Abstract

We used the ratioable fluorescent dye mag-fura-5 to measure intracellular free Zn²⁺([Zn²⁺]_i) in cultured neocortical neurons exposed to neurotoxic concentrations of Zn²⁺ in concert with depolarization or glutamate receptor activation and identified four routes of Zn²⁺ entry. Neurons exposed to extracellular Zn²⁺ plus high K⁺ responded with a peak cell body signal corresponding to a [Zn²⁺]_i of 35–45 nm. This increase in [Zn²⁺]_i was attenuated by concurrent addition of Gd³⁺, verapamil, ω-conotoxin GVIA, or nimodipine, consistent with Zn²⁺ entry through voltage-gated Ca²⁺channels. Furthermore, under conditions favoring reverse operation of the Na⁺–Ca²⁺ exchanger, Zn²⁺ application induced a slow increase in [Zn²⁺]_i and outward whole-cell current sensitive to benzamil–amiloride. Thus, a second route of Zn²⁺ entry into neurons 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 to removal of extracellular Na⁺, consistent with a third route of entry directly through NMDA receptor-gated channels. The kainate-induced increase 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 Ca²⁺-permeable channels gated by certain subtypes of AMPA or kainate receptors.