Quantitative Analysis of Glutamate Receptors in Glial Cells from the Cortex of GFAP/EGFP Mice Following Ischemic Injury: Focus on NMDA Receptors

Abstract

Cortical glial cells contain both ionotropic and metabotropic glutamate receptors. Despite several efforts, a comprehensive analysis of the entire family of glutamate receptors and their subunits present in glial cells is still missing. Here, we provide an overall picture of the gene expression of ionotropic (AMPA, kainate, NMDA) and the main metabotropic glutamate receptors in cortical glial cells isolated from GFAP/EGFP mice before and after focal cerebral ischemia. Employing single-cell RT-qPCR, we detected the expression of genes encoding subunits of glutamate receptors in GFAP/EGFP-positive (GFAP/EGFP+) glial cells in the cortex of young adult mice. Most of the analyzed cells expressed mRNA for glutamate receptor subunits, the expression of which, in most cases, even increased after ischemic injury. Data analyses disclosed several classes of GFAP/EGFP+ glial cells with respect to glutamate receptors and revealed in what manner their expression correlates with the expression of glial markers prior to and after ischemia. Furthermore, we also examined the protein expression and functional significance of NMDA receptors in glial cells. Immunohistochemical analyses of all seven NMDA receptor subunits provided direct evidence that the GluN3A subunit is present in GFAP/EGFP+ glial cells and that its expression is increased after ischemia. In situ and in vitro Ca2+ imaging revealed that Ca2+ elevations evoked by the application of NMDA were diminished in GFAP/EGFP+ glial cells following ischemia. Our results provide a comprehensive description of glutamate receptors in cortical GFAP/EGFP+ glial cells and may serve as a basis for further research on glial cell physiology and pathophysiology.

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Acknowledgments

This study was supported by the Grants GA CR P304/12/G069 and GACR 13-02154S from the Grant Agency of the Czech Republic, GAUK 604212 from the Grant Agency of the Charles University in Prague, CZ.1.05/1.1.00/02.0109 from the European Regional Development Fund, and CZ.1.07/2.3.00/30.0045 from the European Social Fund and the state budget of the Czech Republic.

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The authors declare that they have no conflict of interest.

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Correspondence to Miroslava Anderova.

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Online Resource 1

Complete list of Spearman correlation coefficients between genes coding glutamate receptor subunits and markers of NG2 glia/astrocytes in control mice (CTRL) and mice 7 and 14 days after MCAo (D7, D14). The correlation coefficients were calculated from all cells including those without gene expression

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Dzamba, D., Honsa, P., Valny, M. et al. Quantitative Analysis of Glutamate Receptors in Glial Cells from the Cortex of GFAP/EGFP Mice Following Ischemic Injury: Focus on NMDA Receptors. Cell Mol Neurobiol 35, 1187–1202 (2015). https://doi.org/10.1007/s10571-015-0212-8

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Keywords

  • Astrocytes
  • NG2 glia
  • MCAo
  • Single-cell RT-qPCR
  • Calcium imaging