Glutamate-induced intracellular acidification of cultured hippocampal neurons demonstrates altered energy metabolism resulting from Ca²⁺ loads

1. Glutamate-evoked increases in intracellular free H⁺ concentration ([H⁺](i)) were recorded from single rat hippocampal neurons grown in primary culture with carboxy SNARF-based dual emission microfluorimetry. The possibility that this acidification resulted from altered energy metabolism was investigated. 2. The response to 10 μM glutamate (ΔpH = 0.41 ± 0.14, mean ± SD) was blocked by the N-methyl-D-aspartate (NMDA) receptor antagonist CGS19755 (10 μM) and required extracellular Ca²⁺. 3. Substituting the metabolic inhibitor 2-deoxyglucose for glucose in the extracellular buffer prevented glutamate-induced acidification. 4. Ba²⁺, which carries charge through Ca²⁺ channels, including the Ca²⁺ uniporter on the inner mitochondrial membrane, substituted for Ca²⁺ in mediating glutamate-induced cytoplasmic acidification. 5. Microinjection of ruthenium red, a compound that blocks mitochondrial Ca²⁺ sequestration, significantly inhibited glutamate-induced acidification. 6. The mitochondrial uncoupler, carbonyl cyanide-p-trifluoromethoxyphenyl-hydrazone (FCCP, 0.1 μM), mimicked and partially occluded the glutamate-induced [H⁺](i), increase. 7. These findings indicate that glutamate-induced Ca²⁺ loads are sequestered by mitochondria, uncouple respiration, and produce metabolic acidosis. The glutamate-induced acidification is symptomatic of metabolic stress and may indicate that mitochondria play an important role in glutamate-induced neuronal death.