Using a carbonic anhydrase-based biosensor, the concentration of loosely bound or free zinc (i.e., reactive zinc) was estimated to be 5–20 nM in the extracellular brain fluid (Frederickson et al., 2006b). Given that total zinc in the cerebrospinal fluid is about 150 nM (Hershey et al., 1983), this value suggests that most extracellular zinc also is tightly bound. Establishing the concentration of cytosolic reactive zinc in neurons has been hampered by the technical limitations of using fluorescent indicators for intracellular zinc measurements, as discussed elsewhere (Nolan and Lippard, 2009; Vinkenborg et al., 2010). Attempts in cortical neuron cultures using the dye mag-fura-5 suggested that cytosolic zinc may be found at subnanomolar levels (Sensi et al., 1997). Similar estimates have been obtained in non-neuronal cells with different indicators: ∼0.6 nM Zn²⁺ in HT293 cells by using FluoZin-3 (Krezel and Maret, 2006), and ∼0.4 nM Zn²⁺ in INS-1(832/13) β-cells by using eCALWYs FRET-based sensors (Vinkenborg et al., 2009). Together, the data suggest that the concentration of reactive zinc may be an order of magnitude lower in the cytosol compared with the extracellular brain space. This is noteworthy in view of the fact that the total intracellular zinc concentration largely exceeds that in the extracellular space (∼150 μM; Ehmann et al., 1984). Thus, reactive zinc in the cytosol may represent less than 0.001% of total cellular zinc. Attesting to the importance of controlling the levels of cytosolic zinc, prolonged increases or reductions in intra- or extracellular zinc can compromise neuronal viability (Canzoniero et al., 1999; Bozym et al., 2010).

How circulating zinc, previously absorbed in the gastrointestinal tract, enters the brain or CSF is poorly understood, although it appears to involve active transport mechanisms (Takeda, 2000). Likewise, the homeostatic mechanisms maintaining the zinc gradient across the plasma membrane in neurons are poorly defined, and currently none of the candidate proteins have been directly localized to synapses. The principal mechanism for zinc extrusion in neurons appears to be a secondary active transporter dependent on the Na⁺ and Ca²⁺ electrochemical gradients (Ohana et al., 2004; Qin et al., 2008), but the identity of this exchanger remains elusive. Given that neuronal activity elicits voltage-gated ion fluxes across the plasma membrane, such a zinc extrusion mechanism could potentially be subject to activity-dependent regulation. The zinc transporter ZnT-1 of the Slc30a family was the first plasma membrane protein found to regulate zinc extrusion (Palmiter and Findley, 1995). ZnT-1 is expressed in neurons and glia in several regions of the brain, including some that lack vesicular zinc (Sekler et al., 2002), and its genetic deletion is embryonic lethal (Andrews et al., 2004). Knock-down of ZnT-1 in cultured neurons decreases the rate of zinc efflux (Qin et al., 2009), but it is unclear if ZnT-1 affects the resting level of cytosolic zinc in the presence of an inwardly directed zinc gradient. ZnT-1-dependent zinc efflux is not affected by extracellular cations or ATP depletion, and it may involve oligomerization or association with other proteins (Palmiter and Findley, 1995). In addition, ZnT-1 suppresses the influx of other divalent cations via voltage-gated calcium channels (Ohana et al., 2006; Levy et al., 2009), suggesting additional roles besides zinc transport.

On the other hand, the ZIP-1 and -3 members of the Slc39a protein family have been implicated in the tonic import of extracellular zinc into neurons. ZIP-1 and -3 are highly expressed in CA1 hippocampus, and pyramidal neurons from double-knock-out mice show ∼ 50% reduction in the amount of extracellular zinc uptake (Qian et al., 2011). Assuming that zinc extrusion mechanisms are intact in this mutant mouse, the data indicate a major contribution of ZIP-1 and -3 to neuronal zinc import. The mechanism of zinc transport by ZIP proteins is not fully understood, although it was found to be energy-independent (Gaither and Eide, 2000). Evidence from the bacterial homolog ZIPB indicates that ZIP proteins may act as non-saturable zinc channels allowing passive zinc entry down its concentration gradient (Lin et al., 2010). ZIPB-dependent zinc entry increases with the proton concentration, in keeping with the effect of intracellular pH on neuronal zinc import (Colvin, 2002). Intriguingly, earlier studies found that uptake of Zn⁶⁵ into the neuropil was energy-dependent (Howell et al., 1984), implying that alternative or additional mechanisms may import zinc at synapses.

There is also an interest in the role that the small Zn/Cu-binding protein metallothionein-3 (i.e., MT-3) may play on cytosolic zinc homeostasis. MT-3 has many remarkable biochemical properties with regards to the binding and release of zinc (Maret, 2011), and MT-3 over-expression confers resistance to zinc toxicity in vitro (Palmiter, 1995). An elegant study showed that MT-3 may confer neuroprotection from excitotoxicity by releasing zinc following phosphorylation by PKC, leading to downstream gene expression (Aras et al., 2009). However, MT-3 KO mice thrive under normal conditions and seem to respond normally to systemic zinc overload (Erickson et al., 1997). Hence, the physiological role of neuronal MT-3 in zinc homeostasis is unclear. A safety mechanism that may help maintain the zinc gradient across the plasma membrane is the sequestration of excess zinc into lysosomes. Thus, in CA3 neurons, kainate-induced seizures trigger the accumulation of zinc into lysosomes (Hwang et al., 2008) as well as the up-regulation of the zinc importer ZIP-4 (Emmetsberger et al., 2010). Suggesting a possible link between the two, endocytosis inhibitors prevent the lysosomal degradation of surface ZIP-4 that occurs during zinc overload (Mao et al., 2007).

Biochemically isolated post-synaptic densities (PSD) contain a high amount of zinc (∼4 nmol/mg protein) that supports the structural organization of the PSD (Jan et al., 2002; Baron et al., 2006). Recent data indicate that at least a fraction of this zinc might be reactive or exchangeable, as it can be detected in association with Shank2/3 scaffold protein aggregates in dendritic spines with fluorescent zinc indicators (Grabrucker et al., 2011). In line with this, the same study also showed that zinc chelators can quickly alter the ultrastructural appearance of the PSD in cultured neurons.

Direct measurements of the concentration of reactive zinc in synaptic vesicles are lacking, but simple calculations indicate that it could easily reach high micromolar levels. A single zinc atom would result in a concentration of ∼40 μM within a 0.64 × 10⁻⁵ μm³ synaptic vesicle, which is the average vesicle volume in CA1 boutons (Schikorski and Stevens, 1997). The Neo-Timm’s staining method has been claimed to be able to detect as little as 3–4 zinc atoms (Danscher and Stoltenberg, 2006), which would translate into a minimal concentration of ≥150 μM vesicular zinc. Along these lines, a concentration of ∼1 mM vesicular zinc was inferred by extrapolating from the amount of total zinc found in the hilar region of the hippocampus (Frederickson et al., 1983). On balance, it appears that reactive zinc is several-fold more concentrated within synaptic vesicles than in the cytosol or the extracellular space. The different concentrations likely account for the preferential labeling of synaptic vesicles with zinc probes. Intra-vesicular pH may also affect zinc speciation, releasing it from protein complexes therein and making it more reactive to zinc probes (Vinkenborg et al., 2009). By comparison, the concentration of glutamate in synaptic vesicles ranges from 60 to 210 mM (Clements, 1996). Interestingly, studies in PC12 cells indicate a functional coupling between the vesicular uptake of zinc and glutamate; recombinant murine VGLUT-1 and ZnT-3 were targeted to the same population of synaptic-like microvesicles and reciprocally facilitated their transport activities, perhaps through effects on the transvesicular electrochemical gradient (Salazar et al., 2005). In keeping with current estimates of the cytosolic zinc concentration, ZnT-3 enhanced VGLUT-1-mediated glutamate uptake at low nanomolar zinc levels. Whether ZnT-3 and VGLUT-1 cooperate in a similar manner in synaptic vesicles of the brain has not been examined. However, destroying the vesicular proton gradient with the H⁺-ATPase inhibitor concanamycin A, which prevents glutamate uptake, also reduces pre-synaptic fluorescence of the zinc indicator Zinpyr-1 at putative single MFs (Ketterman and Li, 2008). Intriguingly, functional differences between human and mouse ZnT-3 have been reported. Thus, human, but not mouse, ZnT-3 tends to form dimmers that promote vesicular targeting and stimulate zinc uptake (Salazar et al., 2009).

The concentration of zinc in synaptic vesicles has led to the natural hypothesis that zinc may be co-released with glutamate into the synaptic cleft following vesicular exocytosis. Evidence that neuronal depolarization increases the extracellular level of elemental zinc, either in vivo or in vitro, and in parallel with glutamate, is overwhelming (Howell et al., 1984; Charton et al., 1985; Aniksztejn et al., 1987; Minami et al., 2002). However, no study has yet confirmed the synaptic vesicle origin of this zinc by performing similar experiments in ZnT-3 knock-out mice (which lack any reactive zinc within synaptic vesicles; Cole et al., 1999; Linkous et al., 2008). Other studies have addressed the issue of vesicular zinc release by stimulating brain slices in the presence of fluorescent zinc indicators. Thus, action potential-evoked zinc exocytosis has been reported in CA3 and CA1 synapses by detecting changes in FluoZin-3 fluorescence, an extracellular zinc dye (Qian and Noebels, 2005, 2006; Carter et al., 2011). Importantly, zinc release was found to be highly sensitive to changes in release probability, dependent on vesicular exocytosis, and it was ablated in ZnT-3 KO mice. These are remarkable observations considering the potential limitations of the approach (e.g., ex vivo zinc depletion, inadequate metal contamination or temperature, small fractional volume of the synaptic cleft, optical sensitivity, perturbation of zinc kinetics by the indicator, etc.). Indeed, mixed results have been obtained when using maximal stimulation to compensate for low optical resolution (Li et al., 2001b; Kay, 2003; Datki et al., 2007). The extracellular zinc increase associated with exocytosis should be paralleled by a corresponding decrease in vesicular zinc, and there are some zinc fluorescence measurements from MFs suggesting that this is also the case (Ketterman and Li, 2008). The results suggest a model where released zinc may diffuse into the synaptic cleft, transiently increasing its extracellular concentration in an activity-dependent manner. In keeping with this, vesicular zinc release has been shown to modulate post-synaptic receptors at the MF synapse (Vogt et al., 2000; Pan et al., 2011, see section below). Even more strikingly, intracellular zinc increases in post-synaptic neurons were recently reported following intense axonal depolarization (Suh, 2009; Carter et al., 2011). Again, ZnT-3 deletion abolished the post-synaptic zinc transient, suggesting that vesicular zinc can be trans-synaptically mobilized under some conditions. One intriguing observation from these experiments is the slow decay of the extracellular zinc transient that is triggered by exocytosis. Whether this is a property of individual synapses is still unknown, as zinc transients were not induced with minimal stimulation protocols to activate single MF boutons. The factors governing the synaptic diffusion and clearance of released zinc are not understood. It has been proposed that vesicular zinc may remain bound to synaptic proteins after exocytosis, although the putative synaptic transporters or proteins that may act as fixed buffers are undefined (Vogt et al., 2000; Kay, 2003). It has also been proposed that the high tortuosity of the MF synapse may delay the clearance of zinc (Qian and Noebels, 2005). Direct comparisons of the fluorescence kinetics shown by extracellular indicators for zinc and glutamate (Hires et al., 2008) after release have not been reported.

Another potential source of synaptic zinc is that originating from mitochondria. Virtually all MF terminals and about half of small pre-synaptic boutons contain at least one mitochondrion (Ventura and Harris, 1999; Chang et al., 2006), where it can modulate synaptic plasticity by handling Ca²⁺ and supplying ATP (Cai et al., 2011). Recent evidence in neurons indicates that resting mitochondria may harbor two to threefold higher concentrations of reactive zinc relative to the cytosol (Dittmer et al., 2009). Zinc appears to access the mitochondrial matrix mainly via the Ca²⁺ uniporter, a process dependent on the mitochondrial membrane potential (Malaiyandi et al., 2005; Caporale et al., 2009). The mechanism whereby mitochondrial zinc is extruded is unknown, as no zinc transporters have been localized to mitochondria. In vitro data suggest that large intracellular Ca²⁺ increases, often associated with neuronal injury, may possibly couple to mitochondrial zinc release into the cytosol (Sensi et al., 2003; Dineley et al., 2008; Dittmer et al., 2009).

The synaptic accumulation of reactive zinc and its involvement in several forms of memory suggest a role(s) for zinc in synaptic physiology. Zinc chelators or ZnT-3 deletion do not alter fast excitatory, AMPAR-mediated synaptic transmission evoked by low-frequency stimulation in brain slices (Li et al., 2001a; Lopantsev et al., 2003; Kodirov et al., 2006; Mott et al., 2008; Pan et al., 2011). However, a number of synaptic receptors are known to contain binding sites with nanomolar affinity for extracellular zinc, raising the possibility that zinc modulates their activity in a tonic or phasic manner. In keeping with this, chelation of extracellular zinc increases GABA_AR-, NMDAR-, and kainate receptor-mediated currents at MF synapses (Vogt et al., 2000; Molnar and Nadler, 2001; Ruiz et al., 2004; Mott et al., 2008), potentiates ASIC-mediated currents in cortical neurons (Chu et al., 2004), and reduces glycinergic currents in hypoglossal neurons of the brainstem (Hirzel et al., 2006). Zinc inhibition of NMDARs has been greatly examined in heterologous expression systems and found to operate via two mechanisms: one that is low-affinity and voltage-dependent, and another one that is high-affinity and voltage independent (Paoletti et al., 1997; Traynelis et al., 1998; Choi and Lipton, 1999). Importantly, knock-in mice with point mutations previously implicated in zinc binding to the α1 glycine receptor 1 subunit (Hirzel et al., 2006) or the GluN2A NMDAR subunit (Nozaki et al., 2011) display sensorimotor deficits and impaired pain processing, respectively. Likewise, high-affinity zinc inhibition of Ca_v3.2 channels may limit hyperalgesia in peripheral neurons (Nelson et al., 2007). These findings highlight the importance of zinc modulation of ion channels in clinically relevant responses. However, few of the above studies have examined the origin of the zinc that binds to these ion channels. Using glutamate iontophoresis in CA3 neurons, it has been shown that the increase in NMDAR currents caused by zinc chelators is abolished in ZnT-3 KO mice and is occluded by an increase of similar magnitude triggered by briefly blocking pre-synaptic exocytosis (Vogt et al., 2000). Analogously, zinc chelation no longer enhances kainate receptors in mocha mice with reduced vesicular zinc (Mott et al., 2008). These data indicate that, at least at MFs, tonic vesicular zinc release modulates post-synaptic receptors. At variance with this conclusion, post-synaptic NMDAR currents were found to be of similar amplitude in ZnT-3 KO and control CA3 neurons, although in this case the differential effect of zinc chelation between genotypes or the presence of contaminant zinc was not considered (Lopantsev et al., 2003). Perhaps more importantly, little is known as to the impact of extracellular zinc binding to synaptic ion channels on mnemonic processes or neuronal plasticity. The recent development of GluN2A knock-in mice (Nozaki et al., 2011), which express NMDARs insensitive to nanomolar zinc (H128S substitution) but are presumably still inhibited by higher zinc concentrations, will help establish the role of high-affinity zinc binding on NMDAR-dependent forms of learning and synaptic plasticity.

Ideally, one would like to know the effect of synaptic zinc on synaptic responses elicited by the behavior of interest or to the sort of spike patterns associated with that behavior. This information is not yet available at the electrophysiological level. Converging data from three types of zincergic synapses (the MF-to-CA3, the CA3-to-CA1, and the auditory cortex-to-lateral amygdala synapse) indicate that endogenous zinc is required for the induction of long-term potentiation (i.e., LTP) of AMPA currents, a candidate cellular correlate of learning. Whereas post-synaptic mechanisms have been strongly implicated in the expression of LTP at CA3-to-CA1 synapses, MF-to-CA3 plasticity appears to largely rely on pre-synaptic increases in release probability (Nicoll and Schmitz, 2005). Thus, Lu et al. (2000) showed that the cell-permeable zinc chelators dithizone and DEDTC reversibly block MF LTP. Along these lines, CaEDTA (7.5–10 mM) was also shown to block the induction of MF LTP by two other laboratories (Li et al., 2001a; Huang et al., 2008). Notably, lower CaEDTA concentrations (1–2 mM) or partial (∼50%) removal of vesicular zinc by mutation of the AP-3 δ subunit (Seong et al., 2005) fail to inhibit MF LTP (Lu et al., 2000; Vogt et al., 2000; Li et al., 2001a). In contrast, low concentrations of CaEDTA (1–2 mM) or TPEN (0.1 μM) suffice to inhibit LTP at CA1 and lateral amygdala synapses (Izumi et al., 2006; Kodirov et al., 2006; Takeda et al., 2009). The surprising observation here is that chelator-induced LTP block appears to be complete in CA1 and amygdala, even though only a fraction of their synapses react for vesicular zinc. Paralleling the level of zinc chelation required to affect LTP, application of a high zinc concentration (50–100 μM) is needed to potentiate MF synapses (Li et al., 2001a; Huang et al., 2008), whereas a lower zinc concentration (1–10 μM) enhances LTP in CA1 (Izumi et al., 2006; Takeda et al., 2009; Lorca et al., 2011). Importantly, none of the above studies observed an effect of zinc chelation on basal transmission, paired-pulse modulation, or LTP maintenance, suggesting a role for synaptic zinc in the induction of long-term plasticity. Despite this emerging consensus, not all data are concordant, and others have failed to block MF LTP when using TPEN (20–100 μM; Matias et al., 2006). Collectively, it could be argued that the differential sensitivity to zinc manipulation among synapses grossly correlates with the amount of vesicular zinc that they accumulate. So what, then, is the effect of completely (and selectively) removing vesicular zinc on synaptic plasticity? Surprisingly, only one recent study examined long-term synaptic plasticity in ZnT-3 KO mice, and obtained both confirmatory and unexpected results (Pan et al., 2011). On one hand, the increase in release probability normally observed following tetanus-induced MF LTP was absent in ZnT3 KO mice, confirming a role for vesicular zinc in pre-synaptic plasticity. However, MF LTP was still observed in ZnT3 KO mice, and this was found to be due to disinhibiton of a separate, post-synaptic calcium-dependent mechanism of MF LTP that is normally blocked by zinc in wild type mice (Pan et al., 2011). Thus, in the presence of vesicular zinc, MF LTP is expressed via a pre-synaptic mechanism, but when vesicular zinc is removed, a post-synaptic mechanism is recruited instead, indicating a dual role for zinc. Similar conclusions were reached when using a new, fast-acting extracellular zinc chelator, ZX1. Importantly, both pre- and post-synaptic components of MF LTP were observed in the presence of NMDAR antagonists, differentiating them from a recently described form of MF LTP affecting NMDAR currents (Kwon and Castillo, 2008; Rebola et al., 2008). LTP at other synapses in ZnT3 KO mice remains to be examined. Regarding synaptic plasticity in ZnT3 KO mice, two reported observations require clarification: first, and in contrast to ZX1-perfused wild type mice, post-synaptic calcium chelation does not fully block MF LTP in ZnT3 KO mice; secondly, a basal increase in the frequency of spontaneous MF glutamate release in ZnT3 KO mice has been shown (Lopantsev et al., 2003; Pan et al., 2011). Inducible downregulation of ZnT3 in adult brain will help determine whether these differences reflect homeostatic compensations to gene deletion.

Conclusions on the effects of CaEDTA in LTP are somewhat confounded by its properties and should be confirmed in the future with more selective extracellular zinc chelators, such as Tricine (Paoletti et al., 2009) or ZX1. Given the high-affinity constant of CaEDTA for zinc (K_D = 10⁻¹⁶), its concentration-dependent effects have been attributed to the slow kinetics of zinc chelation, which are largely dictated by the slow off-rate for calcium. Thus, low CaEDTA would remove extracellular zinc at equilibrium but it would fail to intercept much of synaptically released zinc. On the other hand, concentrations of CaEDTA higher than 5 mM (i.e., those that block MF LTP) have been shown to reduce the vesicular zinc content under steady-state conditions (Lavoie et al., 2007; Ketterman and Li, 2008). As CaEDTA is negatively charged at physiological pH and hence unlikely to cross the plasma membrane, the intracellular zinc reduction may reflect a homeostatic response to extracellular chelation of zinc or other cations. It is therefore unclear if high CaEDTA blocks MF LTP by chelating zinc before or following its actual release. The extracellular specificity of alternative zinc chelators may be assessed by quantifying the extent of vesicular zinc depletion when blocking pre-synaptic exocytosis in the presence of the chelator. Future studies should also address the importance of intracellular zinc chelation in pre- and post-synaptic compartments on LTP.

Both learning and repetitive synaptic activation stimulate intracellular signal transduction cascades necessary for memory formation and synaptic plasticity. The involvement of synaptic zinc in some forms of memory and LTP hence suggests that zinc may participate in the mechanisms of neuronal signal transduction. Indeed, three independent groups have recently implicated endogenous zinc in the activation of intracellular signal cascades at MFs (Figure 3; Huang et al., 2008; Besser et al., 2009; Chorin et al., 2011; Sindreu et al., 2011). MF tetanization or direct zinc application (100 μM) in the presence of NMDAR antagonists induces a form of LTP that requires TrkB activity, and is abrogated by CaEDTA (Huang et al., 2008). Whether this type of LTP requires a cAMP-dependent increase in the probability of glutamate release as in classic MF LTP_AMPA has not been shown yet. Providing a possible mechanism, zinc application to cultured neurons activates the cytosolic tyrosine kinase Src, increasing phospho-activation of the TrkB receptor in the absence of its ligand BDNF. It had been previously suggested that zinc activation of TrkB operates through the conversion of pro-BDNF to mature BDNF (Hwang et al., 2005). However, the fact that zinc still potentiates MFs in BDNF KO mice suggests that such mechanism might not be essential. Downstream signals, including the Erk1/2 MAP kinase and PLCγ1 pathways, are also activated by zinc in cultures. Although it remains to be shown that synaptic zinc activates Src–TrkB signaling during induction of this form of MF LTP, the data provide circumstantial evidence that this may be the case. TrkB activation is most prominent in axon terminals (Spencer-Segal et al., 2011), whereas the Src kinase accumulates in NMDAR complexes (Salter and Kalia, 2004). Hence, the pre- or post-synaptic MF localization of this pathway and the route(s) of zinc entry remain to be clarified. Since primary neuron cultures typically fail to stain for vesicular zinc (Li et al., 2003; Love et al., 2005; Grabrucker et al., 2011), these issues may need to be addressed in more intact preparations.

Exogenous zinc application (100 μM) has also been reported to increase phosphorylation of Erk1/2 in CA3 somata of young (P8-P16) mice, an effect that is prevented by the Gαq inhibitor YM-254890 (Besser et al., 2009). Interestingly, brief MF tetanization (10 pulses at 66 Hz) triggers a Gq–dependent post-synaptic calcium increase that is reduced by half in ZnT-3 KO mice, suggesting that synaptic zinc can contribute to G protein-coupled receptor (GPCR) signaling even at a developmental age when synaptic vesicles are only partially stocked with zinc (Frederickson et al., 2006a). Furthermore, zinc-induced Gq signaling leads to rapid Erk-dependent up-regulation of KCC2, a K⁺/Cl⁻ co-transporter, increasing the transmembrane Cl⁻ gradient in CA3 neurons (Chorin et al., 2011). Because KCC2 up-regulation is absent in GPR39 KO mice, this orphan receptor might mediate the metabotropic actions of synaptic zinc. Consistent with this, GPR39 contains extracellular domains that coordinate zinc (Popovics and Stewart, 2011). Interestingly, Erk1/2 activation via TrkB also up-regulates KCC2 as long as PLCγ1 is not co-activated (Rivera et al., 2004). Future studies may clarify the significance of these converging signals to adult synaptic plasticity or memory formation. Intriguingly, similar tetanization regimes and molecular determinants have been associated with the induction of MF LTP_NMDA (Kwon and Castillo, 2008; Rebola et al., 2008). In this regard, another study showed that MF stimulation at 50 Hz triggers a form of short-term plasticity (∼10 min-long) that activates, and is modulated by, the pre-synaptic Erk1/2-Synapsin pathway (Vara et al., 2009). Synapsin is a protein that tethers synaptic vesicles to the actin cytoskeleton to modulate transmitter release (Fdez and Hilfiker, 2006), and shown to interact with the plasticity-related protein Rab3A (Giovedi et al., 2004). Suggesting some behavioral relevance for the above observations, dentate gyrus granule cells can fire in bursts of up to 50 Hz when the animal visits a location corresponding to the place field center of the cell (Jung and Mcnaughton, 1993). More recently, it was found that contextual conditioning induces the pre-synaptic activation of the Erk1/2-Synapsin I pathway at MFs (Sindreu et al., 2011). Furthermore, training-induced activation of Erk1/2 is selectively ablated in MFs of ZnT-3 KO mice; partial inhibition of Erk1/2 restricted to granule cells causes a similar deficit in contextual discrimination as that observed in ZnT-3 KO mice. These data represent the first direct evidence that vesicular zinc regulates neuronal signal transduction associated with memory formation. Mechanistically, ZnT-3 KO mice show disinhibition of MAPK tyrosine phosphatases, suggesting that it may underlie impaired Erk activation. Consistent with this, the authors showed that activity-dependent zinc release couples to phosphatase inhibition and Erk1/2 stimulation at MF terminals. The inhibition of MAPK phosphatases by zinc is in keeping with previous observations in neuron cultures (Ho et al., 2008). Because direct Erk1/2 inhibition has been shown to completely block glutamate release (Jovanovic et al., 2000), zinc inhibition of MAPK phosphatases may regulate other pre-synaptic proteins in addition to Synapsin I. At present, it is unclear how zinc may be recycled back into the pre-synaptic terminal to inhibit phosphatases. The identity of the zinc-sensitive MAPK phosphatase also remains to be shown. Suggesting an important role for tyrosine phosphatases in Erk1/2 regulation, catalyzed dephosphorylation of the tyrosine residue (Y185) appears to be the initial and rate-limiting step in Erk1/2 inactivation (Zhou et al., 2002). Previous studies indicated that zinc can inhibit tyrosine phosphatases with high-affinity by binding to their conserved catalytic domain (Brautigan et al., 1981; Haase and Maret, 2003; Redman et al., 2009). How, then, synaptic zinc regulates select signaling pathways via phosphatase inhibition is an intriguing question.

The aggregate evidence indicates that synaptic zinc is a positive regulator of Erk1/2 at MF synapses, activating it through one or more mechanisms (Figure 3). Whereas zinc-dependent Gq signaling may not require zinc influx, released zinc would need to gain access into the cytosol regulate Src or MAPK phosphatases. At MFs, voltage-gated calcium channels represent the main route for pre- and post-synaptic entry of divalent cations (Reid et al., 2001), and calcium channel inhibition suppresses pre-synaptic Erk1/2 at MFs (Sindreu et al., 2011). In addition, currently undefined zinc importers or co-transporters could promote (re-)uptake of extracellular zinc. Whether, similarly to Erk1/2, activation of TrkB or Gq signaling specifically in granule cells or CA3 neurons supports hippocampus-dependent memory is still not known. Also, the possibility of cross-talk between these zinc-regulated pathways has not been examined. For instance, although basal TrkB activation is normal in ZnT3 KO mice (Sindreu et al., 2011), it is unclear if TrkB further stimulates Erk1/2 during learning. Conclusively establishing the pre- or post-synaptic localization of these various pathways will help draw connections among them. Mechanistically, zinc-activated intracellular cascades will need to be put in context with the previous molecules implicated in MF plasticity, such as the adenylyl cyclases and their downstream targets (Nicoll and Schmitz, 2005). A large body of literature indicates that the Erk1/2 pathway (i.e., Ras–b–Raf–MEK–Erk) acts as a node integrating several upstream signals to control memory and neuronal plasticity, mainly through actions on dendritic proteins and gene transcription (Impey et al., 1999; Thomas and Huganir, 2004; Ye and Carew, 2010). However, there is also evidence that Erk1/2 affects cognition via pre-synaptic mechanisms (Kushner et al., 2005; Cui et al., 2008). Circumstantially, zinc can have opposite effects on MAPK in other cellular models. For instance, zinc can decrease Erk signaling in oocytes by inhibiting Raf, an effect that may be counteracted by ZnT-1 (Jirakulaporn and Muslin, 2004). It remains to be explored if reactive zinc can bidirectionally modulate Erk signaling by engaging various mechanisms in neurons.

A pertinent question is to what extent the effects of synaptic zinc on ion channels and signal transduction cascades are functionally related, and whether they co-occur at individual synapses. At MFs, the effects of zinc on Gq-, TrkB-, and Erk-dependent signaling are all spared by NMDAR blockers, in line with the ability of this synapse to express NMDAR-independent forms of plasticity. Moreover, the effects of synaptic zinc on Erk1/2 activation or TrkB-dependent plasticity are not reproduced at CA1 synapses (Huang et al., 2008; Sindreu et al., 2011), possibly as a result of molecular heterogeneity between synapses. In contrast, blockade of CA1 LTP by zinc chelators has been attributed to the untimely activation (due to disinhibition) of NMDARs upstream of calcineurin, nitric oxide, and p38 (Izumi et al., 2006, 2008). Whether NMDAR-dependent forms of plasticity activate other downstream signals that regulate, or are regulated by, reactive zinc is unclear. CaMKII, PKA, PKC, or Akt are normally activated in ZnT-3 KO hippocampus (Sindreu et al., 2011). However, in vitro studies have shown that zinc unbinding from PKC can trigger the activation of this kinase (Knapp and Klann, 2000; Korichneva et al., 2002), and that constitutively active PKC is, in turn, sufficient to increase the expression of genes containing metal regulatory elements (Aras et al., 2009). Thus, PKC activation during synaptic plasticity could potentially trigger intracellular zinc signaling. The effect of acutely chelating reactive zinc from brain tissue on these pathways remains to be shown. On the other hand, block of LTP at cortico-amygdala synapses by zinc chelators can be rescued by GABA_AR antagonists (Kodirov et al., 2006), implying that zinc can gate LTP via heterosynaptic inhibition of feed-forward interneurons. The authors proposed that such a mechanism may provide spatial control of amygdala LTP between cortical and thalamic afferents.

Future experiments examining how these, and other, mechanisms of zinc action affect cognition are warranted. The deficits in synaptic plasticity and biochemical activation induced by zinc blockade are in agreement with the memory impairments observed in ZnT-3 KO mice or after zinc chelation. While the ZnT-3 KO mouse model has now provided strong evidence for a role of vesicular zinc in cognition, circumstantial evidence suggests that other sources of reactive zinc may also be involved. Altogether, the data we have discussed here lend further support to the hypothesis that synaptic zinc may promote learning and memory through actions on neuronal signal transduction. Traditionally viewed as an inhibitor of glutamatergic transmission, synaptic zinc may turn out to play complementary roles with glutamate in the service of memory and neuronal plasticity: zinc may recruit additional plasticity mechanisms during increased neuronal firing associated with learning, prevent tonic interference from other signaling pathways, or select between converging synaptic contacts to promote memory formation. Many of the recent findings on synaptic zinc had not been anticipated only 5 years ago. Should this trajectory continue, the next few years will reveal interesting new facets of zinc signaling in the brain.