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.Although most cellular Zn²⁺ is sequestered within proteins, stores of loosely bound Zn²⁺ are present in many kinds of cells. This mobile Zn²⁺ pool is believed to mediate cellular processes, including neurotransmission (1). Within the hippocampus, mossy fibers connecting the dentate gyrus (DG) and the CA3 regions contain Zn²⁺ in glutamatergic synaptic vesicles. The precise concentration of Zn²⁺ within the neuronal vesicles is unknown, with upper estimates ranging in the low millimolar range (2, 3). Upon stimulation, Zn²⁺ is believed to be coreleased with glutamate and to modulate glutamatergic synaptic transmission (4, 5). Despite this recent finding, however, much remains to be understood about the dynamics and functional roles of synaptic Zn²⁺.Numerous fluorescent Zn²⁺ probes have been developed for use in biological systems (6, 7), including 6-methoxy-(8-p-toluenesulfonamido)quinoline (8), Zinquin (9), Zinbo-5 (10), and the Zinpyr (ZP) and ZnAF families of probes (11–14). Despite the abundance of fluorescent Zn²⁺ probes, analysis of Zn²⁺ in vivo remains problematic. Many probes bind Zn²⁺ to form complexes with dissociation constants in the nanomolar range; these tight binding affinities lead to rates of Zn²⁺ release that are too slow for time-resolvable measurements. These probes also may act as Zn²⁺ traps, sequestering Zn²⁺ in one region of the cell, and then collecting elsewhere to yield a faulty image of native Zn²⁺ distributions within cells. Reductions in binding affinity can be accomplished via two strategies. First, steric bulk can be installed near the chelating atoms, as was done with two series of methylated Zn²⁺ probes (15, 16). Second, chelating atoms can be removed from the ligand systematically, as was done for probes in the ZnAF series (16), as well as those in the QZ, Zinspy, and ZinAlkylPyr (ZAP) families (17–19).Here we present the synthesis of the probe ZinAlkylPyr-4 (ZAP4) and its 6-carboxylic acid derivative (6-CO₂H-ZAP4). The molecular structure of ZAP4 was modeled after earlier ZAP probes (17), with pentafluorobenzyl groups chosen as the alkyl groups to reduce the basicity of the probe and minimize proton-induced enhancement of the emission. The 6-CO₂H-ZAP4 probe was constructed as a membrane-impermeant Zn²⁺ probe, analogous to the previously reported 6-CO₂H-ZP1 (20).Previous attempts to monitor presynaptic Zn²⁺ dynamics in living brain tissues used membrane-permeant probes to detect intracellular Zn²⁺ from populations of presynaptic terminals at relatively low spatial resolution at the tissue level (21, 22). In addition, activity-dependent extracellular Zn²⁺ release has been monitored with membrane-impermeant probes (16, 23–30). These tissue level-averaged Zn²⁺ imaging methods have limited spatial and temporal resolution and fail to specify the precise location of the relevant synapses.We describe intracellular Zn²⁺ imaging at the single-synapse level in mossy fiber termini of neurons in acute hippocampal slices from adult mice. The technique relies on the membrane-impermeant fluorescent Zn²⁺ probe 6-CO₂H-ZAP4 and two-photon fluorescence microscopy.