Fluorescence imaging study of extracellular zinc at the hippocampal mossy fiber synapse

Although synaptically released, vesicular Zn2+ has been proposed to play a neuromodulatory or neuronal signaling role at the mossy fiber-CA3 synapse, Zn2+ release remains controversial, especially when detected using fluorescent imaging. In the present study, we investigated synaptically released Zn2+ at the mossy fiber (MF) synapse in rat hippocampal slices using three chemically distinct, fluorescent Zn2+, indicators. The indicators employed for this study were cell membrane impermeable (or extracellular) Newport Green KDZn2+ similar to 1 mu M, Zinpyr-4 KDZn2+ similar to 1 nM and FluoZin-3 KDZ2+ similar to 15 nM, chosen, in part, for their distinct dissociation constants. Among the three indicators, FluoZin-3 was also sensitive to Ca2+ KDC2+ similar to 200-300 mu M which was present in the extracellular medium ([Ca2+](0) > 2 mM). Hippocampal slices loaded with either Newport Green or FluoZin-3 showed increases in fluorescence after electrical stimulation of the mossy fiber pathway. These results are consistent with previous studies suggesting the presence of synaptically released Zn2+ in the extracellular space during neuronal activities; however, the rise in FluoZin3 fluorescence observed was complicated by the data that the addition of exogenous Zn2+ onto FluoZin-3 loaded slices gave little change in fluorescence. In the slices loaded with the high-affinity indicator Zinpyr-4, there was little change in fluorescence after mossy fiber activation by electrical stimulation. Further study revealed that the sensitivity of Zinpyr-4 was mitigated by saturation with Zn2+ contamination from the slice. These data suggest that the sensitivity and selectivity of a probe may affect individual outcomes in a given experimental system. (c) 2007 Elsevier Ireland Ltd. All rights reserved.