In vivo calcium imaging of the aging and diseased brain

  • Gerhard Eichhoff
  • Marc Aurel Busche
  • Olga GaraschukEmail author



Over the last decade, in vivo calcium imaging became a powerful tool for studying brain function. With the use of two-photon microscopy and modern labelling techniques, it allows functional studies of individual living cells, their processes and their interactions within neuronal networks. In vivo calcium imaging is even more important for studying the aged brain, which is hard to investigate in situ due to the fragility of neuronal tissue.


In this article, we give a brief overview of the techniques applicable to image aged rodent brain at cellular resolution.


We use multicolor imaging to visualize specific cell types (neurons, astrocytes, microglia) as well as the autofluorescence of the “aging pigment” lipofuscin.


Further, we illustrate an approach for simultaneous imaging of cortical cells and senile plaques in mouse models of Alzheimer’s disease.


In vivo calcium imaging Plaques Alzheimer’s disease Lipofuscin 



This work is supported by grants of the Deutsche Forschungsgemeinschaft (SFB 391 and SFB 596) and the Bundesministerium für Bildung und Forschung (NGFN-2). We are thankful to M. Staufenbiel (Novartis Pharma, Basel, Switzerland) for AD mouse mutants, M. Kerschensteiner and T. Misgeld for CX3CR1-EGFP mice and A. Konnerth for support and helpful discussions. We thank Olympus Europa for providing two-photon-based Fluoview 1000MPE.

Conflict of interest statement

The authors declare that they have no relevant financial or any other interests in this manuscript.


  1. 1.
    Khachaturian ZS. Towards theories of brain aging. In: Kay DW, Burrows GW, editors. Handbook of studies on psychiatry and old age. Amsterdam: Elsevier; 1984. pp 7–30.Google Scholar
  2. 2.
    Toescu EC, Verkhratsky A. The importance of being subtle: small changes in calcium homeostasis control cognitive decline in normal aging. Aging Cell. 2007;6:267–73.PubMedCrossRefGoogle Scholar
  3. 3.
    Thibault O, Gant JC, Landfield PW. Expansion of the calcium hypothesis of brain aging and Alzheimer’s disease: minding the store. Aging Cell. 2007;6:307–17.PubMedCrossRefGoogle Scholar
  4. 4.
    Murchison D, Griffith WH. Calcium buffering systems and calcium signaling in aged rat basal forebrain neurons. Aging Cell. 2007;6:297–305.PubMedCrossRefGoogle Scholar
  5. 5.
    Kirischuk S, Pronchuk N, Verkhratsky A. Measurements of intracellular calcium in sensory neurons of adult and old rats. Neuroscience. 1992;50:947–51.PubMedCrossRefGoogle Scholar
  6. 6.
    Kirischuk S, Verkhratsky A. Calcium homeostasis in aged neurons. Life Sci. 1996;59:451–9.PubMedCrossRefGoogle Scholar
  7. 7.
    Murchison D, Griffith WH. Increased calcium buffering in basal forebrain neurons during aging. J Neurophysiol. 1998;80:350–64.PubMedGoogle Scholar
  8. 8.
    Xiong J, Verkhratsky A, Toescu EC. Changes in mitochondrial status associated with altered Ca2+ homeostasis in aged cerebellar granule neurons in brain slices. J Neurosci. 2002;22:10761–71.PubMedGoogle Scholar
  9. 9.
    Campbell LW, Hao SY, Thibault O, Blalock EM, Landfield PW. Aging changes in voltage-gated calcium currents in hippocampal CA1 neurons. J Neurosci. 1996;16:6286–95.PubMedGoogle Scholar
  10. 10.
    Thibault O, Landfield PW. Increase in single L-type calcium channels in hippocampal neurons during aging. Science. 1996;272:1017–20.PubMedCrossRefGoogle Scholar
  11. 11.
    Murchison D, Griffith WH. High-voltage-activated calcium currents in basal forebrain neurons during aging. J Neurophysiol. 1996;76:158–74.PubMedGoogle Scholar
  12. 12.
    Foster TC, Norris CM. Age-associated changes in Ca2+-dependent processes: relation to hippocampal synaptic plasticity. Hippocampus. 1997;7:602–12.PubMedCrossRefGoogle Scholar
  13. 13.
    LaFerla FM. Calcium dyshomeostasis and intracellular signalling in Alzheimer’s disease. Nat Rev Neurosci. 2002;3:862–72.PubMedCrossRefGoogle Scholar
  14. 14.
    Smith IF, Hitt B, Green KN, Oddo S, LaFerla FM. Enhanced caffeine-induced Ca2+ release in the 3xTg-AD mouse model of Alzheimer’s disease. J Neurochem. 2005;94:1711–8.PubMedCrossRefGoogle Scholar
  15. 15.
    Stutzmann GE, LaFerla FM, Parker I. Ca2+ signaling in mouse cortical neurons studied by two-photon imaging and photoreleased inositol triphosphate. J Neurosci. 2003;23:758–65.PubMedGoogle Scholar
  16. 16.
    Stutzmann GE, Smith I, Caccamo A, Oddo S, LaFerla FM, Parker I. Enhanced ryanodine receptor recruitment contributes to Ca2+ disruptions in young, adult, and aged Alzheimer’s disease mice. J Neurosci. 2006;26:5180–9.PubMedCrossRefGoogle Scholar
  17. 17.
    Stutzmann GE, Smith I, Caccamo A, Oddo S, Parker I, Laferla FM. Enhanced ryanodine-mediated calcium release in mutant PS1-expressing Alzheimer’s mouse models. Ann N Y Acad Sci. 2007;1097:265–77.PubMedCrossRefGoogle Scholar
  18. 18.
    Stutzmann GE, Caccamo A, LaFerla FM, Parker I. Dysregulated IP3 signaling in cortical neurons of knock-in mice expressing an Alzheimer’s-linked mutation in presenilin1 results in exaggerated Ca2+ signals and altered membrane excitability. J Neurosci. 2004;24:508–13.PubMedCrossRefGoogle Scholar
  19. 19.
    Denk W, Strickler JH, Webb WW. Two-photon laser scanning fluorescence microscopy. Science. 1990;248:73–6.PubMedCrossRefGoogle Scholar
  20. 20.
    Svoboda K, Denk W, Kleinfeld D, Tank DW. In vivo dendritic calcium dynamics in neocortical pyramidal neurons. Nature. 1997;385:161–5.PubMedCrossRefGoogle Scholar
  21. 21.
    Helmchen F, Denk W. Deep tissue two-photon microscopy. Nat Methods. 2005;2:932–40.PubMedCrossRefGoogle Scholar
  22. 22.
    Wachowiak M, Cohen LB. Representation of odorants by receptor neuron input to the mouse olfactory bulb. Neuron. 2001;32:723–35.PubMedCrossRefGoogle Scholar
  23. 23.
    Stosiek C, Garaschuk O, Holthoff K, Konnerth A. In vivo two-photon calcium imaging of neuronal networks. Proc Natl Acad Sci U S A. 2003;100:7319–24.PubMedCrossRefGoogle Scholar
  24. 24.
    Garaschuk O, Milos RI, Konnerth A. Targeted bulk-loading of fluorescent indicators for two-photon brain imaging in vivo. Nat Prot. 2006;1:380–6.CrossRefGoogle Scholar
  25. 25.
    Nagayama S, Zeng S, Xiong W, Fletcher ML, Masurkar AV, Davis DJ, et al. In vivo simultaneous tracing and Ca2+ imaging of local neuronal circuits. Neuron. 2007;53:789–803.PubMedCrossRefGoogle Scholar
  26. 26.
    Heim N, Garaschuk O, Friedrich MW, Mank M, Milos RI, Kovalchuk Y, et al. Improved calcium imaging in transgenic mice expressing a troponin-C based biosensor. Nat Methods. 2007;4:127–9.PubMedCrossRefGoogle Scholar
  27. 27.
    Garaschuk O, Griesbeck O, Konnerth A. Troponin C-based biosensors: a new family of genetically encoded indicators for in vivo calcium imaging in the nervous system. Cell Calcium. 2007;42:351–61.PubMedCrossRefGoogle Scholar
  28. 28.
    Diez-Garcia J, Akemann W, Knopfel T. In vivo calcium imaging from genetically specified target cells in mouse cerebellum. Neuroimage. 2007;34:859–69.PubMedCrossRefGoogle Scholar
  29. 29.
    Diez-Garcia J, Matsushita S, Mutoh H, Nakai J, Ohkura M, Yokoyama J, et al. Activation of cerebellar parallel fibers monitored in transgenic mice expressing a fluorescent Ca2+ indicator protein. Eur J Neurosci. 2005;22:627–35.PubMedCrossRefGoogle Scholar
  30. 30.
    Hasan MT, Friedrich RW, Euler T, Larkum ME, Giese G, Both M, et al. Functional fluorescent Ca2+ indicator proteins in transgenic mice under TET control. PLoS Biol. 2004;2:763–75.CrossRefGoogle Scholar
  31. 31.
    Alzheimer A. Ueber eigenartige Krankheitsfaelle des spaeteren Alters. Zeitschrift fuer die gesamte Neurologie und Psychiatrie. 1911;4:356–86.CrossRefGoogle Scholar
  32. 32.
    Walsh DM, Selkoe DJ. Deciphering the molecular basis of memory failure in Alzheimer’s disease. Neuron. 2004;44:181–93.PubMedCrossRefGoogle Scholar
  33. 33.
    Sisodia SS, St George-Hyslop PH. Gamma-secretase, notch, Abeta and Alzheimer’s disease: where do the presenilins fit in. Nat Rev Neurosci. 2002;3:281–90.PubMedCrossRefGoogle Scholar
  34. 34.
    Haass C, Selkoe DJ. Soluble protein oligomers in neurodegeneration: lessons from the Alzheimer’s amyloid beta-peptide. Nat Rev Mol Cell Biol. 2007;8:101–12.PubMedCrossRefGoogle Scholar
  35. 35.
    Games D, Adams D, Alessandrini R, Barbour R, Berthelette P, Blackwell C, et al. Alzheimer-type neuropathology in transgenic mice overexpressing V717F beta-amyloid precursor protein. Nature. 1995;373:523–7.PubMedCrossRefGoogle Scholar
  36. 36.
    Hsiao K, Chapman P, Nilsen S, Eckman C, Harigaya Y, Younkin S, et al. Correlative memory deficits, Abeta elevation, and amyloid plaques in transgenic mice. Science. 1996;274:99–102.PubMedCrossRefGoogle Scholar
  37. 37.
    Sturchler-Pierrat C, Abramowski D, Duke M, Wiederhold KH, Mistl C, Rothacher S, et al. Two amyloid precursor protein transgenic mouse models with Alzheimer disease-like pathology. Proc Natl Acad Sci U S A. 1997;94:13287–92.PubMedCrossRefGoogle Scholar
  38. 38.
    Oddo S, Caccamo A, Shepherd JD, Murphy MP, Golde TE, Kayed R, et al. Triple-transgenic model of Alzheimer’s disease with plaques and tangles: intracellular Abeta and synaptic dysfunction. Neuron. 2003;39:409–21.PubMedCrossRefGoogle Scholar
  39. 39.
    Radde R, Bolmont T, Kaeser SA, Coomaraswamy J, Lindau D, Stoltze L, et al. Abeta42-driven cerebral amyloidosis in transgenic mice reveals early and robust pathology. EMBO Rep. 2006;7:940–6.PubMedCrossRefGoogle Scholar
  40. 40.
    Christie RH, Bacskai BJ, Zipfel WR, Williams RM, Kajdasz ST, Webb WW, et al. Growth arrest of individual senile plaques in a model of Alzheimer’s disease observed by in vivo multiphoton microscopy. J Neurosci. 2001;21:858–64.PubMedGoogle Scholar
  41. 41.
    Bacskai BJ, Hickey GA, Skoch J, Kajdasz ST, Wang Y, Huang GF, et al. Four-dimensional multiphoton imaging of brain entry, amyloid binding, and clearance of an amyloid-beta ligand in transgenic mice. Proc Natl Acad Sci U S A. 2003;100:12462–7.PubMedCrossRefGoogle Scholar
  42. 42.
    McLellan ME, Kajdasz ST, Hyman BT, Bacskai BJ. In vivo imaging of reactive oxygen species specifically associated with thioflavine S-positive amyloid plaques by multiphoton microscopy. J Neurosci. 2003;23:2212–7.PubMedGoogle Scholar
  43. 43.
    Klunk WE, Bacskai BJ, Mathis CA, Kajdasz ST, McLellan ME, Frosch MP, et al. Imaging Abeta plaques in living transgenic mice with multiphoton microscopy and methoxy-X04, a systemically administered Congo red derivative. J Neuropathol Exp Neurol. 2002;61:797–805.PubMedGoogle Scholar
  44. 44.
    Bacskai BJ, Kajdasz ST, Christie RH, Carter C, Games D, Seubert P, et al. Imaging of amyloid-beta deposits in brains of living mice permits direct observation of clearance of plaques with immunotherapy. Nat Med. 2001;7:369–72.PubMedCrossRefGoogle Scholar
  45. 45.
    Stalder M, Phinney A, Probst A, Sommer B, Staufenbiel M, Jucker M. Association of microglia with amyloid plaques in brains of APP23 transgenic mice. Am J Pathol. 1999;154:1673–84.PubMedGoogle Scholar
  46. 46.
    Nimmerjahn A, Kirchhoff F, Kerr JN, Helmchen F. Sulforhodamine 101 as a specific marker of astroglia in the neocortex in vivo. Nat Methods. 2004;1:31–7.PubMedCrossRefGoogle Scholar
  47. 47.
    Jung S, Aliberti J, Graemmel P, Sunshine MJ, Kreutzberg GW, Sher A, et al. Analysis of fractalkine receptor CX(3)CR1 function by targeted deletion and green fluorescent protein reporter gene insertion. Mol Cell Biol. 2000;20:4106–14.PubMedCrossRefGoogle Scholar
  48. 48.
    Hirasawa T, Ohsawa K, Imai Y, Ondo Y, Akazawa C, Uchino S, et al. Visualization of microglia in living tissues using Iba1-EGFP transgenic mice. J Neurosci Res. 2005;81:357–62.PubMedCrossRefGoogle Scholar
  49. 49.
    Aubin JE. Autofluorescence of viable cultured mammalian cells. J Histochem Cytochem. 1979;27:36–43.PubMedGoogle Scholar
  50. 50.
    Porta EA. Pigments in aging: an overview. Ann N Y Acad Sci. 2002;959:57–65.PubMedCrossRefGoogle Scholar
  51. 51.
    Terman A, Brunk UT. Lipofuscin. Int J Biochem Cell Biol. 2004;36:1400–4.PubMedCrossRefGoogle Scholar
  52. 52.
    Brunk UT, Terman A. Lipofuscin: mechanisms of age-related accumulation and influence on cell function. Free Radic Biol Med. 2002;33:611–9.PubMedCrossRefGoogle Scholar
  53. 53.
    Han M, Bindewald-Wittich A, Holz FG, Giese G, Niemz MH, Snyder S, et al. Two-photon excited autofluorescence imaging of human retinal pigment epithelial cells. J Biomed Opt. 2006;11:0105011–3.CrossRefGoogle Scholar
  54. 54.
    Bindewald-Wittich A, Han M, Schmitz-Valckenberg S, Snyder SR, Giese G, Bille JF, et al. Two-photon-excited fluorescence imaging of human RPE cells with a femtosecond Ti:Sapphire laser. Invest Ophthalmol Vis Sci. 2006;47:4553–7.PubMedCrossRefGoogle Scholar
  55. 55.
    Xu C. Two-photon cross sections of indicators. In: Yuste R, Konnerth A, editors. Imaging: a laboratory manual. Cold Spring Harbor: Cold Spring Harbor Press; 2000. pp 19.1–19.9.Google Scholar
  56. 56.
    Garaschuk O, Milos RI, Grienberger C, Marandi N, Adelsberger H, Konnerth A, et al. Optical monitoring of brain function in vivo: from neurons to networks. Pflugers Arch. 2006;453:385–96.PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Gerhard Eichhoff
    • 1
  • Marc Aurel Busche
    • 1
  • Olga Garaschuk
    • 1
    Email author
  1. 1.Institute of NeuroscienceTechnical University of MunichMunichGermany

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