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 Ca(2+) imaging revealed that Ca(2+) 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.

2nd Faculty of Medicine Charles University Prague Czech Republic

Department of Cellular Neurophysiology Institute of Experimental Medicine Academy of Sciences of the Czech Republic Videnska 1083 Prague 4 Czech Republic

Laboratory of Gene Expression Institute of Biotechnology Academy of Sciences of the Czech Republic Prague Czech Republic

Zobrazit více v PubMed

Aronica E, van Vliet EA, Mayboroda OA et al (2000) Upregulation of metabotropic glutamate receptor subtype mGluR3 and mGluR5 in reactive astrocytes in a rat model of mesial temporal lobe epilepsy. Eur J Neurosci 12:2333–2344 PubMed DOI

Aronica E, Leenstra S, Jansen GH et al (2001) Expression of brain-derived neurotrophic factor and tyrosine kinase B receptor proteins in glioneuronal tumors from patients with intractable epilepsy: colocalization with N-methyl-D-aspartic acid receptor. Acta Neuropathol 101:383–392 PubMed

Aronica E, Gorter JA, Ijlst-Keizers H et al (2003) Expression and functional role of mGluR3 and mGluR5 in human astrocytes and glioma cells: opposite regulation of glutamate transporter proteins. Eur J Neurosci 17:2106–2118. doi:10.1046/j.1460-9568.2003.02657.x PubMed DOI

Atlason PT, Garside ML, Meddows E et al (2007) N-Methyl-D-aspartate (NMDA) receptor subunit NR1 forms the substrate for oligomeric assembly of the NMDA receptor. J Biol Chem 282:25299–25307. doi:10.1074/jbc.M702778200 PubMed DOI

Benesova J, Rusnakova V, Honsa P et al (2012) Distinct expression/function of potassium and chloride channels contributes to the diverse volume regulation in cortical astrocytes of GFAP/EGFP mice. PLoS One 7:e29725. doi:10.1371/journal.pone.0029725 PubMed DOI PMC

Brand-Schieber E, Lowery SL, Werner P (2004) Select ionotropic glutamate AMPA/kainate receptors are expressed at the astrocyte-vessel interface. Brain Res 1007:178–182. doi:10.1016/j.brainres.2003.12.051 PubMed DOI

Burnashev N, Khodorova A, Jonas P et al (1992) Calcium-permeable AMPA-kainate receptors in fusiform cerebellar glial cells. Science 256:1566–1570 PubMed DOI

Burnashev N, Zhou Z, Neher E, Sakmann B (1995) Fractional calcium currents through recombinant GluR channels of the NMDA, AMPA and kainate receptor subtypes. J Physiol 485(Pt 2):403–418 PubMed DOI PMC

Cahoy JD, Emery B, Kaushal A et al (2008) A transcriptome database for astrocytes, neurons, and oligodendrocytes: a new resource for understanding brain development and function. J Neurosci 28:264–278. doi:10.1523/JNEUROSCI.4178-07.2008 PubMed DOI PMC

Castillo C, Norcini M, Martin Hernandez LA et al (2013) Satellite glia cells in dorsal root ganglia express functional NMDA receptors. Neuroscience 240:135–146. doi:10.1016/j.neuroscience.2013.02.031 PubMed DOI

Cavara NA, Hollmann M (2008) Shuffling the deck anew: how NR3 tweaks NMDA receptor function. Mol Neurobiol 38:16–26. doi:10.1007/s12035-008-8029-9 PubMed DOI

Chowdhury D, Marco S, Brooks IM et al (2013) Tyrosine phosphorylation regulates the endocytosis and surface expression of GluN3A-containing NMDA receptors. J Neurosci 33(9):4151–4164 PubMed DOI PMC

Conti F, DeBiasi S, Minelli A, Melone M (1996) Expression of NR1 and NR2A/B subunits of the NMDA receptor in cortical astrocytes. Glia 17:254–258. doi:10.1002/(SICI)1098-1136(199607)17:3%3c254:AID-GLIA7%3e3.0.CO;2-0 PubMed DOI

D’Antoni S, Berretta A, Bonaccorso CM et al (2008) Metabotropic glutamate receptors in glial cells. Neurochem Res 33:2436–2443. doi:10.1007/s11064-008-9694-9 PubMed DOI

Dzamba D, Honsa P, Anderova M (2013) NMDA receptors in glial cells: pending questions. Curr Neuropharmacol 11:250–262. doi:10.2174/1570159X11311030002 PubMed DOI PMC

Ehlers MD, Zhang S, Bernhadt JP, Huganir RL (1996) Inactivation of NMDA receptors by direct interaction of calmodulin with the NR1 subunit. Cell 84:745–755 PubMed DOI

Evans RC, Morera-Herreras T, Cui Y et al (2012) The effects of NMDA subunit composition on calcium influx and spike timing-dependent plasticity in striatal medium spiny neurons. PLoS Comput Biol 8:e1002493. doi:10.1371/journal.pcbi.1002493 PubMed DOI PMC

Gottlieb M, Matute C (1997) Expression of ionotropic glutamate receptor subunits in glial cells of the hippocampal CA1 area following transient forebrain ischemia. J Cereb Blood Flow Metab 17:290–300. doi:10.1097/00004647-199703000-00006 PubMed DOI

Haberlandt C, Derouiche A, Wyczynski A et al (2011) Gray matter NG2 cells display multiple Ca PubMed DOI PMC

Haydon P, Carmignoto G (2006) Astrocyte control of synaptic transmission and neurovascular coupling. Physiol Rev. doi:10.1152/physrev.00049.2005 PubMed

Hetman M, Kharebava G (2006) Survival signaling pathways activated by NMDA receptors. Curr Top Med Chem 6:787–799 PubMed DOI

Hollmann M, Hartley M, Heinemann S (1991) Ca PubMed DOI

Honsa P, Pivonkova H, Dzamba D et al (2012) Polydendrocytes display large lineage plasticity following focal cerebral ischemia. PLoS One 7:e36816. doi:10.1371/journal.pone.0036816 PubMed DOI PMC

Káradóttir R, Cavelier P, Bergersen L, Attwell D (2005) NMDA receptors are expressed in oligodendrocytes and activated in ischaemia. Nature 438:1162–1166. doi:10.1038/nature04302.NMDA PubMed DOI PMC

Kehoe LA, Bernardinelli Y, Muller D (2013) GluN3A: an NMDA receptor subunit with exquisite properties and functions. Neural Plast 2013:145387. doi:10.1155/2013/145387 PubMed PMC

Kotermanski SE, Johnson JW (2009) Mg PubMed DOI PMC

Krebs C, Fernandes HB, Sheldon C et al (2003) Functional NMDA receptor subtype 2B is expressed in astrocytes after ischemia in vivo and anoxia in vitro. J Neurosci 23:3364–3372 PubMed PMC

Kukley M, Dietrich D (2009) Kainate receptors and signal integration by NG2 glial cells. Neuron Glia Biol 5:13–20 PubMed DOI

Lalo U, Pankratov Y, Kirchhoff F et al (2006) NMDA receptors mediate neuron-to-glia signaling in mouse cortical astrocytes. J Neurosci 26:2673–2683. doi:10.1523/JNEUROSCI.4689-05.2006 PubMed DOI PMC

Lalo U, Palygin O, Rasooli-Nejad S et al (2014) Exocytosis of ATP from astrocytes modulates phasic and tonic inhibition in the neocortex. PLoS Biol 12:e1001747. doi:10.1371/journal.pbio.1001747 PubMed DOI PMC

Lavezzari G, McCallum J, Dewey CM et al (2004) Subunit-specific regulation of NMDA receptor endocytosis. J Neurosci 24(28):6383–6391 PubMed DOI PMC

Loane DJ, Stoica BA, Faden AI (2012) Metabotropic glutamate receptor-mediated signaling in neuroglia. Wiley Interdiscip Rev Membr Transp Signal 1:136–150. doi:10.1002/wmts.30 PubMed DOI PMC

Matsuda K, Fletcher M, Kamiya Y, Yuzaki M (2003) Specific assembly with the NMDA receptor 3B subunit controls surface expression and calcium permeability of NMDA receptors. J Neurosci 23:10064–10073 PubMed PMC

Matthias K, Kirchhoff F, Seifert G et al (2003) Segregated expression of AMPA-type glutamate receptors and glutamate transporters defines distinct astrocyte populations in the mouse hippocampus. J Neurosci 23:1750–1758 PubMed PMC

Nakahara K, Okada M, Nakanishi S (1997) The metabotropic glutamate receptor mGluR5 induces calcium oscillations in cultured astrocytes via protein kinase C phosphorylation. J Neurochem 69:1467–1475 PubMed DOI

Nolte C, Matyash M, Pivneva T et al (2001) GFAP promoter-controlled EGFP-expressing transgenic mice: a tool to visualize astrocytes and astrogliosis in living brain tissue. Glia 33:72–86 PubMed DOI

Palygin O, Lalo U, Pankratov Y (2011) Distinct pharmacological and functional properties of NMDA receptors in mouse cortical astrocytes. Br J Pharmacol 163:1755–1766. doi:10.1111/j.1476-5381.2011.01374.x PubMed DOI PMC

Paoletti P (2011) Molecular basis of NMDA receptor functional diversity. Eur J Neurosci 33:1351–1365. doi:10.1111/j.1460-9568.2011.07628.x PubMed DOI

Perez-Otano I, Schulteis CT, Contractor A et al (2001) Assembly with the NR1 subunit is required for surface expression of NR3A-containing NMDA receptors. J Neurosci 21:1228–1237 PubMed PMC

Porter JT, McCarthy KD (1995) GFAP-positive hippocampal astrocytes in situ respond to glutamatergic neuroligands with increases in [Ca PubMed DOI

Rusnakova V, Honsa P, Dzamba D et al (2013) Heterogeneity of astrocytes: from development to injury—single cell gene expression. PLoS One 8:e69734. doi:10.1371/journal.pone.0069734 PubMed DOI PMC

Sasaki YF, Rothe T, Premkumar LS et al (2002) Characterization and comparison of the NR3A subunit of the NMDA receptor in recombinant systems and primary cortical neurons. J Neurophysiol 87:2052–2063. doi:10.1152/jn.00531.2001 PubMed

Schipke CG, Ohlemeyer C, Matyash M et al (2001) Astrocytes of the mouse neocortex express functional N-methyl-D-aspartate receptors. FASEB J 15:1270–1272 PubMed

Seifert G, Steinhäuser C (2001) Ionotropic glutamate receptors in astrocytes. Prog Brain Res 132:287–299. doi:10.1016/S0079-6123(01)32083-6 PubMed DOI

Serrano A, Haddjeri N, Lacaille J-C, Robitaille R (2006) GABAergic network activation of glial cells underlies hippocampal heterosynaptic depression. J Neurosci 26:5370–5382. doi:10.1523/JNEUROSCI.5255-05.2006 PubMed DOI PMC

Serrano A, Robitaille R, Lacaille J-C (2008) Differential NMDA-dependent activation of glial cells in mouse hippocampus. Glia 56:1648–1663. doi:10.1002/glia.20717 PubMed DOI

Shigetomi E, Bushong EA, Haustein MD et al (2013) Imaging calcium microdomains within entire astrocyte territories and endfeet with GCaMPs expressed using adeno-associated viruses. J Gen Physiol 141:633–647. doi:10.1085/jgp.201210949 PubMed DOI PMC

Ståhlberg A, Bengtsson M (2010) Single-cell gene expression profiling using reverse transcription quantitative real-time PCR. Methods 50:282–288. doi:10.1016/j.ymeth.2010.01.002 PubMed DOI

Ståhlberg A, Andersson D, Aurelius J et al (2011) Defining cell populations with single-cell gene expression profiling: correlations and identification of astrocyte subpopulations. Nucleic Acids Res 39:e24. doi:10.1093/nar/gkq1182 PubMed DOI PMC

Sun W, McConnell E, Pare J-F et al (2013) Glutamate-dependent neuroglial calcium signaling differs between young and adult brain. Science 339:197–200. doi:10.1126/science.1226740 PubMed DOI PMC

Ulas J, Satou T, Ivins KJ et al (2000) Expression of metabotropic glutamate receptor 5 is increased in astrocytes after kainate-induced epileptic seizures. Glia 30:352–361 PubMed DOI

Verkhratsky A (2009) Neuronismo y reticulismo: neuronal-glial circuits unify the reticular and neuronal theories of brain organization. Acta Physiol (Oxf) 195:111–122. doi:10.1111/j.1748-1716.2008.01926.x PubMed DOI

Verkhratsky A, Kirchhoff F (2007) NMDA receptors in glia. Neuroscientist 13:28–37. doi:10.1177/1073858406294270 PubMed DOI

Vermeiren C, Najimi M, Vanhoutte N et al (2005) Acute up-regulation of glutamate uptake mediated by mGluR5a in reactive astrocytes. J Neurochem 94:405–416. doi:10.1111/j.1471-4159.2005.03216.x PubMed DOI

The correlation between expression profiles measured in single cells and in traditional bulk samples