Wolfgang Walz

Role of astrocytes in the clearance of excess extracellular potassium.

The development of concepts describing potassium clearance mechanisms in the mammalian central nervous system in the last 35 years is reviewed. The pattern of excess potassium in the extracellular space is discussed as are the implications of these potassium levels for neuronal excitability. There is a systematic description of the available evidence for astrocytic involvement in situ. The three possible astrocytic potassium clearance mechanisms are introduced: spatial buffer mechanism; carrier-operated potassium chloride uptake as well as channel-operated potassium chloride uptake. The three mechanisms are compared and their compatibility is discussed. Evidence is now available showing that at least two of these if not all three mechanisms co-exist and complement each other. Finally, it is concluded that these potassium movements are not used as a signal system, only as a homeostatic feedback mechanisms. Such a genuine signal system involving glial elements exists--but it is based on calcium waves.

Controversy surrounding the existence of discrete functional classes of astrocytes in adult gray matter.

Since 1992, it has been possible to record ionic currents from identified astrocytes in situ, using brain slice technology. Brain slice recordings confirm previous in vitro findings that expression of voltage‐gated K+ and Na+ channels are a feature of this cell type. In contrast to cultured astrocytes, most investigators found that astrocytes in situ did not contain detectable, or at very best only low, levels of glial fibrillary acidic protein (GFAP). Structural and immunocytochemical investigations determined that these cells are different from oligodendrocyte precursors. In addition to cells with this current pattern, many but not all investigators found a second pool of astrocytes with no voltage‐gated ion channels and high GFAP content. These two subpopulations of cells were termed complex and passive astrocytes. The existence of passive astrocytes has been questioned because of possible problems with space clamp conditions and spillage of EGTA‐buffered pipette solution around the cells before recordings. Another problem is the fact there is a discrepancy regarding the GFAP content of complex astrocytes. It is of interest that recent immunocytochemical studies suggest the existence of two pools of astrocytes, one with a high GFAP content and one with nondetectable GFAP. Given this, it is tempting to correlate the two (controversial) electrophysiological patterns with immunochemical differences (GFAP) in order to demonstrate two functionally discrete classes of astrocytes in adult gray matter. However, despite evidence that some of the K+ channels may be involved in proliferation, the role of voltage‐gated ion channels in this nonexcitable cell type remains unknown. This is despite the fact that astrocytic Na+ channels show dramatic changes after pathological events, re‐enforcing the notion that the expression of this channel is under tight neuronal control. Several studies suggest that there is a great degree of flexibility and that astrocytes can undergo rapid changes in expression of both membrane ion currents and GFAP. Although it is likely that astrocytes exhibit different structural and membrane properties, this heterogeneity might be a reflection of the flexible plasticity of one astrocyte type under influence of environmental factors rather than of the existence of two distinct and permanent subtypes. GLIA 31:95–103, 2000.

Carrier-mediated KCl accumulation accompanied by water movements is involved in the control of physiological K+ levels by astrocytes.

Potassium accumulation and water transport into mouse astrocytes in primary cultures were investigated when external potassium was increased from 3 to 12 mM. The intracellular potassium content increased by 63% within 50 s of such a change. The increase consisted of a ouabain- and furosemide-sensitive component, both contributing in about the same amounts. Experiments with altered ion composition revealed that the furosemide-sensitive component consisted of a KCl accumulation. Water moved into the astrocytes without delay after such an external K+ increase and increased the cell water by 27%. This water increase was abolished in solutions with reduced Cl- and during application of furosemide. Thus, these results on a KCl uptake accompanied by water movements into astrocytes suggest a potential mechanism by which glial cells in situ can regulate external K+ levels.

Ouabain‐Sensitive and Ouabain‐Resistant Net Uptake of Potassium into Astrocytes and Neurons in Primary Cultures

Abstract: Inhibition of net uptake of 42K by different concentrations of ouabain was studied in primary cultures of astrocytes and in primary cultures of neurons in order to investigate whether there is a pronounced difference between ouabain sensitivity in the two cell types and to determine the genuine magnitudes of the ouabain‐sensitive and the ouabain‐resistant potassium uptakes. In morphologically differentiated astrocytes, obtained after treatment with dibutyryl cyclic AMP (dBcAMP), the sensitivity to ouabain was slightly lower than in neurons, but astrocytes which had not been treated with dBcAMP showed sensitivity similar to the neurons (which likewise were not treated). In the presence of elevated potassium concentrations (12 and 24 mM) ouabain sensitivity was decreased, although only by a factor of 2–3. Accordingly, maximum inhibition of the uptake required under all conditions studied, at most, 1.0 mM ouabain. Like total uptake, this ouabain‐sensitive uptake was several times less intense in neurons than in astrocytes, where it reached its maximum value at an external potassium concentration of 12 mM. Subtraction of the ouabain‐sensitive uptake from the total uptake revealed a considerable ouabain‐resistant uptake. This ouabain‐resistant uptake was studied in detail in the astrocytes, where it was found to increase with increasing potassium concentration over the whole concentration range 3–24 mM and to exceed substantially the maximum amount that can be accumulated by diffusion.

Potassium homeostasis in the ischemic brain

Extracellular [K+] can range within 2.5–3.5 mM under normal conditions to 50–80 mM under ischemic and spreading depression events. Sustained exposure to elevated [K+]o has been shown to cause significant neuronal death even under conditions of abundant glucose supply. Astrocytes are well equipped to buffer this initial insult of elevated [K] through extensive gap junctional coupling, Na+/K+ pump activity (with associated glycogen and glycolytic potential), and endfoot siphoning capability. Their abundant energy availability and alkalinizing mechanisms help sustain Na+/K+ ATPase activity under ischemic conditions. Furthermore, passive K+ uptake mechanisms and water flux mediated through aquaporin‐4 channels in endfoot processes are important energy‐independent mechanisms. Unfortunately, as the length of ischemic episode is prolonged, these mechanisms increase to a point where they begin to have repercussions on other important cellular functions. Alkalinizing mechanisms induce an elevation of [Na+]i, increasing the energy demand of Na+/K+ ATPase and leading to eventual detrimental reversal of the Na+/glutamate− cotransporter and excitotoxic damage. Prolonged ischemia also results in cell swelling and activates volume regulatory processes that release excessive excitatory amino acids, further exacerbating excitotoxic injury. In the days following ischemic injury, reactive astrocytes demonstrate increased cell size and process thickness, leading to improved spatial buffering capacity in regions outside the lesion core where there is better neuronal survival. There is a substantial heterogeneity among reactive astrocytes, with some close to the lesion showing decreased buffering capacity. However, it appears that both Na+/K+ ATPase activity (along with energy production processes) as well as passive K+ uptake mechanisms are upregulated in gliotic tissue outside the lesion to enhance the above‐mentioned homeostatic mechanisms.

Lactate production and release in cultured astrocytes

Intracellular lactate content and release of lactate into the surrounding medium of mouse astrocytes in primary culture was measured using the lactate dehydrogenase method. During culturing the cellular content of astrocytes decreased from 400 to 200 nmol/mg protein. The total lactate released into the extracellular space, however, amounted to 75,000 nmol/mg within 98 h, corresponding to a lactate concentration of 10 mM in the cell culture dish. In another set of experiments, cytotoxic swelling was evoked by exposure of the cells to 60 mM K+, this situation caused a 40% increase in cellular volume and an increase in the KCl content of astrocytes. Within 3 h of a change to 60 mM K+ the intracellular lactate content was increased by 100 nmol/mg (one third) and the lactate release in the extracellular space by about 2000 nmol/mg (twice as high as during exposure to 3 mM K+). However, due to the increased intracellular water content, the lactate concentration inside the cells remained unchanged. It is concluded that astrocytes produce substantial amounts of additional lactate during cytotoxic swelling. This lactate, however, is not increasing the intracellular osmolarity and most of the lactate is released into the extracellular space. Depending on the transmembrane transport mechanism it could have the capability to decrease the strong ion difference and contribute to acid shifts in the extracellular space.

Immunocytochemical evidence for a distinct GFAP-negative subpopulation of astrocytes in the adult rat hippocampus

In order to establish the relative distribution of a GFAP-negative population of astrocytes, and its change in gliotic tissue, sections of the stratum radiatum of the CA1 hippocampal layer of male, adult, Wistar rats were analyzed by immunocytochemical methods. Ten micrometer-thick sections were triple-stained to detect nuclei, glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS). In another set of experiments, the rats received a one-time intraperitoneal injection of kainic acid that caused epileptic seizures. With the use of a behavioral protocol, animals with substantial neuronal loss in the pyramidal layer were selected. Five days after the injection these rats were analyzed similarly to control rats. We find that GFAP-positive cells are a subpopulation of GS-positive cells and that the GFAP-negative subpopulation is quite large (40%). After gliosis the density of GFAP-negative, GS-positive cells stays stable, whereas the GFAP-positive population triples. These experiments confirm electrophysiological experiments showing a distinct, GFAP-negative subset of astrocytes that remains consistent even after injury-induced gliosis and accompanying up-regulation of GFAP.

Intense Furosemide-Sensitive Potassium Accumulation in Astrocytes in the Presence of Pathologically High Extracellular Potassium Levels

An intense K+ accumulation in primary cultures of astrocytes, occurring when external K+ was increased from 5.4 to 54 mM, was investigated. This increase resulted in a doubling of the K+ content within 10 s. Thirty percent of the accumulation was inhibited by furosemide (2 mM). This drug had no effect on the unidirectional influx of K+ at 5.4 mM K+, but when the extracellular K+ concentration was increased, there appeared to be a furosemide-sensitive component of the influx. This component increased with increasing external K+ levels, reaching 44% of the total influx at 72 mM. These results show that astrocytes exhibit an intense furosemide-sensitive K+ accumulation which is activated by K+ levels resembling those occurring in the extracellular compartment during pathological events. Previous studies on a furosemide-sensitive Cl− pump in cultured astrocytes suggest that this accumulation might be via KCl cotransport, which in other systems is involved in volume control.

The Initiation of the Microglial Response

The initial response of microglia to ischemia and ischemia‐like conditions was analyzed in situ and in vitro. After sublethal ischemia in situ, microglia appear activated morphologically, but do not express macrophage‐like antigens. In contrast, neuronal damage induces full expression of immunomolecules in microglia. Additionally, blood‐borne cells readily infiltrate the region of the ischemic core and constitute another source of cells with macrophage‐like expression. Thus, it appears that the microglia are the earliest cells to respond to injury, but their response is graded and complicated by the presence of blood‐borne immune cells. In vitro ischemia‐like conditions caused an irreversible depolarization, ion channel shutdown, and blebbing, indicating that microglia are not equipped to withstand an ischemic insult. Application of ATP alone to microglia produced outward currents and calcium transients and these calcium transients increased when ATP was applied in combination with high potassium. It is known that both outward currents and calcium transients are induced during spreading depression, a feature of focal injury, and this suggests that spreading depression might be one of the initial activators of microglia.

Gene Expression of Aromatic l-Amino Acid Decarboxylase in Cultured Rat Glial Cells

Abstract: Northern blot hybridization was performed to detect aromatic L‐amino acid decarboxylase (AADC) mRNA in primary cultures of astrocytes and C6 glioma cells. The cDNA probe for rat AADC was generated by reverse transcription from rat adrenal gland total RNA and was amplified by the polymerase chain reaction method. AADC mRNA from cultured astrocytes and C6 glioma cells was present as a single band, 2.2 kbp in size, that comigrated with the RNA from rat kidney. Western immunoblot showed a single protein band at 52 kDa for AADC enzyme protein. These findings demonstrate that AADC is expressed in rat glial cells.