Tonic zinc inhibits spontaneous firing in dorsal cochlear nucleus principal neurons by enhancing glycinergic neurotransmission
Introduction
Since the discovery that zinc is loaded into glutamatergic vesicles and is exocytosed with glutamate during synaptic transmission, numerous studies have investigated the role of mobilized zinc in synapses. These studies are consistent with a model whereby zinc serves as an inhibitory neuromodulatory neurotransmitter, inhibiting NMDA receptors (NMDARs), reducing release probability in excitatory synapses, and potentiating glycinergic and GABAergic inhibitory neurotransmission (Xie and Smart, 1991, Hirzel et al., 2006, Nozaki et al., 2011, Pan et al., 2011, Perez-Rosello et al., 2013, Vergnano et al., 2014). Unlike many other neurotransmitter systems, where the actions of tonic levels of neurotransmitters are well studied, the role of tonic zinc remains less understood. Moreover, prior electrophysiological studies are limited and present conflicting results. For example, one study reports that, in mossy fiber synapses, ZnT3-dependent tonic zinc levels modulate NMDARs (Vogt et al., 2000), but more recent work at the same synapses, finds that ambient zinc levels are too low for NMDAR modulation (Vergnano et al., 2014). The role of ambient zinc therefore remains enigmatic.
A major obstacle in determining the role of zinc (synaptic and tonic) has been the lack of a chelator with appropriate kinetic and thermodynamic properties for probing the temporal and spatial changes of mobile zinc at synapses (Radford and Lippard, 2013), as illustrated by the contrasting findings about the role of tonic zinc in previous work. Quite different zinc chelators were applied in these studies: a kinetically slow one (Vogt et al., 2000) and a faster, but low-affinity chelator (Vergnano et al., 2014). To assess the role and origin of tonic zinc we utilized the fast, high-affinity zinc chelator ZX1 (Pan et al., 2011, Radford and Lippard, 2013) as well as transgenic mice lacking ZnT3. We studied the role of tonic zinc on DCN fusiform cells. Fusiform cells generate spontaneous action potentials (Rhode et al., 1983, Hancock and Voigt, 2002, Leao et al., 2012) and, because they are embedded in a zinc-rich nucleus (Frederickson et al., 1988, Rubio and Juiz, 1998, Oertel and Young, 2004), they provide an ideal assay for testing the effects of tonic zinc on neuronal excitability.
Section snippets
Slice preparation
Experiments were conducted according to the methods approved by the Institutional Animal Care and Use Committee of the University of Pittsburgh. Coronal brainstem slices were prepared from ICR mice and ZnT3 knockout (ZnT3 KO) mice (P17–P25). ICR mice were purchased from Harlan and ZnT3 KO mice were purchased from The Jackson Laboratory. The preparation of coronal slices containing DCN has been described in detail previously (Tzounopoulos et al., 2004).
Electrophysiological recordings and analysis
Loose cell-attached voltage-clamp and whole
Tonic zinc decreases spontaneous firing in fusiform cells by enhancing glycinergic neurotransmission
To test whether tonic zinc modulates spontaneous firing in fusiform cells, we examined the effect of ZX1, a high-affinity extracellular zinc chelator (Pan et al., 2011, Radford and Lippard, 2013), on the rate of spontaneous action potentials. In cell-attached recordings, 100 μM ZX1 increased the spontaneous firing rate in fusiform cells; this effect was reversed upon removal of ZX1 from the bath (Figs. 1A, B; ZX1: 148 ± 14% of control, n = 7, p = 0.01 when compared to control; wash out: 95 ± 10%, p = 0.5
Discussion
Our results identify tonic zinc as an endogenous modulator of spontaneous firing in principal DCN neurons. Previous studies established that ~ 50% of fusiform cells are spontaneously active, firing action potentials in the absence of evoked synaptic stimulation. Spontaneous firing is mediated by intrinsic properties (Leao et al., 2012). In particular, fusiform cells with resting membrane potential above or approximately at − 65 mV, which is set by the variable expression of inwardly rectifying
Conclusions
Application of ZX1, an extracellular zinc chelator, reveals that tonic zinc inhibits spontaneous firing in fusiform cells. A GlyR antagonist occluded this inhibition, suggesting that it is mediated by GlyRs. Consistent with this conclusion, tonic zinc enhances glycinergic neurotransmission via pre- and postsynaptic mechanisms. The source of tonic zinc that enhances fusiform cell spontaneous firing rates is not ZnT3-dependent, thus raising the possibility that zinc may influence neuronal
Acknowledgments
This work was supported by funding from the NIH: TT: RO1-DC007905; TPR: F32-DC011664; CTA: F32-DC013734-01A1; and SJL: RO1-GM065519.
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