Peer Review History

Original SubmissionDecember 16, 2019
Decision Letter - Ines Alvarez-Garcia, Editor

Dear Matthieu,

Thank you for submitting your manuscript entitled "PiVR: an affordable and versatile closed-loop platform to study unrestrained sensorimotor behavior" for consideration as a Methods and Resources by PLOS Biology.

Your manuscript has now been evaluated by the PLOS Biology editorial staff as well as by an academic editor with relevant expertise and I am writing to let you know that we would like to send your submission out for external peer review.

However, before we can send your manuscript to reviewers, we need you to complete your submission by providing the metadata that is required for full assessment. To this end, please login to Editorial Manager where you will find the paper in the 'Submissions Needing Revisions' folder on your homepage. Please click 'Revise Submission' from the Action Links and complete all additional questions in the submission questionnaire.

Please re-submit your manuscript within two working days, i.e. by Dec 23 2019 11:59PM.

Login to Editorial Manager here:

During resubmission, you will be invited to opt-in to posting your pre-review manuscript as a bioRxiv preprint. Visit for full details. If you consent to posting your current manuscript as a preprint, please upload a single Preprint PDF when you re-submit.

Once your full submission is complete, your paper will undergo a series of checks in preparation for peer review. Once your manuscript has passed all checks it will be sent out for review.

***Please be aware that, due to the voluntary nature of our reviewers and academic editors, manuscripts may be subject to delays due to their limited availability during the holiday season. Please also note that the journal office will be closed entirely 21st- 29th December inclusive, and 1st January 2020. Thank you for your patience.***

Feel free to email us at if you have any queries relating to your submission.

Kind regards,



Ines Alvarez-Garcia, PhD

Senior Editor

PLOS Biology

Carlyle House, Carlyle Road

Cambridge, CB4 3DN

+44 1223–442810

Revision 1
Decision Letter - Ines Alvarez-Garcia, Editor

Dear Matthieu,

Thank you very much for submitting your manuscript "PiVR: an affordable and versatile closed-loop platform to study unrestrained sensorimotor behavior" for consideration as a Methods and Resources at PLOS Biology. Thank you also for your patience as we completed our editorial process, and please accept my apologies for the delay in providing you with our decision. Your manuscript has been evaluated by the PLOS Biology editors, an Academic Editor with relevant expertise, and by three independent reviewers.

As you will see, the reviewers feel that the system you have developed is novel and useful for the scientific community, however they also raise several issues that would need to be addressed before we can consider your manuscript further for publication. You should make a comprehensive comparison with other methods already available (such as Bonsai, Stytra or FlyPI) and state clearly the advantages and limitations of PiVR. In addition, you should streamline the text and follow the reviewers’ suggestions to improve the structure of the manuscript.

In light of the reviews (attached below), we are pleased to offer you the opportunity to address the [comments/remaining points] from the reviewers in a revised version that we anticipate should not take you very long. We will then assess your revised manuscript and your response to the reviewers' comments and we may consult the reviewers again.

We expect to receive your revised manuscript within 1 month.

Please email us ( if you have any questions or concerns, or would like to request an extension. At this stage, your manuscript remains formally under active consideration at our journal; please notify us by email if you do not intend to submit a revision so that we may end consideration of the manuscript at PLOS Biology.


Your revisions should address the specific points made by each reviewer. Please submit the following files along with your revised manuscript:

1. A 'Response to Reviewers' file - this should detail your responses to the editorial requests, present a point-by-point response to all of the reviewers' comments, and indicate the changes made to the manuscript.

*NOTE: In your point by point response to the reviewers, please provide the full context of each review. Do not selectively quote paragraphs or sentences to reply to. The entire set of reviewer comments should be present in full and each specific point should be responded to individually.

You should also cite any additional relevant literature that has been published since the original submission and mention any additional citations in your response.

2. In addition to a clean copy of the manuscript, please also upload a 'track-changes' version of your manuscript that specifies the edits made. This should be uploaded as a "Related" file type.

*Resubmission Checklist*

When you are ready to resubmit your revised manuscript, please refer to this resubmission checklist:

To submit a revised version of your manuscript, please go to and log in as an Author. Click the link labelled 'Submissions Needing Revision' where you will find your submission record.

Please make sure to read the following important policies and guidelines while preparing your revision:

*Published Peer Review*

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. Please see here for more details:

*PLOS Data Policy*

Please note that as a condition of publication PLOS' data policy ( requires that you make available all data used to draw the conclusions arrived at in your manuscript. If you have not already done so, you must include any data used in your manuscript either in appropriate repositories, within the body of the manuscript, or as supporting information (N.B. this includes any numerical values that were used to generate graphs, histograms etc.). For an example see here:

*Protocols deposition*

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see:

Thank you again for your submission to our journal. We hope that our editorial process has been constructive thus far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.




Ines Alvarez-Garcia, PhD

Senior Editor

PLOS Biology

Carlyle House, Carlyle Road

Cambridge, CB4 3DN

+44 1223–442810


Reviewers’ comments

Rev. 1:

The present study by Tadres and Louis presents a useful and affordable open-source virtual reality setup based on the Raspberry Pi system. The authors present solid data using virtual chemotaxis and phototaxis experiments, confirming that their system works accurately for such purposes. I find this work valuable to the community, especially for the effort to design systems that can be implemented at low cost as well as for teaching platforms.

I have a few comments that I believe would improve the presentation and message of this manuscript and one concern regarding the proposed speed of the system.

First, I have my doubts that the overall close-loop reaction time is correct. The shutter speeds and the image processing time were measured. However, due to the limitations of the bus, there is always a lag between image acquisition and the start of image processing. I have no experience with the Raspberry Pie system, but I have encountered these limitations in top of the line computing systems. Thus, I would test this directly. My suggestion is to use their system at maximal acquisition speed, image short LED flash, and use their computing platform to detect this flash and turn the same LED on for a second time. By looking at the number of frames between the two flashes, the authors could test the speed of the entire system. Although the sampling rate will be limited by the acquisition speed of the camera, the authors will be able to prove if their proposed 20ms processing time is accurate. This is important since the authors correctly claim that "the shorter the delay, the more authentic the virtual reality is perceived." I don't see issues with the experiments presented, but new users will be warned in case the system is slower than the speeds required for their purposes.

My second suggestion is regarding the current structure. The main claim of this paper is to have a VR system that is affordable and reliable; not the experimental results used to benchmark the system. Thus, I would remove the experimental sections from the discussion (Defining that nature of taste-driven responses in adult flies; Exploring the ability of zebrafish [larvae] to orient with minimal graded visual inputs; Exploring the ability of the fruit fly….) and merged them in a minimal form to the experimental results.

Finally, although I truly appreciate and are convinced that the system is useful for particular experiments, it won't be for all. There are limitations on the FOV, camera resolution, speed, etc., that make it indispensable for many labs to engineering their systems. Thus, I would recommend adding this perspective to the discussion. I believe that this will be helpful for labs that thanks to the affordable design will start doing these kinds of VR experiments, but don't have the experience to judge all details.


I would disagree with the first sentence: "Behavior emerges from the conversion of sensory input into motor output". Organisms are not automata, e.g.; there is a whole world of internal states that modulate behavior. Please rephrase.

I would also make a stronger point on why VR systems are important (line 41). You don't need a VR system to stimulate an animal repeatedly. VR systems allow combining the stimulus to the behavior, enabling a precise exploration of response dynamics to sensory stimuli dependent on particular behavioral conditions.

- The paper sometimes uses colloquial terms, e.g., "crack the neuronal code," etc. I would change that language.

- Hz and frames per second are used, I would stick to Hz.

- Reference, e.g., page 213: Schulze, Gomez-Marin (17) -> Schulze et al. (17). Also line 307.

- Get rid of popular in line 220 and 256. Model organisms already imply that they are used widely.

- Line 600 - 608, it is not clear that the (Ci-iii) refers to the supplemental figure.

- Line 195, "field of the view", remove the.

Rev. 2:

In this manuscript, Tadres & Louis present PiVR, a novel hardware designed to perform closed-loop light-based stimulations on small animals. The authors present data based on optogenetic activation of genetically targeted neurons in Drosophila larvae and imagos; they also present a proof of principle using visible light on zebrafish larvae.

PiVR is certainly an interesting device and I can see it becoming popular in the field of Drosophila neuroscience, especially among people interested in developing new paradigms of learning.

The device has some clear strengths and some limitations. The strengths are discussed appropriately and fairly. The limitations not so much and it may be useful to have a more rounded discussion of both so that readers can immediately recognise whether this tool is appropriate for their uses.

Amongst the strengths I would count:

1. The documentation is outstanding.

2. The machine is relatively inexpensive.

3. The basic usage of the machine seems to be easy to implement

Amongst the weaknesses:

1. Some of the proposed usages of PiVR are suboptimal. It is stressed several times in the manuscript that PiVR can be used to acquire videos "offline", for them to be tracked by a different software (such as I doubt this setup can compete, in terms of resolution, speed and even cost with the simpler solution of having an industrial CCD connected to an existing computer.

2. While, in principle, the R in VR stands for any kind of Reality, in fact it is commonly associated to complex visual representations such as projections of objects, patterns, scenarios. PiVR is limited to providing different intensity of lights and therefore its main use-case is going to be optogenetics.

3. It is not clear to me why a reader should prefer PiVR over other already existing alternatives and it may be useful to stress out, perhaps in a table, pros and cons of PiVR vs the "competition". FlyPI , BONSAI, FreeMO-VR are all alternative products with functionalities that overlap the ones of PiVR - the manuscript would benefit from a more straight comparison with them.

I do not have specific comments on the manuscript, besides the fact that I found it perhaps too long and repetitive. I think the strength of this tool is that it does one thing and it does nicely but all this gets somehow lost in a manuscript so discursive and repetitive. The discussion, in particular, is not well focused.

The figures are well presented and clear but perhaps not very focused. figures 2 and 3 read more like a demonstration that "optogenetics works". It would have been more useful to focus on PiVR versatility and show several different use cases rather than only those two very basic ones. Somehow the focus of the figures is optogenetics, not PiVR.

I also had troubles understanding part of figure 4 (F-J) and almost all of figure 5. Perhaps the concepts of slow vs fast dynamic VR can be explained in greater detail with a cartoon and examples of why and how an experimenter may want to use slow or fast dynamic VR could be provided.

Rev. 3:

This paper describes an open, low-cost platform to perform closed-loop behavioral experiments. The intention is to make such experiments more accessible or scalable by removing barriers of cost and development. The utility of the system is thoroughly demonstrated through example experiments in larval and adult Drosophila and zebrafish, which are analyzed to reveal new biological insights.

The resources described in the paper are well designed and described and should be straightforward to apply to many current experimental questions. However, there are several existing platforms with overlapping aims and many of them are not cited or discussed in this paper. Some of these allow much more experimental flexibility than the generation of virtual environments through modulation of a one-dimensional signal that is used in the current paper. Therefore, it is not clear that the method fulfills the criteria required for a resource in PLOS Biology, in that it would enable experiments that were not possible using existing methods. In particular, it would be helpful to cite the following two resources and discuss them in comparison with the PIVR system:

1) Stytra

Stytra is a python-based package, which can be used for animal tracking and closed-loop presentation of stimuli. Implementations with low-cost hardware are possible. Štih V*, Petrucco L*, Kist AM, Portugues R (2019)

Stytra: An open-source, integrated system for stimulation, tracking and closed-loop behavioral experiments. PLOS Computational Biology, doi:10.1371/journal.pcbi.1006699

2) Bonsai.

Bonsai is a software framework that is widely used for designing closed-loop behavior experiments, and can be used in conjunction with low cost hardware.

Bonsai: An event-based framework for processing and controlling data streams. Lopes G, Bonacchi N, Frazão J, Neto JP, Atallah BV, Soares S, Moreira L, Matias S, Itskov PM, Correia PA, Medina RE, Calcaterra L, Dreosti E, Paton JJ, Kampff AR. Frontiers in Neuroinformatics. 2015; 9:7.

Some minor issues:

1) In the introductory discussion on virtual reality in neuroscience, it could be worth citing the productive use of virtual reality in work in zebrafish, in the context of motor adaptation, prey capture and social behavior.

2) The closed-loop experiments based on optogenetic stimulation of the gustatory system in flies have technical similarities with experiments in the following paper, which might be appropriate to cite:

Moreira, J.-M., Itskov, P. M., Goldschmidt, D., Baltazar, C., Steck, K., Tastekin, I., et al. (2019). optoPAD, a closed-loop optogenetics system to study the circuit basis of feeding behaviors. eLife, 8.

3) The terminology used in the zebrafish swimming experiments in not aligned to common usage in the literature, and could be confusing. Individual bursts of movement of the larvae are typically called 'bouts' , and the word 'scoots' is used to refer to a particular type of bout, where the tail oscillations are mostly confined to the caudal region, and which propels the larva forward at a slow speed, without major reorientation or head yaw (also often called 'Slow swims'). Routine turns, by contrast, start with a larger tail movement that reorients the larva, which can be followed by a propulsive phase. Therefore what are called 'scoots' in this paper may encompass both 'scoots' and 'turns' in the terminology of other studies.

4) In the discussion of phototaxis in zebrafish, there are some other papers that perhaps are relevant to cite, because they either describe virtual reality based assays for phototaxis, or discuss the choice of turns vs scoots and the direction of turns. Ahrens, M. B., Huang, K. H., Narayan, S., Mensh, B. D., & Engert, F. (2013). Frontiers in Neural Circuits, 7, 104.

Fernandes, A. M., Fero, K., Arrenberg, A. B., Bergeron, S. A., Driever, W., & Burgess, H. A. (2012). Current Biology, 22(21), 2042-2047. Horstick, E. J., Bayleyen, Y., Sinclair, J. L., & Burgess, H. A. (2017). BMC Biology, 1-16.

Revision 2

Submitted filename: 200306__PointResponse_v2.pdf
Decision Letter - Ines Alvarez-Garcia, Editor

Dear Matthieu,

Thank you for submitting your revised Methods and Resources entitled "PiVR: an affordable and versatile closed-loop platform to study unrestrained sensorimotor behavior" for publication in PLOS Biology. I have now discussed the revision with the team of editors and obtained advice from the original Academic Editor.

We're delighted to let you know that we're now editorially satisfied with your manuscript. However before we can formally accept your paper and consider it "in press", we also need to ensure that your article conforms to our guidelines. A member of our team will be in touch shortly with a set of requests. As we can't proceed until these requirements are met, your swift response will help prevent delays to publication. Please also make sure to address the data and other policy-related requests noted at the end of this email.


Upon acceptance of your article, your final files will be copyedited and typeset into the final PDF. While you will have an opportunity to review these files as proofs, PLOS will only permit corrections to spelling or significant scientific errors. Therefore, please take this final revision time to assess and make any remaining major changes to your manuscript.

NOTE: If Supporting Information files are included with your article, note that these are not copyedited and will be published as they are submitted. Please ensure that these files are legible and of high quality (at least 300 dpi) in an easily accessible file format. For this reason, please be aware that any references listed in an SI file will not be indexed. For more information, see our Supporting Information guidelines:

*Published Peer Review History*

Please note that 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. Please see here for more details:

*Early Version*

Please note that an uncorrected proof of your manuscript will be published online ahead of the final version, unless you opted out when submitting your manuscript. If, for any reason, you do not want an earlier version of your manuscript published online, uncheck the box. Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us as soon as possible if you or your institution is planning to press release the article.

*Protocols deposition*

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see:

*Submitting Your Revision*

To submit your revision, please go to and log in as an Author. Click the link labelled 'Submissions Needing Revision' to find your submission record. Your revised submission must include a cover letter, a Response to Reviewers file that provides a detailed response to the reviewers' comments (if applicable), and a track-changes file indicating any changes that you have made to the manuscript.

Please do not hesitate to contact me should you have any questions.

Best wishes,



Ines Alvarez-Garcia, PhD

Senior Editor

PLOS Biology

Carlyle House, Carlyle Road

Cambridge, CB4 3DN

+44 1223–442810



You may be aware of the PLOS Data Policy, which requires that all data be made available without restriction: I can see that you have deposited your data in Driad (DOI:, but the link doesn't seem to be active and I can't check if the data we need before the manuscript enters production is available. Please either activate it or follow the instructions stated below.

Note that we do not require all raw data (for more information, please also see this editorial: Rather, we ask that all individual quantitative observations that underlie the data summarized in the figures and results of your paper be made available in one of the following forms:

1) Supplementary files (e.g., excel). Please ensure that all data files are uploaded as 'Supporting Information' and are invariably referred to (in the manuscript, figure legends, and the Description field when uploading your files) using the following format verbatim: S1 Data, S2 Data, etc. Multiple panels of a single or even several figures can be included as multiple sheets in one excel file that is saved using exactly the following convention: S1_Data.xlsx (using an underscore).

2) Deposition in a publicly available repository. Please also provide the accession code or a reviewer link so that we may view your data before publication.

Regardless of the method selected, please ensure that you provide the individual numerical values that underlie the summary data displayed in the following figure panels as they are essential for readers to assess your analysis and to reproduce it:

Fig. 3C, F; Fig. 4G, H, J; Fig. S1B, C, D; Fig. S3E, D, I, J; Fig. S4E, F; Fig. S7A, B, C and Fig. S8A, B

NOTE: the numerical data provided should include all replicates AND the way in which the plotted mean and errors were derived (it should not present only the mean/average values).

Please also ensure that figure legends in your manuscript include information on where the underlying data can be found, and ensure your supplemental data file/s has a legend.

Please ensure that your Data Statement in the submission system accurately describes WHERE YOUR DATA CAN BE FOUND.

Revision 3

Submitted filename: Point-by-point_answer_final.pdf
Decision Letter - Ines Alvarez-Garcia, Editor

Dear Dr Louis,

On behalf of my colleagues and the Academic Editor, Tom Baden, I am pleased to inform you that we will be delighted to publish your Methods and Resources in PLOS Biology.

The files will now enter our production system. You will receive a copyedited version of the manuscript, along with your figures for a final review. You will be given two business days to review and approve the copyedit. Then, within a week, you will receive a PDF proof of your typeset article. You will have two days to review the PDF and make any final corrections. If there is a chance that you'll be unavailable during the copy editing/proof review period, please provide us with contact details of one of the other authors whom you nominate to handle these stages on your behalf. This will ensure that any requested corrections reach the production department in time for publication.

Early Version

The version of your manuscript submitted at the copyedit stage will be posted online ahead of the final proof version, unless you have already opted out of the process. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.


We frequently collaborate with press offices. If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximise its impact. If the press office is planning to promote your findings, we would be grateful if they could coordinate with If you have not yet opted out of the early version process, we ask that you notify us immediately of any press plans so that we may do so on your behalf.

We also ask that you take this opportunity to read our Embargo Policy regarding the discussion, promotion and media coverage of work that is yet to be published by PLOS. As your manuscript is not yet published, it is bound by the conditions of our Embargo Policy. Please be aware that this policy is in place both to ensure that any press coverage of your article is fully substantiated and to provide a direct link between such coverage and the published work. For full details of our Embargo Policy, please visit

Thank you again for submitting your manuscript to PLOS Biology and for your support of Open Access publishing. Please do not hesitate to contact me if I can provide any assistance during the production process.

Kind regards,

Alice Musson

Publishing Editor,

PLOS Biology

on behalf of

Ines Alvarez-Garcia,

Senior Editor

PLOS Biology

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 .