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