AMPA/kainate receptor activation blocks K+ currents via internal Na+ increase in mouse cultured stellate astrocytes

Cultured mouse cortical astrocytes of the stellate type were studied by using the patch-clamp technique in whole-cell configuration. The astrocytes express at least two types of outwardly rectifying K⁺ channels which mediate a transient and a sustained current. Activation of AMPA receptors by kainate leads to a substantial blockade of both types of K⁺ currents. The blockade is absent when Na⁺ is withdrawn from the external medium, suggesting that it is caused by constant Na⁺ influx through AMPA receptors. The presence of high Na⁺ solutions in the pipette induces a blockade of both K⁺ currents which is very similar to the blockade induced by kainate, supporting thus the view that the mechanism of the blockade of K⁺ channels by kainate involves Na⁺ increases in the submembrane area. The blockade occurs between 20 and 40 mM [Na⁺]_i, which is within the physiological range of [Na⁺]_i in astrocytes. The data may suggest that the blockade of K⁺ channels by high [Na⁺]_i conditions could provide a mechanism to prevent K⁺ leakage from the astrocytes into the extracellular space during periods of intense neuronal activity. GLIA 20:38-50, 1997. © 1997 Wiley-Liss, Inc.     

Effect of kainate on the membrane conductance of hilar glial precursor cells recorded in the perforated-patch configuration

The effects of kainate on membrane current and membrane conductance were investigated in presumed hilar glial precursor cells of juvenile rats. The perforated-patch configuration was used also to reveal possible second-messenger effects. Kainate evoked an inward current that was accompanied by a biphasic change in membrane conductance in 69% of the cells. An initial conductance increase with a time course similar to that of the inward current was followed by a second delayed conductance increase. This second conductance was absent in whole-cell-clamp recordings, suggesting that it was mediated by a second messenger effect. Analysis of the reversal potentials of the membrane current during both phases of the kainate-induced conductance change revealed that the first conductance increase reflected the activation of AMPA receptors. Several lines of evidence suggest that the delayed second conductance increase was due to the indirect activation of Ca²⁺-dependent K⁺ channels via Ca²⁺-influx through AMPA receptors. (1) the delayed second conductance increase was blocked by Ba²⁺ and the reversal of its underlying current was significantly shifted towards E_K+, suggesting that it is due to the activation of K⁺ channels. (2) The delayed second conductance increase disappeared in a Ca²⁺-free saline buffered with BAPTA, indicating that it depended on Ca²⁺-influx. (3) Co²⁺, Cd²⁺ and nimodipine failed to block the delayed second conductance increase excluding a major contribution of voltage-dependent Ca²⁺ channels. (4) The involvement of metabotropic glutamate receptors also appeared unlikely, because the kainate-induced delayed second conductance increase could not be blocked by a depletion of the intracellular Ca²⁺ stores with the Ca²⁺-ATPase inhibitor thapsigargin, and t-ACPD exerted no effect on membrane current and conductance. We conclude that kainate activates directly AMPA receptors in presumed hilar glial precursor cells. This results in a Ca²⁺ influx that could lead indirectly to the activation of Ca²⁺-dependent K⁺ channels. GLIA 23:35–44, 1998. © 1998 Wiley-Liss, Inc.     

Tetrahydroberberine blocks membrane K channels underlying its inhibition of intracellular message-mediated outward currents in acutely dissociated CA1 neurons from rat hippocampus

In the patch-clamp perforated whole-cell recording mode, tetrahydroberberine (THB), a novel dopamine (DA) receptor antagonist, inhibits not only DA-induced outward K⁺ currents, but also acetylcholine-, caffeine- or strychnine-induced outward current. However, THB does not affect either GABA- or glycine-induced Cl⁻ currents, or non-NMDA receptor agonist-induced cation currents. As expected for a K⁺ channel blocker, THB evokes a downward current deflection accompanied by a decrease of conductance. It is concluded that the direct blockade of membrane K⁺ channels by THB underlies its inhibition of intracellular message-mediated outward currents.     

Na+‐Activated K+ Channels in Rat Supraoptic Neurones

The magnocellular neurosecretory cells (MNCs) of the hypothalamus secrete the neurohormones vasopressin and oxytocin. The systemic release of these hormones depends on the rate and pattern of MNC firing and it is therefore important to identify the ion channels that contribute to the electrical behaviour of MNCs. In the present study, we report evidence for the presence of Na⁺‐activated K⁺ (K_Na) channels in rat MNCs. K_Na channels mediate outwardly rectifying K⁺ currents activated by the increases in intracellular Na⁺ that occur during electrical activity. Although the molecular identity of native K_Na channels is unclear, their biophysical properties are consistent with those of expressed Slick (slo 2.1) and Slack (slo 2.2) proteins. Using immunocytochemistry and Western blot experiments, we found that both Slick and Slack proteins are expressed in rat MNCs. Using whole cell voltage clamp techniques on acutely isolated rat MNCs, we found that inhibiting Na⁺ influx by the addition of the Na⁺ channel blocker tetrodotoxin or the replacement of Na⁺ in the external solution with Li⁺ caused a significant decrease in sustained outward currents. Furthermore, the evoked outward current density was significantly higher in rat MNCs using patch pipettes containing 60 mm Na⁺ than it was when patch pipettes containing 0 mm Na⁺ were used. Our data show that functional K_Na channels are expressed in rat MNCs. These channels could contribute to the activity‐dependent afterhyperpolarisations that have been identified in the MNCs and thereby play a role in the regulation of their electrical behaviour.     

Adult rat optic nerve oligodendrocyte progenitor cells express a distinct repertoire of voltage- and ligand-gated ion channels

Cultured oligodendrocyte progenitor cells derived from the developing central nervous system (CNS) express a pattern of ion channels that is distinct from mature oligodendrocytes and other cell types of the CNS. In the present study, we used the whole-cell patch-clamp technique and the fura-2-based Ca⁺⁺ imaging system to study the ion channel expression of oligodendrocyte progenitor cells derived from the optic nerves of adult rats. We found that the adult oligodendrocyte progenitor cell membrane is dominated by K⁺ currents, both delayed outward and inward rectifying. The inwardly rectifying K⁺ currents were often as large as the outward delayed rectifying K⁺ currents. The delayed rectifying outward currents were partially blocked by 50 mM tetraethylammonium or 1 mM 4-aminopyridine, but not by 2 or 5 mM BaCl₂. This suggests that the delayed rectifier channels expressed by adult progenitor cells are different from those expressed by perinatal cells. Most adult oligodendrocyte progenitor cells showed no or only small A-type K⁺ currents. Both Ca⁺⁺ and Na⁺ channels were also detected in these cells. Furthermore, adult progenitor cells responded to the neurotransmitters GABA and kainate and the pharmacology of these responses indicated that these cells express GABA_A receptors and kainate receptors that are Ca⁺⁺ -permeable. Our study suggests that adult oligodendrocyte progenitor cells are electrophysiologically distinct and that these cells share electrophysiological characteristics with both perinatal progenitor cells and immature oligodendrocytes. © 1995 Wiley-Liss, Inc.     

AMPA receptors shape Ca2+ responses in cortical oligodendrocyte progenitors and CG-4 cells

Intracellular calcium signals triggered by glutamate receptor activation were studied in primary cortical oligodendrocyte lineage cells and in the oligodendrocyte cell line CG-4. Glutamate, kainate, and AMPA (30-300 μM) increased [Ca ²⁺]_i in both types of cells at the stage of oligodendrocyte progenitors (O-2A; GD3⁺) or pro-oligodendroblasts (04⁺). The peak amplitude of Ca²⁺ responses to glutamate receptor agonists was significantly larger in cortical cells. In CG-4 and in cortical cells, the majority (more than 90%) of bipolar GD3⁺ or multipolar 04⁺ cells responded to kamate. In all the cells analyzed, kainate was more efficacious than AMPA and glutamate. The percentage of bipolar or multipolar cells responding to glutamate was significantly lower in the CG-4 cell line than in primary cultures. Cellular responses typical of metabotropic glutamate receptor activation were observed in 20% of the cortical O-2A progenitors, but in none of the CG-4 cells. The AMPA-selective antagonist GYKI 52466 blocked kainate-induced Ca²⁺ responses in cortical O-2A cells. The selective AMPA receptor modulator cyclothiazide (30 μM) greatly potentiated the effects of AMPA (30-100 μM) on [Ca ²⁺]_i in cortical and CG-4 cells. Our findings indicate that Ca²⁺ responses in cells of the oligodendrocyte lineage are primarily shaped by functional AMPA receptors. © 1995 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Inhibition of delayed rectifier K+ conductance in cultured rat cerebellar granule neurons by activation of calcium-permeable AMPA receptors

Activation of AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors in cerebellar granule cells during perforated-patch whole-cell recordings activated an inward current at negative voltages which was followed, after a delay, by the inhibition of an outward potassium current at voltages positive to -20 mV. The activated inward current was inwardly rectifying suggesting that the AMPA receptors were Ca2+-permeable. This was confirmed by direct measurements of intracellular calcium where Ca2+ rises were seen following AMPA receptor activation in Na+-free external solution. Ca2+ rises were equally large in the presence of 100 microM Cd2+ to block voltage-gated Ca2+ channels. Specific voltage-protocols, allowing selective activation of the delayed rectifier potassium current (KV) and the transient A current (KA), showed that kainate inhibited KV, but not to any great extent KA. The inhibition of KV was blocked by the AMPA receptor antagonist CNQX (6-cyano-7-nitroquinoxaline-2,3-dione) and was no longer observed when the KV current was abolished with high concentrations of Ba2+. The responses to kainate were not altered by pre-treating the cells with pertussis toxin, suggesting that the AMPA receptor stimulation of the G-protein Gi cannot account for the effects observed. Replacing extracellular Na+ with choline did not alter the inhibition of KV by kainate, however, removing extracellular Ca2+ reduced the kainate response. The inhibition of KV by kainate was unaffected by the presence of 100 microM Cd2+. The guanylyl cyclase inhibitor, ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one), did not alter kainate inhibition of KV. It is concluded that ion influx (particularly Ca2+ ions) through AMPA receptor channels following receptor activation leads to an inhibition of KV currents in cerebellar granule neurons.     

Analysis of ion channel expression by astrocytes in red nucleus brain stem slices of the rat

The red nucleus (RN) has been widely used to study the formation and remodeling of synaptic connections during development and in post-lesion plasticity. Since glial cells are thought to contribute to synaptic plasticity, and information on functional properties of brain stem glia is missing, we analyzed voltage-gated ion channels as well as glutamate receptors expressed by glial cells of the RN. The patch-clamp technique was applied to identified cells in acute brain stem slices of 5- to 12-day-old rats. Based on their pattern of membrane currents, two types of glial cells could be distinguished. A first type was characterized by passive, symmetrical currents. The second population of cells, which was the focus of the present study, expressed a complex pattern of voltage-gated channels. These cells could be labeled with antibodies against glutamine synthetase and S100β, suggesting an astroglial origin. Depolarizing voltage steps activated transient and delayed rectified K⁺ currents as well as Na⁺ currents. In addition, a subset of cells expressed Ba²⁺ sensitive inward rectifier K⁻ currents activated by hyperpolarization. All “complex” glial cells analyzed possessed ionotropic glutamate receptors of the α-amino-3-hydroxy-5-methyl-4-isoxazoleprorionic acid (AMPA) subtype, while functional kainate and N-methyl-D-aspartate (NMDA) receptors could not be detected. Receptor activation blocked outward rectifying K⁺ currents, similar to previous observations in glial cells of the hippocampus and the corpus callosum. GLIA 19:234–246, 1997. © 1997 Wiley-Liss, Inc.     

Testing competing path models linking the biochemical variables in red blood cells from Li+-treated bipolar patients

Objectives: Red blood cells (RBCs) from Li⁺‐treated bipolar patients have shown abnormalities in intracellular Li⁺ concentration ([Li⁺]_i), Na⁺/Li⁺ exchange rates, and membrane phospholipid levels. Based on Li⁺‐loaded RBC studies, we hypothesized that Li⁺‐treated bipolar patients also have varied intracellular free Mg²⁺ concentrations ([Mg²⁺]_f) as compared with normotensive patients. We addressed how these experimentally determined values are intercorrelated. Assuming that Li⁺ treatment alters these biochemical parameters, we provide hypothetical pathways based upon structural equation modeling statistics. Methods: In RBCs from 30 Li⁺‐treated bipolar patients, we determined [Li⁺]_i, serum [Li⁺] ([Li⁺]_e), Na⁺/Li⁺ exchange parameters, membrane phospholipid levels, [Mg²⁺]_f, and Li⁺ membrane binding affinities. Comprehensive statistical analyses assessed correlations among the biochemical data. We used path analysis statistics to propose potential pathways in which the data were correlated. Results: We found significant correlations within the three Na⁺/Li⁺ exchange parameters and percentage composition of the membrane phospholipids. Additional correlations existed between [Mg²⁺]_f and V_std, K_m, or phospholipid composition, between [Li⁺]_i and percentage of phosphatidylcholine, and between percentage of phosphatidylserine and K_m. Based on these findings, we hypothesized and statistically determined the most probable pathway through which these parameters were intercorrelated. Conclusions: Significant correlations existed between the biochemical parameters that describe the cell membrane abnormality and the Li⁺/Mg²⁺ competition hypotheses. Using path analysis statistics, we identified a biochemical pathway by which Li⁺ may assert its cellular effects. This study serves as an illustrative example how path analysis is a valuable tool in determining the direction of a certain biochemical pathway.     

Hypoxia induced by Na2S2O4 increases [Na]i in mouse glomus cells, an effect depressed by cobalt. Experiments with Na-selective microelectrodes and voltage-clamping

The intracellular sodium concentration ([Na⁺]_i) and resting potential (E_m) of cultured mouse glomus cells (clustered and isolated) were simultaneously measured with intracellular Na⁺-sensitive and conventional, KCl-filled, microelectrodes. Results obtained in clustered and isolated cells were similar. During normoxia (PO₂ 122 Torr), [Na⁺]_i was 12–13 mM corresponding to a Na⁺ equilibrium potential (E_Na) of about 58 mV. Em was about −42 mV. Hypoxia, induced by Na₂S₂O₄ 1 mM (PO₂ 10 Torr), depolarized the cells by about 20 mV, [Na⁺]_i increased by 21 mM and E_Na dropped to about 35 mV. One millimolar of CoCl₂ depressed, or blocked, the effects of Na₂S₂O₄ on [Na⁺]_i but did not affect hypoxic depolarization. Voltage-clamping at −70 mV, while delivering pulses of different amplitudes, produced only small (about 10 pA) and slow TTX-insensitive inward currents. Fast and large (TTX-sensitive) inward currents were not detected. The cell conductance (measured with voltage ramps) was less than 1 nS. It was not affected by hypoxia but was depressed by cobalt. Voltage ramps elicited small inward currents in control and hypoxic solutions that were much smaller than those induced by barium (presumably enhancing calcium currents). Also, normoxic and hypoxic currents had lower thresholds and their troughs were at more negative voltages than in the presence of Ba²⁺. All currents were blocked by 1 mM CoCl₂ suggesting that, at this concentration, cobalt exerted a nonspecific effect on glomus membrane channels. Hypoxia induced a large [Na⁺]_i increase (presumably through inflow), but very small voltage-gated inward currents. Thus, Na⁺ increases (inflow) probably occurred by disturbing a Na⁺/K⁺ exchange mechanism and not by activation of voltage-gated channels.     

Gap junctions equalize intracellular Na+ concentration in astrocytes

Gap junctions between glial cells allow intercellular exchange of ions and small molecules. We have investigated the influence of gap junction coupling on regulation of intracellular Na⁺ concentration ([Na⁺]_i) in cultured rat hippocampal astrocytes, using fluorescence ratio imaging with the Na⁺ indicator dye SBFI (sodium-binding benzofuran isophthalate). The [Na⁺]_i in neighboring astrocytes was very similar (12.0 ± 3.3 mM) and did not fluctuate under resting conditions. During uncoupling of gap junctions with octanol (0.5 mM), baseline [Na⁺]_i was unaltered in 24%, increased in 54%, and decreased in 22% of cells. Qualitatively similar results were obtained with two other uncoupling agents, heptanol and α-glycyrrhetinic acid (AGA). Octanol did not alter the recovery from intracellular Na⁺ load induced by removal of extracellular K⁺, indicating that octanol's effects on baseline [Na⁺]_i were not due to inhibition of Na⁺, K⁺-ATPase activity. Under control conditions, increasing [K⁺]_o from 3 to 8 mM caused similar decreases in [Na⁺]_i in groups of astrocytes, presumably by stimulating Na⁺, K⁺-ATPase. During octanol application, [K⁺]_o-induced [Na⁺]_i decreases were amplified in cells with increased baseline [Na⁺]_i, and reduced in cells with decreased baseline [Na⁺]_i. This suggests that baseline [Na⁺]_i in astrocytes “sets” the responsiveness of Na⁺, K⁺-ATPase to increases in [K⁺]_o. Our results indicate that individual hippocampal astrocytes in culture rapidly develop different levels of baseline [Na⁺]_i when they are isolated from one another by uncoupling agents. In astrocytes, therefore, an apparent function of coupling is the intercellular exchange of Na⁺ ions to equalize baseline [Na⁺]_i, which serves to coordinate physiological responses that depend on the intracellular concentration of this ion. GLIA 20:299–307, 1997. © 1997 Wiley-Liss, Inc.     

Na+—Ca2+ Exchange Currents in Cortical Neurons: Concomitant Forward and Reverse Operation and Effect of Glutamate

Na⁺-Ca²⁺ exchanger-associated membrane currents were studied in cultured murine neocortical neurons, using whole-cell recording combined with intracellular perfusion. A net inward current specifically associated with forward (Na⁺_o-Ca²⁺_i) exchange was evoked at -40 mV by switching external 140 mM Li⁺ to 140 mM Na⁺. The voltage dependence of this current was consistent with that predicted for 3Na⁺:1Ca²⁺ exchange. As expected, the current depended on internal Ca²⁺, and could be blocked by intracellular application of the exchanger inhibitory peptide, XIP. Raising internal Na⁺ from 3 to 20 mM or switching the external solution from 140 mM Li⁺ to 30 mM Na⁺ activated outward currents, consistent with reverse (Na⁺,-Ca²⁺_o) exchange. An external Ca²⁺-sensitive current was also identified as associated with reverse Na⁺-Ca²⁺ exchange based on its internal Na⁺ dependence and sensitivity to XIP. Combined application of external Na⁺ and Ca²⁺ in the absence of internal Na⁺ triggered a 3.3–fold larger inward current than the current activated in the presence of 3 mM internal Na⁺, raising the intriguing possibility that Na⁺-Ca²⁺ exchangers might concurrently operate in both the forward and the reverse direction, perhaps in different subcellular locations. With this idea in mind, we examined the effect of excitotoxic glutamate receptor activation on exchanger operation. After 3–5 min of exposure to 100–200 μM glutamate, the forward exchanger current was significantly increased even when external Na⁺ was reduced to 100 mM, and the external Ca²⁺-activated reverse exchanger current was eliminated.     

The role of inositol trisphosphate on ACh-induced outward currents in bullfrog saccular hair cells

Acetylcholine (ACh) is considered as the most likely candidate for a neurotransmitter of the efferent synapse onto hair cell. In this paper, the nature of this cholinergic receptor mechanism on dissociated bullfrog saccular hair cell was examined by using whole cell recording and Ca²⁺ sensitive fluorophotometric technique. Bothe ACh-induced current and the increase of [Ca²⁺]_i were observed in an oscillatory manner, and were the largest around the basal part of the cell where the efferent synapse is thought to make a contact with the membrane. The reversal potential of ACh-induced current indicated that ACh activated a K⁺ conductance. The ACh-induced current was reversibly blocked by atropine, d-tubocurarine (dTC), apamin, tetraethylammonium (TEA) and quinine. Neither muscarine nor nicotine mimicked the ACh-induced current. When GTPγS was injected into a hair cell, the first ACh application induced an outward current of transient kinetics, but in subsequent trials ACh-induced current lost its decay phase. Intracellularly injectedd-myo-inositol 1,4,5-trisphosphate (InsP₃) generated outward currents. Intracellularly injected heparin suppressed ACh-induced currents, and lithium (Li⁺) increased ACh-induced currents. These results indicate that ACh activates a receptor coupled with a guanine nucleotide binding protein (G-protein) which triggers metabolic cascades of InsP₃ and Ca²⁺leading to the activation of the Ca²⁺-activated K⁺ channel.     

Rectification Properties and Ca2+ Permeability of Glutamate Receptor Channels in Hippocampal Cells

Excitatory amino acids exert a depolarizing action on central nervous system cells through an increase in cationic conductances. Non-NMDA receptors have been considered to be selectively permeable to Na⁺ and K⁺, while Ca²⁺ influx has been thought to occur through the NMDA receptor subtype. Recently, however, the expression of cloned non-NMDA receptor subunits has shown that α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are permeable to Ca²⁺ whenever the receptor lacks a particular subunit (edited GluR-B). The behaviour of recombinant glutamate receptor channels predicts that Ca²⁺ would only permeate through receptors that show strong inward rectification and vice versa, i.e. AMPA receptors with linear current-voltage relationships would be impermeable to Ca²⁺ . Using the whole-cell configuration of the patch-clamp technique, we have studied the Ca²⁺ permeability and the rectifying properties of AMPA receptors, when activated by kainate, in hippocampal neurons kept in culture or acutely dissociated from differentiated hippocampus. Cells were classified according to whether they showed outward rectifying (type I), inward rectifying (type II) or almost linear (type III) current-voltage relationships for kainate-activated responses. AMPA receptors of type I cells (52.2%) were mostly Ca²⁺-impermeable (P_caIP_cs= 0.1) while type II cells (6.5%) expressed Ca²⁺-permeable receptors (P_caIP_cs=0.9).Type III cells (41.3%) showed responses with low but not negligible Ca²⁺ permeability (P_caIP_cs= 0.18). The degree of Ca²⁺ permeability and inward rectification were well correlated in cultured cells, i.e. more inward rectification corresponded to higher Ca²⁺ permeability. In acutely dissociated neurons, the restricted activation of the receptors located either in dendritic or somatic membranes revealed that inward rectifying (i.e. Ca²⁺-permeable) AMPA receptors are preferentially located in the dendritic shaft (i.e. synaptic field). Our results indicate that oligomeric AMPA receptors of different subunit composition are coexpressed in dissimilar proportions in different cells, which would explain the incomplete inward rectification and graded Ca²⁺ permeability. In addition, Ca²⁺-permeable AMPA receptors may exhibit non-homogeneous subcellular distribution.     

Sodium-dependent glutamate uptake as an activator of oxidative metabolism in primary astrocyte cultures from newborn rat

In the present report we describe the effect of glutamate on respiratory activity in primary cultures of astrocytes, derived from cerebral cortex of newborn rat. Glutamate (100 μM) caused an increased oxygen consumption. This effect could not be inhibited by antagonists to the NMDA or AMPA/kainate receptors. Neither trans-ACPD (an agonist to the metabotropic glutamate receptor) nor the Krebs cycle intermediate α-ketoglutarate had any effect on the respiratory rate. An uncontrolled influx of Na⁺, caused by gramicidin, could mimic the glutamate effect on respiratory activity. In addition, the glutamate effect was abolished by addition of ouabain or replacement of Na⁺ by Li⁺ in the perfusion buffer. We conclude that the co-transport of Na⁺, in the Na⁺ -dependent high-affinity glutamate uptake system, mediated the glutamate-induced increase in oxygen consumption through an increased activity of Na⁺/K⁺-ATPases. © 1995 Wiley-Liss, Inc.     

Effects of various cations on the slow K conductance increases induced by carbachol, histamine and dopamine inAplysia neurones

A study was made of the effects of various cations other than K⁺ on three K⁺ conductance increases induced by carbachol, histamine and dopamine in an identified group ofAplysia neurones: the ‘A’ neurones of the cerebral ganglion. The 3 responses were sensitive to alterations of both the extracellular and the intracellular concentrations of Na⁺ and Ca²⁺. In particular, they could be reduced markedly by: (a) lowering [Na]₀ (replacing NaCl by either Tris-HCl, glucosamine chloride, MgCl₂ or sucrose); (b) increasing [Na]_i (by intracellular injection of Na⁺, or by blockade of the Na⁺-K⁺ pump); (c) increasing the extracellular divalent cation concentration; or (d) increasing [Ca]_i⁴.Some of the effects of Na⁺ and divalent cations appear to occur on reaction steps common to the three K⁺ responses, while others probably imply reaction steps specific to one of the systems, since they differ according to the agonist used. The sensitivity to Na⁺ and Ca²⁺ of slow inhibitory responses due entirely to an increase in K⁺ conductance must be taken into account in the interpretation of some slow hyperpolarizing responses previously assumed to involve changes in Na⁺ conductance.     

The ouabain-induced [Ca]i increase in leech Retzius neurones is mediated by voltage-dependent Ca channels

In leech Retzius neurones the inhibition of the Na⁺–K⁺ pump by ouabain causes an increase in the cytosolic free calcium concentration ([Ca²⁺]_i). To elucidate the mechanism of this increase we investigated the changes in [Ca²⁺]_i (measured by Fura-2) and in membrane potential that were induced by inhibiting the Na⁺–K⁺ pump in bathing solutions of different ionic composition. The results show that Na⁺–K⁺ pump inhibition induced a [Ca²⁺]_i increase only if the cells depolarized sufficiently in the presence of extracellular Ca²⁺. Specifically, the relationship between [Ca²⁺]_i and the membrane potential upon Na⁺–K⁺ pump inhibition closely matched the corresponding relationship upon activation of the voltage-dependent Ca²⁺ channels by raising the extracellular K⁺ concentration. It is concluded that the [Ca²⁺]_i increase caused by inhibiting the Na⁺–K⁺ pump in leech Retzius neurones is exclusively due to Ca²⁺ influx through voltage-dependent Ca²⁺ channels.     

Blood monocytes and spleen macrophages differentiate into microglia-like cells on monolayers of astrocytes: Membrane currents

Microglia, the resident macrophages of the central nervous system (CNS), can be distinguished from most other cells of the myelomonocytic lineage by a distinct pattern of memberane currents. In the accompanying paper we have shown that the characteristic morphological feature of microglia, ramification, develops both in microglia and other myelomonocytic cells when they are cocultured with astrocytes. We therefore propose that the electrophysiological properties of microglia also develop under the influence of astrocytes, and moreover, that these properties can also be induced in other cells of the myelomonocytic lineage. Microglia cultured on poly-d-lysine or on a monolayer of fibroblasts possess an inwardly rectifying K⁺ -current only, which is of composite nature. In single-channel recordings two types of K⁺ -channels are found: (i) a non-inactivating channel with a conductance of 43pS, and (ii) an inactivating channel with 32pS. Microglia cultured on a monolayer of astrocytes additionally develop an outward K⁺ -current and a Na⁺ -current. The electric parameters of activation and inactivation of the microglial Na⁺ -current are identical to those of the neuronal Na⁺ -current. Monocytes from peripheral blood and macrophages from spleen exhibit no inward currents. However, when these cells are cocultured with astrocytes they develop microglia-like membrane currents, including the inward and outward K⁺ -rectifyer and the Na⁺ -current. By contrast, on fibroblasts they retain their macrophage current profile. The expression of the microglia-like membrane currents in the monounclear phagocytes is induced by a diffusible factor released from the astrocytes into the culture medium, since monocytes and microglia develop the mature microglial current profile, when cultured in astrocyte conditioned medium. These findings show that the current profile of microglia develops only, when they are in association with astrocytes, and that it is induced in myelomonocytic cells from blood and spleen when these also are associated with astrocytes. These findings add to the growing body of evidence that microglia are derived from the myelmonocytic lineage and indicate that the astrocytes are involved in regulating their morphological and functional properties.     

Single potassium channels in neuropile glial cells of the leech central nervous system

We performed patch-clamp experiments to identify distinct K⁺ channels underlying the high K⁺ conductance and K⁺ uptake mechanism of the neuropile glial cell membrane on the single-channel level. In the soma membrane four different types of K⁺ channels were characterized, which were found to be distributed in clusters. Since no other types of K⁺ channels were observed, these appear to be the complete repertoire of K⁺ channels expressed in the soma region of this cell type. The outward rectifying 42 pS K⁺ channel could markedly contribute to the high K⁺ conductance and the maintenance of the membrane potential, since it shows the highest open probability of all channels. The channel gating occurred in bursts and patch excision decreased the open probability. The outward rectifying 74 pS K⁺ channel was rarely active in the cell-attached configuration; however, patch excision enhanced its open probability considerably. This type of channel may be involved in neuron-glial crosstalk, since it is activated by both depolarizations and increases in the intracellular Ca²⁺ concentration, which are known to be induced by neurotransmitter release following the activation of neurons. The 40 pS and 83 pS K⁺ channels showed inward rectifying properties, suggesting their involvement in the regulation of the extracellular K⁺ content. The 40 pS K⁺ channel could only be observed in the inside-out configuration. The 83 pS channel was activated following patch excision. At membrane potentials more negative than −60 mV, flickering events indicated voltage-dependent gating.     

Developmental regulation of Na+ and K+ conductances in glial cells of mouse hippocampal brain slices

The relative contribution of voltage activated Na⁺ and K⁺ currents to the whole cell current pattern of hippocampal glial cells was analyzed and compared during different stages of postnatal maturation. The patch-clamp technique was applied to identified cells in thin brain slices obtained from animals between postnatal day 5 and 35 (p5-35). We focused on a subpopulation of glial cells in the CA1 stratum radiatum which most probably represents a pool of immature astrocytes, termed “complex” cells. These cells could not be labelled by 01/04 antibodies, but some of the older cells were positively stained for glial fibrillary acidic protein (GFAP). In the early postnatal days, the current pattern of the “complex” cells was dominated by two types of K⁺ outward currents: a delayed rectifier and a transient component. In addition, all cells expressed significant tetrodotoxin (TTX)-sensitive Na⁺ currents. During maturation, the contribution of delayed rectifier and A-type currents significantly decreased. Furthermore, almost all cells after p20 lacked Na⁺ currents. This down-regulation of voltage gated Na⁺ and K⁺ outward currents was accompanied by a substantial increase in passive and inward rectifier K⁺ conductances. We found increasing evidence of electrical coupling between the “complex” cells with continued development. It is concluded that these developmental changes in the electrophysiological properties of “complex” glial cells could be jointly responsible for the well known impaired K⁺ homeostasis in the early postnatal hippocampus. © 1995 Wiley-Liss, Inc.