PONE-D-22-06089OX2R-selective orexin agonism is sufficient to ameliorate cataplexy and sleep/wake fragmentation without inducing drug-seeking behavior in mouse model of narcolepsyPLOS ONE

Dear Dr. Yanagisawa,

Thank you for submitting your manuscript to PLOS ONE. Your manuscript was evaluated by four experts in the field and by myself. After careful consideration, we all feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands, with particular reference to the criteria that experiments, statistics, and other analyses are performed to a high technical standard and are described in sufficient detail and that conclusions are presented in an appropriate fashion and are supported by the data. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. In addition to the points raised by all Reviewers, please also address my additional editor comments below.

Please submit your revised manuscript by May 19 2022 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.

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We look forward to receiving your revised manuscript.

Kind regards,

Alessandro Silvani, M.D., Ph.D.

Academic Editor

PLOS ONE

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Additional Editor Comments:

For clarity and ease of consultation, please report in the manuscript the administered concentrations of AL-OXB and OXA in terms of net peptide. Please indicate the purity and % net peptide content for each peptide. 

Please clarify whether the data fulfilled the requirements for parametric statistical analysis with ANOVA.

Please note that a working URL address to all data underlying the findings or inclusion of the data as supplementary material will be needed before the eventual acceptance of the manuscript for publication.

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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

Reviewer #3: Yes

Reviewer #4: Partly

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: 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: No

Reviewer #3: No

Reviewer #4: No

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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

Reviewer #3: Yes

Reviewer #4: 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 is a very nicely done paper describing the possible roles of the two orexin receptors in reversing the symptoms of narcolepsy. The combination of in vitro and in vivo studies is a great strength of the study, as is the conditioned place preference tests. The paper shows the way to a possible treatment of human narcolepsy.

A limitation of the study is the use of orexin KO mice. Although these mice, developed by some of these investigators, led rather directly to the cause of human narcolepsy, the consensus in the field is that human narcolepsy is due to the loss of orexin neurons, not just the orexin peptide. Orexin neurons contain glutamate, dynorphin and Narp. These transmitters are present in the KO mice, but not in human narcoleptics, so the KO mouse results may not exactly align with the pathology of human narcolepsy. The use of the orexin neuron deletion mice developed by Yamanaka might more closely parallel the human condition and comparison of the results of these two models might shed light on the role of the other transmitters in orexin neurons.

In the introduction, I would suggest moving or deleting "and dreaming." Human cataplexy is generally not accompanied by dreaming unless it progresses into a long REM sleep episode. You may want to position these words after "hallucinations." Histaminergics is misspelled.

A very minor point is that the support and animal study questions ask by PLOS One are not properly addressed in their forms, although all the information is in the manuscript.

Reviewer #2: The authors examined whether orexin 2 receptor selective agonist (AL-OXB) could effectively reduce cataplexy and sleep/wake fragmentation in orexin knockout mice and got affirmative results. In addition, using wild type mice, the authors showed that ICV administration of OXA but not AL-OXB induced conditioned preference indicating less addiction-inducing effect in the latter. The theme of the study is worth examining and overall conclusion seems convincing. However, there are several points to be clarified.

1) Please report the amount of chocolate consumed in the cataplexy experiment (Fig. 2) because orexin is known to induce food seeking. Was there any possibility that possible change in appetite affected the wake/sleep structure?

2) Please specify when of the day pre- and post-test was performed in CPP testing. According to Fig. 5A, it appeared to be during the daytime around ZT3. If it is correct, then mice should be in the resting period and sometimes fall into asleep. Such behavior may distort the preference score. To exclude such possibility, information should be provided about mobility such as the crossing times between the components and/or distance traveled during the observation period.

3) In related to the point #2, why the authors selected to test and inject drugs during the resting period whereas the drugs were injected just before the active (night) period in the sleep study? To examine possible side effect (addiction-inducing effect in this case) of a drug, we should use the same dosing schedule in the main effect (sleep study in this case). Don’t you agree?

Reviewer #3: The manuscript aims at demonstrating that the selective activation of the orexin receptor 2 is sufficient to ameliorate cataplexy and sleep fragmentation in orexin-deficient (ORX-KO) mice without inducing drug seeking behavior as tested using the place preference test.

The originality of this study is to directly compare OXA and AL-OXB in vivo. The authors show that only TMN neurons are activated by AL-OXB (expressing only ORX receptor 2) while OXA activates both, neurons of the TMN and the LC (expressing only ORX receptor 1) as a validation of the selectivity of the compounds for the orexin receptor 2.

Although the study has limits, it is of interest for the design of future therapeutics.

The manuscript is well written.

Comments

• Please add the total number of mice used in the study and for each experiments in the method section.

• Chronic icv injections were performed in only 3 mice. No statistical test can be robust with such small number of individuals. Data should be reported with caution and be mainly descriptive (rather than quantitative). Please mention/discuss it in a “technical limitation” section.

• Figure 1 shows the dose response curves of intracellular CA2+ induced by OXA and L-OXB in vitro. L-OXB is approximately one log more efficient than OXA,. However, drugs are used at the same concentration in vivo. Could the authors comment on it? Please discuss it.

• Figure S1 show an increase in NREM sleep after OXA treatment compared to vehicle at ZT5-7, that is not seen with L-OXB. It might be an additional positive point for L-OXB versus ORXA, but it is not discussed. Could the authors comment on it?

Reviewer #4: This important paper evaluates the efficacy of orexin A (OXA) and AL-orexin B (AL-OXB) in several in vitro and in vivo assays and concludes that Ox2R activation by AL-OXB is sufficient to prevent cataplexy-like episodes and fragmentation of wakefulness in OXKO mice. The work is well-thought out, carefully executed and, with one exception, the results are properly interpreted. That exception is particularly evident on page 17 when the authors conclude the first paragraph with the statement “In other words, the selective activation of OX2R is sufficient to ameliorate both narcoleptic symptoms.” I understand that the authors would like to make this conclusion but, based on the data presented in Figs. 2C, 2D and Fig. 4, the same statement could be made for OX1R since OXA is at least as effective as AL-OXB in all the sleep/wake bioassays shown. The data presented does not exclude this possibility because an Ox1R-selective compound has not been evaluated. As such, I find that statement as well as the title of the paper to be misleading.

The EEG/EMG recordings are scored in 20-sec epochs which is unusually long for rodent studies in which 10-sec epochs are the norm and many labs even score in 4-sec epochs. Although the choice of epoch duration is somewhat arbitrary, longer epochs provide less resolution of the fine structure of sleep architecture. In fact, it is certainly conceivable that all 3 states could occur in a 20-sec epoch. Consider, for example, the sequence NREM-REM-Wake. Consequently, the authors should describe their rationale for such long epochs.

Related to this issue are the determinations of “mean episode duration” as shown in Figs. 2F, 4D and Fig. S1. In fact, Fig. 3 is entirely leveraged on episode duration measurements as the number of state transitions reported in this figure would certainly be larger if 10-sec epochs were used. Because of the fact that rodent sleep is fragmented throughout the 24-h period rather than consolidated as occurs in (most) humans, many sleep researchers establish rules to determine a bout (or episode) of NREM or REM sleep, such as that 3 consecutive epochs of NREM sleep or 2 consecutive epochs of REM sleep must occur before a bout (or episode) is consider to be NREM or REM sleep. The authors do not stipulate any such criteria, which may be due to their use of the long epochs.

Neither OXA and AL-OXB at 1 nmol induce CPP but there’s no data to show whether OXA at 1 nmol could prevent cataplexy-like episodes and fragmentation of wakefulness in OXKO mice. Has OXA been evaluated at that dose or not?

What effect does continuous ICV infusion as shown in Fig. 4 have on cataplexy, if any? It would be interesting to know what effect, if any, continuous ICV infusion of OXA and AL-OXB have on body temperature.

Supp. Fig. 2 suggests that there is a trend toward a rebound in NREM sleep on both the day and subsequent night after cessation of ICV infusion. Although these trends may not be significant on an hourly basis, are they significant over 6-h or 12-h periods? What about NREM delta power?

On page 17, the authors conclude the next paragraph with the statement “despite the stress.” What stress do the authors refer to? This is the first use of the word “stress” in the paper.

On page 20, the authors state “it is likely that higher agonist potency may be required for ameliorating wake fragmentation than suppressing cataplexy.” This is an interesting point that should be developed further.

Figure 4: why are the error bars so large during the infusion? Figure 4D suggests that AL-OXB is weaker than OXA.

Specific comments

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p. 10: “administrated” should be “administered”.

P. 17, last sentence would be better English if the authors changed it to read “These results suggest that the administration of an OX2R-selective agonist in sufficient doses to ameliorate cataplexy and fragmentation of wakefulness does not produce addiction-related behavior.”

p. 21, first line: eliminate “the” after “in”.

The authors are not appropriately listed in Ref 25.

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Reviewer #1: Yes: Jerome Siegel

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

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PONE-D-22-06089R1OX2R-selective orexin agonism is sufficient to ameliorate cataplexy and sleep/wake fragmentation without inducing drug-seeking behavior in mouse model of narcolepsyPLOS ONE

Dear Dr. Yanagisawa,

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. In particular, I encourage you to make minor revisions to your manuscript in order to address the final comments by Reviewer 1 and Reviewer 4. 

Please submit your revised manuscript by Jul 20 2022 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,

Alessandro Silvani, M.D., 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.

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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

Reviewer #3: All comments have been addressed

Reviewer #4: (No Response)

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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)

Reviewer #3: Yes

Reviewer #4: Yes

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

Reviewer #1: Yes

Reviewer #2: (No Response)

Reviewer #3: Yes

Reviewer #4: Yes

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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)

Reviewer #3: Yes

Reviewer #4: Yes

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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)

Reviewer #3: Yes

Reviewer #4: 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: I don't understand what the authors are saying in the second sentence of the Introduction. I suggest shortening it to: "The abnormal gating of REM sleep-related neurophysiological mechanisms, such as REM atonia contributes to cataplexy (sudden bilateral skeletal muscle weakening triggered by a strong emotion) and sleep paralysis."

Dreaming is not confined to REM sleep and is not studied (nor could it be) in this mouse experiment.

Reviewer #2: (No Response)

Reviewer #3: All my comments were answered, I am satisfied by the way they were taken in considerations

Thank you

Reviewer #4: The authors' response re the use of the 20-sec epoch is very misleading and incorrect. For example, in a 20-sec epoch, the following sequence of states commonly occurs: NREM-REM-W. This epoch would be scored as one of the 3 states. The purpose of contiguity rules for shorter 4-sec or 10-sec epochs is to obviate this problem when sleep architecture analyses are reported (e.g., number of bouts and mean bout duration per state). The authors' revised text is thus both facile and misleading and must be corrected before I recommend acceptance.

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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: No

Reviewer #2: Yes: Tomoyuki Kuwaki

Reviewer #3: No

Reviewer #4: No

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[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.

OX2R-selective orexin agonism is sufficient to ameliorate cataplexy and sleep/wake fragmentation without inducing drug-seeking behavior in mouse model of narcolepsy

PONE-D-22-06089R2

Dear Dr. Yanagisawa,

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,

Alessandro Silvani, M.D., 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 #4: 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 #4: (No Response)

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

Reviewer #4: (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 #4: (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 #4: (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 #4: (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 #4: No

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