Astrocytes perform control and regulatory functions in the central nervous system; heterogeneity among them is still a matter of debate due to limited knowledge of their gene expression profiles and functional diversity. To unravel astrocyte heterogeneity during postnatal development and after focal cerebral ischemia, we employed single-cell gene expression profiling in acutely isolated cortical GFAP/EGFP-positive cells. Using a microfluidic qPCR platform, we profiled 47 genes encoding glial markers and ion channels/transporters/receptors participating in maintaining K(+) and glutamate homeostasis per cell. Self-organizing maps and principal component analyses revealed three subpopulations within 10-50 days of postnatal development (P10-P50). The first subpopulation, mainly immature glia from P10, was characterized by high transcriptional activity of all studied genes, including polydendrocytic markers. The second subpopulation (mostly from P20) was characterized by low gene transcript levels, while the third subpopulation encompassed mature astrocytes (mainly from P30, P50). Within 14 days after ischemia (D3, D7, D14), additional astrocytic subpopulations were identified: resting glia (mostly from P50 and D3), transcriptionally active early reactive glia (mainly from D7) and permanent reactive glia (solely from D14). Following focal cerebral ischemia, reactive astrocytes underwent pronounced changes in the expression of aquaporins, nonspecific cationic and potassium channels, glutamate receptors and reactive astrocyte markers.

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

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Reichenbach WH (2005) Astrocytes and ependymal cells. In: Kettenmann H, Ransom BR, editor. Neuroglia. 2 ed. New York: Oxford University Press. 19–36.

Oberheim NA, Takano T, Han X, He W, Lin JH, et al. (2009) Uniquely hominid features of adult human astrocytes. The Journal of neuroscience : the official journal of the Society for Neuroscience 29: 3276–3287. PubMed PMC

Matyash V, Kettenmann H (2010) Heterogeneity in astrocyte morphology and physiology. Brain research reviews 63: 2–10. PubMed

Seifert G, Huttmann K, Binder DK, Hartmann C, Wyczynski A, et al. (2009) Analysis of astroglial K+ channel expression in the developing hippocampus reveals a predominant role of the Kir4.1 subunit. The Journal of neuroscience : the official journal of the Society for Neuroscience 29: 7474–7488. PubMed PMC

Benesova J, Rusnakova V, Honsa P, Pivonkova H, Dzamba D, 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. PubMed PMC

Ge WP, Miyawaki A, Gage FH, Jan YN, Jan LY (2012) Local generation of glia is a major astrocyte source in postnatal cortex. Nature 484: 376–380. PubMed PMC

Freeman MR (2010) Specification and morphogenesis of astrocytes. Science 330: 774–778. PubMed PMC

Zhang Y, Barres BA (2010) Astrocyte heterogeneity: an underappreciated topic in neurobiology. Current opinion in neurobiology 20: 588–594. PubMed

Trotter J, Karram K, Nishiyama A (2010) NG2 cells: Properties, progeny and origin. Brain research reviews 63: 72–82. PubMed PMC

Stallcup WB, Beasley L (1987) Bipotential glial precursor cells of the optic nerve express the NG2 proteoglycan. The Journal of neuroscience : the official journal of the Society for Neuroscience 7: 2737–2744. PubMed PMC

Alonso G (2005) NG2 proteoglycan-expressing cells of the adult rat brain: possible involvement in the formation of glial scar astrocytes following stab wound. Glia 49: 318–338. PubMed

Honsa P, Pivonkova H, Dzamba D, Filipova M, Anderova M (2012) Polydendrocytes display large lineage plasticity following focal cerebral ischemia. PLoS One 7: e36816. PubMed PMC

Robel S, Berninger B, Gotz M (2011) The stem cell potential of glia: lessons from reactive gliosis. Nature reviews Neuroscience 12: 88–104. PubMed

Zelenina M (2010) Regulation of brain aquaporins. Neurochemistry international 57: 468–488. PubMed

Hwang IK, Yoo KY, Li H, Lee BH, Suh HW, et al. (2007) Aquaporin 9 changes in pyramidal cells before and is expressed in astrocytes after delayed neuronal death in the ischemic hippocampal CA1 region of the gerbil. Journal of neuroscience research 85: 2470–2479. PubMed

McKeon RJ, Jurynec MJ, Buck CR (1999) The chondroitin sulfate proteoglycans neurocan and phosphacan are expressed by reactive astrocytes in the chronic CNS glial scar. The Journal of neuroscience : the official journal of the Society for Neuroscience 19: 10778–10788. PubMed PMC

Komitova M, Perfilieva E, Mattsson B, Eriksson PS, Johansson BB (2002) Effects of cortical ischemia and postischemic environmental enrichment on hippocampal cell genesis and differentiation in the adult rat. Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism 22: 852–860. PubMed

Ferraguti F, Corti C, Valerio E, Mion S, Xuereb J (2001) Activated astrocytes in areas of kainate-induced neuronal injury upregulate the expression of the metabotropic glutamate receptors 2/3 and 5. Experimental brain research Experimentelle Hirnforschung Experimentation cerebrale 137: 1–11. PubMed

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

Jacque CM, Vinner C, Kujas M, Raoul M, Racadot J, et al. (1978) Determination of glial fibrillary acidic protein (GFAP) in human brain tumors. Journal of the neurological sciences 35: 147–155. PubMed

Sofroniew MV, Vinters HV (2010) Astrocytes: biology and pathology. Acta neuropathologica 119: 7–35. PubMed PMC

Walz W (2000) Controversy surrounding the existence of discrete functional classes of astrocytes in adult gray matter. Glia 31: 95–103. PubMed

Regan MR, Huang YH, Kim YS, Dykes-Hoberg MI, Jin L, et al. (2007) Variations in promoter activity reveal a differential expression and physiology of glutamate transporters by glia in the developing and mature CNS. The Journal of neuroscience : the official journal of the Society for Neuroscience 27: 6607–6619. PubMed PMC

Mearow KM, Mill JF, Vitkovic L (1989) The ontogeny and localization of glutamine synthetase gene expression in rat brain. Brain research Molecular brain research 6: 223–232. PubMed

Cahoy JD, Emery B, Kaushal A, Foo LC, Zamanian JL, et al. (2008) A transcriptome database for astrocytes, neurons, and oligodendrocytes: a new resource for understanding brain development and function. The Journal of neuroscience : the official journal of the Society for Neuroscience 28: 264–278. PubMed PMC

Kimelberg HK (2004) The problem of astrocyte identity. Neurochemistry international 45: 191–202. PubMed

Klein CA, Zohlnhofer D, Petat-Dutter K, Wendler N (2005) Gene expression analysis of a single or few cells. Current protocols in human genetics/editorial board, Jonathan L Haines [et al] Chapter 11: Unit 11 18. PubMed

Ståhlberg A, Andersson D, Aurelius J, Faiz M, Pekna M, et al. (2011) Defining cell populations with single-cell gene expression profiling: correlations and identification of astrocyte subpopulations. Nucleic acids research 39: e24. PubMed PMC

Raj A, Peskin CS, Tranchina D, Vargas DY, Tyagi S (2006) Stochastic mRNA synthesis in mammalian cells. PLoS biology 4: e309. PubMed PMC

Raj A, van Oudenaarden A (2009) Single-molecule approaches to stochastic gene expression. Annual review of biophysics 38: 255–270. PubMed PMC

Bengtsson M, Hemberg M, Rorsman P, Stahlberg A (2008) Quantification of mRNA in single cells and modelling of RT-qPCR induced noise. BMC molecular biology 9: 63. PubMed PMC

Ståhlberg A, Rusnakova V, Forootan A, Anderova M, Kubista M (2013) RT-qPCR work-flow for single-cell data analysis. Methods 59: 80–88. PubMed

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

Nolte C, Matyash M, Pivneva T, Schipke CG, Ohlemeyer C, 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

Benesova J, Hock M, Butenko O, Prajerova I, Anderova M, et al. (2009) Quantification of astrocyte volume changes during ischemia in situ reveals two populations of astrocytes in the cortex of GFAP/EGFP mice. Journal of neuroscience research 87: 96–111. PubMed

Wahl-Schott C, Biel M (2009) HCN channels: structure, cellular regulation and physiological function. Cell Mol Life Sci 66: 470–494. PubMed PMC

Ståhlberg A, Rusnakova V, Kubista M (2013) The added value of single-cell gene expression profiling. Brief Funct Genomics. PubMed

Sindelka R, Ferjentsik Z, Jonak J (2006) Developmental expression profiles of Xenopus laevis reference genes. Dev Dyn 235: 754–758. PubMed

da Cunha A, Aloyo VJ, Vitkovic L (1991) Developmental regulation of GAP-43, glutamine synthetase and beta-actin mRNA in rat cortical astrocytes. Brain research Developmental brain research 64: 212–215. PubMed

Stanimirovic DB, Ball R, Small DL, Muruganandam A (1999) Developmental regulation of glutamate transporters and glutamine synthetase activity in astrocyte cultures differentiated in vitro. International journal of developmental neuroscience : the official journal of the International Society for Developmental Neuroscience 17: 173–184. PubMed

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. PubMed PMC

Ziak D, Chvatal A, Sykova E (1998) Glutamate-, kainate- and NMDA-evoked membrane currents in identified glial cells in rat spinal cord slice. Physiological research/Academia Scientiarum Bohemoslovaca 47: 365–375. PubMed

Matthias K, Kirchhoff F, Seifert G, Huttmann K, Matyash M, et al. (2003) Segregated expression of AMPA-type glutamate receptors and glutamate transporters defines distinct astrocyte populations in the mouse hippocampus. The Journal of neuroscience : the official journal of the Society for Neuroscience 23: 1750–1758. PubMed PMC

Butt AM, Hamilton N, Hubbard P, Pugh M, Ibrahim M (2005) Synantocytes: the fifth element. Journal of anatomy 207: 695–706. PubMed PMC

Zhou M (2006) Schools GP, Kimelberg HK (2006) Development of GLAST(+) astrocytes and NG2(+) glia in rat hippocampus CA1: mature astrocytes are electrophysiologically passive. Journal of neurophysiology 95: 134–143. PubMed

Nagelhus EA, Horio Y, Inanobe A, Fujita A, Haug FM, et al. (1999) Immunogold evidence suggests that coupling of K+ siphoning and water transport in rat retinal Muller cells is mediated by a coenrichment of Kir4.1 and AQP4 in specific membrane domains. Glia 26: 47–54. PubMed

Connors NC, Adams ME, Froehner SC, Kofuji P (2004) The potassium channel Kir4.1 associates with the dystrophin-glycoprotein complex via alpha-syntrophin in glia. The Journal of biological chemistry 279: 28387–28392. PubMed

Liu H, Yang M, Qiu GP, Zhuo F, Yu WH, et al. (2012) Aquaporin 9 in rat brain after severe traumatic brain injury. Arquivos de neuro-psiquiatria 70: 214–220. PubMed

Sun W, McConnell E, Pare JF, Xu Q, Chen M, et al. (2013) Glutamate-dependent neuroglial calcium signaling differs between young and adult brain. Science 339: 197–200. PubMed PMC

Lyon L, Kew JN, Corti C, Harrison PJ, Burnet PW (2008) Altered hippocampal expression of glutamate receptors and transporters in GRM2 and GRM3 knockout mice. Synapse 62: 842–850. PubMed PMC

Masood K, Besnard F, Su Y, Brenner M (1993) Analysis of a segment of the human glial fibrillary acidic protein gene that directs astrocyte-specific transcription. J Neurochem 61: 160–166. PubMed

Pekny M, Nilsson M (2005) Astrocyte activation and reactive gliosis. Glia 50: 427–434. PubMed

Zamanian JL, Xu L, Foo LC, Nouri N, Zhou L, et al. (2012) Genomic analysis of reactive astrogliosis. The Journal of neuroscience : the official journal of the Society for Neuroscience 32: 6391–6410. PubMed PMC

Hoshi A, Yamamoto T, Shimizu K, Ugawa Y, Nishizawa M, et al. (2012) Characteristics of aquaporin expression surrounding senile plaques and cerebral amyloid angiopathy in Alzheimer disease. J Neuropathol Exp Neurol 71: 750–759. PubMed

McCoy E, Sontheimer H (2010) MAPK induces AQP1 expression in astrocytes following injury. Glia 58: 209–217. PubMed PMC

Rodriguez A, Perez-Gracia E, Espinosa JC, Pumarola M, Torres JM, et al. (2006) Increased expression of water channel aquaporin 1 and aquaporin 4 in Creutzfeldt-Jakob disease and in bovine spongiform encephalopathy-infected bovine-PrP transgenic mice. Acta Neuropathol 112: 573–585. PubMed

Badaut J, Petit JM, Brunet JF, Magistretti PJ, Charriaut-Marlangue C, et al. (2004) Distribution of Aquaporin 9 in the adult rat brain: preferential expression in catecholaminergic neurons and in glial cells. Neuroscience 128: 27–38. PubMed

Badaut J, Regli L (2004) Distribution and possible roles of aquaporin 9 in the brain. Neuroscience 129: 971–981. PubMed

Hara-Chikuma M, Verkman AS (2006) Aquaporin-1 facilitates epithelial cell migration in kidney proximal tubule. J Am Soc Nephrol 17: 39–45. PubMed

McCoy E, Sontheimer H (2007) Expression and function of water channels (aquaporins) in migrating malignant astrocytes. Glia 55: 1034–1043. PubMed PMC

Papadopoulos MC, Verkman AS (2008) Potential utility of aquaporin modulators for therapy of brain disorders. Prog Brain Res 170: 589–601. PubMed PMC

Bezzi P, Carmignoto G, Pasti L, Vesce S, Rossi D, et al. (1998) Prostaglandins stimulate calcium-dependent glutamate release in astrocytes. Nature 391: 281–285. PubMed

Paquet M, Ribeiro FM, Guadagno J, Esseltine JL, Ferguson SS, et al. (2013) Role of metabotropic glutamate receptor 5 signaling and homer in oxygen glucose deprivation-mediated astrocyte apoptosis. Mol Brain 6: 9. PubMed PMC

Durand D, Carniglia L, Caruso C, Lasaga M (2011) Reduced cAMP, Akt activation and p65-c-Rel dimerization: mechanisms involved in the protective effects of mGluR3 agonists in cultured astrocytes. PLoS One 6: e22235. PubMed PMC

Aguado F, Espinosa-Parrilla JF, Carmona MA, Soriano E (2002) Neuronal activity regulates correlated network properties of spontaneous calcium transients in astrocytes in situ. J Neurosci 22: 9430–9444. PubMed PMC

Anderova M, Vorisek I, Pivonkova H, Benesova J, Vargova L, et al. (2011) Cell death/proliferation and alterations in glial morphology contribute to changes in diffusivity in the rat hippocampus after hypoxia-ischemia. J Cereb Blood Flow Metab 31: 894–907. PubMed PMC

Sik A, Smith RL, Freund TF (2000) Distribution of chloride channel-2-immunoreactive neuronal and astrocytic processes in the hippocampus. Neuroscience 101: 51–65. PubMed

Ransom CB, O'Neal JT, Sontheimer H (2001) Volume-activated chloride currents contribute to the resting conductance and invasive migration of human glioma cells. J Neurosci 21: 7674–7683. PubMed PMC

Butenko O, Dzamba D, Benesova J, Honsa P, Benfenati V, et al. (2012) The increased activity of TRPV4 channel in the astrocytes of the adult rat hippocampus after cerebral hypoxia/ischemia. PLoS One 7: e39959. PubMed PMC

Benfenati V, Amiry-Moghaddam M, Caprini M, Mylonakou MN, Rapisarda C, et al. (2007) Expression and functional characterization of transient receptor potential vanilloid-related channel 4 (TRPV4) in rat cortical astrocytes. Neuroscience 148: 876–892. PubMed

Pamenter ME, Perkins GA, McGinness AK, Gu XQ, Ellisman MH, et al. (2012) Autophagy and apoptosis are differentially induced in neurons and astrocytes treated with an in vitro mimic of the ischemic penumbra. PLoS One 7: e51469. PubMed PMC

Neitz A, Mergia E, Eysel UT, Koesling D, Mittmann T (2011) Presynaptic nitric oxide/cGMP facilitates glutamate release via hyperpolarization-activated cyclic nucleotide-gated channels in the hippocampus. Eur J Neurosci 33: 1611–1621. PubMed

Zhao JW, Raha-Chowdhury R, Fawcett JW, Watts C (2009) Astrocytes and oligodendrocytes can be generated from NG2+ progenitors after acute brain injury: intracellular localization of oligodendrocyte transcription factor 2 is associated with their fate choice. The European journal of neuroscience 29: 1853–1869. PubMed

Komitova M, Serwanski DR, Lu QR, Nishiyama A (2011) NG2 cells are not a major source of reactive astrocytes after neocortical stab wound injury. Glia 59: 800–809. PubMed PMC

Patanow CM, Day JR, Billingsley ML (1997) Alterations in hippocampal expression of SNAP-25, GAP-43, stannin and glial fibrillary acidic protein following mechanical and trimethyltin-induced injury in the rat. Neuroscience 76: 187–202. PubMed

Hepp R, Perraut M, Chasserot-Golaz S, Galli T, Aunis D, et al. (1999) Cultured glial cells express the SNAP-25 analogue SNAP-23. Glia 27: 181–187. PubMed

Illarionova NB, Gunnarson E, Li Y, Brismar H, Bondar A, et al. (2010) Functional and molecular interactions between aquaporins and Na,K-ATPase. Neuroscience 168: 915–925. PubMed

Anderova M, Antonova T, Petrik D, Neprasova H, Chvatal A, et al. (2004) Voltage-dependent potassium currents in hypertrophied rat astrocytes after a cortical stab wound. Glia 48: 311–326. PubMed

Bordey A, Lyons SA, Hablitz JJ, Sontheimer H (2001) Electrophysiological characteristics of reactive astrocytes in experimental cortical dysplasia. Journal of neurophysiology 85: 1719–1731. PubMed

Arimura K, Ago T, Kamouchi M, Nakamura K, Ishitsuka K, et al. (2012) PDGF receptor beta signaling in pericytes following ischemic brain injury. Curr Neurovasc Res 9: 1–9. PubMed

Zhu X, Zuo H, Maher BJ, Serwanski DR, LoTurco JJ, et al. (2012) Olig2-dependent developmental fate switch of NG2 cells. Development 139: 2299–2307. PubMed PMC

Marshall CA, Suzuki SO, Goldman JE (2003) Gliogenic and neurogenic progenitors of the subventricular zone: who are they, where did they come from, and where are they going? Glia 43: 52–61. PubMed

Gorski JA, Talley T, Qiu M, Puelles L, Rubenstein JL, et al. (2002) Cortical excitatory neurons and glia, but not GABAergic neurons, are produced in the Emx1-expressing lineage. J Neurosci 22: 6309–6314. PubMed PMC

Machon O, van den Bout CJ, Backman M, Rosok O, Caubit X, et al. (2002) Forebrain-specific promoter/enhancer D6 derived from the mouse Dach1 gene controls expression in neural stem cells. Neuroscience 112: 951–966. PubMed

Prajerova I, Honsa P, Chvatal A, Anderova M (2010) Neural stem/progenitor cells derived from the embryonic dorsal telencephalon of D6/GFP mice differentiate primarily into neurons after transplantation into a cortical lesion. Cell Mol Neurobiol 30: 199–218. PubMed

Honsa P, Pivonkova H, Anderova M (2013) Focal cerebral ischemia induces the neurogenic potential of mouse Dach1-expressing cells in the dorsal part of the lateral ventricles. Neuroscience. PubMed

Benesova J, Hock M, Butenko O, Prajerova I, Anderova M, et al. (2009) Quantification of astrocyte volume changes during ischemia in situ reveals two populations of astrocytes in the cortex of GFAP/EGFP mice. J Neurosci Res 87: 96–111. PubMed

Walz W, Lang MK (1998) Immunocytochemical evidence for a distinct GFAP-negative subpopulation of astrocytes in the adult rat hippocampus. Neurosci Lett. 257(3): 127–30. PubMed

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