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. 1994 Dec 6;91(25):12303–12307. doi: 10.1073/pnas.91.25.12303

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.

M R Witt 1, K Dekermendjian 1, A Frandsen 1, A Schousboe 1, M Nielsen 1
PMCID: PMC45425  PMID: 7527559

Abstract

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 microM), the neuronal responsiveness was largely independent of the glutamate receptor-agonist-induced increase in [Ca2+]i. However, when [Ca2+]i increased above this threshold level, 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.

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Selected References

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