Hostname: page-component-cd9895bd7-8ctnn Total loading time: 0 Render date: 2024-12-27T19:53:55.095Z Has data issue: false hasContentIssue false
  • English
  • Français

Monitoring astrocyte calcium microdomains with improved membrane targeted GCaMP reporters

Published online by Cambridge University Press:  16 December 2010

Eiji Shigetomi ,
Sebastian Kracun  and
Baljit S. Khakh
Eiji Shigetomi
Affiliation:
Department of Physiology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, USA
Sebastian Kracun
Affiliation:
Department of Physiology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, USA
Baljit S. Khakh*
Affiliation:
Department of Physiology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, USA Department of Neurobiology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, USA
*
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: [email protected]
Get access Rights & Permissions [Opens in a new window]

Abstract

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.

Keywords

Glianeuroninteractionssignalling
Type
Research Article
Information
Neuron Glia Biology , Volume 6 , Issue 3 , August 2010 , pp. 183 - 191
Copyright
Copyright © Cambridge University Press 2010

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

REFERENCES

Agulhon, C., Fiacco, T.A. and McCarthy, K.D. (2010) Hippocampal short- and long-term plasticity are not modulated by astrocyte Ca2+ signaling. Science 327, 1250–1254.CrossRefGoogle Scholar
Agulhon, C., Petravicz, J., McMullen, A.B., Sweger, E.J., Minton, S.K., Taves, S.R. et al. (2008) What is the role of astrocyte calcium in neurophysiology? Neuron 59, 932–946.CrossRefGoogle ScholarPubMed
Akerboom, J., Rivera, J.D., Guilbe, M.M., Malavé, E.C., Hernandez, H.H., Tian, L. et al. (2009) Crystal structures of the GCaMP calcium sensor reveal the mechanism of fluorescence signal change and aid rational design. Journal of Biological Chemistry 284, 6455–6464.CrossRefGoogle Scholar
Araque, A., Parpura, V., Sanzgiri, R.P. and Haydon, P.G. (1999) Tripartite synapses: glia, the unacknowledged partner. Trends in Neuroscience 22, 208–215.CrossRefGoogle ScholarPubMed
Atkin, S.D., Patel, S., Kocharyan, A., Holtzclaw, L.A., Weerth, S.H., Schram, V. et al. (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, 212–226.Google ScholarPubMed
Barres, B.A. (2008) The mystery and magic of glia: a perspective on their roles in health and disease. Neuron 60, 430–440.CrossRefGoogle Scholar
Bekar, L.K., He, W. and Nedergaard, M. (2008) Locus coeruleus alpha-adrenergic-mediated activation of cortical astrocytes in vivo. Cerebral Cortex 18, 2789–2795.CrossRefGoogle ScholarPubMed
Benediktsson, A.M., Schachtele, S.J., Green, S.H. and Dailey, M.E. (2005) Ballistic labeling and dynamic imaging of astrocytes in organotypic hippocampal slice cultures. Journal of Neuroscience Methods 141, 41–53.CrossRefGoogle ScholarPubMed
Bers, D.M., Patton, C.W. and Nuccitelli, R. (1994) A practical guide to the preparation of Ca2+ buffers. Methods in Cell Biology 40, 3–29.CrossRefGoogle Scholar
Bowser, D.N. and Khakh, B.S. (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, 4212–4217.CrossRefGoogle ScholarPubMed
Brenner, M., Kisseberth, W.C., Su, Y., Besnard, F. and Messing, A. (1994) GFAP promoter directs astrocyte-specific expression in transgenic mice. Journal of Neuroscience 14, 1030–1037.CrossRefGoogle ScholarPubMed
Clapham, D.E. (2007) Calcium signaling. Cell 131, 1047–1058.CrossRefGoogle ScholarPubMed
Dombeck, D.A., Khabbaz, A.N., Collman, F., Adelman, T.L. and Tank, D.W. (2007) Imaging large-scale neural activity with cellular resolution in awake, mobile mice. Neuron 56, 43–57.CrossRefGoogle ScholarPubMed
Dreosti, E., Odermatt, B., Dorostkar, M.M. and Lagnado, L. (2009) A genetically encoded reporter of synaptic activity in vivo. Nature Methods 6, 883–889.CrossRefGoogle ScholarPubMed
Fellin, T., Pascual, O., Gobbo, S., Pozzan, T., Haydon, P.G. and Carmignoto, G. (2004) Neuronal synchrony mediated by astrocytic glutamate through activation of extrasynaptic NMDA receptors. Neuron 43, 729–743.CrossRefGoogle ScholarPubMed
Fiacco, T.A., Agulhon, C. and McCarthy, K.D. (2009) Sorting out astrocyte physiology from pharmacology. Annual Review of Pharmacology and Toxicology 49, 151–174.CrossRefGoogle ScholarPubMed
Fiacco, T.A., Agulhon, C., Taves, S.R., Petravicz, J., Casper, K.B., Dong, X. et al. (2007) Selective stimulation of astrocyte calcium in situ does not affect neuronal excitatory synaptic activity. Neuron 54, 611–626.CrossRefGoogle Scholar
Fields, R.D. (2004) The other half of the brain. Scientific American 290, 54–61.CrossRefGoogle ScholarPubMed
Fields, R.D. and Burnstock, G. (2006) Purinergic signalling in neuron-glia interactions. Nature Reviews Neuroscience 7, 423–436.CrossRefGoogle ScholarPubMed
Göbel, W., Kampa, B.M. and Helmchen, F. (2007) Imaging cellular network dynamics in three dimensions using fast 3D laser scanning. Nature Methods 4, 73–79.CrossRefGoogle ScholarPubMed
Gordon, G.R., Iremonger, K.J., Kantevari, S., Ellis-Davies, G.C., MacVicar, B.A. and Bains, J.S. (2009) Astrocyte-mediated distributed plasticity at hypothalamic glutamate synapses. Neuron 64, 391–403.CrossRefGoogle ScholarPubMed
Gordon, G.R., Mulligan, S.J. and MacVicar, B.A. (2007) Astrocyte control of the cerebrovasculature. Glia 55, 1214–1221.CrossRefGoogle ScholarPubMed
Gourine, A.V., Kasymov, V., Marina, N., Tang, F., Figueiredo, M.F., Lane, S. et al. (2010) Astrocytes control breathing through pH-dependent release of ATP. Science 329, 571–575.CrossRefGoogle ScholarPubMed
Halassa, M.M., Fellin, T., Takano, H., Dong, J.H. and Haydon, P.G. (2007) Synaptic islands defined by the territory of a single astrocyte. Journal of Neuroscience 27, 6473–6477.CrossRefGoogle ScholarPubMed
Halassa, M.M. and Haydon, P.G. (2010) Integrated brain circuits: astrocytic networks modulate neuronal activity and behavior. Annual Review of Physiology 72, 335–355.CrossRefGoogle ScholarPubMed
Hamilton, N.B. and Attwell, D. (2010) Do astrocytes really exocytose neurotransmitters? Nature Reviews. Neuroscience 11, 227–238.CrossRefGoogle ScholarPubMed
Henneberger, C., Papouin, T., Oliet, S.H. and Rusakov, D.A. (2010) Long-term potentiation depends on release of D-serine from astrocytes. Nature 463, 232–236.CrossRefGoogle ScholarPubMed
Hirase, H., Qian, L., Bartho, P. and Buzsaki, G. (2004) Calcium dynamics of cortical astrocytic networks in vivo. PLoS Biology 2, E96CrossRefGoogle ScholarPubMed
Hires, S.A., Tian, L. and Looger, L.L. (2008) Reporting neural activity with genetically encoded calcium indicators. Brain Cell Biology 36, 69–86.CrossRefGoogle ScholarPubMed
Iadecola, C. and Nedergaard, M. (2007) Glial regulation of the cerebral microvasculature. Nature Neuroscience 10, 1369–1376.CrossRefGoogle ScholarPubMed
Kofuji, P. and Newman, E.A. (2004) Potassium buffering in the central nervous system. Neuroscience 129, 1045–1056.CrossRefGoogle ScholarPubMed
Kotlikoff, M.I. (2007) Genetically encoded Ca2+ indicators: using genetics and molecular design to understand complex physiology. Journal of Physiology 578, 55–67.CrossRefGoogle ScholarPubMed
Lee, C.J., Mannaioni, G., Yuan, H., Woo, D.H., Gingrich, M.B. and Traynelis, S.F. (2007) Astrocytic control of synaptic NMDA receptors. Journal of Physiology 581, 1057–1081.CrossRefGoogle ScholarPubMed
Lee, M.Y., Song, H., Nakai, J., Ohkura, M., Kotlikoff, M.I., Kinsey, S.P. et al. (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, 13232–13237.CrossRefGoogle ScholarPubMed
Lee, S.Y. and Haydon, P.G. (2007) Astrocytic glutamate targets NMDA receptors. Journal of Physiology 581, 887–888.CrossRefGoogle ScholarPubMed
Magistretti, P.J. (2006) Neuron-glia metabolic coupling and plasticity. Journal of Experimental Biology 209, 2304–2311.CrossRefGoogle ScholarPubMed
Nakai, J., Ohkura, M. and Imoto, K. (2001) A high signal-to-noise Ca2+ probe composed of a single green fluorescent protein. Nature Biotechnology 19, 137–141.CrossRefGoogle ScholarPubMed
Nett, W.J., Oloff, S.H. and McCarthy, K.D. (2002) Hippocampal astrocytes in situ exhibit calcium oscillations that occur independent of neuronal activity. Journal of Neurophysiology 87, 528–537.CrossRefGoogle ScholarPubMed
Oberheim, N.A., Takano, T., Han, X., He, W., Lin, J.H., Wang, F. et al. (2009) Uniquely hominid features of adult human astrocytes. Journal of Neuroscience 29, 3276–3287.CrossRefGoogle ScholarPubMed
Parpura, V., Basarsky, T.A., Liu, F., Jeftinija, K., Jeftinija, S. and Haydon, P.G. (1994) Glutamate-mediated astrocyte-neuron signalling. Nature 369, 744–747.CrossRefGoogle ScholarPubMed
Pasti, L., Volterra, A., Pozzan, T. and Carmignoto, G. (1997) Intracellular calcium oscillations in astrocytes: a highly plastic, bidirectional form of communication between neurons and astrocytes in situ. Journal of Neuroscience 17, 7817–7830.CrossRefGoogle ScholarPubMed
Petravicz, J., Fiacco, T.A. and McCarthy, K.D. (2008) Loss of IP3 receptor-dependent Ca2+ increases in hippocampal astrocytes does not affect baseline CA1 pyramidal neuron synaptic activity. Journal of Neuroscience 28, 4967–4973.CrossRefGoogle Scholar
Richler, E., Chaumont, S., Shigetomi, E., Sagasti, A. and Khakh, B.S. (2008) An approach to image activation of transmitter-gated P2X receptors in vitro and in vivo. Nature Methods 5, 87–93.CrossRefGoogle ScholarPubMed
Rodríguez Guilbe, M.M., Alfaro Malavé, E.C., Akerboom, J., Marvin, J.S., Looger, L.L. and Schreiter, E.R. (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, 629–631.CrossRefGoogle ScholarPubMed
Schummers, J., Yu, H. and Sur, M. (2008) Tuned responses of astrocytes and their influence on hemodynamic signals in the visual cortex. Science 320, 1638–1643.CrossRefGoogle ScholarPubMed
Seelig, J.D., Chiappe, M.E., Lott, G.K., Dutta, A., Osborne, J.E., Reiser, M.B. et al. (2010) Two-photon calcium imaging from head-fixed Drosophila during optomotor walking behavior. Nature Methods 7, 535–540.CrossRefGoogle ScholarPubMed
Shigetomi, E., Bowser, D.N., Sofroniew, M.V. and Khakh, B.S. (2008) Two forms of astrocyte calcium excitability have distinct effects on NMDA receptor-mediated slow inward currents in pyramidal neurons. Journal of Neuroscience 28, 6659–6663.CrossRefGoogle ScholarPubMed
Shigetomi, E. and Khakh, B.S. (2009) Measuring near plasma membrane and global intracellular calcium dynamics in astrocytes. Journal of Visualized Experiments 26, 10.3791/1142.Google Scholar
Shigetomi, E., Kracun, S., Sofroniew, M.V. and Khakh, B.S. (2010) A genetically targeted optical sensor to monitor calcium signals in astrocyte processes. Nature Neuroscience 13, 759–766.CrossRefGoogle ScholarPubMed
Smith, S.J. (1992) Do astrocytes process neural information? Progress in Brain Research 94, 119–136.CrossRefGoogle ScholarPubMed
Tallini, Y.N., Ohkura, M., Choi, B.R., Ji, G., Imoto, K., Doran, R. et al. (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, 4753–4758.CrossRefGoogle ScholarPubMed
Tian, L., Hires, S.A., Mao, T., Huber, D., Chiappe, M.E., Chalasani, S.H. et al. (2009) Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators. Nature Methods 6, 875–881.CrossRefGoogle ScholarPubMed
Tritsch, N.X. and Bergles, D.E. (2007) Defining the role of astrocytes in neuromodulation. Neuron 54, 497–500.CrossRefGoogle ScholarPubMed
Wang, Q., Shui, B., Kotlikoff, M.I. and Sondermann, H. (2008) Structural basis for calcium sensing by GCaMP2. Structure 16, 1817–1827.CrossRefGoogle ScholarPubMed
Wang, X., Lou, N., Xu, Q., Tian, G.F., Peng, W.G., Han, X. et al. (2006) Astrocytic Ca2+ signaling evoked by sensory stimulation in vivo. Nature Neuroscience 9, 816–823.CrossRefGoogle ScholarPubMed
Willoughby, D., Wachten, S., Masada, N. and Cooper, D.M. (2010) Direct demonstration of discrete Ca2+ microdomains associated with different isoforms of adenylyl cyclase. Journal of Cell Science 123, 107–117.CrossRefGoogle ScholarPubMed
Zlatkine, P., Mehul, B. and Magee, A.I. (1997) Retargeting of cytosolic proteins to the plasma membrane by the Lck protein tyrosine kinase dual acylation motif. Journal of Cell Science 110, 673–679.CrossRefGoogle Scholar

Shigetomi et al. supplementary movie

Movie 1

Download Shigetomi et al. supplementary movie(Video)
Video 11.5 MB