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

摘要

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.