Regulation of intracellular sodium in cultured rat hippocampal neurones

1. We studied regulation of intracellular Na+ concentration ([Na+](i)) in cultured rat hippocampal neurones using fluorescence ratio imaging of the Na+ indicator dye SBFI (sodium-binding benzofuran isophthalate). 2. In standard CO2/HCO3--buffered saline with 3 mM K+, neurones had a baseline [Na+](i) of 8.9 +/- 3.8 mM (mean +/- S.D.). Spontaneous, transient [Na+](i) increases of 5 mM were observed in neurones on 27% of the coverslips studied. These [Na+](i) increases were often synchronized among nearby neurones and were blocked reversibly by 1 mu M tetrodotoxin (TTX) or by saline containing 10 mM Mg2+, suggesting that they were caused by periodic bursting activity of snynaptically coupled cells. Opening of voltage-gated Na+ channels by application of 50 mu M veratridine caused a TTX-sensitive [Na+](i) increase of 25 mM. 3. Removing extracellular Na+ caused an exponential decline in [Na+](i) to values close to zero within 10 min. Inhibition of Na+,K+-ATPase by removal of extracellular K+ or ouabain application evoked a [Na+](i) increase of 5 mM min(-1). Baseline [Na+](i) was similar in the presence or absence of CO2/HCO3-; switching from CO2/HCO3--free to CO2/HCO3--buffered saline, however, increased [Na+](i) transiently by 3 mM, indicating activation of Na+-dependent Cl--HCO3- exchange. Inhibition of Na+-K+-2Cl(-) cotransport by bumetanide had no effect on [Na+](i). 4. Brief, small changes in extracellular K+ concentration ([K+ ](o)) Influenced neuronal [Na+](i) only weakly. Virtually no change in [Na+](i) was observed with elevation or reduction of [K+](o) by 1 mM. Only 30% of cells reacted to 3 min [K+](o) elevations of up to 5 mM. In contrast, long [K+](o) alterations (greater than or equal to 10 min) to 6 mM or greater slowly changed steady-state [Na+](i) in the majority of cells. 5. Our results indicate several differences between [Na+](i) regulation in cultured hippocampal neurones and astrocytes. Baseline [Na+](i) is lower in neurones compared with astrocytes and is mainly determined by Na+,K+-ATPase, whereas Na+-dependent Cl--HCO3- exchange, Na+-HCO3- cotransport or Na+-K+-2Cl(-) cotransport do not play a significant role. In contrast to glial cells, [Na+](i) of neurones changes only weakly with small alterations in bath [K+](o), suggesting that activity-induced [K+](o) changes in the brain might not significantly influence neuronal Na+,K+-ATPase activity.