COMPLEX CORRELATION BETWEEN EXCITATORY AMINO ACID-INDUCED INCREASE IN THE INTRACELLULAR CA2+ CONCENTRATION AND SUBSEQUENT LOSS OF NEURONAL FUNCTION IN INDIVIDUAL NEOCORTICAL NEURONS IN CULTURE

Primary cultures of cerebral cortical neurons and single-cell imaging of intracellular free Ca2+ concentration ([Ca2+](i)) with the ratiometric dye fura-2 were used to assess excitatory amino acid (EAA)-induced neurotoxicity; the loss of neuronal function as defined by the ability of the cells to respond to K+-induced depolarization by a transient increase in Ca2+ influx was measured. The responsiveness of individual neurons was measured quantitatively as the [Ca2+](i) values of the second KCl (2.KCl) stimulation divided by those of the first KCl (1.KCl) stimulation, giving the value of the ratio (2.KCl/1,KCl). Exposure to EAAs led to an increase in [Ca2+](i), but no simple correlation between the increase in [Ca2+](i) and neuronal responsiveness could be demonstrated. Rather, below a threshold level of [Ca2+](i) (ca. 1 mu M), the neuronal responsiveness was largely independent of the glutamate receptor-agonist-induced increase in [Ca2+](i). However, when [Ca2+](i) increased above this threshold lever, the neurons almost invariably lost the ability to respond to a K+-induced depolarization, particularly after exposure to glutamate. Therefore, the cortical neurons were found to be exceptionally vulnerable to the glutamate-induced loss of function when compared with the effect induced by the glutamate receptor subtype-specific agonists, N-methyl-D-aspartate, quisqualate, and 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl) propionate. The findings suggest that the loss of neuronal membrane polarization precedes plasma membrane disruption and is a sensitive marker of EAA-induced neurodegeneration observed at the single-cell level.