PONE-D-22-26274Dispensability of Oxytocin for Parturition and Maternal Behaviors by Conditional Knockout MicePLOS ONE

Dear Dr. Miyamichi,

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"K.I. was supported by the RIKEN Special Postdoctoral Researchers Program (https://www.riken.jp/en/careers/programs/spdr/),  a grant from the Kao Foundation for Arts and Sciences (https://www.kao-foundation.or.jp/english.html), and Japan society promotion science KAKENHI (19J00403 and 19K16303) (https://www.jsps.go.jp/english/)

K.M. was supported by Japan society promotion science  (20K20589 and 21H02587) (https://www.jsps.go.jp/english/).

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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?

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Reviewer #1: Partly

Reviewer #2: Partly

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

Reviewer #1: Yes

Reviewer #2: No

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Reviewer #1: Yes

Reviewer #2: Yes

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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: The paper by Hagihara et al. reports interesting new findings on the role of oxytocin in regulating parturition, breastfeeding and maternal behavior in female mice. The Authors are leaders in this field of research and have published outstanding papers on oxytocin and reproduction. Here, by selectively deleting oxytocin, or oxytocinergic neurons, in hypothalamic PVN and/or SO nuclei, they confirm that oxytocin is dispensable for parturition and maternal care but also show that the milk ejection role of oxytocin is specifically supported by SO oxytocinergic neurons. Collectively, the paper is well conceived and well written, the methods are adequate. However, some points should be tackled by the Authors before recommending publication.

Major point

In my opinion, the major result of the paper, that oxytocin neurons of the hypothalamic SO nucleus are specifically involved in milk ejection, especially at the onset of lactation, should be confirmed by selectively deleting oxytocin in the PVN. In these mice, according to the Authors’ hypothesis, a normal, or almost-normal, breastfeeding behavior should be observed. This is the most outstanding result of the paper, and the title could be changed or implemented

Minor points

Throughout the manuscript the Authors claim to possible compensatory mechanisms (“genetic compensation”, “enhance the expression of related genes” … which genes?), that in fully oxytocin KO mice could hinder some phenotypes. They should be clearer, and specifically explain what they mean.

In this context (Introduction), why “PVH-specific OT cKO mice show hyperphagic obesity” is not clear to the reader if the Authors do not specify that OT also has a role in food intake and energy balance in adult animals.

The wide readership of the Journal may be not so confident with the mouse models created and studied in the present investigation. In addition to this, the Materials and Method section is at the end of the manuscript. So, a few sentences in the Result section, better describing the procedures used for obtaining the selective conditional KO mice, could render the paper easier to read and meaningful.

When discussing the possible effects of oxytocin on dopaminergic neurons of the ventral tegmental area and rewarding mechanisms, but also concerning data obtained in the present paper, the Authors should also take into consideration the possibility that some phenotypes could be due to a some extent to cutting of afferent, or efferent, projections to, or from, the SO and/or PVN nuclei and to the involvement of glial cells.

Reviewer #2: Oxytocin is necessary for milk let-down and facilitates maternal retrieval in rodents in the transition from nulliparous to parental. The main sources of oxytocin are the paraventricular nucleus of the hypothalamus and the supraoptic nucleus of the hypothalamus. In the mid-1990’s the first knockout studies of oxytocin demonstrated an absence of milk-letdown and impairments in social behavior in congenital whole-body knockouts. In the current report, the authors evaluate the site-specific role of oxytocin using conditional genetic strategies in primiparous female mice.

Strengths:

This is an interesting report that contributes to our understanding of different hypothalamic populations in physiology and behavior.

Major weaknesses:

On line 139-140, the authors state: “ These results suggest that OT+ neurons in the PVH and SO show differential contributions to pup survival.” They do not have a full dataset to support this claim. They do show that conditional deletion of OT from the SO is sufficient to recapitulate deletion from whole body (prior literature) or their conditional deletion from SO AND PVH. There is a missing experiment and result: to support their claim as quoted above, they need to show that conditional deletion of OT from the PVH does not recapitulate the phenotype. This is a likely outcome, but not demonstrated conclusively here.

Additionally, the by using primiparous females during retrieval testing and not virgins, the manuscript fails to capture the impact of oxytocin in the transition from inexperienced to experienced maternal retrieval behavior. It is well-established that inexperienced virgins are likely to ignore pups in their first interactions with them. However, once pregnant and delivered, primiparous dams very quickly learn to retrieve pups. Elevated estrogens from pregnancy and enhanced oxytocin seem to facilitate this process. Once maternal, retrieval behavior is established and less vulnerable to oxytocin manipulations. In this report, the authors have chosen to test their hypothesis in females who have gone through pregnancy- females who will already behave maternally and for which oxytocin is less important. Had they really wanted to evaluate a site specific contribution of the SON or PVN OT to pup retrieval, they should have tested their retrieval behavior in virgins. Contextualizing their findings with others’ work in this area should include a discussion of Rich et al (Rich ME, deCárdenas EJ, Lee HJ, Caldwell HK (2014) Impairments in the Initiation of Maternal Behavior in Oxytocin Receptor Knockout Mice. PLOS ONE 9(6): e98839, and Carcea et al (Carcea, I., Caraballo, N.L., Marlin, B.J. et al. Oxytocin neurons enable social transmission of maternal behaviour. Nature 596, 553–557 (2021). https://doi.org/10.1038/s41586-021-03814-7)

Minor weaknesses:

In the abstract, the language ending on line 38 should be clarified: “We found that cell ablation of oxytocin neurons leads to no additional abnormalities over the oxytocin conditional knockout, suggesting that non-oxytocin ligands expressed by oxytocin neurons have negligible functions” on the responses measured in this report. “on the responses measured in this report” should be added as clarifying language.

The presentation of the figures makes it unclear if the animals used in figures 1 and 2 are the same as in figures 4 and 5. If they are the same, it makes more sense to combine the figures. If they are not the same, then it would be important to show validation of cKO for this additional cohort of animals. If the figures get unwieldy with multiple panels, the following current figure panels seem unnecessary: Figure 1K, Figure 2I, Figure 3H, Figure 4C, Figure 5C

More details of methods should be included instead of solely referencing prior papers. For example, for the ISH, probe details should be included.

It is unclear why the OTflox/- was necessary or an improvement over OTflox/flox. The rationale for this choice should be explained.

How were cells counted? How many sections per side per animal?

“In situ hybridization” would be a better section heading in the methods than “histochemistry”

The statistical analysis for pup growth should be a repeated measures ANOVA assuming that the animals are traceable as individuals overtime.

The cKO model uses oxytocin floxed in the context of an Oxt knockout a allele bred with males who are wild type. This breeding strategy would produce wild-type and heterozygous offspring. It is unclear what the impact of an interaction between pup genotype and mom genotype might be. This should be addressed in the discussion.

In figure 2 the authors use oxytocin in situ hybridization to evaluate the effectiveness of the caspase virus which clearly shows a reduction in oxytocin. However, their claim is that the caspase method reduces the oxytocin cell population rather than just the oxytocin mRNA. Ideally, they would show evidence of cell loss rather than just oxytocin mRNA loss. Is there a significant reduction in the DAPI positive cell density in the PVN and SON?

There appears to be a lot of variability in the pup growth across ages in virus treated females. Given that the remaining OT+ neurons are associated with pup survival, it would be interesting to probe if the remaining oxytocin neurons in the PVN or SON of dams predict pup growth for pups that do survive.

The lack of a virus control is unfortunate.

Were the litters normalized to the same size? Litter size impacts pup growth- too few pups and too many pups reduce per pup weight. This should probably be modeled. This is particularly important due to the influence of ventral stimulation (intensity of which is influence by the number of pups) which stimulates the Ferguson reflex for milk let-down. Is it possible that the KO is less able to modulate pup growth based on litter size?

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Reviewer #2: No

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PONE-D-22-26274R1The Importance of Oxytocin Neurons in the Supraoptic Nucleus for Breastfeeding in MicePLOS ONE

Dear Dr. Miyamichi,

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 point raised during the review process.

Please submit your revised manuscript by Apr 14 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.

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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:

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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.

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Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: No

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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

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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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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 authors have adequately addressed the original reviews. In the new Figure 3 F, the distribution of the data indicate the possibility that the data needs a non-parametric mann-whitney U test rather than a parametric t-test.

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Reviewer #1: No

Reviewer #2: No

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The Importance of Oxytocin Neurons in the Supraoptic Nucleus for Breastfeeding in Mice

PONE-D-22-26274R2

Dear Dr. Miyamichi,

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.

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