Zebrafish pp 333-341 | Cite as

Calcium Imaging of Neuronal Activity in Free-Swimming Larval Zebrafish

Part of the Methods in Molecular Biology book series (MIMB, volume 1451)


Visualization of neuronal activity during animal behavior is a critical step in understanding how the brain generates behavior. In the model vertebrate zebrafish, imaging of the brain has been done mostly by using immobilized fish. Here, we describe a novel method to image neuronal activity of the larval zebrafish brain during prey capture behavior. We expressed a genetically encoded fluorescent calcium indicator, GCaMP, in the optic tectum of the midbrain using the Gal4-UAS system. Tectal activity was then imaged in unrestrained larvae during prey perception. Since larval zebrafish swim only intermittently, detection of the neuronal activity is possible between swimming bouts. Our method makes functional brain imaging under natural behavioral conditions feasible and will greatly benefit the study of neuronal activities that evoke animal behaviors.

Key words

Calcium imaging GCaMP Tectum Prey capture Vision Paramecium Gal4-UAS system 



This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) in Japan: KAKENHI Grant Number 24120521, 25290009, and 25650120.


  1. 1.
    Kettunen P (2012) Calcium imaging in the zebrafish. Adv Exp Med Biol 740:1039–1071CrossRefPubMedGoogle Scholar
  2. 2.
    Semmelhack JL et al (2015) A dedicated visual pathway for prey detection in larval zebrafish. eLife 2014;3:e04878Google Scholar
  3. 3.
    Ritter DA, Bhatt DH, Fetcho JR (2001) In vivo imaging of zebrafish reveals differences in the spinal networks for escape and swimming movements. J Neurosci 21(22):8956–8965PubMedGoogle Scholar
  4. 4.
    Bianco IH, Engert F (2015) Visuomotor transformations underlying hunting behavior in zebrafish. Curr Biol 25(7):831–846CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Nakai J, Ohkura M, Imoto K (2001) A high signal-to-noise Ca(2+) probe composed of a single green fluorescent protein. Nat Biotechnol 19(2):137–141CrossRefPubMedGoogle Scholar
  6. 6.
    Muto A et al (2013) Real-time visualization of neuronal activity during perception. Curr Biol 23(4):307–311CrossRefPubMedGoogle Scholar
  7. 7.
    Muto A et al (2011) Genetic visualization with an improved GCaMP calcium indicator reveals spatiotemporal activation of the spinal motor neurons in zebrafish. Proc Natl Acad Sci U S A 108(13):5425–5430CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Asakawa K, Kawakami K (2008) Targeted gene expression by the Gal4-UAS system in zebrafish. Dev Growth Differ 50(6):391–399CrossRefPubMedGoogle Scholar
  9. 9.
    Asakawa K et al (2008) Genetic dissection of neural circuits by Tol2 transposon-mediated Gal4 gene and enhancer trapping in zebrafish. Proc Natl Acad Sci U S A 105(4):1255–1260CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Kawakami K et al (2010) zTrap: zebrafish gene trap and enhancer trap database. BMC Dev Biol 10:105CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Lister JA et al (1999) Nacre encodes a zebrafish microphthalmia-related protein that regulates neural-crest-derived pigment cell fate. Development 126(17):3757–3767PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Division of Molecular and Developmental Biology, Department of Genetics, National Institute of GeneticsSOKENDAI (The Graduate University for Advanced Studies)MishimaJapan

Personalised recommendations