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