Abstract
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.
