Sex-Related Variations in the Brain Motor-Network Connectivity at Rest during Puberty

Round 1

Reviewer 1 Report

The manuscript entitled “Sex related variations in the brain motor network connectivity at rest during puberty” submitted by Pacheco-Blas et al aims at revealing functional morphological shifts along the cerebellar-mouth and cerebellar-hand motor subnetworks, seemingly, under the presumption that puberty might induce male/female sex differences/biases. Interestingly, their results revealed that puberty-associated functional morphological shifts along both motor cerebellar subnetworks are very much alike in pubertal women and men at the ages of 12 or 16 years; this is true even for those measures that seem to differ statistically in which the overall distribution of the probability density is essentially the same between sexes (but not between ages within sexes mainly for the cerebellar-mouth motor network). These observations provide fundamental support to current scientific trends that clearly show that the female and male brains are more alike than categorically different.  I thus find this work fascinating and welcomed.

Despite my obvious enthusiasm, I would like to pinpoint a handful of niches where improvements must be done before this work can be accepted for publication. They are not minor but major. I will comment on them in the following paragraphs:  

 Abstract:

Instead of beginning with a line referring to technical aspects indeed irrelevant to the main issue explored in the paper, I suggest authors to go directly to the topic of interest. They must briefly describe the phenomenon they are interested in explaining/exploring, and then to introduce the premise or premises of the conceptual model they are trying to address through their work. They could use an introductory phrase such as: “Neurobiologists have long entertained the idea that the brain is fundamentally different between women and men, especially in areas where sexual behavior is presumed to be controlled.  Recent evidence, however, supports otherwise (please review literature published by Daphne Joel and co-workers). Here, we tested whether motor pathways not necessarily involved in sexual functions, differ between men and women in puberty, when hormonal changes may accentuate sexual differences”. Then, the authors can follow the summary already written, and end it with a coherent conclusive sentence. The way they are currently finalizing the summary is overtly irrelevant.

Introduction:

Overall, the introduction seems to me a bit out of focus. I found the first paragraph unnecessary and the second, third, fourth and fifth paragraphs lengthy and disconnected. There is neither a scientific problem clearly identified, nor a conceptual model intended to be evaluated. Fragments of information is all I see. I would recommend strongly to the authors to re-write the introduction following the guides I provided for the abstract.  As it stands now, the introduction lacks scholarly and epistemic quality. There must be, at least, a hypothesis somewhere in the introductory remarks as the natural consequence of introducing a conceptual model with heuristic value.

Materials and Methods:

I noticed that the authors work for the Universidad Nacional Autónoma de México. The data set evaluated, however, was not primarily obtained by the researchers of this institution. There are no explicit credits to the original authors. Is this because the data base is an open source as disclosed in the Data Availability Statement? If so, authors must introduce the link and access code to the data base in the corresponding section.

Please, if possible, disclose inclusion/exclusion criteria and demographic information of the sampled population.

Would it be possible to run a principal component analysis including all the parameters estimated? I believe it would be informative since it might provide a nice visualization on the diversity of the individuals studied.  Besides this, I have no other technical or statistical concerns to comment on.

Results:

I found the figures and text adequate and clear. No concerns whatsoever

Discussion:

Once again, overall, I found the discussion devoid of a central topic; it lacks a plot line pretty much as the introduction. I would suggest authors to concentrate their discussion on the grounds of sexual brain differences (emphasizing the “no major differences side”). The first paragraph is somewhat incomprehensible to me. First, to stand that puberty is the most crucial stage in sexual development because sexual hormones (which, by the way, also have a variety of non-sexual actions) is untenable. Authors, in fact, report data in areas that are not sexual. This first paragraph is rather misleading and misconstrued.  

If puberty (adrenarche and gonadarche) is the essential theme (second and third paragraphs), then, this must be framed adequately in the introduction since (1) it appears that these puberty’s phases influence the decision on what age groups will be included in the study, and (2) authors seemed to be expecting sex and age effects, for an unclear reason. Please, disclose this reason.

The assertion that “The differences observed in our results between the MsN connectivity of males at age 12 and 16 were associated to the high levels of DHEAS and testosterone” (lines 502-503) is speculative and must be tone down since authors did not provide their own data replicating this fact”.

Authors comment that “At the age of 12, there were higher node strength values in the male group than 527 female group. Differential connectivity was expected due to the differential onset of 528 puberty phases [37]” (lines 528-529). This is a rather simple way of looking at biological processes. I could ascribe such differences to distinct individual developmental rates (heterochrony) along their developmental trajectories (principal component analyses could well address this issue since it will reveal the degree of diversity). It is unthinkable that most individuals within age and/or sex groups are developmentally synchronized.  I understand that categorical thinking helps interpreting some of the results, but biology indeed is far from categorical. Diversity must be explored, at least theoretically, in this work. In fact, I see much more similarities between sexes, than disparities, in most of the analyses presented.  

I truly believe that describing the assumed sex differences (that I do not really see) to sex hormones is an over statement. To really address this issue, authors must present their own hormonal data and to run a principal component analysis including all variables. If they could demonstrate that sex categories indeed are present, then their presumptions may have better support. I could do the same assertion to age differences.

It is interesting that authors do noy relay on puberty to explain trends in hubness. They seem comfortable of having replicated a previous study to be confident about their results. However, no explanation is given whatsoever, and we do not know whether the study cited is like theirs. Once again, differential developmental timing may explain hubness trends in this study. Although no sex differences were found…why?

In brief, I would very like to see a more synthetic and focused discussion. Please, while rewriting the discussion, pay attention not only to the values obtained based on central tendency measures, but also to the data distribution. This last measure will provide a less excuses to think categorically and will open the non-categorical, much more variable box of reality.

Conclusion:

Conclusion must be re written. Nothing has been done to claim social influences. We do not even have data on demography!!! Authors comment that MRI acquisition could have impose stress on participants. This is another variable that was not discuss a source of variation. The entire conclusions section is highly speculative. Please, restrict this section to described briefly the most significant findings and provide a sentence on their significance. Avoid speculations and wishful thinking.

Important: English editing is required.

Moderate editing is required 

Author Response

Author Response File: Author Response.docx

Reviewer 2 Report

I would like to extend my appreciation for the excellent work you have presented in your article titled "Puberty-Induced Changes in Motor Network Connectivity: A Study on Sex and Age Variation." Your research has provided valuable insights into the effects of puberty on motor network connectivity in males and females, shedding light on an important aspect of sexual development. I have carefully examined your study and identified several areas that could benefit from further clarification and improvement. please see the document in attached files 

Comments for author File: Comments.docx

minor  revisions for english

Author Response

Reviewer 2

(all changes in the text are in red colour as well as the answers)

I would like to extend my appreciation for the excellent work you have presented in your article titled "Puberty-Induced Changes in Motor Network Connectivity: A Study on Sex and Age Variation." Your research has provided valuable insights into the effects of puberty on motor network connectivity in males and females, shedding light on an important aspect of sexual development. I have carefully examined your study and identified several areas that could benefit from further clarification and improvement. please see the document in attached files 

R.- Your words are greatly appreciated, and we strive to follow your suggestions and observations.

The introduction provides a comprehensive overview of the topic, and the scientific content appears well-organized. However, there are a few suggestions to improve the English and scientific style:

Sentence restructuring: Some sentences could be restructured to improve clarity and flow. For example, in sentence 36, "including the brain in humans" could be rephrased to "including the human brain."

R.- We agree, it has been changed

Sentence fragmentation: In sentence 44, "Although basic cognitive 45 skills develop during childhood" should be combined into one sentence for better readability.

R.-We agree it has been changed

Missing word: In sentence 80, "which 79 is perhaps the most extensively connected" should include the missing word "is" between "which" and "79."

R.-We agree it has been changed

Clarification of information: Some parts, like in sentences 47-51, may benefit from further elaboration or clarification to provide a clearer understanding of the information.

R.-We agree a sentence has been added to clarify the importance of the motor network for motor control. (lines 51-53)

Scientific clarity: In sentence 81, the phrase "a body subjected to continuous hormonal changes as well as 81 receiving constant proprioceptive information" could be further explained to enhance scientific clarity.

R.-We agree, and we add some modifications and a sentence about proprioception. (84-88)

Line 82 Please Write FRMI In All

R.-We agree it has been corrected

Line 96 Please change the reference Sinclair et al., 2015) into the journal guidelines

143-150, graph 1 and table 1 please separate them by a paragraph

R.-We agree Figure 1 has been moved at the end of section 2.5 and Table 1 at the end of section 2.6

Material and methods

Paragraph repetition: There is some repetition in the "Network analysis" subsection (2.7), where the same information is presented multiple times in the paragraph. It appears to be unintentional and could be edited for clarity and conciseness.

R.-We agree. Section 2.7 has undergone revision and correction.

Sentence restructuring: In some parts of the section, sentence structures can be improved to enhance readability and flow.

R.-We apologize and revise the manuscript to improve readability and flow.

Results

One potential area of improvement is the clarity of the text. The section could benefit from better structuring of the paragraphs and sentences. Additionally, some sentences appear to be cut off or incomplete, which may need to be corrected.

R.-We agree and apologize again. MS Ibrahim Ramirez, from the Department of Kinesiology at the University of Waterloo, Canada, has proofread this version and is mentioned in the Acknowledgements section.

Discussion

The discussion could benefit from clearer paragraph divisions to separate distinct topics, Some sentences could be restructured to improve flow and clarity. There are some punctuation errors, such as missing commas or inconsistent use of punctuation.

R.-We agree and apologize again. MS Ibrahim Ramirez, from the Department of Kinesiology at the University of Waterloo, Canada, has proofread this version and is mentioned in the Acknowledgements section.

Author Response File: Author Response.docx

Reviewer 3 Report

General comments

1. The introduction is too general. In it, it is necessary to more explicitly present the concrete research problem and the hypotheses or research questions derived from it (research goals, what we want to achieve with them).

2. The general descriptions are pretty detailed, while the specific ones relating to the research setting could be more organised, complete, and often more comprehensible. The whole article needs a clear common thread.

3. The essence of scientific publications is reproducibility and the possibility of checking the results, so concrete explanations of the research process are necessary.

4. The results must be related to the hypotheses (research questions), and these results must be clearly explained in the Discussion. A connection must be established: Hypothesis - result - explanation of the result and, in Conclusion, what is good/bad, how to eliminate shortcomings and development guidelines.

5. Otherwise, you mention voluntary inhibition of movement; we recognise the activity of the motor network. This premise is probably your primary research question. Still, it needs to be better explained throughout the article. It is confusing and challenging for the average reader, who may not be fully connected with similar research understandable since the entire paper does not have delineated research.

Some particular comments

2.5

The functional Power Atlas was selected for this study [24]. A nilearn python [25] 129 code was used to select 5 mm spheres with centers according to the 264 regions. The time-130 series were extracted using a high pass filter, and motion, WM-CSF and global signal 131 

6. Question: confounds -  how, explain the connection with python code (nilearn is only an open-source library)  and selection process, which time series and which high pass filter, etc. All is a bit confusing, needs additional explanation

2.6

The matrices were thresholded to exclude 135 negative correlation values in all subjects at a density of 0.46. 

7. Question: Explanations needed, how you are thresholded matrices (what mean thresholded in your case), where come from the density of 0.46, etc.

The analysis was then 136 limited to the motor subnetwork - explain why and how you are choose mentioned regions and Pearcon correlation (measures the strength of the linear relationship between two variables - 

8. Question: in your case, which one?, or? for your example.

9. For what is Table 1 important? Is it really necessary to understand your research?? x, y, z - what represents in which unit (unit of measurement??)

2.7

Centrality measures of hubness and leverage were calculated for the entire network 165 for each study group. - what mean study group is one (group) or more (groups) -

10. Question:  explain, Some kind of "conclusion" is needed in this subchapter.

11. You have two 2.7 subchapters so the second one must be 2.8. 

2.8. 

continuous random variable - explanation, PDF - 

12. Question: all subchapter is confusing and needs additional explanations.

The cerebellar mouth and cerebellar-hand MsN were also analyzed as described 197 above. 

13. Question: What does this mean?? Connection with all others?

Results

14. Question: What is in abscise and ordinate in all figures (units, ??)  The Figures must be displayed in their entirety so we can understand them independently! The picture must have a title, and all other comments do not belong on the picture (explanations below it or in the Discussion)

15. In conclusion, when the entire MsN was evaluated, there was a statistically significant 220 difference in the node strength between male and female groups at both ages. That is, by 221 age 12, the male group's node strength values are greater than those in the female group 222 while differences at age 16 are not significant. 

You must decide, is it or is it not significant? This is only one example; check all results and commenters.

Conclusion

The combined sex and age variation we identified in our investigation is interesting 660 because it shows connectivity differences between males and females during early 661 puberty at ages 12 and 16, when a social context begins to influence sexual behavior. - 

16. Question: Otherwise, you mention voluntary inhibition of movement; we recognise the activity of the motor network. This premise is your primary research question. Still, it needs to be better explained throughout the article. It is confusing and challenging for the average reader, who may not be fully connected with similar research understandable since the entire paper does not have delineated research.

Author Response

Reviewer 3

(all changes in the text are in green colour as well as the answers)

Principio del formulario

1. The introduction is too general. In it, it is necessary to more explicitly present the concrete research problem and the hypotheses or research questions derived from it (research goals, what we want to achieve with them).

R.- Firstly, we would like to thank you for your time going through this revision.

From the title of the work “Sex-related variations in the brain motor-network connectivity at rest during puberty”. We exposed in the Introduction the aspects of the literature that we considered important to follow the goals of our study. To start with, there is a difference when we refer to puberty and adolescents, we would like to set it clear that at this point we are not into studying any psychosocial characteristics of the individuals’ data we analyzed.

We modify some sentences to be clearer about our objective at the end of the introduction.

“Our goal is to investigate whether age-sex differences in the so-called state of rest with eyes closed show differences in the brain's motor network connectivity studied by fMRI. Due to the critical role of sexual hormones in human development related to reproductive behaviour, and the fact that proprioceptive stimulation cannot be suppressed while resting. Inactivating voluntary movement is an active state in motor areas related to motor control”.

Our study also aimed to provide a standard that exalts the characteristics of the resting state with eyes closed in adolescents at the onset of puberty when studied by imaging techniques. Considering that there is a growing need to study brain function at this stage of life. Due to the rise in mental illness in adolescents, it is needed to analyze variations when it comes to looking for changes compared to control.

2. The general descriptions are pretty detailed, while the specific ones relating to the research setting could be more organised, complete, and often more comprehensible. The whole article needs a clear common thread.
3. The essence of scientific publications is reproducibility and the possibility of checking the results, so concrete explanations of the research process are necessary.

R.-The data is available to the public and the processing is described as clearly as possible, citing specific references. On page 19 of the Data Availability Statement, it was mentioned that code availability would be shared upon request.

4. The results must be related to the hypotheses (research questions), and these results must be clearly explained in the Discussion. A connection must be established: Hypothesis - result - explanation of the result and, in Conclusion, what is good/bad, how to eliminate shortcomings and development guidelines.

R.-Thank you for your interest that this gets as clear as possible. We take care of making a general discussion of the results in every results section.

Section 3.1 conclusion was written in lines 253-258

Section 3.2 conclusion was written in lines 299-302

Section 3.3 conclusion was written in lines 233-335

Section 3.4 conclusion was written in lines 268-371

Section 3.5 conclusion was written in line   404

Section 3.6 conclusion was written in lines 439-441

Section 3.7 conclusion was written in lines 475-478

We separated four primary areas we wish to explore based on the results in the Discussion section. Each section is discussed separately and is supported by earlier study work from which we provided references. In all situations, the fundamental goal is to provide anatomical context for our network analysis, which is directly connected to neurodevelopment during puberty. 

We aim to make it obvious that we have no preconception for or against finding differences owing to sex, but rather disclose the properties of the motor subnetwork as a benchmark for future research.

We made some changes to the discussion that may help to make it clearer. In addition, the MS I. Ramirez from the Department of Kinesiology at the University of Waterloo proofread the manuscript to make it more legible and fluent.

5. Otherwise, you mention voluntary inhibition of movement; we recognise the activity of the motor network. This premise is probably your primary research question. Still, it needs to be better explained throughout the article. It is confusing and challenging for the average reader, who may not be fully connected with similar research understandable since the entire paper does not have delineated research.

R.-Yes, you are correct. We try to make a confusing concept clearer.

When studying motor-network connections across the entire brain network, it can be highly confusing. Because this subnetwork, which is in charge of motor control, is maybe the most connected network in the entire brain.

In the case of brain imaging research employing a resting state with eyes closed, this network is assumed to be unconnected. That is why we chose to examine it independently and study its variations in the onset of puberty in both sexes.

Some particular comments

2.5

The functional Power Atlas was selected for this study [24]. A nilearn python [25] 129 code was used to select 5 mm spheres with centers according to the 264 regions. The time-130 series were extracted using a high pass filter, and motion, WM-CSF and global signal 131 

6. Question:confounds -  how, explain the connection with python code (nilearn is only an open-source library) and selection process, which time series and which high pass filter, etc. All is a bit confusing, needs additional explanation

R.-We have added a paragraph in section 2.5 (pag. 3) to answer your concern.

“The nilearn function input_data.NiftiSpheresMasker was used to create 5mm sphere masks with a FWHM smoothing filter of 6 mm and a band pass filter between 0.009-0.08Hz, and tr=0.930s. The time series were extracted using the function parcellation.fit_transform with confounds strategy using “high_pass", "motion", "wm_csf" and  "global_signal".

2.6

The matrices were thresholded to exclude 135 negative correlation values in all subjects at a density of 0.46. 

7. Question: Explanations needed, how you are thresholded matrices (what mean thresholded in your case), where come from the density of 0.46, etc.

R.-The sentence was rephrased (page 4, section 2.6) to make it clearer.

“In order to use the highest possible density of only positive values, the correlation matrices were thresholded in order to keep a connectivity density of 0.46.”

The analysis was then 136 limited to the motor subnetwork - explain why and how you are choose mentioned regions and Pearcon correlation (measures the strength of the linear relationship between two variables - 

8. Question: in your case, which one?, or?for your example.

R.-Because of a particular interest to study motor-related regions, the analysis was restricted to atlas’ Networks labeled ‘Cerebellar’, ‘Sensory/Somatomotor Hand’ and ‘Sensory/Somatomotor Mouth’. 

Pearson correlation is widely used and a very standard procedure, thus making it comparable to other works. Also, it’s simple and low computational cost is required.

9. For what is Table 1 important? Is it really necessary to understand your research?? x, y, z - what represents in which unit (unit of measurement??)

 R.-It specifies the coordinates of the regions used in mni coordinates, page 5 line 162 at the end of the table.

2.7

Centrality measures of hubness and leverage were calculated for the entire network 165 for each study group. - what mean study group is one (group) or more (groups) -

 R.-There was a grammar mistake, The line was modified to:

“, a region was considered to be a hub if the hubness score was equal or greater than 2 and at least 45% of the subjects in the group had this region as a hub.”  p5 line 180

10. Question:  explain, Some kind of "conclusion" is needed in this subchapter.

R.-It must be a misunderstanding, we have not made any conclusions in the methods section 

11. You have two 2.7 subchapters so the second one must be 2.8. 

R.- Thank you for your observation, it was corrected

2.8. 

continuous random variable - explanation, PDF - 

12. Question: all subchapter is confusing and needs additional explanations.

R.-Each subchapter in the methods section has a self-explanatory title.

Whenever is needed an extensive explanation on methodology we referred to and specific research work, all listed in the references.

Regarding explanation of PDF, it was defined and described in - p6, section 2.8, line 198

The cerebellar mouth and cerebellar-hand MsN were also analyzed as described 197 above. 

13. Question: What does this mean?? Connection with all others?

 R.-Analogous analysis to the described in section 2.7 was conducted in the cerebellar-mouth and cerebellar-hand networks.

Results 

14. Question: What is in abscise and ordinate in all figures (units, ??) 

R.-Probability density functions (PDF) as well as node strength values has been described under the methods section and are adimensional. They are also defined in the caption figures.

 The Figures must be displayed in their entirety so we can understand them independently! The picture must have a title, and all other comments do not belong on the picture (explanations below it or in the Discussion)

R.-We agree to add Titles inside the Figures. All other information which describes the figure is detailed under the figure caption.

15. In conclusion, when the entire MsN was evaluated, there was a statistically significant 220 difference in the node strength between male and female groups at both ages. That is, by 221 age 12, the male group's node strength values are greater than those in the female group 222 while differences at age 16 are not significant. 

You must decide, is it or is it not significant? This is only one example; check all results and commenters.

R.-We agree it was corrected in line  231

Conclusion

The combined sex and age variation we identified in our investigation is interesting 660 because it shows connectivity differences between males and females during early 661 puberty at ages 12 and 16, when a social context begins to influence sexual behavior. - 

16. Question: Otherwise, you mention voluntary inhibition of movement; we recognise the activity of the motor network. This premise is your primary research question. Still, it needs to be better explained throughout the article. It is confusing and challenging for the average reader, who may not be fully connected with similar research understandable since the entire paper does not have delineated research.

R.-We changed the conclusion to emphasize the goal of this work centre in the activity of the motor network.  We deeply appreciate your feedback.

Submission Date

22 July 2023

Date of this review

04 Aug 2023 15:44:35

Final del formulario

© 1996-2023 MDPI (Basel, Switzerland) unless otherwise stated

Author Response File: Author Response.docx

Reviewer 4 Report

Authors have studied the sex-related variations in the brain motor-network  connectivity at rest during puberty. The paper needs to be improved in the following ways.

1. A proper block diagram is needed for better understanding of the reader.

2. The network analysis is to be written

3. In fig 2 the changes need to be highlighted by different colors

4. It will be good for the readers if the authors can able to identify the changes in male and female along with the physical changes during puberty.

5. The reference are not properly mentioned. 

Needs improvement. 

Author Response

Reviewer 4

(all changes in the text are in navy blue colour as well as the answers)

Authors have studied the sex-related variations in the brain motor-network  connectivity at rest during puberty. The paper needs to be improved in the following ways.

R.- First and foremost, we would like to thank you for your time spent revising, which will result in a better manuscript.

1. A proper block diagram is needed for better understanding of the reader.

R.-We have given description to the flow diagram inside the figure (Figure 1) to make it easier to follow

2. The network analysis is to be written

R.-It was fully described in section 2, under Materials and Methods including all related references.

3. In fig 2 the changes need to be highlighted by different colors

R.-We included Figure 2C, with the Principal Component Analysis (PCA) of each group coloured differently.

4. It will be good for the readers if the authors can able to identify the changes in male and female along with the physical changes during puberty.

R.-It is undeniably interesting, and one of the goals of this work is to establish a standard of healthy adolescents at the start of puberty that could be used to compare physiological and pathophysiological conditions in teenagers, especially those related with behavioural disorders.

5. The reference are not properly mentioned. 

R.-Thank you for your observation. Reference 18 was missing and we corrected this problem.

Round 2

Reviewer 1 Report

I have no further comments or concerns 

Minor English editing 

Author Response

Dear Reviewer 1,

We appreciate all your comments, we will definitely be alert for the analyses that we have pending on the subject.

Reviewer 3 Report

The article has seen improvements and is more understandable and easier to read. Specific answers to the questions are given, some, probably with the author's point of view, less important ones, but not the article is acceptable.

Author Response

Dear reviewer,

Thank you for taking the time and interest to review our manuscript, in you comment;

"The article has seen improvements and is more understandable and easier to read. Specific answers to the questions are given, some, probably with the author's point of view, less important ones, but not the article is acceptable."

Did you mean not or now?

Thank you again,  

Dr. Alicia Ortega