. (LL.82-85). 2. Line 127 and other epoch durations. I would suggest to provide the number of points of the epochs with the durations of 10, 20, and 100 s that were analyzed. Those are important for the evaluation of the errors in Fast Fourier Transforms. According to the suggestion, a phrase of “with 1000 points” was added. (L.131) 3. Line 167. What is the meaning of vertical bars in Panels Ba-Be of Fig. 2? A sentence of “Vertical bars in B indicate relative powers with respect to a common arbitrary unit.” was added in the Figure legends. (L.526) 4. Line 182. 100 sec epoch is not longer than the previously mentioned 1000 sec epoch. Please correct wording. Please also introduce PZT, NTS, and LP in Fig. 3 in the first sentence, such as “Figure 3 shows … and so on”. I think, the reader must observe what is shown in the figure, which is quite complicated. “A 1000 sec epoch” means a total recording duration of this experiment. From this long epoch recording, epochs of 10-20 sec or 100 sec were selected for FFT analyses. According to the suggestion, a sentence of “Figure 3 shows two examples of shorter epoch (~10 sec) recordings for coherence analyses between peripheral cardiorespiratory activities recorded by PZT and central neuronal MUAs in the NTS recorded by glass electrodes.” was added. (LL.187-189) 5. Line 188. I assume Figs. A1 and B1 stand for Fig. 3A1 and 3B1? 6. Line 190. There are no panels A2, A3, A4, B2, B3, and B4 in Fig. 2. I assume those are for Fig. 3? Yes. The relevant figure numbers were carefully checked and all rewritten. (LL.195-209) 7. Line 192. I think "patterns in quality" must be "qualitative patterns" According to the suggestion, the phrase was changed. (L.199) 8. Line 194. What arrows mean in panels A3 and A4 in Fig. 3? Yes. The relevant figure numbers were carefully checked and all rewritten. (LL.195-209) 9. Line 213. Standard deviation for Fig. 4Bb seems incorrect. It is of the order of 1. As the Reviewer pointed out, the digit in the values of the NTS potentials (mV) were wrongly described by one digit. The values were rewritten in the manuscript. The values in the Fig 4B seem to be correct. (LL.220-221) 10. Line 354. Here and further throughout the text: Fig. S1. According to the suggestion, the words were changed throughout the text. :S# fig. � Fig. S#. S# and S## figs. � Figs. S# and S##. 11. Lines 393-395. Bad sentence. Please write more clear sentence or two. The original sentence of “Brainstem neural activity was confirmed to reflect the fluctuational balance between two distinct oscillators (somatic and visceral: respiratory and autonomic <cardiac>: 0–4 Hz delta and 10 Hz theta).” was rewritten to “The brainstem neural activity could represent a fluctuational cooperation generated by two types of distinct oscillator activities (somatic vs. visceral: respiratory vs. autonomic <cardiac>: 0–4 Hz delta vs. 4–10 Hz theta rhythms).”. (LL.401-403) 12. Line 400. The author can use either FFT or spell it out, but not both. FFT has been defined above. According to the suggestion, “the fast Fourier transformation (FFT)” was changed to “the FFT”. (L.408) 13. Lines 558-560. I am not clear about the meaning of the last sentence: It looks like coherence disappear, but panels do not show that signals disappear. The original sentence of “After an apparent cessation of the peripheral and central oscillations, robust signals of delta range almost disappear with theta range signals as seen in (f) – (g).” was changed to “With minor peripheral and central activities of a noise level, robust signals of delta and theta range and their CFC coherence almost disappear as shown in (f) – (g).”. (LL.575-577) 14. Line 613. I would remove "two". According to the suggestion, the word “two” was removed. (L.630) 15. Line 618. (d) and (q) must be removed. (d) and (q) should read (�) and (�). (L.634-635) == I sent the revised version of the original manuscript with “Revised Manuscript with Track Changes” in which changes were shown in red. Thank you for your consideration. I look forward to hearing from you. Sincerely, Yoshinori Kawai, PhD Adati Institute for Brain Study (AIBS), Kawaguchi Saitama JAPAN, 333-0811 E-mail: ibs.stm.kwgt@gmail.com Attachments Attachment Submitted filename: Response_to_Reviewer.docx https://doi.org/10.1371/journal.pone.0289657.r002
14 Jun 2023 Decision Letter - Gennady S. Cymbalyuk, Editor PONE-D-23-04711R1Cross-Frequency Coupling between Slow Harmonics via the Real Brainstem Oscillators: An in vivo Animal StudyPLOS ONE Dear Dr. Kawai, 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. A few minor points are left to be addressed. Please, make sure that the abstract summarizes the most important results and their significance and does not exceed 300 words. Line 199. The phrase "similar patterns in qualitative patterns" must be replaced by "similar qualitative patterns" Figure 3. In the caption, the arrows in panels A3 and A4 should be described or removed. Lines 401-403. Please clarify what brain regions create the two oscillators. Please submit your revised manuscript by Jul 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, Gennady S. Cymbalyuk, 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: All comments have been addressed 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: (No Response) Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: (No Response) 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 #1: (No Response) 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 Response) 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: (No Response) Reviewer #2: The author significantly improved the paper, but some comments were not interpreted correctly. I think the author still needs to make 4 minor corrections. Minor points (lines are numbered as a sequence from the beginning of the paper) 1. Abstract. I suggested to shorten the first part of the Abstract, before the words "In this article …". However, the author moved to the Introduction the sentence started from "In this article …". As the results, Abstract does not describe what is done in the paper. Please shorten the first part of the Abstract from the original submission. I am providing Abstract requirements for PLoS ONE: “The Abstract should: • Describe the main objective(s) of the study • Explain how the study was done, including any model organisms used, without methodological detail • Summarize the most important results and their significance • Not exceed 300 words” 2. Line 199. Phrase "similar patterns in qualitative patterns" must be replaced by "similar qualitative patterns" 3. Figure 3. What arrows mean in panels A3 and A4 in Fig. 3? This question still was not answered. The author needs to remove arrows from the figure, if they are not necessary. 4. Lines 401-403. Still bad sentence. Please explain more clearly what brain regions create two oscillators. It is not clear as it is written in the parentheses. ****** 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: No 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.0289657.r003
Revision 2
17 Jun 2023 Author Response 18 June, 2023 Editorial Office PLOS ONE Journal Dear Editorial Office: I wish to submit a revised version of an Original Research Article (ONE-D-23-04711R1) for publication in PLOS ONE, titled “Cross-Frequency Coupling between Slow Harmonics via the Real Brainstem Oscillators: An in vivo Animal Study.” I appreciate the Reviewers’ careful and encouraging suggestions for revision of this article. In that case, the following revisions or rebuttals (in red in below) were made as our response to comments from the Reviewer. Please, make sure that the abstract summarizes the most important results and their significance and does not exceed 300 words. 1. Abstract. I suggested to shorten the first part of the Abstract, before the words "In this article …". However, the author moved to the Introduction the sentence started from "In this article …". As the results, Abstract does not describe what is done in the paper. Please shorten the first part of the Abstract from the original submission. According to the suggestion, a sentence of “The recorded brain waves represent oscillatory synchrony of electrical activity generated by the varied spatial sizes of neuronal assemblies that are activated simultaneously.” was removed. As indicated in red in the revised manuscript, several words were added to imply an animal model and comply with other recommendations. As the results, I believe that the Abstract summarizes the most important results and their significance more succinctly than before, and confirmed that the abstract does not exceed 300 words (177 words). 2. Line 199. The phrase "similar patterns in qualitative patterns" must be replaced by "similar qualitative patterns" According to the suggestion, the phrase "similar patterns in qualitative patterns" was replaced by "similar qualitative patterns". (L.199) 3. Figure 3. In the caption, the arrows in panels A3 and A4 should be described or removed. According to the suggestion, the phrase “indicated by arrows” was added in the figure legend. (L.545) 4. Lines 401-403. Please clarify what brain regions create the two oscillators. According to the suggestion, a sentence of “The responsible brain regions of the coupling oscillators could be assigned to recurrent networks involving the NTS in the brainstem, especially a C1 neuronal group and the synaptically-connected neighboring areas [22] (Figs. S2 and S3).” was incorporated. (LL.404-406) == I sent the revised version of the original manuscript with “Revised Manuscript with Track Changes” in which changes were shown in red. Thank you for your consideration. I look forward to hearing from you. Sincerely, Yoshinori Kawai, PhD Adati Institute for Brain Study (AIBS), Kawaguchi Saitama JAPAN, 333-0811 E-mail: ibs.stm.kwgt@gmail.com Attachments Attachment Submitted filename: Response_to_Reviewer.docx https://doi.org/10.1371/journal.pone.0289657.r004
24 Jul 2023 Decision Letter - Gennady S. Cymbalyuk, Editor Cross-Frequency Coupling between Slow Harmonics via the Real Brainstem Oscillators: An in vivo Animal Study PONE-D-23-04711R2 Dear Dr. Kawai, 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, Gennady S. Cymbalyuk, 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: All my comments have been properly addressed. I have no further concerns or comments. Best regards. ****** 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.0289657.r005
Formally Accepted
27 Jul 2023 Acceptance Letter - Gennady S. Cymbalyuk, Editor PONE-D-23-04711R2 Cross-Frequency Coupling between Slow Harmonics via the Real Brainstem Oscillators: An in vivo Animal Study Dear Dr. Kawai: 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. Gennady S. Cymbalyuk Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0289657.r006

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