Abstract
Microglial cells are the innate immune cells of the central nervous system. In the healthy adult brain “resting” ramified microglia continuously palpate their environment to monitor the integrity of and to react to any disturbance of brain homeostasis. During injury, inflammation, and in the course of neurodegenerative diseases microglia become activated, proliferate, and release a plethora of cytokines as well as reactive oxygen species. In addition to their well known role in disease, it has become increasingly clear that “resting” microglia also contribute to normal brain physiology, both during postnatal development and in the mature adult brain.
Functional in vivo imaging of microglia first of all captures the morphological changes accompanying microglial transition between “resting” and activated states. In addition, intracellular Ca2+ homeostasis of microglia is believed to be altered between the two states [1–3]. So far, however, microglial Ca2+ signaling was predominantly studied in reduced preparations like brain slices or cell cultures, in which microglia are found in a rather activated state. In this chapter we describe a technique for studying microglial Ca2+ signaling in vivo. Furthermore, we discuss a new approach for visualization of morphological dynamics of microglial cells in vivo at high resolution. This approach utilizes a lectin-based staining technique and is applicable to any deliberate mouse strain at any developmental stage.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Färber K, Kettenmann H (2006) Functional role of calcium signals for microglial function. Glia 54:656–665
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 23:4410–4419
Light AR, Wu Y, Hughen RW, Guthrie PB (2006) Purinergic receptors activating rapid intracellular Ca increases in microglia. Neuron Glia Biol 2:125–138
Prinz M, Priller J, Sisodia SS, Ransohoff RM (2011) Heterogeneity of CNS myeloid cells and their roles in neurodegeneration. Nat Neurosci 14:1227–1235
Soulet D, Rivest S (2008) Microglia. Curr Biol 18:R506–R508
Herculano-Houzel S (2009) The human brain in numbers: a linearly scaled-up primate brain. Front Hum Neurosci 3:31
Block ML, Zecca L, Hong JS (2007) Microglia-mediated neurotoxicity: uncovering the molecular mechanisms. Nat Rev Neurosci 8:57–69
Kettenmann H, Hanisch UK, Noda M, Verkhratsky A (2011) Physiology of microglia. Physiol Rev 91:461–553
Hanisch UK, Kettenmann H (2007) Microglia: active sensor and versatile effector cells in the normal and pathologic brain. Nat Neurosci 10:1387–1394
Nimmerjahn A, Kirchhoff F, Helmchen F (2005) Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo. Science 308:1314–1318
Davalos D, Grutzendler J, Yang G, Kim JV, Zuo Y, Jung S, Littman DR, Dustin ML, Gan WB (2005) ATP mediates rapid microglial response to local brain injury in vivo. Nat Neurosci 8:752–758
Wake H, Moorhouse AJ, Jinno S, Kohsaka S, Nabekura J (2009) Resting microglia directly monitor the functional state of synapses in vivo and determine the fate of ischemic terminals. J Neurosci 29:3974–3980
Tremblay ME, Lowery RL, Majewska AK (2010) Microglial interactions with synapses are modulated by visual experience. PLoS Biol 8:e1000527
Paolicelli RC, Bolasco G, Pagani F, Maggi L, Scianni M, Panzanelli P, Giustetto M, Ferreira TA, Guiducci E, Dumas L, Ragozzino D, Gross CT (2011) Synaptic pruning by microglia is necessary for normal brain development. Science 333:1456–1458
Tremblay ME, Stevens B, Sierra A, Wake H, Bessis A, Nimmerjahn A (2011) The role of microglia in the healthy brain. J Neurosci 31:16064–16069
McLarnon JG (2005) Purinergic mediated changes in Ca2+ mobilization and functional responses in microglia: effects of low levels of ATP. J Neurosci Res 81:349–356
Ransohoff RM, Perry VH (2009) Microglial physiology: unique stimuli, specialized responses. Annu Rev Immunol 27:119–145
Glass CK, Saijo K, Winner B, Marchetto MC, Gage FH (2010) Mechanisms underlying inflammation in neurodegeneration. Cell 140:918–934
Kreutzberg GW (1996) Microglia: a sensor for pathological events in the CNS. Trends Neurosci 19:312–318
Neumann H, Kotter MR, Franklin RJ (2009) Debris clearance by microglia: an essential link between degeneration and regeneration. Brain 132:288–295
Jung S, Aliberti J, Graemmel P, Sunshine MJ, Kreutzberg GW, Sher A, Littman DR (2000) Analysis of fractalkine receptor CX3CR1 function by targeted deletion and green fluorescent protein reporter gene insertion. Mol Cell Biol 20:4106–4114
Hirasawa T, Ohsawa K, Imai Y, Ondo Y, Akazawa C, Uchino S, Kohsaka S (2005) Visualization of microglia in living tissues using Iba1-EGFP transgenic mice. J Neurosci Res 81:357–362
Sasmono RT, Oceandy D, Pollard JW, Tong W, Pavli P, Wainwright BJ, Ostrowski MC, Himes SR, Hume DA (2003) A macrophage colony-stimulating factor receptor-green fluorescent protein transgene is expressed throughout the mononuclear phagocyte system of the mouse. Blood 101:1155–1163
Harrison JK, Jiang Y, Chen S, Xia Y, Maciejewski D, McNamara RK, Streit WJ, Salafranca MN, Adhikari S, Thompson DA, Botti P, Bacon KB, Feng L (1998) Role for neuronally derived fractalkine in mediating interactions between neurons and CX3CR1-expressing microglia. Proc Natl Acad Sci USA 95:10896–10901
Eichhoff G, Brawek B, Garaschuk O (2011) Microglial calcium signal acts as a rapid sensor of single neuron damage in vivo. Biochim Biophys Acta 1813:1014–1024
Rogers JT, Morganti JM, Bachstetter AD, Hudson CE, Peters MM, Grimmig BA, Weeber EJ, Bickford PC, Gemma C (2011) CX3CR1 deficiency leads to impairment of hippocampal cognitive function and synaptic plasticity. J Neurosci 31:16241–16250
Lee S, Varvel NH, Konerth ME, Xu G, Cardona AE, Ransohoff RM, Lamb BT (2010) CX3CR1 deficiency alters microglial activation and reduces beta-amyloid deposition in two Alzheimer’s disease mouse models. Am J Pathol 177:2549–2562
Möller T (2002) Calcium signaling in microglial cells. Glia 40:184–194
Pocock JM, Kettenmann H (2007) Neurotrans-mitter receptors on microglia. Trends Neurosci 30:527–535
Inoue K (2002) Microglial activation by purines and pyrimidines. Glia 40:156–163
Re DB, Przedborski S (2006) Fractalkine: moving from chemotaxis to neuroprotection. Nat Neurosci 9:859–861
Stosiek C, Garaschuk O, Holthoff K, Konnerth A (2003) In vivo two-photon calcium imaging of neuronal networks. Proc Natl Acad Sci USA 100:7319–7324
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 49:365–375
Horikawa K, Yamada Y, Matsuda T, Kobayashi K, Hashimoto M, Matsu-ura T, Miyawaki A, Michikawa T, Mikoshiba K, Nagai T (2010) Spontaneous network activity visualized by ultrasensitive Ca2+ indicators, yellow Cameleon-Nano. Nat Methods 7:729–732
Mank M, Santos AF, Direnberger S, Mrsic-Flogel TD, Hofer SB, Stein V, Hendel T, Reiff DF, Levelt C, Borst A, Bonhoeffer T, Hubener M, Griesbeck O (2008) A genetically encoded calcium indicator for chronic in vivo two-photon imaging. Nat Methods 5:805–811
Tallini YN, Ohkura M, Choi BR, Ji G, Imoto K, Doran R, Lee J, Plan P, Wilson J, Xin HB, Sanbe A, Gulick J, Mathai J, Robbins J, Salama G, Nakai J, Kotlikoff MI (2006) Imaging cellular signals in the heart in vivo: cardiac expression of the high-signal Ca2+ indicator GCaMP2. Proc Natl Acad Sci USA 103:4753–4758
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
Tian L, Hires SA, Mao T, Huber D, Chiappe ME, Chalasani SH, Petreanu L, Akerboom J, McKinney SA, Schreiter ER, Bargmann CI, Jayaraman V, Svoboda K, Looger LL (2009) Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators. Nat Methods 6:875–881
Zhao Y, Araki S, Wu J, Teramoto T, Chang YF, Nakano M, Abdelfattah AS, Fujiwara M, Ishihara T, Nagai T, Campbell RE (2011) An expanded palette of genetically encoded Ca2+ indicators. Science 333:1888–1891
Atkin SD, Patel S, Kocharyan A, Holtzclaw LA, Weerth SH, Schram V, Pickel J, Russell JT (2009) Transgenic mice expressing a cameleon fluorescent Ca2+ indicator in astrocytes and Schwann cells allow study of glial cell Ca2+ signals in situ and in vivo. J Neurosci Methods 181:212–226
Lis H, Sharon N (1998) Lectins: carbohydrate-specific proteins that mediate cellular recognition. Chem Rev 98:637–674
Murphy LA, Goldstein IJ (1977) Five alpha-D-galactopyranosyl-binding isolectins from Bandeiraea simplicifolia seeds. J Biol Chem 252:4739–4742
Streit WJ, Schulte BA, Balentine DJ, Spicer SS (1985) Histochemical localization of galactose-containing glycoconjugates in sensory neurons and their processes in the central and peripheral nervous system of the rat. J Histochem Cytochem 33:1042–1052
Slifkin M, Doyle RJ (1990) Lectins and their application to clinical microbiology. Clin Microbiol Rev 3:197–218
Boucsein C, Kettenmann H, Nolte C (2000) Electrophysiological properties of microglial cells in normal and pathologic rat brain slices. Eur J Neurosci 12:2049–2058
Newell EW, Stanley EF, Schlichter LC (2007) Reversed Na+/Ca2+ exchange contributes to Ca2+ influx and respiratory burst in microglia. Channels (Austin) 1:366–376
Streit WJ, Kreutzberg GW (1987) Lectin binding by resting and reactive microglia. J Neurocytol 16:249–260
Streit WJ (1990) An improved staining method for rat microglial cells using the lectin from Griffonia simplicifolia (GSA I-B4). J Histochem Cytochem 38:1683–1686
Bordey A, Spencer DD (2003) Chemokine modulation of high-conductance Ca2+-sensitive K+ currents in microglia from human hippocampi. Eur J Neurosci 18:2893–2898
Rappert A, Bechmann I, Pivneva T, Mahlo J, Biber K, Nolte C, Kovac AD, Gerard C, Boddeke HW, Nitsch R, Kettenmann H (2004) CXCR3-dependent microglial recruitment is essential for dendrite loss after brain lesion. J Neurosci 24:8500–8509
Bankston PW, Porter GA, Milici AJ, Palade GE (1991) Differential and specific labeling of epithelial and vascular endothelial cells of the rat lung by Lycopersicon esculentum and Griffonia simplicifolia I lectins. Eur J Cell Biol 54:187–195
Moffett JR, Els T, Espey MG, Walter SA, Streit WJ, Namboodiri MA (1997) Quinolinate immunoreactivity in experimental rat brain tumors is present in macrophages but not in astrocytes. Exp Neurol 144:287–301
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
Schwendele B, Brawek B, Hermes M, Garaschuk O (2012) High resolution in vivo imaging of microglia using a versatile non genetically-encoded marker. Eur J Immunol 42:2193–2196
Nimmerjahn A, Kirchhoff F, Kerr JN, Helmchen F (2004) Sulforhodamine 101 as a specific marker of astroglia in the neocortex in vivo. Nat Methods 1:31–37
Kang J, Kang N, Yu Y, Zhang J, Petersen N, Tian GF, Nedergaard M (2010) Sulforhodamine 101 induces long-term potentiation of intrinsic excitability and synaptic efficacy in hippocampal CA1 pyramidal neurons. Neuroscience 169:1601–1609
Garaschuk O (2013) Imaging microcircuit function in healthy and diseased brain. Exp Neurol 242:41–49
Fink S, Kovalchuk Y, Homma R, Schwendele B, Direnberger S, Cohen LB, Griesbeck O, Garaschuk O (2012) In vivo functional imaging of the olfactory bulb at single cell resolution. In: Fellin T, Halassa M (eds) Neuronal network analysis. Humana Press, New York, pp 21–43
Nevian T, Helmchen F (2007) Calcium indicator loading of neurons using single-cell electroporation. Pflugers Arch 454:675–688
Whittemore ER, Korotzer AR, Etebari A, Cotman CW (1993) Carbachol increases intracellular free calcium in cultured rat microglia. Brain Res 621:59–64
Zhang L, McLarnon JG, Goghari V, Lee YB, Kim SU, Krieger C (1998) Cholinergic agonists increase intracellular Ca2+ in cultured human microglia. Neurosci Lett 255:33–36
Biber K, Laurie DJ, Berthele A, Sommer B, Tolle TR, Gebicke-Harter PJ, van Calker D, Boddeke HW (1999) Expression and signaling of group I metabotropic glutamate receptors in astrocytes and microglia. J Neurochem 72:1671–1680
Colton CA, Jia M, Li MX, Gilbert DL (1994) K+ modulation of microglial superoxide production: involvement of voltage-gated Ca2+ channels. Am J Physiol 266:C1650–C1655
Verderio C, Matteoli M (2001) ATP mediates calcium signaling between astrocytes and microglial cells: modulation by IFN-gamma. J Immunol 166:6383–6391
Meyer-Luehmann M, Spires-Jones TL, Prada C, Garcia-Alloza M, de Calignon A, Rozkalne A, Koenigsknecht-Talboo J, Holtzman DM, Bacskai BJ, Hyman BT (2008) Rapid appearance and local toxicity of amyloid-beta plaques in a mouse model of Alzheimer’s disease. Nature 451:720–724
Hefendehl JK, Wegenast-Braun BM, Liebig C, Eicke D, Milford D, Calhoun ME, Kohsaka S, Eichner M, Jucker M (2011) Long-term in vivo imaging of beta-amyloid plaque appearance and growth in a mouse model of cerebral beta-amyloidosis. J Neurosci 31:624–629
Tambuyzer BR, Ponsaerts P, Nouwen EJ (2009) Microglia: gatekeepers of central nervous system immunology. J Leukoc Biol 85:352–370
Yong VW, Rivest S (2009) Taking advantage of the systemic immune system to cure brain diseases. Neuron 64:55–60
Streit WJ, Walter SA, Pennell NA (1999) Reactive microgliosis. Prog Neurobiol 57:563–581
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media New York
About this protocol
Cite this protocol
Brawek, B., Garaschuk, O. (2014). Imaging Morphology and Function of Cortical Microglia. In: Weber, B., Helmchen, F. (eds) Optical Imaging of Neocortical Dynamics. Neuromethods, vol 85. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-785-3_13
Download citation
DOI: https://doi.org/10.1007/978-1-62703-785-3_13
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-784-6
Online ISBN: 978-1-62703-785-3
eBook Packages: Springer Protocols