Differential role of synaptic an extrasynaptic NMDA receptors in glutamate mediated neuronal injury

It has been known for 30 years that high concentrations of glutamate induce cell death in vitro and that similar extracellular concentrations are present in the rodent brain during ischaemia. It was subsequently shown that NMDA receptors mediate most of the glutamate-induced cell death in vitro and in vivo. Taken together, these data suggested that administration of NMDA antagonists in human beings could prevent cell death and confer neuroprotection after stroke. However, discouraging news started to accumulate as the clinical trials were terminated. Despite these developments, the theory of glutamate-induced excitotoxicity, the major power that forced NMDA receptor antagonists into human trials, has not been questioned. So it is possible that a fundamental misunderstanding of the pathophysiological roles of NMDA receptors may have been at the origin of absence of efficacy of glutamate antagonists in the treatment of stroke in Man. In the present study, we investigate the influence of NMDA receptors cellular locations on excitotoxicity.

Materials and methods

All experiments were performed on murine primary cortical cultures containing both neurons and astrocytes prepared from foetal swiss mice at 14–16 days gestation. Cultures were used after 14 days in vitro. Neuronal death was quantified by the measurement of lactate deshydrogenase (LDH) release from damaged cells into the bathing medium. Intracellular free Ca²⁺ measurement were performed by using videomicroscopy, confocal microscopy and calcium sensitive fluorescent probes Fura-2 and Fluo-3.

Results

By blocking GABAA receptor function (with bicuculine at 50 μM + 4-AP at 2.5 mM), cortical neurons fire burst of action potential which resulted in calcium plateaus visualized by videomicroscopy. This increase in intracellular calcium concentration is fully blocked by the co-application of TTX (at 0.5 μM), a compound know to inhibit electrical activity and by the co-application of AP-5, a selective NMDA receptor antagonist. These results demonstrate the pivotal role of synaptic NMDA receptors in the calcium plateaus induced by bicuculine treatment. Next we exposed primary cortical neurons for 24 h to an intense activation of synaptic NMDA receptors by treatments with increasing doses of bicuculine. This treatment did not induce any neurotoxicity. In order to investigate the role of extrasynaptic NMDA receptors in glutamate neurotoxicity, we selectively inactivated synaptic NMDA receptors by exposing neurons to a NMDA receptor open-channel blocker (MK801 at 10 μM) under bicuculline treatment and we performed bath application of increasing concentration of NMDA. In these conditions, we measured a doses dependant increase in intracellular calcium concentration mediated by the selective activation of extrasynaptic NMDA receptors. When applied for 24 h, this conditions induced a dose dependant neurotoxicity. In conclusion, these results demonstrate that synaptic NMDA receptors activation promotes important intracellular calcium concentration increase that do not lead to excitotoxicity. While the activation of extrasynaptic NMDA receptors are directly involved in the neurotoxic effect of glutamate. Therefore a selective targeting of extrasynaptic glutamate receptors may improve the beneficial effect of NMDA antagonists for the treatment of stroke.