Two-photon imaging of Zn[superscript 2+] dynamics in mossy fiber boutons of adult hippocampal slices

Abstract

Mossy fiber termini in the hippocampus accumulate Zn[superscript 2+], which is released with glutamate from synaptic vesicles upon neural excitation. Understanding the spatiotemporal regulation of mobile Zn[superscript 2+] at the synaptic level is challenging owing to the difficulty of visualizing Zn[superscript 2+] at individual synapses. Here we describe the use of zinc-responsive fluorescent probes together with two-photon microscopy to image Zn[superscript 2+] 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 Zn[superscript 2+] 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 Zn[superscript 2+] signal observed at individual presynaptic sites, indicating release of the Zn[superscript 2+] from vesicles in synaptic potentiation. This synapse-level two-photon Zn[superscript 2+] imaging method enables monitoring of presynaptic Zn[superscript 2+] dynamics for improving the understanding of physiological roles of mobile Zn[superscript 2+] in regular and aberrant neurologic function.