NMDA receptor activation induces long-term potentiation of glycine synapses

Michelle L. Kloc; Bruno Pradier; Anda M. Chirila; Julie A. Kauer

doi:10.1371/journal.pone.0222066

Peer Review History

Original SubmissionJuly 5, 2019
6 Aug 2019 Decision Letter - Guangyu Wu, Editor PONE-D-19-19003 NMDA receptor activation induces long-term potentiation of glycine synapses PLOS ONE Dear Dr. Kauer, 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. We would appreciate receiving your revised manuscript by Sep 20 2019 11:59PM. When you are 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. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. 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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. 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(Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: In this study, the authors show that postsynaptic NMDA receptor activation, either via synaptic release of glutamate or exogenous application of NMDA, triggers long lasting potentiation of glycine receptor mediated synaptic events in GABAergic neurons in the dorsal horn. The experiments are quite conclusive and the conclusions are supported by the data presented. I see only a few minor issues that need clarification prior to publication. 1. The authors may have addressed this point in their earlier publications, but I think it is important to demonstrate that the putative glycinergic synaptic responses can be blocked by a classical antagonist such as strychnine 2. Is there an augmentation of glycinergic mIPSCs after NMDA treatment? Do their amplitudes increase as in evoked responses? Reviewer #2: Recommendation: Minor revision Comments to Author: Ref. PONE-D-19-19003 Title: NMDA receptor activation induces long-term potentiation of glycine synapses Overview and general recommendation: This manuscript mechanistically revealed how glycine synapses is potentiated by activation of NMDAR. I found that the paper is clearly written in general and much of it is well described. I feel confident that the authors performed careful and thorough experiments to test their hypothesis. However, the current version needs to be revised for publication due to the following reasons: 1. Missing certain literature citations in discussion part. 2. Please rewrite and emphasis the significance of this study in the manuscript. ****** 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 [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 to be viewed.] 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 us at figures@plos.org. Please note that Supporting Information files do not need this step. Attachments Attachment Submitted filename: review comments_PONE19003.docx https://doi.org/10.1371/journal.pone.0222066.r001
Revision 1
19 Aug 2019 Author Response Responses to the Review Comments to the Author Again - please note that figures can only be seen in the letter we uploaded. We are grateful for the reviewers’ comments, and have addressed each point below: Reviewer #1: In this study, the authors show that postsynaptic NMDA receptor activation, either via synaptic release of glutamate or exogenous application of NMDA, triggers long lasting potentiation of glycine receptor mediated synaptic events in GABAergic neurons in the dorsal horn. The experiments are quite conclusive and the conclusions are supported by the data presented. I see only a few minor issues that need clarification prior to publication. 1. The authors may have addressed this point in their earlier publications, but I think it is important to demonstrate that the putative glycinergic synaptic responses can be blocked by a classical antagonist such as strychnine. Thank you for this comment. We have now added in the methods that our responses are entirely blocked by 1 �M strychnine, and added the reference to a previous PNAS paper in which we showed this. I have attached the figure below for the convenience of the reviewer (Supplementary Figure 3b, Chirila et al., 2014). In addition, in one experiment in our more recent work, strychnine was added after NMDA and the synaptic current was entirely blocked (left-hand figure below). We also added strychnine at the end of 4 experiments in which glycinergic IPSCs were evoked in the trpv1-ChR2 animals – part of Figure 7 in the paper). As can be seen in the graphs (right hand figure above), IPSCs were strongly depressed by strychnine within ten minutes to less than 10% of basal values. We have now added this information to the manuscript, and also cited our previous experiment referring to the Chirila et al. paper. 2. Is there an augmentation of glycinergic mIPSCs after NMDA treatment? Do their amplitudes increase as in evoked responses? We very much appreciate the reviewer’s comment. In the PNAS paper (Chirila et al., 2014) we very much wanted to examine miniature IPSCs; however, under our recording conditions, the minis are so rare that it was very difficult to collect a meaningful number within a reasonable time frame. In that paper, we therefore had to resort to using strontium solutions and a train of electrical stimuli to evoke asynchronous release, in order to produce a sufficient number of events before and after LTP to compare them. In the current manuscript, we felt that this approach would not add much to the paper. To test whether the LTP is mediated by pre- or postsynaptic changes, we measured both paired-pulse ratios (PPR; a sensitive measure of presynaptic probability of release) and exogenous delivery of glycine (a robust measure of postsynaptic sensitivity to glycine). As can be seen in Figure 4, there is zero change on average in PPR, supporting the idea that presynaptic release probability is unchanged, and moreover that the response to bath-applied glycine increases 2-3 fold over the time frame of our experiments, demonstrating that postsynaptic sensitivity is increased enormously after NMDA, consistent with our observations of LTP of glycinergic IPSCs. Given these strong data, to attempt difficult recordings of strontium-evoked asynchronous mIPSCs will not add much to our story. The best we could hope for in my view is to ascertain whether the frequency of minis increased (I am relatively certain that the amplitudes will increase); however, with strontium mIPSCs, claims about frequency are not as convincing as those of amplitude. In summary, we feel that the very large potentiation with simple bath-application of glycine after NMDA LTP, and lack of change in PPR are a sufficiently compelling demonstration of a postsynaptic LTP mechanism, and hope the reviewer will agree. I will also note that in Figure 3i, we report that after NMDA application, IL-1� produces no further potentiation, supporting a common underlying mechanism. We know that IL-1� can increase strontium mIPSCs, and so this figure suggests that NMDA is likely to do so as well. Reviewer #2: Recommendation: Minor revision Comments to Author: Ref. PONE-D-19-19003 Title: NMDA receptor activation induces long-term potentiation of glycine synapses Overview and general recommendation: This manuscript mechanistically revealed how glycine synapses is potentiated by activation of NMDAR. I found that the paper is clearly written in general and much of it is well described. I feel confident that the authors performed careful and thorough experiments to test their hypothesis. However, the current version needs to be revised for publication due to the following reasons: 1. Missing certain literature citations in discussion part. We are grateful to the reviewer for noting this error and have gone carefully over the entire paper with this point in mind. We found a reference that was not included in the discussion (Melzack and Wall, 1965), and have now added this citation. If the reviewer has other specific suggestions for citation issues, we are happy to make revisions. 2. Please rewrite and emphasis the significance of this study in the manuscript. We have made changes in the introduction and discussion to address this important comment. Attachments Attachment Submitted filename: Responses to reviewers.docx https://doi.org/10.1371/journal.pone.0222066.r002
22 Aug 2019 Decision Letter - Guangyu Wu, Editor NMDA receptor activation induces long-term potentiation of glycine synapses PONE-D-19-19003R1 Dear Dr. Kauer, We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements. Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication. Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, 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 enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and 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. With kind regards, Guangyu Wu, PhD Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0222066.r003
Formally Accepted
27 Aug 2019 Acceptance Letter - Guangyu Wu, Editor PONE-D-19-19003R1 NMDA receptor activation induces long-term potentiation of glycine synapses Dear Dr. Kauer: I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, 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. For any other questions or concerns, please email plosone@plos.org. Thank you for submitting your work to PLOS ONE. With kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Guangyu Wu Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0222066.r004

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