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Expression of a linear current-voltage (I-V) relationship (passive) K+ membrane conductance is a hallmark of mature hippocampal astrocytes. However, the molecular identifications of the K+channels underlying this passive conductance remain unknown. We provide the following evidence supporting significant contribution of the two-pore domain K+ channel (K2P) isoforms, TWIK-1 and TREK-1, to this conductance. First, both passive astrocytes and the cloned rat TWIK-1 and TREK-1 channels expressed in CHO cells conduct significant amounts of Cs+ currents, but vary in their relative PCs/PK permeability, 0.43, 0.10, and 0.05, respectively. Second, quinine, which potently inhibited TWIK-1 (IC50 = 85 μM) and TREK-1 (IC50 = 41 μM) currents, also inhibited astrocytic passive conductance by 58% at a concentration of 200 μM. Third, a moderate sensitivity of passive conductance to low extracellular pH (6.0) supports a combined expression of acid-insensitive TREK-1, and to a lesser extent, acid-sensitive TWIK-1. Fourth, the astrocyte passive conductance showed low sensitivity to extracellular Ba2+, and extracellular Ba2+ blocked TWIK-1 channels at an IC50 of 960 μM and had no effect on TREK-1 channels. Finally, an immunocytochemical study showed colocalization of TWIK-1 and TREK-1 proteins with the astrocytic markers GLAST and GFAP in rat hippocampal stratum radiatum. In contrast, another K2P isoform TASK-1 was mainly colocalized with the neuronal marker NeuN in hippocampal pyramidal neurons and was expressed at a much lower level in astrocytes. These results support TWIK-1 and TREK-1 as being the major components of the long-sought K+ channels underlying the passive conductance of mature hippocampal astrocytes. [ABSTRACT FROM AUTHOR]

Accumulating evidence indicates a significant astrocytic involvement in cerebral ischemia neuropathology, but little is known about the immediate astrocytic responses to ischemia insults in terms of electrophysiology and their pathologic implications. We show that astrocytes in acute rat hippocampal slices responded reversibly to more than 30 mins oxygen and glucose deprivation (OGD) treatment with depolarized membrane potentials (Vm) in whole-cell current clamp recording. This depolarization was multiphasic, showing an initial ∼11 mins small-amplitude depolarization plateau, followed by a 6-mins accelerated depolarization, and then a second plateau. Oxygen and glucose deprivation-induced astrocyte Vm depolarization was only marginally inhibited, ∼10%, by inhibition of ionotropic glutamate, γ-aminobutyric acid, purinergic receptors, and glutamate transporters presumed to be present on astrocytes in situ, suggesting increase in extracellular [K+] was primarily responsible for the astrocytic Vm change. The Vm depolarization was five-fold greater when glycolysis was inhibited by iodoacetate in a short 8 mins OGD treatment, suggesting glycolytic ATP is critical in maintaining extracellular K+ homeostasis in the early phase of OGD. Addition of oxidative metabolism inhibitors had much less effect. Cessation of OGD was always followed by a rapid and transient 9 mV astrocyte Vm hyperpolarization relative to the control Vm that was inhibited by ouabain, indicating a reactively enhanced Na+/K+-ATPase activity in post-OGD reperfusion. Altogether, hippocampal astrocytes appear to be electrophysiologically more resistant to acute ischemia insults as compared with neurons, and this should allow astrocytes to rescue endangered neurons in the face of acute ischemia insults via their various homeostatic functions.Journal of Cerebral Blood Flow & Metabolism (2008) 28, 456–467; doi:10.1038/sj.jcbfm.9600545; published online 22 August 2007 [ABSTRACT FROM AUTHOR]

It has been proposed that astrocytes should no longer be viewed purely as support cells for neurons, such as providing a constant environment and metabolic substrates, but that they should also be viewed as being involved in affecting synaptic activity in an active way and, therefore, an integral part of the information-processing properties of the brain. This essay discusses the possible differences between a support and an instructive role, and concludes that any distinction has to be blurred. In view of this, and a brief overview of the nature of the data, the new evidence seems insufficient to conclude that the physiological roles of mature astrocytes go beyond a general support role. I propose a model of mature protoplasmic astrocyte function that is drawn from the most recent data on their structure, the domain concept and their syncytial characteristics, of an independent rather than integrative functioning of the ends of each process where the activities that affect synaptic activity and blood vessel diameter will be concentrated. [ABSTRACT FROM PUBLISHER]

Ubiquitously expressed volume-regulated anion channels (VRACs) are chloride channels which are permeable to a variety of small organic anions, including the excitatory amino acids (EAAs) glutamate and aspartate. Broad spectrum anion channel blockers strongly reduce EAA release in cerebral ischaemia and other pathological states associated with prominent astrocytic swelling. However, it is uncertain whether VRAC serves as a major pathway for EAA release from swollen cells. In the present study, we measured swelling-activated release of EAAs asd-[3H]aspartate efflux, and VRAC-mediated Cl− currents by whole-cell patch clamp in cultured rat astrocytes. We compared the pharmacological profiles of the swelling-activated EAA release pathway and Cl− currents. The expression of candidate Cl− channels was confirmed by RT-PCR. The maxi Cl− channel (p-VDAC) blocker Gd3+, the ClC-2 inhibitor Cd2+, and the MDR-1 blocker verapamil did not affect EAA release or VRAC currents. An antagonist of calcium-sensitive Cl− channels (CaCC), niflumic acid, had little effect on EAA release and only partially inhibited swelling-activated Cl− currents. The phorbol ester PDBu, which blocks ClC-3-mediated Cl− currents, had no effect on VRAC currents and up-regulated EAA release. In contrast, DCPIB, which selectively inhibits VRACs, potently suppressed both EAA release and VRAC currents. Two other relatively selective VRAC inhibitors, tamoxifen and phloretin, also blocked the VRAC currents and strongly reduced EAA release. Taken together, our data suggest that (i) astrocytic volume-dependent EAA release is largely mediated by the VRAC, and (ii) the ClC-2, ClC-3, ClC-4, ClC-5, VDAC, CaCC, MDR-1 and CFTR gene products do not contribute to EAA permeability. [ABSTRACT FROM AUTHOR]

Glia show marked heterogeneity in terms of electrophysiology in the developing brain, and two major types can be identified based on GFAP or NG2 expression. However, it remains to be determined if such an electrophysiological diversity holds for the adult brain and how GFAP and NG2 lineage glia are associated with different electrophysiological phenotypes during the course of development. To address these fundamental questions, we performed in situ whole cell recording from morphologically identified glia from the rat hippocampal CA1 region from postnatal (P) days 1–106 and double-stained postrecorded cells with GLAST and NG2 antibodies. We found glia express mostly voltage-gated outward K+ currents and also have inward Na+ currents in the newborn (P1–P3), but these are no longer present after P22. They consist equally of GLAST(+) and NG2(+) cells in the newborn, but are mainly NG2(+) in juvenile animals (P4–P21). Glia showing voltage-gated outward and inward K+ currents are also present at P1, peak at P5 and decline to a stationary level of ∼10% in the adult. They are GLAST(+) astrocytes from newborn to juvenile but NG2(+) glia in the adult. Electrophysiologically passive glia first appear at P4 and increase to 91% in adults, of which 85% are GLAST(+). These results indicate that glial electrophysiological diversity occurs predominantly in the developing brain. While most glia in the NG2 lineage preserve a certain amount of voltage-gated ion conductances, mature GLAST(+) astrocytes are electrophysiologically passive. [ABSTRACT FROM AUTHOR]

Ubiquitously expressed volume-regulated anion channels (VRACs) are activated in response to cell swelling but may also show limited activity in nonswollen cells. VRACs are permeable to inorganic anions and small organic osmolytes, including the amino acids aspartate, glutamate, and taurine. Several recent reports have demonstrated that neurotransmitters or hormones, such as ATP and vasopressin, induce or strongly potentiate astrocytic whole cell Cl- currents and amino acid release, which are inhibited by VRAC blockers. In the present study, we explored the intracellular signaling mechanisms mediating the effects of ATP on D-[3H]aspartate release via the putative VRAC pathway in rat primary astrocyte cultures. Cells were exposed to moderate (5%) or substantial (30%) reductions in medium osmolarity. ATP strongly potentiated D-[3H]aspartate release in both moderately swollen and substantially swollen cells. These ATP effects were blocked (≥80% inhibition) by intracellular Ca2+ chelation with BAPTA-AM, calmodulin inhibitors, or a combination of the inhibitors of protein kinase C (PKC) and calmodulin-dependent kinase II (CaMK II). In contrast, control D-[3H]aspartate release activated by the substantial hyposmotic swelling showed little (≤25% inhibition) sensitivity to the same pharmacological agents. These data indicate that ATP regulates VRAC activity via two separate Ca2+-sensitive signaling cascades involving PKC and CaMK II and that cell swelling per se activates VRACs via a separate Ca2+/calmodulin-independent signaling mechanism. Ca2+-dependent organic osmolyte release via VRACs may contribute to the physiological functions of these channels in the brain, including astrocyte-tu-neuron intercellular communication. [ABSTRACT FROM AUTHOR]