Dear Dr. Paredes,

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.

Please submit your revised manuscript by May 01 2025 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.

• 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,

Usman Ghafoor

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. Thank you for stating the following financial disclosure:

“Research supported by PAPIIT UNAM IN214524 and CONCYTEQ/CACTI/094/2024 to RP.”

Please state what role the funders took in the study.  If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

If this statement is not correct you must amend it as needed.

Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf.

3. Thank you for stating the following in the Acknowledgments Section of your manuscript:

“The Laboratorio Nacional de Imagenología por Resonancia Magnética (LANIREM) for providing access to the magnetic resonance scanner. Our appreciation also goes to the Unidad de Órtesis y Prótesis de la Escuela Nacional de Estudios Superiores (ENES) Juriquilla and other institutions facilitating participant access. Daniel Arreguín, a PhD student in the Psychology program at the Universidad Nacional Autónoma de México (UNAM), received scholarship support (number 1084993) from the National Council for Humanities, Science and Technology (CONAHCYT). Research supported by PAPIIT UNAM IN214524 and CONCYTEQ/CACTI/094/2024.”

We note that you have provided funding information that is currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

“Research supported by PAPIIT UNAM IN214524 and CONCYTEQ/CACTI/094/2024 to RP.”

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

4. In the online submission form you indicate that your data is not available for proprietary reasons and have provided a contact point for accessing this data. Please note that your current contact point is a co-author on this manuscript. According to our Data Policy, the contact point must not be an author on the manuscript and must be an institutional contact, ideally not an individual. Please revise your data statement to a non-author institutional point of contact, such as a data access or ethics committee, and send this to us via return email. Please also include contact information for the third party organization, and please include the full citation of where the data can be found.

5. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information.

6. We note that there is identifying data in the Supporting Information file <file name>. Due to the inclusion of these potentially identifying data, we have removed this file from your file inventory. Prior to sharing human research participant data, authors should consult with an ethics committee to ensure data are shared in accordance with participant consent and all applicable local laws.

Data sharing should never compromise participant privacy. It is therefore not appropriate to publicly share personally identifiable data on human research participants. The following are examples of data that should not be shared:

-Name, initials, physical address

-Ages more specific than whole numbers

-Internet protocol (IP) address

-Specific dates (birth dates, death dates, examination dates, etc.)

-Contact information such as phone number or email address

-Location data

-ID numbers that seem specific (long numbers, include initials, titled “Hospital ID”) rather than random (small numbers in numerical order)

Data that are not directly identifying may also be inappropriate to share, as in combination they can become identifying. For example, data collected from a small group of participants, vulnerable populations, or private groups should not be shared if they involve indirect identifiers (such as sex, ethnicity, location, etc.) that may risk the identification of study participants.

Additional guidance on preparing raw data for publication can be found in our Data Policy (https://journals.plos.org/plosone/s/data-availability#loc-human-research-participant-data-and-other-sensitive-data) and in the following article: http://www.bmj.com/content/340/bmj.c181.long.

Please remove or anonymize all personal information (<specific identifying information in file to be removed>), ensure that the data shared are in accordance with participant consent, and re-upload a fully anonymized data set. Please note that spreadsheet columns with personal information must be removed and not hidden as all hidden columns will appear in the published file.

Additional Editor Comments:

Based on reviewers comments, it is difficult to accept the mansucript in the current form.

The authors are encouraged to rebuttal and make major changes to the mansucript for re-evaluation.

[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?

Reviewer #1: Partly

Reviewer #2: Partly

**********

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

Reviewer #1: No

Reviewer #2: No

**********

3. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: No

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: No

**********

Reviewer #1: “Cerebellar modifications of brain functional connectivity in individuals with lower limb amputation” by Arreguin, et al., reports on an investigation of functional connectivity differences between a group of 26 individuals with lower limb amputation, compared with 26 matched healthy control participants, using resting-state functional MRI. The reported finding of significant anticorrelation between a spherical region of interest designated as ipsilateral primary motor cortex and a spherical region of interest designated as contralateral cerebellar lobule VI may be of some interest.

However, it is not possible to recommend publication of the manuscript as submitted, for the following reasons:

1. The conclusions stated are not supported by the results presented, as most of the reported results fail to reach statistical significance.

2. The finding of significant anticorrelation does not appear to comport with the definition of functional connectivity, and thus requires proper interpretation.

3. The use of the “network-based statistic” (NBS) methodology to compute only correlations between pairs of regions, rather than larger networks, appears suboptimal.

4. The overall approach—the analytic methodology used, and the interpretation of results—neglects an ample literature indicating that amputation generally results in “massive functional reorganization in somatosensory cortex” (see below).

5. The submission is not in compliance with the PLOS ONE data sharing requirement.

Below we elaborate on these five points, and then list several other concerns.

MAJOR CONCERNS:

1. Most results fail to reach significance.

Table 3 lists "differences in ROI-to-ROI functional connectivity between [groups]" but only the first item survives correction for multiple comparisons, and is therefore statistically significant. The rest are not.

Thus, claims in the abstract that "statistical analysis revealed decreased connectivity in connections…” (note: “connections,” plural) and that "findings ... [highlight] a significant decrease in functional connectivity between the contralateral cerebellum and primary sensorimotor areas…” (note: “areas,” plural) are not supported by the results presented, because only one inter-regional connection was found to differ significantly between groups.

The use of ten regions of interest yields 45 total possible inter-regional connections, so using a p level threshold of 0.05, without correction for multiple comparisons, the analysis of pure noise would be expected to generate on average 0.05 x 45 = 2.25 apparent but not actually significant differences between groups. Of course, repeating this thought experiment with a fresh set of pure noise would yield a similar number of apparent but not truly significant differences between groups—but they would be overwhelmingly likely to be a different set of inter-regional correlations; such results would not be expected to replicate.

Table 3 of the current manuscript lists one correlation that is statistically significant (because it survives correction for multiple comparisons), plus five more, which are not statistically significant. That the authors report five such insignificant correlations, whilst the expected average number is 2.25, is of little interest. These same findings are graphically depicted in Figures 2 and 3; again, most of what is shown here is not statistically significant, and such insignificant findings cannot properly support any scientific conclusions.

Finally, the title of the manuscript is not supported by the results presented. Since only one “connection” is significantly different between the amputee group and the baseline group, the title’s references to “modifications”—plural—is not appropriate.

2. Anticorrelation isn’t connectivity.

The statistically significant finding is of an anticorrelation, but the negative sign—the finding of a negative correlation coefficient—is not mentioned or discussed. Functional connectivity is defined as synchronous neural activity in spatially remote locations, corresponding to correlation. However, the reported finding is an anticorrelation—a correlation coefficient with a negative sign. This does not comport with the definition of functional connectivity. Instead, it resembles the well-known temporal anticorrelation of activity in the default mode network with respect to activity in task-positive regions. The finding is of some interest, but its sign is important—the anticorrelation should be presented and discussed as such, not ignored and treated as if it were a positive correlation.

3. The network-based statistic is questionable when restricted to pairs of regions.

The network-based statistic is analogous to the (more familiar and well-established) cluster-based statistic. The idea of the cluster-based statistic is that rather than testing the null hypothesis at every voxel, we test the null hypothesis for every cluster of potentially active voxels. The advantage is more power; the disadvantage is that we don’t know just where in the cluster the activity is—we know there is activity in the cluster, but not precisely where in the cluster.

The network-based statistic is similar: Instead of testing the null hypothesis at every voxel, we test the null hypothesis for every network of voxels (or, more generally, nodes). This yields more power, but at the expense of not knowing just where within an “active” network the “activity” actually is. We can say the network is active (or significant), but not where within the network the activity or significance resides.

What is key in both cases is the use of clusters or networks of substantial size. It would be odd to use cluster-based statistics but limit analysis to clusters comprised of just two voxels. However, unless this reviewer is mistaken, the present manuscript appears to do this with the network-based statistic. It appears that only correlations between two regions are considered; these are networks comprised of only two nodes. This seems a suboptimal use of the network-based statistic.

4. “Remapping” has been ignored.

It is well-established that “Peripheral denervations of large extents of the sensory epithelium, as a result of limb amputation … can lead to massive functional reorganization in somatosensory cortex…” (from “Thalamic and Cortical Contributions to Neural Plasticity After Limb Amputation,” by Florence, Hackett, and Strata, J. Neurophysiology (2000) https://doi.org/10.1152/jn.2000.83.5.3154

Indeed, the introductory section of the present manuscript (p. 10) refers to “…post-amputation cortical functional reorganization…”

However, the analysis in the present manuscript ignores such functional reorganization, as it is limited to computing correlations between spatially-smoothed MR signal averaged over ten-cm spheres located in fixed positions in neuroanatomical atlas space. The “massive functional reorganization in somatosensory cortex” of the individuals with amputation is simply ignored. This is a suboptimal approach to analysis of these data. See also the final paragraph of the next item.

5. Data not shared.

Per PLOS ONE, data must either be submitted as part of the paper (as supplementary information), which is not practical for large imaging data such as used here, or the data must be placed in a public repository (such as NITRC). Stating that data are "available from" the senior author upon request (per page 6 of the PDF provided for review) is not adequate.

This reviewer endorses the PLOS ONE data sharing requirement, and asks authors to respect it—in general, for the sake of open science, which accelerates scientific progress.

Furthermore, in the present case, given point 4 above, it is to be anticipated that other researchers may seek to re-analyze the data, using, e.g., group spatial independent component analysis or multilayer community detection, to characterize between-group differences in cortical networks. Rather than assuming that cortical representations are fixed—in spite of an ample literature speaking to “massive functional reorganization in somatosensory cortex” of individuals with amputation—and comparing correlations between specified (atlas-based) spherical regions of interest, as the authors have done here, other researchers may be interested in assessing network spatial distribution and power (using spatial independent component analysis, or other algorithms) and/or network graph theoretic characteristics, such as segregation and recruitment coefficients (using multilayer community detection, or other algorithms). This makes data-sharing even more important.

OTHER CONCERNS:

6. Why were “[c]onnectivity differences … assessed controlling for the influence of age?” (pp. 16 & 18) What is the rationale for this? Were the study data also analyzed without controlling for age?

7. There is some concern about the procedure of flipping images of participants with right-side amputation (“images of participants with a right-side amputation underwent hemisphere inversion across the sagittal midline,” p. 14), since none of the pairs of regions of interest that were used are mirror-symmetric with respect to the midline (per Table 1). This suggests that such flipping could bias outcomes.

8. The slice-timing correction appears to have been performed late in the pipeline (p. 14), which raises concerns. Once images have been coregistered, realigned, and unwarped, performing slice timing correction is problematic, since the data no longer respect the acquisition geometry.

9. Re: “The use of NBS, relying on permutation tests, demonstrated reliability and robustness as an alternative…” (p. 19). This claim raises two concerns: A. No results presented in the present manuscript support the assertion that NBS “demonstrated reliability and robustness” in this study. B. It is not clear what “alternative” means here—what is NBS an alternative to? See also point 3 above.

10. The supplementary table indicates that all participants in both groups were right-handed; this should be stated plainly in the manuscript.

11. (minor) Re: “the Pearson correlation test” (p. 15). The Pearson correlation coefficient is not a “test.”

12. (minor) Re: “ROI-ROI connectivity maps were calculated for each participant…” (p. 15). These were matrices, not “maps.”

Reviewer #2: The study investigates functional connectivity alterations in the sensorimotor network of 26 lower limb amputees using resting-state fMRI. Results show decreased connectivity between the contralateral cerebellum and primary sensorimotor areas, suggesting neuroadaptive changes post-amputation. However, the reviewer has the following major concerns regarding the study’s methodology, data interpretation, and statistical analysis:

1. The manuscript does not provide a power analysis or justification for the sample size of 26 amputees. Given the complexity of functional connectivity analyses, this small sample may lack statistical power.

2. The study does not adequately control for potential confounders, such as years of education, prosthesis use, and amputation duration. These factors could significantly impact functional connectivity patterns.

3. The selection of 10 ROIs lacks a clear rationale. Why were these specific coordinates chosen, and how do they compare to previous studies? More details are needed on how these ROIs represent the sensorimotor network.

4. The analysis reports significant findings at p < 0.05 uncorrected, which is problematic given the number of comparisons performed. A stricter correction, such as Bonferroni or permutation testing, should be applied.

5. The manuscript attributes functional connectivity reductions to neuroadaptive processes without considering alternative explanations, such as motion artifacts or scanner-related issues. Additional validation is required.

6. A cross-sectional design limits interpretations. The manuscript should acknowledge that without pre- and post-amputation comparisons, it cannot definitively attribute changes to amputation itself.

7. Some statistical tests, such as those used for connectivity differences, are described in insufficient detail. The authors should clarify whether parametric or non-parametric methods were used consistently.

8. While the findings suggest connectivity changes, the manuscript does not link these to functional or behavioral outcomes. Claims about clinical significance should be supported by direct evidence.

9. Head motion is a major confounder in resting-state fMRI. The manuscript does not report framewise displacement values or whether high-motion participants were excluded.

10. The manuscript states that 5mm ROIs were used but does not justify this choice. Additionally, a 6mm smoothing kernel may dilute regional specificity, affecting results.

11. The manuscript reports hemisphere inversion for right-sided amputees but does not validate whether this step preserves functional connectivity patterns.

12. Some references (e.g., Koziol et al., 2014 on cognition and the cerebellum) do not directly support the claims being made and should be replaced with more relevant citations.

13. The TR and TE values are given, but essential details such as the field of view, voxel size, and number of volumes per run are not clearly reported.

14. The manuscript presents connectivity findings in Figures 2 and 3 but does not provide full statistical details in figure legends (e.g., exact p-values for non-significant findings).

15. The introduction states a broad hypothesis that connectivity should decrease in amputees, but it does not specify which regions or connections are expected to be affected.

**********

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.

<p>Cerebellar functional connectivity alteration in individuals with lower limb amputation

PONE-D-24-44413R1

Dear Dr. Paredes,

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 will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager®  and clicking the ‘Update My Information' link at the top of the page. For questions related to billing, please contact billing support .

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,

Usman Ghafoor

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

The authors have adequately improved the manuscript that warrants its publication.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: (No Response)

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #1: Partly

Reviewer #3: Partly

**********

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

Reviewer #1: I Don't Know

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: No

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #3: Yes

**********

Reviewer #1: Please make the data public, so that other investigators can use it to investigate functional remapping.

Reviewer #3: The authors have thoroughly addressed and resolved all the concerns raised in the previous round of review. They have provided detailed clarifications, strengthened the methodological explanations, and incorporated the suggested revisions throughout the manuscript. Based on the improvements made and the enhanced clarity and rigor now reflected in the revised manuscript, the reviewer is satisfied that the work meets the necessary standards for scholarly publication. Therefore, the reviewer endorses the manuscript for acceptance and recommends proceeding with publication.

**********

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 #3: No

**********