PONE-D-23-41361The bilevel chamber revealed differential involvement of vasopressin and oxytocin receptors in female mouse sexual behaviorPLOS ONE

Dear Dr. Kondo,

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. The manuscript has been assessed by two experts in the field; in particular, I would advise you to pay attention to the comments from Reviewer 1. Please find the detailed critiques in the reviews below.

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We look forward to receiving your revised manuscript.

Kind regards,

Nirakar Sahoo, PhD

Academic Editor

PLOS ONE

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Reviewers' comments:

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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

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: Yes

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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

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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

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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: In the research article entitled “The bilevel chamber revealed differential involvement of vasopressin and oxytocin receptors in female mouse sexual behavior”, the authors attempted to measure the female mice sexual behavior by constructing a new apparatus “bilevel chamber”. Also, they evaluated the effects of the arginine vasopressin (AVP) receptors v1a and v1b, and the oxytocin (OT) receptors on female mice sexual behavior in mice, by making the corresponding knockout female mice. Authors took the advantage of the easy genetic manipulation in mice to overcome the limitations (in comparison to rats) in studying the female mice sexual behavior. However, there are some concerns observed and the following minor and major issues listed.

Comments:

1) page 2:

a) line 10: The aKO females showed decreased rejection-like behavior, based on the discussion on page 20, lines 360-361, and Fig. 4A. Replace “increased” with “decreased”.

2) page 3:

a) line 22: Replace “evolutionary” with “evolutionarily”.

b) lines 31-33: Cite the references 9 and 10 after the word MPOA; or Cite all the references at the end of the sentence.

3) page 4:

a) line 40: The word “typical” is repeated twice. It may be corrected to one of the following:

“a typical diagnostic behavior” (OR) “a typical diagnostic type of behavior”.

b) line 47: Replace “Meanwhile” with “Also, it was reported/discovered that”.

c) line 51: It’s good to provide a figure for showing the sequence and structural similarity between AVP and OT. Otherwise give a reference.

d) lines 53-54: References should be given for lines 53 and 54.

4) page 5:

a) line 70: Use either “the” or “a”. Don’t use both, one after another.

b) line 74: The word “behavior” is repeated twice. This word maybe deleted at the end of the sentence.

5) page 6:

a) line 81: Give the “n” value.

b) lines 89-90: Give the brand/trade names for estradiol and progesterone used in this study.

c) line 89: Correct the “estradiol-17β benzoate” name appropriately to either “17β-estradiol 3-benzoate (EB)” or “17β-estradiol-3-benzoate (EB)”, in order to avoid confusion with the other estradiol benzoate variant “estradiol 17β-benzoate (E2-17B)”.

d) line 96: Replace “be adapt” with “get adapted”.

6) page 7:

a) line 98: Continue the sentence as “…………30 min, whichever is the first.”, if it’s appropriate.

b) lines 101-102: Give the LQ formula in a proper mathematical formula/equation format. Also, if you want to give this in the text, please express this correctly to “A lordosis quotient [LQ, (number of lordosis / total number of mounts and intromissions received) × 100] was calculated………….”.

c) line 103: Modify the sentence to “………..repeated weekly until an LQ ≥ 50% was observed.”.

d) How many weeks did it take to observe an LQ ≥ 50%. It’s better if it’s mentioned here.

e) lines 110-112: How the constrained sideways, due to the narrow depth of the bilevel chamber, help the experimenter better evaluating the female lordosis. Is it by physically forcing/making the female mice body to squeeze as lordosis and/or rise its tail to better heights, due to space constraint. It’s good if it’s explained better.

Also, since it’s an external/instrumental physical/mechanical constraint on the mice body and its free movement, is it considered as disadvantageous for studying the true behavior of the mice. This constraint may act as a stimulus, similar to the male mount, and cause/create a false lordosis. I said false lordosis since it’s not because of the true stimulus (male mount). Is this a con/disadvantage of the newly designed apparatus (bilevel chamber).

7) page 8:

a) line 123: Use “the” after the word “Both”.

b) line 126: Replace “Then, when” with “While”, or with appropriate word.

c) line 128: Continue the sentence as “…………male ejaculation, whichever is the first.”, if it’s appropriate.

d) Why the ordinary cage test was carried out for 30 min (page 7, line 98) and the bilevel chamber test for 20 min (page 8, line 128).

8) page 9:

a) line 138: Remove “were obtained”, if it’s appropriate.

b) line 141: Use “the” after the word “classified”.

c) line 146: Is it possible for intromission event to happen without the lordosis event. How frequently this bout (Intromission without lordosis) was observed. Does this signifies that lordosis is not a necessary event to occur during mice mating/intercourse.

d) line 148: What is the minimum, maximum, average length of different bouts/events explained in this page. How could you measure the events with “> 2 sec” precision.

Also, how did you monitor and count/measure the no. of lordosis events in female mice; and the no. of mount, intromission, and ejaculation events in male mice. If it’s manual monitoring, how accurate is the counting.

e) lines 152-153: Give the IR formula in a proper mathematical formula/equation format. Refer the comment 6b (page 7).

9) page 10:

a) line 156: If the statement “IR was considered equally to LQ” was from the previously published literature, cite the suitable reference for this. And if this is your own observation/statement, explain why you considered this.

b) line 157: Correct appropriately to “……..LQ, as clearly distinguished…….”, if needed.

c) lines 161-163: Why the female lordosis events, and so the true LQ, could not be observed/measured accurately in the ordinary cage. How differently the bilevel chamber help the experimenter to observe the female lordosis events accurately. Just to mention here, the male mount, intromission, and ejaculation events are observed equally well in both the ordinary cage and bilevel chamber apparatus.

And, if the true LQ cannot be measured in the ordinary cage test, why do you consider the “estimated LQ”, measured based on the IR and %Intro, to compare with the true LQ and %Intro calculated in the bilevel chamber test. Why don’t you use the “%Intro” in place of “estimated LQ”.

d) lines 168-169: Correct the sentence appropriately to “……….and intromissions separately revealed that females exhibited………….”.

e) line 172: Replace “behavior” appropriately with “behavioral”, if needed.

10) page 11:

a) lines 180-181: Why the female rejection-like behavior events could not be observed/measured accurately in the ordinary cage. How differently the bilevel chamber help the experimenter to observe the female rejection-like behavior events accurately.

b) line 182: Remove the word “in”.

11) page 12:

a) line 210: Use “and” before the word “dKO”.

b) line 210: Why is the “n” value difference between the WT, aKO, bKO, and dKO mice groups.

12) page 13:

a) line 223: Does this line means that different stimulus males were used in the subsequent/different bilevel chamber tests. Make it clear.

13) page 14:

a) lines 244-245: The LQ formula/equation on page 14, lines 244-245 is different from that on page 7, lines 101-102. Correct this appropriately.

b) line 245: Replace “s” with “sec”, for seconds.

c) Correct the following:

line 245: Mean duration

line 248: Total duration

Fig. 3B: Total duration

14) page 15:

a) lines 251-253: Explain it better. For example, in the test 1 also the lordosis duration was suppressed in bKO females compared to WT females.

b) line 259: Correct appropriately to “…post hoc analyses by Bonferroni test showed…….”.

c) line 269: Figure title should be given in the Fig. 4 legend.

15) page 16:

a) Correct the following:

line 270: Mean % time

line 275: Time

Fig. 4B: % time

b) Lines 280, 283: Why is the “n” value difference between the WT, OTRKO, aKO, bKO, and dKO mice groups.

16) page 17:

a) line 296: Replace “s” with “sec”, for seconds.

b) line 304: How do you explain the effect of test replication.

17) page 18:

a) line 308: Figure title should be given in the Fig. 6 legend.

b) Correct the following:

line 309: Mean % time

line 314: Time

Fig. 6B: % time

c) What is the significance of % time spent with males, in the context of female sexual behavior.

18) page 19:

a) line 328: If the v1b receptor is not essential for lordosis expression, then why bKO showed almost no lordosis. Interpret this in a better way.

b) line 330: “in dKO females, aKO restored lordosis from the suppression by bKO up to the WT level”. How do you justify this hypothesis. Any experimental evidence. And how the dKO showed normal levels of lordosis, while the bKO showed no lordosis. It means there may be elements (other than v1a and v1b receptors) involving in nullifying/inhibiting the effect of bKO in presence of aKO. Can you shed light on this. If you propose that the crosstalk between OT and v1b receptors (page 20, line 356) maybe the underlying cause for this, please explain it better. And if so, how did you propose this hypothesis, and any attempts made for experimental evidence. Any other elements may involve in this ?.

19) page 21:

a) How the OTRKO females exhibit normal lordosis levels, while showing increased rejection-like behavior. What is the stimulus for lordosis in this case, if it’s not the male mount (which is not possible due to rejection by female mice).

b) lines 378-379: Does the “rejection-like behavior” reflect “proceptivity”. Please explain this.

20) Have you attempted to evaluate the effects of AVP, OT, AVP/OT combination, progesterone administrations/treatments in WT, aKO, bKO, dKO, OTRKO mice, similar to the rat lordosis experiments (page 19, lines 333-334; page 20, lines 352-353; page 21, lines 371).

21) Did you monitor and measure the Intromission and ejaculation events in experiments II and III.

22) Figure 2:

a) Replace “Ordinary” with “Ordinary Cage Test”, if appropriate

b) Replace “Bilevel Chamber” with “Bilevel Chamber Test”, if appropriate

23) P values and the corresponding bars should be given in the following figures.

a) Figure 3: Fig. 3B panel: P values and the corresponding bars for all the groups in Test 1.

b) Figure 4: Fig. 4B panel: P values and the corresponding bars for all the groups in Test 1 and 2.

c) Figure 5: Fig. 5A panel: P values and the corresponding bars for all the groups in Test 1 and 2.

Reviewer #2: Authors in their manuscript described a new test for sexual behavior in mice and the role of vasopressin and oxytocin receptors in mice female sexual behavior. While the test itself seems interesting, new findings in sexual behavior can be a good addition to our knowledge of how such social neuropeptides as AVP and OT function. Here are several questions to be asked before considering the manuscript for publication.

1) Figure 3A and b WT upper significance bar misleading, it looks like WT mice have statistical differences with both aKO and bKO, while the difference is only with bKO. Please check and modify the significance bars.

2) While the results of rejection-like behavior are clear, the influence of V1A and V1B receptors on lordosis seems puzzling. May you address more in the discussion why the absence of both receptors has the same results as their presence? While it seems to be some kind of interaction, it’s still unclear how knockout of V1AR can recover the effects of knockout of V1BR.

3) Why you choose a different strain for experiment I? (C57BL6 for II and III is clear to me, because KO mice are based on the same strain)

4) You used the terms receptivity and proceptivity in the conclusion and briefly used them in the discussion; May you clearly state in the introduction what you mean by those terms?

5) May you indicate in your methods part how you clean the Believel chamber at the end of the test in order to remove odor influence between tests?

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Reviewer #1: No

Reviewer #2: No

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Submitted filename: Review_Report_PONE-D-23-41361.pdf

PONE-D-23-41361R1The bilevel chamber revealed differential involvement of vasopressin and oxytocin receptors in female mouse sexual behaviorPLOS ONE

Dear Dr. Kondo,

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.

The reviewer 1 provided a few minor comments that need to be addressed, apart from that, all looks good. Please submit your revised manuscript by Jun 15 2024 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 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,

Nirakar Sahoo, PhD

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.

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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: All comments have been addressed

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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: I Don't Know

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: 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: Yes

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: In the revised version of the manuscript entitled “The bilevel chamber revealed differential involvement of vasopressin and oxytocin receptors in female mouse sexual behavior”, the authors made several edits as per the reviewers’ comments. I appreciate that the authors addressed all the issues raised by the reviewers, made all the suggested/necessary corrections throughout the text and figures, which improved the manuscript a lot. I think that the newly included extended discussions at multiple places improved the general understandability of the manuscript. These insertions also shed light on the future prospective and directions of the research in mice sexual behavior, regulatory mechanism(s), and the AVP & OT roles. I hope the “Bilevel chamber” devised in this research will be a useful tool in the future research. And I believe in my understanding that the revised manuscript maybe in good shape for publication with the following few minor modifications.

Minor Comments:

Line 56: Reframe the sentence to correct “with of nine”.

Line 95: Remove the extra space before the word “two”.

General comment: Please correct the page numbers in the responses to the reviewers’ comments, if the author responses along with the reviewer comments will be published as a separate section in the published manuscript.

Reviewer #2: The authors did a good job of addressing my comments and giving satisfactory answers. Overall I recommend accepting the manuscript at this stage.

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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: MADHUSUDHANARAO KATIKI

Reviewer #2: No

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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

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Submitted filename: PONE_D2341361R1_Review1_Report.pdf

The bilevel chamber revealed differential involvement of vasopressin and oxytocin receptors in female mouse sexual behavior

PONE-D-23-41361R2

Dear Dr. Kondo,

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.

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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,

Nirakar Sahoo, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: