Abstract
In experimentally amenable organism models, several different physiological techniques have been developed to functionally record the neuronal activity, with the goal to map the neuronal circuitry and elucidate the neural code underlying major neurophysiological functions, such as olfaction, vision, learning and memory, sleep, locomotor activity, to name but a few. Apart from electrophysiological approaches, the main approach is optical imaging, principally based on the detection of changes in calcium concentration using fluorescent probes/sensors. The first generation of sensors was based on detecting calcium activity using fluorescent dye markers. The second generation, based on the development of genetically encoded fluorescent probes has allowed to precisely target the neurons of interest. However, because all of these approaches based on fluorescence require light excitation, deep structures of the brain still remain difficult to record. This means that the development of other alternative or complementary techniques is still worthwhile. Recently a novel bioluminescence approach has been developed, allowing to functionally map, in vivo, neuronal activity and circuitry. The aim of this chapter is to describe detailed protocols, from the genesis and the use of the GFP-aequorin probe, the setup, the recording and the analysis methods to perform in vivo functional brain imaging in Drosophila. Some original results that have been revealed by this new approach are also presented as well as discussion about the biological signification of the detected and recorded Ca2+-activity. Finally, advantages and constraints of using this approach compared to others are discussed.
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Miyawaki A (2005) Innovations in the imaging of brain functions using fluorescent proteins. Neuron 48:189–199
Griesbeck O (2004) Fluorescent proteins as sensors for cellular functions. Curr Opin Neurobiol 14:636–641
Garaschuk O, Milos RI, Grienberger C, Marandi N, Adelsberger H, Konnerth A (2006) Optical monitoring of brain function in vivo: from neurons to networks. Pflugers Arch 453:385–396
Dombeck DA, Khabbaz AN, Collman F, Adelman TL, Tank DW (2007) Imaging large-scale neural activity with cellular resolution in awake, mobile mice. Neuron 56:43–57
Kerr R, Lev-Ram V, Baird G, Vincent P, Tsien RY, Schafer WR (2000) Optical imaging of calcium transients in neurons and pharyngeal muscle of C. elegans. Neuron 26:583–594
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 12:1877–1884
Fiala A, Spall T (2003) In vivo calcium imaging of brain activity in Drosophila by transgenic cameleon expression. Sci STKE 174:PL6
Nakai J, Ohkura M, Imoto K (2001) A high signal-to-noise Ca2+ probe composed of a single green fluorescent protein. Nat Biotechnol 19:137–141
Wang Y, Wright NJ, Guo H, Xie Z, Svoboda K, Malinow R, Smith DP, Zhong Y (2001) Genetic manipulation of the odor-evoked distributed neural activity in the Drosophila mushroom body. Neuron 29:267–276
Wang Y, Guo HF, Pologruto TA, Hannan F, Hakker I, Svoboda K, Zhong Y (2004) Stereotyped odor-evoked activity in the mushroom body of Drosophila revealed by green fluorescent protein-based Ca2+ imaging. J Neurosci 24:6507–6514
Wang JW, Wong AM, Flores J, Vosshall LB, Axel R (2003) Two-photon calcium imaging reveals an odor-evoked map of activity in the fly brain. Cell 112:271–282
Yu D, Baird GS, Tsien RY, Davis RL (2003) Detection of calcium transients in Drosophila mushroom body neurons with camgaroo reporters. J Neurosci 23:64–72
Yu D, Ponomarev A, Davis RL (2004) Altered representation of the spatial code for odors after olfactory classical conditioning; memory trace formation by synaptic recruitment. Neuron 42:437–449
Yu D, Keene AC, Srivatsan A, Waddell S, Davis RL (2005) Drosophila DPM neurons form a delayed and branch-specific memory trace after olfactory classical conditioning. Cell 123:945–957
Yu D, Akalal DB, Davis RL (2006) Drosophila alpha/beta mushroom body neurons form a branch-specific, long-term cellular memory trace after spaced olfactory conditioning. Neuron 52:845–855
Higashijima S, Masino MA, Mandel G, Fetcho JR (2003) Imaging neuronal activity during zebrafish behavior with a genetically encoded calcium indicator. J Neurophysiol 90:3986–3997
Hara M, Bindokas V, Lopez JP, Kaihara K, Landa LR Jr, Harbeck M, Roe MW (2004) Imaging endoplasmic reticulum calcium with a fluorescent biosensor in transgenic mice. Am J Physiol Cell Physiol 287:C932–C938
Hasan MT, Friedrich RW, Euler T, Larkum ME, Giese G, Both M, Duebel J, Waters J, Bujard H, Griesbeck O, Tsien RY, Nagai T, Miyawaki A, Denk W (2004) Functional fluorescent Ca2+ indicator proteins in transgenic mice under TET control. PLoS Biol 2:e163
Nagai T, Yamada S, Tominaga T, Ichikawa M, Miyawaki A (2004) Expanded dynamic range of fluorescent indicators for Ca2+ by circularly permuted yellow fluorescent proteins. Proc Natl Acad Sci USA 101:10554–10559
Vincent P, Maskos U, Charvet I, Bourgeais L, Stoppini L, Leresche N, Changeux JP, Lambert R, Meda P, Paupardin-Tritsch D (2006) Live imaging of neural structure and function by fibred fluorescence microscopy. EMBO Rep 11:1154–1161
Reiff DF, Ihring A, Guerrero G, Isacoff EY, Joesch M, Nakai J, Borst A (2005) In vivo performance of genetically encoded indicators of neural activity in flies. J Neurosci 25:4766–4778
Shimomura O, Johnson FH (1978) Peroxidized coelenterazine, the active group in the photoprotein aequorin. Proc Natl Acad Sci USA 75:2611–2615
Shimomura O, Musicki B, Kishi Y (1989) Semi-synthetic aequorins with improved sensitivity to Ca2+ ions. Biochem J 261:913–920
Shimomura O, Musicki B, Kishi Y, Inouye S (1993) Light-emitting properties of recombinant semi-synthetic aequorins and recombinant fluorescein-conjugated aequorin for measuring cellular calcium. Cell Calcium 14:373–378
Leclerc C, Webb SE, Daguzan C, Moreau M, Miller AL (2000) Imaging patterns of calcium transients during neural induction in Xenopus laevis embryos. J Cell Sci 113:3519–3529
Gilland E, Miller AL, Karplus E, Baker R, Webb SE (1999) Imaging of multicellular large-scale rhythmic calcium waves during zebrafish gastrulation. Proc Natl Acad Sci USA 96:157–161
Rosay P, Davies SA, Yu Y, Sözen A, Kaiser K, Dow JA (1997) Cell-type specific calcium signalling in a Drosophila epithelium. J Cell Sci 110:1683–1692
Rosay P, Armstrong JD, Wang Z, Kaiser K (2001) Synchronized neural activity in the Drosophila memory centers and its modulation by amnesiac. Neuron 30:759–770
Torfs H, Poels J, Detheux M, Dupriez V, Van Loy T, Vercammen L, Vassart G, Parmentier M, Vanden Broeck J (2002) Recombinant aequorin as a reporter for receptor-mediated changes of intracellular Ca2+-levels in Drosophila S2 cells. Invert Neurosci 4:119–124
Kerr M, Davies SA, Dow JA (2004) Cell-specific manipulation of second messengers; a toolbox for integrative physiology in Drosophila. Curr Biol 14:1468–1474
MacPherson MR, Pollock VP, Kean L, Southall TD, Giannakou ME, Broderick KE, Dow JA, Hardie RC, Davies SA (2005) Transient receptor potential-like channels are essential for calcium signaling and fluid transport in a Drosophila epithelium. Genetics 169:541–1552
Baubet V, Le Mouellic H, Campbell AK, Lucas-Meunier E, Fossier P, Brulet P (2000) Chimeric green fluorescent protein-aequorin as bioluminescent Ca2+ reporters at the single-cell level. Proc Natl Acad Sci USA 97:7260–7265
Gorokhovatsky AY, Marchenkov VV, Rudenko NV, Ivashina TV, Ksenzenko VN, Burkhardt N, Semisotnov GV, Vinokurov LM, Alakhov YB (2004) Fusion of Aequorea victoria GFP and aequorin provides their Ca2+-induced interaction that results in red shift of GFP absorption and efficient bioluminescence energy transfer. Biochem Biophys Res Commun 320:703–711
Rogers KL, Stinnakre J, Agulhon C, Jublot D, Shorte SL, Kremer EJ, Brulet P (2005) Visualization of local Ca2+ dynamics with genetically encoded bioluminescent reporters. Eur J Neurosci 3:597–610
Curie T, Rogers KL, Colasante C, Brûlet P (2007) Red-shifted aequorin-based bioluminescent reporters for in vivo imaging of Ca2+ signaling. Mol Imaging 6:30–42
Rogers KL, Picaud S, Roncali E, Boisgard R, Colasante C, Stinnakre J, Tavitian B, Brûlet P (2007) Non-invasive in vivo imaging of calcium signaling in mice. PLoS One 2:e974
Rogers KL, Martin JR, Renaud O, Karplus E, Nicola MA, Nguyen M, Picaud S, Shorte SL, Brûlet P (2008) EMCCD based bioluminescence recording of single-cell Ca2+. J Biomed Opt 13:1–10
Martin JR, Rogers KL, Chagneau C, Brûlet P (2007) In vivo bioluminescence imaging of Ca2+ signalling in the brain of Drosophila. PLoS One 2:e275
Murmu MS, Stinnakre J, Martin JR (2010) Presynaptic Ca2+-stores contribute to odor-induced response in Drosophila olfactory receptor neurons. J Exp Biol 213:4163–4173
Murmu MS, Stinnakre J, Réal E, Martin JR (2011) Calcium-stores mediate adaptation in axon terminals of Olfactory Receptor Neurons in Drosophila. BMC Neurosci 12:105
Adams MD et al (2000) The genome sequence of Drosophila melanogaster. Science 287:2185–2195
Brand AH, Perrimon N (1993) Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118:401–415
Elliott DA, Brand AH (2008) The GAL4 system: a versatile system for the expression of genes. Methods Mol Biol 420:79–95
Lai SL, Lee T (2006) Genetic mosaic with dual binary transcriptional systems in Drosophila. Nat Neurosci 9:703–709
Potter CJ, Tasic B, Russler EV, Liang L, Luo L (2010) The Q system: a repressible binary system for transgene expression, lineage tracing, and mosaic analysis. Cell 141:536–548
Sweeney ST, Broadie K, Keane J, Niemann H, O’Kane CJ (1995) Targeted expression of tetanus toxin light chain in Drosophila specifically eliminates synaptic transmission and causes behavioral defects. Neuron 14:341–351
Martin JR, Keller A, Sweeney ST (2002) Targeted expression of tetanus toxin: a new tool to study the neurobiology of behavior. Adv Genet 47:1–47
Kitamoto T (2001) Conditional modification of behavior in Drosophila by targeted expression of a temperature-sensitive shibire allele in defined neurons. J Neurobiol 47:81–92
Pulver SR, Pashkovski SL, Hornstein NJ, Garrity PA, Griffith LC (2009) Temporal dynamics of neuronal activation by Channelrhodopsin-2 and TRPA1 determine behavioral output in Drosophila larvae. J Neurophysiol 101:3075–3088
Peabody NC, Pohl JB, Diao F, Vreede AP, Sandstrom DJ, Wang H, Zelensky PK, White BH (2009) Characterization of the decision network for wing expansion in Drosophila using targeted expression of the TRPM8 channel. J Neurosci 29:3343–3353
White B, Osterwalder T, Keshishian H (2001) Molecular genetic approaches to the targeted suppression of neuronal activity. Curr Biol 11:R1041–R1053
Hodge JJ (2009) Ion channels to inactivate neurons in Drosophila. Front Mol Neurosci 2:13
Aso Y, Grubel K, Busch S, Friedrich AB, Siwanowicz I, Tanimoto H (2009) The mushroom body of adult Drosophila characterized by GAL4 drivers. J Neurogenet 23:156–172
Ashburner M (1989) Drosophila, A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
Roberts DB (1998) Drosophila, a practical approach. Oxford University Press, Oxford
Gu H, O’Dowd DK (2006) Cholinergic synaptic transmission in adult Drosophila Kenyon cells in situ. J Neurosci 26:265–272
Agulhon C, Platel JC, Kolomiets B, Forster V, Picaud S, Brocard J, Faure P, Brulet P (2007) Bioluminescent imaging of Ca2+ activity reveals spatiotemporal dynamics in glial networks of dark-adapted mouse retina. J Physiol 583:945–958
Miller AL, Karplus E, Jaffe LF (1994) Imaging (Ca2+) i with aequorin using a photon imaging detector. Methods Cell Biol 40:305–338
Kazama H, Wilson RI (2008) Homeostatic matching and nonlinear amplification at identified central synapses. Neuron 58:401–413
Yaksi E, Wilson RI (2010) Electrical coupling between olfactory glomeruli. Neuron 67:1034–1047
Heisenberg M (2003) Mushroom body memoir: from maps to models. Nat Rev Neurosci 4:266–275
Davis RL (2005) Olfactory memory formation in Drosophila: from molecular to systems neuroscience. Annu Rev Neurosci 28:275–302
Davis RL (2011) Traces of Drosophila memory. Neuron 70:8–19
Strauss R, Heisenberg M (1993) A higher control center of locomotor behavior in the Drosophila brain. J Neurosci 13:1852–1861
Strauss R (2002) The central complex and the genetic dissection of locomotor behaviour. Curr Opin Neurobiol 12:633–638
Martin JR, Raabe T, Heisenberg M (1999) Central complex substructures are required for the maintenance of locomotor activity in Drosophila melanogaster. J Comp Physiol A 185:277–288
Martin JR, Faure F, Ernst R (2002) The power law distribution for walking-time intervals correlates with the ellipsoid-body in Drosophila. J Neurogenet 15:1–15
Renn SC, Armstrong JD, Yang M, Wang Z, An X, Kaiser K, Taghert PH (1999) Genetic analysis of the Drosophila ellipsoid body neuropil: organization and development of the central complex. J Neurobiol 41:189–207
Joiner WJ, Crocker A, White BH, Sehgal A (2006) Sleep in Drosophila is regulated by adult mushroom bodies. Nature 441:757–760
Renn SC, Park JH, Rosbash M, Hall JC, Taghert PH (1999) A pdf neuropeptide gene mutation and ablation of PDF neurons each cause severe abnormalities of behavioral circadian rhythms in Drosophila. Cell 99:791–802
Bellen HJ (1998) The fruit fly: a model organism to study the genetics of alcohol abuse and addiction? Cell 93:909–912
Wolf FW, Heberlein U (2003) Invertebrate models of drug abuse. J Neurobiol 54:161–178
Bilen J, Bonini NM (2005) Drosophila as a model for human neurodegenerative disease. Annu Rev Genet 39:153–171
Muqit MM, Feany MB (2002) Modelling neurodegenerative diseases in Drosophila: a fruitful approach? Nat Rev Neurosci 3:237–243
Martin JR, Ollo R (1996) A new Drosophila Ca2+/calmodulin-dependent protein kinase (Caki) is localized in the central nervous system and implicated in walking speed. EMBO J 15:1865–1876
Markova SV, Vysotski ES, Blinks JR, Burakova LP, Wang BC, Lee J (2002) Obelin from the bioluminescent marine hydroid Obelia geniculata: cloning, expression, and comparison of some properties with those of other Ca2+-regulated photoproteins. Biochemistry 41:2227–2236
Bakayan A, Vaquero CF, Picazo F, Llopis J (2011) Red fluorescent protein-aequorin fusions as improved bioluminescent Ca2+ reporters in single cells and mice. PLoS One 6(5):e19520
Manjarrés IM, Chamero P, Domingo B, Molina F, Llopis J, Alonso MT, García-Sancho J (2008) Red and green aequorins for simultaneous monitoring of Ca2+ signals from two different organelles. Pflugers Arch 455:961–970
Barron AB (2000) Anaesthetising Drosophila for behavioural studies. J Insect Physiol 2000(46):439–442
Martin JR (2003) Locomotor activity: a complex behavioural trait to unravel. Behav Processess 64:145–160
Acknowledgments
I’m indebted to P. Brûlet and his colleagues, K. Rogers, and S. Picaud, who have primarily developed the bioluminescence approach. I also thank the different past and present members of my laboratory (students and Post-Docs: E. Carbognin, M.S. Murmu, E. Real, P. Pavot, A. Khammari and L. Mellottée) who have participated in the development of the bioluminescence approach or recording neuronal activity, in Drosophila. I also thanks D. Nässel for the critical reading of the manuscript, as well as the French “Agence National pour la Recherche” (ANR-Neuroscience), the NERF (Neuropôle Ile-de France), and the CNRS, for their precious financial support.
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Martin, JR. (2012). In Vivo Functional Brain Imaging Using a Genetically Encoded Ca2+-Sensitive Bioluminescence Reporter, GFP-Aequorin. In: Martin, JR. (eds) Genetically Encoded Functional Indicators. Neuromethods, vol 72. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-014-4_1
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DOI: https://doi.org/10.1007/978-1-62703-014-4_1
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