Biomedical subjects

H Kettenmann

Publications and source records attributed to H Kettenmann.

At least 19 recordsLinked to original sources

PubMed1992

Bradykinin receptors in cultured astrocytes from neonatal rat brain are linked to physiological responses.

Specific binding sites for bradykinin (BK) have recently been demonstrated on astrocytes of primary cultures from neonatal rat brain. In this study we demonstrate that BK induces membrane currents in concert with an elevation of [Ca2+]i. In 67% of astrocytes, BK induced an inward current as determined with the perforated patch-clamp technique in the whole-cell recording configuration. In a small population of astrocytes (20%), a BK-activated outward current was observed, while in the remainder of the cells (13%) no apparent current responses were detected. As recorded by fura-2 microfluorimetry, the peptide induced a transient rise of [Ca2+/bdi even when the extracellular calcium was removed. In the majority of astrocytes, the selective B1-agonist des-Arg9-BK elicited physiological responses with a much lower potency, indicating that the BK receptors are predominantly of the B2 subtype. A minor population of astrocytes was present which only responded to des-Arg9-BK.

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PubMed1992

Calcium entry through kainate receptors and resulting potassium-channel blockade in Bergmann glial cells.

Glutamate receptors, the most abundant excitatory transmitter receptors in the brain, are not restricted to neurons; they have also been detected on glial cells. Bergmann glial cells in mouse cerebellar slices revealed a kainate-type glutamate receptor with a sigmoid current-to-voltage relation, as demonstrated with the patch-clamp technique. Calcium was imaged with fura-2, and a kainate-induced increase in intracellular calcium concentration was observed, which was blocked by the non-N-methyl-D-aspartate (NMDA) glutamate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and by low concentrations of external calcium, indicating that there was an influx of calcium through the kainate receptor itself. The entry of calcium led to a marked reduction in the resting (passive) potassium conductance of the cell. Purkinje cells, which have glutamatergic synapses, are closely associated with Bergmann glial cells and therefore may provide a functionally important stimulus.

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PubMed1992

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.

Action Potentials↗

PubMed1992

An immortalized cell line expresses properties of activated microglial cells.

Murine cultured microglial cells were immortalized after infection with a v-raf/v-myc recombinant retrovirus. This immortalized cell line (BV-2) shares properties with body macrophages with respect to the antigen profile, their phagocytic capacity and antimicrobial activity. BV-2 cells are not constitutively able to kill tumor cells in vitro, but acquire antitumor activity following an increase in [Ca++]i. BV-2 cells, like microglial cells, are however, distinct from peripheral macrophages by their expression of inwardly rectifying K+ channels in concert with a lack in outwardly rectifying K+ channels and the formation of spineous processes. The BV-2 cell line thus represents a suitable model for in vitro studies of activated microglial cells.

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PubMed1992

A fast perfusion system for single cell physiology optimized for microscopes with water immersion objectives.

A perfusion system was constructed which allows the fast application of different solutes underneath a water immersion objective. The perfusion system is mounted into the immersion objective by milling a slot into the frontal metal plate of the lens holder. It consists of a five-channel pipette fixed to the objective and solution reservoirs gated by computer controlled magnetic valves. Up to five different solutions can be applied to the specimen under study. The solution between objective and specimen is completely exchanged after 1-2 s as determined from fluorescence measurements. This arrangement is optimized for [Ca2+] measurements with a fluorescence measurement system in tissue slices, where upright microscopes are required. It offers the advantage of saving a micromanipulator for the perfusion pipette and facilitates a fast, reproducible and precise positioning of the perfusion system.

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PubMed1991

Inverse but not full benzodiazepine agonists modulate recombinant alpha 6 beta 2 gamma 2 GABAA receptors in transfected human embryonic kidney cells.

We compared the modulation of GABA (gamma-aminobutyric acid)-activated currents by benzodiazepines in recombinant GABAA receptors containing either one of two alpha subunits, alpha 1 or alpha 6. Lüddens et al. (Nature, 346 (1990) 648-651) have previously demonstrated that the alpha 6 subunit is part of a cerebellar receptor subtype which selectively binds Ro15-4513, an antagonist of alcohol-induced motor ataxia. Here we report that the imidazobenzodiazepine Ro15-4513 (ethyl 8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo-(1,5-a) (1,4)benzodiazepine-3-carboxylate) reduced GABA-activated currents in recombinant alpha 6 beta 2 gamma 2 and alpha 1 beta 2 gamma 2 receptors, thus acting consistently as an inverse agonist. Moreover, another well characterized negative modulator, DMCM (methyl-4-ethyl-6,7-dimethoxy-beta-carboline-3-carboxylate), also reduces GABA activated-currents in both receptors. In contrast, flunitrazepam (FNZM), a benzodiazepine agonist, increases GABA-activated currents in alpha 1 beta 2 gamma 2 receptors, but not in alpha 6 beta 2 gamma 2 receptors. This study lends further support to the hypothesis that the binding sites of full and partial inverse agonists are different.

Action Potentials↗

PubMed1991

Characteristics of activity-dependent potassium accumulation in mammalian peripheral nerve in vitro.

Ion-sensitive microelectrodes were used to study the behavior of extracellular ions in rat sciatic nerve during and following activity. Nerve stimulation produced increases in [K+]o that were dependent upon the frequency and duration of stimulation; no change in extracellular pH occurred with stimulation. Increases in [K+]o depended on axonal discharge since they were blocked by inhibiting sodium channels with tetrodotoxin. At 22 degrees C, stimulation could induce increases in [K+]o of several mM; at 36 degrees C, stimulation rarely produced increases in [K+]o greater than 1 mM. Stimulated increases in [K+]o dissipated very slowly (i.e. t 1/2 = 50-100 s) and the rate of dissipation was not significantly affected by anoxia, changes in temperature, changes in extracellular pH, or the application of a blocker of Na+, K(+)-ATPase (ouabain) or a K+ channel blocker (Ba2+). In comparison to the central nervous system, neural activity in rat sciatic nerve produced smaller increases in [K+]o and these increases dissipated much more slowly. The primary mechanism of K+ dissipation appeared to be diffusion, probably facilitated by the larger extracellular space in peripheral nerve compared to the central nervous system, but impeded by diffusion barriers imposed by the blood-nerve barrier.

Action Potentials↗

PubMed1991

Activation of neurokinin receptors modulates K+ and Cl- channel activity in cultured astrocytes from rat cortex.

Short application of the neurokinin receptor agonist substance P (SP) leads to a biphasic depolarization of astrocytes cultured from rat cortex. The rapid and transient depolarizing event lasted few seconds, the slow one several minutes. In some cells, only the slow depolarizing component was observed. During the slow depolarizing event, the sensitivity of the membrane potential for a change in the K+ gradient decreased, indicating a decrease in the relative K+ permeability of the membrane. The rapid SP-induced depolarization could be reversed, when the membrane potential was depolarized to about 0 mV by elevation of the extracellular K+ concentration, indicating a reversal potential close to the Cl- equilibrium potential. When the membrane was clamped close to the resting membrane potential using the whole-cell patch-clamp technique, SP induced a biphasic inward current with a similar time course as the SP-induced membrane depolarization. Evaluating current-to-voltage curves indicated a conductance decrease during the slow inward current with a reversal potential of the SP-dependent current close to the K+ equilibrium potential. The mean open time of single K+ channels, measured in the cell-attached configuration of the patch-clamp technique, decreased after application of SP. In contrast, the mean open time of single Cl- channels increased. We conclude that activation of neurokinin receptors in astrocytes modulates the activity of K+ and Cl- channels, leading to a complex depolarization of the membrane potential.

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PubMed1991

Single K+ channel properties in cultured mouse Schwann cells: conductance and kinetics.

Cultured Schwann cells are characterized by a strong outward rectification of the membrane; the threshold of the outward currents is close to the resting membrane potential of about -50 mV (Gray et al.: In Ritchie, Keynes (eds): Ion Channels in Neural Membranes. New York: Alan R. Liss, Inc., pp 145-157, 1986). These outward currents show up a heterogeneity among the cultured Schwann cells: some cells displayed inactivating, others non-inactivating outward currents (Hoppe et al.: Pflügers Arch 415:22-28, 1989). In this study we characterized the single channel currents using the patch-clamp technique in the intact patch recording configuration. The conductance of all recorded channels was 10-12 pS (5.6 mM [K+]o). These channels were K+ selective since changes in extracellular [K+] resulted in changes of the reversal potential as predicted for an exclusively K+ selective pore. The reversal potentials also predicted an intracellular [K+] of 60 mM indicating that the K+ equilibrium potential is slightly negative to the membrane potential. Analysis of the kinetic behavior of the channels resolved two different types of behaviour: 40% inactivated during a depolarizing voltage step, the others showing no sign of inactivation. The analysis of open probability and gating properties in the steady state showed up more differences between these two channel types: mean open probability peaked at about 10 mV for inactivating channels, while it continuously increased for non-inactivating channels. The inactivation time constants of averaged single channel and whole cell currents were similar and showed both a similar voltage dependency. We conclude that cultured Schwann cells express either two types of K+ channels with similar conductance or a channel which can acquire two functional states and that these channels can account for the different types of K+ currents observed in these cells.

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PubMed1991

K+ channel properties in cultured mouse Schwann cells: dependence on extracellular K+.

In cultured Schwann cells, single-channel and whole-cell K+ currents can be activated by depolarizing the membrane to values more negative than -50 mV. In elevated extracellular K+ concentration ([K+]o), however, single-channel activity and whole-cell currents could be recorded at more negative potentials. Thus, the threshold of current activation was shifted to more negative potentials. This shift in the activation threshold was only observed with normal (50-60 mM) intracellular [K+] levels; it was not apparent when [K+]i was elevated to 145 mM. The control of [K+]o on the gating properties of K+ channels may serve to enhance the capability of the Schwann cell to take up [K+]o and thus may serve for [K+] homeostasis in the peripheral nerve.

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PubMed1991

A subpopulation of bone marrow-derived macrophage-like cells shares a unique ion channel pattern with microglia.

Rat microglia share a number of antigenic, functional, and morphological similarities with macrophages from other tissues, but are characterized by a distinctly different pattern of ion channels in the cellular membrane (Kettenmann et al., J Neurosci Res 26:278-287, 1990). Macrophages typically express outward and inward K+ currents. In contrast, microglia lack outward currents and only show inwardly rectifying K+ currents, regardless of the isolation or cultivation method employed for microglia. In this study we demonstrate that a subpopulation of bone marrow-derived macrophage-like cells possesses inward rectifier K+ currents, but no outward currents and thus with regard to the electrophysiological characteristics closely resembles microglia. A second population of bone marrow-derived macrophage-like cells shows the usual channel pattern described for other body macrophages. Our results strengthen the hypothesis that in the bone marrow distinct pools of precursor cells exist, possibly reflecting an early differential lineage determination for body and brain macrophages, i.e., microglia.

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PubMed1991

Two populations of glial cells from fish optic nerve/tract with distinct electrophysiological properties.

The electrophysiological properties of the two major glial cell types in cultures from the regenerating goldfish optic nerve/tract were studied with patch-clamp techniques. Spindle-shaped cells express myelin proteins. These oligodendrocyte-like cells possess outwardly rectifying currents, do not show glutamate activated currents and are rarely electrically coupled to neighboring cells. Cells of epitheloid morphology probably represent astrocytes. They are GFAP-positive and do not exhibit myelin proteins. These cells have glutamate activated currents, display a linear current to voltage relationship and are extensively electrically coupled thus displaying properties similar to mammalian astrocytes.

4-Aminopyridine↗

PubMed1991

Effect of steroids on gamma-aminobutyrate-induced currents in cultured rat astrocytes.

Cultured astrocytes from rat cortex respond to the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) by the activation of Cl- channels [Bormann J, Kettenmann H (1988) Proc Natl Acad Sci USA 85:9336-9340]. The glial response shares many pharmacological properties with those mediated by neuronal GABAA receptors, but differs in its sensitivity to inverse benzodiazepine agonists [Backus KH, Kettenmann H, Schachner M (1988) Glia 1:132-140]. To compare glial GABA receptors further with their neuronal counterparts, we analysed the effect of steroids, which have recently been shown to modulate neuronal GABAA-receptor-mediated responses, on GABA-induced currents in astrocytes. The agonist allotetrahydrodeoxycorticosterone (THDOC) at concentrations of 100 nM and 1 microM enhanced GABA-evoked (with 10 microM GABA) currents up to 115% and 162.4% of controls respectively. The antagonist dehydroisoandrosterone 3-sulphate (DHEAS) at concentrations of 1 microM, and 100 microM depressed GABA-evoked (10 microM) currents to 72%, 42.8% and 21.4% of controls respectively. The steroids were less effective at higher GABA concentrations. 100 microM DHEAS directly elicited a membrane current, while THDOC (1 microM) did not exert any direct response. This study demonstrates that steroids modulate GABA-evoked currents and thus may interfere with any of the functions of glial GABA receptors that are at present under discussion.

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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.

Animals↗

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.

Animals↗

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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