Glutamate-induced changes in intracellular free Ca2+ concentration ([Ca2+]i) were recorded in single rat hippocampal neurons grown in primary culture by employing the Ca2+ indicator indo-1 and a dual-emission microfluorimeter. The [Ca2+]i was monitored in neurons exposed to 100 microM glutamate for 5 min and for an ensuing 3 hr period. Ninety-two percent (n = 64) of these neurons buffered the glutamate-induced Ca2+ load back to basal levels after removal of the agonist; thus, the majority of cells had not lost the ability to regulate [Ca2+]i at this time. However, following a variable delay, in 44% (n = 26) of the neurons that buffered glutamate-induced Ca2+ loads to basal levels, [Ca2+]i rose again to a sustained plateau and failed to recover. The changes in [Ca2+]i that occur during glutamate-induced delayed neuronal death can be divided into three phases: (1) a triggering phase during which the neuron is exposed to glutamate and the [Ca2+]i increases to micromolar levels, followed by (2) a latent phase during which the [Ca2+]i recovers to a basal level, and (3) a final phase that begins with a gradual rise in the [Ca2+]i that reaches a sustained plateau from which the neuron does not recover. This delayed Ca2+ overload phase correlated significantly with cell death. The same sequence of events was also observed in recordings from neuronal processes. The delayed Ca2+ increase and subsequent death were dependent upon the presence of extracellular Ca2+ during glutamate exposure. Calcium influx during the triggering phase resulted from the activation of both NMDA and non-NMDA receptors as indicated by studies using receptor antagonists and ion substitution. Treatment with TTX (1 microM) or removal of extracellular Ca2+ for a 30 min window following agonist exposure failed to prevent the delayed Ca2+ overload. The delayed [Ca2+]i increase could be reversed by removing extracellular Ca2+, indicating that it resulted from Ca2+ influx. The three phases defined by changes in the [Ca2+]i during glutamate-induced neuronal toxicity suggest three distinct targets to which neuroprotective agents may be directed.

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 microM glutamate (delta pH = 0.41 +/- 0.14, mean +/- SD) was blocked by the N-methyl-D-aspartate (NMDA) receptor antagonist CGS19755 (10 microM) 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 microM), 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.

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