Biomedical subjects

H Kettenmann

Publications and source records attributed to H Kettenmann.

At least 127 records · Page 7Linked to original sources

PubMed1991

Glutamate and GABA receptors in vertebrate glial cells.

Glial cells of the central nervous system express receptors for the main inhibitory and excitatory neurotransmitters, GABA and glutamate. The glial GABA and glutamate receptors share many properties with the neuronal GABAA and kainate/quisqualate receptors, but are molecularly and, in some aspects, pharmacologically distinct from their neuronal counterparts. The functional role of these receptors is as yet speculative: They have been proposed to control proliferation of astrocytes, serve to balance ion changes at GABAergic synapses, or they could enable the glial cell to detect neuronal synaptic activity.

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PubMed1991

Physiological properties of oligodendrocytes during development.

The electrical properties of oligodendrocytes during their development in cell culture were analyzed by combining two techniques: cell identification with cell-type and stage-specific antibodies and the patch-clamp technique. The transition from the bipotential precursor cell, which can still develop into astrocytes and oligodendrocytes, into an oligodendrocyte results in a marked change in the ion channel pattern. During this developmental transition, voltage-activated Na+ and several types of K+ currents disappear, whereas a comparatively passive, inwardly rectifying K+ current becomes dominant. GABAA receptor-mediated Cl- currents and a pH-activated Na+ current are down-regulated at this transition but are still present at all developmental stages. In contrast, electrical coupling develops only in oligodendrocytes. This change in the channel repertoire could reflect the transition of a cell in a mobile, mitotic, plastic state (the glial precursor) to a more differentiated specialized state (the oligodendrocyte).

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PubMed1991

Developmental changes in the membrane current pattern, K+ buffer capacity, and morphology of glial cells in the corpus callosum slice.

Recent studies indicated that glial cells in tissue culture can express a variety of different voltage-gated channels, while little is known about the presence of such channels in glial cells in vivo. We used a mouse corpus callosum slice preparation, in which after postnatal day 5 (P5) more than 99% of all perikarya belong to glial cells (Sturrock, 1976), to study the current patterns of glial cells during their development in situ. We combined the patch-clamp technique with intracellular labeling using Lucifer yellow (LY) and subsequent ultrastructural characterization. In slices of mice from P6 to P8, we predominantly found cells expressing delayed-rectifier K+ currents. They were similar to those described for cultured glial precursor cells (Sontheimer et al., 1989). A-type K+ currents or Na+ currents were not or only rarely observed, in contrast to cultured glial precursors. LY labeling revealed that numerous thin processes extended radially from the perikaryon of these cells, and ultrastructural observations suggested that they resemble immature glial cells. In slices of older mice (P10-13), when myelination of the corpus callosum has already commenced, many cells were characterized by an almost linear current-voltage relationship. This current pattern was similar to cultured oligodendrocytes (Sontheimer et al., 1989). Most processes of LY-filled cells with such a current profile extended parallel to each other. Electron microscopy showed that these processes surround thick, unmyelinated axons. We suggest that cells with oligodendrocyte-type electrophysiology are promyelinating oligodendrocytes. In contrast to cultured oligodendrocytes, membrane currents of promyelinating oligodendrocytes in the slice decayed during the voltage command. This decay was due not to inactivation, but to a marked change in the potassium equilibrium potential within the voltage jump. This implies that, in the more mature corpus callosum, small membrane polarizations in a physiological range can lead to extensive changes in the K+ gradient across the glial membrane within a few milliseconds.

3,3'-Diaminobenzidine↗

PubMed1990

Swelling-induced changes in electrophysiological properties of cultured astrocytes and oligodendrocytes. I. Effects on membrane potentials, input impedance and cell-cell coupling.

We have studied the membrane potential responses of cultured oligodendrocytes and astrocytes during changes in the osmolarity of the bathing solution. Oligodendrocytes responded with a de- or hyperpolarization when changing to hypo- or hyperosmolar medium, consistent with dilution or concentration of intracellular [K+], respectively. Astrocytes responded with a similar hyperpolarization in hyperosmolar medium that could also be explained by an increase in intracellular [K+]. In hypoosmolar medium, however, a much larger depolarization of astrocytes was observed. These data imply that in astrocytes, in contrast to oligodendrocytes, swelling activates specific channels, which might be involved in volume control. Dual-impalement studies in single cells were used to measure effects of medium hypotonicity on cell input resistance, while dual-impalement studies on neighboring cells were used to measure changes in cell-cell coupling. In oligodendrocytes an increase in apparent cell input resistance and electrical coupling was found. In cultured astrocytes there was no change in apparent cell input resistance but an apparent decrease in cell-cell electrical coupling when cultures were exposed to the lowest medium osmolarity examined (-100 mM NaCl). However, because of the unavoidable contribution of cell-cell electrical coupling to the measurement of membrane impedance by current injection into a single cell a lack of change in apparent cell resistance could be due to offsetting changes in cell-cell coupling.

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PubMed1990

Swelling-induced changes in electrophysiological properties of cultured astrocytes and oligodendrocytes. II. Whole-cell currents.

Using whole cell patch-clamp recordings we have found that swelling cultured cerebrocortical astrocytes or mouse spinal cord oligodendrocytes by perfusing them with hypotonic medium induced inward currents at the normal resting potential of -60 mV. The currents in the oligodendrocytes were always less than for astrocytes. We examined the reversal potentials of these responses by rapidly jumping the holding potential to different values and measuring the currents. We found that the hypotonic medium-induced conductance increase was always preceded by a conductance decrease in the case of oligodendrocytes, but only sometimes preceded by a conductance decrease in cultured astrocytes. The reversal potential of the conductance increase for astrocytes was around -40 mV, while the conductance decrease had a more negative reversal potential of -60 mV or less. For oligodendrocytes the reversal potential for the conductance increase was around -50 mV while the conductance decrease had a reversal potential of -90 mV or less. This suggests that K+ conductance decreased in the initial phase, while the conductance increase was due to additional channel openings. Ion substitution experiments in the case of the astrocytes showed that the reversal potential was shifted to a more positive value when medium K+ was increased, but was unaffected when Na+ was substituted by N-methyl-D-glucamine or Cl- by D-glucuronate, when corrected for liquid junction potential changes. Thus, the channels opened in these cells are likely to include non-specific cation channels. It is of interest that the two cells show a difference in their responses, and in the case of astrocytes these are likely to be involved in the regulatory volume decrease processes documented in these cells.

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PubMed1990

Differential benzodiazepine pharmacology of mammalian recombinant GABAA receptors.

We compared gamma-aminobutyric acid (GABA)-activated currents and their modulation by benzodiazepines in cultured human cells transfected with complementary desoxyribonucleic acid (cDNA) encoding different GABAA receptor subunits. Flunitrazepam, a benzodiazepine agonist which potentiates GABA responses in both neurons and astrocytes was only effective in receptors containing the gamma 2 subunit (alpha 1 beta 1 gamma 2 and alpha 5 beta 1 gamma 2). The beta-carboline methyl-4-ethyl-6,7-dimethoxy-beta-carboline-3-carboxylate (DMCM) decreased GABA-activated currents in receptors composed of alpha 1 beta 1 gamma 1 and alpha 1 beta 1 gamma 2 subunits but increased GABA-activated currents in receptors containing the alpha 5 subunit (alpha 5 beta 1 gamma 1 and alpha 5 beta 1 gamma 2). These results strongly suggest that flunitrazepam and DMCM do not act on isosteric sites and that differences in the responsiveness of GABAA receptors to these compounds are based on different subunit compositions of GABAA receptors.

Carbolines↗

PubMed1990

Cultured glial precursor cells from mouse cortex express two types of calcium currents.

Glial precursor cells of the oligodendrocyte lineage express two types of Ca2+ channels which can be distinguished by their peak activation. Ca2+ currents were recorded using the patch-clamp technique in the whole cell configuration and were found to be similar to the low and the high voltage activated Ca2+ channel described in neurons. While the majority of precursor cells expressed only one of the two current types respectively, in 30% of cells both types of current could be identified. These Ca2+ channels might play an important role in signal transduction during target recognition of the developing glial cell.

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PubMed1990

Intracellular pH shifts capable of uncoupling cultured oligodendrocytes are seen only in low HCO3- solution.

Electrical coupling between cultured mouse oligodendrocytes was transiently blocked when pHi was decreased below about 6.5 using the NH4+ prepulse method. This uncoupling could, however, only be achieved if the dominant pHi regulating mechanism in these cells, the Na+/HCO3- cotransporter, was blocked by lowering bath [HCO3-]. Under this condition, an NH4+ prepulse caused pHi to decrease toward the passive distribution for H+ (i.e., about pH 6.2). In the presence of normal bath [HCO3-] an NH4+ prepulse did not decrease pHi below 6.5 even when the second pHi regulating mechanism, the Na+/H+ exchanger, was blocked by amiloride, and consequently oligodendrocytes could not be uncoupled. Increasing CO2, which uncouples glial cells in situ (Connors et al: J. Neurosci. 4:1324-1330, 1984), did not uncouple cultured oligodendrocytes in the presence of normal bath [HCO3-], but did cause uncoupling in low [HCO3-] solution. These results indicate that electrical coupling between cultured oligodendrocytes is sensitive to pHi; in normal bath [HCO3-], however, the pHi regulation of these cells is so effective that standard techniques for intracellular acidification are unable to lower pHi to levels which cause the closure of oligodendrocyte gap junctions.

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PubMed1990

Electrical coupling, without dye coupling, between mammalian astrocytes and oligodendrocytes in cell culture.

Evidence of electrical and dye coupling between oligodendrocytes and astrocytes was sought in cultures of mouse spinal cord. Cell identity was verified using cell specific antigenic markers. In most experiments current was injected into oligodendrocytes while recording voltage in nearby astrocytes. Nine of 17 oligodendrocyte-astrocyte cell pairs showed weak electrical coupling; the average estimated coupling ratio was 0.03 +/- 0.06 (cf. 0.11 for oligodendrocyte-oligodendrocyte and 0.44 for astrocyte-astrocyte pairs; Kettenmann and Ransom: Glia, 1: 64-73, 1988). Application of 0.5 mM BaCl2 or 44.6 mM CsCl depolarized astrocytes and oligodendrocytes and was estimated to increase the coupling ratio between these cells 3-5-fold; these effects were rapid in onset and completely reversible. In 5 of 7 cases, oligodendrocyte-astrocyte pairs that appeared uncoupled in normal solution exhibited coupling during Ba++ or Cs+ exposure. The actions of these cations are believed to be mediated by blockade of glial K+ channels. Depolarization, per se, as induced by increasing [K+]o, did not increase coupling ratio. The fluorescent dye lucifer yellow (LY) was injected into 10 oligodendrocytes, 8 of which were electrically coupled to nearby astrocytes, and never passed into astrocytes in detectable quantities. Likewise, astrocytes injected with LY stained other astrocytes, but never oligodendrocytes. These findings document the presence of weak electrical coupling between astrocytes and oligodendrocytes, in the absence of dye coupling. Weak coupling of this sort could subserve metabolic interactions between these cells mediated by the passage of small but important molecules such as cyclic AMP, but would not allow strong electrical interactions. If such coupling among glial cells is widespread, it would constitute a "metabolic syncytium" that could serve to coordinate glial behavior.

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PubMed1990

Cultured microglial cells have a distinct pattern of membrane channels different from peritoneal macrophages.

Microglia are the source of the resident macrophages of the brain and thus belong to one of the most reactive cell types in cerebral tissue. They are attributed to have an important role in a number of pathological conditions, such as multiple sclerosis, viral infections like AIDS, and in lethal or sublethal injuries of neurons where the blood-brain barrier is left intact (Streit et al., 1988; McGeer et al., 1988; Gendelman et al., 1989). Microglia share a number of macrophage characteristics but so far lack a distinguishing positive marker. In this study it is shown that microglia are distinguished from other macrophages by a unique pattern of ion channels. We compared membrane currents of microglial cells with those from peritoneal macrophages cultured under identical conditions. Although in macrophages a delayed outward K+ current was previously described (Randriamampita and Trautmann, 1987), microglial cells lacked any specific outward current. Instead, these cells were characterized by large inwardly rectifying currents, activated by hyperpolarizing voltage steps. The reversal potential in different K+ gradients and the sensitivity of the current to to Ba2+, TEA, and 4-AP indicates that this current is K+ selective. In single-channel recordings, a 30 pS K+ selective channel similar to the classical inward rectifier K+ channel was observed. Thus, the expression of membrane channels served not only to distinguish microglia from other cells inside and outside the brain, e.g., blood macrophages, but also suggests a unique functional state of this cell population.

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PubMed1990

Intracellular Na+ activity in cultured mouse oligodendrocytes.

Na(+)-selective double-barrelled microelectrodes were used to measure the intracellular Na+ activity (aiNa) and membrane potential (Em) in oligodendrocytes from cultures of embryonic mouse spinal cord. In Na(+)-free solutions aiNa rapidly fell from its baseline of about 15 mM to values below 1 mM. Elevation of the K+ concentration in the bath ([K+]o) from 5.4 to 15 or 50 mM elicited an aiNa decrease of 4.7 or 9.0 mM, respectively. Ouabain blocked the aiNa decrease in response to 50 mM K+ by 37%. Bath application of 1 mM glutamate resulted in a membrane depolarization of 4.5 mV and a concomitant rise of aiNa by 8.6 mM. aiNa increased by approximately 11 mM after washout of a solution containing 20 mM NH4+. This aiNa increase was not blocked by amiloride, excluding a major contribution of a Na+/H+ antiporter. We conclude that, in cultured oligodendrocytes, transmembraneous Na+ movements are involved in pH regulation, glutamate causes an influx of Na+, and that the Na+/K+ pump and passive KCl uptake contribute to K+ accumulation.

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PubMed1989

Importance of a novel GABAA receptor subunit for benzodiazepine pharmacology.

Neurotransmission effected by GABA (gamma-aminobutyric acid) is predominantly mediated by a gated chloride channel intrinsic to the GABAA receptor. This heterooligomeric receptor exists in most inhibitory synapses in the vertebrate central nervous system (CNS) and can be regulated by clinically important compounds such as benzodiazepines and barbiturates. The primary structures of GABAA receptor alpha- and beta-subunits have been deduced from cloned complementary DNAs. Co-expression of these subunits in heterologous systems generates receptors which display much of the pharmacology of their neural counterparts, including potentiation by barbiturates. Conspicuously, however, they lack binding sites for, and consistent electrophysiological responses to, benzodiazepines. We now report the isolation of a cloned cDNA encoding a new GABAA receptor subunit, termed gamma 2, which shares approximately 40% sequence identity with alpha- and beta-subunits and whose messenger RNA is prominently localized in neuronal subpopulations throughout the CNS. Importantly, coexpression of the gamma 2 subunit with alpha 1 and beta 1 subunits produces GABAA receptors displaying high-affinity binding for central benzodiazepine receptor ligands.

Amino Acid Sequence↗

PubMed1989

Sequence and expression of human GABAA receptor alpha 1 and beta 1 subunits.

The deduced amino acid sequences of cDNA clones encoding human GABAA receptor alpha 1 and beta 1 subunits are presented. The human subunits display very high levels of sequence identity with the corresponding bovine receptor subunits. The cloned human GABAA receptor subunits induce the formation of GABA-gated chloride channels when expressed in mammalian cells.

Amino Acid Sequence↗

PubMed1989

GABA triggers a Cl- efflux from cultured mouse oligodendrocytes.

gamma-Aminobutyric acid (GABA) has been shown to depolarize the membranes of astrocytes and oligodendrocytes taken from different tissues and species. The mechanism mediating this depolarization was identified, in cultured rat brain astrocytes, as an activation of GABA receptor-linked Cl- channels. A subpopulation of cultured oligodendrocytes from mouse spinal cord also responded to GABA with a membrane depolarization. In the present study we demonstrate that, in oligodendrocytes, the depolarization was accompanied by a decrease in intracellular Cl- activity [( Cl-]i) as measured with ion-selective microelectrodes. At rest, [Cl-]i was elevated above the passive distribution, and upon application of GABA, [Cl-]i decreased towards the level of passive distribution. Furosemide blocked the Cl- inward carrier which led to a passive Cl- distribution; in the presence of furosemide, GABA no longer elicited a membrane depolarization or a change in [Cl-]i. We conclude that oligodendrocytes, which were depolarized by GABA, expressed GABA-activated Cl- channels. Oligodendrocytes which were unresponsive to GABA demonstrated a non-passive Cl- distribution indicating that they did express inward-directed Cl- carriers, but no GABA-activated Cl- channels.

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PubMed1989

Activation of substance P receptors leads to membrane potential responses in cultured astrocytes.

Cultured astrocytes from rat cortex and spinal cord responded with different types of membrane potential changes upon brief (10 seconds) applications of the natural neurokinin agonists substance P and neurokinin A. The most prominent type of response was a long-lasting membrane depolarization. In some cells, an initial rapid depolarization followed by a partial repolarization preceded the slow depolarizing event. Few astrocytes responded with a hyperpolarization of the membrane. Selective agonists at the NK-1 receptive site, substance P-methyl ester (SP-OME) and septide, mimicked the response to the natural neurokinins as did DiMe-C7, a selective NK-3 receptor agonist. A putative neurokinin antagonist, (D-Arg1,D-Pro2,D-Trp7,9,Leu11)SP (DADPDT) partially blocked membrane potential responses induced by substance P, SP-OME, septide, DiMe-C7, and NKA. The authors conclude that astrocytes express NK-1 and NK-3 receptors, which upon activation affect the electrical properties of these cells.

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PubMed1989

Pharmacological characterization of the glutamate receptor in cultured astrocytes.

Cultured astrocytes from neonatal rat cerebral hemispheres are depolarized by the excitatory neurotransmitter glutamate. In this study we have used selective agonists of different neuronal glutamate receptor subtypes, namely, the N-methyl-D-aspartate (NMDA), kainate, and quisqualate type, to characterize pharmacologically the glutamate receptor in astrocytes. The agonists of the neuronal quisqualate receptor, alpha-amino-3-hydroxy-5-methyl-4-isoxazole-4-propionic acid (AMPA) and quisqualate, depolarized the membrane. Kainate, an agonist of the neuronal kainate receptor, depolarized astrocytes more effectively than quisqualate. Combined application of kainate and quisqualate depolarized astrocytes to a level which was intermediate to that evoked by quisqualate and kainate individually. Agonists activating the neuronal NMDA receptor, namely NMDA and quinolinate, were ineffective. Application of NMDA did not alter the membrane potential even in combination with glycine or in Mg2+-free solution, conditions under which neuronal NMDA receptor activation is facilitated. The nonselective agonists L-cysteate, L-homocysteate, and beta-N-oxalylamino-L-alanine (BOAA) mimicked the effect of glutamate. Dihydrokainate, a blocker of glutamate uptake, did not, and several antagonists of neuronal glutamate receptors only slightly affect the glutamate response. These findings suggest that astrocytes express one type of glutamate receptor which is activated by both kainate and quisqualate, lending further support to the notion that cultured astrocytes express excitatory amino acid receptors which have some pharmacological similarities to their neuronal counterparts.

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PubMed1989

Carrier-mediated Cl- transport in cultured mouse oligodendrocytes.

We studied the steady state and the regulation of intracellular Cl- activity (aCl-i) and the mechanisms of KCl uptake in cultured oligodendrocytes from mouse spinal cord using Cl(-)-selective microelectrodes. The majority of oligodendrocytes actively accumulated Cl- above passive distribution (2-3 mM), few cells showed a passive Cl- distribution. To identify the carriers mediating Cl- uptake, oligodendrocytes were maintained in a solution with low extracellular Cl- concentration ([Cl-]o) which resulted in a rapid decrease in aCl-i. The recovery of aCl-i above its passive distribution in normal [Cl-]o was blocked in the absence of Na+ or in the presence of furosemide and of bumetanide, which has been reported to inhibit Na+/K+/Cl- cotransport. We therefore conclude that Cl- uptake is primarily due to the activity of a Na+K+/Cl- transport system. Cl- uptake above passive distribution was not affected in HCO3(-)-free solution or in the presence of SITS and DIDS, indicating that Cl-/HCO3- exchange is not involved in Cl- uptake by oligodendrocytes. Elevation of [K+]o induced an increase in aCl-i and, as shown earlier, intracellular K+ activity. This K+-induced Cl- uptake was not blocked by bumetanide, furosemide, SITS, or DIDS, suggesting that under conditions of raised [K+]o the combined uptake of K+ and Cl- is not mediated by a carrier, but can be explained by the entry through channels driven by Donnan forces.

Animals↗

PubMed1989

Glial cells of the oligodendrocyte lineage express proton-activated Na+ channels.

Neurons and oligodendrocytes, but not type I astrocytes and Schwann cells, generate large Na+ currents in response to a step increase of [H+]. Proton-activated Na+ channels are the first cationic channels expressed in neuronal precursor cells from the mammalian brain. Glial precursor cells cultured from mouse brain are also capable of generating Na+ currents in response to step acidification (INa(H]. With further development along the oligodendrocyte lineage, this property is retained, whereas voltage-activated Na+ and K+ currents disappear. Comparing INa(H) of oligodendrocytes with INa(H) of their precursor cells did not reveal a difference in current amplitude, suggesting a higher density of INa(H) channels on the (smaller) precursor cells. The properties of INa(H) in glial precursor cells and oligodendrocytes are similar to those of neurons, with respect to activation conditions, time course, and the effect of extracellular Ca2+ concentrations. The results are consistent with previous observations which showed that oligodendrocytes partially preserve their chemically activated, but completely lose their voltage-activated, ion channels.

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