Distinct expression/function of potassium and chloride channels contributes to the diverse volume regulation in cortical astrocytes of GFAP/EGFP mice.

Jana Benesova,Pavel Honsa,David Dzamba,Helena Pivonkova,Vendula Rusnakova,Miroslava Anderova,Mikael Kubista

doi:10.1371/journal.pone.0029725

Abstract

Recently, we have identified two astrocytic subpopulations in the cortex of GFAP-EGFP mice, in which the astrocytes are visualized by the enhanced green–fluorescent protein (EGFP) under the control of the human glial fibrillary acidic protein (GFAP) promotor. These astrocytic subpopulations, termed high response- (HR-) and low response- (LR-) astrocytes, differed in the extent of their swelling during oxygen-glucose deprivation (OGD). In the present study we focused on identifying the ion channels or transporters that might underlie the different capabilities of these two astrocytic subpopulations to regulate their volume during OGD. Using three-dimensional confocal morphometry, which enables quantification of the total astrocytic volume, the effects of selected inhibitors of K+ and Cl− channels/transporters or glutamate transporters on astrocyte volume changes were determined during 20 minute-OGD in situ. The inhibition of volume regulated anion channels (VRACs) and two-pore domain potassium channels (K2P) highlighted their distinct contributions to volume regulation in HR-/LR-astrocytes. While the inhibition of VRACs or K2P channels revealed their contribution to the swelling of HR-astrocytes, in LR-astrocytes they were both involved in anion/K+ effluxes. Additionally, the inhibition of Na+-K+-Cl− co-transporters in HR-astrocytes led to a reduction of cell swelling, but it had no effect on LR-astrocyte volume. Moreover, employing real-time single-cell quantitative polymerase chain reaction (PCR), we characterized the expression profiles of EGFP-positive astrocytes with a focus on those ion channels and transporters participating in astrocyte swelling and volume regulation. The PCR data revealed the existence of two astrocytic subpopulations markedly differing in their gene expression levels for inwardly rectifying K+ channels (Kir4.1), K2P channels (TREK-1 and TWIK-1) and Cl− channels (ClC2). Thus, we propose that the diverse volume changes displayed by cortical astrocytes during OGD mainly result from their distinct expression patterns of ClC2 and K2P channels.

Highlights

The generation of brain edema, leading to an intracranial pressure increase and brain herniation, markedly alters the course and treatment of cerebral ischemia
In the present study we have demonstrated that the two previously described cortical astrocytic populations – HR- and low response- (LR-)astrocytes – respond differently to Cl2 and K+ channel inhibitors when applied during oxygen-glucose deprivation (OGD)
In accordance with the pharmacological experiments, quantitative single-cell polymerase chain reaction (PCR) profiling revealed that cortical astrocytes are heterogeneous with respect to the gene expression profiles of the ion channels/transporters that participate in their volume regulation

Summary

Introduction

The generation of brain edema, leading to an intracranial pressure increase and brain herniation, markedly alters the course and treatment of cerebral ischemia. Astrocytes have been reported to contribute predominantly to edema formation. They support neuronal function and provide for the maintenance of ionic and neurotransmitter homeostasis under physiological conditions; they may contribute to the worsening of brain damage under pathological conditions. The uptake of ions and amino acids may turn into an excessive osmolyte influx leading to the generation of edema, which in turn contributes to further ischemic damage. Astrocytic Na+-dependent glutamate uptake, which is responsible for the termination of synaptic transmission and helps to protect neurons from the excitotoxic activity of glutamate, may reverse as a consequence of the ionic dis-balance accompanying energy depletion during severe ischemia and contribute to an increase of extracellular glutamate concentrations [2]. K+-Cl2 (KCC) and Na+-K+-Cl2 (NKCC) co-transporters, the major regulators of neuronal and astrocytic Cl2 and Na+

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