Abstract
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.
Highlights
Astrocytes comprise a heterogeneous cell type with several subgroups; for review see [1]
EGFP+ cells from glial fibrillary acidic protein (GFAP)/EGFP transgenic mice are today routinely used by several research groups [4,35] and characterizing their gene expression profile is highly desirable
For preamplification and further analysis we selected EGFP+ cells that were positive for the astrocytic markers Glul and/or Eaat2 and EGFP+ cells that were positive for Cspg4 and/or platelet-derived growth factor a receptor (Pdgfra), which are markers of NG2 glia
Summary
Astrocytes comprise a heterogeneous cell type with several subgroups; for review see [1]. In our recent work we demonstrated the presence of two distinct subpopulations of astrocytes that respond differently to oxygen-glucose deprivation, probably due to their different expression of chloride channels (ClC2), inwardly rectifying K+ channels (KIR4.1) and K2P channels, such as TREK-1 and TWIK-1 [5]. NG2 glia were shown to be progenitors of some groups of reactive astrocytes [11,12], which appear after CNS injury This process, termed astrogliosis, is characterized by the increased expression of glial fibrillary acidic protein (GFAP) and Nestin, followed by the elevated expression of vimentin [13]. Since non-glial cells express some of these markers, detecting a combination of markers is needed for the reliable identification of astrocytes [26]
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