Excitatory to inhibitory synaptic ratios are unchanged at presymptomatic stages in multiple models of ALS

Calum Bonthron; Sarah Burley; Matthew J. Broadhead; Vanya Metodieva; Seth G. N. Grant; Siddharthan Chandran; Gareth B. Miles

doi:10.1371/journal.pone.0306423

Peer Review History

Original SubmissionFebruary 22, 2024
16 May 2024 Decision Letter - Stephen D. Ginsberg, Editor PONE-D-24-06359Excitatory to inhibitory synaptic ratios are unchanged at presymptomatic stages in multiple models of ALS PLOS ONE Dear Dr. Miles, 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 Jun 30 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols. We look forward to receiving your revised manuscript. Kind regards, Stephen D. Ginsberg, Ph.D. Section 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 in your Competing Interests section: "None." Please complete your Competing Interests on the online submission form to state any Competing Interests. If you have no competing interests, please state ""The authors have declared that no competing interests exist."", as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now This information should be included in your cover letter; we will change the online submission form on your behalf. 3. When completing the data availability statement of the submission form, you indicated that you will make your data available on acceptance. We strongly recommend all authors decide on a data sharing plan before acceptance, as the process can be lengthy and hold up publication timelines. Please note that, though access restrictions are acceptable now, your entire data will need to be made freely accessible if your manuscript is accepted for publication. This policy applies to all data except where public deposition would breach compliance with the protocol approved by your research ethics board. If you are unable to adhere to our open data policy, please kindly revise your statement to explain your reasoning and we will seek the editor's input on an exemption. Please be assured that, once you have provided your new statement, the assessment of your exemption will not hold up the peer review process. 4. 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. Additional Editor Comments: After careful consideration by 2 Reviewers and an Academic Editor, all of the critiques of both Reviewers must be addressed in detail in a revision to determine publication status. If you are prepared to undertake the work required, I would be pleased to reconsider my decision, but revision of the original submission without directly addressing the critiques of the Reviewers does not guarantee acceptance for publication in PLOS ONE. If the authors do not feel that the queries can be addressed, please consider submitting to another publication medium. A revised submission will be sent out for re-review. The authors are urged to have the manuscript given a hard copyedit for syntax and grammar. ============================== 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 ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: I Don't Know ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: No ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: This paper details an analysis of excitatory vs inhibitory synapses in the spinal cord, primary co-cultures and iPSC for SOD1G93A and C9orf72 expansion models. Overall they have not found any differences. They acknowledge that there are things that they have not tested regarding functionality but it is clear there are no overt differences in these models with the presence of an ALS related mutation. In general, given the negative message in this paper i have no problem with the results that have been presented, although their significance is hard given how unclear the data is around this as a definitive cause of ALS in fully symptomatic models. That said it is important that this is published given the large amount of work that is involved in producing such data so that the field can see that there are no differences. The only thing that I would like to see would be the inclusion of some details of the phenotypes of the mice that they are using. Whilst i am aware most of these are published models, it would be better to have the details in this paper so that people can see how early these are being studied before symptoms. Again for the iPSC, give some phenotype details so that it is clear what is being actually looked at here in terms of time frames etc. Given these are validated models, do these iPSC show phenotypes that should be taken into account. Reviewer #2: This is an interesting manuscript analyzing the balance of excitation and inhibition at presymptomatic stages in models of ALS. The manuscript presents some important data, however major revisions are needed. It seems surprising that the E:I ratio is shown first, followed by the counts of the different synapses. It is expected that the synapses have already been counted, in order to get the ratio..? Why not show these data first, and then the ratio as the last part? In fact, it should be clarified in details how exactly the ratio was calculated, as that is not clear in the present version. Page 12 line 274 – 279 it is written: “For brevity, +N and +A refer to neurons and astrocytes, respectively, which are positive for the SOD1G93A or C9BAC500 mutation. -N and -A refers to these cell types generated from negative littermates that do not express ALS mutations. This produced 4 conditions in which to investigate non-cell autonomous effects on E:I ratios (+N/+A, +N/-A, -N/+A, -N/-A) (see Fig 1B).” Based on this explanation, it would be most suitable to display the data derived from -N/-A conditions first in the figures, since that would be close to normal situation, with no mutations expressed. Usually, when preparing figures, the wild-type or control situation is displayed first, followed by the different interventions of increasing complexity. This comment is relevant for all figures. Similarly, Figure 3 also shows the mutant first followed by the control. This should also be the other way around. Figure legends could be improved by adding a short sentence in the beginning, as sort of a header. This would help the reader get the conclusion from each figure. Page 5, line 123. Please specify the specific “C57bl/6” background, as there is more than one strain of these mice. Page 13 line 299 refers to “Figure 1D”, but there is no panel 1D. Similar in the Figure legend above on page 12. Again, this comment is also relevant for Figure 2. Please correct. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No 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. ============================== https://doi.org/10.1371/journal.pone.0306423.r001
Revision 1
5 Jun 2024 Author Response Response to Reviewer We would like to thank both reviewers for their careful consideration of the manuscript, and for acknowledging the importance of our findings for the field. We have now amended the manuscript in light of the helpful reviewer comments. We believe the changes made have improved the manuscript and hope it will satisfy the requirements for publication. In our responses below, and when referring to lines or sections, we refer to changes in the ‘tracked changes’ version of the manuscript. Reviewer 1: “The only thing that I would like to see would be the inclusion of some details of the phenotypes of the mice that they are using. Whilst I am aware most of these are published models, it would be better to have the details in this paper so that people can see how early these are being studied before symptoms.” • We agree that providing a more thorough background on the phenotypes of the mouse models is highly valuable and aids in the interpretation and understanding of our findings. We have therefore provided more substantial information regarding the phenotypes of the mice in our revised Discussion section [431-444]. “Again for the iPSC, give some phenotype details so that it is clear what is being actually looked at here in terms of time frames etc. Given these are validated models, do these iPSC show phenotypes that should be taken into account.” • Similarly, we have provided more extensive discussion of the phenotypes of iPSC-derived MNs [509-515]. Notably we have included mention of a hyperexcitability phenotype we (and others) have previously seen in iPSC-derived MNs at the timepoint used in our current study. This provides further support for our conclusion that any observed hyperexcitable phenotype is unlikely to be driven by biases in excitatory versus inhibitory synapse formation [396]. Reviewer 2: “It seems surprising that the E:I ratio is shown first, followed by the counts of the different synapses. It is expected that the synapses have already been counted, in order to get the ratio..? Why not show these data first, and then the ratio as the last part? In fact, it should be clarified in details how exactly the ratio was calculated, as that is not clear in the present version.” • We quantified the number of excitatory and inhibitory synapses in each individual image, then an E:I value was produced from these values (no. excitatory synapses / no. inhibitory synapses). The ratios for each image were then averaged across mouse or cell line. A more detailed explanation of E:I quantification has been added in the revised Results section [287-289]. • We appreciate that these data could be presented in either order (i.e. raw synapse numbers followed by ratios or vice versa). We wanted to present the ratio data first because we think it provides the clearest and simplest summary of the data, and speaks most directly to the main question we set in our project; whether the balance between excitatory and inhibitory synapses is altered at early stages of disease. Subsequently, we show the number of excitatory and inhibitory synapses (normalised to either the area or the number of DAPI nuclei) to verify that the static E:I ratios observed were not masking a scaled increase / decrease of excitatory and inhibitory synapses. We have added a justification for why we present the ratios followed by synapse densities in our revised Results section [285-287]. “Based on this explanation, it would be most suitable to display the data derived from -N/-A conditions first in the figures, since that would be close to normal situation, with no mutations expressed. Usually, when preparing figures, the wild-type or control situation is displayed first, followed by the different interventions of increasing complexity. This comment is relevant for all figures.” • All relevant figures (Fig. 1, 2, 3, 4, 5 and Supp. Fig. 1) have been amended accordingly. “Figure legends could be improved by adding a short sentence in the beginning, as sort of a header. This would help the reader get the conclusion from each figure.” • All figure and supplementary figure legends now include an initial title sentence. “Page 5, line 123. Please specify the specific “C57bl/6” background, as there is more than one strain of these mice.” • We have now included full details of the mouse lines [124-128]. “Page 13 line 299 refers to “Figure 1D”, but there is no panel 1D. Similar in the Figure legend above on page 12. Again, this comment is also relevant for Figure 2. Please correct.” • We thank the reviewer for noticing this error. Panel denotation for Figure 1D and 2D have been added to the figures. Attachments Attachment Submitted filename: Response to Reviewers .docx https://doi.org/10.1371/journal.pone.0306423.r002
18 Jun 2024 Decision Letter - Stephen D. Ginsberg, Editor Excitatory to inhibitory synaptic ratios are unchanged at presymptomatic stages in multiple models of ALS PONE-D-24-06359R1 Dear Dr. Miles, 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. If you have any questions relating to publication charges, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Stephen D. Ginsberg, Ph.D. Section Editor PLOS ONE Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed Reviewer #2: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The authors have addressed all the comments and i am happy to proceed this to publication. Well Done! Reviewer #2: The comments have satisfactorily been addressed. The manuscript has been significantly improved by the revision. The reviewer has no further comments. ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No https://doi.org/10.1371/journal.pone.0306423.r003
Formally Accepted
28 Jun 2024 Acceptance Letter - Stephen D. Ginsberg, Editor PONE-D-24-06359R1 PLOS ONE Dear Dr. Miles, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. 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. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Stephen D. Ginsberg Section Editor PLOS ONE https://doi.org/10.1371/journal.pone.0306423.r004

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .