PONE-D-21-31823Ultrasound stimulation of the motor cortex during tonic muscle contractionPLOS ONE

Dear Dr. Heimbuch,

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.

==============================

Both reviewers provided detailed comments that need to be addressed. There should be more throughout discussion of relevant published papers (two were pointed out by the reviewers). The rationale for Experiment 2 as to why the chosen parameters are expected to produce offline effects should be more clearly stated. There is significant limitation with the low number of subjects which should be acknowledged or increase the number of subjects.

==============================

Please submit your revised manuscript by Dec 30 2021 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,

Robert Chen

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. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability.

"Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized.

Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access.

We will update your Data Availability statement to reflect the information you provide in your cover letter.

3. 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.

[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: Yes

Reviewer #2: Partly

**********

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

Reviewer #1: Yes

Reviewer #2: 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: 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: No

**********

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: Please see attached document for my review.

Reviewer #2: Heimbuch et al., examine the effect of TUS of M1 on corticospinal excitability and inhibition as measured by EMG. In Experiment 1 TUS was applied during (i.e., “online”) voluntary muscle contraction and EMG was assessed with a focus on the cortical silent period (CSP), compared to TMS induced CSPs. In Experiment 2, TMS motor evoked potentials (MEP) were measured before and after the same TUS protocol applied at rest to assess after-effects (i.e., “offline” changes) in corticospinal excitability. While the justification for Experiment 1 is clear, the justification for the protocol in Experiment 2 (with relatively long inter-trial intervals) is unclear, as there is no indication in the literature that such a protocol with TUS (nor TMS) would result in offline effects that outlast the stimulation. Nonetheless, Experiment 1 provides interesting null results about the (lack of) TUS effects on inhibitory interneurons at previously used parameters, which are relevant for the TUS community. However, the methods section requires several clarifications, and a number of statements seem to oversimplify conclusions from previous literature. I have the following specific comments.

1. Line 64 – we don’t know whether these intensities are unsafe. They just haven’t been used in humans yet.

2. Lines 61 to 69 – would be good to cite Fomenko et al. 2020 and Xia et al. 2021 which also looked at TUS effects on M1.

3. Lines 70 to 77 – it would be helpful to discuss the effects (or lack thereof) of TUS on SICI (Legon et al. 2018 and Fomenko et al. 2020).

4. Lines 81 to 82 – better to state explicitly here already that Experiment 2 is about the after-effects (offline effects) of TUS

5. In Experiment 1, was the order of TUS vs TMS blocks counterbalanced? Were there any sham/ control trials between TUS or TMS trials?

6. Lines 105-115 – It sounds like the motor hotspot search was entirely based on a couple for predefined targets and not really optimized iteratively to find the best coil position and orientation for good, stable, and well-formed MEPs. Is it possible that the optimal TME MEP hotspot was not determined and application of them more focal TUS at those locations was therefore ineffective? Please show the MEP waveform averages for all subjects in the supplement.

7. Lines 129 to 136 – was the TMS-induced MEP measured before and after an entire block of 20 TUS trials?

8. Lines 137 to 144 – What specific coupling medium was used (gel, gel pads, water bladder, etc.) and how was good coupling ensured in the presence of hair?

9. Line 139 – is the focus 3 cm long, or 3cm away from the transducer face? Please provide both values, in addition to the width of the focus.

10. Line 153 – what attenuation factor is used to estimate in the intracranial intensity? Was it the equation on line 254? Please also provide the Ispta.

11. Lines 169 to 182 – the descriptions of cSP onset and offset determination is difficult to understand. For instance, why would the ‘first rising EMG value’ be used as onset? Please try to use a figure to explain this. And also mention whether similar methods have been used in other papers.

12. Line 189 – why the mean of the two flanking valleys instead of the larger valley? Does this method have any advantages, and has it been previously used?

13. Line 210 – what is the criteria for determining ‘silence’? Is it different from the threshold used to determine cSP onset and offset?

14. Please specify the exact MR acquisition parameters for the anatomical MRIs that were used for neuronavigation and acoustic simulation. Please also provide examples of the skull masks overlayed on the subject specific MRIs.

15. Line 279 to 282 – what about the TUS trials?

16. Line 283 – can you please elaborate how ‘spike rate’ is measured? Is this the ‘rate of EMG peaks’ described earlier? Does this measure have any specific physiological significance?

17. Line 396 – it is likely that some early studies recorded responses to the acoustic rather than neuromodulatory effects of TUS. However, recent papers have confirmed that neuromodulation is also present (see for eg. Mohammadjavadi et al. 2019, Brain Stimulation). I recommend adding some nuance to this statement. On the topic of sensory confounds, did any participants in your study report auditory and/ or somatosensory perception accompanying the TUS trials? Have they been asked explicitly?

18. Lines 408 to 409 – again, I recommend adding some nuance to this statement. While TUS may preferentially stimulate inhibitory neurons at some parameter combinations, there is also evidence that it can stimulate excitatory neurons (see Yu et al. 2021, Nature Communications).

19. Fig. S3 – it seems like this figure shows fewer peaks in the TMS compared to TUS trials. I presume the amplitude of the peaks where higher in TMS?

20. Fig. 5 – Why are MEP AUCs shown on the y-axis as if they only had a very limited number of discrete values? Please alternatively also show (and used for statistics) the more well-established MEP peak-to-peak amplitudes.

21. Fig. 7. – from the caption it does not become clear what the difference between (a) and (b) is supposed to be. In (a), both TUS beams seem to have “hit” the central sulcus in the hand knob, if anything more post-centrally, whereas in (b) it looks like the focal spot is very anterior and rather in the premotor cortex. None of the presented foci seem to be particularly well targeting the anterior wall of the central sulcus where M1 corticospinal output neurons are located. Simulated acoustic pressure maps for all targets and should be presented for all subjects in the Supplement.

22. Fig. 7 – it looks like the skull image for the subj-01 is not complete. Maybe the FOV was cut-off? This has major implications for the quality of the simulations. Please discuss these limitations.

23. Some information seem to be missing in the data availability statement: “All EMG files are available from the XXX database (url: XXX). Code to run acoustic simulations is available via the MATLAB package TUSX (www.tusx.org).”

**********

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

[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.

Attachments

Attachment

Submitted filename: PONE-D-21-31823_review.docx

PONE-D-21-31823R1Ultrasound stimulation of the motor cortex during tonic muscle contractionPLOS ONE

Dear Dr. Heimbuch,

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.

Both reviewers felt that the paper has improved but there are still points that needs to be addressed. The control experiment is reasonable. The experiment should be performed or at a minimum the issues discussed.

Please submit your revised manuscript by Apr 15 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,

Robert Chen

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.

[Note: HTML markup is below. Please do not edit.]

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: (No Response)

**********

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

**********

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

Reviewer #1: Yes

Reviewer #2: 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

**********

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

**********

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: Review was uploaded as an attachment.

Reviewer #2: Tha authors have adequately addressed most of my concerns. I have only some minor comments remaining:

1. The heading exposure vs. excitability is confusing. A suggestion is to say ‘association between exposure and excitability’ instead.

2. Can you please provide some supporting evidence for this statement –

‘In a scenario of on ongoing voluntary motor activity, a subset of interneurons in M1 will have already been recruited, so perhaps the channels or cells that tUS would have affected are already being actively modulated by the time tUS energies into the tissue (e.g. channels have already been opened).’

For instance, is there evidence from TMS studies suggesting that inhibitory interneurons are already active during a voluntary muscle contraction?

3. Please include the information about counter-balancing and sham in the manuscript.

**********

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

[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.

Attachments

Attachment

Submitted filename: Round2_PONE-D-21-31823_review.docx

Ultrasound stimulation of the motor cortex during tonic muscle contraction

PONE-D-21-31823R2

Dear Dr. Heimbuch,

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,

Robert Chen

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: