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H Kettenmann

Publications and source records attributed to H Kettenmann.

162 records · Page 9Linked to original sources
PubMed1985

Monoclonal cell surface antibodies do not produce short-term effects on electrical properties of mouse oligodendrocytes in culture.

Eleven monoclonal antibodies (O1-O11) directed against the surface of oligodendrocytes were applied individually or in combination during measurement of membrane potential, input resistance and K+-pump activity in explant cultures of mouse spinal cord. Antibody binding to oligodendrocytes was verified by indirect immunofluorescence. None of the antibodies affected the electrical properties studied. On the basis of these observations, it is possible to identify oligodendrocytes immunocytologically prior to electrophysiological characterization.

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PubMed1985

Cultured astrocytes form a syncytium after maturation.

The formation of functional gap junctions between astrocytes was investigated during differentiation of these cells in culture. Precursor cells of GFA (glial fibrillary acidic) protein-positive astrocytes were cultured in a chemically defined medium as a homogeneous population. These cells were rarely coupled to one neighbour, as revealed by electrical and dye coupling and never formed a large syncytium, as investigated by injection and spread of Lucifer Yellow. Differentiation with respect to GFA protein accumulation can be induced in these cells by culturing in horse serum-containing medium. The formation of functional junctions developed within 2 weeks in about 20% of the cells. Coupled cells formed a large syncytium. When the astrocytes were co-cultured with primary cerebellar cells (consisting predominantly of small neurons) after the switch to serum-containing medium the percentage of coupled astrocytes increased to about 65%. Again the coupled cells formed a large syncytium. Since no physical contact was possible between the astrocyte cultures and the primary cerebellar cells the stimulation of coupling had to be signalized by soluble factor(s).

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PubMed1985

Pharmacological properties of gamma-aminobutyric acid-, glutamate-, and aspartate-induced depolarizations in cultured astrocytes.

Differentiated glial fibrillary acidic protein-positive astrocytes in homogeneous cultures of early postnatal rat cerebral hemispheres respond by membrane depolarization to gamma-aminobutyric acid (GABA), glutamate, and aspartate with a threshold concentration of approximately 10(-5) M. The GABA-induced depolarization is antagonized by two blockers of the neuronal GABAA receptor, picrotoxin and bicuculline, but is not affected by the uptake blockers beta-alanine or nipecotic acid. An agonist of the GABAA receptor, muscimol, produces a dose-response curve similar to that of GABA, whereas the agonist of the GABAB receptor, baclofen, did not alter the membrane potential. When repetitive pulses of GABA are given to one cell, its responsiveness depends on the time interval between pulses. Within 30 sec after termination of the first pulse the cell remains unresponsive to the second pulse. With increased time intervals between the pulses, reactivity toward GABA recovers. Five minutes after the first pulse the cell regains 75% of its initial depolarization peak. Aspartate results in a depolarization similar in size and time course to that induced by glutamate. The glutamate agonists, quisqualate and ibotenate, and kainate are less potent than glutamate. N-Methyl-D-aspartate has no effect on the membrane potential of astrocytes. The pharmacological features of the glutamate response are therefore similar to those of the receptor mediating neuronal glutamate transport.

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PubMed1984

Aspartate, glutamate and gamma-aminobutyric acid depolarize cultured astrocytes.

Cultures of differentiated, glial fibrillary acidic protein-positive astrocytes from early postnatal rat cerebral hemispheres respond with depolarization of 2-36 mV to glutamate, gamma-aminobutyric acid (GABA) and aspartate but not to glycine or taurine. While GABA resulted in a transient depolarization, the effect of glutamate and aspartate persisted during the application. Since neurons were not present in these cultures a contribution of transmitter-mediated K+ release from adjacent neurons could be excluded. The depolarization triggered by these neurotransmitters is therefore an intrinsic reaction of astrocytes.

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PubMed1984

Depolarization of cultured oligodendrocytes by glutamate and GABA.

Subpopulations of cultured oligodendrocytes from mouse spinal cord respond with depolarization to glutamate and GABA. Heterogeneity in the oligodendrocyte population was indicated by the observation that some cells respond to both GABA and glutamate, while others respond to only one and some are not responsive to either. Depolarizations are not mediated by an increase of [K+]o released from neurons. The response to GABA was blocked in Na+-free solution and is not accompanied by a change in input resistance. Several other neurotransmitters did not induce changes in membrane potential.

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PubMed1984

Some properties of single potassium channels in cultured oligodendrocytes.

K+ channels were studied in oligodendrocytes in cultures of mouse spinal cord. Single channel currents were measured using the gigaseal technique. The conductance of the channels varied greatly i.e. from 6 to 125 pS (38 +/- 28 SD, N = 21). In some patches there were up to three current levels of the same size. At -70 mV the open state probability was 0.51 +/- 0.17 and the average duration of an opening 70 +/- 20 ms for 4 channels with conductance from 16-57 pS. These analyses exclude brief flickering (less than 2 ms) or long closed periods (seconds to minutes). These times were not markedly affected by pulling the patch off the cell or by superfusing the isolated patch with media containing 10 mmol X 1(-1) TEA or EGTA without Ca2+. At membrane potentials between -90 and -30 mV there was a small but consistent effect of depolarization to increase the open state probability. Large positive or negative voltage steps decreased the open state probability. Current voltage measurements on intact cells showed a striking decrease in membrane conductance at these large membrane potentials. The leakage conductance of the patch also exhibited some K+ selectivity. The oligodendrocyte membrane appears to contain about one K+ channel per 5 micron 2. The known electrical properties of cultured oligodendrocytes can essentially be explained by the distribution and properties of these K+ channels.

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PubMed1984

Electrical properties of oligodendrocytes in culture.

The electrical properties of immunocytologically identified oligondendrocytes from embryonic mouse spinal cord maintained in culture for 3 to 6 weeks studied by passing current and recording potential changes with two separate intracellular electrodes. The average input resistance was 3.3 M omega and ranged from 0.7 to 16 M omega (n = 35). The input resistance increased by 19% with depolarization and decreased by 9% with hyperpolarization of 25 mV. The membrane time constant determined from the slope of the late exponential tail was 3.45 +/- 2.5 ms SD (n = 15). The specific membrane resistance of three cells was determined by a simplified square pulse analysis combined with measurement of membrane area. Membrane area was estimated from photomicrographs of cells injected with Lucifer Yellow CH and stained with the cell surface-reactive antibody 04 and from electron micrographs. An average specific membrane resistance of 1.3 X 10(3) omega cm2 and specific capacitance of 1.7 mu F/cm2 were calculated. Increasing [K+]o depolarized the cells and decreased the input resistance and the time constant.

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PubMed1984

gamma-Aminobutyric acid directly depolarizes cultured oligodendrocytes.

gamma-Aminobutyric acid (GABA) depolarizes in a dose-dependent manner approximately one-third of all immunologically identified oligodendrocytes in cultures of mouse spinal cord. Measurements of [K+]o indicate that the response to GABA is not due to K+ released from active neurons. The depolarization is not accompanied by a change in cell input resistance. Replacement of sodium in the bathing solution abolishes the entire response, whereas ouabain only inhibits the repolarization phase. Current clamp experiments with two separate intracellular electrodes show that the depolarization increases at more positive potentials while the repolarization increases at more negative potentials. Bicuculline and picrotoxin but not nipecotic acid reduce the GABA effect. Pentobarbital and chlordiazepoxid also reduce the GABA-induced depolarization. Muscimol produces a depolarization similar to that of GABA. Heterogeneity in the oligodendrocyte population is indicated by the observation that some cells respond to both GABA and glutamate, while others respond only to one and some are not responsive to either.

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PubMed1983

Antibody L1 ejected from a micropipette identifies neurons without altering electrical activity.

Antibody L1 which reacts specifically with the cell surface of central nervous system neurons was pressure ejected from a micropipette into the vicinity of a cultured neuron, or applied to the bathing fluid during intracellular recording of activity. No alterations in membrane potential, shape of action potential, firing rates and postsynaptic activities were observed. Binding of antibody was observed by indirect immunofluorescence after injection of Lucifer Yellow. Bath application of antibody resulted in a uniform neuronal staining over the entire culture, whereas pressure ejected antibodies were limited to neuronal structures within about 200 micron of the cell recorded from. Live, L1 antigen-positive neurons could be identified by indirect immunofluorescence prior to recording.

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PubMed1983

Intracellular SITS injection dye-uncouples mammalian oligodendrocytes in culture.

When the blue fluorescing dye SITS (4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulfonic acid disodium salt) is injected into one of a pair of electrically and dye-coupled oligodendrocytes it does not cross the intercellular junctions but remains in the injected cell. Moreover, the fluorescent dye Lucifer Yellow CH (LY), which normally crosses these intercellular junctions after injection, does not diffuse into a SITS-injected cell. Thus, intracellular SITS injection leads to dye-uncoupling. SITS injection does not eliminate electrical-coupling.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1983

Coupling among identified cells in mammalian nervous system cultures.

Cell coupling can be demonstrated among groups of oligodendrocytes, astrocytes, and fibroblasts or fibroblast-like cells, but not for dorsal root ganglion neurons, Schwann cells, or macrophages in mouse or rat cell cultures. Neurons were identified by their ability to generate action potentials. Non-neuronal cells were recognized immunologically by their ability to react with a variety of rhodamine-labeled cell type-specific antibodies. Intracellular injection of the gap junction permeable fluorescent dye Lucifer Yellow and ionic current were used to establish the presence of coupling among these identified cells. Coupling under the culture conditions used in this study does not represent a random membrane interaction between closely apposed cells but rather a form of communication among restricted populations.

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PubMed1983

Exclusive potassium dependence of the membrane potential in cultured mouse oligodendrocytes.

Membrane potential, conductance, and intracellular potassium concentration were measured in oligodendrocytes in 3- to 10-week-old cultures of embryonic mouse spinal cord. After intracellular recording the cells were first injected with Lucifer Yellow and then stained by immunofluorescence using rhodamine-labeled monoclonal antibody 01 specific for oligodendrocyte cell surfaces. The membrane potential of these identified oligodendrocytes was in mV -66 +/- 4.3 SD; it could be reversibly reduced almost to zero by the addition of ouabain. Changes in external K+ but not Na+, Ca++, or Cl- changed the membrane potential. A 10-fold increase in extracellular potassium concentration ([K+]0) depolarized the cell by about 52 mV. This is less than the 61 mV predicted by the Nernst equation for a K+ electrode assuming a constant intracellular potassium concentration ([K+]i). However, when [K+]i was measured with an ion-selective electrode during the increase in [K+]0 it was found to rise. The Nernst equation for K+ accurately predicts the oligodendrocyte membrane potential when the increase in [K+]i is taken into account. Oligodendrocytes may be described as accurate K+ electrodes with a variable reference solution.

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PubMed1982

Immunocytochemical cell identification in nervous system culture combined with intracellular injection of a blue fluorescing dye (SITS).

SITS, a stilbene isothiocyanate derivative, was injected by iontophoresis into neurons and oligodendrocytes in cultures of mouse spinal cord. The dye readily stains the entire cell. When excited with ultraviolet light it emits a blue fluorescence. Thus, SITS-injected cells may be distinguished from those injected with Lucifer yellow CH. Furthermore, the blue cells can be identified in culture after the cells have been labeled with two different antibodies using rhodamine (TRITC) or fluorescein (FITC) as fluorochromes by the use of appropriate filter combinations.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1982

Single potassium channel currents in cultured mouse oligodendrocytes.

Single channel currents were recorded from isolated membrane patches of cultured mouse oligodendrocytes using the giga-seal technique. The observed conductance of the channel was 71 +/- 34 pS. Isolated patches contained 1 to 4 channels with similar conductances and kinetics. Closed times of the channel varied from less than 1 msec to many minutes. The open state was always interrupted by flickering to the closed state. The kinetics of opening and closing appeared insensitive to voltage steps of up to +/- 75 mV from the resting level of the membrane potential, but could be affected by very large voltage steps. The observed changes in channel current in response to changes of potassium concentration on either side of the membrane indicate a high selectivity for potassium. The results show a membrane with constant macroscopic permeability that contains channels which open and close.

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PubMed1982

Cell puncturing with a step motor driven manipulator with simultaneous measurement of displacement.

A small angle stepping motor was used for construction of a micropositioner. Linear movements are produced by direct coupling of the rotor axis to high precision microdrive. The linearly moving system is constructed from stainless steal prismatic guides with hardened surfaces and permits precise steps in the 100 nm range. Extreme reduction of the moving masses and minimal friction of the radial thrust bearing enables strong acceleration of the electrode. During simultaneous measurements of step performance motoneurons in the frog spinal cord, CA 1 cells of hippocampal brain slices and glia cells in tissue culture were punctured with single electrodes (tip less than l micron and double barrelled ion-sensitive microelectrodes (phi 1,5-2 micron). In all three preparations, cell penetration could be performed by means of both types of electrodes with a high yield when the step velocity reached or exceeded 4 mm/s. Steps with lower velocity resulted in less successful cell penetrations and were accompanied by typical dimpling effects. The results indicate that a critical velocity is required for cell puncturing with a minimum of damage.

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PubMedDate not supplied

Cell-based delivery of cytokines allows for the differentiation of a doxycycline inducible oligodendrocyte precursor cell line in vitro.

BACKGROUND: Stem cells, having the property of self renewal, offer the promise of lifelong repair of damaged tissue. However, somatic tissue-committed primary stem cells are rare and difficult to expand in vitro. Genetically modified stem-like cells with the ability to expand conditionally provide a valuable tool with which to study stem cell biology, especially the cellular events of proliferation and differentiation. In addition, stem cells may be appropriate candidates for therapeutic applications. METHODS: Double transgenic mice possesing SV40 T antigen (Tag) under the control of the reverse tetracycline-transactivator (rtTA) were used to establish cell lines. One brain cell line was partially characterized by DNA sequencing, morphology, antigen expression using flow cytometry, confocal microscopy, and electrophysiology using the patch clamp technique. Cell cycle analysis was performed using propidium iodide staining; cell viability and H3-thymidine incorporation assays. The ability of this cell line to differentiate was assessed by confocal microscopy following co-culture with stem cells secreting cytokines. RESULTS: We report here the establishment and partial characterization of a cell line derived from the brain tissue of rtTA-SV40 Tag transgenic mice. Analysis of the morphology and antigen markers has shown that this cell line mimics some aspects of primary glial precursors. The results of electrophysiology are consistent with this and suggest that the cell line is derived from O2A glial precursor cells. Cell cycle progression of this cell line is doxycycline-dependent. In the absence of doxycycline, cells become apoptotic. Differentiation into mature type 2 astrocytes and (precursor) oligodendrocytes can be induced upon withdrawal of doxycycline and addition of epithelial stem cells secreting cytokine, such as hIL3 (human Interleukine 3) or hIL6 to the culture. In contrast, co-culturing with hCNTF (human Ciliary NeuroTrophic Factor)-secreting epithelial stem cells did not induce them to mature into progeny cell types. CONCLUSION: The differentiation of this O2A glial precursor line does not occur automatically in culture. Additional external help is required from the cell-based delivery of appropriate transgenic cytokines. Withdrawal of doxycycline from the culture medium removes the proliferation signals and induces a fatal outcome.

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PubMedDate not supplied

Magnetic labeling of activated microglia in experimental gliomas.

Microglia, as intrinsic immunoeffector cells of the central nervous system (CNS), play a very sensitive, crucial role in the response to almost any brain pathology where they are activated to a phagocytic state. Based on the characteristic features of activated microglia, we investigated whether these cells can be visualized with magnetic resonance imaging (MRI) using ultrasmall superparamagnetic iron oxides (USPIOs). The hypothesis of this study was that MR microglia visualization could not only reveal the extent of the tumor, but also allow for assessing the status of immunologic defense. Using USPIOs in cell culture experiments and in a rat glioma model, we showed that microglia can be labeled magnetically. Labeled microglia are detected by confocal microscopy within and around tumors in a typical border-like pattern. Quantitative in vitro studies revealed that microglia internalize amounts of USPIOs that are significantly higher than those incorporated by tumor cells and astrocytes. Labeled microglia can be detected and quantified with MRI in cell phantoms, and the extent of the tumor can be seen in glioma-bearing rats in vivo. We conclude that magnetic labeling of microglia provides a potential tool for MRI of gliomas, which reflects tumor morphology precisely. Furthermore, the results suggest that MRI may yield functional data on the immunologic reaction of the CNS.

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