Calcium-sensitive fluorescent dyes can report increases in intracellular free zinc concentration in cultured forebrain neurons

Abstract

High concentrations of Zn2+ are found in presynaptic terminals of excitatory neurons in the CNS. Zn2+ can be released during synaptic activity and modulate postsynaptic receptors, but little is known about the possibility that Zn2+ may enter postsynaptic cells and produce dynamic changes in the intracellular Zn2+ concentration ([Zn2+](i)). We used fura-2 and magfura-2 to detect the consequences of Zn2+ influx in cultured neurons under conditions that restrict changes in intracellular Ca2+ and Mg2+ concentrations. The resulting ratio changes for both dyes were reversed completely by the Zn2+ chelator, N,N,N',N'-tetrakis(2- pyridylmethyl)ethylenediamine, indicating that these dyes are measuring changes in [Zn2+](i). We found that fura-2 was useful in measuring small increases in [Zn2+](i) associated with exposure to Zn2+ alone that may be mediated by a Na+/Ca2+ exchanger. Magfura-2, which has a lower affinity for Zn2+, was more useful in measuring larger agonist-stimulated increases in [Zn2+](i). The coapplication of 300 μM Zn2+ and 100 μM glutamate/10 μM glycine resulted in a [Zn2+](i) increase that was ~40-100 nM in magnitude and could be inhibited by the NMDA receptor antagonist, MK-801 (30 μM), or extracellular Na+. This suggests that Zn2+ influx can occur through at least two different pathways, leading to varying increases in [Zn2+](i). These findings demonstrate the feasibility of measuring changes in [Zn2+](i) in neurons.