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