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 μM tetrodotoxin (TTX) or by saline containing 10 mM Mg²⁺, suggesting that they were caused by periodic bursting activity of synaptically coupled cells. Opening of voltage-gated Na⁺ channels by application of 50 μM 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 (≤ 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⁺]₀, suggesting that activity-induced [K⁺]_o changes in the brain might not significantly influence neuronal Na⁺,K⁺-ATPase activity.