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.

  • Article
  • Published:

TRPA1 channels regulate astrocyte resting calcium and inhibitory synapse efficacy through GAT-3

Abstract

Astrocytes contribute to the formation and function of synapses and are found throughout the brain, where they show intracellular store–mediated Ca2+ signals. Here, using a membrane-tethered, genetically encoded calcium indicator (Lck-GCaMP3), we report the serendipitous discovery of a new type of Ca2+ signal in rat hippocampal astrocyte-neuron cocultures. We found that Ca2+ fluxes mediated by transient receptor potential A1 (TRPA1) channels gave rise to frequent and highly localized 'spotty' Ca2+ microdomains near the membrane that contributed appreciably to resting Ca2+ in astrocytes. Mechanistic evaluations in brain slices showed that decreases in astrocyte resting Ca2+ concentrations mediated by TRPA1 channels decreased interneuron inhibitory synapse efficacy by reducing GABA transport by GAT-3, thus elevating extracellular GABA. Our data show how a transmembrane Ca2+ source (TRPA1) targets a transporter (GAT-3) in astrocytes to regulate inhibitory synapses.

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: Spotty Ca2+ signals in rat hippocampal astrocyte-neuron cocultures.
Figure 2: Evidence that TRPA1 channels mediate spotty Ca2+ signals in cocultures.
Figure 3: TRPA1 channels regulate basal Ca2+ in cocultures and astrocytes in slices.
Figure 4: Buffering astrocyte intracellular Ca2+ levels decreases mIPSC amplitudes in interneurons but not pyramidal neurons in hippocampal slices.
Figure 5: Effect of the TRPA1 channel blocker (HC 030031) on mIPSCs arriving onto pyramidal neurons and interneurons in the hippocampus.
Figure 6: Role of GAT-3 GABA transporters.
Figure 7: Astrocyte dialysis with BAPTA regulates GAT-3 in astrocytes.
Figure 8: The effects of HC 030031 and astrocyte BAPTA dialysis are abolished in the Trpa1−/− mice.

Similar content being viewed by others

References

  1. Halassa, M.M. & Haydon, P.G. Integrated brain circuits: astrocytic networks modulate neuronal activity and behavior. Annu. Rev. Physiol. 72, 335–355 (2010).

    Article  CAS  Google Scholar 

  2. Attwell, D. et al. Glial and neuronal control of brain blood flow. Nature 468, 232–243 (2010).

    Article  CAS  Google Scholar 

  3. Henneberger, C., Papouin, T., Oliet, S.H. & Rusakov, D.A. Long-term potentiation depends on release of D-serine from astrocytes. Nature 463, 232–236 (2010).

    Article  CAS  Google Scholar 

  4. Agulhon, C., Fiacco, T.A. & McCarthy, K.D. Hippocampal short- and long-term plasticity are not modulated by astrocyte Ca2+ signaling. Science 327, 1250–1254 (2010).

    Article  CAS  Google Scholar 

  5. Sontheimer, H. Voltage-dependent ion channels in glial cells. Glia 11, 156–172 (1994).

    Article  CAS  Google Scholar 

  6. Bomben, V.C., Turner, K.L., Barclay, T.T. & Sontheimer, H. Transient receptor potential canonical channels are essential for chemotactic migration of human malignant gliomas. J. Cell. Physiol. 226, 1879–1888 (2011).

    Article  CAS  Google Scholar 

  7. Hires, S.A., Tian, L. & Looger, L.L. Reporting neural activity with genetically encoded calcium indicators. Brain Cell Biol. 36, 69–86 (2008).

    Article  CAS  Google Scholar 

  8. 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 

  9. Shigetomi, E., Kracun, S. & Khakh, B.S. Monitoring astrocyte calcium microdomains with improved membrane targeted GCaMP reporters. Neuron Glia Biol. 6, 183–191 (2010).

    Article  Google Scholar 

  10. Shigetomi, E., Kracun, S., Sofroniew, M.V. & Khakh, B.S. A genetically targeted optical sensor to monitor calcium signals in astrocyte processes. Nat. Neurosci. 13, 759–766 (2010).

    Article  CAS  Google Scholar 

  11. 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 

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

    Google Scholar 

  13. Clapham, D.E. Transient receptor potential (TRP) channels. in Encyclopedia of Neuroscience Vol. 9 (ed. Squire, L.R.) 1109–1133 (Academic, Oxford, UK, 2009).

    Chapter  Google Scholar 

  14. McNamara, C.R. et al. TRPA1 mediates formalin-induced pain. Proc. Natl. Acad. Sci. USA 104, 13525–13530 (2007).

    Article  CAS  Google Scholar 

  15. Jordt, S.E. et al. Mustard oils and cannabinoids excite sensory nerve fibres through the TRP channel ANKTM1. Nature 427, 260–265 (2004).

    Article  CAS  Google Scholar 

  16. Bandell, M. et al. Noxious cold ion channel TRPA1 is activated by pungent compounds and bradykinin. Neuron 41, 849–857 (2004).

    Article  CAS  Google Scholar 

  17. Karashima, Y. et al. Bimodal action of menthol on the transient receptor potential channel TRPA1. J. Neurosci. 27, 9874–9884 (2007).

    Article  CAS  Google Scholar 

  18. 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 

  19. Xiang, Y. et al. Light-avoidance-mediating photoreceptors tile the Drosophila larval body wall. Nature 468, 921–926 (2010).

    Article  CAS  Google Scholar 

  20. García-Añoveros, J. & Nagata, K. TRPA1. Handb. Exp. Pharmacol. 179, 347–362 (2007).

    Article  Google Scholar 

  21. Wu, L.J., Sweet, T.B. & Clapham, D.E. International Union of Basic and Clinical Pharmacology. LXXVI. Current progress in the mammalian TRP ion channel family. Pharmacol. Rev. 62, 381–404 (2010).

    Article  CAS  Google Scholar 

  22. Reeves, A.M., Shigetomi, E. & Khakh, B.S. Bulk loading of calcium indicator dyes to study astrocyte physiology: key limitations and improvements using morphological maps. J. Neurosci. 31, 9353–9358 (2011).

    Article  CAS  Google Scholar 

  23. Kuchibhotla, K.V., Lattarulo, C.R., Hyman, B.T. & Bacskai, B.J. Synchronous hyperactivity and intercellular calcium waves in astrocytes in Alzheimer mice. Science 323, 1211–1215 (2009).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  25. Kwan, K.Y. et al. TRPA1 contributes to cold, mechanical, and chemical nociception but is not essential for hair-cell transduction. Neuron 50, 277–289 (2006).

    Article  CAS  Google Scholar 

  26. Soeller, C. & Cannell, M.B. Analysing cardiac excitation-contraction coupling with mathematical models of local control. Prog. Biophys. Mol. Biol. 85, 141–162 (2004).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  28. Overstreet, L.S., Jones, M.V. & Westbrook, G.L. Slow desensitization regulates the availability of synaptic GABAA receptors. J. Neurosci. 20, 7914–7921 (2000).

    Article  CAS  Google Scholar 

  29. Glykys, J. & Mody, I. Activation of GABAA receptors: views from outside the synaptic cleft. Neuron 56, 763–770 (2007).

    Article  CAS  Google Scholar 

  30. Lee, S. et al. Channel-mediated tonic GABA release from glia. Science 330, 790–796 (2010).

    Article  CAS  Google Scholar 

  31. Ribak, C.E., Tong, W.M. & Brecha, N.C. GABA plasma membrane transporters, GAT-1 and GAT-3, display different distributions in the rat hippocampus. J. Comp. Neurol. 367, 595–606 (1996).

    Article  CAS  Google Scholar 

  32. Chiu, C.S. et al. Number, density, and surface/cytoplasmic distribution of GABA transporters at presynaptic structures of knock-in mice carrying GABA transporter subtype 1-green fluorescent protein fusions. J. Neurosci. 22, 10251–10266 (2002).

    Article  CAS  Google Scholar 

  33. Deken, S.L., Wang, D. & Quick, M.W. Plasma membrane GABA transporters reside on distinct vesicles and undergo rapid regulated recycling. J. Neurosci. 23, 1563–1568 (2003).

    Article  CAS  Google Scholar 

  34. Wang, D. & Quick, M.W. Trafficking of the plasma membrane gamma-aminobutyric acid transporter GAT1. Size and rates of an acutely recycling pool. J. Biol. Chem. 280, 18703–18709 (2005).

    Article  CAS  Google Scholar 

  35. Macia, E. et al. Dynasore, a cell-permeable inhibitor of dynamin. Dev. Cell 10, 839–850 (2006).

    Article  CAS  Google Scholar 

  36. 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 

  37. Nilius, B., Prenen, J. & Owsianik, G. Irritating channels: the case of TRPA1. J. Physiol. (Lond.) 589, 1543–1549 (2011).

    Article  CAS  Google Scholar 

  38. Chen, J. et al. Pore dilation occurs in TRPA1 but not in TRPM8 channels. Mol. Pain 5, 3 (2009).

    PubMed  PubMed Central  Google Scholar 

  39. Zurborg, S., Yurgionas, B., Jira, J.A., Caspani, O. & Heppenstall, P.A. Direct activation of the ion channel TRPA1 by Ca2+. Nat. Neurosci. 10, 277–279 (2007).

    Article  CAS  Google Scholar 

  40. Wang, Y.Y., Chang, R.B., Waters, H.N., McKemy, D.D. & Liman, E.R. The nociceptor ion channel TRPA1 is potentiated and inactivated by permeating calcium ions. J. Biol. Chem. 283, 32691–32703 (2008).

    Article  CAS  Google Scholar 

  41. Xu, H., Delling, M., Jun, J.C. & Clapham, D.E. Oregano, thyme and clove-derived flavors and skin sensitizers activate specific TRP channels. Nat. Neurosci. 9, 628–635 (2006).

    Article  CAS  Google Scholar 

  42. Golovina, V.A. Visualization of localized store-operated calcium entry in mouse astrocytes. Close proximity to the endoplasmic reticulum. J. Physiol. (Lond.) 564, 737–749 (2005).

    Article  CAS  Google Scholar 

  43. Grimaldi, M., Maratos, M. & Verma, A. Transient receptor potential channel activation causes a novel form of [Ca2+]I oscillations and is not involved in capacitative Ca2+ entry in glial cells. J. Neurosci. 23, 4737–4745 (2003).

    Article  CAS  Google Scholar 

  44. Malarkey, E.B., Ni, Y. & Parpura, V. Ca2+ entry through TRPC1 channels contributes to intracellular Ca2+ dynamics and consequent glutamate release from rat astrocytes. Glia 56, 821–835 (2008).

    Article  Google Scholar 

  45. Shirakawa, H. et al. Transient receptor potential canonical 3 (TRPC3) mediates thrombin-induced astrocyte activation and upregulates its own expression in cortical astrocytes. J. Neurosci. 30, 13116–13129 (2010).

    Article  CAS  Google Scholar 

  46. Everaerts, W. et al. The capsaicin receptor TRPV1 is a crucial mediator of the noxious effects of mustard oil. Curr. Biol. 21, 316–321 (2011).

    Article  CAS  Google Scholar 

  47. Song, I., Savtchenko, L. & Semyanov, A. Tonic excitation or inhibition is set by GABAA conductance in hippocampal interneurons. Nat. Commun. 2, 376 (2011).

    Article  Google Scholar 

  48. Ortinski, P.I. et al. Selective induction of astrocytic gliosis generates deficits in neuronal inhibition. Nat. Neurosci. 13, 584–591 (2010).

    Article  CAS  Google Scholar 

  49. Moss, F.J. et al. GABA transporter function, oligomerization state, and anchoring: correlates with subcellularly resolved FRET. J. Gen. Physiol. 134, 489–521 (2009).

    Article  CAS  Google Scholar 

  50. Hill, K. & Schaefer, M. Ultraviolet light and photosensitising agents activate TRPA1 via generation of oxidative stress. Cell Calcium 45, 155–164 (2009).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors are indebted to T.J. O'Dell and M.V. Sofroniew for discussions during the course of these experiments. The authors are grateful to E. Toulme for assistance with western blots, to M. Hamby for tips on astrocyte-enriched cultures and to S. Kracun for molecular biology help and discussions. Thanks to M. Nedergaard for discussions during the early stages of this project. Special thanks to members of the Astrocyte Biology and Biophysics Affinity Group at UCLA for discussions. Thanks to A. Patapoutian (mouse TRPA1; The Scripps Research Institute), D. Julius (rat TRPA1; University of California San Francisco) and Y. Gwack (mCherry; University of California Los Angeles) for sharing plasmids. Thanks to N. Brecha (University of California Los Angeles) for GAT-1 and GAT-3 antibodies. Our work was supported mainly by US National Institutes of Health National Institute of Neurological Disorders and Stroke grant NS060677 and partly by grants NS071292 and NS063186, the Whitehall Foundation and a Stein-Oppenheimer Endowment Award (to B.S.K.).

Author information

Authors and Affiliations

Authors

Contributions

E.S. and X.T. carried out the experiments with guidance from B.S.K. B.S.K. directed the research project. K.Y.K. and D.P.C. generated the knockout mice. B.S.K., E.S. and X.T. generated the figures. B.S.K. wrote the paper and all authors contributed to the final version.

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 1–15, Supplementary Tables 1–3 (PDF 2263 kb)

Supplementary Video 1

Spotty Ca2+ signals in a representative astrocyte in cocultures (AVI 55382 kb)

Supplementary Video 2

Spotty Ca2+ signals are abolished upon application of Ca2+-free buffers in astrocytes in cocultures (AVI 64171 kb)

Supplementary Video 3

HC 030031, a specific blocker of TRPA1 channels, largely reduces spotty Ca2+ signals in astrocytes in cocultures (AVI 97534 kb)

Supplementary Video 4

Spotty Ca2+ signals are preserved in astrocytes in cocultures transfected with control siRNA (AVI 22160 kb)

Supplementary Video 5

Spotty Ca2+ signals are reduced in astrocytes in cocultures transfected with TRPA1 siRNA (AVI 22160 kb)

Supplementary Video 6

A low concentration of AITC increases astrocyte spotty Ca2+ signals in cocultures (AVI 22160 kb)

Supplementary Video 7

Spotty Ca2+ signals in control astrocytes in cocultures (AVI 18468 kb)

Supplementary Video 8

Overexpression of mouse TRPA1 channels increases spotty Ca2+ signals in astrocytes in cocultures (AVI 18468 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Shigetomi, E., Tong, X., Kwan, K. et al. TRPA1 channels regulate astrocyte resting calcium and inhibitory synapse efficacy through GAT-3. Nat Neurosci 15, 70–80 (2012). https://doi.org/10.1038/nn.3000

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nn.3000

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