Two-photon imaging of Zn2+ dynamics in mossy fiber boutons of adult hippocampal slices

Mossy fiber termini in the hippocampus accumulate Zn2+, which is released with glutamate from synaptic vesicles upon neural excitation. Understanding the spatiotemporal regulation of mobile Zn2+ at the synaptic level is challenging owing to the difficulty of visualizing Zn2+ at individual synapses. Here we describe the use of zinc-responsive fluorescent probes together with two-photon microscopy to image Zn2+ dynamics mediated by NMDA receptor-dependent long-term potentiation induction at single mossy fiber termini of dentate gyrus neurons in adult mouse hippocampal slices. The membrane-impermeant fluorescent Zn2+ probe, 6-CO2H-ZAP4, was loaded into presynaptic vesicles in hippocampal mossy fiber termini upon KCl-induced depolarization, which triggers subsequent endocytosis and vesicular restoration. Local tetanic stimulation decreased the Zn2+ signal observed at individual presynaptic sites, indicating release of the Zn2+ from vesicles in synaptic potentiation. This synapse-level two-photon Zn2+ imaging method enables monitoring of presynaptic Zn2+ dynamics for improving the understanding of physiological roles of mobile Zn2+ in regular and aberrant neurologic function.