PONE-D-22-16820Selective optogenetic activation of NaV1.7-expressing afferents in NaV1.7-ChR2 mice induces nocifensive behavior without affecting responses to mechanical and thermal stimuliPLOS ONE

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==============================The reviewers are convinced of the usefulness of the novel tool you developed, however they asked for better characterization of both the mouse line (integration specificity, locus characterization, expression faithfulness, etc. ) and depth of behavior characterization. Please address all concerns of reviewers in your revised manuscript.

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Tudor C. Badea, M.D., M.A., Ph.D.

Academic Editor

PLOS ONE

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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?

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: Partly

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

Reviewer #1: I Don't Know

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: In this study, the authors generated a new Nav-1.7-iCre knocking mice using CRISPR and crossed it to Ai32 reporter mice to have ChR2 expressed in Cre+ mouse DRG neurons. The authors then conducted VFH, thermal reflex, and peripheral light induced spontaneous behaviors and chamber preference assays. Overall, it's a new mouse line that is worth to be published, so the field is aware of the line and experimental results. However, there are major issues that the authors will need to fix during revision.

1) the manuscript title is misleading (or wrong). The mechanical and thermal tests (no deficits) have nothing to do with ontogenetic activation of Nav-1.7-Cre+ neurons. These are baseline behaviors. Thus, the title must be changed.

2) There is little data showing characterizing of the new Nav1.7-Cre line. The illustration showing the genomic structure, but no PCR or sequencing validating insert as anticipated. There is no co-staining of reporter (GFP) with endogenous Nav1.7 (or double in situ) or other common DRG neuron markers, CGRP, IB4, NF200. For the DRG section images, only GFP, and there is no quantification!!!

3) For peripheral light triggered paw withdrawal reflex, how do the authors know that it indicates pain response? Fos immunostaining with spinal cord sections after blue light stimuli would help to strengthen this point.

Other suggestion is to combine figures. Each current figure contains only one panel.

In addition to the experiments/data, the writing is also problematic. The description of generating Nav1.7 mouse line should be a main result section (the first part), which was put in the method instead. The results part overall is too short and over simplified. On the other hand, the discussion is lengthy and some parts (like Mrgd-ChR2 mice) are even irrelevant. The entire manuscript will need to be re-written.

Reviewer #2: The authors generated and characterized four genotypes of mice expressing ChR2 in Nav1.7-expressing primary afferent neurons. The four different genotypes show no difference compared to wild type animals in acute pain behaviour when exposed to von Frey filament stimulation and plantar test. However, the authors demonstrated nocifensive behaviour of these animals when stimulated with blue light, in a light intensity-dependent manner. The knock-in animals also displayed conditioned place aversion to blue light, as they spent less time in a room with a blue LED-illuminated floor, compared to a green LED-illuminated one. This work described a novel and valuable animal model to investigate further the role of the TTX-sensitive voltage-gated sodium channel Nav1.7 in a variety of pathological pain states. The experiments are clearly described and the conclusions are in line with the experimental results. This reviewer has only a few reservations concerning the general presentation of the experimental results and particularly the characterization of ChR-EYFP in DRG sections as presented in Fig. 6.

1. As a general observation, the Results section is extremely succinct, and the overall description of the data lacks quantitative detail. A more in-depth presentation of the actual results is warranted. For example, the authors limit their description of the nocifensive behaviour elicited by blue light to paw withdrawal (in Fig.3) and they do not discuss other manifestations which are generally associated with pain behaviour (paw licking, jumping, vocalization, etc…). Moreover, an important control is missing from the data shown in Fig.3: the experimenters should have used light of a different frequency (yellow light, at 590 nm, for example) at the highest light intensity used in the experiment, to rule out a ChR-independent effect of strong illumination on the animal behaviour. Finally, the light intensity used in the experiments illustrated in Fig.3 should be provided in mW/mm2, not in mW.

2. There is no attempt to quantify and characterise in more detail the expression of ChR-EYFP in DRG cell bodies in the sections illustrated in Fig.6. More information should be provided in terms of what percentage of neurons present YFP fluorescence, what is their average size, etc… It would also be of interest to dissect the expression of ChR-EYFP in peptidergic versus non-peptidergic neurons, using an anti-CGRP and/or an anti-P2X3 antibody, for instance.

3. It would be of interest to investigate and confirm the trafficking of ChR to peripheral and central endings of Nav1.7-expressing peripheral nociceptors, by monitoring YFP fluorescence in skin and spinal cord sections.

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Reviewer #1: No

Reviewer #2: No

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Selective optogenetic activation of NaV1.7-expressing afferents in NaV1.7-ChR2 mice induces nocifensive behavior without affecting responses to mechanical and thermal stimuli

PONE-D-22-16820R1

Dear Dr. MARUTA,

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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Kind regards,

Tudor C. Badea, M.D., M.A., 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 #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: Yes

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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: The authors have responded in a satisfying manner to this reviewer's queries. The paper can be published in the revised form.

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Reviewer #2: No

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