1. We probed for the expression of electrogenic Na+/HCO3- co-transport in cultured mammalian astrocytes by recording voltage and current changes induced by bath application of HCO3-, with the use of patch-clamp electrophysiology. Application of 25 mM HCO3-, at a constant pHo, to astrocytes bathed in a nominally HCO3(-)-free solution, produced an abrupt and reversible change in membrane potential ranging from +3 to -30 mV [-11.8 +/- 9.34 (SD) mV]; 55% of cells showed relatively large hyperpolarizing responses (-18.8 +/- 6.23 mV), whereas 45% showed only small shifts in membrane potential (range of -5 to +3 mV; -1.9 +/- 1.96 mV). 2. The size of the HCO3(-)-induced hyperpolarization was strongly related to the cell's initial resting membrane potential in HCO3(-)-free solution; the larger responses were seen in cells with relatively low resting membrane potentials (-48.5 +/- 9.4 mV), and the smaller responses were seen in cells with more negative potentials (-68.1 +/- 6.5 mV). The membrane potentials of hippocampal astrocytes were highly variable in HCO3(-)-free solution (range -38 to -80 mV; -60.9 +/- 12.53); this variability was greatly reduced in HCO3(-)-containing solution (range -59 to -82 mV; -68.5 +/- 4.8). 3. The magnitude of the HCO3(-)-induced response was less strongly correlated with cell input resistance, which was higher in the larger responder cells than in the small responders. However, the differences in input resistance were insufficient to account for the different HCO3(-)-induced responses observed. 4. In the presence of extracellular Ba2+, which by blocking K+ conductance depolarized cells by 30-50 mV, cells that initially showed a small response, showed a large and completely reversible hyperpolarization (-18.4 +/- 6.13 mV) to application of 25 mM HCO3-. In Na(+)-free solution, the HCO3(-)-induced hyperpolarization was reduced by 66%, and the response was not sustained, as in Na(+)-containing solution. Removal of extracellular Cl- had no effect on the HCO3- response The stilbene derivative 4,4'-diisothiocyano-2,2'-stilbene disulfonate (DIDS), a blocker of anion transport, eliminated the HCO3(-)-induced hyperpolarization. Blockers of Na+/K+ ATPase and Na(+)-H+ exchange were without effect. These observations indicated the presence of an electrogenic Na+/HCO3- co-transporter in hippocampal astrocytes. 6. Voltage-clamp recording demonstrated that the HCO3(-)-induced hyperpolarization was caused by outward currents averaging 335 +/- 104 pA. The reversal potential of the HCO3(-)-induced current ranged between -80 and -99 mV with an average = -86.1 +/- 6.2 mV.(ABSTRACT TRUNCATED AT 400 WORDS)

Summary: Purpose: We studied Na' channel expression and the ability to generate action potential (AP)-like responses in primary cultures of human astrocytes by whole cell patchclamp recording techniques.Methods: Tissue samples from 22 patients with various classifications of temporal lobe epilepsy (TLE) were plated to form separate astrocyte cultures from three regions; the hippocampus, parahippocampus, and anterolateral temporal neocortex.Results: The resting membrane potential of seizure focus astiocytes (MTLE, mesial TLE) was significantly depolarized (approximately -55 mV) as compared with cortical astrocytes (-80 mV). Hippocampal astrocytes from other substrates for TLE (MaTLE, mass-associated TLE; PTLE, paradoxical TLE) in which the hippocampus is not the seizure focus displayed resting membrane potentials similar to those of neocortical astrocytes (approximately -75 mV). Astrocytes from the seizure focus (MTLE) displayed much larger tetrodotoxin (TTX)-sensitive Na' currents with -66-fold higher Na' channel density (113.5 -c 17.41 pA/pf) than that of comparison neocortical astrocytes (1.7 r 3.7 pA/pf) or than that of the hippocampal and parahippocampal astrocytes of the MaTLE and PTLE groups. As a consequence of this higher channel density, seizure focus astrocytes were capable of generating AP-like responses. However, at the resting potential, most Na' channels are inactive and no spontaneous firing was observed. In contrast, astrocytes in the comparison neocortex from all groups and the hippocampus and parahippocampus from the MaTLE and PTLE groups could not fire AP-like responses even after large current injections.Conclusions: The function of Na' channels in these astrocytes is unclear. However, the marked differences in seizure focus astrocytes as compared with cortical and nonseizure focus hippocampal astrocytes suggest a more active role for astrocytes associated with hyperexcitable neurons at a seizures focus.