Currents In Glial Cells Research Articles

A potassium channel-linked mechanism of glial cell swelling in the postischemic retina

The cellular mechanisms underlying glial cell swelling, a central cause of edema formation in the brain and retina, are not yet known. Here, we show that glial cells in the postischemic rat retina, but not in control retina, swell upon hypotonic stress. Swelling of control cells could be evoked when their K + channels were blocked. After transient ischemia, glial cells strongly downregulated their K + conductance and their prominent Kir4.1 protein expression at blood vessels and the vitreous body. In contrast, the expression of the aquaporin-4 (AQP4) (water channel) protein was only slightly altered after ischemia. Activation of D 2 dopaminergic receptors prevents the hypotonic glial cell swelling. The present results elucidate the coupling of transmembraneous water fluxes to K + currents in glial cells and reveal the role of altered K + channel expression in the development of cytotoxic edema. We propose a mechanism of postischemic glial cell swelling where a downregulation of their K + conductance prevents the emission of intracellularly accumulated K + ions, resulting in osmotically driven water fluxes from the blood into the glial cells via aquaporins. Inhibition of these water fluxes may be beneficial to prevent ischemia-evoked glial cell swelling.

GABAA receptor agonists modulate K currents in adult hippocampal glial cells in situ

Glial cells are known for their role in development and expression of GABA receptors. However, there seems to be a lack of in situ studies characterizing GABA receptor expression and function in glial cells from early development to adulthood. Consequently, we examined GABA receptor expression on rat hippocampal glial cells in both neonatal and adult slices using the whole-cell patch-clamp technique. Glial cells in adult and neonatal slices exhibit responses to muscimol (1 mM; GABAA), but not baclofen (1 mM; GABAB), demonstrating that receptor electrophysiology remains qualitatively similar in glial cells throughout development. Adult muscimol current densities however, do show a decrease in size to approximately 36% of the neonatal response. Muscimol responses were found to be sensitive to bicuculline, suggesting that they are mediated by GABAA receptors. In addition to receptor currents, muscimol causes a concomitant long-term blockade of outward K+ currents in glial cells of both neonatal and adult slices. Comparisons of percentage peak blockade in adult and neonatal glial cells show no significant difference. However, when comparing average absolute conductance blockade, we see that adult glial cells display a significantly smaller response than neonatal and cultured astrocytes. Therefore, although the percentage blockade of outward currents remains consistent throughout development, neonatal glial cells display a larger physiological effect. Thus, it can be concluded that, although the complex GABA response in glial cells is affected by development, the receptor current and secondary blockade are a basic mechanism for neuronal–glial interaction throughout life. GLIA 26:129–138, 1999. © 1999 Wiley-Liss, Inc.

GABAA receptor agonists modulate K+ currents in adult hippocampal glial cells in situ

Glial cells are known for their role in development and expression of GABA receptors. However, there seems to be a lack of in situ studies characterizing GABA receptor expression and function in glial cells from early development to adulthood. Consequently, we examined GABA receptor expression on rat hippocampal glial cells in both neonatal and adult slices using the whole-cell patch-clamp technique. Glial cells in adult and neonatal slices exhibit responses to muscimol (1 mM; GABAA), but not baclofen (1 mM; GABAB), demonstrating that receptor electrophysiology remains qualitatively similar in glial cells throughout development. Adult muscimol current densities however, do show a decrease in size to approximately 36% of the neonatal response. Muscimol responses were found to be sensitive to bicuculline, suggesting that they are mediated by GABAA receptors. In addition to receptor currents, muscimol causes a concomitant long-term blockade of outward K+ currents in glial cells of both neonatal and adult slices. Comparisons of percentage peak blockade in adult and neonatal glial cells show no significant difference. However, when comparing average absolute conductance blockade, we see that adult glial cells display a significantly smaller response than neonatal and cultured astrocytes. Therefore, although the percentage blockade of outward currents remains consistent throughout development, neonatal glial cells display a larger physiological effect. Thus, it can be concluded that, although the complex GABA response in glial cells is affected by development, the receptor current and secondary blockade are a basic mechanism for neuronal-glial interaction throughout life.

Qualitative analysis of membrane currents in glial cells from normal and gliotic tissue in situ: down-regulation of Na + current and lack of P 2 purinergic responses

To date, the electrophysiological properties of glial cells located in reactive scar tissue are unknown. To address this issue two subtypes of hippocampal glial cells, located in thin vital slices of normal or gliotic brain tissue, were analysed for their voltage controlled ion channels using the patch-clamp technique. Reactive gliosis was induced in adult rats by a single peritoneal injection of kainic acid. The intensity of the following seizures was rated ascending from 1 to 6. Rats which exhibited seizures of level 3 or higher showed, within three days, a marked loss of pyramidal cells (>60% in CA1 and CA3) and an increase in the density of glial fibrillary acidic protein immunostaining, representing an apparent increase in the number and size of astrocytes in all layers of the hippocampal CA1 subfield. Reactive and normal astrocytes of one subtype, electrophysiologically characterized by time-independent potassium currents, did not significantly differ in membrane potential and potassium conductivity. Glutamine synthetase-positive, but mostly glial fibrillary acidic protein-negative, glial cells (presumably representing immature astrocytes) were also included in this study. This subtype of glial cells showed several voltage- and time-dependent potassium currents and, under control conditions, tetrodotoxin-sensitive voltage-gated Na + channels, which were almost completely lost after reactive gliosis. Another part of this study focuses on the sensitivity of reactive and control glial cells for extracellular ATP. Several in vitro studies suggest that P 2 purinergic receptors on glial cells could trigger the induction of reactive gliosis. In contrast to results described on cultured astrocytes, we found in situ that hippocampal glial cells were not sensitive to ATP or stable P 2 receptor agonists in control or in gliotic brain slices. In summary, the presence of at least two different subtypes of hippocampal astrocytes was demonstrated for control as well as for gliotic brain tissue. A dramatic down-regulation of tetrodotoxin-sensitive sodium channels in one subpopulation of reactive astrocytes was shown. This result supports the hypothesis that the presence of active neurons could be required to maintain glial voltage-gated sodium channels. Furthermore, we conclude that there is no longtime expression of P2 purinoceptors on hippocampal astrocytes in situ, and therefore the involvement of astrocytic ATP receptors in the genesis of reactive gliosis is unlikely.

Potassium currents in cultured glia of the frog optic nerve

The processes that participate in clearing increases in [K+]o produced by active neurons include KCl uptake, Na pump stimulation, and spatial buffering. The latter process requires glial cells to carry: 1) inward K+ currents in regions where K+ is elevated at a glial membrane potential more negative than EK; and 2) outward K+ currents at normal K+ and glial membrane potential more positive than EK (Orkand et al: J Neurophysiol 29:788, 1966). Techniques for isolation and culturing glial cells brought new possibilities for studying ionic channels involved in spatial buffering. However, they raised the question of the extent to which the properties of ionic channels are changed due to the process of culturing when glial cells are exposed to an artificial environment and deprived of direct interaction with neurons. We studied potassium currents in glial cells from the frog optic nerve that were cultured for 1-8 days. At 24-48 h, cells exhibited an inwardly rectifying Cs+ blocked current (IK(IN)) that increased in amplitude and shifted its threshold of activation to EK when [K+]o was increased from 3 to 6 or 10 mM. IK(IN), diminished after 3 days in culture and virtually disappeared after 5 days. At 24-48 h, a potassium delayed rectifier current (IKD) was relatively small but became large at 3 days, and was practically the only current present after 5 days. IKD was activated at -8.5 +/- 0.58 mV(SE, n = 48) and 58 +/- 2.2% (SE, n = 48) blocked by 20 mM tetraethylammonium. The results of this study support the idea that the inward rectifying potassium channels (Kir) are responsible for carrying K+ into glial cells whenever [K+]o increases. However, the delayed rectifier potassium channels (KD) cannot provide the pathway for outward K+ current during spatial buffering, and another mechanism must be involved in this process. Our study provides further evidence that culture conditions can greatly influence functional expression of ionic channels in glial cells.

Potassium currents in cultured glia of the frog optic nerve

The processes that participate in clearing increases in [K+]o produced by active neurons include KCl uptake, Na pump stimulation, and spatial buffering. The latter process requires glial cells to carry: 1) inward K+ currents in regions where K+ is elevated at a glial membrane potential more negative than EK; and 2) outward K+ currents at normal K+ and glial membrane potential more positive than EK (Orkand et al: J Neurophysiol 29:788, 1966). Techniques for isolation and culturing glial cells brought new possibilities for studying ionic channels involved in spatial buffering. However, they raised the question of the extent to which the properties of ionic channels are changed due to the process of culturing when glial cells are exposed to an artificial environment and deprived of direct interaction with neurons. We studied potassium currents in glial cells from the frog optic nerve that were cultured for 1–8 days. At 24–48 h, cells exhibited an inwardly rectifying Cs' blocked current (IK(IN)) that increased in amplitude and shifted its threshold of activation to EK when [K+]o was increased from 3 to 6 or 10 mM. IK(IN) diminished after 3 days in culture and virtually disappeared after 5 days. At 24–48 h, a potassium delayed rectifier current (IKD) was relatively small but became large at 3 days, and was practically the only current present after 5 days. IKD was activated at −8.5 ± 0.58 mV (SE, n = 48) and 58 ± 2.2% (SE, n = 48) blocked by 20 mM tetraethylammonium. The results of this study support the idea that the inward rectifying potassium channels (Kir) are responsible for carrying K+ into glial cells whenever [K+]o increases. However, the delayed rectifier potassium channels (KD) cannot provide the pathway for outward K+ current during spatial buffering, and another mechanism must be involved in this process. Our study provides further evidence that culture conditions can greatly influence functional expression of ionic channels in glial cells. © 1996 Wiley-Liss, Inc.

Expression of glycine receptor subunits in glial cells of the rat spinal cord.

We previously demonstrated that the inhibitory neurotransmitter glycine induced membrane currents in glial cells from rat spinal cord. In this present study, the patch-clamp technique was combined with the reverse transcription-mediated PCR to analyze the glycine receptor-subunit expression in individual glial cells of rats age 3-18 days. Using the patch-clamp technique in the whole-cell configuration, glial cells were identified by their membrane current pattern and tested for responsiveness to glycine. Subsequently, the cytoplasm was harvested followed by reverse transcription of total cytoplasmic RNA. Subunit-specific cDNA fragments were amplified and analyzed by agarose gel electrophoresis, Southern blotting, and sequencing. In all cell types investigated, transcripts of the alpha1 subunit, but not of alpha 2 or alpha 3 subunits, were detected. In addition, about one-half the glial cells analyzed contained beta-subunit mRNA. These results illustrate that glial cells of rat spinal cord express functional glycine receptors in contrast to cultured glial cells. Glial cells are in intimate contact with synaptic regions making it likely that these nonneuronal receptors may be activated during glycinergic transmission and may trigger yet unknown responses in the glial cells.

Lysophosphatidylcholine Inhibits NMDA-Induced Currents by a Mechanism Independent of Phospholipase A2-Mediated Protein Kinase C Activation in Hippocampal Glial Cells

Lysophosphatidylcholine (LysoPC), which is formed by phospholipase A2 (PLA2)-mediated phosphatidylcholine hydrolysis, is involved in enhancement of the diacylglycerol- or phorbol ester-dependent protein kinase C (PKC) activation. in the present study, the effect of lysoPC on NMDA-evoked currents was examined by whole-cell patches in cultured rat hippocampal glial cells. NMDA activated the receptor channel and produced currents in glial cells. LysoPC reversibly inhibited the NMDA-gated currents by 43% and this inhibition was not affected by a selective PKC inhibitor, GF109203X. These results indicate that lysoPC inhibits NMDA-induced currents by a mechanism independent of PLA2-mediated PKC activation. A possible explanation for the effect could be that lysoPC directly alters channel-gating kinetics of NMDA receptor or interacts with membrane components surrounding NMDA receptors.

Kainate activates Ca(2+)-permeable glutamate receptors and blocks voltage-gated K+ currents in glial cells of mouse hippocampal slices.

Glial cells in the CA1 stratum radiatum of the hippocampus of 9- to 12-day-old mice show intrinsic responses to glutamate due to the activation of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)/kainate receptors. In the present study we have focused on a subpopulation of the hippocampal glial cells, the "complex" cells, characterized by voltage-gated Na+ and K+ channels. Activation of glutamate receptors in these cells led to two types of responses, the activation of a cationic conductance, and a longer-lasting blockade of voltage-gated K+ channels. In particular, the transient (inactivating) component of the outwardly rectifying K+ current was diminished by kainate. Concomitantly, as described in Bergmann glial cells, kainate also elevated cytosolic Ca2+. This increase was due to an influx via the glutamate receptor itself. In contrast to Bergmann glial cells, the cytosolic Ca2+ increase was not a link to the K+ channel blockade, since the blockade occurred in the absence of the Ca2+ signal and, vice versa, an increase in cytosolic Ca2+ induced by ionomycin did not block the transient K+ current. We conclude that glutamate receptor activation leads to complex and variable changes in different types of glial cells; the functional importance of these changes is as yet unresolved.

Sodium and Calcium Currents in Glial Cells of the Mouse Corpus Callosum Slice.

We studied Na+ and Ca2+ currents in glial cells during the development of the corpus callosum in situ. Glioblasts and oligodendrocytes from frontal brain slices of postnatal day (P) 3 to P18 mice were identified based on morphological and ultrastructural features after characterization of the currents with the patch-clamp technique. Slices from P3 - P8 mice contained predominantly glioblasts with immature morphological features. These cells showed Na+ and Ca2+ currents, but the population with these currents decreased between P3 and P8. Na+ currents were blocked in Na+-free bathing solution and in the presence of tetrodotoxin, Ca2+ currents were only observed when a high concentration of extracellular Ba2+ was present. The cells from the corpus callosum of P10 - P18 mice predominantly had morphological features of oligodendrocytes. In these cells, which in some cases were shown to form myelin, neither Na+ nor Ca2+ currents were detected. To compare these in situ results with those from the electrophysiologically and immunocytochemically well-characterized cultured glial cells, we determined the expression pattern of stage-specific antigens in the corpus callosum in situ. The first O4 antigen-positive glial precursors were observed at P1, the earliest stage examined. The oligodendrocytic antigens O7 and O10 appeared at P6 and P14, respectively, and prominent labelling with the corresponding markers was seen at P12 and P18, respectively. Despite the existence of numerous mature, O10-positive oligodendrocytes at P18, which expressed Ca2+ channels in vitro, we failed to detect Ca2+ currents in situ at this stage.

GABA- and glutamate-activated currents in glial cells of the mouse corpus callosum slice.

Whole-cell transmitter-activated currents were recorded with the patch-clamp technique from glial cells in thin frontal brain slices of the corpus callosum. In slices from 6- to 8-day-old mice, glioblasts were predominantly found, while oligodendrocytes were predominant in slices from 10- to 13-day-old mice. These developmental stages could be readily distinguished by their K+ channel pattern and their morphology and ultrastructural features. Both cell types expressed GABA and glutamate receptors in this in situ preparation. GABA responses showed similarities to those described for GABAA receptors, i.e., they were mimicked by muscimol, blocked by bicuculline, and enhanced by pentobarbital. Glutamate responses showed similarities to those of the kainate/quisqualate receptor subtype. The amplitude of GABA-activated currents recorded in oligodendrocytes was significantly smaller than that from glioblasts, while glutamate responses did not show marked differences in either cell type.

Spatial buffering of light-evoked potassium increases by retinal Müller (glial) cells.

Activity-dependent variations in extracellular potassium concentration in the central nervous system may be regulated, in part, by potassium spatial buffering currents in glial cells. The role of spatial buffering in the retina was assessed by measuring light-evoked potassium changes in amphibian eyecups. The amplitude of potassium increases in the vitreous humor was reduced to approximately 10 percent by 50 micromolar barium, while potassium increases in the inner plexiform layer were largely unchanged. The decrease in the vitreal potassium response was accurately simulated with a numerical model of potassium current flow through Müller cells, the principal glial cells of the retina. Barium also substantially increased the input resistance of Müller cells and blocked the Müller cell-generated M-wave, indicating that barium blocks the potassium channels of Müller cells. Thus, after a light-evoked potassium increase within the retina, there is a substantial transfer of potassium from the retina to the vitreous humor by potassium current flow through Müller cells.