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Neuroscience 158 (2009) 126–136

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EVIEW

INC AT GLUTAMATERGIC SYNAPSES

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. PAOLETTI,* A. M. VERGNANO, B. BARBOUR AND. CASADO

aboratoire de Neurobiologie, CNRS UMR 8544, Ecole Normaleupérieure, 46 rue d’Ulm, 75005 Paris, France

bstract—It has long been known that the mammalian fore-rain contains a subset of glutamatergic neurons that se-uester zinc in their synaptic vesicles. This zinc may beeleased into the synaptic cleft upon neuronal activity. Extra-ellular zinc has the potential to interact with and modulateany different synaptic targets, including glutamate recep-

ors and transporters. Among these targets, NMDA receptorsppear particularly interesting because certain NMDA recep-or subtypes (those containing the NR2A subunit) containllosteric sites exquisitely sensitive to extracellular zinc. Thexistence of these high-affinity zinc binding sites raises theossibility that zinc may act both in a phasic and tonic mode.hanges in zinc concentration and subcellular zinc distribu-

ion have also been described in several pathological condi-ions linked to glutamatergic transmission dysfunctions.owever, despite intense investigation, the functional signif-

cance of vesicular zinc remains largely a mystery. In thiseview, we present the anatomy and the physiology of thelutamatergic zinc-containing synapse. Particular emphasis

s put on the molecular and cellular mechanisms underlyinghe putative roles of zinc as a messenger involved in excita-ory synaptic transmission and plasticity. We also highlighthe many controversial issues and unanswered questions.inally, we present and compare two widely used zinc chela-ors, CaEDTA and tricine, and show why tricine should bereferred to CaEDTA when studying fast transient zinc ele-ations as may occur during synaptic activity. © 2009 IBRO.ublished by Elsevier Ltd. All rights reserved.

ey words: glutamate, zinc, synapse, chelator, excitotoxicity,pilepsy.

Contentsocalization of brain “chelatable zinc” 126s zinc released during neuronal activity? 127inc targets at excitatory synapses 128inc in excitatory synaptic transmission and plasticity 130inc and epilepsy 131inc and excitotoxicity 131anipulating extracellular zinc concentrations using chelators

132onclusion 133cknowledgments 134eferences 134

Corresponding author. Tel: �33-144-323894.-mail address: paoletti@biologie.ens.fr (P. Paoletti).bbreviations: AMPAR, AMPA receptor; LTP, long-term potentiation;

pF, mossy fiber; NMDAR, NMDA receptor; NTD, N-terminal domain;SD, postsynaptic density.

306-4522/09 © 2009 IBRO. Published by Elsevier Ltd. All rights reserved.oi:10.1016/j.neuroscience.2008.01.061

126

LOCALIZATION OF BRAIN “CHELATABLEZINC”

he divalent cation Zn2� is the second most prevalentrace element in the body (after iron). It has myriad func-ional roles throughout the body and it has been knownince the 1930s to be a vital part of a healthy diet. Althoughhe vast majority (�95%) of zinc ions are trapped withinroteins as structural or catalytic co-factors, in the mam-alian brain there is a pool of zinc that is less tightly boundnd that can be revealed by histochemical techniques,uch as the Timm’s sulfide-silver staining procedure, asell as with the fluorescent probes zinquin or TSQ (6-ethoxy-8-p-toluene sulfonamide quinoline; Fredericksont al., 2000, 2005). The distribution of this loosely-boundinc (or “chelatable zinc”) is remarkable for two reasons:rst, it is mainly restricted to the forebrain (Fig. 1) and,econd, at the ultrastructural level, histochemically reac-ive zinc is localized almost exclusively within synapticesicles of a subset of glutamatergic axon terminals (Fig. 2).

Zinc-containing axon terminals are found throughouthe telencephalon, being particularly abundant in the neo-ortex, pyriform cortex, hippocampus, striatum and amyg-ala (Fig. 1) (Perez-Clausell and Danscher, 1985; Fred-rickson, 1989; Frederickson et al., 2000; Valente et al.,002; Danscher and Stoltenberg, 2005). Zinc-containingbers are generally not long range. There are virtually noinc-containing fibers that project from cortical regions to

ower brain structures. Similarly, ascending fibers that runrom the thalamus, brainstem or spinal structures to higherrain structures are also devoid of synaptic zinc. Thisistribution is probably best illustrated by the conspicu-usly weak zinc staining in cortex layer IV, where therimary thalamocortical afferents terminate. Equally strik-

ng is the fact that large layer V pyramidal cells (such as theetz cells in the primary motor cortex), which are therincipal source of efferents for all the motor-related sub-ortical structures, never contain “chelatable zinc” in theirerminals. Instead, zinc-containing neurons form a com-lex and elaborate associational network that intercon-ects most of cerebral cortices and limbic structures. Thus,or instance, most amygdalar nuclei send zinc-containingbers to neocortical and allocortical regions but are alsoensely innervated by zinc-containing boutons originating

n those regions. The same applies to the perirhinal cortex.nother structure that contains high levels of zinc is theippocampus, a region important for learning and memory.esicular zinc can be detected in each component of the

olysynaptic circuit that includes: i) perforant path projec-

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P. Paoletti et al. / Neuroscience 158 (2009) 126–136 127

ions from the entorhinal cortex to the dentate gyrus; ii)ossy fiber (MF) projections from granule cells to hilareurons and pyramidal CA3 cells; iii) projections from CA3o CA1 pyramidal neurons; iv) projections from CA1 to theubiculum. In this circuit, MFs are unique in that theyontain exceptionally large amounts of zinc in their termi-als, making this pathway an attractive system to studyossible roles for synaptic zinc (see below).

Cytosolic zinc is transported into small, clear and roundynaptic vesicles by the neuronal-specific zinc transporternT3 (also named Slc30a3; Palmiter et al., 1996; Wenzelt al., 1997). ZnT3 belongs to a large family of zinc trans-orters (the ZnTs), which all facilitate zinc efflux from theytoplasm either into various intracellular compartments orcross the plasma membrane (McMahon and Cousins,998; Palmiter and Huang, 2004). ZnT3 and the vesicularlutamate transporter Vglut1 are found in the same vesicleopulation (Salazar et al., 2005) and the vesicular zinconcentration has been shown to be determined by thebundance of ZnT3 protein (Cole et al., 1999). Thus,

ig. 1. Histochemical reactive zinc in the brain. Sagittal section of railver staining of zinc by the Timm-Danscher method. The blue is Nissayer IV, see text) and hippocampus (in particular the h and the MFmygdalar neurons. Am, amygdala; ao, accessory olfactory bulb; cp,Reprinted, with permission, from Frederickson et al., 2000.)

ig. 2. Zinc-containing bouton in the human cerebral cortex. Theostmortem gas-autometallography method of Danscher was used tohow the zinc (silver grains) in the boutons of human cortex. Thetaining is in a type I bouton apposed to a typical asymmetric synaptic

apecialization. S, dendritic spine. (Reprinted, with permission, fromrederickson et al., 2000.)

rains of mice carrying a targeted disruption of the ZnT3ene (ZnT3 KO mice) completely lack “chelatable zinc”Cole et al., 1999). Zinc-positive terminals represent only aubset of the glutamatergic boutons in the brain. Interest-

ngly, in the rat hippocampus, it was recently shown thathile all giant MF terminals contain vesicular zinc, aboutalf of the boutons in the stratum radiatum of the CA1egion stain for zinc (Sindreu et al., 2003).

IS ZINC RELEASED DURING NEURONALACTIVITY?

ecause “chelatable zinc” is accumulated into synapticesicles that also contain the neurotransmitter glutamate, itas been surmised that zinc, together with glutamate,ould be released in the extracellular medium during neu-onal activity. Early studies in the 1980s showed thattrong stimulation of the hippocampal MFs, which showhe strongest zinc staining in the brain, elevates the con-entration of zinc measured in the perfusate, suggestinghat endogenous zinc can indeed be released from thesebers (Assaf and Chung, 1984; Howell et al., 1984; An-

ksztejn et al., 1987). This release is both calcium andepolarization-dependent, in agreement with a vesicularelease mechanism. Additional clues for zinc release byippocampal MFs came from studies showing that intensenon-physiological) stimulation leads to an almost com-lete disappearance of zinc staining in these fibers (Slov-

ter, 1985; Frederickson et al., 1988). However, it is onlyecently, by combining zinc imaging and electrophysiolog-cal recordings, that more direct evidence for a quantalo-release of zinc and glutamate has been provided (Vogtt al., 2000; Li et al., 2001a; Molnar and Nadler, 2001;eno et al., 2002; Qian and Noebels, 2005, 2006). Among

he most convincing studies are those performed by Qian

ter autometallographic staining. The tan–brown–black staining is the. Note the dense zinc staining in the Am, striatum (cp), cortex (exceptalso that the neuropil staining in the cp originates from cortical andputamen; h, hilus of dentate gyrus; s, subiculum; IV, cortical layer IV.

t brain afl stainings). Note

nd Noebels using the membrane-impermeant zinc-selec-

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P. Paoletti et al. / Neuroscience 158 (2009) 126–136128

ive fluorescent dye FluoZin-3 (a derivative of the calciumrobes fluo-3/4; Gee et al., 2002). In acute hippocampallices, these authors have shown that zinc exocytosis cane reliably detected after individual action potentials notnly at the zinc-enriched MF synapses, but also at CA3–A1 synapses, despite their lesser zinc content. The lackf zinc signal in slices from ZnT3 KO mice confirmed theesicular origin of the released zinc (Qian and Noebels,005, 2006).

While there is little doubt that during synaptic activityesicular zinc is exocytosed, there is no consensus abouthe amplitude and time course of zinc concentrationseached in the synaptic cleft (knowledge of both is impor-ant to evaluate zinc action). At hippocampal MF synapses,he most studied zinc-containing synapses in the brain,stimates differ by several orders of magnitude, rangingrom 10 nM to over 10 �M. Using the fluorescent dyenAF-2, Ueno et al. (2002) estimated that, following tetanictimulation, synaptic zinc levels could reach up to 30 �M.his study further suggested that zinc may spread up to00 �m from its release site (zinc spillover) allowing foristant heterosynaptic modulation. These results are ques-

ionable, however, because the probe ZnAF-2 has beenhown since then to be membrane permeant and enterynaptic vesicles (Kay and Toth, 2006). More convincingly,y studying the ability of released zinc to inhibit NMDAeceptor (NMDAR) –mediated synaptic currents, Vogt etl. (2000) estimated that, at MF-CA3 synapses, zinc couldeach tens of �M possibly peaking around 100 �M (seelso Molnar and Nadler, 2001). In these experiments, the

ack of effect of MF stimulation on neighboring associa-ional/commissural synapses recorded in parallel arguesgainst the zinc spillover hypothesis. Using Newportreen as a zinc-selective fluorescent dye, Li et al. (2001a)stimated that in the hilar region of the dentate gyrus,imilar high zinc concentrations (�10 �M) are achievedollowing strong stimulation of the MFs (100 Hz, 5 s). Muchower values, however, have been proposed by others. Inheir imaging study, Qian and Noebels (2005) observedtrong fluorescent zinc signals using FluoZin-3 but notsing FluoZin-2, a zinc indicator with a much lower affinityor zinc (Kd�3 �M vs. 15 nM), indicating that only submi-romolar zinc concentrations might be reached. Similarly,y comparing the fluorescence of a series of improvednAFs dyes (fluorescein derivatives) covering a largepectrum of zinc sensitivities, Komatsu et al. (2005) esti-ated the released zinc concentration following K�-in-uced depolarization to be on the order of 1 �M in theentate gyrus hilus but not more than 100 nM in CA1 andA3 regions. The mere fact that zinc is released uponeuronal activity has even been recently challenged byay and Toth in experiments using fluorometric measure-ents on brain slices. These authors suggest that duringxocytosis, zinc is exposed to the extracellular space butoes not diffuse in the extracellular space, instead remain-

ng tightly bound to some as yet unidentified presynapticomponents. In this scenario, zinc would be “externalized”ather than released and zinc elevations in the synaptic

left would not exceed a few nM, even at hippocampal MF a

ynapses (Kay, 2003, 2006; Kay and Toth, 2006). Thesendings are obviously at odds with the findings from manyther groups, requiring additional studies be performed.nowing the magnitude and duration of the zinc concen-

ration transient in the synaptic cleft after exocytosis islearly of key importance in evaluating the potential neu-omodulatory roles of this divalent cation.

ZINC TARGETS AT EXCITATORY SYNAPSES

here are multiple mechanisms by which extracellular zincould modulate fast excitatory glutamatergic transmission.oth ionotropic glutamate receptors (iGluRs) and gluta-ate transporters are sensitive to extracellular zinc (Smartt al., 2004; Frederickson et al., 2005). However, depend-

ng on the nature of the target, zinc may either boost orepress the synaptic response. In addition, the potency ofinc varies considerably between the different targets.

The best characterized synaptic zinc targets areMDARs. In the late 80s, Peters et al. (1987) and West-rook and Mayer (1987) discovered that zinc, at low mi-romolar concentrations, selectively inhibits NMDAR-me-iated responses recorded from cultured hippocampaleurons. The subsequent analysis of the mechanism ofinc inhibition revealed that, unlike Mg2�, which acts as ahannel blocker, the major effect of Zn2� ions on NMDARctivity is to produce a voltage-independent non-competi-ive (allosteric) inhibition, seen as a decrease in the chan-el’s open probability (Mayer et al., 1989; Christine andhoi, 1990; Legendre and Westbrook, 1990). At higheroncentrations (�20 �M), zinc can also produce a voltage-ependent inhibition of NMDAR currents, probably byinding at the Mg2�-blocking site inside the pore. How-ver, the voltage-dependence of the Zn2� block is lowerhan that of Mg2�, presumably because zinc can permeateMDAR channels more easily than Mg2� (Legendre andestbrook, 1990; Paoletti et al., 1997).

The cloning of NMDAR subunits opened the way forhe study of the zinc modulations of the different subtypesf NMDARs. As expected from studies on native receptors,ecombinant NMDARs are inhibited by zinc through a dualechanism, voltage-independent and voltage-dependent,

he former being of higher affinity. The range of zinc sen-itivity was, however, unexpected. Depending on the re-eptor subunit composition, voltage-independent zinc sen-itivity (IC50) was shown to span more than three orders ofagnitude: from low nM for NR1/NR2A receptors, to 1 �M

or NR1/NR2B receptors and �10 �M for NR1/NR2C andR1/NR2D receptors (Table 1; and see Paoletti et al.,997; Chen et al., 1997; Traynelis et al., 1998; Rachline etl., 2005). In fact, NMDARs containing the NR2A subunita subunit widely expressed in the adult brain) have suchigh zinc sensitivity that ambient zinc levels present asontaminants in standard solutions are sufficient to consti-utively inhibit these receptors, whether recombinant orative (Paoletti et al., 1997). This exquisite zinc sensitivityf NR2A-containing receptors has two important conse-uences: first, zinc chelators and/or zinc-buffered solutions

re required for proper zinc sensitivity measurements (see

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elow); second, in vivo, synaptic zinc may exert its modu-atory effects not only during fast phasic synaptic releases originally thought but also under a tonic mode. In otherords, under normal resting conditions, the NR2A-specificinc binding site might be partially occupied by zinc and theMDARs correspondingly inhibited (see below). Another

nteresting property of the NR2A-specific high-affinity zincnhibition is that it is not complete, with 20–40% of the

aximal NR1/NR2A currents remaining at saturating zinconcentrations. Residual currents are even larger for re-eptors incorporating two different types of NR2 subunitsa NR2A and non-NR2A subunit; Hatton and Paoletti,005). The partial nature of high-affinity zinc antagonismay be seen as a way to spare some NMDAR activity evenuring substantial rises in extracellular zinc, as may occururing intense neuronal activity. Interestingly, the maximal

evel of zinc inhibition can be modified by tyrosine kinaserc phosphorylation of the receptor (Zheng et al., 1998).his may permit adjustment of the level of NMDAR activity

ollowing activation of intracellular signal transductionascades.

The binding site accounting for the NR2A-specific high-ffinity zinc modulation has been characterized in detail. Itas been mapped to the large N-terminal domain (NTD) ofhe NR2A subunit that forms a discrete modulatory domainreceding the glutamate-binding domain (Choi and Lipton,999; Fayyazuddin et al., 2000; Paoletti et al., 2000; Lowt al., 2000). In this modulatory domain, a few key residueshat are likely to directly coordinate the Zn2� ion have beendentified. Their location in a 3D model suggests that zincinds a central cleft and promotes domain closure uponhe ligand (Venus-flytrap mechanism; Paoletti et al., 2000).he mechanism that transduces this conformationalhange into closure of the channel gate has been recentlyissected out and resembles very much the mechanismsnderlying AMPA and kainate receptor desensitizationGielen et al., 2008). Finally, the NTD of the NR2B subunit,ut not that of NR2C and NR2D subunits, also forms a zincinding site but of much lower affinity, accounting for the

ow micromolar zinc inhibition of NR1/NR2B receptorsRachline et al., 2005). Thus, the most abundant NMDAR

able 1. Zinc effects on recombinant glutamate receptors and transp

arget Subtype Effect

MDARs NR1/NR2A Inhibition

NR1/NR2B

NR1/NR2C

NR1/NR2D

All subtypes InhibitionMPARs Homomeric GluR1 No effect

Homomeric GluR3 Potentiation

ainate receptors Heteromeric GluR6/KA2 Inhibitionransporters EAAT1 Inhibition

EAAT2 No effect

ND, not determined. For references see text.

ubtypes (those incorporating NR2A and/or NR2B sub- n

nits) contain “zinc sensors” capable of detecting extracel-ular zinc over a wide concentration range (low nM to lowM) depending on their NR2 subunit composition.

AMPA receptors (AMPARs), which co-localize withMDARs in the postsynaptic membrane of glutamatergicynapses, are also sensitive to extracellular zinc. How-ver, little is known about the effects of zinc on theseeceptors. From the few studies available, it appears thatinc modulation of AMPARs differs from the zinc modula-ion of NMDARs in at least two important aspects (Table): first, the effects are opposite, zinc acting chiefly as annhancer of AMPARs (AMPAR inhibition can be observedut only at very high zinc concentrations in the mM range);econd, AMPARs are less sensitive to zinc, being appar-ntly unaffected by submicromolar zinc concentrationsMayer et al., 1989; Rassendren et al., 1990). The GluR3ubunit seems necessary for the zinc modulation, sinceomomeric GluR3 receptor currents are potentiated byinc, while homomeric GluR1 receptor currents are notDreixler and Leonard, 1994). Responsiveness to zinc islso affected by the alternative splicing, with flop variants,ut not flip variants, conferring resistance to zinc modula-ion (Shen and Yang, 1999). Almost nothing is knownbout zinc interactions with kainate receptors besides theact that recombinant GluR6/KA2 receptors are inhibited in

non voltage-dependent manner by low �M zinc concen-rations (Fukushima et al., 2003). Since AMPA and kainateeceptor subunits all possess NTDs, these domains mayarbor, like in the NMDAR subunits NR2A and NR2B, zincinding sites. This possibility remains to be explored.

Glutamate transporters, which are important for clear-ng glutamate from the synaptic cleft, are also modulatedy exogenous applications of extracellular zinc. Zinc inhib-

ts salamander retinal Müller cell glutamate transport withelatively high affinity (IC50 �700 nM; Spiridon et al.,998). On recombinant glutamate transporters, zinc, atoncentrations around 10 �M, acts as a non-competitiveartial antagonist of glutamate transport by EAAT1 (exci-atory amino-acid transporter 1), but has no effect on trans-ort by EAAT2 (Table 1) (Vandenberg et al., 1998). The

nhibition of glutamate uptake by zinc could be harmful to

y EC50 or IC50 (�M) Comments

Voltage-independent and non-competitiveinhibition. High-affinity NR2A-specificinhibition not total (max. inhibition�60–80%). For NR2A-containingreceptors, measured using tricine-buffered zinc solutions.

�60 mV) Voltage-dependent pore blockFlip variants more sensitive than flop

variants. No zinc buffer used. Inhibitionat very high zinc concentrations (lowmM).

No zinc buffer used.Non-competitive partial inhibition (max.

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P. Paoletti et al. / Neuroscience 158 (2009) 126–136130

evels. However, the fact that zinc also inhibits the releasef glutamate that occurs by reversed operation of gluta-ate transporters during ischemic insults could counter-alance this effect and attenuate excitotoxicity (Spiridon etl., 1998).

Finally, high-voltage-activated calcium channels thatediate calcium-dependent neurotransmitter release at

entral synapses are also inhibited by �M zinc concentra-ions (Magistretti et al., 2003; Sun et al., 2007). Theirocalization on the presynaptic membrane close to theesicular release points makes them plausible targets forxocytosed zinc.

ZINC IN EXCITATORY SYNAPTICTRANSMISSION AND PLASTICITY

s described above, at certain glutamatergic synapses ofhe forebrain, zinc fulfils most of the prerequisites for anndogenous modulator of synaptic transmission: presencef zinc inside synaptic vesicles and presence of multipleynaptic targets, most noticeably NMDARs. In addition,iven the central importance of NMDARs in cognitive func-ions (learning and memory) as well as in numerous patho-ogical states of the CNS (stroke, pain, schizophrenia),here has always been intense speculation that zinc couldct as a critical neural messenger in healthy and diseasedtates of the brain through its ability to regulate NMDARctivity. Yet, despite clear indications that endogenousrain zinc is involved in glutamate-induced toxicity (excito-oxicity) under pathological conditions (Choi and Koh, 1998nd see below), there has been no clear demonstration ofrole of zinc in synaptic transmission under physiological

onditions.Obviously, the question of the synaptic role of zinc is

inked to that concerning the amount of zinc releaseduring synaptic activity. Authors concluding that little or noiffusible zinc is released into the synaptic cleft (seebove) propose that zinc acts mainly under a tonic mode.inc ions would “stick” to cellular membranes forming azinc veneer.” The degree of tonic modulation would beetermined by the number of zinc ions in the veneer, whichould accumulate slowly and progressively with synapticctivity and decline as zinc is removed from the veneerKay, 2003; Kay et al., 2006). According to this scenario,inc would only be present at low (nM) concentrations athe external surface of postsynaptic membranes. The dis-overy that numerous ion channels and neurotransmittereceptors including NMDARs and the two major inhibitoryABA-A and glycine receptors contain high affinity (nM)inc binding sites makes the “zinc veneer” modulationypothesis a plausible one (Paoletti et al., 1997; Suwa etl., 2001; Smart et al., 2004). Several other groups, how-ver, have presented evidence for a more classical phasicype modulation of synaptic transmission by zinc. Becausehe hippocampal MFs are by far the most zinc-enrichedegion of the brain, the role of endogenous zinc has beenhoroughly explored at synapses engaging MFs.

Vogt and colleagues (2000) have analyzed the gluta-

atergic inputs made by MFs onto CA3 pyramidal neurons p

n acute slices using the zinc chelator CaEDTA. Theseuthors reached three important conclusions: first, thatmbient zinc spontaneously released from MFs constitu-ively occupies the NR2A-specific high-affinity zinc bindingite of NMDARs located in the stratum lucidum (where MFerminals are); second, that during low frequency phasicctivity, zinc levels reached in the synaptic cleft would beigh enough (10–100 �M) to engage the lower affinityoltage-dependent zinc binding site on NMDARs; third,hat there would be no zinc spillover to the neighboringtratum radiatum (where associational/commissural inputsre). Similar conclusions were reached by Molnar andadler (2001) for the MF recurrent synapses onto dentateranule cells. According to Ueno et al. (2002), however,inc could diffuse from stratum lucidum to the proximal partf the stratum radiatum (but not to the distal part). Yet, inhis latter study, zinc spillover was best observed whenigh-frequency tetanic stimulations were used. Conflictingata regarding the levels of zinc reached in the synapticleft after a single stimulation have been obtained by Lu etl. (2000). In their conditions, CaEDTA increases theMDA component of the EPSC at the MF/CA3 synapse involtage-independent manner, indicating that just the

igh-affinity (nM) zinc binding site of NMDARs is occupieduring low frequency phasic activity. The reasons for theiscrepancy between the results of Vogt et al. and Lu et al.emain unclear. A case can be made for active zinc trans-ort systems, which may be sensitive to slight experimen-al differences, such as temperature. The role of zinc inasal synaptic transmission has also been explored at thechaeffer collateral inputs onto CA1 pyramidal neurons

Izumi et al., 2006). There, addition of CaEDTA has noffect on NMDAR EPSPs, suggesting that tonic zinc inhi-ition of synaptic NMDARs does not occur at these syn-pses. Surprisingly, in the same study and at the sameynapses, it was shown that CaEDTA could affect a formf metaplasticity, an effect that was attributed to the relieff tonic zinc inhibition of supposedly extrasynapticMDARs (Izumi et al., 2006).

The study of synaptic plasticity has often been carriedut concomitantly with that of the role of zinc in basalynaptic transmission. Here again there is little consensus.he zinc-dependence of long-term potentiation (LTP) at

he synapses between MFs and CA3 pyramidal cells, aorm of plasticity that does not depend on NMDAR activa-ion, is still a subject of debate. Some data argue against apecific role of zinc. Thus, MF/CA3 LTP is unaffected inlices from mocha mutant mice, in which insertion of ZnT3roteins in synaptic vesicles is deficient (Vogt et al., 2000).aution must be taken with this model however sinceesicular zinc in mocha mice is reduced but not absent (inontrast to ZnT3 KO mice; Stoltenberg et al., 2004). Fur-hermore, some authors have reported that chelating zinceither extracellular or intracellular) does not prevent LTPnduction (Xie and Smart, 1994; Vogt et al., 2000). How-ver, contradictory results have been presented by Lu etl. (2000). In their hands, chelating extracellular zinc (usingmM CaEDTA) does not prevent LTP induction, but de-

leting vesicular zinc, using the membrane-permeant zinc

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P. Paoletti et al. / Neuroscience 158 (2009) 126–136 131

helators DEDTC or dithizone, does. These data suggesthat synaptic zinc is required for proper LTP induction.hey also support a presynaptic role for zinc in MF/CA3TP. Li et al. (2001b) have presented a third set of partiallyonflicting results. They show that the membrane-imper-eant zinc chelator CaEDTA does prevent MF/CA3 LTPxpression, but only when used at high concentrations10 mM). These authors propose that at MF/CA3 syn-pses, zinc behaves as a transcellular messenger re-

eased from presynaptic vesicles and acting on postsyn-ptic cytoplasmic targets. Such targets may be found in theostsynaptic density (PSD). There, zinc binds the scaffold-

ng protein Shank3 and promotes its packaging into largeD-sheets. By these means, zinc may modulate the struc-ure and the function of the PSD, allowing the expressionf synaptic plasticity (Gundelfinger et al., 2006).

Besides hippocampus, another brain region where zincas been shown to participate in synaptic plasticity is theinc-enriched amygdala, a structure involved in fear learn-ng. Principal neurons of the lateral amygdalar nucleuseceive glutamatergic inputs from both auditory cortex anduditory thalamus. Interestingly, only cortical inputs areinc-containing and display LTP under conditions of intactABAergic inhibition. By studying the effects of zinc ch-lators and exogenous zinc applications, Kodirov and col-

eagues (2006) showed that synaptically released zincnables LTP at cortico-amygdalar synapses by depressingeed-forward inhibition of principal neurons. This may hap-en after spillover of zinc from excitatory to neighboring

nhibitory synapses (Kodirov et al., 2006).Finally, it is worth noting that most of the studies per-

ormed so far have focused on postsynaptic receptors asargets for synaptic zinc. However, there is increasingvidence that neurotransmitter receptors, and in particular

onotropic glutamate receptors, are also present on pre-ynaptic terminals. These receptors would appear to beptimally located to be targets for vesicular zinc. This is thease, in particular, for kainate receptors, which form auto-eceptors at the zinc-enriched hippocampal MF terminalsPinheiro et al., 2007), but also potentially for NMDARs inhe cortex (Berretta and Jones, 1996; Sjostrom et al.,003). The hypothesis that presynaptic glutamate recep-ors are a locus of zinc action remains to be tested.

ZINC AND EPILEPSY

everal observations point to a role of endogenous syn-ptic zinc in the pathophysiology of epilepsy (Frederick-on, 1989). First, there is a striking overlap of seizure-rone limbic regions and Timm-positive territories (i.e. hip-ocampus, amygdala). Second, manipulating brain zincffects seizure susceptibility. Third, CNS zinc levels arebnormal in seizure-prone animals and seizures canause substantial modifications of cerebral zinc levels.evertheless, whether zinc is a pro- or anti-convulsantgent remains unclear.

In mice, dietary and congenital deficiencies of zinc aressociated with increased susceptibility to seizures (Fuka-

ori and Itoh, 1990; Feller et al., 1991; Takeda et al., c

003). Similarly, zinc chelators (such as DEDTC) are pro-onvulsant (Mitchell and Barnes, 1993). In line with thesendings, ZnT3 KO mice are more prone to limbic seizureslicited by kainic acid, suggesting that the net effect ofippocampal zinc on acute seizures in vivo is to dampenxcitability (Cole et al., 2000). Thus, in the hippocampus,elease of vesicular zinc (mostly from the MFs) may serveo protect this structure from paroxysmal seizure activity. Aechanistic explanation commonly put forward to account

or these neuroprotective effects of zinc is an inhibition ofMDAR activity induced by direct zinc binding on these

eceptors (see above). This hypothesis has, however, noteen proven. In addition, there are alternative explanationso a direct antagonism of NMDARs for the anti-convulsantffects of zinc, such as an increased GABA-mediated in-ibition due to increased GABA release (Ben-Ari andherubini, 1991; Xie and Smart, 1991).

In contradiction with the previous results, others haveeported pro-epileptic effects of zinc. Pei and Koyama1986) have shown that intrahippocampal injection of zincauses seizures in rabbits. By comparing zinc levels ofpilepsy-prone and epilepsy-resistant rat strains, Flynn etl. (2007) found that the levels of synaptic zinc were sig-ificantly lower in the epilepsy-resistant rats, suggestinghat zinc excess may facilitate the development of sei-ures. Possible explanations for these pro-convulsive ef-ects of zinc could be a diminished inhibitory synapticransmission (through direct zinc inhibition of GABA-Aeceptors; Smart et al., 2004) and/or enhanced AMPARctivity (through direct zinc potentiation of these receptors;ee above).

ZINC AND EXCITOTOXICITY

xcitotoxicity is a process during which excessive gluta-ate release causes over-activation of glutamate recep-

ors, accumulation of intracellular calcium and eventuallyeuronal death. NMDARs are known to be central in thisrocess, in particular because of their high calcium perme-bility (Kemp and McKernan, 2002). Excitotoxicity occursuring cerebral ischemia and in neurodegenerative disor-ers such as Parkinson’s and Huntington’s diseases. Inevere epilepsy, intense seizure activity can also lead toMDAR-mediated cell injury. In turn, strong activity oflutamatergic pathways, as observed during excitotoxicvents, should favor synaptic zinc release. Accordingly,any reports have proposed that brain vesicular zinc is

nvolved in glutamate-induced cell death (Frederickson etl., 2005). Nevertheless, the role of zinc in excitotoxicityemains controversial.

Several studies have shown that exogenously appliedinc, at high concentrations (�100 �M), is potentially neu-otoxic both in vivo and in vitro. This may be due to zincntry into the cells, which would induce mitochondrial dys-unction and the production of reactive oxygen speciesChoi and Koh, 1998). Indeed, in areas with zinc-rich fi-ers, following seizures, transient forebrain ischemia orrauma, there is depletion of presynaptic zinc pools, while

helatable endogenous zinc selectively accumulates in

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P. Paoletti et al. / Neuroscience 158 (2009) 126–136132

amaged neurons. This has led to the “translocation”ypothesis, in which presynaptic zinc is first released intohe extracellular space and then enters postsynaptic neu-ons, mainly through calcium-permeable GluR2-lackingMPARs (Frederickson et al., 1989; Koh et al., 1996;eiss and Sensi, 2000). However, the fact that zinc accu-ulates in degenerating neurons of ZnT3 KO mice (Lee etl., 2000) and in experiments performed with extracellularinc chelators (Lavoie et al., 2007) strongly argues againsthe vesicular origin of cytoplasmic zinc. Zinc accumulationay rather originate from intracellular zinc stores (Lavoiet al., 2007).

Other evidence supports the possibility of a neuropro-ective action of synaptic zinc. Thus, low �M concentra-ions of zinc have been shown to protect neurons fromxcitotoxic insults induced by glutamate or NMDA (Choind Koh, 1998). Chelation of vesicular zinc in vivo or initro lowers the threshold for stimulation-induced seizuresnd increases kainic acid-induced neuronal deathDominguez et al., 2003). Moreover, ZnT3 KO mice, whichack vesicular zinc, show enhanced neuronal damage fol-owing limbic seizures compared with WT animals (Cole etl., 2000; but see Lee et al., 2000). In these studies, theeuroprotective effects of zinc have usually been attributedo the direct inhibition of NMDARs by extracellular zinc.rotection against neurodegeneration by small elevationsf intracellular zinc has also been proposed (Côté et al.,005).

In conclusion, zinc may have contrasting effects on cellurvival, depending on its concentration and whether itcts from inside or outside the cell. There is little doubt thattrong cytosolic zinc accumulation is deleterious for neu-ons, but the source of this zinc may not be the Timm-tained vesicular zinc but rather intracellular zinc pools. Inontrast, extracellular zinc, at concentrations below a cer-ain threshold, may protect against neuronal death by lim-ting NMDAR overactivation.

MANIPULATING EXTRACELLULAR ZINCCONCENTRATIONS USING CHELATORS

any of the past studies on CNS zinc have relied in part orotally on the use of zinc chelators. Zinc chelators areowerful tools but they have limitations. Inappropriate usef certain zinc chelators and/or erroneous interpretation ofata obtained with these agents may explain some of thenumerous) discrepancies in the field.

Compounds that chelate zinc avidly (but also othereavy metal ions) are plenty. Some, however, are knowno pass cell membranes easily and thus cannot be used toodify zinc levels selectively in the extracellular compart-ent. This is the case of DEDTC (diethyldithiocarbamiccid), dithizone or TPEN (N,N,N=,N=-tetrakis2-pyridylmeth-lethyenediamine). Only compounds that are membrane-mpermeant can be properly used to remove free zinc (andther heavy metals) selectively from the extracellular me-ium. The best known are EDTA, EGTA and DTPA (dieth-lenetriaminepentaacetic acid). These agents, however,

ave an important drawback for the study of the role of zinc s

n synaptic transmission: they also chelate calcium, an ionhat is usually present in mM concentrations in the extra-ellular media and that is required for exocytosis. To cir-umvent this problem, Koh et al. (1996) introduced the usef CaEDTA–disodium EDTA saturated with equimolara2�. Since EDTA has a much higher affinity for Zn2� than

or Ca2� (absolute Kd�10�16.4 M vs. 10�10.6 M; Dawsont al., 1986), applying low mM concentrations of CaEDTAllows effective chelation of extracellular zinc at equilib-ium, without reducing extracellular Ca2� (Koh et al., 1996;i et al., 2001; Lavoie et al., 2007). However, here again,xperimental use of CaEDTA encounters some limitations:

) it acts as an all-or-none chelator allowing the comparisonf physiological (unknown zinc concentration) and zinc-ree situations only; ii) it is a slow chelator, partly becausea2� (and H�) must unbind for Zn2� to bind. The kineticsf zinc chelation in a medium containing CaEDTA predicthat in 1 or 10 mM CaEDTA, 100 �M extracellular zincould be reduced by only 6% (to 94 �M) or half (to54 �M), respectively, after 60 ms (Fig. 3). Contradicting

he results of Li et al. (2001b), CaEDTA appears thereforenefficient to prevent a transient zinc rise as it might occururing fast synaptic transmission (see also Kay, 2003).

To chelate extracellular zinc on a timescale compatibleith rapid synaptic transmission, we propose the use of

ricine (N-[Tris(hydroxymethyl)methyl]glycine), a well-char-cterized pH buffer that we first introduced as a zinc bufferdecade ago (Paoletti et al., 1997). Tricine combinesany interesting properties: first, it has very good solubility

n aqueous solutions; second, it has an intermediate affin-ty for zinc (absolute Kd�10�5.0 M; Vieles et al., 1972;aoletti et al., 1997); third, it does not bind Ca2� and Mg2�

or very poorly; absolute Kd�10�1.2 M; Dawson et al.,986). Thus, tricine has all the required features to act asrapid zinc-specific chelator devoid of side effects on

ynaptic release. This should allow effective and specificuffering of rapid zinc transients. Calculations predict thatuch is the case. In 1 or 10 mM tricine, 100 �M extracel-ular zinc would be reduced by �40% (to 61 �M) or by

99% (to 1.2 �M), respectively, after only 5 �s (Fig. 3).inally, in contrast to other zinc chelators, tricine does notct like an all-or-none chelator but can be used (usually at0 mM) to impose known extracellular zinc concentrations

n the 1 nM-10 �M range, the range of interest for the studyf synaptic zinc targets (Paoletti et al., 1997; see alsoayyazuddin et al., 2000). Tricine-based zinc-buffered so-

utions are now commonly used for quantitative in vitrotudies of the zinc sensitivity of various receptors andhannels (see for instance, Traynelis et al., 1998; Suwa etl., 2001; Chu et al., 2004). Tricine could also be used inrain slices or in vivo preparations. One should keep inind, however, that perturbing endogenous zinc concen-

rations may produce pleiotropic effects, because of thebundance of potential synaptic zinc targets (see above).his is a general problem for any zinc chelator, implying

hat caution is needed in the interpretations of the ob-

erved effects.

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P. Paoletti et al. / Neuroscience 158 (2009) 126–136 133

CONCLUSION

everal decades after the discovery of brain “chelatableinc” (Maske, 1955) and its localization in synaptic vesiclesHaug, 1967), the importance of this metal ion in brainunction is still disputed. Zinc seems to satisfy severalriteria for a neural messenger: it is stored in synapticesicles, is possibly released upon depolarization and canct at various membrane targets. The selective associationf zinc with glutamate-containing synaptic vesicles sug-ests that zinc might be a co-transmitter at some excitatoryynapses. Several reasons, however, make the study ofrain zinc a difficult task. The first is that perturbing endog-nous zinc concentrations, as done in many past studies,roduces pleiotropic effects, as expected from the abun-ance of potential zinc targets. Extracellular zinc can mod-late the activity of both glutamate receptors and trans-orters. It also has the potential to act as a modulator of

nhibitory neurotransmission by affecting GABA- and gly-ine-mediated currents. In addition, zinc potentially influ-nces transmitter release by acting on presynaptic recep-ors or channels. Effects of zinc are thus expected to beultiple and complex, both increasing and decreasingembrane excitability. Tools for manipulating zinc levels

0.0 0.5 1.0 1.5 2

020

4060

8010

0

time (s)

[Zn]

free

(µM

)

10 mM CaEDTA1 mM CaEDTA10 mM tricine1 mM tricine

A

ig. 3. Fast kinetics of zinc chelation by tricine but not by CaEDTA. SaEDTA or tricine, in the following solutions (total amounts in mM). Sa 1, Mg 1, tricine 10. Dashed blue: Ca 1, Mg 1, tricine 1. The sameas a much higher affinity for zinc than tricine, tricine is five orders ofinc from a solution (at equilibrium), while tricine is more appropriatericine is also well-suited for buffering zinc at equilibrium levels in the n000). The Zn, Ca, Mg, H, EDTA, tricine reaction system was repreonstants for (unprotonated) EDTA were (M�1 s�1): Zn 2.4�109, Complexes were (s�1): Zn 1�10�7, Ca 0.15, Mg 12, H 0.5. The assoc�109, Mg 1�109, H 1�1010. The dissociation rate constants for tricodeled direct attack of the CaEDTA complex by Zn, according to a�1 s�1. Without this reaction, chelation of zinc by EDTA in the presen

han shown. The reaction mixture was assumed to be perfectly bufferetrength were made. For EDTA, the association rate constants were takere obtained by dividing the association rate constants by the follow986). For tricine, the association rate constants were fixed to 1�109

btained as for EDTA using the following stability constants: Zn 105,t al., 1997). The system of equations was solved numerically within thodesolve” library (Setzer, 2006), which is based upon the LSODA soethod with local error control and switches automatically between sol

rst allowed to equilibrate for 1�105 seconds in the absence of zinc,olerance 10-fold had a negligible effect on the solution.

lso have important drawbacks. Most zinc-chelating m

gents also chelate calcium, thus interfering with synapticransmission. Those which do not affect calcium levelssuch as CaEDTA) are often too slow to affect transientises in synaptic zinc. The finding that depleting the vesic-lar zinc by genetic ablation of the ZnT3 gene results in noarked phenotype other than a moderate increase in sus-

eptibility to epileptic seizures (Cole et al., 2000), mightesult from the simultaneous effects of zinc on inhibitionnd excitation and the possibility of compensatory mech-nisms. Another hurdle in studying synaptic action of zinc

s that the levels of zinc reached in the synaptic cleftollowing neuronal activity are unknown. While many stud-es have reported levels in the 1–100 �M range, recenteports have challenged this view, suggesting that zinclevations are rather in the low nM range. The issue of theinc concentrations in the extracellular space is a key one,ince it will determine which of the putative synaptic targetsre likely to be affected by zinc. It should also indicatehether zinc acts in a phasic or tonic mode (or both).

Despite these difficulties, there are good reasons toxpect that the situation may improve in the near future.he use of new experimental tools to study brain synapticinc should help greatly. This is the case with tricine, a

0 5 10 15 20

020

4060

8010

0

time (µs)

[Zn]

free

(µM

)

s showing the rate at which 100 �M added zinc is buffered by eithera 11, Mg 1, EDTA 10. Dashed red: Ca 2, Mg 1, EDTA 1. Solid blue:

ns are shown on different time scales in A and B. Although CaEDTAe faster as a buffer. CaEDTA is therefore more suitable for removingring rapid zinc transients of the sort expected from synaptic release.r and low micromolar ranges (Paoletti et al., 1997; Fayyazuddin et al.,s a system of first-order differential equations. The association rate, Mg 6�109, H 1�1010. The dissociation rate constants for EDTAe constants for (unprotonated) tricine were (M�1 s�1): Zn 1�109, Calexes were (s�1): Zn 1�104, Ca 6�107, Mg 6�107, H 70. We also

ttack by Cu (Hering and Morel, 1988), with a rate constant of 1�103

ess Ca would require unbinding of calcium and would be even slower3. No corrections for temperature (from room to physiological) or ionicduced) from Hering and Morel (1988). The dissociation rate constantsity constants: Zn 1016.4, Ca 1010.6, Mg 108.7, H 1010.3 (Dawson et al.,a and Mg and to 1�1010 for H. The dissociation rate constants wereMg 101.2, H 108.15 (Vieles et al., 1972; Dawson et al., 1986; Paolettianalysis environment (R Development Core Team, 2007), using the

dhakrishnan and Hindmarsh, 1993). This uses an adaptive step-sizehods suitable for stiff and non-stiff problems. The reaction system wass then added (at t�0 in the figures) at 100 �M. Tightening the error

.0

B

imulationolid red: Csimulatiomagnitudfor buffeanomolasented aa 6�109

iation ratine compsimilar ace of exc

d at pH 7.en (or deing stabilfor Zn, CCa 101.2,e GNU Rlver (Ra

ution metwhich wa

olecule with physico-chemical properties appropriate for

fTaoheab

AA

A

A

B

B

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C

C

C

C

C

C

C

D

D

D

D

F

F

F

F

F

F

F

F

F

F

G

G

G

H

H

H

H

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I

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K

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P. Paoletti et al. / Neuroscience 158 (2009) 126–136134

ast and selective chelation of synaptically-released zinc.his is also the case with genetically modified mice withlterations in specific zinc targets. The recent identificationf zinc binding sites on different neurotransmitter receptorsas opened the way for creating such animals (see Hirzelt al., 2006). Novel zinc dyes with improved properties arelso to be expected. Maybe the time has finally arrived forrain zinc to reveal its secrets.

cknowledgments—This work was supported by INSERM (P.P.),NR (P.P. and B.B.) and Région Ile-de-France (A.M.V.).

REFERENCES

niksztejn L, Charton G, Ben-Ari Y (1987) Selective release of endog-enous zinc from the hippocampal mossy fibers in situ. Brain Res404:58–64.

ssaf SY, Chung SH (1984) Release of endogenous Zn2� from braintissue during activity. Nature 308:734–736.

en-Ari Y, Cherubini E (1991) Zinc and GABA in developing brain.Nature 353:220.

erretta N, Jones RS (1996) Tonic facilitation of glutamate release bypresynaptic N-methyl-D-aspartate autoreceptors in the entorhinalcortex. Neuroscience 75:339–344.

hen N, Moshaver A, Raymond LA (1997) Differential sensitivity ofrecombinant N-methyl-D-aspartate receptor subtypes to zinc inhi-bition. Mol Pharmacol 51:1015–1023.

hoi DW, Koh JY (1998) Zinc and brain injury. Annu Rev Neurosci21:347–375.

hoi YB, Lipton SA (1999) Identification and mechanism of action oftwo histidines residues underlying high-affinity Zn2� inhibition ofthe NMDA receptor. Neuron 23:171–180.

hristine CW, Choi DW (1990) Effect of zinc on NMDA receptor-mediated channel currents in cortical neurons. J Neurosci 10:108–116.

hu XP, Wemmie JA, Wang WZ, Zhu XM, Saugstad JA, Price MP,Simon RP, Xiong ZG (2004) Subunit-dependent high-affinity zincinhibition of acid-sensing ion channels. J Neurosci 24:8678–8689.

ole TB, Robbins CA, Wenzel HJ, Schwartzkroin PA, Palmiter RD(2000) Seizures and neuronal damage in mice lacking vesicularzinc. Epilepsy Res 39:153–169.

ole TB, Wenzel HJ, Kafer KE, Schwartzkroin PA, Palmiter RD (1999)Elimination of zinc from synaptic vesicles in the intact mouse brainby disruption of the ZnT3 gene. Proc Natl Acad Sci U S A 96:1716–1721.

ôté A, Chiasson M, Peralta MR 3rd, Lafortune K, Pellegrini L, Toth K(2005) Cell type-specific action of seizure-induced intracellular zincaccumulation in the rat hippocampus. J Physiol 566:821–837.

anscher G, Stoltenberg M (2005) Zinc-specific autometallographic invivo selenium methods: tracing of zinc-enriched (ZEN) terminals,ZEN pathways, and pools of zinc ions in a multitude of other ZENcells. J Histochem Cytochem 53:141–153.

awson RMC, Elliott DC, Elliott WH, Jones KM (1986) Data for bio-chemical research. New York: Oxford Science.

ominguez MI, Blasco-Ibanez JM, Crespo C, Marques-Mari AI, Mar-tinez-Guijarro FJ (2003) Zinc chelation during non-lesioning over-excitation results in neuronal death in the mouse hippocampus.Neuroscience 116:791–806.

reixler JC, Leonard JP (1994) Subunit-specific enhancement of glu-tamate receptor responses by zinc. Brain Res Mol Brain Res22:144–150.

ayyazuddin A, Villaroel A, Le Goff A, Lerma J, Neyton J (2000) Fourresidues of the extracellular N-terminal domain of the NR2A sub-unit control high-affinity Zn2� binding to NMDA receptors. Neuron

25:683–694.

eller DJ, Tso-Olivas DY, Savage DD (1991) Hippocampal mossyfiber zinc deficit in mice genetically selected for ethanol withdrawalseizure susceptibility. Brain Res 545:73–79.

lynn C, Brown CE, Galasso SL, McIntyre DC, Teskey GC, Dyck RH(2007) Zincergic innervation of the forebrain distinguishes epilep-sy-prone from epilepsy-resistant rat strains. Neuroscience 144:1409–1414.

rederickson CJ (1989) Neurobiology of zinc and zinc-containing neu-rons. Int Rev Neurobiol 31:145–238.

rederickson CJ, Hernandez MD, Goik SA, Morton JD, McGinty JF(1988) Loss of zinc staining from hippocampal mossy fibers duringkainic acid induced seizures: a histofluorescence study. Brain Res446:383–386.

rederickson CJ, Hernandez MD, McGinty JF (1989) Translocation ofzinc may contribute to seizure-induced death of neurons. BrainRes 480:317–321.

rederickson CJ, Koh JY, Bush AI (2005) The neurobiology of zinc inhealth and disease. Nat Rev Neurosci 6:449–462.

rederickson CJ, Suh SW, Silva D, Thompson RB (2000) Importanceof zinc in the central nervous system: the zinc-containing neurons.J Nutr 130:1471–1483.

ukahori M, Itoh M (1990) Effects of dietary zinc status on seizuresusceptibility and hippocampal zinc content in the El (epilepsy)mouse. Brain Res 529:16–22.

ukushima T, Shingai R, Ogurusu T, Ichinose M (2003) Inhibition ofwillardiine-induced currents through rat GluR6/KA-2 kainate recep-tor channels by zinc and other divalent cations. Neurosci Lett349:107–110.

ee KR, Zhou ZL, Qian WJ, Kennedy R (2002) Detection and imagingof zinc secretion from pancreatic beta-cells using a new fluorescentzinc indicator. J Am Chem Soc 124:776–778.

ielen M, Le Goff A, Stroebel D, Johnson JW, Neyton J, Paoletti P(2008) Structural rearrangements of NR1/NR2A NMDA receptorsduring allosteric inhibition. Neuron 57:80–93.

undelfinger ED, Boeckers TM, Baron MK, Bowie JU (2006) A role forzinc in postsynaptic density asSAMbly and plasticity? Trends Bio-chem Sci 31:366–373.

atton CJ, Paoletti P (2005) Modulation of triheteromeric NMDA re-ceptors by N-terminal domain ligands. Neuron 46:261–274.

aug FM (1967) Electron microscopical localization of the zinc inhippocampal mossy fibre synapses by a modified sulfide silverprocedure. Histochemie 8:355–368.

ering JG, Morel FMM (1988) Kinetics of trace metal complexation:role of alkaline-earth metals. Environ Sci Technol 22:1469–1478.

irzel K, Muller U, Latal AT, Hulsmann S, Grudzinska J, Seeliger MW,Betz H, Laube B (2006) Hyperekplexia phenotype of glycine re-ceptor alpha1 subunit mutant mice identifies Zn(2�) as an essen-tial endogenous modulator of glycinergic neurotransmission. Neu-ron 52:679–690.

owell GA, Welch MG, Frederickson CJ (1984) Stimulation-induceduptake and release of zinc in hippocampal slices. Nature 308:736–738.

zumi Y, Auberson YP, Zorumski CF (2006) Zinc modulates bidirec-tional hippocampal plasticity by effects on NMDA receptors. J Neu-rosci 26:7181–7188.

ay AR (2003) Evidence for chelatable zinc in the extracellular spaceof the hippocampus, but little evidence for synaptic release of Zn.J Neurosci 23:6847–6855.

ay AR (2006) Imaging synaptic zinc: promises and perils. TrendsNeurosci 29:200–206.

ay AR, Neyton J, Paoletti P (2006) A startling role for synaptic zinc.Neuron 52:572–574.

ay AR, Toth K (2006) Influence of location of a fluorescent zinc probein brain slices on its response to synaptic activation. J Neuro-physiol 95:1949–1956.

emp JA, McKernan RM (2002) NMDA receptor pathway as drug

targets. Nat Neurosci 5:1039–1042.

K

K

K

L

L

L

L

L

L

L

M

M

M

M

M

M

P

P

P

P

P

P

P

P

Q

Q

R

R

R

R

S

S

S

S

S

S

S

S

S

S

S

T

T

P. Paoletti et al. / Neuroscience 158 (2009) 126–136 135

odirov SA, Takizawa S, Joseph J, Kandel ER, Shumyatsky GP,Bolshakov VY (2006) Synaptically released zinc gates long-termpotentiation in fear conditioning pathways. Proc Natl Acad SciU S A 103:15218–15223.

oh JY, Suh SW, Gwag BJ, He YY, Hsu CY, Choi DW (1996) The roleof zinc in selective neuronal death after transient global cerebralischemia. Science 272:1013–1016.

omatsu K, Kikuchi K, Kojima H, Urano Y, Nagano T (2005) Selectivezinc sensor molecules with various affinities for Zn2�, revealingdynamics and regional distribution of synaptically released Zn2� inhippocampal slices. J Am Chem Soc 127:10197–10204.

avoie N, Peralta MR 3rd, Chiasson M, Lafortune K, Pellegrini L,Seress L, Toth K (2007) Extracellular chelation of zinc does notaffect hippocampal excitability and seizure-induced cell death inrats. J Physiol 578:275–289.

ee JY, Cole TB, Palmiter RD, Koh JY (2000) Accumulation of zinc indegenerating hippocampal neurons of ZnT3-null mice after sei-zures: evidence against synaptic vesicle origin. J Neurosci 20:RC79.

egendre P, Westbrook GL (1990) The inhibition of single N-methyl-D-aspartate-activated channels by zinc ions on cultured rat neu-rones. J Physiol 429:429–449.

i Y, Hough CJ, Suh SW, Sarvey JM, Frederickson CJ (2001a) Rapidtranslocation of Zn(2�) from presynaptic terminals into postsynap-tic hippocampal neurons after physiological stimulation. J Neuro-physiol 86:2597–2604.

i Y, Hough CJ, Frederickson CJ, Sarvey JM (2001b) Induction ofmossy fiber ¡ Ca3 long-term potentiation requires translocation ofsynaptically released Zn2�. J Neurosci 21:8015–8025.

ow CM, Zheng F, Lyuboslavsky P, Traynelis SF (2000) Moleculardeterminants of coordinated proton and zinc inhibition of N-methyl-D-aspartate receptors. Proc Natl Acad Sci U S A 97:11062–11067.

u YM, Taverna FA, Tu R, Ackerley CA, Wang YT, Roder J (2000)Endogenous Zn(2�) is required for the induction of long-termpotentiation at rat hippocampal mossy fiber-CA3 synapses. Syn-apse 38:187–197.

agistretti J, Castelli L, Taglietti V, Tanzi F (2003) Dual effect of Zn2�

on multiple types of voltage-dependent Ca2� currents in ratpalaeocortical neurons. Neuroscience 117:249–264.

aske H (1955) Uber den topochemischen Nachweis von Zink imAmmonshorn verschiedener saugetiere. Naturwissenschaften 42:424.

ayer ML, Vyklicky LJ, Westbrook GL (1989) Modulation of excitatoryamino acid receptors by group IIB metal cations in cultured mousehippocampal neurons. J Physiol 415:329–350.

cMahon RJ, Cousins RJ (1998) Mammalian zinc transporters. J Nutr128:667–670.

itchell CL, Barnes MI (1993) Proconvulsant action of diethyldithio-carbamate in stimulation of the perforant path. Neurotoxicol Teratol15:165–171.

olnar P, Nadler JV (2001) Synaptically-released zinc inhibits N-methyl-D-aspartate receptor activation at recurrent mossy fibersynapses. Brain Res 910:205–207.

almiter RD, Cole TB, Quaife CJ, Findley SD (1996) ZnT-3, a putativetransporter of zinc into synaptic vesicles. Proc Natl Acad Sci U S A93:14934–14939.

almiter RD, Huang L (2004) Efflux and compartmentalization of zincby members of the SLC30 family of solute carriers. Pflugers Arch447:744–751.

aoletti P, Ascher P, Neyton J (1997) High-affinity zinc inhibition ofNMDA NR1-NR2A receptors. J Neurosci 17:5711–5725.

aoletti P, Perin-Dureau F, Fayyazuddin A, Le Goff A, Callebaut I,Neyton J (2000) Molecular organization of a zinc binding N-termi-nal modulatory domain in a NMDA receptor subunit. Neuron28:911–925.

ei YQ, Koyama I (1986) Features of seizures and behavioral changesinduced by intrahippocampal injection of zinc sulfate in the rabbit:

a new experimental model of epilepsy. Epilepsia 27:183–188.

erez-Clausell J, Danscher G (1985) Intravesicular localization of zincin rat telencephalic boutons. A histochemical study. Brain Res337:91–98.

eters S, Koh J, Choi DW (1987) Zinc selectively blocks the action ofN-methyl-D-aspartate on cortical neurons. Science 236:589–593.

inheiro PS, Perrais D, Coussen F, Barhanin J, Bettler B, Mann JR,Malva JO, Heinemann SF, Mulle C (2007) GluR7 is an essentialsubunit of presynaptic kainate autoreceptors at hippocampalmossy fiber synapses. Proc Natl Acad Sci U S A 104:12181–12186.

ian J, Noebels JL (2005) Visualization of transmitter release with zincfluorescence detection at the mouse hippocampal mossy fibresynapse. J Physiol 566:747–758.

ian J, Noebels JL (2006) Exocytosis of vesicular zinc reveals persis-tent depression of neurotransmitter release during metabotropicglutamate receptor long-term depression at the hippocampal CA3-CA1 synapse. J Neurosci 26:6089–6095.

Development Core Team (2007) A language and environment forstatistical computing. Vienna, Austria: R Foundation for StatisticalComputing. ISBN 3-900051-07-0, URL http://www.R-project.org.

achline J, Perin-Dureau F, Le Goff A, Neyton J, Paoletti P (2005) Themicromolar zinc-binding domain on the NMDA receptor subunitNR2B. J Neurosci 25:308–317.

adhakrishnan K, Hindmarsh AC (1993) Description and use ofLSODE, the Livermore Solver for Ordinary Differential Equations.LLNL report UCRL-ID-113855, December 1993.

assendren FA, Lory P, Pin JP, Nargeot J (1990) Zinc has oppositeeffects on NMDA and non-NMDA receptors expressed in Xenopusoocytes. Neuron 4:733–740.

alazar G, Craige B, Love R, Kalman D, Faundez V (2005) Vglut1 andZnT3 co-targeting mechanisms regulate vesicular zinc stores inPC12 cells. J Cell Sci 118:1911–1921.

etzer RW (2006) Odesolve: solvers for ordinary differential equa-tions. R package version 0.5–16.

hen Y, Yang XL (1999) Zinc modulation of AMPA receptors may berelevant to splice variants in carp retina. Neurosci Lett 259:177–180.

indreu CB, Varoqui H, Erickson JD, Perez-Clausell J (2003) Boutonscontaining vesicular zinc define a subpopulation of synapses withlow AMPAR content in rat hippocampus. Cereb Cortex 13:823–829.

jostrom PJ, Turrigiano GG, Nelson SB (2003) Neocortical LTD viacoincident activation of presynaptic NMDA and cannabinoid recep-tors. Neuron 39:641–654.

loviter RS (1985) A selective loss of hippocampal mossy fiber Timmstain accompanies granule cell seizure activity induced by per-forant path stimulation. Brain Res 330:150–153.

mart TG, Hosie AM, Miller PS (2004) Zn2� ions: modulators of excitatoryand inhibitory synaptic activity. Neuroscientist 10:432–442.

piridon M, Kamm D, Billups B, Mobbs P, Attwell D (1998) Modulationby zinc of the glutamate transporters in glial cells and conesisolated from the tiger salamander retina. J Physiol 506( Pt 2):363–376.

toltenberg M, Nejsum LN, Larsen A, Danscher G (2004) Abundanceof zinc ions in synaptic terminals of mocha mutant mice: zinctransporter 3 immunohistochemistry and zinc sulphide autometal-lography. J Mol Histol 35:141–145.

un HS, Hui K, Lee DW, Feng ZP (2007) Zn2� sensitivity of high- andlow-voltage activated calcium channels. Biophys J 93:1175–1183.

uwa H, Saint-Amant L, Triller A, Drapeau P, Legendre P (2001)High-affinity zinc potentiation of inhibitory postsynaptic glycinergiccurrents in the zebrafish hindbrain. J Neurophysiol 85:912–925.

akeda A, Hirate M, Tamano H, Nisibaba D, Oku N (2003) Suscepti-bility to kainate-induced seizures under dietary zinc deficiency.J Neurochem 85:1575–1580.

raynelis SF, Burgess MF, Zheng F, Lyuboslavsky P, Powers JL(1998) Control of voltage-independent zinc inhibition of NMDA

receptors by the NR1 subunit. J Neurosci 18:6163–6175.

U

V

V

V

V

W

W

W

X

X

Z

P. Paoletti et al. / Neuroscience 158 (2009) 126–136136

eno S, Tsukamoto M, Hirano T, Kikuchi K, Yamada MK, NishiyamaN, Nagano T, Matsuki N, Ikegaya Y (2002) Mossy fiber Zn2�

spillover modulates heterosynaptic N-methyl-D-aspartate receptoractivity in hippocampal CA3 circuits. J Cell Biol 158:215–220.

alente T, Auladell C, Perez-Clausell J (2002) Postnatal developmentof zinc-rich terminal fields in the brain of the rat. Exp Neurol174:215–229.

andenberg RJ, Mitrovic AD, Johnston GA (1998) Molecular basis fordifferential inhibition of glutamate transporter subtypes by zincions. Mol Pharmacol 54:189–196.

ieles P, Frezou C, Galsomias J, Bonniol A (1972) Etude physico-chimique de la tricine et de ses complexes avec les ions detransition Co II, Ni II, Cu II, Zn II. J Chim Phys Physiochim Biol69:869–874.

ogt K, Mellor J, Tong G, Nicoll R (2000) The actions of synapticallyreleased zinc at hippocampal mossy fiber synapses. Neuron

26:187–196.

eiss JH, Sensi SL (2000) Ca2�-Zn2� permeable AMPA or kainatereceptors: possible key factors in selective neurodegeneration.Trends Neurosci 23:365–371.

enzel HJ, Cole TB, Born DE, Schwartzkroin PA, Palmiter RD (1997)Ultrastructural localization of zinc transporter-3 (ZnT-3) to synapticvesicle membranes within mossy fiber boutons in the hippocampusof mouse and monkey. Proc Natl Acad Sci U S A 94:12676–12681.

estbrook GL, Mayer ML (1987) Micromolar concentrations of Zn2�

antagonize NMDA and GABA responses of hippocampal neurons.Nature 328:640–643.

ie X, Smart TG (1994) Modulation of long-term potentiation in rat hip-pocampal pyramidal neurons by zinc. Pflugers Arch 427:481–486.

ie XM, Smart TG (1991) A physiological role for endogenous zinc inrat hippocampal synaptic neurotransmission. Nature 349:521–524.

heng F, Gingrich MB, Traynelis SF, Conn PJ (1998) Tyrosine kinasepotentiates NMDA receptor currents by reducing tonic zinc inhibi-

tion. Nat Neurosci 1:185–191.

(Accepted 8 January 2008)(Available online 15 February 2008)