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 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.
Witt
M R
MR
Department of Biochemistry, Sct. Hans Hospital, Roskilde, Denmark.
Dekermendjian
K
K
Frandsen
A
A
Schousboe
A
A
Nielsen
M
M
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000502:physiology*; D018846:Excitatory Amino Acids / Q000494:pharmacology*; D005333:Fetus; D018698:Glutamic Acid / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D051379:Mice; D016202:N-Methylaspartate / Q000494:pharmacology; D009474:Neurons / Q000187:drug effects / Q000502:physiology*; D011189:Potassium Chloride / Q000494:pharmacology; D016318:Quisqualic Acid / Q000494:pharmacology; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid / Q000494:pharmacology
D018846:Excitatory Amino Acids; D018698:Glutamic Acid; D016202:N-Methylaspartate; D011189:Potassium Chloride; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; D016318:Quisqualic Acid; D002118:Calcium