Using Primary Neuron Cultures of Drosophila to Analyze Neuronal Circuit Formation and Function

  • Andreas Prokop
  • Barbara Küppers-Munther
  • Natalia Sánchez-Soriano
Protocol
Part of the Neuromethods book series (NM, volume 69)

Abstract

For many decades, primary neuron cultures of Drosophila have been used complementary to work in vivo. Primary cultures were instrumental for the analysis of physiological properties of Drosophila neurons and synapses, and they were used for the analysis of developmental processes. Recent developments have established Drosophila primary neurons based on Schneider’s culture media as a means to investigate the neuronal cytoskeleton, opening up novel opportunities for research into cellular mechanisms of axonal growth, synapse formation, and perhaps even neuronal degeneration. These cell cultures provide readouts for cytoskeletal dynamics that are difficult or impossible to access in vivo, and which turned out to be highly conserved with mammalian or other vertebrate neurons. Therefore, the same genetic manipulations in Drosophila can now be studied synergistically in culture and in vivo, to address cell biological principles of neuronal circuit formation and function. Here, we describe in detail how these cell cultures are generated and discuss principal considerations for the experimental design and the solution of common problems. Furthermore, we describe in detail how to generate Schneider’s media with adjustable inorganic ion concentrations. These media have been shown to promote the physiological maturation of neurons, thus expanding the use of the primary neuron cultures into the synaptic stage. The culture strategies described here recapitulate in vivo development with impressive accuracy and provide a promising means for Drosophila research on neuronal development and function.

Key words

Drosophila Cell culture Primary neurons Axonal growth Neurogenesis Synapse formation Cytoskeleton 

References

  1. 1.
    Arimura N, Kaibuchi K (2007) Neuronal polarity: from extracellular signals to intracellular mechanisms. Nat Rev Neurosci 8:194–205PubMedCrossRefGoogle Scholar
  2. 2.
    Craig AM, Graf ER, Linhoff MW (2006) How to build a central synapse: clues from cell culture. Trends Neurosci 29:8–20PubMedCrossRefGoogle Scholar
  3. 3.
    Lowery LA, van Vactor D (2009) The trip of the tip: understanding the growth cone machinery. Nat Rev Mol Cell Biol 10:332–343PubMedCrossRefGoogle Scholar
  4. 4.
    Olmsted JB, Carlson K, Klebe R, Ruddle F, Rosenbaum J (1970) Isolation of microtubule protein from cultured mouse neuroblastoma cells. Proc Natl Acad Sci U S A 65:129–136PubMedCrossRefGoogle Scholar
  5. 5.
    Rasko I, Georgieva M, Farkas G, Santha M, Coates J, Burg K, Mitchell DL, Johnson RT (1993) New patterns of bulk DNA repair in ultraviolet irradiated mouse embryo carcinoma cells following differentiation. Somat Cell Mol Genet 19:245–255PubMedCrossRefGoogle Scholar
  6. 6.
    Liu T, Sims D, Baum B (2009) Parallel RNAi screens across different cell lines identify generic and cell type-specific regulators of actin organization and cell morphology. Genome Biol 10:R26PubMedCrossRefGoogle Scholar
  7. 7.
    Ui K, Nishihara S, Sakuma M, Togashi S, Ueda R, Miyata Y, Miyake T (1994) Newly established cell lines from Drosophila larval CNS express neural specific characteristics. In Vitro Cell Dev Biol Anim 30A:209–216PubMedCrossRefGoogle Scholar
  8. 8.
    Song HJ, Stevens CF, Gage FH (2002) Neural stem cells from adult hippocampus develop essential properties of functional CNS neurons. Nat Neurosci 5:438–445PubMedGoogle Scholar
  9. 9.
    Schwartz PH, Bryant PJ, Fuja TJ, Su H, O’Dowd DK, Klassen H (2003) Isolation and characterization of neural progenitor cells from post-mortem human cortex. J Neurosci Res 74:838–851PubMedCrossRefGoogle Scholar
  10. 10.
    Seecof R, Alleaume N, Teplitz R, Gerson I (1971) Differentiation of neurons and myocytes in cell cultures made from Drosophila gastrulae. Exp Cell Res 69:161–173PubMedCrossRefGoogle Scholar
  11. 11.
    Banker G, Goslin K (1988) Developments in neuronal cell culture. Nature 336:185–186PubMedCrossRefGoogle Scholar
  12. 12.
    Banker G, Goslin K (1998) Culturing nerve cells, 2nd edn. MIT Press, Cambridge, MAGoogle Scholar
  13. 13.
    Beadle DJ (2006) Insect neuronal cultures: an experimental vehicle for studies of physiology, pharmacology and cell interactions. Invert Neurosci 6:95–103PubMedCrossRefGoogle Scholar
  14. 14.
    Rohrbough J, O’Dowd DK, Baines RA, Broadie K (2003) Cellular bases of behavioral plasticity: establishing and modifying synaptic circuits in the Drosophila genetic system. J Neurobiol 54:254–271PubMedCrossRefGoogle Scholar
  15. 15.
    Lüer K, Technau GM (1992) Primary culture of single ectodermal precursors of Drosophila reveals a dorsoventral prepattern of intrinsic neurogenic and epidermogenic capabilities at the early gastrula stage. Development 116:377–385PubMedGoogle Scholar
  16. 16.
    Lüer K, Technau GM (2009) Single cell cultures of Drosophila neuroectodermal and mesectodermal central nervous system progenitors reveal different degrees of developmental autonomy. Neural Dev 4:30PubMedCrossRefGoogle Scholar
  17. 17.
    Brody T, Odenwald WF (2000) Programmed transformations in neuroblast gene expression during Drosophila CNS lineage development. Dev Biol 226:34–44PubMedCrossRefGoogle Scholar
  18. 18.
    Ceron J, Tejedor FJ, Moya F (2006) A primary cell culture of Drosophila postembryonic larval neuroblasts to study cell cycle and asymmetric division. Eur J Cell Biol 85:567–575PubMedCrossRefGoogle Scholar
  19. 19.
    Kim YT, Wu CF (1996) Reduced growth cone motility in cultured neurons from Drosophila memory mutants with a defective cAMP cascade. J Neurosci 16:5593–5602PubMedGoogle Scholar
  20. 20.
    Kraft R, Levine RB, Restifo LL (1998) The steroid hormone 20-hydroxyecdysone enhances neurite growth of Drosophila mushroom body neurons isolated during metamorphosis. J Neurosci 18:8886–8899PubMedGoogle Scholar
  21. 21.
    Küppers B, Sánchez-Soriano N, Letzkus J, Technau GM, Prokop A (2003) In developing Drosophila neurones the production of gamma-amino butyric acid is tightly regulated downstream of glutamate decarboxylase translation and can be influenced by calcium. J Neurochem 84:939–951PubMedCrossRefGoogle Scholar
  22. 22.
    Sánchez-Soriano N, Löhr R, Bottenberg W, Haessler U, Kerassoviti A, Knust E, Fiala A, Prokop A (2005) Are dendrites in Drosophila homologous to vertebrate dendrites? Dev Biol 288:126–138PubMedCrossRefGoogle Scholar
  23. 23.
    Katsuki T, Ailani D, Hiramoto M, Hiromi Y (2009) Intra-axonal patterning: intrinsic compartmentalization of the axonal membrane in Drosophila neurons. Neuron 64:188–199PubMedCrossRefGoogle Scholar
  24. 24.
    Küppers-Munther B, Letzkus J, Lüer K, Technau G, Schmidt H, Prokop A (2004) A new culturing strategy optimises Drosophila primary cell cultures for structural and functional analyses. Dev Biol 269:459–478PubMedCrossRefGoogle Scholar
  25. 25.
    Bai J, Sepp KJ, Perrimon N (2009) Culture of Drosophila primary cells dissociated from gastrula embryos and their use in RNAi screening. Nat Protoc 4:1502–1512PubMedCrossRefGoogle Scholar
  26. 26.
    Sánchez-Soriano N, Travis M, Dajas-Bailador F, Goncalves-Pimentel C, Whitmarsh AJ, Prokop A (2009) Mouse ACF7 and Drosophila short stop modulate filopodia formation and microtubule organisation during neuronal growth. J Cell Sci 122:2534–2542PubMedCrossRefGoogle Scholar
  27. 27.
    Sánchez-Soriano N, Gonçalves-Pimentel C, Beaven R, Haessler U, Ofner L, Ballestrem C, Prokop A (2010) Drosophila growth cones: a genetically tractable platform for the analysis of axonal growth dynamics. Dev Neurobiol 70:58–71PubMedGoogle Scholar
  28. 28.
    Matusek T, Gombos R, Szecsenyi A, Sánchez-Soriano N, Czibula A, Pataki C, Gedai A, Prokop A, Rasko I, Mihaly J (2008) Formin proteins of the DAAM subfamily play a role during axon growth. J Neurosci 28:13310–13319PubMedCrossRefGoogle Scholar
  29. 29.
    Gonçalves-Pimentel C, Gombos R, Mihály J, Sánchez-Soriano N, Prokop A (2011) Dissecting regulatory networks of filopodia formation in a Drosophila growth cone model. PLoS One 6:e18340PubMedCrossRefGoogle Scholar
  30. 30.
    Prokop A, Sánchez-Soriano N, Gonçalves-Pimentel C, Molnár I, Kalmár T, Mihály J (2011) DAAM family members leading a novel path into formin research. Commun Integr Biol 4:538–542PubMedGoogle Scholar
  31. 31.
    Sánchez-Soriano N, Tear G, Whitington P, Prokop A (2007) Drosophila as a genetic and cellular model for studies on axonal growth. Neural Dev 2:9PubMedCrossRefGoogle Scholar
  32. 32.
    Conde C, Caceres A (2009) Microtubule assembly, organization and dynamics in axons and dendrites. Nat Rev Neurosci 10:319–332PubMedCrossRefGoogle Scholar
  33. 33.
    Pak CW, Flynn KC, Bamburg JR (2008) Actin-binding proteins take the reins in growth cones. Nat Rev Neurosci 9:136–147PubMedCrossRefGoogle Scholar
  34. 34.
    Insall RH, Machesky LM (2009) Actin dynamics at the leading edge: from simple machinery to complex networks. Dev Cell 17:310–322PubMedCrossRefGoogle Scholar
  35. 35.
    Benitez-King G, Ramirez-Rodriguez G, Ortiz L, Meza I (2004) The neuronal cytoskeleton as a potential therapeutical target in neurodegenerative diseases and schizophrenia. Curr Drug Targets CNS Neurol Disord 3:515–533PubMedCrossRefGoogle Scholar
  36. 36.
    Hirth F (2010) Drosophila melanogaster in the study of human neurodegeneration. CNS Neurol Disord Drug Targets 9:504–523PubMedGoogle Scholar
  37. 37.
    Papanikolopoulou K, Skoulakis EM (2011) The power and richness of modelling tauopathies in Drosophila. Mol Neurobiol 44:122–133PubMedCrossRefGoogle Scholar
  38. 38.
    Schneider I (1964) Differentiation of larval Drosophila eye-antennal discs in vitro. J Exp Zool 156:91–104PubMedCrossRefGoogle Scholar
  39. 39.
    Stewart BA, Atwood HL, Renger JJ, Wang J, Wu CF (1994) Improved stability of Drosophila larval neuromuscular preparations in haemolymph-like physiological solutions. J Comp Physiol A 175:179–191PubMedCrossRefGoogle Scholar
  40. 40.
    Küppers-Munther B (2004) Optimierung und Verwendung embryonaler Primärkulturen von Drosophila zur Untersuchung der Bildung. Struktur und Funktion von Synapsen, Johannes Gutenberg-University, MainzGoogle Scholar
  41. 41.
    Glauert AM (1991) Fixation, dehydration and embedding of biological specimens, vol 3, 8th edn. North Holland Publishing Group, AmsterdamGoogle Scholar
  42. 42.
    Rogers SL, Rogers GC, Sharp DJ, Vale RD (2002) Drosophila EB1 is important for proper assembly, dynamics, and positioning of the mitotic spindle. J Cell Biol 158:873–884PubMedCrossRefGoogle Scholar
  43. 43.
    Prokop A, Technau GM (1993) Cell transplantation. In: Hartley D (ed) Cellular interactions in development: a practical approach. Oxford University Press, London, pp 33–57Google Scholar
  44. 44.
    Shields G, Dübendorfer A, Sang JH (1975) Differentiation in vitro of larval cell types from early embryonic cells of Drosophila melanogaster. J Embryol Exp Morphol 33:159–175PubMedGoogle Scholar
  45. 45.
    Dübendorfer A, Eichenberger-Glinz S (1980) Development and metamorphosis of larval and adult tissues of Drosophila in vitro. In: Kurstak E, Maramorosch K, Dübendorfer A (eds) Invertebrate systems in vitro. Elsevier, Amsterdam, pp 169–185Google Scholar
  46. 46.
    Matsuura R, Tanaka H, Go MJ (2004) Distinct functions of Rac1 and Cdc42 during axon guidance and growth cone morphogenesis in Drosophila. Eur J Neurosci 19:21–31PubMedCrossRefGoogle Scholar
  47. 47.
    Reichardt L, Prokop A (2011) Introduction: the role of extracellular matrix in nervous system development and maintenance. Dev Neurobiol 71:883–888PubMedCrossRefGoogle Scholar
  48. 48.
    Broadie K, Baumgartner S, Prokop A (2011) Extracellular matrix and its receptors in Drosophila neural development. Dev Neurobiol 71:1102–1130PubMedCrossRefGoogle Scholar
  49. 49.
    Takagi Y, Nomizu M, Gullberg D, MacKrell AJ, Keene DR, Yamada Y, Fessler JH (1996) Conserved neuron promoting activity in Drosophila and vertebrate laminin alpha1. J Biol Chem 271:18074–18081PubMedCrossRefGoogle Scholar
  50. 50.
    Campos-Ortega JA, Hartenstein V (1997) The embryonic development of Drosophila melanogaster. Springer, BerlinGoogle Scholar
  51. 51.
    Doe CQ (1992) Molecular markers for identified neuroblasts and ganglion mother cells in the Drosophila central nervous system. Development 116:855–863PubMedGoogle Scholar
  52. 52.
    Budnik V, Gorczyca M, Prokop A (2006) Selected methods for the anatomical study of Drosophila embryonic and larval neuromuscular junctions. In: Budnik V, Ruiz-Cañada C (eds) The fly neuromuscular junction: structure and function—international review of neurobiology. Elsevier Academic Press, San Diego, pp 323–374CrossRefGoogle Scholar
  53. 53.
    Wu C-F, Suzuki N, Poo M (1983) Dissociated neurons from normal and mutant Drosophila larval central nervous system in cell culture. J Neurosci 3:1888–1899PubMedGoogle Scholar
  54. 54.
    Su H, O’Dowd DK (2003) Fast synaptic currents in Drosophila mushroom body Kenyon cells are mediated by alpha-bungarotoxin-­sensitive nicotinic acetylcholine receptors and picrotoxin-sensitive GABA receptors. J Neurosci 23:9246–9253PubMedGoogle Scholar
  55. 55.
    Prokop A, Technau GM (1994) Normal function of the mushroom body defect gene of Drosophila is required for the regulation of the number and proliferation of neuroblasts. Dev Biol 161:321–337PubMedCrossRefGoogle Scholar
  56. 56.
    Prokop A (1999) Integrating bits and pieces—synapse formation in Drosophila embryos. Cell Tissue Res 297:169–186PubMedCrossRefGoogle Scholar
  57. 57.
    Schmidt H, Rickert C, Bossing T, Vef O, Urban J, Technau GM (1997) The embryonic central nervous system lineages of Drosophila melanogaster. II. Neuroblast lineages derived from the dorsal part of the neuroectoderm. Dev Biol 198:186–204CrossRefGoogle Scholar
  58. 58.
    Truman JW, Bate CM (1988) Spatial and temporal patterns of neurogenesis in the CNS of Drosophila melanogaster. Dev Biol 125:145–157PubMedCrossRefGoogle Scholar
  59. 59.
    Prokop A, Technau GM (1991) The origin of postembryonic neuroblasts in the ventral nerve cord of Drosophila melanogaster. Development 111:79–88PubMedGoogle Scholar
  60. 60.
    Truman JW, Talbot WS, Fahrbach SE, Hogness DS (1994) Ecdysone receptor expression in the CNS correlates with stage-specific responses to ecdysteroids during Drosophila and Manduca development. Development 120:219–234PubMedGoogle Scholar
  61. 61.
    Truman JW, Schuppe H, Shepherd D, Williams DW (2004) Developmental architecture of adult-specific lineages in the ventral CNS of Drosophila. Development 131:5167–5184PubMedCrossRefGoogle Scholar
  62. 62.
    Feiguin F, Llamazares S, Gonzalez C (1998) Methods in Drosophila cell cycle biology. Curr Top Dev Biol 36:279–291PubMedCrossRefGoogle Scholar
  63. 63.
    Technau G, Heisenberg M (1982) Neural reorganization during metamorphosis of the corpora pedunculata in Drosophila melanogaster. Nature 295:405–407PubMedCrossRefGoogle Scholar
  64. 64.
    Donady JJ, Seecof RL (1972) Effect of the gene lethal (1) myospheroid on Drosophila embryonic cells in vitro. In Vitro 8:7–12PubMedCrossRefGoogle Scholar
  65. 65.
    Kuroda Y (1974) In vitro activity of cells from genetically lethal embryos of Drosophila. Nature 252:40–41PubMedCrossRefGoogle Scholar
  66. 66.
    Wu C (1988) Neurogenetic studies of Drosophila central nervous system neurons in culture. Academic, New YorkGoogle Scholar
  67. 67.
    Lee T, Luo L (1999) Mosaic analysis with a repressible neurotechnique cell marker for studies of gene function in neuronal morphogenesis. Neuron 22:451–461PubMedCrossRefGoogle Scholar
  68. 68.
    Theodosiou NA and Xu T (1998) Use of FLP/FRT system to study Drosophila development. Methods 14, 355–365PubMedCrossRefGoogle Scholar
  69. 69.
    Duffy JB (2002) GAL4 system in Drosophila: a fly geneticist’s Swiss army knife. Genesis 34:1–15PubMedCrossRefGoogle Scholar
  70. 70.
    Küppers B, Sánchez-Soriano N, Prokop A (2001) Regulation of GABA in the developing CNS of Drosophila embryos. In: Paper presented at neurobiology of Drosophila, Cold Spring Harbor, New YorkGoogle Scholar
  71. 71.
    Brody T, Odenwald WF (2005) Regulation of temporal identities during Drosophila neuroblast lineage development. Curr Opin Cell Biol 17:672–675PubMedCrossRefGoogle Scholar
  72. 72.
    Sicaeros B, Campusano JM, O’Dowd DK (2007) Primary neuronal cultures from the brains of late stage Drosophila pupae. J Vis Exp 4:200. doi:10.3791/200 PubMedGoogle Scholar
  73. 73.
    O’Dowd DK (1995) Voltage-gated currents and firing properties of embryonic Drosophila neurons grown in a chemically defined medium. J Neurobiol 27:113–126PubMedCrossRefGoogle Scholar
  74. 74.
    Lüer K, Schmidt H, Technau GM (1998) Development of neuronal and glial properties within lineages derived from CNS midline precursors in single cell culture. In: Elsner N, Wehner R (eds) New neuroethology on the move; Proceedings of the 26th Göttingen ­neurobiology conference, Stuttgart, New York, p 696Google Scholar
  75. 75.
    Löhr R, Godenschwege T, Buchner E, Prokop A (2002) Compartmentalisation of central neurons in Drosophila: a new strategy of mosaic analysis reveals localisation of pre-synaptic sites to specific segments of neurites. J Neurosci 22:10357–10367PubMedGoogle Scholar
  76. 76.
    Luo L, Liao YJ, Jan LY, Jan YN (1994) Distinct morphogenetic functions of similar small GTPases: Drosophila Drac1 is involved in axonal outgrowth and myoblast fusion. Genes Dev 8:1787–1802PubMedCrossRefGoogle Scholar
  77. 77.
    Mlodzik M, Baker NE, Rubin GM (1990) Isolation and expression of scabrous, a gene regulating neurogenesis in Drosophila. Genes Dev 4:1848–1861PubMedCrossRefGoogle Scholar
  78. 78.
    Oh HW, Campusano JM, Hilgenberg LG, Sun X, Smith MA, O’Dowd DK (2008) Ultrastructural analysis of chemical synapses and gap junctions between Drosophila brain neurons in culture. Dev Neurobiol 68:281–294PubMedCrossRefGoogle Scholar
  79. 79.
    Masur SK, Kim YT, Wu CF (1990) Reversible inhibition of endocytosis in cultured neurons from the Drosophila temperature-sensitive mutant shibire ts1. J Neurogenet 6:191–206PubMedCrossRefGoogle Scholar
  80. 80.
    Seecof RL, Teplitz RL, Gerson I, Ikeda K, Donady JJ (1972) Differentiation of neuromuscular junctions in cultures of embryonic Drosophila cells. Proc Natl Acad Sci U S A 69:566–570PubMedCrossRefGoogle Scholar
  81. 81.
    Berke BA, Lee J, Peng IF, Wu CF (2006) Sub-cellular Ca2+ dynamics affected by voltage- and Ca2+-gated K+ channels: regulation of the soma-growth cone disparity and the quiescent state in Drosophila neurons. Neuroscience 142:629–644PubMedCrossRefGoogle Scholar
  82. 82.
    Berke B, Wu CF (2002) Regional calcium regulation within cultured Drosophila neurons: effects of altered cAMP metabolism by the learning mutations dunce and rutabaga. J Neurosci 22:4437–4447PubMedGoogle Scholar
  83. 83.
    Wu CF, Sakai K, Saito M, Hotta Y (1990) Giant Drosophila neurons differentiated from cytokinesis-arrested embryonic neuroblasts. J Neurobiol 21:499–507PubMedCrossRefGoogle Scholar
  84. 84.
    Yao W-D, Rusch J, Poo MM, Wu C-F (2000) Spontaneous acetylcholine secretion from developing growth cones of Drosophila central neurons in culture: effects of cAMP-pathway mutations. J Neurosci 20:2626–2637PubMedGoogle Scholar
  85. 85.
    Dent EW, Gupton SL, Gertler FB (2011) The growth cone cytoskeleton in axon outgrowth and guidance. Cold Spring Harb Perspect Biol 3(3):a001800PubMedCrossRefGoogle Scholar
  86. 86.
    Fanti Z, Martinez-Perez ME, De-Miguel FF (2010) NeuronGrowth, a software for automatic quantification of neurite and filopodial dynamics from time-lapse sequences of digital images. Dev Neurobiol 71:870–881CrossRefGoogle Scholar
  87. 87.
    Stepanova T, Smal I, van Haren J, Akinci U, Liu Z, Miedema M, Limpens R, van Ham M, van der Reijden M, Poot R et al (2010) History-dependent catastrophes regulate axonal microtubule behavior. Curr Biol 20:1023–1028PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Andreas Prokop
    • 1
  • Barbara Küppers-Munther
    • 2
    • 3
  • Natalia Sánchez-Soriano
    • 1
  1. 1.Wellcome Trust Centre for Cell-Matrix ResearchUniversity of ManchesterManchesterUK
  2. 2.CellartisGöteborgSweden
  3. 3.Systems Biology Research CenterUniversity SkövdeSkövdeSweden

Personalised recommendations