Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Technical Report
  • Published:

A genetically targeted optical sensor to monitor calcium signals in astrocyte processes

Abstract

Calcium signaling is studied as a potential form of astrocyte excitability that may control astrocyte involvement in synaptic and cerebrovascular regulation. Fundamental questions remain unanswered about astrocyte calcium signaling, as current methods can not resolve calcium in small volume compartments, such as near the cell membrane and in distal cell processes. We modified the genetically encoded calcium sensor GCaMP2 with a membrane-tethering domain, Lck, increasing the level of Lck-GCaMP2 near the plasma membrane tenfold as compared with conventional GCaMP2. Using Lck-GCaMP2 in rat hippocampal astrocyte-neuron cocultures, we measured near-membrane calcium signals that were evoked pharmacologically or by single action potential–mediated neurotransmitter release. Moreover, we identified highly localized and frequent spontaneous calcium signals in astrocyte somata and processes that conventional GCaMP2 failed to detect. Lck-GCaMP2 acts as a genetically targeted calcium sensor for monitoring calcium signals in previously inaccessible parts of astrocytes, including fine processes.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Simultaneous imaging of global and near-membrane calcium in astrocytes.
Figure 2: Quantification of spontaneous and pharmacologically evoked calcium signals measured by TIRF and EPI microscopy.
Figure 3: Design and characterization of Lck-GCAMP2.
Figure 4: ATP-evoked calcium signals in astrocytes measured with Lck-GCaMP2.
Figure 5: Responses of astrocytes expressing Lck-GCaMP2 during EFS of neurons.
Figure 6: Spontaneous calcium signals measured with Lck-GCaMP2.
Figure 7: Spotty calcium signals measured in astrocyte processes.
Figure 8: Microdomain signals are a result of transmembrane calcium flux.

Similar content being viewed by others

References

  1. Kofuji, P. & Newman, E.A. Potassium buffering in the central nervous system. Neuroscience 129, 1045–1056 (2004).

    Article  CAS  Google Scholar 

  2. Magistretti, P.J. Neuron-glia metabolic coupling and plasticity. J. Exp. Biol. 209, 2304–2311 (2006).

    Article  CAS  Google Scholar 

  3. Sofroniew, M.V. Molecular dissection of reactive astrogliosis and glial scar formation. Trends Neurosci. 32, 638–647 (2009).

    Article  CAS  Google Scholar 

  4. Araque, A., Parpura, V., Sanzgiri, R.P. & Haydon, P.G. Tripartite synapses: glia, the unacknowledged partner. Trends Neurosci. 22, 208–215 (1999).

    Article  CAS  Google Scholar 

  5. Iadecola, C. & Nedergaard, M. Glial regulation of the cerebral microvasculature. Nat. Neurosci. 10, 1369–1376 (2007).

    Article  CAS  Google Scholar 

  6. Gordon, G.R., Mulligan, S.J. & MacVicar, B.A. Astrocyte control of the cerebrovasculature. Glia 55, 1214–1221 (2007).

    Article  Google Scholar 

  7. Cornell-Bell, A.H., Finkbeiner, S.M., Cooper, M.S. & Smith, S.J. Glutamate induces calcium waves in cultured astrocytes: long-range glial signaling. Science 247, 470–473 (1990).

    Article  CAS  Google Scholar 

  8. Hirase, H., Qian, L., Bartho, P. & Buzsaki, G. Calcium dynamics of cortical astrocytic networks in vivo. PLoS Biol. 2, e96 (2004).

    Article  Google Scholar 

  9. Wang, X. et al. Astrocytic Ca(2+) signaling evoked by sensory stimulation in vivo. Nat. Neurosci. 9, 816–823 (2006).

    Article  CAS  Google Scholar 

  10. Bekar, L.K., He, W. & Nedergaard, M. Locus coeruleus alpha-adrenergic–mediated activation of cortical astrocytes in vivo. Cereb. Cortex 18, 2789–2795 (2008).

    Article  Google Scholar 

  11. Dombeck, D.A., Khabbaz, A.N., Collman, F., Adelman, T.L. & Tank, D.W. Imaging large-scale neural activity with cellular resolution in awake, mobile mice. Neuron 56, 43–57 (2007).

    Article  CAS  Google Scholar 

  12. Göbel, W., Kampa, B.M. & Helmchen, F. Imaging cellular network dynamics in three dimensions using fast 3D laser scanning. Nat. Methods 4, 73–79 (2007).

    Article  Google Scholar 

  13. Schummers, J., Yu, H. & Sur, M. Tuned responses of astrocytes and their influence on hemodynamic signals in the visual cortex. Science 320, 1638–1643 (2008).

    Article  CAS  Google Scholar 

  14. Oberheim, N.A. et al. Uniquely hominid features of adult human astrocytes. J. Neurosci. 29, 3276–3287 (2009).

    Article  CAS  Google Scholar 

  15. Fiacco, T.A., Agulhon, C. & McCarthy, K.D. Sorting out astrocyte physiology from pharmacology. Annu. Rev. Pharmacol. Toxicol. 49, 151–174 (2009).

    Article  CAS  Google Scholar 

  16. Bushong, E.A., Martone, M.E., Jones, Y.Z. & Ellisman, M.H. Protoplasmic astrocytes in CA1 stratum radiatum occupy separate anatomical domains. J. Neurosci. 22, 183–192 (2002).

    Article  CAS  Google Scholar 

  17. Halassa, M.M., Fellin, T., Takano, H., Dong, J.H. & Haydon, P.G. Synaptic islands defined by the territory of a single astrocyte. J. Neurosci. 27, 6473–6477 (2007).

    Article  CAS  Google Scholar 

  18. Gordon, G.R. et al. Astrocyte-mediated distributed plasticity at hypothalamic glutamate synapses. Neuron 64, 391–403 (2009).

    Article  CAS  Google Scholar 

  19. Porter, J.T. & McCarthy, K.D. Hippocampal astrocytes in situ respond to glutamate released from synaptic terminals. J. Neurosci. 16, 5073–5081 (1996).

    Article  CAS  Google Scholar 

  20. Parpura, V. et al. Glutamate-mediated astrocyte-neuron signaling. Nature 369, 744–747 (1994).

    Article  CAS  Google Scholar 

  21. Pasti, L., Volterra, A., Pozzan, T. & Carmignoto, G. Intracellular calcium oscillations in astrocytes: a highly plastic, bidirectional form of communication between neurons and astrocytes in situ. J. Neurosci. 17, 7817–7830 (1997).

    Article  CAS  Google Scholar 

  22. Perea, G., Navarrete, M. & Araque, A. Tripartite synapses: astrocytes process and control synaptic information. Trends Neurosci. 32, 421–431 (2009).

    Article  CAS  Google Scholar 

  23. Lee, C.J. et al. Astrocytic control of synaptic NMDA receptors. J. Physiol. (Lond.) 581, 1057–1081 (2007).

    Article  CAS  Google Scholar 

  24. Fellin, T. et al. Neuronal synchrony mediated by astrocytic glutamate through activation of extrasynaptic NMDA receptors. Neuron 43, 729–743 (2004).

    Article  CAS  Google Scholar 

  25. Fiacco, T.A. et al. Selective stimulation of astrocyte calcium in situ does not affect neuronal excitatory synaptic activity. Neuron 54, 611–626 (2007).

    Article  CAS  Google Scholar 

  26. Petravicz, J., Fiacco, T.A. & McCarthy, K.D. Loss of IP3 receptor–dependent Ca2+ increases in hippocampal astrocytes does not affect baseline CA1 pyramidal neuron synaptic activity. J. Neurosci. 28, 4967–4973 (2008).

    Article  CAS  Google Scholar 

  27. Tritsch, N.X. & Bergles, D.E. Defining the role of astrocytes in neuromodulation. Neuron 54, 497–500 (2007).

    Article  CAS  Google Scholar 

  28. Barres, B.A. The mystery and magic of glia: a perspective on their roles in health and disease. Neuron 60, 430–440 (2008).

    Article  CAS  Google Scholar 

  29. Agulhon, C. et al. What is the role of astrocyte calcium in neurophysiology? Neuron 59, 932–946 (2008).

    Article  CAS  Google Scholar 

  30. Lee, S.Y. & Haydon, P.G. Astrocytic glutamate targets NMDA receptors. J. Physiol. (Lond.) 581, 887–888 (2007).

    Article  CAS  Google Scholar 

  31. Shigetomi, E., Bowser, D.N., Sofroniew, M.V. & Khakh, B.S. Two forms of astrocyte calcium excitability have distinct effects on NMDA receptor-mediated slow inward currents in pyramidal neurons. J. Neurosci. 28, 6659–6663 (2008).

    Article  CAS  Google Scholar 

  32. Nett, W.J., Oloff, S.H. & McCarthy, K.D. Hippocampal astrocytes in situ exhibit calcium oscillations that occur independent of neuronal activity. J. Neurophysiol. 87, 528–537 (2002).

    Article  Google Scholar 

  33. Grosche, J. et al. Microdomains for neuron-glia interaction: parallel fiber signaling to Bergmann glial cells. Nat. Neurosci. 2, 139–143 (1999).

    Article  CAS  Google Scholar 

  34. Bush, T.G. et al. Fulminant jejuno-ileitis following ablation of enteric glia in adult transgenic mice. Cell 93, 189–201 (1998).

    Article  CAS  Google Scholar 

  35. Pascual, O. et al. Astrocytic purinergic signaling coordinates synaptic networks. Science 310, 113–116 (2005).

    Article  CAS  Google Scholar 

  36. Jaiswal, J.K. & Simon, S.M. Imaging single events at the cell membrane. Nat. Chem. Biol. 3, 92–98 (2007).

    Article  CAS  Google Scholar 

  37. Parpura, V. & Haydon, P.G. Physiological astrocytic calcium levels stimulate glutamate release to modulate adjacent neurons. Proc. Natl. Acad. Sci. USA 97, 8629–8634 (2000).

    Article  CAS  Google Scholar 

  38. Nakai, J., Ohkura, M. & Imoto, K. A high signal-to-noise Ca2+ probe composed of a single green fluorescent protein. Nat. Biotechnol. 19, 137–141 (2001).

    Article  CAS  Google Scholar 

  39. Tallini, Y.N. et al. 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 (2006).

    Article  CAS  Google Scholar 

  40. Mao, T., O' Connor, D.H., Scheuss, V., Nakai, J. & Svoboda, K. Characterization and subcellular targeting of GCaMP-type genetically encoded calcium indicators. PLoS One 3, e1796 (2008).

    Article  Google Scholar 

  41. Zlatkine, P., Mehul, B. & Magee, A.I. Retargeting of cytosolic proteins to the plasma membrane by the Lck protein tyrosine kinase dual acylation motif. J. Cell Sci. 110, 673–679 (1997).

    CAS  PubMed  Google Scholar 

  42. Benediktsson, A.M., Schachtele, S.J., Green, S.H. & Dailey, M.E. Ballistic labeling and dynamic imaging of astrocytes in organotypic hippocampal slice cultures. J. Neurosci. Methods 141, 41–53 (2005).

    Article  Google Scholar 

  43. Tian, L. et al. Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators. Nat. Methods 6, 875–881 (2009).

    Article  CAS  Google Scholar 

  44. Shigetomi, E. & Khakh, B.S. Measuring near plasma membrane and global intracellular calcium dynamics in astrocytes. J. Vis. Exp. 26, 10.3791/1142 (2009).

    Google Scholar 

  45. Richler, E., Chaumont, S., Shigetomi, E., Sagasti, A. & Khakh, B.S. An approach to image activation of transmitter-gated P2X receptors in vitro and in vivo. Nat. Methods 5, 87–93 (2008).

    Article  CAS  Google Scholar 

  46. Goldman, J.E. & Abramson, B. Cyclic AMP-induced shape changes of astrocytes are accompanied by rapid depolymerization of actin. Brain Res. 528, 189–196 (1990).

    Article  CAS  Google Scholar 

  47. Oberheim, N.A. et al. Loss of astrocytic domain organization in the epileptic brain. J. Neurosci. 28, 3264–3276 (2008).

    Article  CAS  Google Scholar 

  48. Cahoy, J.D. et al. A transcriptome database for astrocytes, neurons, and oligodendrocytes: a new resource for understanding brain development and function. J. Neurosci. 28, 264–278 (2008).

    Article  CAS  Google Scholar 

  49. Atkin, S.D. et al. 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 (2009).

    Article  CAS  Google Scholar 

  50. Reyes, R.C. & Parpura, V. The trinity of Ca2+ sources for the exocytotic glutamate release from astrocytes. Neurochem. Int. 55, 2–8 (2009).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank L. Looger for a preprint of a manuscript on GCaMP3, A. Sagasti for the GCaMP2 plasmid, D.E. Bergles for insightful discussions and for the Lck domain plasmid, and M. Simon for MrgA1-EGFP. Our work was supported by the National Institutes of Health (NS060677 and NS057624), the Whitehall Foundation, a S&R Foundation Ryuji Ueno Award for Ion Channels or Barrier Function Research and a Stein-Oppenheimer Foundation Endowment Award (B.S.K.). E.S. was partly supported by the Uehara Memorial Foundation Fellowship of Japan.

Author information

Authors and Affiliations

Authors

Contributions

E.S. carried out all of the imaging experiments. S.K. carried out all of the cloning experiments. All of the authors contributed to the writing of the manuscript. E.S. and B.S.K. constructed the figures. B.S.K. directed the research with feedback from M.V.S.

Corresponding author

Correspondence to Baljit S Khakh.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8 and Supplementary Table 1 (PDF 1262 kb)

Supplementary Video 1

Movie of two astrocytes expressing cyotsolic GCaMP2. (AVI 7511 kb)

Supplementary Video 2

Movie of astrocytes challenged with ATP. (AVI 2256 kb)

Supplementary Video 3

Movie of an astrocyte expressing Lck-GCaMP2. (AVI 8066 kb)

Supplementary Video 4

Original movie of the astrocyte shown in Supplementary Video 3. (AVI 8066 kb)

Supplementary Video 5

Movie of an astrocyte expressing Lck-GCaMP2 imaged with EPI microscopy. (AVI 16621 kb)

Supplementary Video 6

Movie of an astrocyte expressing Lck-GFP imaged with EPI microscopy. (AVI 16621 kb)

Supplementary Video 7

Movie of a neuron expressing Lck-GCaMP2 imaged with EPI microscopy. (AVI 9503 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Shigetomi, E., Kracun, S., Sofroniew, M. et al. A genetically targeted optical sensor to monitor calcium signals in astrocyte processes. Nat Neurosci 13, 759–766 (2010). https://doi.org/10.1038/nn.2557

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nn.2557

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing