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Optical Calcium Imaging Using DNA-Encoded Fluorescence Sensors in Transgenic Fruit Flies, Drosophila melanogaster

  • Shubham Dipt
  • Thomas Riemensperger
  • André Fiala
Part of the Methods in Molecular Biology book series (MIMB, volume 1071)

Abstract

The invention of protein-based fluorescent biosensors has paved the way to target specific cells with these probes and visualize intracellular processes not only in isolated cells or tissue cultures but also in transgenic animals. In particular, DNA-encoded fluorescence proteins sensitive to Ca2+ ions are often used to monitor changes in intracellular Ca2+ concentrations. This is of particular relevance in neuroscience since the dynamics of intracellular Ca2+ concentrations represents a faithful correlate for neuronal activity, and optical Ca2+ imaging is commonly used to monitor spatiotemporal activity across populations of neurons. In this respect Drosophila provides a favorable model organism due to the sophisticated genetic tools that facilitate the targeted expression of fluorescent Ca2+ sensor proteins. Here we describe how optical Ca2+ imaging of neuronal activity in the Drosophila brain can be carried out in vivo using two-photon microscopy. We exemplify this technique by describing how to monitor odor-evoked Ca2+ dynamics in the primary olfactory center of the Drosophila brain.

Key words

Optical Ca2+ imaging Neuronal activity Drosophila melanogaster Two-photon microscopy G-CaMP Olfactory coding 

Notes

Acknowledgments

This work was supported by the Deutsche Forschungsgemeinschaft (SPP1392, FI 821/2-1, and SFB 889/B4) and the German Federal Ministry for Education and Research via the Bernstein Center for Computational Neuroscience Göttingen B01, grant number 01GQ1005A.

References

  1. 1.
    Miyawaki A (2003) Fluorescence imaging of physiological activity in complex systems using GFP-based probes. Curr Opin Neurobiol 13:591–596PubMedCrossRefGoogle Scholar
  2. 2.
    Looger LL, Griesbeck O (2012) Genetically encoded neural activity indicators. Curr Opin Neurobiol 22:18–23PubMedCrossRefGoogle Scholar
  3. 3.
    Miyawaki A (2005) Innovations in the imaging of brain functions using fluorescent proteins. Neuron 48:189–199PubMedCrossRefGoogle Scholar
  4. 4.
    Willoughby D, Cooper DM (2008) Live-cell imaging of cAMP dynamics. Nat Methods 5:29–36PubMedCrossRefGoogle Scholar
  5. 5.
    Mutoh H, Perron A, Akemann W, Iwamoto Y, Knöpfel T (2011) Optogenetic monitoring of membrane potentials. Exp Physiol 96:13–18PubMedCrossRefGoogle Scholar
  6. 6.
    Tian L, Akerboom J, Schreiter ER, Looger LL (2012) Neural activity imaging with genetically encoded calcium indicators. Prog Brain Res 196:79–94PubMedCrossRefGoogle Scholar
  7. 7.
    Dreosti E, Lagnado L (2011) Optical reporters of synaptic activity in neural circuits. Exp Physiol 96:4–12PubMedCrossRefGoogle Scholar
  8. 8.
    Berridge MJ (1998) Neuronal calcium signaling. Neuron 21:13–26PubMedCrossRefGoogle Scholar
  9. 9.
    Miyawaki A, Llopis J, Heim R, McCaffery JM, Adams JA, Ikura M, Tsien RY (1997) Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature 388:882–887PubMedCrossRefGoogle Scholar
  10. 10.
    Romoser VA, Hinkle PM, Persechini A (1997) Detection in living cells of Ca2+-dependent changes in the fluorescence emission of an indicator composed of two green fluorescent protein variants linked by a calmodulin-binding sequence. A new class of fluorescent indicators. J Biol Chem 272:13270–13274PubMedCrossRefGoogle Scholar
  11. 11.
    Mank M, Griesbeck O (2008) Genetically encoded calcium indicators. Chem Rev 108:1550–1564PubMedCrossRefGoogle Scholar
  12. 12.
    Riemensperger T, Pech U, Dipt S, Fiala A (2012) Optical calcium imaging in the nervous system of Drosophila melanogaster. Biochim Biophys Acta 1820:1169–1178PubMedCrossRefGoogle Scholar
  13. 13.
    Bachmann A, Knust E (2008) The use of P-element transposons to generate transgenic flies. Methods Mol Biol 420:61–77PubMedCrossRefGoogle Scholar
  14. 14.
    Venken KJ, Simpson JH, Bellen HJ (2011) Genetic manipulation of genes and cells in the nervous system of the fruit fly. Neuron 72:202–230PubMedCrossRefGoogle Scholar
  15. 15.
    Brand AH, Perrimon N (1993) Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118:401–415PubMedGoogle Scholar
  16. 16.
    Vosshall LB, Stocker RF (2007) Molecular architecture of smell and taste in Drosophila. Annu Rev Neurosci 30:505–533PubMedCrossRefGoogle Scholar
  17. 17.
    Hallem EA, Carlson JR (2006) Coding of odors by a receptor repertoire. Cell 125:143–160PubMedCrossRefGoogle Scholar
  18. 18.
    Fiala A, Spall T, Diegelmann S, Eisermann B, Sachse S, Devaud JM, Buchner E, Galizia CG (2002) Genetically expressed cameleon in Drosophila melanogaster is used to visualize olfactory information in projection neurons. Curr Biol 12:1877–1884PubMedCrossRefGoogle Scholar
  19. 19.
    Denk W, Strickler JH, Webb WW (1990) Two-photon laser scanning fluorescence microscopy. Science 248:73–76PubMedCrossRefGoogle Scholar
  20. 20.
    Larsson MC, Domingos AI, Jones WD, Chiappe ME, Amrein H, Vosshall LB (2004) Or83b encodes a broadly expressed odorant receptor essential for Drosophila olfaction. Neuron 43:703–714PubMedCrossRefGoogle Scholar
  21. 21.
    Tian L, Hires SA, Mao T, Huber D, Chiappe ME, Chalasani SH, Petreanu L, Akerboom J, McKinney SA, Schreiter ER, Bargmann CI, Jayaraman V, Svoboda K, Looger LL (2009) Imaging neural activity in worms, flies and mice with improved G-CaMP calcium indicators. Nat Methods 6:875–881PubMedCrossRefGoogle Scholar
  22. 22.
    Estes PS, Roos J, van der Bliek A, Kelly RB, Krishnan KS, Ramaswami M (1996) Traffic of dynamin within individual Drosophila synaptic boutons relative to compartment-specific markers. J Neurosci 16:5443–5456PubMedGoogle Scholar
  23. 23.
    Strutz A, Völler T, Riemensperger T, Fiala A, Sachse S (2012) Calcium imaging of neural activity in the olfactory system of Drosophila. In: Martin JR (ed) Genetically encoded functional indicators. Springer Neuromethods 72:43–70Google Scholar
  24. 24.
    Fiala A, Spall T (2003) In vivo calcium imaging of brain activity in Drosophila by transgenic cameleon expression. Sci STKE (174):PL6Google Scholar
  25. 25.
    Olsson SB, Kuebler LS, Veit D, Steck K, Schmidt A, Knaden M, Hansson BS (2011) A novel multicomponent stimulus device for use in olfactory experiments. J Neurosci Methods 195:1–9PubMedCrossRefGoogle Scholar
  26. 26.
    Thévenaz P, Ruttimann UE, Unser M (1998) A pyramid approach to subpixel registration based on intensity. IEEE Trans Image Process 7:27–41PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2014

Authors and Affiliations

  • Shubham Dipt
    • 1
  • Thomas Riemensperger
    • 1
  • André Fiala
    • 1
  1. 1.Johann-Friedrich-Blumenbach-Institute for Zoology and Anthropology and Molecular Neurobiology of BehaviorGeorg-August-Universität GöttingenGöttingenGermany

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