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

Abstract

Mossy fiber termini in the hippocampus accumulate Zn 2+ , which is released with glutamate from synaptic vesicles upon neural excitation. Understanding the spatiotemporal regulation of mobile Zn 2+ at the synaptic level is challenging owing to the difficulty of visualizing Zn 2+ at individual synapses. Here we describe the use of zinc-responsive fluorescent probes together with two-photon microscopy to image Zn 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 2+ probe, 6-CO 2 H-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 2+ signal observed at individual presynaptic sites, indicating release of the Zn 2+ from vesicles in synaptic potentiation. This synapse-level two-photon Zn 2+ imaging method enables monitoring of presynaptic Zn 2+ dynamics for improving the understanding of physiological roles of mobile Zn 2+ in regular and aberrant neurologic function.zinc ion | metalloneurochemistry A lthough most cellular Zn 2+ is sequestered within proteins, stores of loosely bound Zn 2+ are present in many kinds of cells. This mobile Zn 2+ 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 Zn 2+ in glutamatergic synaptic vesicles. The precise concentration of Zn 2+ within the neuronal vesicles is unknown, with upper estimates ranging in the low millimolar range (2, 3). Upon stimulation, Zn 2+ 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 Zn 2+ .Numerous fluorescent Zn 2+ 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)(12)(13)(14). Despite the abundance of fluorescent Zn 2+ probes, analysis of Zn 2+ in vivo remains problematic. Many probes bind Zn 2+ to form complexes with dissociation constants in the nanomolar range; these tight binding affinities lead to rates of Zn 2+ release that are too slow for time-resolvable measurements. These probes also may act as Zn 2+ traps, sequestering Zn 2+ in one region of the cell, and then collecting elsewhere to yield a faulty image of native Zn 2+ 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 Zn 2+ 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...

A far-red emitting probe for unambiguous detection of mobile zinc in acidic vesicles and deep tissue

“…This staining is similar to that obtained recently using probe 6-CO 2 H–ZAP4. 21 Vesicular zinc in the brain is loaded by the ZnT3 (Slc30A3) transporter and this gene has been successfully knocked out in transgenic mice. 22 …”

“…Additionally, we co-stained hippocampal slices with SpiroZin2 and the membrane-permeable green fluorescent Zn 2+ sensor ZP1, which was used previously to image mobile zinc in mossy fiber boutons. 21 …”

Activation of group II metabotropic glutamate receptors blocks zinc release from hippocampal mossy fibers

“…Another possible contribution for the observed inhibition is the reduction of action potential-induced calcium influx into presynaptic terminals by DCG-IV, followed by the suppression of transmitter release. Previous observations indicate that mGlu receptor 2 activation can inhibit zinc relase [ 30 ] and N-type voltage-gated calcium channels [ 31 , 32 ], which mediate glutamate release. Down-regulation of these channels, which are involved in synaptic transmission at the mossy fiber-CA3 synapses in the hippocampus [ 33 ], may result in the suppression of calcium-dependent glutamate release.…”

Postsynaptic zinc potentiation elicited by KCl depolarization at hippocampal mossy fiber synapses

“…fibers. It has been shown that zinc is released from these fibers into the extracellular medium when electrical stimuli are delivered (Li et al 2001a;Quinta-Ferreira et al 2004;Khan et al 2014;Vergnano et al 2014) and that it enters to postsynaptic neurons following intense electrical or chemical stimulation (Vogt et al 2000;Li et al 2001aLi et al , 2001bUeno et al 2002;Ketterman and Li 2008). The exposition of the slices to a high concentration of exogenous potassium causes an enhancement of the measured fluorescence signals, considered to be associated with postsynaptic zinc changes (Li et al 2001b;Ketterman and Li 2008).…”