title: Regulation of intracellular sodium in cultured rat hippocampal neurones.
abstract: 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 microM 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 microM 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 (> or = 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.
authors.affiliation: Department of Neurology, Yale University School of Medicine, New Haven, CT 06520, USA. crose@biomed.med.yale.edu
authors.forename: C R
authors.initials: CR
authors.lastname: Rose
authors.forename: B R
authors.initials: BR
authors.lastname: Ransom
mesh_terms: D000818:Animals; D001572:Benzofurans / Q000378:metabolism; D001639:Bicarbonates / Q000494:pharmacology; D002034:Bumetanide / Q000494:pharmacology; D002245:Carbon Dioxide / Q000494:pharmacology; D002478:Cells, Cultured; D018920:Coculture Techniques; D004232:Diuretics / Q000494:pharmacology; D004988:Ethers, Cyclic / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D006624:Hippocampus / Q000166:cytology / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000378:metabolism; D009457:Neuroglia / Q000378:metabolism; D009474:Neurons / Q000378:metabolism*; D011188:Potassium / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D012964:Sodium / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000378:metabolism; D013779:Tetrodotoxin / Q000494:pharmacology; D014701:Veratridine / Q000494:pharmacology
chemical_list: D001572:Benzofurans; D001639:Bicarbonates; D004232:Diuretics; D004988:Ethers, Cyclic; D005456:Fluorescent Dyes; D002034:Bumetanide; C061665:sodium-binding benzofuran isophthalate; D002245:Carbon Dioxide; D013779:Tetrodotoxin; D014701:Veratridine; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D008274:Magnesium; D011188:Potassium