Astrocyte uncoupling as a cause of human temporal lobe epilepsy.

Glial cells are now recognized as active communication partners in the central nervous system, and this new perspective has rekindled the question of their role in pathology. In the present study we analysed functional properties of astrocytes in hippocampal specimens from patients with mesial temporal lobe epilepsy without (n = 44) and with sclerosis (n = 75) combining patch clamp recording, K(+) concentration analysis, electroencephalography/video-monitoring, and fate mapping analysis. We found that the hippocampus of patients with mesial temporal lobe epilepsy with sclerosis is completely devoid of bona fide astrocytes and gap junction coupling, whereas coupled astrocytes were abundantly present in non-sclerotic specimens. To decide whether these glial changes represent cause or effect of mesial temporal lobe epilepsy with sclerosis, we developed a mouse model that reproduced key features of human mesial temporal lobe epilepsy with sclerosis. In this model, uncoupling impaired K(+) buffering and temporally preceded apoptotic neuronal death and the generation of spontaneous seizures. Uncoupling was induced through intraperitoneal injection of lipopolysaccharide, prevented in Toll-like receptor4 knockout mice and reproduced in situ through acute cytokine or lipopolysaccharide incubation. Fate mapping confirmed that in the course of mesial temporal lobe epilepsy with sclerosis, astrocytes acquire an atypical functional phenotype and lose coupling. These data suggest that astrocyte dysfunction might be a prime cause of mesial temporal lobe epilepsy with sclerosis and identify novel targets for anti-epileptogenic therapeutic intervention.

Versatile and simple approach to determine astrocyte territories in mouse neocortex and hippocampus.

BACKGROUND: Besides their neuronal support functions, astrocytes are active partners in neuronal information processing. The typical territorial structure of astrocytes (the volume of neuropil occupied by a single astrocyte) is pivotal for many aspects of glia-neuron interactions. METHODS: Individual astrocyte territorial volumes are measured by Golgi impregnation, and astrocyte densities are determined by S100ß immunolabeling. These data are compared with results from conventionally applied methods such as dye filling and determination of the density of astrocyte networks by biocytin loading. Finally, we implemented our new approach to investigate age-related changes in astrocyte territories in the cortex and hippocampus of 5- and 21-month-old mice. RESULTS: The data obtained by our simplified approach based on Golgi impregnation were compared to previously published dye filling experiments, and yielded remarkably comparable results regarding astrocyte territorial volumes. Moreover, we found that almost all coupled astrocytes (as indicated by biocytin loading) were immunopositive for S100ß. A first application of this new experimental approach gives insight in age-dependent changes in astrocyte territorial volumes. They increased with age, while cell densities remained stable. In 5-month-old mice, the overlap factor was close to 1, revealing little or no interdigitation of astrocyte territories. However, in 21-month-old mice, the overlap factor was more than 2, suggesting that processes of adjacent astrocytes interdigitate. CONCLUSION: Here we verified the usability of a simple, versatile method for assessing astrocyte territories and the overlap factor between adjacent territories. Second, we found that there is an age-related increase in territorial volumes of astrocytes that leads to loss of the strict organization in non-overlapping territories. Future studies should elucidate the physiological relevance of this adaptive reaction of astrocytes in the aging brain and the methods presented in this study might be a powerful tool to do so.

Albumin is taken up by hippocampal NG2 cells and astrocytes and decreases gap junction coupling.

PURPOSE: Dysfunction of the blood-brain barrier (BBB) and albumin extravasation have been suggested to play a role in the etiology of human epilepsy. In this context, dysfunction of glial cells attracts increasing attention. Our study was aimed to analyze in the hippocampus (1) which cell types internalize albumin injected into the lateral ventricle in vivo, (2) whether internalization into astrocytes impacts their coupling and expression of connexin 43 (Cx43), and (3) whether expression of Kir4.1, the predominating astrocytic K(+) channel subunit, is altered by albumin. METHODS: The patch-clamp method was combined with single cell tracer filling to investigate electrophysiologic properties and gap junction coupling (GJC). For cell identification, mice with cell type-specific expression of a fluorescent protein (NG2kiEYFP mice) and immunohistochemistry were employed. Semiquantitative real time polymerase chain reaction (RT-PCR) allowed analysis of Kir4.1 and Cx43 transcript levels. KEY FINDINGS: We show that fluorescently labeled albumin is taken up by astrocytes, NG2 cells, and neurons, with NG2 cells standing out in terms of the quantity of uptake. Within 5 days postinjection (dpi), intracellular albumin accumulation was largely reduced suggesting rapid degradation. Electrophysiologic analysis of astrocytes and NG2 cells revealed no changes in their membrane properties at either time point. However, astrocytic GJC was significantly decreased at 1 dpi but returned to control levels within 5 dpi. We found no changes in hippocampal Cx43 transcript expression, suggesting that other mechanisms account for the observed changes in coupling. Kir4.1 mRNA was regulated oppositely in the CA1 stratum radiatum, with a strong increase at 1 dpi followed by a decrease at 5 dpi. SIGNIFICANCE: The present study demonstrates that extravasal albumin in the hippocampus induces rapid changes of astrocyte function, which can be expected to impair ion and transmitter homeostasis and contribute to hyperactivity and epileptogenesis. Therefore, astrocytes may represent alternative targets for antiepileptic therapeutic approaches.

Impact of aquaporin-4 channels on K+ buffering and gap junction coupling in the hippocampus.

Aquaporin-4 (AQP4) is the main water channel in the brain and primarily localized to astrocytes where the channels are thought to contribute to water and K(+) homeostasis. The close apposition of AQP4 and inward rectifier K(+) channels (Kir4.1) led to the hypothesis of direct functional interactions between both channels. We investigated the impact of AQP4 on stimulus-induced alterations of the extracellular K(+) concentration ([K(+)](o)) in murine hippocampal slices. Recordings with K(+)-selective microelectrodes combined with field potential analyses were compared in wild type (wt) and AQP4 knockout (AQP4(-/-)) mice. Astrocyte gap junction coupling was assessed with tracer filling during patch clamp recording. Antidromic fiber stimulation in the alveus evoked smaller increases and slower recovery of [K(+)](o) in the stratum pyramidale of AQP4(-/-) mice indicating reduced glial swelling and a larger extracellular space when compared with control tissue. Moreover, the data hint at an impairment of the glial Na(+)/K(+) ATPase in AQP4-deficient astrocytes. In a next step, we investigated the laminar profile of [K(+)](o) by moving the recording electrode from the stratum pyramidale toward the hippocampal fissure. At distances beyond 300 µm from the pyramidal layer, the stimulation-induced, normalized increases of [K(+)](o) in AQP4(-/-) mice exceeded the corresponding values of wt mice, indicating facilitated spatial buffering. Astrocytes in AQP4(-/-) mice also displayed enhanced tracer coupling, which might underlie the improved spatial re- distribution of [K(+)](o) in the hippocampus. These findings highlight the role of AQP4 channels in the regulation of K(+) homeostasis.

Role of astroglial connexin30 in hippocampal gap junction coupling.

The impact of connexin30 (Cx30) on interastrocytic gap junction coupling in the normal hippocampus is matter of debate; reporter gene analyses indicated a weak expression of Cx30 in the mouse hippocampus. In contrast, mice lacking connexin43 (Cx43) in astrocytes exhibited only 50% reduction in coupling. Complete uncoupling of hippocampal astrocytes in mice lacking both Cx30 and Cx43 suggested that Cx30 participates in interastrocytic gap junction coupling in the hippocampus. With comparative reporter gene assays, immunodetection, and cre/loxP-based reporter approaches we demonstrate that Cx30 is more abundant than previously thought. The specific role of Cx30 in interastrocytic coupling has never been investigated. Employing tracer coupling analyses in acute slices of Cx30 deficient mice here we show that Cx30 makes a substantial contribution to interastrocytic gap junctional communication in the mouse hippocampus.

Analysis of astroglial K+ channel expression in the developing hippocampus reveals a predominant role of the Kir4.1 subunit.

Astrocytes in different brain regions display variable functional properties. In the hippocampus, astrocytes predominantly express time- and voltage-independent currents, but the underlying ion channels are not well defined. This ignorance is partly attributable to abundant intercellular coupling of these cells through gap junctions, impeding quantitative analyses of intrinsic membrane properties. Moreover, distinct types of cells with astroglial properties coexist in a given brain area, a finding that confused previous analyses. In the present study, we investigated expression of inwardly rectifying (Kir) and two-pore-domain (K2P) K+ channels in astrocytes, which are thought to be instrumental in the regulation of K+ homeostasis. Freshly isolated astrocytes were used to improve space-clamp conditions and allow for quantitative assessment of functional parameters. Patch-clamp recordings were combined with immunocytochemistry, Western blot analysis, and semiquantitative transcript analysis. Comparative measurements were performed in different CA1 subregions of astrocyte-targeted transgenic mice. While confirming weak Ba2+ sensitivity in situ, our data demonstrate that in freshly isolated astrocytes, the main proportion of membrane currents is sensitive to micromolar Ba2+ concentrations. Upregulation of Kir4.1 transcripts and protein during the first 10 postnatal days was accompanied by a fourfold increase in astrocyte inward current density. Hippocampal astrocytes from Kir4.1-/- mice lacked Ba2+-sensitive currents. In addition, we report functional expression of K2P channels of the TREK subfamily (TREK1, TREK2), which mediate astroglial outward currents. Together, our findings demonstrate that Kir4.1 constitutes the pivotal K+ channel subunit and that superposition of currents through Kir4.1 and TREK channels underlies the "passive" current pattern of hippocampal astrocytes.

Connexin expression by radial glia-like cells is required for neurogenesis in the adult dentate gyrus.

In the adult dentate gyrus, radial glia-like cells represent putative stem cells generating neurons and glial cells. Here, we combined patch-clamp recordings, biocytin filling, immunohistochemistry, single-cell transcript analysis, and mouse transgenics to test for connexin expression and gap junctional coupling of radial glia-like cells and its impact on neurogenesis. Radial glia-like cells were identified in mice expressing EGFP under control of the nestin and gfap promoters. We show that a majority of Radial glia-like cells are coupled and express Cx43. Neuronal precursors were not coupled. Mice lacking Cx30 and Cx43 in GFAP-positive cells displayed almost complete inhibition of proliferation and a significant decline in numbers of radial glia-like cells and granule neurons. Inducible virus-mediated ablation of connexins in the adult hippocampus also reduced neurogenesis. These findings strongly suggest the requirement of connexin expression by radial glia-like cells for intact neurogenesis in the adult brain and point to possible communication pathways of these cells.

Ultrastructural and functional characterization of satellitosis in the human lateral amygdala associated with Ammon's horn sclerosis.

The amygdala displays neuronal cell loss and gliosis in human temporal lobe epilepsy (TLE). Therefore, we investigated a certain type of gliosis, called satellitosis, in the lateral amygdala (LA) of TLE patients with Ammon's horn sclerosis (AHS, n = 15) and non-AHS (n = 12), and in autopsy controls. Satellite cells were quantified using light and electron microscopy at the somata of Nissl-stained and glutamic acid decarboxylase-negative projection neurons, and their functional properties were studied using electrophysiology. Non-AHS cases suffered from ganglioglioma, cortical dysplasia, Sturge-Weber syndrome, astrocytoma WHO III-IV, Rasmussen's encephalitis, cerebral infarction and perinatal brain damage. TLE cases with AHS had a more prominent satellitosis as compared to non-AHS and/or autopsy cases, which correlated with epilepsy duration but not age. At ultrastructural level, the predominant type of satellite cells occurring in both AHS and non-AHS cases displayed a dark cytoplasm and an irregularly shaped dark nucleus, whereas perineuronal glial cells with a light cytoplasm and light oval nucleus were much rarer. Satellite cells expressed time- and voltage-dependent transmembrane currents as revealed by patch-clamp recordings typical for 'complex' glia, although only 44% of satellite cells were immunostained for the chondroitin sulfate proteoglycan NG2. Together, the perineuronal cells described here were a heterogenous cell population regarding their NG2 expression, although they resembled NG2 cells rather than bona fide oligodendrocytes and astrocytes based on their ultrastructural and electrophysiological characteristics. Thus, perineuronal satellitosis as studied in the LA seems to be a hallmark of AHS-associated TLE pathology in patients suffering from intractable epilepsy.

The proapoptotic BCL-2 homology domain 3-only protein Bim is not critical for acute excitotoxic cell death.

Prolonged and repetitive epileptic activity is causally linked to neuronal cell death in the brain and is most marked in vulnerable subfields of the hippocampus. The Bcl-2 family protein Bim, a proapoptotic member of the BCL-2 homology domain 3-only subfamily, has been implicated as an important mediator of neuronal cell damage in various pathological conditions, although its role in epilepsy-associated cell death is not understood. We performed intrahippocampal stereotaxic injections of the glutamate analog kainic acid as an in vivo model of acute excitotoxicity to assess neuronal injury in Bim-deficient and control wild-type mice. A variety of cell death parameters including chromatin condensation, TdT-mediated dUTP nick end labeling, and caspase-3 activity was assessed. We found no differences in the extent of hippocampal neuronal death parameters between the 2 groups. Moreover, electroencephalographic recordings after kainic acid injection revealed indistinguishable patterns of seizure activity in Bim-deficient and wild-type animals. These in vivo and histological data suggest that Bim is not critically involved in excitotoxicity-induced acute neuronal cell injury.

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.

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.

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.

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.

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.

AMPA receptor-mediated modulation of inward rectifier K+ channels in astrocytes of mouse hippocampus.

Astrocytes and neurons are tightly associated and recent data suggest a direct signaling between neuronal and glial cells in vivo. To further analyze these interactions, the patch-clamp technique was combined with single-cell RT-PCR in acute hippocampal brain slices. Subsequent to functional analysis, the cytoplasm of the same cell was harvested to perform transcript analysis and identify subunits that underlie inwardly rectifying K+ currents (I(Kir)) in astrocytes of the CA1 stratum radiatum. Transcripts encoding Kir2.1, Kir2.2, or Kir2.3, were encountered in a majority of cells, while Kir4.1 was less frequent. Further investigation revealed that glial Kir channels are rapidly inhibited upon activation of AMPA-type glutamate receptors, most probably due a receptor-mediated influx of Na+, which plugs the channels from the intracellular side. A transient inhibition of I(Kir) in astrocytes in response to neuronal glutamate release and glial AMPA receptor activation represents a further, so far undetected mechanism to balance neuronal excitability.