Excitatory amino acid‐induced alterations of cytoplasmic free Ca²⁺ individual cerebellar granule neurons: Role in neurotoxicity

Abstract

The effects of glutamate on intracellular free Ca2+, [Ca2+]i, and neurotoxicity were compared in cerebellar granule neurons in vitro. [Ca2+]i was measured with fura-2 and digital fluorescence imaging microscopy; neurotoxicity was monitored using a vital dye and colorimetric analysis. Glutamate produced dose-dependent increases in [Ca2+]i, which tended to be transient for glutamate concentrations in a range of 0.01-0.5 microM and sustained for higher levels of glutamate. The ED50 for the [Ca2+]i response to glutamate was 6 microM. The LD50 for glutamate-induced neurotoxicity was similar, i.e., 10 microM. The effect of glutamate on [Ca2+]i was greatly diminished when external Ca2+ was removed and blocked by Mg2+ or N-methyl-D-aspartate (NMDA)-type receptor antagonists. The latter conditions as well as preloading granule neurons with the intracellular Ca2+ chelator quin2 largely prevented glutamate cytotoxicity. The neurotoxic effect of glutamate required incubations with the stimulus for 10-20 min at 25 degrees C. Withdrawal of glutamate after this period was accompanied by a prolonged alteration in [Ca2+]i. Pretreatment of the cells with the ganglioside GM1 reduced this late increase in [Ca2+]i as well as the neurotoxic effects of glutamate. This indicates that glutamate-induced neurotoxicity results from a composite of diverse temporal alterations in Ca2+ homeostasis and that blunting any of these components reduces excitotoxicity.

The use of antioxidants to prevent glutamate-induced derangement of calcium ion metabolism in rat cerebral cortex synaptosomes

“…Some authors do not believe these reactions to be involved, assigning the leading role to the activation of protein kinases and phosphatases [9,14,27]. Other authors hold that activation of free-radial reactions triggered by calcium ions entering cells after interaction of glutamate with ionotropic NMDA receptors is a necessary stage in mediating the neurotoxicity of glutamate [24,26,37].…”

“…This increase is initiated by glutamate released from nerve terminals into the intercellular space at high concentrations and acting on a variety of glutamate receptors. The leading role appears to be played by the interaction of glutamate with NMDA receptors [8,13,14,26,27]. The biochemical mechanisms underlying derangements of the intracellular Ca 2+ concentration as a result of this interaction remain unclear.…”

Characterization of glutamate toxicity in cultured rat cerebellar granule neurons at reduced temperature

“…In cultured rat cerebellar granule neurons, glutamate excitotoxicity is also mediated by Ca ‫םם‬ entry through NMDA receptors (11,12). Cerebellar granule neurons differ, however, in their relative resistance to these glutamate-induced effects.…”

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.

“…This is underlined by the finding that depolarization per se is not the cause of neuronal degeneration, since high concentrations of K+ are not toxic (refs. 22 (28). Alternatively, the level of…”

“…Although the amplitude of the EAAinduced increase in [Ca2e]i (28,29) or the time to recover from the plateau level (30) has been found to be predictive for subsequent cell death, such a distinct correlation has not always been reported (19,31,32) except for studies that showed that the duration of the agonist application and the source of Ca2+ were determinants of neuronal death (24,33).…”