Expression of a linear current-voltage (I-V) relationship (passive) Kϩ membrane conductance is a hallmark of mature hippocampal astrocytes. However, the molecular identifications of the K ϩ channels underlying this passive conductance remain unknown. We provide the following evidence supporting significant contribution of the two-pore domain K ϩ channel (K 2P ) 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 ϩ currents, but vary in their relative P Cs /P K permeability, 0.43, 0.10, and 0.05, respectively. Second, quinine, which potently inhibited TWIK-1 (IC 50 ϭ 85 M) and TREK-1 (IC 50 ϭ 41 M) currents, also inhibited astrocytic passive conductance by 58% at a concentration of 200 M. 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 Ba 2ϩ , and extracellular Ba 2ϩ blocked TWIK-1 channels at an IC 50 of 960 M 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 K 2P 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 ϩ channels underlying the passive conductance of mature hippocampal astrocytes.

Mammalian protoplasmic astrocytes are extensively coupled through gap junction channels but the biophysical properties of these channels under physiological and ischemic conditions in situ are not well defined. Using confocal morphometric analysis of biocytin-filled astrocytic syncytia in rat hippocampal CA1 stratum radiatum we found that each astrocyte directly couples, on average, to 11 other astrocytes with a mean interastrocytic distance of 45 microm. Voltage-independent and bidirectional transjunctional currents were always measured between directly coupled astrocyte pairs in dual voltage-clamp recordings, but never from astrocyte-NG2 glia or astrocyte-interneuron pairs. The electrical coupling ratio varied considerably among astrocytes in developing postnatal day 14 rats (P14, 0.5-12.4%, mean = 3.6%), but became more constant in young adult P21 rats (0.18-3.9%, mean = 1.6%), and the coupling ratio declined exponentially with increasing pair distance. Electrical coupling was not affected by short-term oxygen-glucose deprivation (OGD) treatment, but showed delayed inhibition in an acidic extracellular pH of 6.4. Combination of acidic pH (6.4) and OGD, a condition that better represents cerebral ischemia in vivo, accelerated the inhibition of electrical coupling. Our results show that, under physiological conditions, 20.7-24.2% of K(+) induced currents can travel from any astrocytic soma in CA1 stratum radiatum to the gap junctions of the nearest neighbor astrocytes, but this should be severely inhibited as a consequence of the OGD and acidosis seen in the ischemic brain.

Whole-cell patch clamp recording has been successfully used in identifying the voltage-dependent gating and conductance properties of ion channels in a variety of cells. However, this powerful technique is of limited value in studying low membrane resistance cells, such as astrocytes in situ, because of the inability to control or accurately measure the real amplitude of command voltages. To facilitate the study of ionic conductances of astrocytes, we have developed a dual patch recording method which permits membrane current and membrane potential to be simultaneously recorded from astrocytes in spite of their extraordinarily low membrane resistance. The utility of this technique is demonstrated by measuring the voltage-dependent activation of the inwardly rectifying K+ current abundantly expressed in astrocytes and multiple ionic events associated with astrocytic GABAA receptor activation. This protocol can be performed routinely in the study of astrocytes. This method will be valuable for identifying and characterizing the individual ion channels that orchestrate the electrical activity of low membrane resistance cells.