PONE-D-22-16443A Stable Five Days of Hypolocomotion and Abnormal Swimming Pattern in 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine (MPTP)-Induced Zebrafish Model of Parkinson’s DiseasePLOS ONE

Dear Dr. Mohamed,

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.

Dear Dr Wael Mohamed,

You will find enclose 4 reviews for your manuscript. They all find merit to your work. From their commentaries, you will see that no additional experiments are presently required, but we will ask you to carefully take into consideration all their questions and remarks, and more precisely to rigorously document responses to reviewers 3 and 4 comments.

Sincerely,

Prof Bruno Giros, PhD

McGill University

Academic Editor

Please submit your revised manuscript by Sep 08 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,

Bruno Giros, 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. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.

3. Your ethics statement should only appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please move it to the Methods section and delete it from any other section. Please ensure that your ethics statement is included in your manuscript, as the ethics statement entered into the online submission form will not be published alongside your manuscript. 

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

Reviewer #3: Yes

Reviewer #4: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: 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

Reviewer #3: Yes

Reviewer #4: No

**********

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

**********

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 work presented by Khairiah Razali et al. aims at describing the impact of MPTP treatment in zebrafish. They analyzed the effect of the drug on different locomotor assays in zebrafish before and after 24h and 96 h. They suggested that the treatment induced locomotor deficits that may be relevant to Parkinson’s disease. This topic is important since there is a lack of in vivo preclinical model that are mimicking human pathology, thus pertaining the development of therapeutic strategies. Overall, the experiment are well conducted and the results are of importance.

However, I have some recommendations.

1) The authors claim that treatment with MPTP would induce visual deficit that could explain some of the locomotor behavior. In order to address this important question, I would suggest the authors to realize optokinetic response experiment to determine if MPTP treatment affects visual acuity.

2) The observed phenotype with the turn angle, the meander and the angular velocity necessitate prosper discussion with relevant citations in order to strengthen their conclusion on the Parkinson’s disease phenotype.

Reviewer #2: A good manuscript.

Title needs some rephrasing

Discussion needs to address better some other toxicological and biochemical mechanism involved in this

Results are decent and well worked

Introduction looks ok.

Methodology also fine, with no flaws detected.

Reviewer #3: General comments (page numbers refer to the numbers at the bottom of the docx-file, line numbers to the numbers on the left margin of the document):

In the submitted MS Razali and coworkers report the behavioral change by a single dose of MPTP intraperitoneally administered to the adult zebrafish 1 and 4 days after injection. This is a methodologically sound and thorough study and the novel insight is basically the stability of behavioral changes between these time points, except a loss of increased freezing bouts at 4 days (see also specific point to page 12 below). This finding might be important for the use of the MPTP-model in zebrafish in the context of neuroprotective studies considering the well-known reversibility of the neurodegeneration induced by MPTP in this species. Due to the focus of this rather methodologically circumscribed study, all discussions of mechanistic aspects of MPTP in the zebrafish are redundant (page 18, line 417 to page 20, line 458), which means that the discussion should be shortened considerably and shifted to a more thorough comparison of the specific behavioral findings to that reported in the literature (e.g. comparison to the study of Anichtchik OV et al., J. Neurochem. 2004).

Specific points:

Page 3, line 54:

Who says that cholinergic neurons are lost in the substantia nigra in PD?

Page 3, line 55:

A review on the promise Zebrafish in PD is not an appropriate review on the basic characteristics of the pathology of PD

Page 8, line 176-182 or page 9, line 196-202:

Please specify the geometry of the IN and OUT zone within the recording tank.

Page 9, line 196-202:

Please specify the definition of bouts in the context of freezing behavior.

Page 9, line 196-202:

Please specify the definition of frequency in the IN zone in the context of swimming pattern.

Page 12, line 262-264, page 12, line 273-275 (legend to Fig.5):

Is there no statistical significance between 96h and 24h in Fig.5b for MPTP-injected fish?

Page 13, line 287 (legend to Fig.6):

Please specify for frequency in the IN zone during which time the numbers are counted (similarly to the way the number of freezing bouts are described in the legend to Fig.5b).

Page 18, line 422/423:

The wording suggests that reference 36 (Miskitsh & Chacko, 2014) is reporting data about MPTP, which is not the case

Page 18, line 424 to page 19, line 429:

It is unclear why just reference 37 (Risiglione et al., 2022) is especially relevant for the degenerating effect of MPTP on dopaminergic neurons, a neurotoxic mechanism elucidated by studies decades earlier.

Page 19, line 440-441:

Reference 38 (Zinger et al., 2011), a review about neuroinflammation and kynurenine in PD, is not an appropriate reference for the indirect pathway in basal ganglia, first described decades earlier.

Page 19, line 450-452, page 20, line 454-456:

Reference 41 (Huang et al. Parkinson’s disease 2017) is not a specific rodent study, but a review about changes in the MPTP mouse model.

Page 23-26, References:

Please give more complete information for cited books.

Reviewer #4: This study investigates the neurobehavioral toxicity of a single i.p. injection of MPTP, a model chemical for Parkinson's disease, in adult zebrafish. The main merit of this study is a complete characterization of behavioral endpoints at 0, 24h, and 96h post MPTP injection. The behavioral analysis was rather comprehensive and in great details. The main pitfalls are 1) both male and female adult fish were used in this study, but no mention about whether there was any sex difference in all these behavioral parameters; and 2) lack of histopathological examination of the MPTP-treated brain to confirm any DA neuron loss, though authors acknowledged this in their Discussion. A minor comment is that there are a few recent MPTP studies using the zebrafish were not cited and incorporated in the Discussion, for example: https://pubs.acs.org/doi/abs/10.1021/acschemneuro.2c00089

https://www.sciencedirect.com/science/article/abs/pii/S0161813X22000997?via%3Dihub

**********

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

Reviewer #3: No

Reviewer #4: 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.

Characterization of Neurobehavioral Pattern in A Zebrafish 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine (MPTP)-Induced Model: A 96-hour Behavioral Study

PONE-D-22-16443R1

Dear Dr. Mohamed,

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,

Bruno Giros, 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 #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

Reviewer #4: 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 #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: 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

Reviewer #3: Yes

Reviewer #4: 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

Reviewer #3: Yes

Reviewer #4: 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: Accept. The answers are ok. Ms looks ok for publication now, in my humble opinion.

Accept. The answers are ok. Ms looks ok for publication now, in my humble opinion.

Reviewer #3: All my suggestions were met by appropriate changes. The Discussion increased by about 30 lines; if the Editor considers that a problem, the mechanistic aspects that I already considered redundant in my review of the original submission could be shortened, e.g. page 21, line 474-line 485.

Reviewer #4: The authors have adequately addressed all my previous comments. I have no further comments for this revised version.

**********

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

Reviewer #3: No

Reviewer #4: No

**********