MEASUREMENT OF INTRACELLULAR FREE ZINC IN LIVING CORTICAL-NEURONS - ROUTES OF ENTRY

We used the ratioable fluorescent dye mag-fura-5 to measure intracellu lar free Zn2+ ([Zn2+](i)) in cultured neocortical neurons exposed to n eurotoxic concentrations of Zn2+ in concert with depolarization or glu tamate receptor activation and identified four routes of Zn2+ entry. N eurons exposed to extracellular Zn2+ plus high K+ responded with a pea k cell body signal corresponding to a [Zn2+](i) of 35-45 nM. This incr ease in [Zn2+](i) was attenuated by concurrent addition of Gd3+, verap amil, omega-conotoxin GVIA, or nimodipine, consistent with Zn2+ entry through voltage-gated Ca2+ channels. Furthermore, under conditions fav oring reverse operation of the Na+-Ca2+ exchanger, Zn2+ application in duced a slow increase in [Zn2+](i) and outward whole-cell current sens itive to benzamil-amiloride. Thus, a second route of Zn2+ entry into n eurons may be via transporter-mediated exchange with intracellular Na. Both NMDA and kainate also induced rapid increases in neuronal [Zn2](i). The NMDA-induced increase was only partly sensitive to Gd3+ or t o removal of extracellular Na+, consistent with a third route of entry directly through NMDA receptor-gated channels. The kainate-induced in crease was highly sensitive to Gd3+ or Na+ removal in most neurons but insensitive in a minority subpopulation (''cobalt-positive cells''), suggesting that a fourth route of neuronal Zn2+ entry is through the C a2+-permeable channels gated by certain subtypes of AMPA or kainate re ceptors.