Elsevier

Neurobiology of Disease

Volume 81, September 2015, Pages 14-19
Neurobiology of Disease

Tonic zinc inhibits spontaneous firing in dorsal cochlear nucleus principal neurons by enhancing glycinergic neurotransmission

https://doi.org/10.1016/j.nbd.2015.03.012Get rights and content

Abstract

In many synapses of the CNS, mobile zinc is packaged into glutamatergic vesicles and co-released with glutamate during neurotransmission. Following synaptic release, the mobilized zinc modulates ligand- and voltage-gated channels and receptors, functioning as an inhibitory neuromodulator. However, the origin and role of tonic, as opposed to phasically released, zinc are less well understood. We investigated tonic zinc in the dorsal cochlear nucleus (DCN), a zinc-rich, auditory brainstem nucleus. Our results show that application of a high-affinity, extracellular zinc chelator (ZX1) enhances spontaneous firing in DCN principal neurons (fusiform cells), consistent with inhibition of this neuronal property by tonic zinc. The enhancing effect was prevented by prior application of strychnine, a glycine receptor antagonist, suggesting that ZX1 interferes with zinc-mediated modulation of spontaneous glycinergic inhibition. In particular, ZX1 decreased the amplitude and the frequency of glycinergic miniature inhibitory postsynaptic currents in fusiform cells, from which we conclude that tonic zinc enhances glycinergic inhibitory neurotransmission. The observed zinc-mediated inhibition in spontaneous firing is present in mice lacking the vesicular zinc transporter (ZnT3), indicating that non-vesicular zinc inhibits spontaneous firing. Noise-induced increase in the spontaneous firing of fusiform cells is crucial for the induction of tinnitus. In this context, tonic zinc provides a powerful break of spontaneous firing that may protect against pathological run-up of spontaneous activity in the DCN.

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.

References (49)

  • A.S. Nakashima et al.

    Dynamic, experience-dependent modulation of synaptic zinc within the excitatory synapses of the mouse barrel cortex

    Neuroscience

    (2010)
  • D. Oertel et al.

    What's a cerebellar circuit doing in the auditory system?

    Trends Neurosci.

    (2004)
  • E. Pan et al.

    Vesicular zinc promotes presynaptic and inhibits postsynaptic long-term potentiation of mossy fiber-CA3 synapse

    Neuron

    (2011)
  • R.J. Radford et al.

    Chelators for investigating zinc metalloneurochemistry

    Curr. Opin. Chem. Biol.

    (2013)
  • D.M. Ramirez et al.

    Differential regulation of spontaneous and evoked neurotransmitter release at central synapses

    Curr. Opin. Neurobiol.

    (2011)
  • J.P. Rauschecker et al.

    Tuning out the noise: limbic–auditory interactions in tinnitus

    Neuron

    (2010)
  • C.K. Su et al.

    Three-dimensional printed sample load/inject valves enabling online monitoring of extracellular calcium and zinc ions in living rat brains

    Anal. Chim. Acta

    (2014)
  • A.M. Vergnano et al.

    Zinc dynamics and action at excitatory synapses

    Neuron

    (2014)
  • K. Vogt et al.

    The actions of synaptically released zinc at hippocampal mossy fiber synapses

    Neuron

    (2000)
  • H. Wang et al.

    Plasticity at glycinergic synapses in dorsal cochlear nucleus of rats with behavioral evidence of tinnitus

    Neuroscience

    (2009)
  • P.F. Apostolides et al.

    Regulation of interneuron excitability by gap junction coupling with principal cells

    Nat. Neurosci.

    (2013)
  • P.F. Apostolides et al.

    Superficial stellate cells of the dorsal cochlear nucleus

    Front. Neural Circ.

    (2014)
  • P.F. Apostolides et al.

    Chemical synaptic transmission onto superficial stellate cells of the mouse dorsal cochlear nucleus

    J. Neurophysiol.

    (2014)
  • A.B. Bloomenthal et al.

    Biphasic modulation of the strychnine-sensitive glycine receptor by Zn2 +

    Mol. Pharmacol.

    (1994)
  • Cited by (24)

    • The Function and Regulation of Zinc in the Brain

      2021, Neuroscience
      Citation Excerpt :

      Finally, zinc chelation prevents endogenous potentiation of GlyRs, resulting in increases spontaneous firing in DCN principal neurons. Interestingly, this effect is independent of ZnT3 (Perez-Rosello et al., 2015), suggesting a separate tonic source of zinc can also mediate extracellular zinc signaling. This tonic zinc pool may be generated and regulated by other zinc transporters, such as ZnT1, which, as mentioned earlier is located on the plasma membrane and can influence NMDA receptor function (Krall et al., 2020).

    • The ion channels and synapses responsible for the physiological diversity of mammalian lower brainstem auditory neurons

      2019, Hearing Research
      Citation Excerpt :

      Their firing is initiated by a persistent sodium current (INaP) which depolarizes the membrane reaching the activation threshold of the transient sodium current and the T-type calcium current, and controlled by BK and SK calcium-dependent potassium currents (Kim and Trussell, 2007). Most cartwheel neurons fire action potential spontaneously (Kim and Trussell, 2007; Zugaib et al., 2016) which produces a strong tonic glycinergic inhibition on the fusiform neurons, detected as a high frequency of spontaneous inhibitory post-synaptic potentials (Roberts and Trussell, 2010; Lu and Trussell, 2016; Perez-Rosello et al., 2015; Zugaib et al., 2016). Because of their firing pattern, morphology, connectivity and molecular markers, cartwheel neurons are considered to be correlated to the cerebellar Purkinje neurons (Wouterlood and Mugnaini, 1984; Berrebi and Mugnaini, 1988; Zhang and Oertel, 1993a).

    • Zinc transporter 3 (ZnT3) and vesicular zinc in central nervous system function

      2017, Neuroscience and Biobehavioral Reviews
      Citation Excerpt :

      Experiments on slices containing hypoglossal motoneurons show that tonic zinc levels are sufficient to potentiate spontaneous inhibitory postsynaptic currents (IPSCs) (Hirzel et al., 2006). More recently, it has been observed that tonic extracellular zinc has an inhibitory effect in the DCN (Perez-Rosello et al., 2015). Here, chelating zinc with ZX1 disinhibited the principal fusiform neurons, increasing their spontaneous firing rate.

    • Metals and neurodegeneration

      2015, Neurobiology of Disease
    View all citing articles on Scopus
    View full text