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Helmut Kettenmann

Publications and source records attributed to Helmut Kettenmann.

At least 55 records · Page 3Linked to original sources
PubMed2003

Neuronal or glial progeny: regional differences in radial glia fate.

The precursor function of the ubiquitous glial cell type in the developing central nervous system (CNS), the radial glia, is largely unknown. Using Cre/loxP in vivo fate mapping studies, we found that radial glia generate virtually all cortical projection neurons but not the interneurons originating in the ventral telencephalon. In contrast to the cerebral cortex, few neurons in the basal ganglia originate from radial glia, and in vitro lineage analysis revealed intrinsic differences in the potential of radial glia from the dorsal and ventral telencephalon. This shows that the progeny of radial glia not only differs profoundly between brain regions but also includes the majority of neurons in some parts of the CNS.

Animals↗
PubMed2003

DMACM-caged adenosine nucleotides: ultrafast phototriggers for ATP, ADP, and AMP activated by long-wavelength irradiation.

The development of new photocleavable adenosine nucleotides based on the photochemistry of [7-(dimethylamino)coumarin-4-yl]methyl (DMACM) esters is described. The phototriggers liberate adenosine triphosphate (ATP), diphosphate, and monophosphate upon UV/Vis irradiation between 334 and 405 nm. The efficiency of photocleavage at long wavelengths is high as a result of a combination of appropriate quantum yields and intensive absorptivities. By using time-resolved fluorescence spectroscopy, we determined a lower limit of 1.6 x 10(9) s(-1) for the rate constant of the release of ATP from DMACM-caged ATP. The favorable properties of DMACM-caged ATP were confirmed in physiological studies by confocal laser scanning microscopy. We were able to uncage DMACM-caged ATP in cultures of mouse astrocytes and in brain tissue slices from mice and were also able to measure the effect of photoreleased ATP on the cellular response of astrocytes, namely the ability of the ATP to evoke Ca(2+) ion waves.

Adenosine Diphosphate↗
PubMed2003

Segregated expression of AMPA-type glutamate receptors and glutamate transporters defines distinct astrocyte populations in the mouse hippocampus.

Recent data have suggested the existence of direct signaling pathways between glial cells and neurons. Here we report the coexistence of distinct types of cells expressing astrocyte-specific markers within the hippocampus that display diverse morphological, molecular, and functional profiles. Usage of transgenic mice with GFAP promoter-controlled enhanced green fluorescent protein (EGFP) expression allowed the identification of astroglial cells after fresh isolation or in brain slices. Combining patch-clamp recordings and single-cell reverse transcription-PCR, we distinguished two morphologically distinct types of EGFP-positive cells, one expressing glutamate transporters and the other expressing ionotropic glutamate receptors. None of the EGFP-positive cells coexpressed glutamate receptors and transporters. Subpopulations of glutamate receptor-bearing EGFP-positive cells expressed AN2, the mouse homolog of the rat NG2 proteoglycan or transcripts for excitatory amino acid carrier 1, a neuronal glutamate transporter. Our data demonstrate the presence of distinct, independent populations of cells with astroglial properties in the developing hippocampus that can differently modulate neuronal signaling pathways. The observed heterogeneity of cells with GFAP promoter-regulated EGFP expression and S100beta/GFAP immunoreactivity challenges the hitherto accepted definition of astrocytes.

Amino Acid Transport System X-AG↗
PubMed2003

GABAA receptor-expressing astrocytes in the supraoptic nucleus lack glutamate uptake and receptor currents.

An important function of astrocytes is the clearance of excess extracellular glutamate via specific carriers whose expression has become an astrocytic marker. In the present study, we found that a large population of astrocytes in the supraoptic nucleus (SON) of the rat hypothalamus lacks glutamate uptake currents and receptor responses but expresses GABAA receptors. Patch clamp recordings in acute hypothalamic slices that included the SON showed typical astrocytic membrane currents and demonstrated that GABA, via GABAA receptor activation, triggered a conductance increase with the reversal potential close to the Cl- equilibrium potential and a decrease in resting K+ conductance. Intracellular labeling with Lucifer Yellow revealed that these cells had a radial glia-like morphology, with cell bodies lined up along the base of the brain and long processes traversing the nucleus; they were not dye-coupled. Parallel immunocytochemical labelings showed that they expressed strong GABAA receptor and glial fibrillary acidic protein (GFAP) immunoreactivities. In addition, our electrophysiological and morphological analyses revealed another population of astrocytes in this nucleus, located next to the subarachnoid space. They were less numerous than the radial type, had a round morphology and few processes, and were dye-coupled. Unlike the radial astrocytes, they showed little immunoreactivity for GABAA receptor or GFAP. Moreover, they did not respond to GABA but to glutamate, a response that was partially mimicked by aspartate, indicating glutamate transporter expression. Taken together, our observations add to growing evidence illustrating heterogeneity of astrocytes in the adult brain, a heterogeneity that reflects striking differences in form and function of astrocytic populations in regions as discrete as the SON of the hypothalamus.

Action Potentials↗
PubMed2003

Subpopulation of nestin-expressing progenitor cells in the adult murine hippocampus shows electrophysiological and morphological characteristics of astrocytes.

Based on the expression of glial fibrillary acidic protein (GFAP), a recent hypothesis considered stem or progenitor cells in the adult hippocampus to be a type of astrocyte. In a complementary approach, we used transgenic mice expressing green fluorescent protein (GFP) under the promoter for nestin, an intermediate filament present in progenitor cells, to demonstrate astrocytic features in nestin-GFP-positive cells. Morphologically, two subpopulations of nestin-GFP-positive cells were distinguishable; one had an elaborate tree of processes in the granule cell layer and expression of GFAP (but not of S100beta, another astrocytic marker). Electron microscopy revealed vascular end feet of nestin-positive cells, further supporting astrocytic differentiation. Electrophysiological examination of nestin-GFP-positive cells on acutely isolated hippocampal slices showed passive current characteristics of astrocytes in one subset of cells. Among the nestin-GFP-expressing cells with lacking astrocytic features, two cell types could be identified electrophysiologically: cells with delayed-rectifying potassium currents and a very small number of cells with sodium currents, potentially representing signs of the earliest steps of neuronal differentiation.

Age Factors↗
PubMed2003

Astrocytes from mouse brain slices express ClC-2-mediated Cl- currents regulated during development and after injury.

Chloride channels are important for astrocytic volume regulation and K+ buffering. We demonstrate functional expression of a hyperpolarization-activated Cl- current in a subpopulation of astrocytes in acute slices or after fresh isolation from adult brain of GFAP/EGFP transgenic animals in which astrocytes are selectively labeled. When Na+ and K+ were substituted with NMDG+ and Cs+ in extra- and intracellular solutions, an inward current was observed at negative membrane potentials. The current displayed features as described for a Cl- current characterized in cultured astrocytes: it activated time dependently at potentials negative to -40 mV, displayed no inactivation within 1 s, and was inhibited reversibly by submicromolar concentrations of Cd2+. The current was not detectable in astrocytes from ClC-2 knockout mice, indicating that the ClC-2 chloride channel generated the conductance. Current density was significantly lower in a corresponding population of astrocytes isolated from immature brain and in reactive astrocytes within a lesion site.

Animals↗
PubMed2003

Purinergic receptors on microglial cells: functional expression in acute brain slices and modulation of microglial activation in vitro.

Microglial cells are the pathologic sensors in the brain. ATP released from damaged cells is a candidate for signalling neural injury to microglia. Moreover, ATP is an extracellular messenger for propagating astrocyte activity in the form of Ca2+ waves. To test for the functional expression of purinoreceptors in microglial cells we employed the patch-clamp technique in acute slices of adult mouse brain. ATP triggered a nonselective cationic and a K+ current. Pharmacological screening with purinergic ligands indicated the presence of P2Y1 and P2Y2/4 receptors linked to the activation of a K+ current and P2X receptors, including P2X7, linked to the activation of a nonselective cationic current. These findings suggest that microglial cells in situ express different purinergic receptors with distinct sensitivity and functional coupling. To test for the involvement of purinoreceptors in microglial activation, we stimulated cultured microglial cells with lipopolysaccharide and measured the release of tumour necrosis factor alpha, interleukin-6, interleukin-12 and macrophage inflammatory protein 1alpha, induction of K+ outward currents and nitric oxide release. All these parameters were reduced in the presence of purinergic ligands, indicating that purinergic receptor activation attenuated indicators of microglial activation.

Adenosine Triphosphate↗
PubMed2002

Bergmann glial cells form distinct morphological structures to interact with cerebellar neurons.

It is well established that Bergmann glial cells closely interact with neuronal elements in the molecular layer of the cerebellum. We reconstructed dye-labeled Bergmann glial cells from electron microscopic serial sections and identified their contact sites with neurons as "glial microdomains" (Grosche et al. [1999] Nature Neurosci. 2:139-143). In the present paper we describe these structures in more detail, and show that 1) immature Bergmann fibers up to postnatal day 7 are smooth and lack appendages but contain several large mitochondria at sites where the first indications of growing side branches are observed; 2) Bergmann fibers from cerebella at postnatal day 30 form two types of outgrowths, short simple thorns and longer complex appendages; 3) each of the latter (i.e., a glial microdomain) is in contact with only a few synapses and nonsynaptic neuronal excrescences; 4) every given region of the neuropil is occupied by (at least) two interdigitating glial microdomains; 5) the synaptic clefts are entirely surrounded by glial protrusions, whereas the extrasynaptic surfaces and small axons are only partially covered; and 6) many small neuronal excrescenses without vesicles are completely ensheathed by glial caps, representing novel glial-neuronal structures of unknown function (glial thimbles). Computational modelling of the microdomains indicates that each is electrotonically independent of the stem process from which it arises, as well as of neighbouring domains. We assume that the glial microdomain is a morphological unit to compartmentalize ensembles of synapses, serving to synchronize local synaptic activity.

Aging↗
PubMed2002

Secondary lymphoid tissue chemokine (CCL21) activates CXCR3 to trigger a Cl- current and chemotaxis in murine microglia.

Microglial cells represent the major immunocompetent element of the CNS and are activated by any type of brain injury or disease. A candidate for signaling neuronal injury to microglial cells is the CC chemokine ligand CCL21, given that damaged neurons express CCL21. Investigating microglia in acute slices and in culture, we demonstrate that a local application of CCL21 for 30 s triggered a Cl(-) conductance with lasted for tens of minutes. This response was sensitive to the Cl(-) channel blockers 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid and 4-acetamide-4'-isothiocyanatostilbene, 2,2'-disulfonic acid. Moreover, CCL21 triggered a chemotaxis response, which was sensitive to Cl(-) channel blockers. In microglial cells cultured from CCR7 knockout mice, CCL21 produced the same type of Cl(-) current as well as a chemotaxis response. In contrast, in microglial cells from CXCR3 knockout mice, CCL21 triggered neither a Cl(-) conductance nor a chemotaxis response after CCL21 application. We conclude that the CCL21-induced Cl(-) current is a prerequisite for the chemotaxis response mediated by the activation of CXCR3 but not CCR7 receptors, indicating that in brain CCL21 acts via a different receptor system than in lymphoid organs.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2002

Interferon-gamma differentially modulates the release of cytokines and chemokines in lipopolysaccharide- and pneumococcal cell wall-stimulated mouse microglia and macrophages.

During bacterial infections of the CNS, activated microglia could support leucocyte recruitment to the brain through the synthesis of cyto- and chemokines. In turn, invading leucocytes may feedback on microglial cells to influence their chemokine release pattern. Here, we analyzed the capacity of interferon-gamma (IFNgamma) to serve as such a leucocyte-to-microglia signal. Production of cyto- and chemokines was stimulated in mouse microglia cultures by treatments with lipopolysaccharide (LPS) from Gram-negative Escherichia coli or cell walls from Gram-positive Streptococcus pneumoniae (PCW). IFNgamma presence during the stimulation (0.1-100 ng/mL) modulated the patterns of LPS- and PCW-induced cyto- and chemokine release in a dose-dependent, potent and complex manner. While amounts of TNFalpha and IL-6 remained nearly unchanged, IFNgamma enhanced the production of IL-12, MCP-1 and RANTES, but attenuated that of KC, MIP-1alpha and MIP-2. Release modulation was obtained with IFNgamma preincubation (treatment of cells before LPS or PCW administration), coincubation and even delayed addition to an ongoing LPS or PCW stimulation. Together the changes observed for the microglial chemokine release under IFNgamma would shift the chemoattractive profile from favouring neutrophils to a preferential attraction of monocytes and T lymphocyte populations--as actually seen during the course of bacterial meningitis. The findings support the view of activated microglia as a major intrinsic source for an instant production of a variety of chemokines and suggest that leucocyte-derived IFNgamma could potentially regulate the microglial chemokine release pattern.

Animals↗
PubMed2001

Astrocyte Ca2+ waves trigger responses in microglial cells in brain slices.

Pathologic impacts in the brain lead to a widespread activation of microglial cells far beyond the site of injury. Here, we demonstrate that glial Ca2+ waves can trigger responses in microglial cells. We elicited Ca2+ waves in corpus callosum glial cells by electrical stimulation or local adenosine triphosphate (ATP) ejection in acute brain slices. Macroglial cells, but not microglia, were bulk-loaded with Ca2+-sensitive dyes. Using a transgenic animal in which astrocytes were labeled by the enhanced green fluorescence protein (EGFP) allowed us to identify the reacting cell populations: the wave activated a Ca2+ response in both astrocytes and non-astrocytic glial cells and spread over hundreds of micrometers even into the adjacent cortical and ventricular cell layers. Regenerative ATP release and subsequent activation of metabotropic purinergic receptors caused the propagation of the glial Ca2+ wave: the wave was blocked by the purinergic receptor antagonist Reactive Blue 2 and was not affected by the gap junction blocker octanol, but enhanced in Ca2+ free saline. To test whether microglial cells respond to the wave, microglial cells were labeled with a dye-coupled lectin and membrane currents were recorded with the patch-clamp technique. When the wave passed by, a current with the characteristics of a purinergic response was activated. Thus, Ca2+ waves in situ are not restricted to astrocytic cells, but broadly activate different glial cell types.

Adenosine Triphosphate↗
PubMed2001

Requirement of functional ryanodine receptor type 3 for astrocyte migration.

Astrocyte motility plays an important role in the response of the brain to injury and during regeneration. We used two in vitro assays, a wound-healing model and a chemotaxis assay, to study mechanisms that control astrocyte motility. Ryanodine receptors (RyR), intracellular calcium-release channels, modulate intracellular Ca2+ levels, and also motility: 1) blocking RyR with antagonizing concentration of ryanodine (200 microM) strongly attenuated motility and 2) motility of astrocytes cultured from homozygous RyR type 3 knockout mice was impaired strongly compared with wild-type. In contrast, MIP-1a-induced chemotaxis was neither impaired in the presence of ryanodine nor in the cells from the knockout animals. Reverse transcription-polymerase chain reaction (RT-PCR) analysis combined with Western blotting and immunocytochemistry confirmed the expression of RyR type 3, but not type 1 or 2 in cultured and acutely isolated astrocytes. RyR in astrocytes are linked to Ca2+ signaling because the RyR agonist 4-chloro-m-cresol induced a release of Ca2+ from intracellular stores. These results indicate that astrocytes express only RyR type 3 and that this receptor is important for controlling astrocyte motility.

Animals↗
PubMed1992

Ca2+ Channel Expression in the Oligodendrocyte Lineage.

The development of oligodendrocytes from their precursor cells through different developmental stages can be studied in vitro. These stages can be distinguished by specific monoclonal antibodies and by a characteristic K+ channel profile. In this study we demonstrate that the occurrence of Ca2+ currents also undergoes marked changes during the development of mouse oligodendrocytes. Immature precursor cells which can develop into astrocytes or oligodendrocytes expressed two different types of voltage-activated Ca2+ channels. The expression of Ca2+ channels in precursor cells was strongly correlated with the expression of Na+ channels. When cells started to express the O1 antigen and were committed to the oligodendrocyte lineage, Ca2+ and Na+ currents could no longer be detected. Large Ca2+ currents were, however, recorded later in the development of the oligodendrocytes, correlated with the expression of the O10 antigen. The Ca2+ channels were classified as high and low voltage-activated Ca2+ channels according to their range of activation, and are further described by their kinetic and pharmacological properties.

Journal Article↗
PubMed1992

GABA Triggers a [Ca2+]i Increase in Murine Precursor Cells of the Oligodendrocyte Lineage.

The development of oligodendrocytes from their precursor cells can be studied in vitro by using a culture system in which cells can be identified at different developmental stages. We used this culture system to compare the effect of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) on intracellular Ca2+ concentration ([Ca2+]i) using fura-2 fluorescence systems. Application of GABA evoked transient [Ca2+]i increases in precursor cells. In contrast, [Ca2+]i levels were not affected in oligodendrocytes, which were identified by their positive labelling with the antibody O1. The precursor cells, identified by a lack of O1 staining, responded to GABA in the concentration range between 10-6 and 10-4 M. Since muscimol mimicked and bicuculline as well as picrotoxin blocked the GABA response, we conclude that the response is mediated by activation of GABAA receptors. The involvement of Ca2+ channels is inferred from the observation that the [Ca2+]i changes could be blocked by nifedipine or by omitting Ca2+ from the bath solution. Both GABAA receptors and Ca2+ channels have been previously identified on these precursor cells with the aid of the patch-clamp technique. We thus propose the following mechanism to explain our observations: the Cl- efflux via the GABA receptor depolarizes precursor cells, and this depolarization leads to activation of Ca2+ channels, resulting in an influx of Ca2+ and the observed rise in cytosolic [Ca2+]. Although its physiological importance is speculative, this event could serve as a signal from GABAergic neurons to glial precursor cells.

Journal Article↗
PubMed1992

Sodium and Calcium Currents in Glial Cells of the Mouse Corpus Callosum Slice.

We studied Na+ and Ca2+ currents in glial cells during the development of the corpus callosum in situ. Glioblasts and oligodendrocytes from frontal brain slices of postnatal day (P) 3 to P18 mice were identified based on morphological and ultrastructural features after characterization of the currents with the patch-clamp technique. Slices from P3 - P8 mice contained predominantly glioblasts with immature morphological features. These cells showed Na+ and Ca2+ currents, but the population with these currents decreased between P3 and P8. Na+ currents were blocked in Na+-free bathing solution and in the presence of tetrodotoxin, Ca2+ currents were only observed when a high concentration of extracellular Ba2+ was present. The cells from the corpus callosum of P10 - P18 mice predominantly had morphological features of oligodendrocytes. In these cells, which in some cases were shown to form myelin, neither Na+ nor Ca2+ currents were detected. To compare these in situ results with those from the electrophysiologically and immunocytochemically well-characterized cultured glial cells, we determined the expression pattern of stage-specific antigens in the corpus callosum in situ. The first O4 antigen-positive glial precursors were observed at P1, the earliest stage examined. The oligodendrocytic antigens O7 and O10 appeared at P6 and P14, respectively, and prominent labelling with the corresponding markers was seen at P12 and P18, respectively. Despite the existence of numerous mature, O10-positive oligodendrocytes at P18, which expressed Ca2+ channels in vitro, we failed to detect Ca2+ currents in situ at this stage.

Journal Article↗
PubMed1991

The Neural Cell Adhesion Molecule (N-CAM) Modulates K+ Channels in Cultured Glial Precursor Cells.

Application of antibodies against the neural cell adhesion molecule (N-CAM) to O4-positive murine glial precursor cells in vitro results in a reduction of two distinct K+ currents measured using the whole cell patch clamp technique. Both the A-type and delayed rectifier K+ currents are reduced in amplitude within a few minutes of the application of poly- or monoclonal antibodies against N-CAM. This effect is not due to the binding of any antibody to the surface of the glial precursor cells because monoclonal antibody directed against the O4 surface antigen, or polyclonal antibodies directed against liver cell membranes (which also bind to the surface of glial precursor cells), do not affect membrane currents. Activators of protein kinase C, such as phorbol esters or diacylglycerol, also induce changes in potassium currents that appear, both in magnitude and kinetics, to be similar to those induced by antibodies against N-CAM. In contrast, activation of G proteins upregulates K+ currents. Glial precursor cells thus respond to triggering of N-CAM by altering channel properties. These observations suggest that adhesive events between neural cells can influence the intracellular ionic milieu.

Journal Article↗
PubMed1991

Single Channel Recording from Glial Cells on the Untreated Surface of the Frog Optic Nerve.

The patch clamp technique has been used to record single channel currents from the untreated surface of the intact frog optic nerve after the meninges and basal lamina have been mechanically removed. Cells filled via dialysis with Lucifer yellow (LY) from the patch pipette had a typical astrocyte morphology and were dye-coupled to adjacent astrocytes. This is consistent with the electron-microscopic observation that all the cells on the surface of this nerve are astrocytes. Two types of ion channels were studied in detached patches. One, identified as a K+ channel, had a conductance of 88 +/- 4 (S.E.) n=9 pS and an equilibrium potential of -59 +/- 8 mV in physiological K+ solutions. The steady-state open probability was not significantly altered by changing the membrane potential. A second channel had a large conductance of 300 - 1200 pS, a reversal potential of approximately 0 mV in symmetrical and non-symmetrical solutions, and was open only in the voltage range of +/-20 mV. These are the characteristics of a large anionic channel described in other preparations including cultured astrocytes.

Journal Article↗