Imaging of Astrocytic Activity in Living Rodents

  • Norio Takata
  • Yoshiaki Shinohara
  • Masamichi Ohkura
  • Tsuneko Mishima
  • Junichi Nakai
  • Hajime Hirase
Part of the Neuromethods book series (NM, volume 85)


Mounting evidence from in vitro experiments supports bidirectional interactions between astrocytes and neurons, suggesting glial involvement in neuronal information processing in the brain. Elevation of the cytosolic calcium ion (Ca2+) concentration has been suggested to be important for gliotransmission; however, the study of Ca2+ dynamics in cerebral cortical astrocytes in vivo became possible only recently. Here, we describe a set of procedures to monitor Ca2+ concentration fluctuations in a population of astrocytes or in astrocytic processes using in vivo two-photon microscopy. Simultaneous recording of neuronal activity by electrodes and astrocytic activity by imaging is a promising way to reveal the nature of neuron–glia interactions in the brain of living rodents.

Key words

Bolus loading Adenovirus G-CaMP Astrocytes Astrocytic processes Cerebral cortex 


  1. 1.
    Nedergaard M, Ransom B, Goldman SA (2003) New roles for astrocytes: redefining the functional architecture of the brain. Trends Neurosci 26:523–530PubMedCrossRefGoogle Scholar
  2. 2.
    Karlsen AS, Pakkenberg B (2011) Total numbers of neurons and glial cells in cortex and basal ganglia of aged brains with Down syndrome—a stereological study. Cereb Cortex 21:2519–2524PubMedCrossRefGoogle Scholar
  3. 3.
    Mishima T, Sakatani S, Hirase H (2005) In vivo intracellular recording and subsequent morphological visualization of rat neocortical astrocytes. Soc Neurosci. Abstract, 385.4Google Scholar
  4. 4.
    Verkharatsky A, Butt A (2007) Glial neurobiology. Wiley, Chichester, West SussexCrossRefGoogle Scholar
  5. 5.
    Tasaki I (1939) The electro-saltatory transmission of the nerve impulse and the effect of narcosis upon the nerve fiber. Am J Physiol 127:211–227Google Scholar
  6. 6.
    Huxley AF, Stampfli R (1949) Evidence for saltatory conduction in peripheral myelinated nerve fibres. J Physiol 108:315–339Google Scholar
  7. 7.
    Nishiyama A, Chang A, Trapp BD (1999) NG2+ glial cells: a novel glial cell population in the adult brain. J Neuropathol Exp Neurol 58:1113–1124PubMedCrossRefGoogle Scholar
  8. 8.
    Aloisi F (2001) Immune function of microglia. Glia 36:165–179PubMedCrossRefGoogle Scholar
  9. 9.
    Hanisch UK, Kettenmann H (2007) Microglia: active sensor and versatile effector cells in the normal and pathologic brain. Nat Neurosci 10:1387–1394PubMedCrossRefGoogle Scholar
  10. 10.
    Magistretti PJ, Pellerin L, Rothman DL, Shulman RG (1999) Energy on demand. Science 283:496–497PubMedCrossRefGoogle Scholar
  11. 11.
    Wyss MT, Jolivet R, Buck A, Magistretti PJ, Weber B (2011) In vivo evidence for lactate as a neuronal energy source. J Neurosci 31:7477–7485PubMedCrossRefGoogle Scholar
  12. 12.
    Grosche J, Matyash V, Moller T, Verkhratsky A, Reichenbach A, Kettenmann H (1999) Microdomains for neuron-glia interaction: parallel fiber signaling to Bergmann glial cells. Nat Neurosci 2:139–143PubMedCrossRefGoogle Scholar
  13. 13.
    Chao TI, Rickmann M, Wolff JR (2002) The synapse-astrocyte boundary: anatomical basis for an integrative role of glia in synaptic transmission. In: Volterra A, Magistretti P, Haydon PG (eds) Tripartite synapses: synaptic transmission with glia. Oxford University Press, New York, pp 3–23Google Scholar
  14. 14.
    Ventura R, Harris KM (1999) Three-dimensional relationships between hippocampal synapses and astrocytes. J Neurosci 19:6897–6906PubMedGoogle Scholar
  15. 15.
    Bushong EA, Martone ME, Jones YZ, Ellisman MH (2002) Protoplasmic astrocytes in CA1 stratum radiatum occupy separate anatomical domains. J Neurosci 22:183–192PubMedGoogle Scholar
  16. 16.
    Oberheim NA, Takano T, Han X, He W, Lin JH, Wang F, Xu Q, Wyatt JD, Pilcher W, Ojemann JG, Ransom BR, Goldman SA, Nedergaard M (2009) Uniquely hominid features of adult human astrocytes. J Neurosci 29:3276–3287PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Parpura V, Basarsky TA, Liu F, Jeftinija K, Jeftinija S, Haydon PG (1994) Glutamate-mediated astrocyte-neuron signalling. Nature 369:744–747PubMedCrossRefGoogle Scholar
  18. 18.
    Guthrie PB, Knappenberger J, Segal M, Bennett MV, Charles AC, Kater SB (1999) ATP released from astrocytes mediates glial calcium waves. J Neurosci 19:520–528PubMedGoogle Scholar
  19. 19.
    Araque A, Parpura V, Sanzgiri RP, Haydon PG (1999) Tripartite synapses: glia, the unacknowledged partner. Trends Neurosci 22:208–215PubMedCrossRefGoogle Scholar
  20. 20.
    Volterra A, Meldolesi J (2005) Astrocytes, from brain glue to communication elements: the revolution continues. Nat Rev Neurosci 6:626–640PubMedCrossRefGoogle Scholar
  21. 21.
    Moldestad O, Karlsen P, Molden S, Storm JF (2009) Tracheotomy improves experiment success rate in mice during urethane anesthesia and stereotaxic surgery. J Neurosci Methods 176:57–62PubMedCrossRefGoogle Scholar
  22. 22.
    Takata N, Hirase H (2008) Cortical layer 1 and layer 2/3 astrocytes exhibit distinct calcium dynamics in vivo. PLoS One 3:e2525PubMedCentralPubMedCrossRefGoogle Scholar
  23. 23.
    Yang G, Pan F, Parkhurst CN, Grutzendler J, Gan WB (2010) Thinned-skull cranial window technique for long-term imaging of the cortex in live mice. Nat Protoc 5:201–208PubMedCrossRefGoogle Scholar
  24. 24.
    Olesen SP (1987) Leakiness of rat brain microvessels to fluorescent probes following craniotomy. Acta Physiol Scand 130:63–68PubMedCrossRefGoogle Scholar
  25. 25.
    Stosiek C, Garaschuk O, Holthoff K, Konnerth A (2003) In vivo two-photon calcium imaging of neuronal networks. Proc Natl Acad Sci USA 100:7319–7324PubMedCrossRefGoogle Scholar
  26. 26.
    Nimmerjahn A, Kirchhoff F, Kerr JN, Helmchen F (2004) Sulforhodamine 101 as a specific marker of astroglia in the neocortex in vivo. Nat Methods 1:31–37PubMedCrossRefGoogle Scholar
  27. 27.
    Sullivan MR, Nimmerjahn A, Sarkisov DV, Helmchen F, Wang SS (2005) In vivo calcium imaging of circuit activity in cerebellar cortex. J Neurophysiol 94:1636–1644PubMedCrossRefGoogle Scholar
  28. 28.
    Miyawaki A, Llopis J, Heim R, McCaffery JM, Adams JA, Ikura M, Tsien RY (1997) Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature 388:882–887Google Scholar
  29. 29.
    Muto A, Ohkura M, Kotani T, Higashijima S, Nakai J, Kawakami K (2011) Genetic visualization with an improved GCaMP calcium indicator reveals spatiotemporal activation of the spinal motor neurons in zebrafish. Proc Natl Acad Sci USA 108:5425–5430PubMedCrossRefGoogle Scholar
  30. 30.
    Metzner W, Koch C, Wessel R, Gabbiani F (1998) Feature extraction by burst-like spike patterns in multiple sensory maps. J Neurosci 18:2283–2300PubMedGoogle Scholar
  31. 31.
    Takata N, Mishima T, Hisatsune C, Nagai T, Ebisui E, Mikoshiba K, Hirase H (2011) Astrocyte calcium signaling transforms cholinergic modulation to cortical plasticity in vivo. J Neurosci 31:18155–18165PubMedCrossRefGoogle Scholar
  32. 32.
    Pasti L, Zonta M, Pozzan T, Vicini S, Carmignoto G (2001) Cytosolic calcium oscillations in astrocytes may regulate exocytotic release of glutamate. J Neurosci 21:477–484PubMedGoogle Scholar
  33. 33.
    Fiacco TA, McCarthy KD (2006) Astrocyte calcium elevations: properties, propagation, and effects on brain signaling. Glia 54:676–690PubMedCrossRefGoogle Scholar
  34. 34.
    Petravicz J, Fiacco TA, McCarthy KD (2008) Loss of IP3 receptor-dependent Ca2+ increases in hippocampal astrocytes does not affect baseline CA1 pyramidal neuron synaptic activity. J Neurosci 28:4967–4973Google Scholar
  35. 35.
    Shigetomi E, Bowser DN, Sofroniew MV, Khakh BS (2008) Two forms of astrocyte calcium excitability have distinct effects on NMDA receptor-mediated slow inward currents in pyramidal neurons. J Neurosci 28:6659–6663PubMedCentralPubMedCrossRefGoogle Scholar
  36. 36.
    Hirase H, Qian L, Bartho P, Buzsaki G (2004) Calcium dynamics of cortical astrocytic networks in vivo. PLoS Biol 2:E96PubMedCentralPubMedCrossRefGoogle Scholar
  37. 37.
    Sasaki T, Kuga N, Namiki S, Matsuki N, Ikegaya Y (2011) Locally synchronized astrocytes. Cereb Cortex 21:1889–1900PubMedCrossRefGoogle Scholar
  38. 38.
    Fellin T, Pascual O, Gobbo S, Pozzan T, Haydon PG, Carmignoto G (2004) Neuronal synchrony mediated by astrocytic glutamate through activation of extrasynaptic NMDA receptors. Neuron 43:729–743PubMedCrossRefGoogle Scholar
  39. 39.
    Hoogland TM, Kuhn B, Gobel W, Huang W, Nakai J, Helmchen F, Flint J, Wang SS (2009) Radially expanding transglial calcium waves in the intact cerebellum. Proc Natl Acad Sci USA 106:3496–3501PubMedCrossRefGoogle Scholar
  40. 40.
    Kuga N, Sasaki T, Takahara Y, Matsuki N, Ikegaya Y (2011) Large-scale calcium waves traveling through astrocytic networks in vivo. J Neurosci 31:2607–2614PubMedCrossRefGoogle Scholar
  41. 41.
    Schummers J, Yu H, Sur M (2008) Tuned responses of astrocytes and their influence on hemodynamic signals in the visual cortex. Science 320:1638–1643PubMedCrossRefGoogle Scholar
  42. 42.
    Nimmerjahn A, Mukamel EA, Schnitzer MJ (2009) Motor behavior activates Bergmann glial networks. Neuron 62:400–412PubMedCentralPubMedCrossRefGoogle Scholar
  43. 43.
    Dombeck DA, Khabbaz AN, Collman F, Adelman TL, Tank DW (2007) Imaging large-scale neural activity with cellular resolution in awake, mobile mice. Neuron 56:43–57PubMedCentralPubMedCrossRefGoogle Scholar
  44. 44.
    Wang X, Lou N, Xu Q, Tian GF, Peng WG, Han X, Kang J, Takano T, Nedergaard M (2006) Astrocytic Ca2+ signaling evoked by sensory stimulation in vivo. Nat Neurosci 9:816–823PubMedCrossRefGoogle Scholar
  45. 45.
    Bekar LK, He W, Nedergaard M (2008) Locus coeruleus alpha-adrenergic-mediated activation of cortical astrocytes in vivo. Cereb Cortex 18:2789–2795PubMedCrossRefGoogle Scholar
  46. 46.
    Petzold GC, Albeanu DF, Sato TF, Murthy VN (2008) Coupling of neural activity to blood flow in olfactory glomeruli is mediated by astrocytic pathways. Neuron 58:897–910PubMedCentralPubMedCrossRefGoogle Scholar
  47. 47.
    Takata N, Nagai T, Ozawa K, Oe Y, Mikoshiba K, Hirase H (2013) Cerebral blood flow modulation by Basal forebrain or whisker stimulation can occur independently of large cytosolic Ca2+ signaling in astrocytes. PLoS One 8:e66525Google Scholar
  48. 48.
    Yang Y, Ge W, Chen Y, Zhang Z, Shen W, Wu C, Poo M, Duan S (2003) Contribution of astrocytes to hippocampal long-term potentiation through release of D-serine. Proc Natl Acad Sci USA 100:15194–15199PubMedCrossRefGoogle Scholar
  49. 49.
    Perea G, Araque A (2007) Astrocytes potentiate transmitter release at single hippocampal synapses. Science 317:1083–1086PubMedCrossRefGoogle Scholar
  50. 50.
    Henneberger C, Papouin T, Oliet SH, Rusakov DA (2010) Long-term potentiation depends on release of D-serine from astrocytes. Nature 463:232–236PubMedCentralPubMedCrossRefGoogle Scholar
  51. 51.
    Agulhon C, Fiacco TA, McCarthy KD (2010) Hippocampal short- and long-term plasticity are not modulated by astrocyte Ca2+ signaling. Science 327:1250–1254Google Scholar
  52. 52.
    Kanemaru K, Okubo Y, Hirose K, Iino M (2007) Regulation of neurite growth by spontaneous Ca2+ oscillations in astrocytes. J Neurosci 27:8957–8966PubMedCrossRefGoogle Scholar
  53. 53.
    Haber M, Zhou L, Murai KK (2006) Cooperative astrocyte and dendritic spine dynamics at hippocampal excitatory synapses. J Neurosci 26:8881–8891PubMedCrossRefGoogle Scholar
  54. 54.
    Nishida H, Okabe S (2007) Direct astrocytic contacts regulate local maturation of dendritic spines. J Neurosci 27:331–340PubMedCrossRefGoogle Scholar
  55. 55.
    Shigetomi E, Kracun S, Sofroniew MV, Khakh BS (2010) A genetically targeted optical sensor to monitor calcium signals in astrocyte processes. Nat Neurosci 13:759–766PubMedCentralPubMedCrossRefGoogle Scholar
  56. 56.
    Hirase H, Nikolenko V, Goldberg JH, Yuste R (2002) Multiphoton stimulation of neurons. J Neurobiol 51:237–247PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Norio Takata
    • 1
  • Yoshiaki Shinohara
    • 1
  • Masamichi Ohkura
    • 2
  • Tsuneko Mishima
    • 1
  • Junichi Nakai
    • 3
  • Hajime Hirase
    • 1
  1. 1.RIKEN Brain Science InstituteWakoJapan
  2. 2.Saitama University, Brain Science Institute SaitamaWakoJapan
  3. 3.Saitama University Brain Science Institute, SaitamaWakoJapan

Personalised recommendations