Biomedical subjects

H Kettenmann

Publications and source records attributed to H Kettenmann.

At least 109 records · Page 6Linked to original sources

PubMed1993

Patch-clamp recording from Müller (glial) cell endfeet in the intact isolated retina and acutely isolated Müller cells of mouse and guinea-pig.

Müller cells span through the entire retina and terminate with the formation of endfeet at the vitreous body. These endfeet are thought to be specialized for maintaining the K+ homeostasis in the retina based on the assumption that voltage signals can passively spread from the cell body to the endfeet. We employed the patch-clamp technique to study the physiological properties of these endfeet in a retinal wholemount preparation from guinea-pig or mouse. After assessing one endfoot with the patch pipette and establishing the whole cell recording configuration, a membrane area which approximately matched the size of one endfoot and proximal process could be voltage-clamped. This morphological correlation could be established by filling the cytoplasm with the fluorescent dye Lucifer Yellow via the patch-pipette. The morphological, immunocytochemical and ultrastructural inspection of the recorded cells revealed that mouse Müller cell endfeet were connected by only a thin stalk to the proximal process. In contrast, guinea-pig endfeet were connected by thick stalks. The endfoot current in the mouse was dominated by a voltage and time-independent K+ conductance. In contrast, in some of the recordings from guinea-pig, delayed and inwardly rectifying K+ currents were observed. These voltage-gated currents were more frequently observed or were facilitated when the membrane area under voltage clamp was increased, blocking the passive K+ currents by Ba2+ in both, mouse and guinea-pig. We thus assume that the voltage-gated currents were not in the endfeet membrane, but rather in the proximal process and could thus be better activated in the guinea-pig with its thicker stalk or after increasing the membrane area under voltage clamp control. Similar results were obtained in freshly isolated Müller cells; in contrast to the cells from the wholemount the voltage-gated currents were more frequently observed. These studies demonstrate that the Müller cell endfoot of the mouse with its vascularized retina is an electrically isolated unit and that voltage signals do not spread to the proximal process. Such a property would, however, be required for the redistribution of K+ via spatial buffer currents. In contrast, guinea-pig Müller glial cells with their stout morphological connection between endfoot and proximal process are better suited to fulfil this task.

Animals↗

PubMed1993

Current potentiation by diazepam but not GABA sensitivity is determined by a single histidine residue.

The GABAA/benzodiazepine receptor is the principal inhibitory neurotransmitter receptor in the mammalian brain and is assembled from sequence-related subunits, such as alpha 1 beta 2 gamma 2. In contrast to alpha 1 beta 2 gamma 2 receptors, alpha 6 beta 2 gamma 2 receptors fail to exhibit high-affinity binding of allosteric positive modulators of GABA-activated chloride currents. The critical determinant responsible for this difference in ligand binding was previously traced to a position in the extracellular domain of the two alpha subunits (alpha 1 His100 and alpha 6 Arg 101). We now show by patch clamp analysis that this amino acid exchange also determines the diazepam potentiation. Thus, alpha 1(Arg101)beta 2 gamma 2 receptors do not, but alpha 6(His100)beta 2 gamma 2 receptors do exhibit diazepam potentiation. However, the same extracellular determinant is not responsible for the increased GABA sensitivity of alpha 6 beta 2 gamma 2 receptors relative to alpha 1 beta 2 gamma 2 receptors as revealed by electrophysiological analysis and by differential GABA sensitivity of [35S]TBPS binding.

Amino Acid Sequence↗

PubMed1993

NMDA-activated currents in Bergmann glial cells.

NMDA receptors play a crucial role in synaptic plasticity of the central nervous system and were thought to be exclusive to neurones. In this study we provide evidence that Bergmann glial cells from mouse cerebellar slices show intrinsic responses to NMDA. As in neurones, NMDA increased membrane conductance and the responses were blocked by the NMDA antagonist ketamine, but not by the non-NMDA glutamate receptor antagonist CNQX. In contrast to responses in neurones, the current voltage relation of the glial NMDA-induced current was linear, reversed at -40 mV, currents were not blocked by Mg2+ or enhanced by glycine and NMDA did not induce an increase in cytosolic Ca2+ as recorded with a fura-2 imaging system. These data imply the presence of distinct NMDA receptors on Bergmann glial cells; these glial receptors could be the substitute for complex neurone-glia interactions in the cerebellum.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed1993

Extracellular ATP activates a cation conductance and a K+ conductance in cultured microglial cells from mouse brain.

Microglial cells have important functions during regenerative processes after brain injury. It is well established that they rapidly respond to damage to the brain tissue. Stages of activation are associated with changes of cellular properties such as proliferation rate or expression of surface antigens. Yet, nothing is known about signal substances leading to the rapid changes of membrane properties, which may be required to initiate the transition from one cell stage into another. From our present study, using the patch-clamp technique, we report that cultured microglial cells obtained from mouse or rat brain respond to extracellularly applied ATP with the activation of a cation conductance. Additionally, in the majority of cells an outwardly directed K+ conductance was activated with some delay. Since ADP, AMP, and adenosine (in descending order) were less potent or ineffective in inducing the cation conductance, the involvement of a P2 purinergic receptor is proposed. The receptor activation is accompanied by an increase of cytosolic Ca2+ as determined by a fura-2-based Ca(2+)-imaging system. This ATP receptor could enable microglial cells to respond to transmitter release from nerve endings with ATP as a transmitter or cotransmitter or to the death of cells with resulting leakage of ATP.

Adenosine↗

PubMed1993

Membrane properties of ameboid microglial cells in the corpus callosum slice from early postnatal mice.

Microglial cells in culture are distinct from neurons, macroglial cells, and macrophages of tissues other than brain with respect to their membrane current pattern. To assess these cells in the intact tissue, we have applied the patch-clamp technique to study membrane currents in microglial cells from acute, whole brain slices of 6-9-d-old mice in an area of microglial cell invasion, the cingulum. As strategies to identify microglial cells prior to or after recording, we used binding and incorporation of Dil-acetylated low-density lipoproteins, binding of fluorescein isothiocyanate-coupled IgG via microglial Fc-receptors, and ultrastructural characterization. As observed previously for cultured microglial cells, depolarizing voltage steps activate only minute if any membrane currents, while hyperpolarizing voltage steps induced large inward currents. These currents exhibited properties of the inwardly rectifying K+ channel in that the reversal potential depended on the transmembrane K+ gradient, inactivation time constants decreased with hyperpolarization, and the current was blocked by tetraethylammonium (50 mM). This study represents the first attempt to assess microglial cells in situ using electrophysiological methods. It opens the possibility to address questions related to the function of microglial cells in the intact CNS.

4-Aminopyridine↗

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.

Animals↗

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.

Animals↗

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.

Animals↗

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.

Animals↗

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.

Animals↗

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.

Animals↗

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.

Animals↗

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.

Animals↗

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.

Animals↗