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Monitoring astrocyte calcium microdomains with improved membrane targeted GCaMP reporters

  • Eiji Shigetomi (a1), Sebastian Kracun (a1) and Baljit S. Khakh (a1) (a2)
  • DOI:
  • Published online: 16 December 2010

Astrocytes are involved in synaptic and cerebrovascular regulation in the brain. These functions are regulated by intracellular calcium signalling that is thought to reflect a form of astrocyte excitability. In a recent study, we reported modification of the genetically encoded calcium indicator (GECI) GCaMP2 with a membrane-tethering domain, Lck, to generate Lck-GCaMP2. This GECI allowed us to detect novel microdomain calcium signals. The microdomains were random and ‘spotty’ in nature. In order to detect such signals more reliably, in the present study we further modified Lck-GCaMP2 to carry three mutations in the GCaMP2 moiety (M153K, T203V within EGFP and N60D in the CaM domain) to generate Lck-GCaMP3. We directly compared Lck-GCaMP2 and Lck-GCaMP3 by assessing their ability to monitor several types of astrocyte calcium signals with a focus on spotty microdomains. Our data show that Lck-GCaMP3 is between two- and four-times better than Lck-GCaMP2 in terms of its basal fluorescence intensity, signal-to-noise and its ability to detect microdomains. The use of Lck-GCaMP3 thus represents a significantly improved way to monitor astrocyte calcium signals, including microdomains, and will facilitate detailed exploration of their molecular mechanisms and physiological roles.

Corresponding author
Correspondence should be addressed to: Baljit S. Khakh, Department of Physiology, David Geffen School of Medicine, University of California, Los Angeles, 10833 Le Conte Avenue, 53-359 CHS, Los Angeles, CA 90095-1751USA phone: 310 825 6258 fax: 310 206 5661 email:
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This list contains references from the content that can be linked to their source. For a full set of references and notes please see the PDF or HTML where available.

C. Agulhon , T.A. Fiacco and K.D. McCarthy (2010) Hippocampal short- and long-term plasticity are not modulated by astrocyte Ca2+ signaling. Science 327, 12501254.

C. Agulhon , J. Petravicz , A.B. McMullen , E.J. Sweger , S.K. Minton , S.R. Taves (2008) What is the role of astrocyte calcium in neurophysiology? Neuron 59, 932946.

J. Akerboom , J.D. Rivera , M.M. Guilbe , E.C. Malavé , H.H. Hernandez , L. Tian (2009) Crystal structures of the GCaMP calcium sensor reveal the mechanism of fluorescence signal change and aid rational design. Journal of Biological Chemistry 284, 64556464.

A. Araque , V. Parpura , R.P. Sanzgiri and P.G. Haydon (1999) Tripartite synapses: glia, the unacknowledged partner. Trends in Neuroscience 22, 208215.

S.D. Atkin , S. Patel , A. Kocharyan , L.A. Holtzclaw , S.H. Weerth , V. Schram (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. Journal of Neurscience Methods 181, 212226.

B.A. Barres (2008) The mystery and magic of glia: a perspective on their roles in health and disease. Neuron 60, 430440.

L.K. Bekar , W. He and M. Nedergaard (2008) Locus coeruleus alpha-adrenergic-mediated activation of cortical astrocytes in vivo. Cerebral Cortex 18, 27892795.

A.M. Benediktsson , S.J. Schachtele , S.H. Green and M.E. Dailey (2005) Ballistic labeling and dynamic imaging of astrocytes in organotypic hippocampal slice cultures. Journal of Neuroscience Methods 141, 4153.

D.M. Bers , C.W. Patton and R. Nuccitelli (1994) A practical guide to the preparation of Ca2+ buffers. Methods in Cell Biology 40, 329.

D.N. Bowser and B.S. Khakh (2007) Two forms of single vesicle astrocyte exocytosis imaged with total internal reflection fluorescence microscopy. Proceedings of the National Academy of Sciences of the U.S.A. 104, 42124217.

D.E. Clapham (2007) Calcium signaling. Cell 131, 10471058.

D.A. Dombeck , A.N. Khabbaz , F. Collman , T.L. Adelman and D.W. Tank (2007) Imaging large-scale neural activity with cellular resolution in awake, mobile mice. Neuron 56, 4357.

E. Dreosti , B. Odermatt , M.M. Dorostkar and L. Lagnado (2009) A genetically encoded reporter of synaptic activity in vivo. Nature Methods 6, 883889.

T. Fellin , O. Pascual , S. Gobbo , T. Pozzan , P.G. Haydon and G. Carmignoto (2004) Neuronal synchrony mediated by astrocytic glutamate through activation of extrasynaptic NMDA receptors. Neuron 43, 729743.

T.A. Fiacco , C. Agulhon and K.D. McCarthy (2009) Sorting out astrocyte physiology from pharmacology. Annual Review of Pharmacology and Toxicology 49, 151174.

T.A. Fiacco , C. Agulhon , S.R. Taves , J. Petravicz , K.B. Casper , X. Dong (2007) Selective stimulation of astrocyte calcium in situ does not affect neuronal excitatory synaptic activity. Neuron 54, 611626.

R.D. Fields (2004) The other half of the brain. Scientific American 290, 5461.

R.D. Fields and G. Burnstock (2006) Purinergic signalling in neuron-glia interactions. Nature Reviews Neuroscience 7, 423436.

W. Göbel , B.M. Kampa and F. Helmchen (2007) Imaging cellular network dynamics in three dimensions using fast 3D laser scanning. Nature Methods 4, 7379.

G.R. Gordon , K.J. Iremonger , S. Kantevari , G.C. Ellis-Davies , B.A. MacVicar and J.S. Bains (2009) Astrocyte-mediated distributed plasticity at hypothalamic glutamate synapses. Neuron 64, 391403.

G.R. Gordon , S.J. Mulligan and B.A. MacVicar (2007) Astrocyte control of the cerebrovasculature. Glia 55, 12141221.

A.V. Gourine , V. Kasymov , N. Marina , F. Tang , M.F. Figueiredo , S. Lane (2010) Astrocytes control breathing through pH-dependent release of ATP. Science 329, 571575.

M.M. Halassa , T. Fellin , H. Takano , J.H. Dong and P.G. Haydon (2007) Synaptic islands defined by the territory of a single astrocyte. Journal of Neuroscience 27, 64736477.

M.M. Halassa and P.G. Haydon (2010) Integrated brain circuits: astrocytic networks modulate neuronal activity and behavior. Annual Review of Physiology 72, 335355.

N.B. Hamilton and D. Attwell (2010) Do astrocytes really exocytose neurotransmitters? Nature Reviews. Neuroscience 11, 227238.

C. Henneberger , T. Papouin , S.H. Oliet and D.A. Rusakov (2010) Long-term potentiation depends on release of D-serine from astrocytes. Nature 463, 232236.

H. Hirase , L. Qian , P. Bartho and G. Buzsaki (2004) Calcium dynamics of cortical astrocytic networks in vivo. PLoS Biology 2, E96

S.A. Hires , L. Tian and L.L. Looger (2008) Reporting neural activity with genetically encoded calcium indicators. Brain Cell Biology 36, 6986.

C. Iadecola and M. Nedergaard (2007) Glial regulation of the cerebral microvasculature. Nature Neuroscience 10, 13691376.

P. Kofuji and E.A. Newman (2004) Potassium buffering in the central nervous system. Neuroscience 129, 10451056.

M.I. Kotlikoff (2007) Genetically encoded Ca2+ indicators: using genetics and molecular design to understand complex physiology. Journal of Physiology 578, 5567.

C.J. Lee , G. Mannaioni , H. Yuan , D.H. Woo , M.B. Gingrich and S.F. Traynelis (2007) Astrocytic control of synaptic NMDA receptors. Journal of Physiology 581, 10571081.

M.Y. Lee , H. Song , J. Nakai , M. Ohkura , M.I. Kotlikoff , S.P. Kinsey (2006) Local subplasma membrane Ca2+ signals detected by a tethered Ca2+ sensor. Proceedings of the National Academy of Sciences of the U.S.A. 103, 1323213237.

S.Y. Lee and P.G. Haydon (2007) Astrocytic glutamate targets NMDA receptors. Journal of Physiology 581, 887888.

P.J. Magistretti (2006) Neuron-glia metabolic coupling and plasticity. Journal of Experimental Biology 209, 23042311.

J. Nakai , M. Ohkura and K. Imoto (2001) A high signal-to-noise Ca2+ probe composed of a single green fluorescent protein. Nature Biotechnology 19, 137141.

N.A. Oberheim , T. Takano , X. Han , W. He , J.H. Lin , F. Wang (2009) Uniquely hominid features of adult human astrocytes. Journal of Neuroscience 29, 32763287.

V. Parpura , T.A. Basarsky , F. Liu , K. Jeftinija , S. Jeftinija and P.G. Haydon (1994) Glutamate-mediated astrocyte-neuron signalling. Nature 369, 744747.

J. Petravicz , T.A. Fiacco and K.D. McCarthy (2008) Loss of IP3 receptor-dependent Ca2+ increases in hippocampal astrocytes does not affect baseline CA1 pyramidal neuron synaptic activity. Journal of Neuroscience 28, 49674973.

E. Richler , S. Chaumont , E. Shigetomi , A. Sagasti and B.S. Khakh (2008) An approach to image activation of transmitter-gated P2X receptors in vitro and in vivo. Nature Methods 5, 8793.

M.M. Rodríguez Guilbe , E.C. Alfaro Malavé , J. Akerboom , J.S. Marvin , L.L. Looger and E.R. Schreiter (2008) Crystallization and preliminary X-ray characterization of the genetically encoded fluorescent calcium indicator protein GCaMP2. Acta Crystallographica Section F, Structural Biology and Crystalization Communications 64, 629631.

J. Schummers , H. Yu and M. Sur (2008) Tuned responses of astrocytes and their influence on hemodynamic signals in the visual cortex. Science 320, 16381643.

J.D. Seelig , M.E. Chiappe , G.K. Lott , A. Dutta , J.E. Osborne , M.B. Reiser (2010) Two-photon calcium imaging from head-fixed Drosophila during optomotor walking behavior. Nature Methods 7, 535540.

E. Shigetomi , D.N. Bowser , M.V. Sofroniew and B.S. Khakh (2008) Two forms of astrocyte calcium excitability have distinct effects on NMDA receptor-mediated slow inward currents in pyramidal neurons. Journal of Neuroscience 28, 66596663.

E. Shigetomi , S. Kracun , M.V. Sofroniew and B.S. Khakh (2010) A genetically targeted optical sensor to monitor calcium signals in astrocyte processes. Nature Neuroscience 13, 759766.

S.J. Smith (1992) Do astrocytes process neural information? Progress in Brain Research 94, 119136.

Y.N. Tallini , M. Ohkura , B.R. Choi , G. Ji , K. Imoto , R. Doran (2006) Imaging cellular signals in the heart in vivo: Cardiac expression of the high-signal Ca2+ indicator GCaMP2. Proceedings of the National Academy of Sciences of the U.S.A. 103, 47534758.

L. Tian , S.A. Hires , T. Mao , D. Huber , M.E. Chiappe , S.H. Chalasani (2009) Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators. Nature Methods 6, 875881.

N.X. Tritsch and D.E. Bergles (2007) Defining the role of astrocytes in neuromodulation. Neuron 54, 497500.

Q. Wang , B. Shui , M.I. Kotlikoff and H. Sondermann (2008) Structural basis for calcium sensing by GCaMP2. Structure 16, 18171827.

X. Wang , N. Lou , Q. Xu , G.F. Tian , W.G. Peng , X. Han (2006) Astrocytic Ca2+ signaling evoked by sensory stimulation in vivo. Nature Neuroscience 9, 816823.

D. Willoughby , S. Wachten , N. Masada and D.M. Cooper (2010) Direct demonstration of discrete Ca2+ microdomains associated with different isoforms of adenylyl cyclase. Journal of Cell Science 123, 107117.

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