Optogenetically induced cellular habituation in non-neuronal cells

Mattia Bonzanni; Nicolas Rouleau; Michael Levin; David L. Kaplan

doi:10.1371/journal.pone.0227230

Peer Review History

Original SubmissionSeptember 5, 2019
2 Dec 2019 Decision Letter - Mark S. Shapiro, Editor PONE-D-19-25035 Optogenetically induced cellular habituation in non-neuronal cells PLOS ONE Dear Dr. Kaplan, 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 Jan 16 2020 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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I would very strongly recommend following the recommendations of the reviewer, if the authors decide to re-submit to the journal. [Note: HTML markup is below. Please do not edit.] 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 ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: 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 ****** 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 ****** 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: A submitted manuscript on “Optogenetically induced cellular habituation in non-neuronal cells” is an interesting and important research work in this area. Authors have presented a meaningful research finding related to optogenetic modulation of the ionic current in a non-neuronal cells and its affect on habituation response. I enjoyed reading this research with lots of information and, nicely written and presented. However, It would be great to repeat key experiments of the habituation aspects with the stable cell lines of HEK expressing ChR variant. In order to visualize full “ Global” cellular response of the optogenetic modulated ionic current on habituation in the non-neuronal cells, authors may consider to perform comparative transcriptomic/ proteomics analysis. This might give a clue about the effect of optogenetically modulated ionic current associated with unknown gene regulation and/ or protein-protein interactions/ protein levels. ****** 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 [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. https://doi.org/10.1371/journal.pone.0227230.r001
Revision 1
7 Dec 2019 Author Response Response to Reviewer Inputs: 1) “We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements.” Accordingly, we edited the manuscript. In particular, we added a supplementary figure (Suppl. Fig.3) and removed the sentence. 1) “Has the statistical analysis been performed appropriately and rigorously? I do not know” In order to clarify the performed statistical analysis, we added the following sentences (in red) in the “Statistical Analysis” section: “Data were analyzed with Clampfit10 (Axon) and Origin Pro 9. To test the impact of the stimulation features on the habituation profile, we compared the mean percentage of depolarization at the steady state and the mean tau of habituation (τH) at different conditions. These two parameters are sufficient to uniquely describe a mono-exponential profile. Data were compared using either One-Way ANOVA followed by Fisher's LSD post-hoc test or Student’s T-test; significance level was set to p=0.05. Data outliers were excluded using Tukey's method. Data were collected from different transfection experiments ranging from a minimum of four to a maximum of twelve.” 2) “However, it would be great to repeat key experiments of the habituation aspects with the stable cell lines of HEK expressing ChR2 variant. In order to visualize full “Global” cellular response of the optogenetic modulated ionic current on habituation in the non-neuronal cells, authors may consider to perform comparative transcriptomic/ proteomics analysis. This might give a clue about the effect of optogenetically modulated ionic current associated with unknown gene regulation and/ or protein-protein interactions/ protein levels.” We thank the reviewer for the comment; as correctly pointed out, our data do not explore the molecular response of the system. Even if it appears to be a limitation in the manuscript, we purposely omitted any molecular investigations. Indeed, the primary aim of the project was to test phenotypic rather than mechanistic similarities between organism-level behavioral and single cell manifestations of habituation. Also at organism level, the molecular pathways are multiple and rarely generalize. As we suggested in the Discussion section, habituation, similarly to positive/negative feedbacks, must be considered a biological strategy to deal with repetitive stimulation and thus studied at the phenotypic level. Even if the reviewer’s suggestions are appropriate, we do feel that they do not apply in the presented cellular model. In this light, we would like to explain our reasons: • Conceptual perspective: As we verbalized in the Discussion section, even if not fully supported by our limited dataset, we do believe that habituations systems do not necessarily share mechanistic processes. It follows that the significance of any molecular insights will be limited to this unique and non-physiological system. It follows that the transcriptomic/proteomics analysis will lack translatability to other systems. As we pointed out in the Limitation Section, the choice of the presented model was dictated by methodological rather than biological considerations. We thus feel that any molecular insight could not be generalized to more relevant systems. • Technical perspectives: Considering the fast onset of the habituation effect (<2 seconds), we are confident that the main effect is due to the modulation of the activity and/or recruitment of the existing repertoire of proteins already present at the time of stimulation. Similarly, in neuronal substrates the short term learning effects are due to either cytosolic events, such as increase of cAMP and phosphorylation events, or the insertion in the membrane of pre-existing pools of proteins (such as AMPA receptors in the early events of LTP). Transcriptional and protein changes occur later and are also associated with a different phenotypic response (short- vs long-term effects). Attachments Attachment Submitted filename: Mattia Response to Reviewers rev dk final.docx https://doi.org/10.1371/journal.pone.0227230.r002
16 Dec 2019 Decision Letter - Mark S. Shapiro, Editor Optogenetically induced cellular habituation in non-neuronal cells PONE-D-19-25035R1 Dear Dr. Kaplan, 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, Mark S. Shapiro Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0227230.r003
Formally Accepted
10 Jan 2020 Acceptance Letter - Mark S. Shapiro, Editor PONE-D-19-25035R1 Optogenetically induced cellular habituation in non-neuronal cells Dear Dr. Kaplan: 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. Mark S. Shapiro Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0227230.r004

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