PONE-D-24-35662Electroconvulsive shock and transcranial magnetic stimulation do not alter the survival or spine density of adult-born striatal neuronsPLOS ONE

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Reviewer's Comment:

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: Yes

Reviewer #3: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: 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: Gaertner et al have investigated the effect of two neurostimulation techniques on the survival or adult born striatal neurons. The study specifically studies the effect of the stimulation on neurons born prior to the stimulation regime. Although the study has some small sample sizes with some animals only have 1 neuron counted for dendritic spine density, it is well designed and well written. Publishing negative results is critical and informative and the authors should be congratulated on publishing these negative findings.

I only have minor corrections that should be easy to address.

Methods:

- please provide an estimate of the electric field induced or at least a dB/dT value for the rTMS stimulation. Presumably a biphasic sine wave was used?

- please check whether the duration listed (189 seconds) for iTBS is correct.

- Was there a rationale for delivering rTMS every second day? rTMS is most commonly given daily for a period. An alternative protocol is fine to use, but a description of how that rationale was chosen would be useful.

- please provide the NA for the objectives used.

- was there a reason 1-6 cells was counted per animal for dendritic spines? Counting 1 neuron vs 6 neurons is likely to have a large impact on the interpretation of the results.

Discussion:

- It would be helpful to discuss the impact of the following on the results and their interpretation.

(1) the use of an rTMS coil that stimulates the entire brain and at least some of the spinal cord if not most of the peripheral nerves. This coil is known to activate neurons in the spinal cord of rats, and would therefore be reasonable to assume is not focal at all for the mouse brain.

(2) the use of a non-standard rTMS protocol. Potentially a lack of effect could be due to the spacing of the stimulation. While the results are interesting, noting that other rTMS protocols could still have potential is worth noting.

Reviewer #2: The ms by Gaertner and colleagues describes the effects of ECT and rTMS on the survival and spine density of adult-born striatal neurons in mice. Both techniques did not alter striatal neurons survival and structural plasticity with the adopted stimulation patterns, which still needs to be pointed out.

Though of interest, the ms suffers from an unclear vocabulary, thus needing a strong language revision.

I have some major and few minor concerns.

Major points:

Introduction: Recent literature of rTMS effects on hippocampus, mPFC and M1 structural plasticity reported a stimulation-dependent modulation in mice; high-frequency rTMS increased dendritic plasticity in prefrontal and motor cortex, while 1 Hz stimulation induced structural plasticity in mature granule cells, as also dendritic complexity of newly generated neurons, though not increasing DG progenitor cells proliferation. These data shuld be discussed.

Methods: animals should be be better described in the initial paragraph.

Methods: Why did the average motor threshold was determined with a separate group of animals?

Results: better to specify what the markers RFP, DARPP-32 and NeuN are used for.

Minor points:

Introduction: The sentence "rTMS and electroconvulsive shock (ECS), the animal model of ECT,..." is not clear at all. Please rephrase.

Introduction: The sentence "More recent studies indicate that neurogenesis in the ventral striatum is increased in response to chronic pain" needs more details.

Methods: The sentence "Sham-stimulated mice were restrained and treated identically with the exception that the stimulation intensity" is not at all clear!! Please rephrase.

Methods: specify the microscope and objective used for the acquisition of images

Figure legend 3: modify "Each colored circle" with "each dot".

Reviewer #3: In their manuscript, Gaertner et al have investigated the effects of electroconvulsive shock (ECT) and 10Hz repetitive transcranial magnetic stimulation (rTMS) on the survival of adult born striatal neurons in the mouse brain. The author’s have used a transgenic mouse line to label adult-born neurons via tamoxifen injection, and beginning from 8 days later, applied ECT or iTBS rTMS for 10 sessions over 3 weeks. Neither ECT or rTMS induced changes in the amount of surviving adult-born striatal neurons or the density of dendritic spines. The manuscript is written well the findings are of interest to the field. My comments are detailed below:

Major comments

-The authors describe that they administered rTMS using a Cool-40 coil at 115% of motor threshold. Can the authors please provide detail on the estimated magnetic field or electric field at the surface of the cortex in their model. Additionally, there is evidence to suggest that this coil may lack focality and stimulate regions of the brain and spinal cord outside of the targeted region (https://doi.org/10.1111/ner.12387 ). Some acknowledgements of the non-focality of this coil should be included in the methods or discussion.

-Were animals that were delivered ECT anaesthetised for the duration of the stimulation or was this conducted in non-anesthetised animals? If it was conducted in anesthetised animals, can details of the anaesthesia protocol please be detailed.

-Can the authors please provide information on why the stimulation protocol was chosen. Why was stimulation delivered every second day? Also, why was tissue collected 3 days after the final stimulation day? Some discussion of how this stimulation protocol compares to other stimulation protocols and how the timing of tissue processing may have contributed to the results should be included.

-The discussion does not discuss the implications of the results and how these findings contribute to our current understanding of brain stimulation. Others studies using rTMS have demonstrated that it can modulate subcortical areas (https://doi.org/10.1016/j.crneur.2022.100033 ) and alter neurogenesis (ref 9 in the manuscript). Some discussion on how the current study fits in with these previous studies and its significance should be expanded on.

-It is interesting that the authors chose to include both male and female animals in their design. Noting the small sample size when subdividing the treatment groups by sex, the authors may want to include some quantitative details of this output (e.g. a table showing differences between sex in their treatment groups) as there seems to be a small difference between sex in Fig 2v iTBS which may be important to note for future studies. Nonetheless, some acknowledgement on the effects of sex on their results should be included in the discussion.

Minor comments

-The reference list seems to be duplicated and also slightly different between both lists. This should be amended.

-A reference should be supplied for the Allen mouse brain atlas (page 4).

-The enlarged images in the figures do not contain a scale bar (e.g. 1c, 1e, 2b-e, etc). Can a scale bar for these higher magnification images please be included.

-The y-axis for the bar graphs in the included figures should be a consistent scale (e.g. fig 2v).

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Reviewer #1: Yes:  Alex Tang

Reviewer #2: No

Reviewer #3: Yes:  Jamie Beros

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.

PONE-D-24-35662R1Electroconvulsive shock and transcranial magnetic stimulation do not alter the survival or spine density of adult-born striatal neurons

PLOS ONE

Dear Dr. Snyder,

Thank you for resubmitting your work to PLOS ONE. Please make the corrections posed by the Reviewers so I can render a decision on this manuscript.

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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: (No Response)

Reviewer #2: All comments have been addressed

Reviewer #3: (No Response)

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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 #1: Partly

Reviewer #2: Yes

Reviewer #3: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: 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 #1: Yes

Reviewer #2: Yes

Reviewer #3: (No Response)

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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 #1: Yes

Reviewer #2: Yes

Reviewer #3: 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 #1: -The authors state the induced e field for their coil was 180V/m as per the modelling results from the Opitz lab. However, that simulation was done with an input of 100uA/us. This is not the input used in the current study, so the estimate of 180V/m is not correct.

- The authors maintain that the coil used is focal however I still strongly disagree. The coil is larger than the mouse head, with the windings sitting over the spinal cord unless the coil was offset such that the coil was overhanging at the front of the head to avoid the spinal cord. This would however distort the e-field. Moreover, the e-field is not restricted to a small portion of the cortex, it stimulates essentially the whole brain as well. Therefore, I maintain that the authors need to acknowledge the lack of focality in their discussion and make some mention of how this would impact their results.

- I accept the comment about low numbers being due to low number of adult born cells. This should be mentioned in the discussion as a limitation.

Reviewer #2: The Authors cleared all my concerns. I only have one minor issue: please modify "noninvasive" with "non-invasive"

Reviewer #3: I would like to thank the authors for considering and replying to the reviewer comments. Most of the comments are adequately addressed and the manuscript with the new inclusions read well. I have minor comments below that I believe still need to be addressed:

-The authors have replied to the reviewer’s comment and refute the claim that the coil used in the study does not stimulate the spinal cord as stated in the original coil validation paper and cite an additional modelling paper. The manuscript and figure that the author’s provided to substantiate their claims show that other areas across the surface of the brain (e.g. lateral cortex, cerebellum and olfactory bulbs) display an increased electric field with this coil, showing that areas outside of the targeted area can be affected. My comment was intended to consider how the focality of the applied stimulation (what/how many areas of the brain are being stimulated by the coil) led to/did not lead to the outcomes observed in the study rather than if it specifically stimulates the spinal cord. I still believe that it is important for the authors to at least acknowledge the claim in the original validation paper that the coil may stimulate areas outside of the targeted area within their manuscript. The authors can discuss/include their reasoning refuting the focality claim there if they wish.

-For the new sentence “While we did not have sufficient group sizes to investigate sex differences, it is worth noting that ECS-treated males had more than twice as many new neurons than their sham counterparts, but the two groups of females were virtually identical”, can the authors please include the mean value from these groups (treatment vs sham) in this sentence to aid in the comparison.

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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: Yes:  Alex Tang

Reviewer #2: No

Reviewer #3: Yes:  Jamie Beros

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.

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Section Editor

PLOS ONE

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Electroconvulsive shock and transcranial magnetic stimulation do not alter the survival or spine density of adult-born striatal neurons

PONE-D-24-35662R2

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