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+∼1 μM, Zinpyr-4 KDZn2+∼1 nMKDZn2+∼1 nM and FluoZin-3 KDZn2+∼15 nMKDZn2+∼15 nM, chosen, in part, for their distinct dissociation constants. Among the three indicators, FluoZin-3 was also sensitive to Ca2+KDCa2+∼200–300 μM which was present in the extracellular medium ([Ca2+]o > 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 FluoZin-3 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.