Explorations of hippocampal zinc using zinc-selective fluorescent dyes

Zn is a transition metal that fulfills many roles in mammalian cells, from structural support for many proteins, to second messenger and enzymatic cofactors. Specific neuronal terminals in the hippocampus contain higher Zn concentrations than other brain cells, but it is still unclear as to why Zn accumulates there. Since Zn is co-packaged with the neurotransmitter glutamate in synaptic vesicles, one possibility is that it gets released during neurotransmission. To study zinc uptake in the cytoplasm and the possibility of Zn release, we employed different fluorescent Zn indicators. These dyes passively cross the cell membrane and become fluorescent upon zinc binding. We found that the extracellular concentration of zinc and therefore zinc influx into the cell is limited by the presence of phosphate, which induces zinc precipitation by forming insoluble zinc-phosphate salts. Zinc solubility and influx is increased by the application of histidine to the extracellular medium. We also found that exogenously applied zinc in the presence of a zinc ionophore seems to translocate in vesicles and cytoplasmic compartments. Zinc seems to be very tightly buffered as it enters the cytoplasm, since transient increases in fluorescence (as observed during Ca2+ influx into the cytoplasm) are not observed. Our data also seems to indicate that zinc is not being freely released in the extracellular space, but is being externalized instead.