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H Kettenmann

Publications and source records attributed to H Kettenmann.

At least 91 records · Page 5Linked to original sources
PubMed1995

Expression of functional GABAA receptors in neuroendocrine gastropancreatic cells.

Gastropancreatic neuroendocrine cells synthesize large amounts of gamma-aminobutyric acid (GABA). This amino acid neurotransmitter appears to be stored in and released from, vesicles similar to small synaptic vesicles. So far, the function of GABA in gastropancreatic, neuroendocrine cells has not been clarified. Previous work suggested that only pancreatic, glucagon-producing alpha 2 cells contain functional GABAA receptors. Using subunit-specific antibodies in sections of human antral mucosa, a human gastrinoma and rat pancreas, we show that expression of GABAA receptors is abundant in gastropancreatic, neuroendocrine cells. Using the patch-clamp technique in the whole-cell mode we demonstrate that both the rat insulinoma cell line RIN 38 and the amphicrine cell line AR42J express functional GABAA receptors, which are characterized by a relatively low benzodiazepine and Zn2+ sensitivity and by an insensitivity to the inverse benzodiazepine agonist 6,7-alpha-methoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM). In contrast to neurons, activation of GABAA receptors leads to a membrane depolarization. This depolarization presumably activates voltage-gated Ca2+ channels, resulting in an increase in cytosolic Ca2+ concentration, [Ca2+]i, as shown with the fluorimetric dye fura-2. The combination of GABA release, GABAA receptor activation and the [Ca2+]i increase could constitute an autocrine mechanism, modulating the release of hormones such as gastrin, insulin and somatostatin.

Animals↗
PubMed1995

Modulation of potassium currents in cultured murine microglial cells by receptor activation and intracellular pathways.

The electrophysiological properties of ameboid microglia from rodent brain are dominated by inwardly rectifying potassium channels and by the lack of outward currents. This channel pattern results in a distinct physiological behavior: depolarizing events, e.g. following adenosine triphosphate receptor activation, can lead to a long lasting membrane depolarization. Here we address the question whether this resting K+ channel activity can be modulated. Intracellular application of guanosine 5'-O-(3-thiotriphosphate) induced an outward current and led to a complete disappearance of the inward current inward rectifier potassium current as measured with the patch clamp technique. Moreover, an elevation in cytosolic calcium concentration (to 1.6 microM) via intracellular perfusion reversibly blocked the inward current. The inhibition of inward currents by guanosine 5'-O-(3-thiotriphosphate) could be enhanced by additional adenosine triphosphate receptor activation. Adenosine triphosphate or tumor necrosis factor receptor activation alone could lead to a transient partial block of the inward rectifier and to the transient appearance of a delayed outward current. We conclude that the activity of the microglia K+ channels and thus the physiological behavior of microglia can be modulated on a time scale of seconds by receptor activation and distinct intracellular pathways.

Adenosine Triphosphate↗
PubMed1995

Physiology of Bergmann glial cells.

While Bergmann glial cells play an important role in the development of the cerebellum they were thought to serve as passive insulators of the Purkinje cell dendritic tree and its synaptic connections. New results challenge this view and demonstrate that Bergmann glial cells are equipped with a large repertoire of receptors allowing them to sense the activity of synapses. These receptors have distinct biophysical and pharmacological features activating second-messenger pathways in the Bergmann glial cells. It is evident that the synapse has to be viewed as consisting of three elements, the presynaptic and postsynaptic region and the glial ensheathment. All three elements of this synaptic complex may undergo plastic changes as a prerequisite for central nervous system plasticity. Glial cells could interfere with synaptic transmission by communicating with neurons via the extracellular space, e.g., by modulating ion concentrations or transmitter levels in the cleft (Fig. 6).

Animals↗
PubMed1995

Developmental regulation of ion channels and receptors on glial cells.

The electrophysiological properties of glial precursor cells and oligodendrocytes were studied in an intact tissue preparation, the corpus callosum slice, with the patch-clamp technique. The pattern of voltage-gated currents exhibited by different cell types in this white matter tract was compared with that from cultured cells of the O-2A lineage. Precursor cells from the in vitro and the in situ preparation were strikingly similar in most aspects. In contrast, oligodendrocytes in the intact tissue differed from their culture correlates and displayed the ability to redistribute excess K+. To study glial transmitter receptors in neuronglial interactions during development, Bergmann glial cells were investigated in a slice preparation of the cerebellum.

Animals↗
PubMed1995

GABAA/benzodiazepine receptors in acutely isolated hippocampal astrocytes.

The properties of GABA receptor-mediated responses were examined in noncultured astrocytes, acutely isolated from the mature rat hippocampus. Whole-cell patch clamping revealed a GABA-activated Cl- conductance that was mimicked by the GABAA receptor agonist muscimol and depressed by the GABAA antagonists bicuculline and picrotoxin. The GABAA-activated currents were potentiated by the barbiturate pentobarbital and the benzodiazepine diazepam. The benzodiazepine inverse agonist DMCM either enhanced or depressed the astrocytic GABAA-mediated responses, suggesting receptor heterogeneity with respect to pharmacologic profiles. In addition, GABA evoked an increase in [Ca2+]n measured by indo-1 fluorometry, which was depressed in the presence of verapamil or picrotoxin. A GABAA-induced depolarization, therefore, causes Ca2+ influx through voltage-gated Ca2+ channels. The expression and subcellular localization of GABAA receptors and its subunits were examined using immunohistochemical and fluorescent benzodiazepine binding techniques. Polyclonal antisera raised against the GABAA/benzodiazepine receptor, which recognizes multiple subunit isoforms, labeled receptors on the astrocytic cell body and most large processes. In contrast, antisera generated against either alpha 1 or beta 1 subunit peptides revealed immunoreactivity predominantly on a subset of processes. To determine the subcellular distribution of membrane-bound receptors, a fluorescent benzodiazepine derivative was superfused over live astrocytes and visualized with laser-scanning confocal microscopy. Specific fluorescence was distributed in discrete clusters on the cell soma and a subset of distal processes. Collectively, these data support the view that astrocytes, like neurons, express GABAA receptors and target subunit isoforms to distinct cellular localizations. Astrocytic GABAA receptors may be involved in both [Cl-]o and [pH]o homeostasis, and a GABA-evoked increase in [Ca2+]i could serve as a signal between GABAergic neurons and astrocytes.

Amino Acid Sequence↗
PubMed1995

ATP-induced cytoplasmic calcium mobilization in Bergmann glial cells.

ATP receptor mediated Ca2+ signaling was recorded from Bergmann glial cells in cerebellar slices obtained from mice of different ages (postnatal days 6 to 45). To measure the cytoplasmic concentration of Ca2+ ([Ca2+]in), either individual cells were loaded with the Ca(2+)-sensitive probes using the whole cell patch clamp technique or slices were incubated with the dye and the microfluorimetric system was focused on individual cells. Signals were recorded either with single-detector microfluorimetry of the dye fura-2 or by confocal laser scanning microfluorimetry (fluo-3-based recordings). Extracellular application of 100 microns ATP caused a transient elevation of [Ca2+]in, which amplitude was significantly higher in Bergmann glial cell processes as compared with their soma. The rank order of potency for the purinoreceptor agonists was: ADP > or = ATP > UTP >> AMP = adenosine = alpha, beta-methylene-ATP. ATP-triggered Ca2+ transients were reversibly inhibited by the P2 purinoreceptor agonist suramin (100 microM). The involvement of P2 metabotropic receptors is inferred by the observation that ATP mediated cytoplasmic Ca2+ transients were not associated with a measurable change in membrane conductance. The [Ca2+]in increase was due to release from inositol-1,4,5-trisphosphate (InsP3)-sensitive intracellular stores since responses were still observed in Ca(2+)-free extracellular solutions and were irreversibly blocked by the inhibitor of the sarco(endo)plasmic reticulum Ca2+ ATPase, thapsigargin, and by the competitive inhibitor of the InsP3-gated intracellular Ca2+ channels heparin. Intracellular dialysis altered the refilling process of the InsP3-sensitive stores, suggesting that cytoplasmic factors control ATP mediated Ca2+ signalling.

Adenosine Triphosphate↗
PubMed1994

Astroglia-released factor shows similar effects as benzodiazepine inverse agonists.

Media conditioned by cultured neonatal cerebral cortex microexplants (CCM) or astrocytes (ACM) contain low molecular weight (< 1,000 Da) substance(s) which inhibits the gamma aminobutyric acid (GABA)-induced inward current recorded in cerebellar granule cells and hippocampal neurons in culture using the whole-cell patch-clamp technique. This effect is specific for CCM and ACM, as medium conditioned by PC12 cells (PC12CM) does not affect the GABA response of these cells. It is also specific for GABA-induced currents because glutamate-induced currents do not change either in amplitude or in shape in the presence of CCM or ACM. The inhibitory effect on the GABA response in cerebellar granule cells of both ACM and CCM could be suppressed by flumazenil, a specific benzodiazepine (BZD) antagonist and could be mimicked by two BZD inverse agonists. These data thus demonstrate the presence of a BZD inverse agonist-like activity in CCM and ACM. This effect of ACM on different neuronal cell types was heterogenous since no detectable effect could be observed on the GABA-induced current in GABA-responsive dorsal root ganglion (DRG) neurons, presumably reflecting a functional heterogeneity of the GABAA receptors present in these different neuronal subsets. By the release of such an endogenous BZD inverse agonist-like activity, glia cells could possibly modulate GABAA receptor-mediated responses.

Animals↗
PubMed1994

Extracellular ATP-induced currents in astrocytes: involvement of a cation channel.

Whole-cell currents were measured with the perforated patch clamp technique in cultured rat astrocytes to analyze the underlying ionic mechanism for a P2-purinoceptor-mediated depolarization. ATP (100 microM) induced an inward current with a mean amplitude of 130 pA and an EC50 of 17 microM. The response desensitized during a 1 min application. Replacement of extracellular Na+ with NMDG or K+ abolished the ATP-evoked inward current. Replacement of Na+ with choline, however, resulted in an ATP-evoked response of one-third the amplitude in normal solution. This is indicative of a cation rather than Na+ channel. However, due to difficulties in voltage-clamping these gap junction-coupled cells at voltages different from the membrane resting potential, the current reversal potential could not be determined. Measurements with K(+)-sensitive microelectrodes showed that 100 microM ATP lowered the intracellular K+ concentration. Replacement of extracellular Ca2+ or Cl- did not alter the ATP-induced inward currents. Fura-2 imaging experiments revealed a transient rise of the intracellular Ca2+ concentration during ATP application. Removal of extracellular Ca2+ did not influence the peak response; it did, however, shorten the time course. These results and previous observations that the permeability changes are caused by a P2x receptor are indicative of an ATP-sensitive cation conductance. In addition, cytoplasmic Ca2+ is increased by mobilization from intracellular stores, and by additional influx across the cell membrane. Extracellular ATP released by neurons could evoke K+ release from astrocytes as well as be a mediator for cation changes that signal cell activation processes when released by damaged cells.

Adenosine Triphosphate↗
PubMed1994

In vitro and in vivo characterisation of glial cells immortalised with a temperature sensitive SV40 T antigen-containing retrovirus.

An oncogene-carrying replication-defective retrovirus was used to establish immortalised lines of murine glial cells. Primary cultures of early postnatal cerebellar cells were infected with a retrovirus based on the Murine Moloney Leukemia Virus containing a temperature-sensitive mutant of the Simian Virus 40 large T antigen (SV40 T) oncogene and a gene coding for resistance to the antibiotic G418. Infected cells were selected in G418 and after several in vitro passages cells expressing the O4 antigen were established as a cell line. At a later time point O4-positive single-cell clones were established. Two different types of clones were obtained: 1) "plastic" clones consisting of cells which initially had a morphological and antigenic phenotype of young glial precursor cells but which gradually lost these features, and 2) "stable" cell clones including a clone with the immunological and electrophysiological characteristics of Schwann cells. Culture of the latter cells in the presence of 1 mM dibutyryl cyclic adenosine monophosphate for a period of at least 10 days induced a change in shape and a shift in antigen expression towards a more "differentiated" maturation stage. When the SV40 T O4-positive immortalised cell line isolated on the cell sorter was transplanted into demyelinated lesions in adult rats, cells were observed ensheathing axons and forming limited amounts of PNS-type myelin. Glial cells immortalised with a temperature-sensitive mutant of the SV40 T oncogene thus retain many physiological properties of their primary culture counterparts and can be induced to undergo limited differentiation in vitro and in vivo. These cell lines, which represent immature CNS glia or Schwann cells, are providing useful tools for investigating the role of cell surface antigens involved in neuron-glial interactions.

Animals↗
PubMed1994

Properties of GABA and glutamate responses in identified glial cells of the mouse hippocampal slice.

In this study, the patch-clamp technique was applied to brain slices to test for the presence of GABAA and glutamate receptors in glial cells of an intact tissue preparation, the hippocampus from 9-12 day old mice. Two types of glial cells were studied in the CA1 stratum pyramidale, termed passive and complex cells, which were distinct by their characteristic pattern of voltage-dependent currents. Both cell types were previously identified as glial by combining electrophysiology with ultrastructural inspection (Steinhüser et al., 1992, Eur J Neurosci 4:472-484). A subpopulation of passive cells was positive, all complex cells were negative for immunocytochemical staining against glial fibrillary acidic protein, a marker of mature astrocytes. In both cell types, GABA activated currents compatible with GABAA-receptor mediated responses. The glutamate response in complex and in most of the passive cells was mediated by a ligand-gated ion channel and closely matched the pharmacology of the kainate receptor. Activation of glutamate receptors led to a transient decrease of the resting K+ conductance in complex cells and to an irreversible decrease in the passive cells. In three passive cells, glutamate-activated currents were most likely dominated by an electrogenic uptake. In a small group of passive cells NMDA-activated currents were observed. This study provides evidence that glial cells from an intact tissue express receptors for the most abundant transmitters in the central nervous system, glutamate, and GABA.

Animals↗
PubMed1994

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.

Animals↗
PubMed1994

AMPA/kainate receptor activation in murine oligodendrocyte precursor cells leads to activation of a cation conductance, calcium influx and blockade of delayed rectifying K+ channels.

Studies during the last few years have shown that glial cells can express a large repertoire of neurotransmitter receptors. In this study, we have characterized the properties of a glutamate receptor in oligodendrocytes and their precursor cells from cultures of mouse brain, using the patch-clamp technique to measure ligand-activated currents and a fura-2 imaging system to determine changes in free cytosolic Ca2+ concentration ([Ca2+]i). The precursor cells were identified by their characteristic morphology and their voltage-gated currents as described previously [Sontheimer H. et al. (1989) Neuron 2, 1135-1145]. The ligands kainate, domoate and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA), as well as L-glutamate but not trans-1-amino-1,3-cyclopentanedicarboxylate elicited inward currents at a holding potential of -70 mV and the antagonist 6-cyano-7-nitroquinoxaline-2,3-dione blocked the glutamate- and kainate-induced response reversibly, indicating the expression of an AMPA/kainate-type glutamate receptor. The response is due to the activation of a cationic conductance as revealed by analysing the reversal potential of the kainate-activated current. Receptor activation is accompanied by two additional responses: (i) an increase in [Ca2+]i mediated by depolarization and a subsequent activation of voltage-gated Ca2+ channels and (ii) a transient blockade of a delayed rectifying K+ current, but not of the A-type K+ current. The blockade of the K+ current was not due to the increase in [Ca2+]i since it was also observed in Ca(2+)-free bathing solution when no increase in [Ca2+]i was detectable after exposure to kainate. In contrast to precursor cells, oligodendrocytes responded weakly or not at all to glutamate or related ligands. We conclude that glutamate activates a complex pattern of physiological events in the glial precursor cells, which may play a role during the differentiation process of these cells.

Animals↗
PubMed1994

Developmental regulation of voltage-gated K+ channel and GABAA receptor expression in Bergmann glial cells.

Bergmann glial cells are closely associated with neurons: during development they provide guiding structures for migrating granule cells and in the adult cerebellum they display intimate interactions with Purkinje cells. In this study, we have addressed the question of whether such changes in neuronal-glial interactions during development are accompanied by variations in the membrane properties of Bergmann glial cells. We used a mouse cerebellum slice preparation to study membrane currents of the Bergmann glial cells at various stages of development in situ using the patch-clamp technique. The distinct morphology of Bergmann glial cells was revealed by Lucifer yellow injections during recording. While Bergmann glial cells in mice of postnatal day 20 (P20) to P30 have thick processes with arborized, irregularly shaped leaf-like appendages, the processes of cells from younger mice (P5-P7) are thinner and smoother. This morphological maturation is accompanied by a variation in voltage-gated currents. In cells from P5 to P7, delayed outward- and inward-rectifying K+ currents were recorded, while older Bergmann glial cells were characterized by, large, voltage- and time-independent K+ currents. In addition, application of GABA induces two effects, a rapid activation of a Cl- conductance and a longer-lasting decrease in the (resting) K+ conductance. Both effects were mediated by benzodiazepine-insensitive GABAA receptors. Responses in cells of P5-P7 mice were large as compared to the small or even undetectable responses in P20-P30 cells. These GABAA receptors were characterized immunohistochemically in mice and rat brain sections with five subunit-specific antibodies. Bergmann glial cells exhibit a distinct but transient immunoreactivity for the GABAA receptor alpha 2-, alpha 3-, and delta-subunits. Staining is maximal between P7 and P10 and decreases gradually thereafter. In contrast, antibodies to the alpha 1- and beta 2,3-subunits fail to decorate Bergmann glial cells, although they yield a prominent staining of both the Purkinje cells and the granule cells. These changes in the Bergmann glial cell membrane properties and GABAA receptor expression suggest a transition between functional states during development of the Bergmann glial cells.

Aging↗
PubMed1993

Identification of neuropeptide Y receptors in cultured astrocytes from neonatal rat brain.

Specific binding sites for neuropeptide Y could be demonstrated in primary cultures of astrocytes from neonatal rat brain. Neuropeptide Y binding was saturable, reversible, and temperature dependent as revealed by saturation studies and kinetic experiments. Scatchard analysis of equilibrium binding data indicated a single population of high-affinity binding sites with respective KD and Bmax values of 0.43 nM and 6.9 fmol/2.7 x 10(5) cells. Physiological responses induced by neuropeptide Y could be detected in a distinct subpopulation of cultured astrocytes on the basis of two criteria: 1) electrophysiological responses and 2) single cell measurements of changes in [Ca2+]i. In that fraction of cells responding (20-70%, varying among cultures from different preparations), brief application of neuropeptide Y led to a membrane potential depolarization, lasting several minutes. When the membrane was clamped close to the resting membrane potential using the whole-cell patch-clamp technique, neuropeptide Y induced an inward current with a similar time course as the neuropeptide Y-induced membrane depolarization. As detected by single cell microfluorimetric (fura-2) measurements neuropeptide Y induced an increase of [Ca2+]i which was caused by the entry of extracellular Ca2+. Both the [Ca2+]i increase and the electrophysiological responses were unaffected by pretreatment of the astrocytes with pertussis toxin.

Adenosine Diphosphate Ribose↗
PubMed1993

Benzodiazepine-induced motor impairment linked to point mutation in cerebellar GABAA receptor.

The selectively outbred alcohol-non-tolerant (ANT) rat line is highly susceptible to impairment of postural reflexes by benzodiazepine agonists such as diazepam. ANT cerebella are generally devoid of diazepam-insensitive high-affinity binding of the benzodiazepine [3H]Ro15-4513, whereas in non-selected strains such binding marks a granule-cell-specific GABAA (gamma-aminobutyric acid) receptor containing the alpha 6 subunit. A critical determinant for diazepam insensitivity of this 'wild-type' cerebellar GABAA receptor is an arginine residue in alpha 6 position 100, where other alpha subunits carry a histidine. Here we report that the alpha 6 gene of ANT rats is expressed at wild-type levels but carries a point mutation generating an arginine-to-glutamine substitution at position 100. In consequence, alpha 6(Q100)beta 2 gamma 2 receptors show diazepam-mediated potentiation of GABA-activated currents and diazepam-sensitive binding of [3H]Ro15-4513. Our results suggest that cerebellar motor control may be a distinct behavioural correlate of the alpha 6-subunit-containing GABAA receptor subtype.

Affinity Labels↗
PubMed1993

Electrophysiological behavior of microglia.

The present knowledge of voltage- and ligand-activated ion channels of cultured microglial cells is described and its relevance is discussed. All microglial cells cultured from rat or mouse brain express an inward rectifying K+ channel but no outward currents. This expression is not changed by the length of the cultivation period, nor is it different in freshly isolated cells. It makes the microglial cells distinct from peritoneal macrophages, which possess an outward rectifying K+ channel. In bone marrow, 2 populations of cells could be distinguished electrophysiologically, one with the channel pattern of macrophages and one with that of microglial cells. This finding is interesting in light of the fact that it is presently hypothesized that the differentiation of monocytes into microglia takes place exclusively during embryonic development but not in the adult. The available data thus support the hypothesis that within the bone marrow a population of macrophage precursor cells exists with a possible lineage relationship to brain macrophages. The lack of outward currents in the microglial cells has the functional consequence that even a small inward current leads to a large membrane depolarization, since K+ outward currents are not activated with the depolarization. The microglial cell is thus very sensitive to depolarizing events. We found that ATP induced an inward current and an increase in the conductance, whereas ADP, AMP, and adenosine did not. These relative potencies indicate that microglia possess a P2 purinoceptor linked to an ion channel. The amplitude of the inward current elicited by ATP is about 80 pA and is sufficient to depolarize microglial cells close to 0 mV.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1993

Development of cell-cell coupling among cells of the oligodendrocyte lineage.

The development of functional gap-junctional communication was studied in cells of the oligodendrocyte lineage. The presence of cell-cell coupling was determined by the passage of current between cells using a double whole-cell patch-clamp system or by injecting the low molecular weight dye Lucifer yellow into individual cells via a patch pepette and observing the diffusion of the dye into adjacent cells. The developmental stage of the cells under study was determined using antibodies to specific surface markers (04, 01, and 010) that characterize cells of sequential maturity along the oligodendrocyte lineage (Kuhlmann-Krieg et al., 1988; Sommer and Schachner, 1981; 1982). Both stages of precursor cells of this lineage, O4+ and O4-, almost never showed dye or electrical coupling, even though they were in close physical contact with other cells. The O1-positive oligodendrocytes with simple morphology were also noncoupled, with only few exceptions. In contrast, more than 40 percent of more mature, O10-positive oligodendrocytes showed cell-cell coupling detectable with both dye and current injection. Thus, the formation of gap junctions between cells of the oligodendrocyte lineage does not occur with, but some time after, the commitment of the cell to becoming an oligodendrocyte.

Animals↗