No abstract

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.

Astrocytes in different brain regions display variable functional properties. In the hippocampus, astrocytes predominantly express timeand 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 (K 2P ) K ϩ channels in astrocytes, which are thought to be instrumental in the regulation of K ϩ 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 Ba 2ϩ sensitivity in situ, our data demonstrate that in freshly isolated astrocytes, the main proportion of membrane currents is sensitive to micromolar Ba 2ϩ 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 Kir4.1Ϫ/Ϫ mice lacked Ba 2ϩ -sensitive currents. In addition, we report functional expression of K 2P channels of the TREK subfamily (TREK1, TREK2), which mediate astroglial outward currents. Together, our findings demonstrate that Kir4.1 constitutes the pivotal K ϩ channel subunit and that superposition of currents through Kir4.1 and TREK channels underlies the "passive" current pattern of hippocampal astrocytes.

IntroductionRecent work on glial cell physiology has disclosed that these cells are much more actively involved in brain information processing than hitherto thought. This new insight stimulates a new view according to which the active brain has to be regarded as an integrated circuit of interactive neurons and glial cells. Astrocytes in particular are now regarded as direct communication partners of neurons, by dynamically interacting with synapses through the uptake and release of neurotransmitters and receptor-mediated intracellular Ca 2+ signalling (for reviews, see Haydon, 2001;Newman, 2003;Volterra and Steinhäuser, 2004;Schipke and Kettenmann, 2004). Intriguingly, a distinct subset of glial cells in the hippocampus was reported to receive direct synaptic input from glutamatergic and GABAergic neurons. These glial cells expressed the proteoglycan, NG2, and on this basis were regarded as oligodendrocyte precursor cells (OPCs) (Bergles et al., 2000;Lin and Bergles, 2003). However, the identity of these cells needs further consideration because the specificity of NG2 as an OPC marker becomes increasingly questionable. Current work suggests that NG2 cells comprise a distinct, heterogeneous type of neuroglial cells (Nishiyama et al., 2002;Stallcup, 2002;Greenwood and Butt, 2003;Aguirre et al., 2004;Peters, 2004).Using transgenic mice expressing green fluorescent protein under control of the human GFAP promoter (hGFAP/EGFP mice), we have recently reported a co-existence of two types of glial cells in the hippocampus, distinguishable from each other by mutually exclusive expression of glutamate transporters (GluT type) and ionotropic glutamate receptors (GluR cells). GluT type cells were extensively coupled via gap junctions and contacted blood vessels, thus matching properties of classical astrocytes. By contrast, GluR cells lacked junctional coupling and did not enwrap capillaries (Matthias et al., 2003;Wallraff et al., 2004). Moreover, GluR cells co-expressed S100␤, a common astrocyte marker, NG2, as well as neuronal genes, and hence escaped classification into neurons, astrocytes, or oligodendrocytes.Here we used the hGFAP/EGFP transgenic animal to identify distinct types of glial cells in live slices. We combined ultrastructural analysis and post-recording immunocytochemistry to test whether the two populations of hGFAP/EGFP-positive glial cells in the hippocampus receive synaptic input. Electron microscopic inspection identified synapse-like structures with EGFP-positive postsynaptic compartments. Patch clamp recordings revealed stimulus- 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 synaps...

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.