Confocal microscopic visualization of MK-801-induced cytoplasmic vacuoles in vitro

We examined neuroprotective and neurotoxic effects of the NMDA antagonist, MK-801, in primary cell cultures derived from embryonic mouse neocortex. Brief deprivation of oxygen and glucose, or direct application of N-methyl- D-aspartate (NMDA), resulted in acute neuronal swelling followed by neuronal death during the next day. This excitotoxic neuronal injury could be blocked by inclusion of a wide variety of NMDA antagonists in the cell culture medium. MK-801 attenuated neuronal death in the low micromolar range; 1 to 10 μM concentrations were sufficient to maximally reduce injury from NMDA toxicity, oxygen deprivation, or combined deprivation of oxygen and glucose. MK-801 alone caused no apparent toxicity at these concentrations in exposures of 24 to 48 hours. However, 24-hour exposure to 100 μM MK-801 resulted in appearance of cytoplasmic vacuoles, which could be visualized with immunofluorescence against the microtubule associated protein, MAP2, together with laser scanning confocal microscopy. Thus, at concentrations sufficient to block NMDA receptors, MK-801 is neuroprotective rather than neurotoxic for cortical neurons in vitro. This model system may provide a method to examine cellular mechanisms underlying the neurotoxicity of MK-801 at very high concentrations.