Intracellular consequences of glutamate receptor activation in mouse hippocampal neurons : an imaging based study

Abstract

Intracellular consequences of glutamate receptor activation were investigated in mouse hippocampal neurons using fluorescent probes for the measurement of Ca²⁺ (Fluo-3 AM; Fluo-5N AM), mitochondrial DΨ (TMRE) and superoxide (Het) in conjunction with confocal laser scanning microscopy (CLSM). The excessive activation of glutamate receptors is thought to be a common feature of neuronal death in a wide spectrum of neurological disorders. The intracellular events that link toxic glutamate receptor activation to eventual cell death are not well understood, but are likely to involve to loss of Ca²⁺ homeostasis, mitochondrial dysfunction and free radical damage.

Glutamate acts on a large number of ionotropic and metabotropic receptor subtypes. The selective activation of NMDA receptors was particularly damaging, causing neuronal cell death in over 80% of cultured mouse hippocampal neurons 24 hours after drug application. In contrast, agonists at other glutamate receptor subtypes including kainate and ACPD caused only 14% and 7% cell death respectively. Doses of glutamate receptor agonist were selected to elicit equivalent "early" neuronal Ca²⁺ increases are measured by Fluo-3 AM in the first 200 seconds following drug addition. Early neuronal Ca²⁺ increases did not correlate with eventual neuronal death, suggesting that differential routes of Ca²⁺ increase could have different pathological consequences for neurons. Experiments were then conducted to investigate intracellular events downstream of glutamate receptor activation that could lead to neuronal death.

The same doses of glutamate receptor agonists had profoundly different effects on mitochondrial DΨ, which correlated to neuronal death, NMDA caused a robust increase in TMRE fluorescence indicative of mitochondrial depolarization, whereas kainate, ACPD, AMPA and quisqualate had no significant effect on mitochondrial DΨ despite causing equivalent early Ca²⁺ increases. All the glutamate receptor agonists caused increased neuronal superoxide production as measured by increased Het fluorescence.

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