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...

NG2 cells, the fourth type of glia in the mammalian CNS, receive synaptic input from neurons. The function of this innervation is unknown yet. Postsynaptic changes in intracellular Ca2+-concentration ([Ca2+]i) might be a possible consequence. We employed transgenic mice with fluorescently labeled NG2 cells to address this issue. To identify Ca2+-signaling pathways we combined patch-clamp recordings, Ca2+-imaging, mRNA-transcript analysis and focal pressure-application of various substances to identified NG2-cells in acute hippocampal slices. We show that activation of voltage-gated Ca2+-channels, Ca2+-permeable AMPA-receptors, and group I metabotropic glutamate-receptors provoke [Ca2+]i-elevations in NG2 cells. The Ca2+-influx is amplified by Ca2+-induced Ca2+-release. Minimal electrical stimulation of presynaptic neurons caused postsynaptic currents but no somatic [Ca2+]i elevations, suggesting that [Ca2+]i elevations in NG2 cells might be restricted to their processes. Local Ca2+-signaling might provoke transmitter release or changes in cell motility. To identify structural prerequisites for such a scenario, we used electron microscopy, immunostaining, mRNA-transcript analysis, and time lapse imaging. We found that NG2 cells form symmetric and asymmetric synapses with presynaptic neurons and show immunoreactivity for vesicular glutamate transporter 1. The processes are actin-based, contain ezrin but not glial filaments, microtubules or endoplasmic reticulum. Furthermore, we demonstrate that NG2 cell processes in situ are highly motile. Our findings demonstrate that gray matter NG2 cells are endowed with the cellular machinery for two-way communication with neighboring cells.