Technical Reference #96

96.

N-Methyl-D-Aspartate Induces A Rapid; Reversible; And Calcium-Dependent Intracellular Acidosis In Cultured Fetal Rat Hippocampal Neurons Irwin; R.P.; Zin; S.Z.; Long; R.T.;, National Institute of Mental Health, The Journal of Neuroscience, 14(96), (1994)
Link To Paper

Abstract:
The ability of NMDA to alter intracellular pH (pHi) was studied in fetal rat hippocampal neurons and glia using the pH-sensitive fluorescent indicator 2'7'-bis-(2-carboxyethyl)-5-(and-6)- carboxyfluorescein (BCECF). Brief exposure (60 sec) of hippocampal neurons to NMDA (2.5-250 microM) results in a rapid and in most cells reversible reduction in pHi with full recovery to baseline pHi values taking several minutes following removal of NMDA. In contrast little or no change in pHi was observed in glial cells exposed to these same concentrations of NMDA. The NMDA-induced acidification of neurons was concentration and time dependent with an EC50 of 39 microM and Emax (delta pH) of -0.53. More prolonged exposure to NMDA (> or = 10 min) resulted in a more prolonged reduction in pHi values over the ensuing 20 min observation period. The intracellular acidification resulting from NMDA exposure of hippocampal neurons was blocked by the NMDA receptor antagonist 3-((+/-)-2-carboxypiperazin-4-yl)-propyl-1- phosphonic acid (CPP). Moreover removal of extracellular Ca2+ eliminated both the selective NMDA-induced elevation in [Ca2+]i and the reduction in pHi indicating that Ca2+ influx may be required for the decrease in pHi induced by NMDA receptor activation. Finally the NMDA- induced reduction in pHi was not significantly attenuated when extracellular [H+] was decreased by increasing extracellular pH to 8.0. The latter suggests that an intracellular source of H+ is responsible for the NMDA-induced reduction in neuronal pHi. The reduction in neuronal pHi induced by NMDA receptor activation may mediate some of the physiological and (or) pathophysiological actions of glutamate.

Materials & Methods:
ley rat embryos were maintained in primary culture as described by Segal(l983). Briefly hippocampal tissue was rapidly dissected and mechanically disrupted and the cell suspension plated onto poly-L-lysine (Sigma St. Louis MO)-coated glass-bottom 35 mm culture dishes (MatTek Ashland MA) containing Modified Eagle’s Medium with Earle’s salts (Gibco Laboratories Grand Island NY) supplemented with 10% fetal calf serum 10% horse serum 6000 gm/liter glucose and 2 mM glutamine. Cells were incubated in a humidified atmosphere containing 5% CO and 95% air. Culture media lackina fetal calf serum but containing- 5% horse serum were added 7 d aft& plating. All experiments were carried out using cells maintained 7- 14 d in vitro. Perfusion solutions. Cultures were washed three times.

Microscopic Technique
Phase Contrast Microscopy

Cell Type(s)
Neurons - Hippocampal