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Microglia pp 243–265Cite as

Labeling Microglia with Genetically Encoded Calcium Indicators

Part of the Methods in Molecular Biology book series (MIMB,volume 2034)

Abstract

Genetically encoded calcium indicators (GECIs) have become widely used for Ca2+ imaging in cultured cells as well as in living organisms. Transduction of microglia with viral vectors encoding GECIs provides a convenient means to label microglia for in vivo Ca2+ imaging. We describe a method using microglia-specific microRNA-9-regulated viral vector, to label microglial cells with a ratiometric GECI (Twitch-2B). This method enables longitudinal recording of both transient and sustained elevations of Ca2+ in microglia in live animals.

Key words

  • Microglia
  • Genetically encoded calcium indicator
  • MicroRNA
  • Calcium imaging
  • Transduction
  • Basal Ca2+ level

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References

  1. Kettenmann H, Hanisch UK, Noda M, Verkhratsky A (2011) Physiology of microglia. Physiol Rev 91(2):461–553. https://doi.org/10.1152/physrev.00011.2010

    CAS  CrossRef  PubMed  Google Scholar 

  2. Brawek B, Garaschuk O (2013) Microglial calcium signaling in the adult, aged and diseased brain. Cell Calcium 53(3):159–169. https://doi.org/10.1016/j.ceca.2012.12.003

    CAS  CrossRef  PubMed  Google Scholar 

  3. Hoffmann A, Kann O, Ohlemeyer C, Hanisch UK, Kettenmann H (2003) Elevation of basal intracellular calcium as a central element in the activation of brain macrophages (microglia): suppression of receptor-evoked calcium signaling and control of release function. J Neurosci 23(11):4410–4419

    CAS  CrossRef  Google Scholar 

  4. Eichhoff G, Brawek B, Garaschuk O (2011) Microglial calcium signal acts as a rapid sensor of single neuron damage in vivo. Biochim Biophys Acta 1813(5):1014–1024. https://doi.org/10.1016/j.bbamcr.2010.10.018

    CAS  CrossRef  PubMed  Google Scholar 

  5. Inoue K (2002) Microglial activation by purines and pyrimidines. Glia 40(2):156–163. https://doi.org/10.1002/glia.10150

    CrossRef  PubMed  Google Scholar 

  6. Re DB, Przedborski S (2006) Fractalkine: moving from chemotaxis to neuroprotection. Nat Neurosci 9(7):859–861. https://doi.org/10.1038/nn0706-859

    CAS  CrossRef  PubMed  Google Scholar 

  7. Cui YH, Le Y, Zhang X, Gong W, Abe K, Sun R, Van Damme J, Proost P, Wang JM (2002) Up-regulation of FPR2, a chemotactic receptor for amyloid beta 1–42 (A beta 42), in murine microglial cells by TNF alpha. Neurobiol Dis 10(3):366–377

    CAS  CrossRef  Google Scholar 

  8. Brawek B, Garaschuk O (2017) Monitoring in vivo function of cortical microglia. Cell Calcium 64:109–117. https://doi.org/10.1016/j.ceca.2017.02.011

    CAS  CrossRef  PubMed  Google Scholar 

  9. Hanisch UK, Kettenmann H (2007) Microglia: active sensor and versatile effector cells in the normal and pathologic brain. Nat Neurosci 10(11):1387–1394. https://doi.org/10.1038/nn1997

    CAS  CrossRef  PubMed  Google Scholar 

  10. Stosiek C, Garaschuk O, Holthoff K, Konnerth A (2003) In vivo two-photon calcium imaging of neuronal networks. Proc Natl Acad Sci U S A 100(12):7319–7324. https://doi.org/10.1073/pnas.1232232100

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  11. Garaschuk O (2013) Imaging microcircuit function in healthy and diseased brain. Exp Neurol 242:41–49. https://doi.org/10.1016/j.expneurol.2012.02.009

    CrossRef  PubMed  Google Scholar 

  12. 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(3):385–396. https://doi.org/10.1007/s00424-006-0150-x

    CAS  CrossRef  PubMed  Google Scholar 

  13. Tian L, Hires SA, Looger LL (2012) Imaging neuronal activity with genetically encoded calcium indicators. Cold Spring Harb Protoc 2012(6):647–656. https://doi.org/10.1101/pdb.top069609

    CrossRef  PubMed  Google Scholar 

  14. Perez Koldenkova V, Nagai T (2013) Genetically encoded Ca2+ indicators: properties and evaluation. Biochim Biophys Acta 1833(7):1787–1797. https://doi.org/10.1016/j.bbamcr.2013.01.011

    CAS  CrossRef  PubMed  Google Scholar 

  15. Seifert S, Pannell M, Uckert W, Farber K, Kettenmann H (2011) Transmitter- and hormone-activated Ca2+ responses in adult microglia/brain macrophages in situ recorded after viral transduction of a recombinant Ca2+ sensor. Cell Calcium 49(6):365–375. https://doi.org/10.1016/j.ceca.2011.03.005

    CAS  CrossRef  PubMed  Google Scholar 

  16. Wendeln AC, Degenhardt K, Kaurani L, Gertig M, Ulas T, Jain G, Wagner J, Hasler LM, Wild K, Skodras A, Blank T, Staszewski O, Datta M, Centeno TP, Capece V, Islam MR, Kerimoglu C, Staufenbiel M, Schultze JL, Beyer M, Prinz M, Jucker M, Fischer A, Neher JJ (2018) Innate immune memory in the brain shapes neurological disease hallmarks. Nature 556(7701):332–338. https://doi.org/10.1038/s41586-018-0023-4

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  17. Tay TL, Mai D, Dautzenberg J, Fernandez-Klett F, Lin G, Sagar DM, Drougard A, Stempfl T, Ardura-Fabregat A, Staszewski O, Margineanu A, Sporbert A, Steinmetz LM, Pospisilik JA, Jung S, Priller J, Grun D, Ronneberger O, Prinz M (2017) A new fate mapping system reveals context-dependent random or clonal expansion of microglia. Nat Neurosci 20(6):793–803. https://doi.org/10.1038/nn.4547

    CAS  CrossRef  PubMed  Google Scholar 

  18. Gee JM, Smith NA, Fernandez FR, Economo MN, Brunert D, Rothermel M, Morris SC, Talbot A, Palumbos S, Ichida JM, Shepherd JD, West PJ, Wachowiak M, Capecchi MR, Wilcox KS, White JA, Tvrdik P (2014) Imaging activity in neurons and glia with a Polr2a-based and cre-dependent GCaMP5G-IRES-tdTomato reporter mouse. Neuron 83(5):1058–1072. https://doi.org/10.1016/j.neuron.2014.07.024

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  19. Pozner A, Xu B, Palumbos S, Gee JM, Tvrdik P, Capecchi MR (2015) Intracellular calcium dynamics in cortical microglia responding to focal laser injury in the PC::G5-tdT reporter mouse. Front Mol Neurosci 8:12. https://doi.org/10.3389/fnmol.2015.00012

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  20. Tvrdik P, Kalani MYS (2017) In vivo imaging of microglial calcium signaling in brain inflammation and injury. Int J Mol Sci 18(11):E2366. https://doi.org/10.3390/ijms18112366

    CAS  CrossRef  PubMed  Google Scholar 

  21. Farber K, Kettenmann H (2006) Functional role of calcium signals for microglial function. Glia 54(7):656–665. https://doi.org/10.1002/glia.20412

    CrossRef  PubMed  Google Scholar 

  22. Mank M, Griesbeck O (2008) Genetically encoded calcium indicators. Chem Rev 108(5):1550–1564. https://doi.org/10.1021/cr078213v

    CAS  CrossRef  PubMed  Google Scholar 

  23. Thestrup T, Litzlbauer J, Bartholomaus I, Mues M, Russo L, Dana H, Kovalchuk Y, Liang Y, Kalamakis G, Laukat Y, Becker S, Witte G, Geiger A, Allen T, Rome LC, Chen TW, Kim DS, Garaschuk O, Griesinger C, Griesbeck O (2014) Optimized ratiometric calcium sensors for functional in vivo imaging of neurons and T lymphocytes. Nat Methods 11(2):175–182. https://doi.org/10.1038/nmeth.2773

    CAS  CrossRef  PubMed  Google Scholar 

  24. Garaschuk O, Griesbeck O (2009) Monitoring calcium levels with genetically encoded indicators. In: Verkhratsky A, Petersen O (eds) Calcium measurement methods, vol 43. Humana, New York, pp 101–117

    CrossRef  Google Scholar 

  25. Jakobsson J, Lundberg C (2006) Lentiviral vectors for use in the central nervous system. Mol Ther 13(3):484–493. https://doi.org/10.1016/j.ymthe.2005.11.012

    CAS  CrossRef  PubMed  Google Scholar 

  26. Brawek B, Liang Y, Savitska D, Li K, Fomin-Thunemann N, Kovalchuk Y, Zirdum E, Jakobsson J, Garaschuk O (2017) A new approach for ratiometric in vivo calcium imaging of microglia. Sci Rep 7(1):6030. https://doi.org/10.1038/s41598-017-05952-3

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  27. Brown BD, Gentner B, Cantore A, Colleoni S, Amendola M, Zingale A, Baccarini A, Lazzari G, Galli C, Naldini L (2007) Endogenous microRNA can be broadly exploited to regulate transgene expression according to tissue, lineage and differentiation state. Nat Biotechnol 25(12):1457–1467. https://doi.org/10.1038/nbt1372

    CAS  CrossRef  PubMed  Google Scholar 

  28. Sachdeva R, Jonsson ME, Nelander J, Kirkeby A, Guibentif C, Gentner B, Naldini L, Bjorklund A, Parmar M, Jakobsson J (2010) Tracking differentiating neural progenitors in pluripotent cultures using microRNA-regulated lentiviral vectors. Proc Natl Acad Sci U S A 107(25):11602–11607. https://doi.org/10.1073/pnas.1006568107

    CrossRef  PubMed  PubMed Central  Google Scholar 

  29. Akerblom M, Sachdeva R, Quintino L, Wettergren EE, Chapman KZ, Manfre G, Lindvall O, Lundberg C, Jakobsson J (2013) Visualization and genetic modification of resident brain microglia using lentiviral vectors regulated by microRNA-9. Nat Commun 4:1770. https://doi.org/10.1038/ncomms2801

    CAS  CrossRef  PubMed  Google Scholar 

  30. Goverdhana S, Puntel M, Xiong W, Zirger JM, Barcia C, Curtin JF, Soffer EB, Mondkar S, King GD, Hu J, Sciascia SA, Candolfi M, Greengold DS, Lowenstein PR, Castro MG (2005) Regulatable gene expression systems for gene therapy applications: progress and future challenges. Mol Ther 12(2):189–211. https://doi.org/10.1016/j.ymthe.2005.03.022

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  31. De Palma M, Montini E, Santoni de Sio FR, Benedicenti F, Gentile A, Medico E, Naldini L (2005) Promoter trapping reveals significant differences in integration site selection between MLV and HIV vectors in primary hematopoietic cells. Blood 105(6):2307–2315. https://doi.org/10.1182/blood-2004-03-0798

    CAS  CrossRef  PubMed  Google Scholar 

  32. Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116(2):281–297

    CAS  CrossRef  Google Scholar 

  33. Qin JY, Zhang L, Clift KL, Hulur I, Xiang AP, Ren BZ, Lahn BT (2010) Systematic comparison of constitutive promoters and the doxycycline-inducible promoter. PLoS One 5(5):e10611. https://doi.org/10.1371/journal.pone.0010611

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  34. Damdindorj L, Karnan S, Ota A, Hossain E, Konishi Y, Hosokawa Y, Konishi H (2014) A comparative analysis of constitutive promoters located in adeno-associated viral vectors. PLoS One 9(8):e106472. https://doi.org/10.1371/journal.pone.0106472

    CrossRef  PubMed  PubMed Central  Google Scholar 

  35. Holtmaat A, Bonhoeffer T, Chow DK, Chuckowree J, De Paola V, Hofer SB, Hubener M, Keck T, Knott G, Lee WC, Mostany R, Mrsic-Flogel TD, Nedivi E, Portera-Cailliau C, Svoboda K, Trachtenberg JT, Wilbrecht L (2009) Long-term, high-resolution imaging in the mouse neocortex through a chronic cranial window. Nat Protoc 4(8):1128–1144. https://doi.org/10.1038/nprot.2009.89

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  36. Goldey GJ, Roumis DK, Glickfeld LL, Kerlin AM, Reid RC, Bonin V, Schafer DP, Andermann ML (2014) Removable cranial windows for long-term imaging in awake mice. Nat Protoc 9(11):2515–2538. https://doi.org/10.1038/nprot.2014.165

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  37. Perillo EP, McCracken JE, Fernee DC, Goldak JR, Medina FA, Miller DR, Yeh HC, Dunn AK (2016) Deep in vivo two-photon microscopy with a low cost custom built mode-locked 1060 nm fiber laser. Biomed Opt Express 7(2):324–334. https://doi.org/10.1364/BOE.7.000324

    CrossRef  PubMed  PubMed Central  Google Scholar 

  38. Chan KY, Jang MJ, Yoo BB, Greenbaum A, Ravi N, Wu WL, Sanchez-Guardado L, Lois C, Mazmanian SK, Deverman BE, Gradinaru V (2017) Engineered AAVs for efficient noninvasive gene delivery to the central and peripheral nervous systems. Nat Neurosci 20(8):1172–1179. https://doi.org/10.1038/nn.4593

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  39. Tervo DG, Hwang BY, Viswanathan S, Gaj T, Lavzin M, Ritola KD, Lindo S, Michael S, Kuleshova E, Ojala D, Huang CC, Gerfen CR, Schiller J, Dudman JT, Hantman AW, Looger LL, Schaffer DV, Karpova AY (2016) A designer AAV variant permits efficient retrograde access to projection neurons. Neuron 92(2):372–382. https://doi.org/10.1016/j.neuron.2016.09.021

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  40. Deo C, Lavis LD (2018) Synthetic and genetically encoded fluorescent neural activity indicators. Curr Opin Neurobiol 50:101–108. https://doi.org/10.1016/j.conb.2018.01.003

    CAS  CrossRef  PubMed  Google Scholar 

  41. Tiscornia G, Singer O, Verma IM (2006) Production and purification of lentiviral vectors. Nat Protoc 1(1):241–245. https://doi.org/10.1038/nprot.2006.37

    CAS  CrossRef  PubMed  Google Scholar 

  42. Kutner RH, Zhang XY, Reiser J (2009) Production, concentration and titration of pseudotyped HIV-1-based lentiviral vectors. Nat Protoc 4(4):495–505. https://doi.org/10.1038/nprot.2009.22

    CAS  CrossRef  PubMed  Google Scholar 

  43. Cribbs AP, Kennedy A, Gregory B, Brennan FM (2013) Simplified production and concentration of lentiviral vectors to achieve high transduction in primary human T cells. BMC Biotechnol 13:98. https://doi.org/10.1186/1472-6750-13-98

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  44. Poynter G, Huss D, Lansford R (2009) Generation of high-titer lentivirus for the production of transgenic quail. Cold Spring Harbor Protoc 2009(1):pdb prot5117. https://doi.org/10.1101/pdb.prot5117

    CrossRef  Google Scholar 

  45. Jiang W, Hua R, Wei M, Li C, Qiu Z, Yang X, Zhang C (2015) An optimized method for high-titer lentivirus preparations without ultracentrifugation. Sci Rep 5:13875. https://doi.org/10.1038/srep13875

    CrossRef  PubMed  PubMed Central  Google Scholar 

  46. Baekelandt V, Eggermont K, Michiels M, Nuttin B, Debyser Z (2003) Optimized lentiviral vector production and purification procedure prevents immune response after transduction of mouse brain. Gene Ther 10(23):1933–1940. https://doi.org/10.1038/sj.gt.3302094

    CAS  CrossRef  PubMed  Google Scholar 

  47. Andermann ML, Kerlin AM, Reid RC (2010) Chronic cellular imaging of mouse visual cortex during operant behavior and passive viewing. Front Cell Neurosci 4:3. https://doi.org/10.3389/fncel.2010.00003

    CrossRef  PubMed  PubMed Central  Google Scholar 

  48. Andermann ML, Kerlin AM, Roumis DK, Glickfeld LL, Reid RC (2011) Functional specialization of mouse higher visual cortical areas. Neuron 72(6):1025–1039. https://doi.org/10.1016/j.neuron.2011.11.013

    CAS  CrossRef  PubMed  Google Scholar 

  49. Kerlin AM, Andermann ML, Berezovskii VK, Reid RC (2010) Broadly tuned response properties of diverse inhibitory neuron subtypes in mouse visual cortex. Neuron 67(5):858–871. https://doi.org/10.1016/j.neuron.2010.08.002

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  50. Sofroniew NJ, Flickinger D, King J, Svoboda K (2016) A large field of view two-photon mesoscope with subcellular resolution for in vivo imaging. elife 5:e14472. https://doi.org/10.7554/eLife.14472

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  51. Ji N (2017) Adaptive optical fluorescence microscopy. Nat Methods 14(4):374–380. https://doi.org/10.1038/nmeth.4218

    CAS  CrossRef  PubMed  Google Scholar 

  52. Saini M, Singh Y, Arora P, Arora V, Jain K (2015) Implant biomaterials: a comprehensive review. World J Clin Cases 3(1):52–57. https://doi.org/10.12998/wjcc.v3.i1.52

    CrossRef  PubMed  PubMed Central  Google Scholar 

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Acknowledgments

We thank Dr. Wilhelm Pfleging from Karlsruhe Institute of Technology for the preparation of microengineered cover glasses for our project, which was funded by Karlsruhe Nano Micro Facility (KNMF Proposal #2013-009-001195) and Ariane Kaupp for help with graphics. This work was supported by VolkswagenStiftung Grant 90233 and DFG GA654/13-1 to O.G.

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  1. Department of Neurophysiology, Institute of Physiology, Eberhard Karls University of Tübingen, Tübingen, Germany

    Yajie Liang & Olga Garaschuk

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  1. Yajie Liang
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  2. Olga Garaschuk
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Correspondence to Olga Garaschuk .

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Editors and Affiliations

  1. Department of Neurophysiology, Institute of Physiology, Eberhard Karls University of Tübingen, Tübingen, Germany

    Prof. Dr. Olga Garaschuk

  2. Faculty of Biology, Medicine and Health The University of Manchester, , Center for Basic and Translational Neuroscience, Faculty of Health and Medical Sciences University of Copenhagen Copenhagen, Denmark, Achucarro Center for Neuroscience IKERBASQUE Basque Foundation for Science Bilbao, Spain, Manchester, UK

    Dr. Alexei Verkhratsky

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Liang, Y., Garaschuk, O. (2019). Labeling Microglia with Genetically Encoded Calcium Indicators. In: Garaschuk, O., Verkhratsky, A. (eds) Microglia. Methods in Molecular Biology, vol 2034. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9658-2_18

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  • DOI: https://doi.org/10.1007/978-1-4939-9658-2_18

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