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In vitro imaging of primary neural cell culture from Drosophila

Nature Protocols volume 8, pages 958–965 (2013)Cite this article

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Abstract

Cell culture systems are widely used for molecular, genetic and biochemical studies. Primary cell cultures of animal tissues offer the advantage that specific cell types can be studied in vitro outside of their normal environment. We provide a detailed protocol for generating primary neural cell cultures derived from larval brains of Drosophila melanogaster. The developing larval brain contains stem cells such as neural precursors and intermediate neural progenitors, as well as fully differentiated and functional neurons and glia cells. We describe how to analyze these cell types in vitro by immunofluorescent staining and scanning confocal microscopy. Cell type–specific fluorescent reporter lines and genetically encoded calcium sensors allow the monitoring of developmental, cellular processes and neuronal activity in living cells in vitro. The protocol provides a basis for functional studies of wild-type or genetically manipulated primary neural cells in culture, both in fixed and living samples. The entire procedure takes ∼3 weeks.

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Figure 1: Illustration of important steps and typical equipment used in the protocol.
Figure 2: Primary neural cell cultures derived from Drosophila larval brains.

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References

  1. Campos-Ortega, J. & Hartenstein, V. The Embryonic Development of Drosophila melanogaster 2nd edn. (Springer, 1997).

  2. Schneider, I. Cell lines derived from late embryonic stages of Drosophila melanogaster. J. Embryol. Exp. Morphol. 27, 353–365 (1972).

    CAS  PubMed  Google Scholar 

  3. Echalier, G. & Ohanessian, A. Isolation, in tissue culture, of Drosophila melangaster cell lines. Serie D: Sciences naturelles 268, 1771–1773 (1969).

    CAS  Google Scholar 

  4. Echalier, G. Drosophila Cells in Culture. (Academic Press, 1997).

  5. Dobbelaere, J. et al. A genome-wide RNAi screen to dissect centriole duplication and centrosome maturation in Drosophila. PLoS Biol. 6, e224 (2008).

    Article  Google Scholar 

  6. Mohr, S., Bakal, C. & Perrimon, N. Genomic screening with RNAi: results and challenges. Annu. Rev. Biochem. 79, 37–64 (2010).

    Article  CAS  Google Scholar 

  7. Castoreno, A.B. et al. Small molecules discovered in a pathway screen target the Rho pathway in cytokinesis. Nat. Chem. Biol. 6, 457–463 (2010).

    Article  CAS  Google Scholar 

  8. Zhang, Y. et al. Expression in aneuploid Drosophila S2 cells. PLoS Biol. 8, e1000320 (2010).

    Article  Google Scholar 

  9. Gonzalez, M. et al. Generation of stable Drosophila cell lines using multicistronic vectors. Sci. Rep. 1, 75 (2011).

    Article  Google Scholar 

  10. Simcox, A. et al. Efficient genetic method for establishing Drosophila cell lines unlocks the potential to create lines of specific genotypes. PLoS Genet. 4, e1000142 (2008).

    Article  Google Scholar 

  11. Donady, J.J. & Seecof, R.L. Effect of the gene lethal (1) myospheroid on Drosophila embryonic cells in vitro. In Vitro 8, 7–12 (1972).

    Article  CAS  Google Scholar 

  12. Prokop, A., Kuppers-Munther, B. & Sanchez-Soriano, N. Using primary neuron cultures of Drosophila to analyze neuronal circuit formation and function. in The Making and Un-making of Neuronal Circuits Ch. 10 (ed. Hassan, B.A.) 225–248 (Humana Press, 2012).

  13. Grosskortenhaus, R., Pearson, B.J., Marusich, A. & Doe, C.Q. Regulation of temporal identity transitions in Drosophila neuroblasts. Dev. Cell 8, 193–202 (2005).

    Article  CAS  Google Scholar 

  14. Kraft, R. et al. Phenotypes of Drosophila brain neurons in primary culture reveal a role for fascin in neurite shape and trajectory. J. Neurosci. 26, 8734–8747 (2006).

    Article  CAS  Google Scholar 

  15. Kraft, R., Levine, R.B. & Restifo, L.L. The steroid hormone 20-hydroxyecdysone enhances neurite growth of Drosophila mushroom body neurons isolated during metamorphosis. J. Neurosci. 18, 8886–8899 (1998).

    Article  CAS  Google Scholar 

  16. Seecof, R.L., Teplitz, R.L., Gerson, I., Ikeda, K. & Donady, J. Differentiation of neuromuscular junctions in cultures of embryonic Drosophila cells. Proc. Natl. Acad. Sci. USA 69, 566–570 (1972).

    Article  CAS  Google Scholar 

  17. Sicaeros, B., Campusano, J.M. & O'Dowd, D.K. Primary neuronal cultures from the brains of late-stage Drosophila pupae. J. Vis. Exp. 200 (2007).

  18. Luer, K. & Technau, G.M. Single-cell cultures of Drosophila neuroectodermal and mesectodermal central nervous system progenitors reveal different degrees of developmental autonomy. Neural Develop. 4, 30 (2009).

    Article  Google Scholar 

  19. Gu, H. et al. Cav2-type calcium channels encoded by cac regulate AP-independent neurotransmitter release at cholinergic synapses in adult Drosophila brain. J. Neurophysiol. 101, 42–53 (2009).

    Article  CAS  Google Scholar 

  20. Su, H. & O'Dowd, D.K. Fast synaptic currents in Drosophila mushroom body Kenyon cells are mediated by α-bungarotoxin–sensitive nicotinic acetylcholine receptors and picrotoxin-sensitive GABA receptors. J. Neurosci. 23, 9246–9253 (2003).

    Article  CAS  Google Scholar 

  21. Campusano, J.M., Su, H., Jiang, S.A., Sicaeros, B. & O'Dowd, D.K. nAChR-mediated calcium responses and plasticity in Drosophila Kenyon cells. Dev. Neurobiol. 67, 1520–1532 (2007).

    Article  CAS  Google Scholar 

  22. Riemensperger, T., Pech, U., Dipt, S. & Fiala, A. Optical calcium imaging in the nervous system of Drosophila melanogaster. Biochim. Biophys. Acta 1820, 1169–1178 (2012).

    Article  CAS  Google Scholar 

  23. Liu, Z., Celotto, A.M., Romero, G., Wipf, P. & Palladino, M.J. Genetically encoded redox sensor identifies the role of ROS in degenerative and mitochondrial disease pathogenesis. Neurobiol. Dis. 45, 362–368 (2012).

    Article  CAS  Google Scholar 

  24. Wu, C.F., Suzuki, N. & Poo, M.M. Dissociated neurons from normal and mutant Drosophila larval central nervous system in cell culture. J. Neurosci. 3, 1888–1899 (1983).

    Article  CAS  Google Scholar 

  25. Ceron, J., Tejedor, F.J. & Moya, F. A primary cell culture of Drosophila postembryonic larval neuroblasts to study cell cycle and asymmetric division. Eur. J. Cell Biol. 85, 567–575 (2006).

    Article  CAS  Google Scholar 

  26. Moraru, M., Egger, B., Bao, D.B. & Sprecher, S.G. Analysis of cell identity, morphology, apoptosis and mitotic activity in a primary neural cell culture system in Drosophila. Neural Develop. 7, 14 (2012).

    Article  Google Scholar 

  27. Berger, C. et al. FACS purification and transcriptome analysis of Drosophila neural stem cells reveals a role for Klumpfuss in self-renewal. Cell Rep. 2, 407–418 (2012).

    Article  CAS  Google Scholar 

  28. Tian, L. et al. Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators. Nat. Methods 6, 875–881 (2009).

    Article  CAS  Google Scholar 

  29. Ashburner, M., Golic, K.G. & Hawley, R.S. Drosophila: a Laboratory Handbook 2nd edn. (Cold Spring Harbor Laboratory Press, 2005).

  30. Python, F. & Stocker, R.F. Adult-like complexity of the larval antennal lobe of D. melanogaster despite markedly low numbers of odorant receptor neurons. J. Comparative Neurol. 445, 374–387 (2002).

    Article  Google Scholar 

  31. Park, J.H. et al. Differential regulation of circadian pacemaker output by separate clock genes in Drosophila. Proc. Natl. Acad. Sci. USA 97, 3608–3613 (2000).

    Article  CAS  Google Scholar 

  32. Poeck, B., Triphan, T., Neuser, K. & Strauss, R. Locomotor control by the central complex in Drosophila—An analysis of the tay bridge mutant. Dev. Neurobiol. 68, 1046–1058 (2008).

    Article  CAS  Google Scholar 

  33. Egger, B., Boone, J.Q., Stevens, N.R., Brand, A.H. & Doe, C.Q. Regulation of spindle orientation and neural stem cell fate in the Drosophila optic lobe. Neural Develop. 2, 1 (2007).

    Article  Google Scholar 

  34. Prokop, A., Bray, S., Harrison, E. & Technau, G.M. Homeotic regulation of segment-specific differences in neuroblast numbers and proliferation in the Drosophila central nervous system. Mech. Dev. 74, 99–110 (1998).

    Article  CAS  Google Scholar 

  35. Thacker, S.A., Bonnette, P.C. & Duronio, R.J. The contribution of E2F-regulated transcription to Drosophila PCNA gene function. Curr. Biol. 13, 53–58 (2003).

    Article  CAS  Google Scholar 

  36. Langevin, J. et al. Lethal giant larvae controls the localization of notch-signaling regulators numb, neuralized, and Sanpodo in Drosophila sensory-organ precursor cells. Curr. Biol. 15, 955–962 (2005).

    Article  CAS  Google Scholar 

  37. Akerboom, J. et al. Optimization of a GCaMP calcium indicator for neural activity imaging. J. Neurosci. 32, 13819–13840 (2012).

    Article  CAS  Google Scholar 

  38. Weng, M., Komori, H. & Lee, C.Y. Identification of neural stem cells in the Drosophila larval brain. Methods Mol. Biol. 879, 39–46 (2012).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank the Bloomington Stock Center, K. Matthews, Y. Bellaiche and L. Looger for fly lines, and the DHSB for antibodies. Special thanks to our colleagues at the Department of Biology, the Institute of Developmental and Cell Biology and the Sprecher lab for helpful discussions. This work was funded by grant no. PP00P3_123339 from the Swiss National Science Foundation to S.G.S., the Novartis Foundation for Biomedical Research to S.G.S. and by the Swiss University Conference (SUK/CUS) to B.E.

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Author notes

  1. Manuela Moraru

    Present address: Present address: ISREC, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.,

Authors and Affiliations

  1. Department of Biology, Institute of Cell and Developmental Biology, University of Fribourg, Fribourg, Switzerland

    Boris Egger, Lena van Giesen, Manuela Moraru & Simon G Sprecher

Authors
  1. Boris Egger
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Contributions

B.E., L.v.G. and M.M. performed the experiments. M.M., B.E., L.v.G. and S.G.S. developed the protocol. B.E. and S.G.S. wrote the paper.

Corresponding author

Correspondence to Simon G Sprecher.

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The authors declare no competing financial interests.

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Egger, B., van Giesen, L., Moraru, M. et al. In vitro imaging of primary neural cell culture from Drosophila. Nat Protoc 8, 958–965 (2013). https://doi.org/10.1038/nprot.2013.052

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