Biomedical subjects

Christian Steinhäuser

Publications and source records attributed to Christian Steinhäuser.

At least 19 recordsLinked to original sources

PubMed2006

Viscoelastic properties of individual glial cells and neurons in the CNS.

One hundred fifty years ago glial cells were discovered as a second, non-neuronal, cell type in the central nervous system. To ascribe a function to these new, enigmatic cells, it was suggested that they either glue the neurons together (the Greek word "gammalambdaiotaalpha" means "glue") or provide a robust scaffold for them ("support cells"). Although both speculations are still widely accepted, they would actually require quite different mechanical cell properties, and neither one has ever been confirmed experimentally. We investigated the biomechanics of CNS tissue and acutely isolated individual neurons and glial cells from mammalian brain (hippocampus) and retina. Scanning force microscopy, bulk rheology, and optically induced deformation were used to determine their viscoelastic characteristics. We found that (i) in all CNS cells the elastic behavior dominates over the viscous behavior, (ii) in distinct cell compartments, such as soma and cell processes, the mechanical properties differ, most likely because of the unequal local distribution of cell organelles, (iii) in comparison to most other eukaryotic cells, both neurons and glial cells are very soft ("rubber elastic"), and (iv) intriguingly, glial cells are even softer than their neighboring neurons. Our results indicate that glial cells can neither serve as structural support cells (as they are too soft) nor as glue (because restoring forces are dominant) for neurons. Nevertheless, from a structural perspective they might act as soft, compliant embedding for neurons, protecting them in case of mechanical trauma, and also as a soft substrate required for neurite growth and facilitating neuronal plasticity.

Animals↗

PubMed2006

Classification of projection neurons and interneurons in the rat lateral amygdala based upon cluster analysis.

Neurons in the rat lateral amygdala in situ were classified based upon electrophysiological and molecular parameters, as studied by patch-clamp, single-cell RT-PCR and unsupervised cluster analyses. Projection neurons (class I) were characterized by low firing rates, frequency adaptation and expression of the vesicular glutamate transporter (VGLUT1). Two classes were distinguished based upon electrotonic properties and the presence (IB) or absence (IA) of vasointestinal peptide (VIP). Four classes of glutamate decarboxylase (GAD67) containing interneurons were encountered. Class III reflected "classical" interneurons, generating fast spikes with no frequency adaptation. Class II neurons generated fast spikes with early frequency adaptation and differed from class III by the presence of VIP and the relatively rare presence of neuropeptide Y (NPY) and somatostatin (SOM). Class IV and V were not clearly separated by molecular markers, but by membrane potential values and spike patterns. Morphologically, projection neurons were large, spiny cells, whereas the other neuronal classes displayed smaller somata and spine-sparse dendrites.

Amygdala↗

PubMed2006

The impact of astrocytic gap junctional coupling on potassium buffering in the hippocampus.

Astrocytic gap junctions have been suggested to contribute to spatial buffering of potassium in the brain. Direct evidence has been difficult to gather because of the lack of astrocyte-specific gap junction blockers. We obtained mice with coupling-deficient astrocytes by crossing conditional connexin43-deficient mice with connexin30(-/-) mice. Similar to wild-type astrocytes, genetically uncoupled hippocampal astrocytes displayed negative resting membrane potentials, time- and voltage-independent whole-cell currents, and typical astrocyte morphologies. Astrocyte densities were also unchanged. Using potassium-selective microelectrodes, we assessed changes in potassium buffering in hippocampal slices of mice with coupling-deficient astrocytes. We demonstrate that astrocytic gap junctions accelerate potassium clearance, limit potassium accumulation during synchronized neuronal firing, and aid in radial potassium relocation in the stratum lacunosum moleculare. Furthermore, slices of mice with coupling-deficient astrocytes displayed a reduced threshold for the generation of epileptiform events. However, it was evident that radial relocation of potassium in the stratum radiatum was not dependent on gap junctional coupling. We suggest that the perpendicular array of individual astrocytes in the stratum radiatum makes these cells ideally suited for spatial buffering of potassium released by pyramidal cells, independent of gap junctions. In general, a surprisingly large capacity for K+ clearance was conserved in mice with coupling-deficient astrocytes, indicating that gap junction-dependent processes only partially account for K+ buffering in the hippocampus.

Animals↗

PubMed2006

Functional changes in astroglial cells in epilepsy.

Epilepsy comprises a group of disorders characterized by the periodic occurrence of seizures, and pathologic specimens from patients with temporal lobe epilepsy demonstrate marked reactive gliosis. Since recent studies have implicated glial cells in novel physiological roles in the CNS, such as modulation of synaptic transmission, it is plausible that glial cells may have a functional role in the hyperexcitability characteristic of epilepsy. Indeed, alterations in distinct astrocyte membrane channels, receptors and transporters have all been associated with the epileptic state. This review integrates the current evidence regarding astroglial dysfunction in epilepsy and the potential underlying mechanisms of hyperexcitability. Functional understanding of the cellular and molecular alterations of astroglia-dependent hyperexcitability will help to clarify the physiological role of astrocytes in neural function as well as lead to the identification of novel therapeutic targets.

Animals↗

PubMed2006

Astrocyte dysfunction in neurological disorders: a molecular perspective.

Recent work on glial cell physiology has revealed that glial cells, and astrocytes in particular, are much more actively involved in brain information processing than previously thought. This finding has stimulated the view that the active brain should no longer be regarded solely as a network of neuronal contacts, but instead as a circuit of integrated, interactive neurons and glial cells. Consequently, glial cells could also have as yet unexpected roles in the diseased brain. An improved understanding of astrocyte biology and heterogeneity and the involvement of these cells in pathogenesis offers the potential for developing novel strategies to treat neurological disorders.

Amyotrophic Lateral Sclerosis↗

PubMed2005

Molecular and functional properties of neurons in the human lateral amygdala.

Neuronal properties were investigated through patch-clamp recording in situ in surgical specimens of the human lateral amygdala (LA) obtained from patients with intractable temporal lobe epilepsy. Projection neurons displayed spiny dendrites, action potentials with varying degree of frequency adaptation, and an inwardly rectifying K+ (Kir) conductance coupled to GABA(B) receptors. In interneurons, dendrites were spineless or sparsely spiny, action potentials were shorter than those in projection neurons and often occurred spontaneously, and GABA(B) receptor-mediated responses were lacking. Single-cell RT-PCR demonstrated expression of Kir channel subunits Kir3.1 and Kir3.2 and of vesicular glutamate transporters VGLUT1 and VGLUT2 in projection neurons. It is concluded that projection neurons and interneurons of the human LA can be distinguished based upon morphological, electrophysiological, and molecular biological criteria. The most striking difference relates to the expression of postsynaptic GABA(B) receptors coupled to Kir3 channels in projection neurons and the lack of functional GABA(B) receptors in interneurons.

Action Potentials↗

PubMed2005

Synaptic transmission onto hippocampal glial cells with hGFAP promoter activity.

Glial cells increasingly gain importance as part of the brain's communication network. Using transgenic mice expressing green fluorescent protein (EGFP) under the control of the human GFAP promoter, we tested for synaptic input to identified glial cells in the hippocampus. Electron microscopic inspection identified synapse-like structures with EGFP-positive postsynaptic compartments. Sub-threshold stimulation to Schaffer collaterals resulted in stimulus-correlated, postsynaptic responses in a subpopulation of EGFP-positive cells studied with the patch-clamp technique in acute slices. This cell population can be recognized by its distinct morphology and has been termed GluR cells in a preceding study. These cells are distinct from the classical astrocytes due to their antigen profile and functional properties, but also lack characteristic features of oligodendrocytes or neurons. GluR cells also received spontaneous synaptic input. Stimulus-correlated and spontaneous responses were quantitatively analysed by ascertaining amplitude distributions, failure rates, kinetics as well as pharmacological properties. The data demonstrate that GABAergic and glutamatergic neurons directly synapse onto GluR cells and suggest a low number of neuronal release sites. These data demonstrate that a distinct type of glial cells is integrated into the synaptic circuit of the hippocampus, extending the finding that synapse-based brain information processing is not a property exclusive to neurons.

Animals↗

PubMed2004

Glial modulation of synaptic transmission in the hippocampus.

For many years, astrocytes and oligodendrocytes were considered the inert partners of neurons in the central nervous system (CNS), but several recent studies have dramatically challenged this view. Glial cells express a large number of different voltage- and ligand-gated ion channels (Verkhratsky and Steinhäuser. Brain Res Rev 32:380-412, 2000) as well as G-protein-coupled receptors (Verkhratsky et al. Physiol Rev 78:99-141, 1998)-machinery necessary to sense and respond to neuronal activity. These findings raised the fundamental question as to whether glial receptors are stimulated under physiological conditions, and what sorts of events are triggered by such activation. During the early 1990s, P. Haydon and colleagues made the seminal observation that [Ca(2+)](i) rises in cultured astrocytes are associated with the release of glutamate, which suggested that astrocytes respond to activation and play active modulatory roles in intercellular communication (Parpura et al. Nature 369:744-747, 1994). Subsequent studies performed in situ confirmed and extended this initial observation. In this review, we will focus specifically on the hippocampus and sum up evidence of bidirectional communication between astrocytes and neurons emerging from recent studies using acute slice preparations.

Animals↗

PubMed2004

Astrocytes contain a vesicular compartment that is competent for regulated exocytosis of glutamate.

Astrocytes establish rapid cell-to-cell communication through the release of chemical transmitters. The underlying mechanisms and functional significance of this release are, however, not well understood. Here we identify an astrocytic vesicular compartment that is competent for glutamate exocytosis. Using postembedding immunogold labeling of the rat hippocampus, we show that vesicular glutamate transporters (VGLUT1/2) and the vesicular SNARE protein, cellubrevin, are both expressed in small vesicular organelles that resemble synaptic vesicles of glutamatergic terminals. Astrocytic vesicles, which are not as densely packed as their neuronal counterparts, can be observed in small groups at sites adjacent to neuronal structures bearing glutamate receptors. Fluorescently tagged VGLUT-containing vesicles were studied dynamically in living astrocytes by total internal reflection fluorescence (TIRF) microscopy. After activation of metabotropic glutamate receptors, astrocytic vesicles underwent rapid (milliseconds) Ca(2+)- and SNARE-dependent exocytic fusion that was accompanied by glutamate release. These data document the existence of a Ca(2+)-dependent quantal glutamate release activity in glia that was previously considered to be specific to synapses.

Animals↗

PubMed2004

Enhanced relative expression of glutamate receptor 1 flip AMPA receptor subunits in hippocampal astrocytes of epilepsy patients with Ammon's horn sclerosis.

Astrocytes express ionotropic glutamate receptors (GluRs), and recent evidence suggests that these receptors contribute to direct signaling between neurons and glial cells in vivo. Here, we have used functional and molecular analyses to investigate receptor properties in astrocytes of human hippocampus resected from patients with pharmacoresistant temporal lobe epilepsy (TLE). Histopathological analysis allowed us to distinguish two forms of epilepsy: Ammon's horn sclerosis (AHS) and lesion-associated TLE. Human hippocampal astrocytes selectively expressed the AMPA subtype of ionotropic glutamate receptors. Single-cell RT-PCR found preferential expression of the subunits GluR1 and GluR2 in human astrocytes, and the expression patterns were similar in patients with AHS and lesion-associated epilepsy. The AMPA receptor-specific modulators, cyclothiazide (CTZ) and 4-[2-(phenylsulfonylamino)ethylthio]-2,6-difluoro-phenoxyacetamide (PEPA), were used to investigate splice variant expression. Astrocytes of sclerotic specimens displayed a slower dissociation of CTZ from the receptor and a lower ratio of current potentiation by PEPA to potentiation by CTZ, suggesting enhanced expression of flip receptor variants in AHS versus lesion-associated epilepsy. Real-time PCR and restriction analysis substantiated this presumption by identifying elevated flip-to-flop mRNA ratios of GluR1 in single astrocytes of AHS specimens. These findings imply that in AHS, glutamate may lead to prolonged depolarization of astrocytes, thereby facilitating the generation or spread of seizure activity.

Alternative Splicing↗

PubMed2004

Distinct types of astroglial cells in the hippocampus differ in gap junction coupling.

Previous studies have shown that two subpopulations of cells with astrocytic properties coexist in the mouse hippocampus, which display distinct morphological and functional characteristics, specifically a nonoverlapping expression of either AMPA-type glutamate receptors (GluR cells) or glutamate transporters (GluT cells). Use of transgenic mice with hGFAP promoter-controlled EGFP expression and patch-clamp recordings allow reliable identification of the two cell types in hippocampal slices. Extending functional characterization, we report here the complete lack of gap junctional tracer coupling in GluR cells, while GluT cells are shown to be extensively coupled. This distinction is valid in immature as well as adult animals. Analysis of transgenic mice expressing beta-Gal under regulatory elements of the Cx43 promoter revealed the absence of Cx43 in GluR cells. Experiments using gap junction blockers demonstrated that passive currents, displayed primarily by GluT cells, do not reflect intercellular coupling but are attributable to intrinsic membrane properties of individual cells. This study supports the notion that the two subpopulations of hGFAP-EGFP-positive cells represent distinct cell types with contrasting physiological properties. Since GluR cells do not participate in the astrocytic gap junctional network, their functional role must be different from spatial buffering of ions or signaling molecules, i.e., properties generally assigned to astrocytes.

Amino Acid Transport System X-AG↗

PubMed2003

Connexin 47 (Cx47)-deficient mice with enhanced green fluorescent protein reporter gene reveal predominant oligodendrocytic expression of Cx47 and display vacuolized myelin in the CNS.

To further characterize the recently described gap junction gene connexin 47 (Cx47), we generated Cx47-null mice by replacing the Cx47 coding DNA with an enhanced green fluorescent protein (EGFP) reporter gene, which was thus placed under control of the endogenous Cx47 promoter. Homozygous mutant mice were fertile and showed no obvious morphological or behavioral abnormalities. Colocalization of EGFP fluorescence and immunofluorescence of cell marker proteins revealed that Cx47 was mainly expressed in oligodendrocytes in highly myelinated CNS tissues and in few calcium-binding protein S100beta subunit-positive cells but not in neurons or peripheral sciatic nerve. This corrects our previous conclusion that Cx47 mRNA is expressed in brain and spinal cord neurons (Teubner et al., 2001). Cx47 protein was detected by Western blot analysis after immunoprecipitation in CNS tissues of wild-type mice but not in heart or Cx47-deficient tissues. Electron microscopic analysis of CNS white matter in Cx47-deficient mice revealed a conspicuous vacuolation of nerve fibers, particularly at the site of the optic nerve where axons are first contacted by oligodendrocytes and myelination starts. Initial analyses of Cx32/Cx47-double-deficient mice showed that these mice developed an action tremor and died on average at 51 d after birth. The central white matter of these double-deficient mice exhibited much more abundant vacuolation in nerve fibers than mice deficient only in Cx47.

Animals↗

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

Accelerated hippocampal spreading depression and enhanced locomotory activity in mice with astrocyte-directed inactivation of connexin43.

Using a human glial fibrillary acidic protein (hGFAP) promoter-driven cre transgene, we have achieved efficient inactivation of a floxed connexin43 (Cx43) gene in astrocytes of adult mice. The loss of Cx43 expression was monitored in a cell-autonomous manner via conditional replacement of the Cx43-coding region by a lacZ reporter gene. In this way, we bypassed the early postnatal lethality previously reported for Cx43 null mice and characterized the phenotypic consequences of Cx43 deficiency in the CNS. Mice lacking Cx43 in astrocytes were viable and showed no evidence of either neurodegeneration or astrogliosis. Spreading depression (SD) is a pathophysiological phenomenon observed in the CNS that is characterized by a propagating wave of depolarization followed by neuronal inactivation. Inhibitors of gap junctional communication have previously been shown to block initiation and propagation of SD. In contrast, we observed an increase in the velocity of hippocampal SD in the stratum radiatum of mice lacking Cx43 in astrocytes. In the same brain subregion, dye-coupling experiments revealed a reduction in overall astrocytic intercellular communication by approximately 50%. This strongly suggests separate and different neuronal and glial contributions of gap junctional intercellular communication to SD. Concomitant with increased velocity of spreading depression, we observed enhanced locomotory activity in mice lacking Cx43 in astrocytes.

Animals↗

PubMed2003

Changes in splice variant expression and subunit assembly of AMPA receptors during maturation of hippocampal astrocytes.

Astrocytes in the hippocampus express glutamate receptors of the AMPA subtype. An increasing body of evidence suggests a contribution of astroglial AMPA receptors to a direct signaling between neurons and glial cells in vivo. Here, we have combined functional analysis with singlecell RT-PCR to investigate whether hippocampal astrocytes express Ca(2+)-permeable AMPA receptors. We show that by postnatal day 5, a mosaic of Ca(2+)-permeable and less Ca(2+)-permeable AMPA receptors coexists in individual astrocytes, while receptors with a more uniform, low divalent permeability dominate in older cells. Moreover, we report an upregulation of the flip form of the GluR2 subunit during maturation, while the splicing status of GluR1 and GluR4 remains unchanged. Due to its specific properties, Ca(2+)-permeable AMPA receptors in astrocytes might strengthen neuron-to-glia signaling and enable proper formation of structural and functional connections between glial cells and glutamatergic synapses in the developing hippocampus.

Alternative Splicing↗

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

Seizures preferentially stimulate proliferation of radial glia-like astrocytes in the adult dentate gyrus: functional and immunocytochemical analysis.

Kainate-induced seizures increase hippocampal neurogenesis. Glial fibrillary acidic protein-positive astrocytes with radial processes in the dentate gyrus share many of the characteristics of radial glia and appear to act as precursor cells for adult dentate neurogenesis. Using the chemoconvulsant kainate and transgenic mice with human glial-fibrillary acidic protein (hGFAP) promoter-controlled enhanced green fluorescent protein (EGFP) expression, we examined the proliferation, morphology and electrophysiological properties of astrocytes in the neurogenic subgranular zone of the dentate gyrus in control animals and upon the induction of seizure-induced cell proliferation, three days post-kainate. EGFP-positive cells with and without radial processes could easily be distinguished. Kainate treatment caused a significant increase in the total number of proliferating EGFP-positive cells, particularly a tenfold elevation in the number of proliferating radial glia-like astrocytes, and also caused a preferential shift in the dividing cell population towards cells expressing EGFP. Immunohistochemical analysis revealed a surprisingly low proportion of cells coexpressing the astroglial marker S100beta and EGFP. Kainate increased the number of EGFP-positive, S100beta-positive and S100beta-positive-EGFP-positive astrocytes in the subgranular zone. We also report a subset of faintly EGFP-positive cells expressing markers of early neuronal differentiation. Patch-clamp analysis revealed the presence of three functionally different populations of EGFP-positive cells in both kainate and control tissue. We conclude that there is an early increase in proliferating radial glia-like astrocytes in the dentate after kainate-induced seizures, consistent with a recruitment of precursors for seizure-induced neurogenesis.

Animals↗

PubMed2003

Functional network integration of embryonic stem cell-derived astrocytes in hippocampal slice cultures.

Embryonic stem (ES) cells provide attractive prospects for neural transplantation. So far, grafting strategies in the CNS have focused mainly on neuronal replacement. Employing a slice culture model, we found that ES cell-derived glial precursors (ESGPs) possess a remarkable capacity to integrate into the host glial network. Following deposition on the surface of hippocampal slices, ESGPs actively migrate into the recipient tissue and establish extensive cell-cell contacts with recipient glia. Gap junction-mediated coupling between donor and host astrocytes permits widespread delivery of dye from single donor cells. During maturation, engrafted donor cells display morphological, immunochemical and electrophysiological properties that are characteristic of differentiating native glia. Our findings provide the first evidence of functional integration of grafted astrocytes, and depict glial network integration as a potential route for widespread transcellular delivery of small molecules to the CNS.

Animals↗