Abstract
Intracellular calcium signals in neurons frequently derive from the stimulation of G protein-coupled receptors (GPCR) by neurotransmitters, neuropeptides, and neurohormones. GPCR stimulation in neurons leads to generation of inositol 1,4,5-trisphosphate (IP3), which in turn activates endoplasmic reticulum (ER)-localized IP3 receptors. The IP3 receptor (IP3R) is a ligand-gated Ca2+ channel, which releases Ca2+ from ER stores. In Drosophila neurons it has been shown that depletion of ER Ca2+ store is followed by store-operated Ca2+ entry (SOCE) through STIM and Orai, the ER Ca2+ sensor and the plasma membrane Ca2+ channel respectively. The elucidation of this Ca2+ signaling pathway in neurons has in part been possible due to the ease of genetic manipulation in Drosophila, which has allowed neuron-specific knockdown of various proteins of interest. This has been followed by standardization of conditions for culturing neurons from dissected brains of the relevant genotypes, such that they could be used for robust Ca2+ measurements by imaging with standard Ca2+ indicator dyes. Protocols for measurement of IP3-mediated Ca2+ release, passive depletion of ER Ca2+ store, and SOCE in primary cultures of Drosophila neurons are described here.
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References
Saito M, Wu CF (1991) Expression of ion channels and mutational effects in giant Drosophila neurons differentiated from cell division-arrested embryonic neuroblasts. J Neurosci 11(7):2135–2150
Wu CF, Sakai K, Saito M, Hotta Y (1990) Giant Drosophila neurons differentiated from cytokinesis-arrested embryonic neuroblasts. J Neurobiol 21(3):499–507
Seecof RL, Unanue RL (1968) Differentiation of embryonic Drosophila cells in vitro. Exp Cell Res 50(3):654–660
Shields G, Sang JH (1970) Characteristics of five cell types appearing during in vitro culture of embryonic material from Drosophila melanogaster. J Embryol Exp Morphol 23(1):53–69
Savakis C, Demetri G, Cherbas P (1980) Ecdysteroid-inducible polypeptides in a Drosophila cell line. Cell 22(3):665–674
Sang JH (1981) Drosophila cells and cell lines. In: Maramorosch K (ed) Advances in cell culture, vol 1. Academic Press, Inc., New York, pp 125–182
Moscona A (1961) Rotation-mediated histogenetic aggregation of dissociated cells. A quantifiable approach to cell interactions in vitro. Exp Cell Res 22:455–475
Wu CF, Suzuki N, Poo MM (1983) Dissociated neurons from normal and mutant Drosophila larval central nervous system in cell culture. J Neurosci 3(9):1888–1899
Schneider I (1964) Differentiation of larval Drosophila eye-antennal discs in vitro. J Exp Zool 156:91–103
Schneider I, Blumenthal AB (1978) Drosophila cell and tissue culture. In: Ashburner M, Wright TRF (eds) The genetics and biology of Drosophila, vol IIa. Academic Press, Inc., New York, pp 265–315
Banerjee S et al (2006) Compensation of inositol 1,4,5-trisphosphate receptor function by altering sarco-endoplasmic reticulum calcium ATPase activity in the Drosophila flight circuit. J Neurosci 26(32):8278–8288
Venkiteswaran G, Hasan G (2009) Intracellular Ca2+ signaling and store-operated Ca2+ entry are required in Drosophila neurons for flight. Proc Natl Acad Sci U S A 106(25):10326–10331
Chakraborty S, Hasan G (2012) Functional complementation of Drosophila itpr mutants by rat Itpr1. J Neurogenet 26(3–4):328–337
Chakraborty S et al (2016) Mutant IP3 receptors attenuate store-operated Ca2+ entry by destabilizing STIM–Orai interactions in Drosophila neurons. J Cell Sci 15; 129(20):3903–3910
Bird GS, DeHaven WI, Smyth JT, Putney JW (2008) Methods for studying store-operated calcium entry. Methods 46(3):204–212
Hogan PG, Lewis RS, Rao A (2010) Molecular basis of calcium signaling in lymphocytes: STIM and ORAI. Annu Rev Immunol 28:491–533
Akerboom J et al (2009) Crystal structures of the GCaMP calcium sensor reveal the mechanism of fluorescence signal change and aid rational design. J Biol Chem 284(10):6455–6464
Tian L et al (2009) Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators. Nat Methods 6(12):875–881
Phelps CB, Brand AH (1998) Ectopic gene expression in Drosophila using GAL4 system. Methods 14(4):367–379
Akerboom J et al (2013) Genetically encoded calcium indicators for multi-color neural activity imaging and combination with optogenetics. Front Mol Neurosci 6:2
Wachowiak M et al (2013) Optical dissection of odor information processing in vivo using GCaMPs expressed in specified cell types of the olfactory bulb. J Neurosci 33(12):5285–5300
Hong JH, Min CH, Jeong B, Kojiya T, Morioka E, Nagai T, Ikeda M, Lee KJ (2009)(2010) Intracellular calcium spikes in rat suprachiasmatic nucleus neurons induced by BAPTA-based calcium dyes. PLoS One 5(3):e9634. https://doi.org/10.1371/journal.pone.0009634
Jiang SA, Campusano JM, Su H, O’Dowd DK (2005) Drosophila mushroom body Kenyon cells generate spontaneous calcium transients mediated by PLTX-sensitive calcium channels. J Neurophysiol 94(1):491–500
Sicaeros B, Campusano JM, O’Dowd DK (2007) Primary neuronal cultures from the brains of late stage Drosophila pupae. J Vis Exp 4:200
Chakraborty S and Hasan G (2017) Spontaneous Ca2+ Influx in Drosophila Pupal Neurons Is Modulated by IP3-Receptor Function and Influences Maturation of the Flight Circuit. Front Mol Neurosci 10:111.
Grynkiewicz G, Poenie M, Tsien RY (1985) A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260(6):3440–3450
Hardie RC (1996) INDO-1 measurements of absolute resting and light-induced Ca2+ concentration in Drosophila photoreceptors. J Neurosci 16(9):2924–2933
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Chakraborty, S., Hasan, G. (2018). Store-Operated Ca2+ Entry in Drosophila Primary Neuronal Cultures. In: Penna, A., Constantin, B. (eds) The CRAC Channel. Methods in Molecular Biology, vol 1843. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8704-7_11
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DOI: https://doi.org/10.1007/978-1-4939-8704-7_11
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