A new method of recording from the giant fiber of Drosophila melanogaster shows that the strength of its auditory inputs remains constant with age

Jonathan M. Blagburn

doi:10.1371/journal.pone.0224057

Peer Review History

Original SubmissionOctober 3, 2019
25 Nov 2019 Decision Letter - Gennady Cymbalyuk, Editor PONE-D-19-27717 A new method of recording from the giant fiber of Drosophila melanogaster shows that the strength of its auditory inputs remains constant with age PLOS ONE Dear Dr Blagburn, 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. Line 28: Please, revise for clarity the sentence describing the 40% decrease and the following few sentences. Line 44: "motor neuron" or "motoneuron". Also line 284. Line 49: I think you can delete the "somewhat uncoordinated" Line 63: The shakeb should be designated as innexins and with a reference. Line 70. Eliminate quotations around love song and please describe the courtship song in a sentence or two for non-Drosophila readers Line 84: Consider using "the strength of the connection between auditory input and GF" Line 96: "Changes" in food probably does not mean different types of food? Please clarify in the text. Please, explain at line 163 the relationship between sound intensity and particle velocity. Are they linear? Some incite for readers who is not familiar with particle velocity might be beneficial. Line 164: Sound duration and frequency should be given. See also line 197: What does i.e. mean here? Were different frequencies used? Line 166 and following: Maybe this could be improved a bit by explaining the general idea at the beginning of the paragraph: that an electrical stimulus activates optical pathways that are kept subthreshold, but enable the auditory stimulus to become suprathreshold and elicit GF activity. This would also help for later (line 384). Line 208. Remove about. The latency should be directly measured and not roughly estimated. Line 209 - 210. The phrase in the parentheses feels like a sentence fragment. Please revise it. Line 267. Remove "in passing". It's either been reported or not. Line 289. Remove the whole sentence stating that a direct measurement of GF activity would be more advantageous. It's unnecessary and such direct measurements have their own limitations (equipment expenses, more difficult, invasive, etc). Line 350. 1ms delay to what? Line 404 / 405: Maybe this sentence would be better if split into two. Split sentence in line 406 into smaller statements. Line 427: It's not clear why this is a combined results / discussion section. The discussion part clearly starts in line 427, so why not call it discussion here? (just a suggestion) Line 441. This half sentence needs to be moved to line 433. Figure 4. Remove the figure title. Perhaps swap it for the y-axis and use no figure title. Figure 5. There are a lot of non GFN spikes in these recordings that don't seem reliably time locked to stimulation. Perhaps mentioning that these are from other non-identified neurons should be mentioned in the figure legend. We would appreciate receiving your revised manuscript by Jan 09 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. To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols 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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. We look forward to receiving your revised manuscript. Kind regards, Gennady Cymbalyuk, Ph.D. Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. 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 http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf [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: Yes Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 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: No Reviewer #2: 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: I enjoyed reading the manuscript by Jonathan Blackburn examining the changes in signal propagation through the giant fiber system over time in Drosophila. The paper introduces an elegant means of dissociating several synaptic steps in the giant fiber pathway by using sharp extracellular electrodes at several recording sites to deduce pre-synaptic activity in auditory and visual pathways from action potentials generated in the GFN. The data generally support the conclusion that JON sensitivity changes over the course of the flies lifetime while the giant fiber neuron response to auditory input remains constant. A small caveat is that high variability in the JON to GFN synapse strength may drown out small statistical effects. Essentially, it is difficult to prove there is no change over time as the change may simply be below statistical resolution. The study uses reasonable sample sizes to try to mitigate this risk. Overall, the study of synaptic aging is of high interest and this novel approach could facilitate identifying differences in synaptic maturation in a what that is more high throughput than direct synaptic physiology. I do believe the manuscript could be improved eliminating some of the more informal writing style and reducing the use of quotations. Below are some additional specific suggestions. 1) Line 70. Eliminate quotations around love song and please describe the courtship song in a sentence or two for non-Drosophila readers 2) Line 74. I don't think it's correct to refer to the adult as hatching. I believe this is eclosion. 3) I understand the complexity in measuring particle velocity and thus why the authors reported dB in arbitrary units. Perhaps the authors though could make a statement at line 163 about the relationship between sound intensity and particle velocity. Are they linear? Does a double in sound intensity result in at least a near doubling in particle velocity. Some incite for readers who don't think about particle velocity might be beneficial. 4) Line 208. Remove about. The latency should be directly measured and not roughly estimated. 5) Line 209 - 210. The phrase in the parentheses feels like a sentence fragment. Please revise it. 6) Figure 4. Remove the figure title. Perhaps swap it for the y-axis and use no figure title. 7) Line 267. Remove "in passing". It's either been reported or not. 8) Line 289. Remove the whole sentence stating that a direct measurement of GF activity would be more advantageous. It's unnecessary and such direct measurements have their own limitations (equipment expenses, more difficult, invasive, etc). 9) Figure 5. There are a lot of non GFN spikes in these recordings that don't seem reliably time locked to stimulation. Perhaps mentioning that these are from other non-identified neurons should be mentioned in the figure legend. 10) For the diptheria toxin experiments, was there any independent validation that the toxin killed these neurons? Co-expression of GFP with the toxin is a reasonable what to do this. 11) The sentence in line 406 is a very long run on sentence and was difficult to understand. Simply chop it up into smaller more comprehend-able statements. 12) Line 439, eliminate "never". Just state, "because JON PSP's do not go above threshold in this configuration ... 13). Please revise the writing of point (5) starting on line 439 of the manuscript. It is a really long sentence and it causes confusion. I am not sure that somatic recordings are very useful for assuming spike thresholds as the spike initiation site might be very far from the soma. I simply did not understand point (5). Reviewer #2: This is a very well written manuscript. I have only minor comments. The manuscript describes how the sensitivity of auditory pathways in flies changes with age, but that their output connection to a pair of descending giant fibers does not. This is shown using an elegant 'trick' to measure the connection strength between the auditory neurons and the giant fibers: An additional electrical stimulus activates visual pathways that converge onto the same giant fibers but this input to the giant fibers is kept subthreshold. Only visual and auditory stimulus together 'sum up' and elicit action potentials in the giant fibers. The experiments are well described and the conclusions are well formulated. The author has a very nice way of discussing alternative interpretations and laying out arguments for and against these interpretations. Line 28: The sentence describing the 40% decrease is difficult to understand. Similarly, the following few sentences could be better. They make sense once one has read the whole paper, but as a first time reader they are difficult. The end of the introduction gives a nicer summary of the results, for example. Line 44: "motor neuron" or "motoneuron". Also line 284. Line 49: I think you can deleted the "somewhat uncoordinated" Line 63: The shakeb should be designated as innexins and with a reference. Line 84: I suggest to use "the strength of the connection between auditory input and GF" Line 96: "Changes" in food probably does not mean different types of food? Please clarify in the text. Line 164: Sound duration and frequency should be given. See also line 197: What does i.e. mean here? Were different frequencies used? Line 166 and following: Maybe this could be improved a bit by explaining the general idea at the beginning of the paragraph: that an electrical stimulus activates optical pathways that are kept subthreshold, but enable the auditory stimulus to become suprathreshold and elicit GF activity. This would also help for later (line 384). Line 350. 1ms delay to what? Line 404 / 405: Maybe this sentence would be better if split into two. Line 427: It's not clear to me why this is a combined results / discussion section. The discussion part clearly starts in line 427, so why not call it discussion here? (just a suggestion) Line 441. This half sentence needs to be moved to line 433. Figure 6: the averaged inset needs a scale bar and state the number of averaged responses. Figure 6B: How do we know that the large spike is not the 'usual' GF spike, while the smaller spike before is just a subthreshold reflection of synaptic input? Maybe this could be stated clearly in the text. ****** 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: Yes: Wolfgang Stein [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.0224057.r001
Revision 1
3 Dec 2019 Author Response PONE-D-19-27717 Response to Reviewers. Line 28: Please, revise for clarity the sentence describing the 40% decrease and the following few sentences. These sentences have been rewritten Line 44: "motor neuron" or "motoneuron". Also line 284. Corrected to “motor neuron” Line 49: I think you can delete the "somewhat uncoordinated" Deleted Line 63: The shakeb should be designated as innexins and with a reference. This has been done as requested Line 70. Eliminate quotations around love song and please describe the courtship song in a sentence or two for non-Drosophila readers As requested Line 84: Consider using "the strength of the connection between auditory input and GF" As suggested Line 96: "Changes" in food probably does not mean different types of food? Please clarify in the text. Clarified to: “changed to fresh food vials weekly” Please, explain at line 163 the relationship between sound intensity and particle velocity. Are they linear? Some incite for readers who is not familiar with particle velocity might be beneficial. I had incorrectly used the term “sound intensity” rather loosely here – it has a more precise meaning in acoustics, to do with power per unit area. I have changed this to “sound level” throughout. As I understand it, in the near-field conditions used here, sound particle velocity is apparently not directly proportional to sound pressure, as it is at greater distances from the source. I have made this clear in the Methods. Line 164: Sound duration and frequency should be given. See also line 197: What does i.e. mean here? Were different frequencies used? After further research, it appears that use of the term frequency has little meaning when applied to a sound transient or impulse, so I removed any references to the stimulus frequency, only referring to its initial peak duration and amplitude. Line 166 and following: Maybe this could be improved a bit by explaining the general idea at the beginning of the paragraph: that an electrical stimulus activates optical pathways that are kept subthreshold, but enable the auditory stimulus to become suprathreshold and elicit GF activity. This would also help for later (line 384). As requested. Line 208. Remove about. The latency should be directly measured and not roughly estimated. Done Line 209 - 210. The phrase in the parentheses feels like a sentence fragment. Please revise it. Done Line 267. Remove "in passing". It's either been reported or not. Done Line 289. Remove the whole sentence stating that a direct measurement of GF activity would be more advantageous. It's unnecessary and such direct measurements have their own limitations (equipment expenses, more difficult, invasive, etc). Good point. Done Line 350. 1ms delay to what? This was expressed badly before. I was referring to the delay from the TTL pulse at the trace onset. This has been made clearer in the text. Line 404 / 405: Maybe this sentence would be better if split into two. Duly split. Split sentence in line 406 into smaller statements. Done Line 427: It's not clear why this is a combined results / discussion section. The discussion part clearly starts in line 427, so why not call it discussion here? (just a suggestion) You are absolutely right – I have done as suggested. Line 441. This half sentence needs to be moved to line 433. Done as suggested Figure 4. Remove the figure title. Perhaps swap it for the y-axis and use no figure title. Good suggestion Figure 5. There are a lot of non GFN spikes in these recordings that don't seem reliably time locked to stimulation. Perhaps mentioning that these are from other non-identified neurons should be mentioned in the figure legend. Mentioned. Review Comments to the Author Reviewer #1: 1) Line 70. Eliminate quotations around love song and please describe the courtship song in a sentence or two for non-Drosophila readers Done 2) Line 74. I don't think it's correct to refer to the adult as hatching. I believe this is eclosion. Absolutely correct! Fixed. 3) I understand the complexity in measuring particle velocity and thus why the authors reported dB in arbitrary units. Perhaps the authors though could make a statement at line 163 about the relationship between sound intensity and particle velocity. Are they linear? Does a double in sound intensity result in at least a near doubling in particle velocity. Some incite for readers who don't think about particle velocity might be beneficial. I had used the term “sound intensity” rather loosely here – it has a more precise meaning in acoustics, to do with power per unit area. I have changed this to “sound level” throughout. Unfortunately, in the near-field conditions used here, sound particle velocity is apparently not directly proportional to sound pressure, as it is at greater distances from the source. I have made this clear in the Methods. 4) Line 208. Remove about. The latency should be directly measured and not roughly estimated. As above, done 5) Line 209 - 210. The phrase in the parentheses feels like a sentence fragment. Please revise it. As above, done 6) Figure 4. Remove the figure title. Perhaps swap it for the y-axis and use no figure title. Done 7) Line 267. Remove "in passing". It's either been reported or not. Done 8) Line 289. Remove the whole sentence stating that a direct measurement of GF activity would be more advantageous. It's unnecessary and such direct measurements have their own limitations (equipment expenses, more difficult, invasive, etc). Done 9) Figure 5. There are a lot of non GFN spikes in these recordings that don't seem reliably time locked to stimulation. Perhaps mentioning that these are from other non-identified neurons should be mentioned in the figure legend. Done 10) For the diptheria toxin experiments, was there any independent validation that the toxin killed these neurons? Co-expression of GFP with the toxin is a reasonable what to do this. This would have been the most elegant way to show that the GF was completely missing and I was originally planning to do this. However the loss of many stocks after hurricane Maria prevented me from doing these particular experiments. I did however look at the neck connective from the diphtheria toxin expressing animals and was unable to see the GF axons with Nomarski optics, when they are normally very prominent. I have now mentioned this in the Results. It must be pointed out that it is not necessary that the GF be completely eliminated, just poisoned, in order for it to stop spiking. I have also altered the word “ablated” to “poisoned”. 11) The sentence in line 406 is a very long run on sentence and was difficult to understand. Simply chop it up into smaller more comprehend-able statements. Done 12) Line 439, eliminate "never". Just state, "because JON PSP's do not go above threshold in this configuration ... Done 13). Please revise the writing of point (5) starting on line 439 of the manuscript. It is a really long sentence and it causes confusion. I am not sure that somatic recordings are very useful for assuming spike thresholds as the spike initiation site might be very far from the soma. I simply did not understand point (5). This part has been rewritten, and the threshold estimate moved to point 1. The point about the actual value of spike threshold is well taken, however, all these estimates are made from the point of view of the soma, since our only measurements of EPSPs come from patch recordings. Reviewer #2: Line 28: The sentence describing the 40% decrease is difficult to understand. Similarly, the following few sentences could be better. They make sense once one has read the whole paper, but as a first time reader they are difficult. The end of the introduction gives a nicer summary of the results, for example. This last part of the abstract has been rewritten to hopefully be clearer. Line 44: "motor neuron" or "motoneuron". Also line 284. Corrected Line 49: I think you can deleted the "somewhat uncoordinated" Deleted Line 63: The shakeb should be designated as innexins and with a reference. This has been done Line 84: I suggest to use "the strength of the connection between auditory input and GF" As suggested Line 96: "Changes" in food probably does not mean different types of food? Please clarify in the text. Clarified to mean fresh food vials weekly Line 164: Sound duration and frequency should be given. See also line 197: What does i.e. mean here? Were different frequencies used? After further research, it appears that use of the term frequency has little meaning when applied to a sound transient or impulse, so I removed any references to the stimulus frequency, only referring to its initial peak duration and amplitude. Line 166 and following: Maybe this could be improved a bit by explaining the general idea at the beginning of the paragraph: that an electrical stimulus activates optical pathways that are kept subthreshold, but enable the auditory stimulus to become suprathreshold and elicit GF activity. This would also help for later (line 384). Edited as requested Line 350. 1ms delay to what? This was expressed badly before. I was referring to the delay from the TTL pulse at the trace onset. This has been made clearer in the text. Line 404 / 405: Maybe this sentence would be better if split into two. Done Line 427: It's not clear to me why this is a combined results / discussion section. The discussion part clearly starts in line 427, so why not call it discussion here? (just a suggestion) They have been split into two sections, as suggested. Line 441. This half sentence needs to be moved to line 433. Edited Figure 6: the averaged inset needs a scale bar and state the number of averaged responses. Added to the figure and legend. Figure 6B: How do we know that the large spike is not the 'usual' GF spike, while the smaller spike before is just a subthreshold reflection of synaptic input? Maybe this could be stated clearly in the text. The small subthreshold response to eye stimulation could indeed also be from the visual circuit’s synaptic inputs to the GF, although I suspect that these are likely to be too small. I have tried to clarify this in the text. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0224057.r002
16 Dec 2019 Decision Letter - Gennady Cymbalyuk, Editor A new method of recording from the giant fiber of Drosophila melanogaster shows that the strength of its auditory inputs remains constant with age PONE-D-19-27717R1 Dear Dr. Blagburn, 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. 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With kind regards, Gennady Cymbalyuk, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): 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 #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: (No Response) ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: (No Response) ****** 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: (No Response) ****** 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: (No Response) ****** 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 Author has adequately addressed the issues that I raised in the previous version of the manuscript. As a result, the current version is now clearer and better supported. Reviewer #2: (No Response) ****** 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: Yes: Wolfgang Stein https://doi.org/10.1371/journal.pone.0224057.r003
Formally Accepted
17 Dec 2019 Acceptance Letter - Gennady Cymbalyuk, Editor PONE-D-19-27717R1 A new method of recording from the giant fiber of Drosophila melanogaster shows that the strength of its auditory inputs remains constant with age Dear Dr. Blagburn: 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. Gennady Cymbalyuk Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0224057.r004

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