PONE-D-23-30357Pharmacological intervention of behavioural traits of autism spectrum disorder in a prenatal valproic acid-induced mouse model of autismPLOS ONE

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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: Partly

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: I Don't Know

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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

Reviewer #3: 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: No

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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: - A Figure is needed to show the affinities and selectivity of test compound betahistine.

- When discussing the involvement of histaminergic and/or cholinergic neurotransmission in the observed effects for betahistine, cite the following siginificant studies in your discussion;

1) Eissa N, Al Awad M, Venkatachalam K, Jayaprakash P, Thomas SD, Zhong S, Stark H, Sadek B. Simultaneous antagonism at H3R/D2R/D3R reduces autism-like self-grooming and aggressive behaviors by mitigating MAPK activation in mice. Int. J. Mol. Sci. 2023; 24:526.

2) Saad AK, Akour A, Mahboob A, AbuRuz S, Sadek B. Role of brain modulators in neurodevelopment: focus on autism spectrum disorder and associated comorbidities. Pharmaceuticals 2022, 15:612.

3) Eissa N, Venkatachalam K, Jayaprakash P, Falkenstein M, Frank A, Reiner-Link D, Stark H, Sadek B. The multi-targeting ligand ST-2223 with histamine H3 receptor and dopamine D2/D3 receptor antagonist properties mitigates autism-like repetitive behaviors and brain oxidative stress in mice. Int. J. Mol. Sci. 2021; 22(4), 1947.

4) Eissa N, Azimullah S, Jayaprakash P, Jayaraj RL, Reiner D, Ojha SK, Beiram R, Stark H, Łażewska D, Kieć-Kononowicz K, Sadek B. The dual-active histamine H3 receptor antagonist and acetylcholine esterase inhibitor E100 alleviates autistic-like behaviors and oxidative stress in valproic acid induced autism in mice. Int. J. Mol. Sci. 2020; 21(11), 3996.

- References to justify the selected and used doses of all test compounds are missing.

-Quality of all Figures should be improved, were difficult to read.

Reviewer #2: 1. The authors chose to treat the rats from PND44 to 64. Why this particular time window, frequency and duration of treatment? What is the authors' hypothesis when treating after weaning, when the neurodevelopmental period is mostly completed? Of note, relieving effects of treatment observed in nearly young adults are encouraging.

2. The term ‘Alteration’ and ‘alternation’ is written interchangeably in Y maze in methods and results section. It should be spontaneous alternation and not alteration. Correct it.

3. Data representation: histograms with scatter plots are highly recommended to allow the reader to assess the animal numbers per group and the dispersion of individual data at a glance.

4. Autistic mice: avoid using such wording; use the terms "VPA-exposed mice" instead.

5. In abstract it is mentioned that “After administration of the experimental doses, various locomotor tests: Open Field, Hole-Board, Hole Cross tests and behavioural tests: Y-Maze Spontaneous Alternation Test were performed and compared with positive and negative control groups”. What is the positive control group that is mentioned in this statement, which is nowhere mentioned in the rest of manuscript?

6. What is the significance of combining betahistine and rasagiline, why were both the drugs given in combination in T1 group? Kindly add the significance in the manuscript.

7. Authors have concluded that drugs have potential to ameliorate short-term memory deficits, it is recommended to perform other behavioral parameters for memory assessment like, Morris water maze, Radial Arm maze and Novel object recognition, to have better glance into the cognitive outcomes.

8. Furthermore it is recommended to report the histopathological changes in the appropriate regions of the brain of VPA exposed mice, using hematoxylin and eosin stain and Nissl stain, and also estimation of levels of acetyl choline and histamine in the VPA exposed mice and treated mice is highly recommended. These basic investigations will make the preliminary findings from this study more reliable and convincing for further research into these molecules and their mechanisms in ASD.

Reviewer #3: 1. In Lines 23 and 26 leave space after ‘p’ to be consistent. Apply this to the results section too.

2. Sometimes the authors mentioned autism and other times ASD. Please be consistent.

3. Why were the 2 mice genders used? Will this not have any impact on the behavior results?

4. I suggest that the second group of healthy offspring from the healthy mothers (NC2) to be Betahistine dihydrochloride of 3mg/kg in combination with 0.8mg/kg of Rasagiline to ensure that there are no off-target effects due to the combination of the drugs. This is important as especially there is no absence of in vitro profile of the combination of these drugs. Why the current NC2 is used?

5. Is there any results for the effects of Rasagiline only in VPA-induced mouse of ASD on all behavioral tests studied?

6. Line 343 mentions ‘In the study evidence in support of the claim was observed in the Negative control 2 group (NC2) whereby no significant change in short-term memory retention when compared with the Control group (C). To test for the neuroprotective effect of rasagiline, the effect of rasagiline only should be tested on an ASD mouse model of ASD. This group is missed.

7. The quality of all the figures is poor. Figures with high resolution need to be added.

8. In Figure 1, add the full name of the behavioral tests or mention them in the figure caption.

9. In Line 223, 224, and 343 the control and negative control 1 should all be in small letters as it is throughout the paper.

10. Elaboration is required on how betahistine showed anxiolytic and donepezil showed anxiogenic properties at different doses.

11. In Line 286 remove the bracket.

12. In Lines 292 and 292 ‘embryonic’ should be in small letters.

13. In 294 remove the extra space.

14. The authors should mention VPA-induced mice, not valproic acid-induced mice throughout the paper. The abbreviations should be described the first time in the text only.

15. In line 404, rephrase the sentence. There are no types of ASD patients.

16. Please check the manuscript for the English language.

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Reviewer #1: Yes: Bassem Sadek

Reviewer #2: No

Reviewer #3: Yes: Nermin Eissa

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Submitted filename: Comments PONE-D-23-30357.docx

Pharmacological intervention of behavioural traits and brain histopathology of prenatal valproic acid-induced mouse model of autism

PONE-D-23-30357R1

Dear Dr. Neelotpol,  

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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 #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 #2: Partly

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

Reviewer #2: 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 #2: 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 #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 #2: Authors have addressed all the queries and with the histopathological data, the data is more convincing. However, authors could have provided the histopathological data for each interventional group.

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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 #2: Yes: Kajal Rawat

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