Abstract

A long-standing objective in neuroscience has been to image distributed neuronal activity in freely behaving animals. Here we introduce NeuBtracker, a tracking microscope for simultaneous imaging of neuronal activity and behavior of freely swimming fluorescent reporter fish. We showcase the value of NeuBtracker for screening neurostimulants with respect to their combined neuronal and behavioral effects and for determining spontaneous and stimulus-induced spatiotemporal patterns of neuronal activation during naturalistic behavior.

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References

  1. 1.

    et al. Nat. Methods 13, 325–328 (2016).

  2. 2.

    & Curr. Opin. Neurobiol. 22, 18–23 (2012).

  3. 3.

    & Curr. Opin. Neurobiol. 32, 78–86 (2015).

  4. 4.

    , , & Neuron 81, 1328–1343 (2014).

  5. 5.

    et al. Nat. Methods 11, 883–884 (2014).

  6. 6.

    et al. Nature 485, 471–477 (2012).

  7. 7.

    , , , & Curr. Biol. 23, 307–311 (2013).

  8. 8.

    , , , & Nat. Neurosci. 13, 513–520 (2010).

  9. 9.

    et al. Zebrafish 10, 70–86 (2013).

  10. 10.

    & Annu. Rev. Neurosci. 40, 125–147 (2017).

  11. 11.

    et al. Nat. Chem. Biol. 12, 559–566 (2016).

  12. 12.

    et al. Proc. Natl. Acad. Sci. USA 113, E1074–E1081 (2016).

  13. 13.

    , & Nat. Methods 13, 569–572 (2016).

  14. 14.

    , , , & Cell Syst. 1, 338–348 (2015).

  15. 15.

    & Front. Behav. Neurosci. 10, 160 (2016).

  16. 16.

    et al. Proc. Natl. Acad. Sci. USA 110, 19579–19584 (2013).

  17. 17.

    et al. Nat. Methods 12, 1039–1046 (2015).

  18. 18.

    et al. Nat. Methods 11, 727–730 (2014).

  19. 19.

    Nat. Methods 8, 811–819 (2011).

  20. 20.

    , & Neuron 86, 106–139 (2015).

  21. 21.

    & Front. Syst. Neurosci. 8, 39 (2014).

  22. 22.

    et al. PLoS One 8, e64429 (2013).

  23. 23.

    , & Pattern Recognit. 46, 1415–1432 (2013).

  24. 24.

    , , , & Dev. Dyn. 203, 253–310 (1995).

  25. 25.

    et al. Dev. Biol. 397, 1–17 (2015).

  26. 26.

    , , , & Curr. Biol. 27, 1288–1302 (2017).

  27. 27.

    et al. J. Neurosci. 33, 15235–15247 (2013).

  28. 28.

    et al. Nat. Methods 7, 634–636 (2010).

  29. 29.

    et al. PLoS One 7, e40508 (2012).

  30. 30.

    , , & Science 322, 1065–1069 (2008).

  31. 31.

    , , , & Opt. Express 21, 21010–21026 (2013).

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Acknowledgements

We thank K. Kawakami and A. Muto (National Institute of Genetics (Japan)) for sharing tg(gSA2AzGFF49A;UAS:GCaMP7a) and tg(gSA2AzGFF152B), here referred to as GR152:Gal4, which was provided to us by R. Portugues and L. Knogler (Max Planck Institute of Neurobiology), K. Asakawa (National Institute of Genetics (Japan)) for providing tg(mnGFF7;UAS:eGFP), J. Ngai (University of California, Berkeley) for tg(OMP4; UAS:GCaMP1.6), M. Ahrens (Janelia Research Campus) for sharing tg(HuC:H2B-GCaMP6s), H. Baier and K. Slanchev (Max Planck Institute of Neurobiology) for sharing tg(HuC:Gal4;UAS:eGFP) and tg(HuC:Gal4;UAS:GCaMP6s), and L. Godinho (Technical University of Munich) for providing tg(HuC:Gal4;UAS:GCaMP6s). We are grateful to J. Huisken and F. Fahrbach for providing assistance on building the SPIM; J. Fuchs assisted with the SPIM setup and experiment. We thank B. Wolfrum, P. Rinklin, J. Rebling, D. Razansky and the central machine shop of Helmholtz Zentrum Muenchen for help with the design and production of the arenas; H. Rolbieski for administrative support and help with data organization; T. Durovic and J. Ninkovic for assistance with the two-photon microscopy experiments; M. Fabiszak, C. Penningroth and L. Garrett for valuable comments on the manuscript. We are grateful for support from the European Research Council under grant agreements ERC-StG: 311552 (G.G.W., A.L., P.S.) and the Helmholtz Alliance ICEMED (G.G.W.).

Author information

Affiliations

  1. Institute of Biological and Medical Imaging, Helmholtz Zentrum München, Munich, Germany.

    • Panagiotis Symvoulidis
    • , Antonella Lauri
    • , Michele Cappetta
    • , Anja Stelzl
    • , Maximilian Koch
    • , Carlos Cruz Perez
    • , Ahne Myklatun
    • , Andriy Chmyrov
    • , Vasilis Ntziachristos
    •  & Gil G Westmeyer
  2. Institute of Developmental Genetics, Helmholtz Zentrum München, Munich, Germany.

    • Panagiotis Symvoulidis
    • , Antonella Lauri
    • , Michele Cappetta
    • , Anja Stelzl
    • , Carlos Cruz Perez
    • , Ahne Myklatun
    • , Wolfgang Wurst
    •  & Gil G Westmeyer
  3. Department of Nuclear Medicine, Technical University of Munich, Munich, Germany.

    • Panagiotis Symvoulidis
    • , Antonella Lauri
    • , Michele Cappetta
    • , Steffen Schneider
    • , Ahne Myklatun
    •  & Gil G Westmeyer
  4. Computer Aided Medical Procedures, Technical University of Munich, Munich, Germany.

    • Anca Stefanoiu
    •  & Tobias Lasser
  5. Institute of Neuroscience, Technical University of Munich, Munich, Germany.

    • Hongbo Jia
  6. Champalimaud Centre for the Unknown, Lisbon, Portugal.

    • Sabine Renninger
  7. Chair for Biological Imaging, Technical University of Munich, Munich, Germany.

    • Andriy Chmyrov
    •  & Vasilis Ntziachristos

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Contributions

P.S. designed and implemented NeuBtracker with important contributions from H.J., M.K. and A.C.; programmed control software; conducted neurobehavioral imaging experiments including SPIM; analyzed data; and wrote the manuscript. A.L. designed and implemented neurobehavioral and two-photon experiments, conducted p-ERK imaging experiments, analyzed data, created illustrations and wrote the manuscript. A.S. and T.L. implemented image registration routines and improved tracking algorithm and analyzed data together with S.S. M.C. carried out the first implementation of tracking algorithm and performed original image and signal processing. C.C.P. carried out the initial implementation of SPIM. A.M. conducted photoaversion experiments on immobilized fish. A.S. was responsible for animal husbandry and provided support for neurobehavioral imaging. S.R. generated tg(HuC:GCaMP6s). W.W. and V.N. supported the project and provided feedback on the manuscript. G.G.W. conceived and supervised the work, analyzed data and wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Gil G Westmeyer.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–13 and Supplementary Table 1.

  2. 2.

    Life Sciences Reporting Summary

    Life Sciences Reporting Summary.

Zip files

  1. 1.

    Supplementary Software

    NeuBtracker software suite - data acquisition and processing routines

Videos

  1. 1.

    NeuBtracker: an open-source imaging platform for interrogating neurobehavioral dynamics in freely behaving fish

    Overview of the design, features, data acquisition and processing pipeline of NeuBtracker