Routes of zinc entry in mouse cortical neurons: role in zinc-induced neurotoxicity Philippe Marin, Maurice Israe Èl, 1 Jacques Glowinski and Joe ÈlPre Âmont Chaire de Neuropharmacologie, INSERM U114, Colle Áge de France, 11, Place Marcelin Berthelot, 75231 Paris Cedex 05, France 1 CNRS UPR 9040, Laboratoire de Neurobiologie Cellulaire et Mole Âculaire, 1, Avenue de la Terrasse, 91198 Gif sur Yvette Cedex, France Keywords: excitotoxicity, glutamate, NMDA, TSQ Abstract ExposureofcentralneuronstoZn 2+ triggersneuronaldeath.TheroutesofZn 2+ entrywereinvestigatedinlivingcorticalneuronsfrom the mouse using the speci®c Zn 2+ ¯uorescent dye N-(6-methoxy-8-quinolyl)-p-toluene sulphonamide (TSQ), which preferentially detects membrane-bound Zn 2+ . Exposure of cortical neurons to increasing concentrations of Zn 2+ (1±100 mM) induced a progressive increase in the ¯uorescence of TSQ. This ¯uorescence signal was not attenuated by the permeation of plasma membrane with digitonin. Accordingly, the major part of TSQ ¯uorescence (two-thirds) was associated to the particulate fraction of cortical neurons exposed to Zn 2+ . These results suggest that Zn 2+ detected with TSQ in neurons is mainly bound to membranes. TSQ ¯uorescence measured in neurons exposed to 3 mM Zn 2+ was enhanced by Na + -pyrithione, a Zn 2+ ionophore, a-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 mM Zn 2+ was only decreased by NMDA receptor antagonists, whereas it remained unaltered in the presence of antagonists of AMPA receptors or L-type voltage-gated Ca 2+ channels. Zn 2+ entry through NMDAreceptorsdidnotcontributetoZn 2+ -inducedneuronaldeath,asitwaspreventedbyantagonistsofNMDAreceptorsonlywhen they were added after the Zn 2+ exposure. Finally, Zn 2+ induced a delayed accumulation of extracellular glutamate which might be responsible for the delayed NMDA receptor activation that leads to neuronal death. Introduction Zn 2+ is an essential trace element and plays a key role in the function of many enzymes and the control of gene expression. Zn 2+ is present in large amounts in discrete areas of the mammalian brain. The highest concentrations were found in the hippocampus and the cerebral cortex where Zn 2+ is primarily localized in nerve endings of glutamatergic neurons (Frederickson etal., 1983; 1987; Pe Ârez- Clauser & Danscher, 1985). Zn 2+ is released together with glutamate upon neuronal activity into the extracellular space where it can reach a concentration of several hundred micromolar (Assaf & Chung, 1984; Howell etal., 1984). Released Zn 2+ is an important modulator of inhibitory and excitatory synaptic transmissions. Indeed, Zn 2+ potentiates neuroexcitation mediated by a-amino-3-hydroxy-5- methylisoxazole-4-propionic acid (AMPA) receptors, whereas it blocks N-methyl-D-aspartate (NMDA) receptors via a dual mechan- ism involving a voltage-dependent or -independent inhibition, depending on its concentration (Peters etal., 1987; Christine & Choi, 1990; Rassendren etal., 1990; Paoletti etal., 1997). Micromolar concentrations of Zn 2+ also block g-aminobutyric acid (GABA) receptor-mediated responses (Westbrook & Mayer, 1987). High Zn 2+ concentrations trigger neuronal death (Yokoyama etal., 1986; Choi etal., 1988; Koh & Choi, 1994; Manev etal., 1997), an effect which is potentiated by AMPA receptor activation (Weiss etal., 1993; Freund & Reddig, 1994). Zn 2+ has also been implicated in several pathological disorders: (i) Zn 2+ in¯ux into neurons appears to be a key mechanism underlying neuronal death after transient global cerebral ischaemia (Koh etal., 1996); and (ii) it could also contribute to neuronal loss during Alzheimer's disease by either interacting with enzymes or proteins, including transcription factors, that are critical for neuronal survival, or by promoting aggregation of b-amyloid protein (for review, see Cuajungco & Lees, 1997). Several routes of Zn 2+ entry, including Zn 2+ in¯ux through voltage-gated Ca 2+ channels, NMDA and Ca 2+ -permeable AMPA receptors, have been implicated in Zn 2+ toxicity (Weiss etal., 1993; Koh & Choi, 1994; Manev etal., 1997). However, little is known about the intracellular homeostasis of Zn 2+ and its subcellular distribution. In two recent studies, mag-fura-5, fura-2 and mag-fura-2 have been used as Zn 2+ -sensitive ¯uorescent dyes to detect changes in intracellular free Zn 2+ in cultured neurons. Due to the lack of speci®city of these dyes for Zn 2+ , most of these experiments were performed in the absence of extracellular Ca 2+ and Mg 2+ , and changes in intracellular free Zn 2+ were only detected in the presence of very high concentrations of Zn 2+ (Sensi etal., 1997, Cheng & Reynolds, 1998). Histochemical experiments using the Zn 2+ -speci®c ¯uorescent dye N-(6-methoxy-8-quinolyl)-p-toluene sulphonamide (TSQ) have also provided information about the concentration and location of Zn 2+ in brain tissue (Frederickson etal., 1987). In this study, we have used the Zn 2+ ¯uorescent dye TSQ, which interacts mainly with membrane-bound Zn 2+ , to investigate the routes of Zn 2+ entry in living cortical neurons. Additional experiments were performed to investigate the role of these routes of entry and the contribution of glutamate in Zn 2+ -induced neurotoxicity. Correspondence: Dr J. Pre Âmont, as above. E-mail: premont@infobiogen.fr Received 23 April 1999, revised 12 August 1999, accepted 2 September 1999 European Journal of Neuroscience, Vol. 12, pp. 8±18, 2000 Ó European Neuroscience Association