Routes of zinc entry in mouse cortical neurons: role in zinc-induced neurotoxicity.

Exposure of central neurons to Zn2+ triggers neuronal death. The routes of Zn2+ entry were investigated in living cortical neurons from the mouse using the specific Zn2+ fluorescent dye N-(6-methoxy-8-quinolyl)-p-toluene sulphonamide (TSQ), which preferentially detects membrane-bound Zn2+. Exposure of cortical neurons to increasing concentrations of Zn2+ (1-100 microM) induced a progressive increase in the fluorescence of TSQ. This fluorescence signal was not attenuated by the permeation of plasma membrane with digitonin. Accordingly, the major part of TSQ fluorescence (two-thirds) was associated to the particulate fraction of cortical neurons exposed to Zn2+. These results suggest that Zn2+ detected with TSQ in neurons is mainly bound to membranes. TSQ fluorescence measured in neurons exposed to 3 microM Zn2+ was enhanced by Na+-pyrithione, a Zn2+ ionophore, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), N-methyl-D-aspartate (NMDA) or KCl-induced depolarization. However, in the absence of any treatment, TSQ labelling of neurons exposed to 3 microM Zn2+ was only decreased by NMDA receptor antagonists, whereas it remained unaltered in the presence of antagonists of AMPA receptors or L-type voltage-gated Ca2+ channels. Zn2+ entry through NMDA receptors did not contribute to Zn2+-induced neuronal death, as it was prevented by antagonists of NMDA receptors only when they were added after the Zn2+ exposure. Finally, Zn2+ induced a delayed accumulation of extracellular glutamate which might be responsible for the delayed NMDA receptor activation that leads to neuronal death.