Glutamate-induced calcium transient triggers delayed calcium overload and neurotoxicity in rat hippocampal neurons

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 μm 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 μM) 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.