PONE-D-20-08431

Induction of fatigue-like behavior by pelvic irradiation of male mice alters cognitive behaviors and BDNF expression.

PLOS ONE

Dear Dr. Saligan,

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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: Partly

Reviewer #2: Yes

Reviewer #3: Partly

Reviewer #4: Partly

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: 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: No

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: 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: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: 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: The aim of this study is to clarify whether there is connection between fatique-like behavior and cognitive behavioral changes in mice and compare BDNF levels in brain.

There are several major issues with this current paper, regarding organization, data interpretation and visualization.

It is not clear why researchers used normal mice and targeted them with pelvic irradiation instead of using prostate cancer mouse model which is commercially available. If prostate cancer patients receiving radiation treatment have specific cognitive deficits with cancer-related fatigue and this needs to be investigated, the reason to use normal mice to show relationship between BDNF and CRF should be explained. Prostate cancer mouse model receiving pelvic irradiation and normal mouse received irradiation probably will give different response.

Although general structure of the manuscript is just fine, some issues must be revisited

Page2, Line 51-57, entire paragraf should be rewritten and organized.

In the entire manuscript there is no definition of BDNF. Appreviation is not described.

There is no data about weight measurement in the manuscript. During the experimention, weight loss may contribute to the behavioral assesments.

Page 3, Line 89: Is there any data for elevated 0-maze as mentioned?

Page 4, Line 132 and page 7, Line 254: There is no data for WB provided in my copy.

Page7, Line 261: It is not clear whether previous study is published or not , no reference was given. May be those data should be included in this manuscript.

Without Wb data, it is difficult to conclude the relationship BDNF level, irradiation and cognitive performance.

Reviewer #2: This is an interesting and timely study. The following issues should be addressed:

1. Please describe how shielding was performed for the localized radiation exposure. Please also indicate the details with regard to the irradiator used.

2. “Data from 8 mice were excluded from VWRA analysis due a 50% or greater drop in recorded VWRA totals during the baseline recording period.” It is not clear what this means; please explain.

3. Please refer to the time line to indicate the sequence of the behavioral tests and the interval between them in the methods section.

4. Please do not use the word “sacrificed” as standard for most journals now.

5. There are limitations to analyze whole brain levels of BDNF as more regional changes would be anticipated to play a role in wheel running and performance in the behavioral tests used in this study.

6. The reduced wheel running activity could affect BDNF levels in these animals; so what is a consequence of radiation and what is a consequence of reduced running seems hard to distinguish without a locked wheel control. This should be acknowledged and discussed.

7. It would be good to analyze whether wheel running in the light and/or dark periods was affected and whether there were circadian changes.

8. There seems no need to show the data as a percent of baseline with these striking group differences; please show actual data that is not normalized as well.

9. It seems better to only show the significant correlations and include the other data as supplementary table or figure panels.

10. The correlation shown in Figure 4F does not seem very convincing and might be driven by some individual data points.

Reviewer #3: The authors describe the effect of pelvic irradiation of male mice on cognitive behaviors and whole brain BDNF contents.

The paper showed that pelvic irradiation produced a reduced rate of spontaneous alteration in Y maze test and expression changes in mature/pro BDNF levels in whole brain. Although this study contains interesting information, mechanistic information may be less focused.

1)Why did authors examine protein BDNF levels in whole brain?

It has been reported that central BDNF exhibits opposite actions depending on the site of actions. For example, in preclinical studies, inflammation promotes reduced BDNF in the PFC and hippocampus, as well as increased BDNF in the NAc of the brain, resulting in a

depression-like phenotype in rodents (Zhang et al. Curr Neuropharmacol. 2016;14(7):721-31.)

If they try to suggest the mechanistic correlation between cognitive/emotional function and BDNF expression, it is strongly recommended to examine the BDNF levels in at least either hippocampus, PFC, or NAc in the pelvic irradiation model. Or they could additionally examine inflammatory status (ex. cytokines, microglia activation) in whole brain levels as they discussed in the Discussion (Page 7, L274: A more plausible cause of behavioral changes is a systemic inflammatory response…).

2)The Fig. 4D, 4F, 4H seems to show pro BDNF in irradiation animals. Why are the expression plots of proBDNF in each Figure so different? (The animals number could be 4D = 4F + 4H)

-It is recommended to include both irradiation (closed circle) and sham (open circle) animals in correlation plots.

-Please show the number of animals in each Figure.

3) In Fig 2I, VWRA (active time) correlates with Y maze's spotaneous alterlation. In Fig.4F, proBDNF is inversely correlated with Y maze's spotaneous alterlation. So why does VWRA not correlate with proBDNF in Fig4D? Please discuss this point.

Reviewer #4: This manuscript describes the assessment of fatigue and cognitive functions in a mouse model of pelvic irradiation. The premise of the study stems from findings in cancer patients, especially from prostate cancer patients, that fatigue is a common finding following radiation therapy and that there may or may not be an associated cognitive decline. Voluntary running wheel exercise was used before and after irradiation to assess the extent of fatigue resulting from irradiation. The mice were also assessed for working spatial memory, anxiety, reversal learning, and tissue levels of proBDNF and mBDNF in the brain. The authors observed a significant level of fatigue in the irradiated cohort which was supported by a significant reduction in voluntary wheel running activities following irradiation. Associated with the reduced voluntary activity was an impaired performance in the spontaneous alteration behavior in the Y maze exploration. Most interesting was the outcome in the reversal learning test. The authors showed that, without constrained by time, the irradiated mice were able to learn and perform to the same level of accuracy as sham controls in obtaining food pellets in the reversal phase of the nose pole/food reward learning paradigm. Consistent with the voluntary wheel running outcome, irradiated mice showed a significant reduction in ambulatory activities in the reversal learning arena, and the reduced activity correlated with reduced food intake. The authors also observed a significant reduction of mBDNF in the brains of irradiated mice, which may be a result of reduced overall physical activity. The studies are carefully designed and the results are relevant to a significant problem in cancer patients. The study results should be of interest to clinician and investigators in the radiation and cancer therapy field in general. Several issues warrant further clarification from the authors.

1. Given that importance of the results from reversal learning, it would have been nice to have a separate test to confirm the results. In the absence of such confirmation, authors should discuss what future studies may be employed to strengthen the conclusion in the Discussion.

2. It was not clear initially that two separate sets of mice were used for the voluntary wheel running/OF/Y maze and the reversal learning and that mice in the voluntary wheel running group only went through OF or Y maze, but not both arena tests. Providing these information earlier in the Methods section would have been helpful.

3. Elevated zero maze was not performed (or at least the data were not provided), but was mentioned in line 89.

4. There was a mention OF and Y maze tests were performed between ZT7 and ZT10 (line 89-90). Please clarify what is ZT7 and ZT10.

5. The reversal learning started on Day 5 (or 3 days after irradiation), but it was not clear how many days were devoted to the reversal phase. Was there a proficiency criterion to reach before the test was terminated?

6. As a related question – there were two more days of training after irradiation. Were the performance from the irradiated mice comparable to the sham controls, and how was the performance compared to the pre-irradiation phase?

7. To measure performance without time constraint, the authored chose to analyze performance during the first 1600 nose poke in the reversal phase. What was the rationale for selecting 1600 nose pokes as the cutoff?

8. It will be helpful if the authors can indicate on the timescale (Figures E and F) which segments are the dark phase and which are the light phase. Likewise, because the time required to complete 1600 nose pokes in the reversal phase is different between sham and irradiated animals, it will be helpful for the authors to provide that information.

9. Figures 3G and 3J are a percentage over a percentage (performance during the reversal phase over performance during the first night of training). It is also not clear if 3J uses the average performance over the entire reversal phase, or just during the first 1600 nose pokes, and normalizes it to the first 1371 nose pokes. It may be helpful in visualizing the data if the authors can plot out the performance during the first night or the first 1600 nose pokes of the reversal learning and performance of the first night or the first 1371 nose pokes, respectively, during the training phase for side-by-side comparisons.

10. The authors put sample sizes in 2A and 3A, but did not provide the same information for Y maze, OF, and BDNF plots. For consistency, these information should be provided.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: Yes: Ting Ting Huang

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PONE-D-20-08431R1

Induction of fatigue-like behavior by pelvic irradiation of male mice alters cognitive behaviors and BDNF expression.

PLOS ONE

Dear Dr. Saligan,

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.

As you will note Reviewer 2 has some additional minor comments that need to be addressed before the manuscript can be accepted. When the corrections are made, they will be reviewed by me and there is no need for additional review.

Please submit your revised manuscript by Jul 30 2020 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: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Michelle M. Adams, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

I have also received Reviewer 1's comments indicating that the manuscript is now suitable for acceptance.

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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: (No Response)

Reviewer #3: All comments have been addressed

Reviewer #4: 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

Reviewer #3: (No Response)

Reviewer #4: Yes

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

Reviewer #2: Yes

Reviewer #3: (No Response)

Reviewer #4: 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

Reviewer #3: (No Response)

Reviewer #4: 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

Reviewer #3: (No Response)

Reviewer #4: 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: While the authors have been responsive, the following changes are required to the figures:

1. Fig. 2. remove panels C, E, and F to suppl data as there is no significance; put the number of mice in the figure legend; in the G panel, show only the significant correlation and mention the other two in the text or figure legend.

2. Fig. 3. remove panels E, F, H, I, J to suppl data as there is no significance; put the number of mice in the figure legend; in the D panel, show only the two significant correlation and mention the third one in the text or figure legend.

3. Fig. 4. remove panel C to suppl data; panel D, show only the significant correlations; put the mouse numbers in the figure legend.

Reviewer #3: (No Response)

Reviewer #4: The authors have satisfactorily addressed all comments. There are two minor issues that require authors' attention:

1. Figure 3C, Y axis. This is normalized data (normalized to food pellets consumed on the day before irradiation). Consequently, the Y axis should be labeled as Food Pellets (% baseline).

2. Figure S5 D&E. Here the authors label Y axis as Open Time. Is this time in the center of the OF arena? If so, Center Time should be used to be consistent with labels used in Figures 2 and S2.

3. Figure S3. Can the authors provide a scale bar for the color scheme? The descriptive language "with darker colors representing more pellets, light colors representing fewer pellets" is not very helpful.

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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.

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Reviewer #2: No

Reviewer #3: No

Reviewer #4: Yes: Ting-Ting Huang

[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.

Induction of fatigue-like behavior by pelvic irradiation of male mice alters cognitive behaviors and BDNF expression.

PONE-D-20-08431R2

Dear Dr. Saligan,

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.

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Kind regards,

Michelle M. Adams, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: