PONE-D-23-22233Oxytocinergic Projection from the Hypothalamus to Supramammillary Nucleus Drives Recognition Memory in MicePLOS ONE

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PLOS ONE

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Additional Editor Comments:

The findings are interesting. However, please provide the more convincing image demonstrating the projections of SuM. The reviewers' comments are needed to be addressed carefully, especially ones from Reviewer 1.

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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: Partly

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

Reviewer #1: Yes

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: No

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: This manuscript describes results of experiments testing the influence of oxytocin projection neurons from periventricular nucleus (PVN) to the supramammillary nucleus (SuM) on long-term memory for objects. The excitatory DREADDs receptor, hM3Di was expressed in oxytocin neurons of the PVN, mice were subsequently administered systemic CNO and tested for the influence on c-Fos expression in SuM, spatial working in a Y-maze task, and object recognition memory. Systemic CNO enhanced the preference of the mice to explore the novel object during a test session conducted 72 h after the initial training session. A follow-up study demonstrated a similar enhancing effect after mice received local infusion of CNO into the SuM. The authors conclude that oxytocin projection neurons of the PVN play a significant role in object recognition memory. There is compelling evidence from prior reports that oxytocin influences social memory, and converging evidence has defined circuits supporting this effect. The present manuscript expands on this literature by demonstrating the involvement of oxytocin in a different form of recognition memory. The results are interesting and the breadth of the approach here is commendable. There are some issues outlined below that should be addressed.

1. The details of how the object recognition task was conducted are missing. (A) For example, page 7 (lines 174-175) indicates that object A and B were used, and line 177 indicates that two object A's were presented during the training session. The object recognition task is based on spontaneous responses of the mice, and one must be careful to counterbalance the objects presented. For example, it would be important for the training session to present two object A's to half of the mice, and two object B's to the other half of the mice. The authors state that there were no differences in preference between the objects; however, counterbalancing objects presented during training and testing brings rigor to the approach.

(B) The measure used to assess memory was preference ratio, which suffers from a limited range: chance performance in 50%, and substantial preference for the novel object is rarely more than 75%. A better measure of test session memory performance is the Discrimination Ratio, calculated by taking the difference score (time exploring object B - time exploring object A) and dividing the result by the total time spent exploring both objects. Discrimination ratio scores better account for overall differences in total object exploration between individual mice, and the data tend to have a wider range of scores than that of preference ratio. The discrimination ratio is a well accepted and more rigorous measure of object memory than preference ratio. The authors are encouraged to present their test session object recognition data as discrimination ratio scores.

(C) Page 7, line 182 states that the "Data was manually analyzed." The authors should provide more detail as to what this means. How was object exploration scored, what constituted object exploration, and who scored the data? How was scientific rigor upheld while manual analysis was occurring?

2. Details are missing as to how the c-Fos counts were conducted and analyzed. How many sections from each mouse were counted for each of the regions (CA1, CA2, CA3, ... etc.)? Were the counts conducted blind? c-Fos counts were normalized by the area of the respective region. However, how was this determined given the need to control for the thickness of tissue and the differences in the size of the respective structures across sections? Also, background varies across a piece of tissue and across slides, so what approach was taken to achieve consistency across slides?

3. The c-Fos data were analyzed by multiple t-tests - that is, an unpaired t-test (saline- vs. CNO-treated) for normalized c-Fos counts in each region. The authors should consider that conducting multiple t-tests carries the risk of yielding a significant outcome by chance. Given that the data were normalized (though see comment #2 above), why not instead conduct a two-way RMANOVA (treatment group as between-subjects variable, and region of interest as the repeated measures/within-subjects variable)?

4. The manuscript refers to Table 1, but this information was not provided.

Minor issues

a) page 6, line 153: the statement that "...dorsal, but not ventral Hipp[ocampus] is involved in cognitive function..." is not well supported by the literature. For example see: Jin & Maren (2015) Scientific Reports; Okuyama et al. (2016) Science; Contreras et al. (2018) Hippocampus; Rao et al, (2019) Cell Reports; Jimenez et al. (2020) Nature Communications; to name a few example references.

b) the manuscript contains an overwhelming number of acronyms (OXT, ORM, SuM, PVN, ABC, CNO, NORT, AD, etc.), which makes some of the sentences challenging to understand. This reviewer suggests limiting the acronyms to a minimal number of terms that are used throughout.

c) page 10, line 249-252: This sentence is quite difficult to understand. Please revise.

d) page 11, line 267: "competed" should be revised to "compared"

e) page 13, line 314-315: The statement "...our result suggested that OXTergic neuron activity may be involved in each stage of AD pathological progression." is not supported by the results described in the current manuscript.

Reviewer #2: 1. As stated by the author in the introduction, "Projections of oxytocin (OXT) neurons exhibit extensive distribution across regions associated with cognitive functions. For instance, prior studies have revealed that OXT neurons project to the hippocampus (Hip), perirhinal cortex, entorhinal cortex (Ent), and supramammillary nucleus (SuM)." However, the findings from immunohistochemical staining (Fig. 1) merely illustrate the fibers within the paraventricular nucleus (PVN) and supramammillary nucleus (SuM). It would be advantageous to incorporate outcomes from other cerebral regions, notably the hippocampus (DG).

2. Regarding c-Fos staining and quantification subsequent to CNO treatment, it is advisable for the authors to undertake additional verification to ascertain that the identified c-Fos positive neurons post-treatment indeed are OXTergic neurons.

3. Does the localized administration of CNO to the SuM have an impact on the DG? While the results section does not explicitly address the SuM-DG pathway, it extensively delves into this topic in the discussion. It would be better for the authors to substantiate their conceptual arguments in the discussion by presenting relevant empirical findings.

4. In the discussion section (page 11, line 270), the authors put forth the assertion that “Thus, we propose that activating DG and SuM neurons enhance ORM by activating OXT neurons.” It would be judicious for the authors to complement this proposal by providing evidential support. Specifically, they could present data that demonstrate the comparable effects observed in “Effects of activation of OXTergic neurons in SuM by NORT”, where local activation of oxytocinergic fibers in the DG produced similar outcomes.

5. In this study, only male mice were used as the subjects. Could potential distinctions arise in female mice?

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Reviewer #1: Yes: Robert W. Stackman Jr.

Reviewer #2: No

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Oxytocinergic Projection from the Hypothalamus to Supramammillary Nucleus Drives Recognition Memory in Mice

PONE-D-23-22233R1

Dear Dr. Saitoh,

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,

Peng Zhong, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Please make minor revision based on Reviewer 1's comments.

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: 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: (No Response)

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

Reviewer #1: Yes

Reviewer #2: (No Response)

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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: (No Response)

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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: (No Response)

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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: The revised manuscript is improved with the changes that the authors have made. A few issues remain to be addressed.

1. Measures of effect size should be included for each significant statistical value.

2. In response to the critique that the NORT results would be better analyzed as measures of discrimination ratio (difference score divided by total object exploration) instead of preference ratio. The authors seem to have disregarded this message and have now included difference score measures in the supplementary material. Both preference ratio and difference score measures suffer from the issue of high variability between groups and between individual mice. That is the reason for the original message that discrimination ratio measures represent a more rigorous measure of object memory in the NORT task. The discrimination ratio scores have a wider range of possible scores and generally have a reduced variability. Thus, analyses of NORT discrimination ratios tends to lead to fewer false interpretations of results.

3. The c-Fos counts indicate no differences in any of the examined areas after NORT between mice receiving saline or CNO. This result is a bit surprising given several reports of increased c-Fos in hippocampal cell layers and related regions 90 min after the training or test session of the NORT task (see Beer et al.; Mendez et al 2015a, 2015b; Cinalli et al. 2020; Bernstein et al., 2019; Barbosa et al., 2013, etc.). Given those reports, one might have expected differential counts between cingulate and CA1 and CA3, for example. As well differences might have been expected in the CNO treated mice if the drug was expected to have indirectly increased activity of hippocampal neurons. The authors should provide more interpretative information regarding these issues.

Reviewer #2: (No Response)

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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: Robert Stackman Jr.

Reviewer #2: No

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