Detection of zinc translocation into apical dendrite of CA1 pyramidal neuron after electrical stimulation

https://doi.org/10.1016/j.jneumeth.2008.09.016Get rights and content

Abstract

Translocation of the endogenous cation zinc from presynaptic terminals to postsynaptic neurons after brain insult has been implicated as a potential neurotoxic event. Several studies have previously demonstrated that a brief electrical stimulation is sufficient to induce the translocation of zinc from presynaptic vesicles into the cytoplasm (soma) of postsynaptic neurons. In the present work I have extended those findings in three ways: (i) providing evidence that zinc translocation occurs into apical dendrites, (ii) presenting data that there is an apparent translocation into apical dendrites when only a zinc-containing synaptic input is stimulated, and (iii) presenting data that there is no zinc translocation into apical dendrite of ZnT3 KO mice following electrical stimulation.

Hippocampal slices were preloaded with the “trappable” zinc fluorescent probe, Newport Green. After washout, a single apical dendrite in the stratum radiatum of hippocampal CA1 area was selected and focused on. Burst stimulation (100 Hz, 500 μA, 0.2 ms, monopolar) was delivered to either the adjacent Schaffer-collateral inputs (zinc-containing) or to the adjacent temporo-ammonic inputs (zinc-free) to the CA1 dendrites.

Stimulation of the Schaffer collaterals increased the dendritic fluorescence, which was blocked by TTX, low-Ca medium, or the extracellular zinc chelator, CaEDTA. Stimulation of the temporo-ammonic pathway caused no significant rise in the fluorescence. Genetic depletion of vesicular zinc by ZnT3 KO showed no stimulation-induced apical dendrite zinc rise.

The present study provides evidence that synaptically released zinc translocates into postsynaptic neurons through the apical dendrites of CA1 pyramidal neurons during physiological synaptic activity.

Introduction

Excessive levels of synaptically released zinc are directly implicated in the phenomenon of excitotoxicity. When brain tissue is reperfused with oxygen and glucose after being deprived of these metabolic substrates for short periods of time, considerable levels of excitatory amino acid transmitters (e.g., glutamate) and Zn2+ are released into the synaptic cleft. Thereafter, Zn2+ appears in the neuron somata within an hour (Frederickson et al., 2006). The first evidence of zinc’s role in excitotoxic neuron death was obtained in rats suffering from kainic acid-induced seizures (Frederickson et al., 1988), a finding that capitalized on earlier evidence of zinc’s toxicity to cultured neurons (Yokoyama et al., 1986). That result was soon replicated (Frederickson et al., 1989), and extended to the models of ischemia (Tonder et al., 1990, Koh et al., 1996, Calderone et al., 2004), seizure (Suh et al., 2001), head trauma (Suh et al., 2000b, Suh et al., 2006) and hypoglycemia (Suh et al., 2004, Suh et al., 2007). In all cases the basic finding was that zinc disappeared from presynaptic boutons and subsequently appeared selectively in dying postsynaptic neurons during excitotoxic events (Frederickson, 1989, Frederickson et al., 2000, Frederickson et al., 2005). Weiss and co-workers have shown that zinc ingress into neurons during excitotoxic conditions is via three channels: an NMDA-gated channel, a voltage-dependent channel, and an AMPA/KA-gated channel (Yin and Weiss, 1995, Sensi et al., 1999, Yin et al., 1999, Weiss and Sensi, 2000).

Previous zinc “translocation” studies both after in vivo excitotoxic event (Frederickson et al., 1989, Tonder et al., 1990, Koh et al., 1996, Suh et al., 2000b, Suh et al., 2007) and after in vitro exogenous zinc application (Yokoyama et al., 1986) suggests the possibility that synaptically released zinc can enter postsynaptic neurons even under physiological conditions. To identify Zn2+ as a trans-synaptic messenger, it is important to demonstrate that the influx of extracellular free Zn2+ into intracellular compartments is attained under near-physiological conditions of neuronal activation, i.e. after electrical stimulation at physiological rates. Although the Zn2+-selective fluorescent dye N-(6-methoxy-8-quinolyl)-p-toluene sulfonamide (TSQ) has been used to locate pools of chelatable Zn2+ (Frederickson et al., 1987), its lipophilic nature and toxicity make it unsuitable for quantitative measurements of Zn2+ in living cells. Two sulfonamide derivatives of quinoline, N-(6-methoxy-8-quinolyl)-p-carboxy benzoyl-sulfonamide (TFL) and Zinquin, have allowed quantitative measurement of Zn2+ in hippocampal slices (Budde et al., 1997), but these load poorly into neurons (Sensi et al., 1997). Previously, mag-fura-5 and mag-fura-2 have been used to measure changes in Zn2+ after K+-induced depolarization or glutamate receptor activation in cultured central neurons (Sensi et al., 1997, Cheng and Reynolds, 1998). However, the known sensitivity of mag-fura-5 (Kd ∼20 μM for Ca2+, 2.6 mM for Mg2+) or mag-fura-2 (Kd ∼225 nM for Ca2+) was a major limitation in these studies (Grynkiewicz et al., 1985, Simons, 1993, Petrozzino et al., 1995, Stork and Li, 2006).

All previous theories were based on the hypothesis that Zn2+ is released from Zn2+-containing boutons, travels across the synaptic cleft, and then modulates postsynaptic neuronal receptor function or even enters postsynaptic neurons. Previously the Li group demonstrated that electrical stimulation induced Zn2+ release from the hippocampal hilus and this released Zn2+ can be translocated into soma of postsynaptic neurons (Li et al., 2001, Bastian and Li, 2007, Ketterman and Li, 2008). However, translocation of released Zn2+ into apical dendrite of postsynaptic neuron has not been previously observed and characterized. Therefore this study aims to determine whether presynaptically released zinc ions enter into the apical dendrite of CA1 pyramidal neuron during normal, physiological synaptic activity.

Section snippets

Materials

Newport Green, tetrakis-(2-pyridylmethyl) ethylenediamine (TPEN) and Pluronic F-127 were obtained from Molecular Probes (Eugene, OR). Ethylenediaminetetraacetic acid disodium-calcium salt (CaEDTA) and tetrodotoxin (TTX) were obtained from Sigma (St. Louis, MO).

Acute hippocampal slice

Conventional acutely prepared brain slices were used. For these, male Sprague–Dawley rats (100–150 g) or C57/Bl6 wild type mice or ZnT3 KO mice of the same strain (30–45 g) were anesthetized by halothane inhalation (5%), decapitated, and

Detection of vesicular zinc release by cell-impermeable Newport Green

To verify that our electrical stimulation method and zinc sensing probes are properly working in this study, electrical stimulation-induced vesicular zinc release was tested by cell-impermeable Newport Green in the hippocampal hilus using acute hippocampal slice. Electrical stimulation and imaging was performed during perfusion flow stop. Images were captured every minute for 5 min. In this experiment, a bipolar electrode was used. Three minutes after electrical stimulation, fluorescence signals

Discussion

The pivotal question of the present study is whether one could monitor the zinc translocation into apical dendrites of postsynaptic neurons following physiological stimulation. The present study firstly demonstrates the apparent translocation of zinc into apical dendrites of CA1 pyramidal neurons after electrical stimulation.

To address above question, either cell-permeable or cell-impermeable Newport Green was employed. Non-ratiometric, Newport Green (NG) has several advantages for monitoring

Acknowledgments

This author gratefully acknowledges the skillful technical assistance of Yaping Zeng and Kathy Frederickson, and critical comments of Dr. Christopher Frederickson. This author also acknowledges Aaron M. Hamby’s help in editing this manuscript.

References (47)

  • C.J. Frederickson et al.

    Concentrations of extracellular free zinc (pZn)e in the central nervous system during simple anesthetization, ischemia and reperfusion

    Exp Neurol

    (2006)
  • G. Grynkiewicz et al.

    A new generation of Ca2+ indicators with greatly improved fluorescence properties

    J Biol Chem

    (1985)
  • J.Y. Lee et al.

    Zinc released from metallothionein-iii may contribute to hippocampal CA1 and thalamic neuronal death following acute brain injury

    Exp Neurol

    (2003)
  • V. Nadler et al.

    Distinctive structural requirement for the binding of uncompetitive blockers (phencyclidine-like drugs) to the NMDA receptor

    Eur J Pharmacol

    (1990)
  • J.J. Petrozzino et al.

    Micromolar Ca2+ transients in dendritic spines of hippocampal pyramidal neurons in brain slice

    Neuron

    (1995)
  • T.J. Simons

    Measurement of free Zn2+ ion concentration with the fluorescent probe mag-fura-2 (furaptra)

    J Biochem Biophys Methods

    (1993)
  • S.W. Suh et al.

    Release of synaptic zinc is substantially depressed by conventional brain slice preparations

    Brain Res

    (2000)
  • S.W. Suh et al.

    Evidence that synaptically-released zinc contributes to neuronal injury after traumatic brain injury

    Brain Res

    (2000)
  • N. Tonder et al.

    Possible role of zinc in the selective degeneration of dentate hilar neurons after cerebral ischemia in the adult rat

    Neurosci Lett

    (1990)
  • K. Vogt et al.

    The actions of synaptically released zinc at hippocampal mossy fiber synapses

    Neuron

    (2000)
  • J.H. Weiss et al.

    Ca2+–Zn2+ permeable AMPA or kainate receptors: possible key factors in selective neurodegeneration

    Trends Neurosci

    (2000)
  • M. Yokoyama et al.

    Brief exposure to zinc is toxic to cortical neurons

    Neurosci Lett

    (1986)
  • E. Aizenman et al.

    Induction of neuronal apoptosis by thiol oxidation: putative role of intracellular zinc release

    J Neurochem

    (2000)
  • Cited by (0)

    View full text