Zebrafish pp 307-320 | Cite as

Correlating Whole Brain Neural Activity with Behavior in Head-Fixed Larval Zebrafish

  • Michael B. Orger
  • Ruben PortuguesEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1451)


We present a protocol to combine behavioral recording and imaging using 2-photon laser-scanning microscopy in head-fixed larval zebrafish that express a genetically encoded calcium indicator. The steps involve restraining the larva in agarose, setting up optics that allow projection of a visual stimulus and infrared illumination to monitor behavior, and analysis of the neuronal and behavioral data.

Key words

Whole-brain imaging Behavior 2-Photon microscopy Zebrafish 



The authors would like to acknowledge the contribution of colleagues Claudia Feierstein and Florian Engert to the development of the methods described in this protocol. MBO was supported by a Marie Curie Career Integration Grant, PCIG09-GA-2011-294049. RP was supported by the Max Planck Society.


  1. 1.
    Fetcho JR, O’Malley DM (1997) Imaging neuronal networks in behaving animals. Curr Opin Neurobiol 7:832–838CrossRefPubMedGoogle Scholar
  2. 2.
    Kettunen P (2012) Calcium imaging in the zebrafish. Adv Exp Med Biol 740:1039–1071CrossRefPubMedGoogle Scholar
  3. 3.
    Ahrens MB, Li JM, Orger MB et al (2012) Brain-wide neuronal dynamics during motor adaptation in zebrafish. Nature 485:471–477PubMedPubMedCentralGoogle Scholar
  4. 4.
    Ahrens MB, Orger MB, Robson DN et al (2013) Whole-brain functional imaging at cellular resolution using light-sheet microscopy. Nat Methods 10:413–420CrossRefPubMedGoogle Scholar
  5. 5.
    Wolf S, Supatto W, Debrégeas G et al (2015) Whole-brain functional imaging with two-photon light-sheet microscopy. Nat Methods 12:379–380CrossRefPubMedGoogle Scholar
  6. 6.
    Portugues R, Feierstein CE, Engert F et al (2014) Whole-brain activity maps reveal stereotyped, distributed networks for visuomotor behavior. Neuron 81:1328–1343CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Renninger SL, Orger MB (2013) Two-photon imaging of neural population activity in zebrafish. Methods 62:255–267CrossRefPubMedGoogle Scholar
  8. 8.
    Niell CM, Smith SJ (2005) Functional imaging reveals rapid development of visual response properties in the zebrafish tectum. Neuron 45:941–951CrossRefPubMedGoogle Scholar
  9. 9.
    O'Malley DM, Sankrithi NS, Borla MA et al (2004) Optical physiology and locomotor behaviors of wild-type and nacre zebrafish. Methods Cell Biol 76:261–284CrossRefPubMedGoogle Scholar
  10. 10.
    Portugues R, Engert F (2011) Adaptive locomotor behavior in larval zebrafish. Front Syst Neurosci 5:72CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Lister JA, Robertson CP, Lepage T et al (1999) Nacre encodes a zebrafish microphthalmia-related protein that regulates neural-crest-derived pigment cell fate. Development 126:3757–3767PubMedGoogle Scholar
  12. 12.
    Severi K, Portugues R, Marques J et al (2014) Neural control and modulation of swimming speed in the larval zebrafish. Neuron 83(3):692–707CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Orger MB, Kampff AR, Severi KE et al (2008) Control of visually guided behavior by distinct populations of spinal projection neurons. Nat Neurosci 11:327–333CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Miri A, Daie K, Burdine RD et al (2011) Regression-based identification of behavior-encoding neurons during large-scale optical imaging of neural activity at cellular resolution. J Neurophysiol 105:964–980CrossRefPubMedGoogle Scholar
  15. 15.
    Nestares O, Heeger DJ (2000) Robust multiresolution alignment of MRI brain volumes. Magn Reson Med 43:705–715CrossRefPubMedGoogle Scholar
  16. 16.
    Mukamel EA, Nimmerjahn A, Schnitzer MJ (2009) Automated analysis of cellular signals from large-scale calcium imaging data. Neuron 63:747–760CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Romano SA, Pietri T, Pérez-Schuster V et al (2015) Spontaneous neuronal network dynamics reveal circuit’s functional adaptations for behavior. Neuron 85:1070–1085CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Kim CK, Miri A, Leung LC et al (2014) Prolonged, brain-wide expression of nuclear-localized GCaMP3 for functional circuit mapping. Front Neural Circ 8:138Google Scholar
  19. 19.
    Akerboom J, Chen TW, Wardill TJ et al (2012) Optimization of a GCaMP calcium indicator for neural activity imaging. J Neurosci 32:13819–13840CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Straw AD (2008) Vision egg: an open-source library for realtime visual stimulus generation. Front Neuroinformatics 2Google Scholar
  21. 21.
    Brainard DH (1997) The psychophysics toolbox. Spat Vis 10:433–436CrossRefPubMedGoogle Scholar
  22. 22.
    Page-Mccaw PS, Chung SC, Muto A et al (2004) Retinal network adaptation to bright light requires tyrosinase. Nat Neurosci 7:1329–1336CrossRefPubMedGoogle Scholar
  23. 23.
    Kubo F, Hablitzel B, Maschio MD et al (2014) Functional architecture of an optic flow-responsive area that drives horizontal eye movements in zebrafish. Neuron 81:1344–1359CrossRefPubMedGoogle Scholar
  24. 24.
    Bianco IH, Engert F (2015) Visuomotor transformations underlying hunting behavior in zebrafish. Curr Biol 25:831–846CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Huber D, Gutnisky DA, Peron S et al (2012) Multiple dynamic representations in the motor cortex during sensorimotor learning. Nature 484:473–478CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Champalimaud Neuroscience ProgrammeChampalimaud Centre for the UnknownLisbonPortugal
  2. 2.Max Planck Institute of Neurobiology, Sensorimotor Control Research GroupMartinsriedGermany

Personalised recommendations