Independent mechanisms of potassium clearance by astrocytes in gliotic tissue

Abstract

The "glial impairment hypothesis" states that astrocytes which change from normal into the reactive type lose their ability to clear extracellular K+, which in turn leads to hyperexcitability in the gliotic tissue. As this hypothesis was never proven or disproven, the question of glial efficiency in K+ clearance in gliotic tissue is still controversial, mainly due to the lack of direct measurements of the intracellular K+ concentration of reactive astrocytes. In order to investigate K+ accumulation by glial cells of gliotic tissue, we used hippocampal slices. Adult rats, previously treated with kainic acid, exhibited loss of neurons and gliosis in the CA1 layer of the hippocampus within 3 days. After this time period, double-barrelled microelectrodes were used to inject Lucifer yellow into cells of the stratum radiatum of the CA1 subfield in 400-microm-thick hippocampal slices. These cells had electrophysiological and morphological characteristics of astrocytes. Most injected cells (70%) were dye-coupled to other cells and were glial fibrillary acidic protein (GFAP)-positive (80%). We found, however, that GFAP-positive cells were dye-coupled not only to each other, but also to GFAP-negative cells. In another set of experiments, we investigated the glial membrane potential during reduction of the extracellular Cl-concentration and the use of the Cl- channel blocker 4,4'-diisothiocyanostilbene-2,2' disulphonic acid (DIDS). The results suggest that reactive astrocytes have a significant resting Cl- conductance. K+-selective microelectrodes were used to analyze the intracellular glial K+ concentration. When the extracellular K+ concentration was increased from 3.5 mM to 10 mM, the intracellular K+ concentration increased by 23 mM. Experiments in which different ion transport systems were blocked with ouabain and DIDS suggest that this increase is dependent on two mechanisms, which can substitute each other: the Na+, K+-ATPase and passive K+ and anion fluxes. Inhibition of either of the two mechanisms did not block the K+ uptake. If, however, the Na+, K+-ATPase and Cl- channels were inhibited at the same time, the net accumulation of K+ was blocked. It appears, therefore, that astrocytes in the gliotic stratum radiatum of the hippocampal slice have the capacity to limit increases in extracellular K+ that are produced by hyperactive surviving hippocampal neurons by passive mechanisms and hence independently of blood and oxygen supply.

Chloride/anion channels in glial cell membranes

“…A third channel, ClC-2 has been connected to the more permanent transformation of astrocytes from normal to reactive subtypes (Ferroni et al, 1995). This was demonstrated in cultured cells, but there is also in vivo evidence for a changed chloride conductance after gliosis (Walz and Wuttke, 1999). Reactive gliosis involves an increase in shape and stellation of these astrocytes, but not necessarily proliferation (Jabs et al, 1999).…”

“…MacVicar et al (1989) found a reversal potential for the GABA A response, in reactive astrocytes of the hippocampal slice, that was more positive than the membrane potential, indicating an active chloride accumulation. Walz and Wuttke (1999) described a resting conductance, whose pharmacological blockade hyperpolarized the membrane of reactive astrocytes in the hippocampal slice, also indicating an active accumulation of chloride. Ballanyi et al (1987) recorded intracellularly from glial cells in slices of guinea-pig olfactory cortex and found evidence for passively distributed chloride ions, contradicting all other in situ and in vitro findings.…”

“…Walz and Wuttke (1999) analyzed the contribution of a chloride conductance to the resting membrane potential of reactive astrocytes in the gliotic hippocampus of rats. These investigators used pharmacological and ion substitution experiments to conclude that a substantial chloride resting permeability exists of approximately P Cl /P K ϭ 0.3.…”

“…Most of the chloride influx during KCl accumulation seems to be due to carrier activity (Walz, 2000). However, in reactive astrocytes, there are two potassium mechanisms working in parallel (Walz and Wuttke, 1999). One is the transmembrane sodium cycle involving Na/K pumping and uptake by the Na,K,2Cl carrier, which leads to KCl accumulation and cycling of Na across the membrane without changing its concentration much.…”

“…Therefore, the expression of new chloride current(s) in reactive astrocytes appears to be necessary for the functional adjustment of these cells to their new environment after injury rather than a requirement for these changes. The new functions may be connected to potassium homeostasis (Walz and Wuttke, 1999) and/or osmosensitivity (Fava et al, 2001). …”