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.

Characterization of Panglial Gap Junction Networks in the Thalamus, Neocortex, and Hippocampus Reveals a Unique Population of Glial Cells.

The thalamus plays important roles as a relay station for sensory information in the central nervous system (CNS). Although thalamic glial cells participate in this activity, little is known about their properties. In this study, we characterized the formation of coupled networks between astrocytes and oligodendrocytes in the murine ventrobasal thalamus and compared these properties with those in the hippocampus and cortex. Biocytin filling of individual astrocytes or oligodendrocytes revealed large panglial networks in all 3 gray matter regions. Combined analyses of mice with cell type-specific deletion of connexins (Cxs), semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and western blotting showed that Cx30 is the dominant astrocytic Cx in the thalamus. Many thalamic astrocytes even lack expression of Cx43, while in the hippocampus astrocytic coupling is dominated by Cx43. Deletion of Cx30 and Cx47 led to complete loss of panglial coupling, which was restored when one allele of either Cxs was present. Immunohistochemistry revealed a unique antigen profile of thalamic glia and identified an intermediate cell type expressing both Olig2 and Cx43. Our findings further the emerging concept of glial heterogeneity across brain regions.

Dual reporter approaches for identification of Cre efficacy and astrocyte heterogeneity.

Gene inactivation reporters are powerful tools to circumvent limitations of the widely used Cre/loxP system of conditional mutagenesis. With new conditional transgenic mouse lines expressing the enhanced cyan fluorescent protein (ECFP) instead of connexin43 (Cx43) after Cre-mediated recombination, we demonstrate dual reporter approaches to simultaneously examine astrocyte subpopulations expressing different connexins, identify compensatory up-regulation within gene families, and quantify Cre-mediated deletion at the allelic level. Analysis of a newly generated Cx43 knock-in ECFP mouse revealed an unexpected heterogeneity of Cx43-expressing astrocytes across brain areas.

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.

Quality control of astrocyte-directed Cre transgenic mice: the benefits of a direct link between loss of gene expression and reporter activation.

Cre recombinase activity for cell-type restricted deletion of floxed target genes (i.e., flanked by Cre recognition loxP-sites) is often measured by separate matings with recombination-activated reporter gene mice. Using a floxed Gja1 (Cx43) allele, we demonstrate the benefits of a direct link between reporter gene expression and target gene deletion to overcome critical limitations of the Cre/loxP system. The widely used human glial fibrillary acidic protein (hGFAP)-Cre transgene exhibits variable recombination activity and requires postexperimental validation. Such quality control is essential to correlate the extent of Cre-mediated Gja1 ablation with phenotypical alterations and to maintain the activity status of hGFAP-Cre in transgenic mouse colonies. We present several strategies to control for the fidelity of hGFAP-Cre mediated recombination. (c) 2008 Wiley-Liss, Inc.

Changes in the proliferative capacity of NG2 cell subpopulations during postnatal development of the mouse hippocampus.

Besides astrocytes and oligodendrocytes, NG2 proteoglycan-expressing cells (NG2 glia) represent a third subtype of macroglia in the brain. Originally described as oligodendrocyte precursor cells, they feature several characteristics not expected from mere progenitor cells, including synaptic connections with neurons. There is accumulating evidence that the properties of NG2 glia differ between different brain regions and developmental stages. To further analyze this proposed heterogeneity, we studied electrophysiological properties, transcript and protein expression, distribution and proliferative capacity of NG2 glia during postnatal development, focusing on the hippocampus and corpus callosum. All NG2 glia displayed a 'complex' current pattern consisting of voltage- and time-dependent in- and outward currents. In juvenile mice, Kir current densities and rectification index were highly variable and on average significantly lower than in adult animals. Single cell RT-PCR analyses of electrophysiologically characterized cells demonstrated that different glial genes were expressed at variable extent, independent of developmental stage and genetic background. In the hippocampus proper and the corpus callosum, the density of NG2 glia was highest at postnatal days (P)10-12, decreased by ~50 % at P25-35 and then remained stable in adults (P80-90). Interestingly, co-expression of NG2 and S100ß, a marker for mature astrocytes, increased from 7 % at P10-12 to 27 % at P25-35 in the hippocampus proper, and then dropped again in the stratum radiatum at P80-90. In the dentate gyrus and corpus callosum, co-expression of NG2 and S100ß was very low (3 %) and constant throughout development. Age-related differences were also observed with Ki-67, a proliferation marker. In NG2 glia of the CA1 region, its expression decreased from 16 % at P10-12 to 9 % (P25-35) and then 3 % (P80-90). Triple-stainings revealed that Ki-67 was also expressed in 2-3 % of NG2/S100ß-positive cells in the juvenile and mature stratum radiatum, indicating that the latter, in contrast to S100ß-positive astrocytes, still host proliferative potential. Taken together, we found significant differences in transcript and protein expression, electrophysiological properties and proliferative capacity of NG2 glia in the mouse forebrain, suggesting the co-existence of several subpopulations of NG2 glia. Our data thus support the idea of a substantial regional and developmental heterogeneity in this subtype of macroglia.

Dysbalance of astrocyte calcium under hyperammonemic conditions.

Increased brain ammonium (NH4(+)/NH3) plays a central role in the manifestation of hepatic encephalopathy (HE), a complex syndrome associated with neurological and psychiatric alterations, which is primarily a disorder of astrocytes. Here, we analysed the influence of NH4(+)/NH3 on the calcium concentration of astrocytes in situ and studied the underlying mechanisms of NH4(+)/NH3-evoked calcium changes, employing fluorescence imaging with Fura-2 in acute tissue slices derived from different regions of the mouse brain. In the hippocampal stratum radiatum, perfusion with 5 mM NH4(+)/NH3 for 30 minutes caused a transient calcium increase in about 40% of astrocytes lasting about 10 minutes. Furthermore, the vast majority of astrocytes (∼ 90%) experienced a persistent calcium increase by ∼ 50 nM. This persistent increase was already evoked at concentrations of 1-2 mM NH4(+)/NH3, developed within 10-20 minutes and was maintained as long as the NH4(+)/NH3 was present. Qualitatively similar changes were observed in astrocytes of different neocortical regions as well as in cerebellar Bergmann glia. Inhibition of glutamine synthetase resulted in significantly larger calcium increases in response to NH4(+)/NH3, indicating that glutamine accumulation was not a primary cause. Calcium increases were not mimicked by changes in intracellular pH. Pharmacological inhibition of voltage-gated sodium channels, sodium-potassium-chloride-cotransporters (NKCC), the reverse mode of sodium/calcium exchange (NCX), AMPA- or mGluR5-receptors did not dampen NH4(+)/NH3-induced calcium increases. They were, however, significantly reduced by inhibition of NMDA receptors and depletion of intracellular calcium stores. Taken together, our measurements show that sustained exposure to NH4(+)/NH3 causes a sustained increase in intracellular calcium in astrocytes in situ, which is partly dependent on NMDA receptor activation and on release of calcium from intracellular stores. Our study furthermore suggests that dysbalance of astrocyte calcium homeostasis under hyperammonemic conditions is a widespread phenomenon, which might contribute to the disturbance of neurotransmission during HE.

Germ-line recombination activity of the widely used hGFAP-Cre and nestin-Cre transgenes.

Herein we demonstrate with PCR, immunodetection and reporter gene approaches that the widely used human Glial Fibrillary Acidic Protein (hGFAP)-Cre transgene exhibits spontaneous germ-line recombination activity in leading to deletion in brain, heart and tail tissue with high frequency. The ectopic activity of hGFAP-Cre requires a rigorous control. We likewise observed that a second widely used nestin-Cre transgene shows germ-line deletion. Here we describe procedures to identify mice with germ-line recombination mediated by the hGFAP-Cre and nestin-Cre transgenes. Such control is essential to avoid pleiotropic effects due to germ-line deletion of loxP-flanked target genes and to maintain the CNS-restricted deletion status in transgenic mouse colonies.

Satellite glial cells in sensory ganglia: their possible contribution to inflammatory pain.

Neurons in dorsal root ganglia (DRG) are surrounded by an envelope of satellite glial cells (SGCs). Little is known about SGC physiology and their interactions with neurons. In this work, we investigated changes in mouse DRG neurons and SGC following the induction of inflammation in the hind paw by the injection of complete Freund's adjuvant (CFA). The electrophysiological properties of neurons were characterized by intracellular electrodes. Changes in coupling mediated by gap junctions between SGCs were monitored using intracellular injection of the fluorescent dye Lucifer yellow. Pain was assessed with von Frey hairs. We found that two weeks after CFA injection there was a 38% decrease in the threshold for firing an action potential in DRG neurons, consistent with neuronal hyperexcitability. Injection of Lucifer yellow into SGCs revealed that, compared with controls, coupling by gap junctions among SGCs surrounding adjacent neurons increased 2.7-, 3.2-, and 2.5-fold one week, two weeks, and one month, respectively, after CFA injection. In SGCs enveloping neurons that project into the inflamed paw this effect was more enhanced (5.4-fold). Interneuronal coupling was augmented by up to 7% after CFA injection. Pain threshold in the injected paw decreased by 13%, 16%, and 11% compared with controls at one week, two weeks, and one month, respectively, after CFA injection. Intraperitoneal injection of the gap junction blocker carbenoxolone prevented the inflammation-induced decrease in pain threshold. The results show that augmented glial coupling is one of the major events occurring in DRG following inflammation. The elevation in pain threshold after carbenoxolone administration provides indirect support for the idea that augmented intercellular coupling might contribute to chronic pain.