Synaptic Zn²⁺ Inhibits Neurotransmitter Release by Promoting Endocannabinoid Synthesis

Slice preparation

Experiments were conducted according to the methods approved by the Institutional Animal Care and Use Committee of the University of Pittsburgh. Coronal brainstem slices were prepared from either sex ICR, mZnR wild type (mZnR WT) and knockout (mZnR KO), and ZnT3 wild type (ZnT3 WT) and knockout (ZnT3 KO) mice (P17–P25). The preparation of coronal slices containing DCN has been described in detail (Tzounopoulos et al., 2004).

Electrophysiology

Fluorescence Imaging

For Ca²⁺ imaging, slices were loaded with Fura-2 AM (25 μM, TefLabs) for 20 min. at room temperature, in the presence of 0.02% pluronic acid, and then washed in ACSF for at least 20 min (Besser et al., 2009). For extracellular Zn²⁺ imaging, Newport Green (2 μM) was added to the slice. Slices were then loaded in a microscope chamber and perfused with PO₄-free ACSF to avoid Zn²⁺ precipitation (Chorin et al., 2011). Fluorescent imaging measurements were acquired every 3 s (Imaging Workbench 4, INDEC BioSystems; and polychrome monochromator, TILL Photonics) using a 10X objective (Olympus BX51) with 4 × 4 binning of the image (SensiCam, PCO). Regions of interest (ROI) were chosen throughout the DCN molecular layer that was not covered by the stimulating electrode, and traces from 4 ROIs were averaged for each slice. Cell bodies are surrounded by our selected ROIs, thus, the observed Fura-2 fluorescence changes mostly represent the result of Ca²⁺ rises in neuronal cell bodies. Fura-2 fluorescence signals are represented as a ratio of the signal obtained using excitation of 340 nm/380 nm wavelengths and a 510 nm emission bandpass filter (Chroma Technology). Newport Green fluorescent signals were normalized to the initial fluorescent level (F₀) and the signal is represented as F/F₀. Bar graphs represent the averaged difference in the fluorescent signal averaged over n slices as indicated. The stimulating electrode was placed on the molecular layer of the DCN and a train of 100 pulses at 100 Hz was applied (Master-8 stimulator unit, A.M.P.I.). When required, the initial baseline was used to calculate a linear regression curve that was subtracted from the trace.

2-AG quantification

A modified isotopic dilution liquid chromatography/tandem mass spectrometry (LC/MS/MS) method (Zhang et al., 2010) was used to determine 2-AG in dorsal cochlear nucleus slices. DCN slices were incubated at 37° C for ~30 min to allow the tissue to recover after the cutting procedure (similarly to electrophysiological experiments). 2-AG concentrations were measured in control samples and samples exposed to Zn²⁺ (100 μM, 5 min) in the presence or absence of U73122 (5μM). In experiments in which U73122 was used, slices were pretreated with this drug or vehicle for 30 min. After this pretreatment, both control and treated slices were transferred to a chamber with either normal solution or Zn²⁺ containing solution for 5 min (U73122 was also present during the 5 min treatment). Samples were then immediately flash-frozen and stored in liquid nitrogen. The tissues were later thawed and homogenized in 20 μl of H₂0 with 0.02 % trifluoroacetic acid and 2 μl aliquots were used for protein concentration determinations using the Bradford method. Then, 180 μl cold acetonitrile was added to the samples previously spiked with 9 pmoles 2-AG-d8 internal standard and followed by homogenization. The samples were centrifuged at 14000g for 10 min at 4°C and the supernatant transferred into vials. Brain homogenates at a 5 mg tissue/ml were used to perform quantification quality controls by spiking samples with internal standard and 300 pmol of 1-AG and 2-AG per mg of tissue. Samples were thawed and homogenized in the presence of deuterated 2-AG internal standard, thus controlling for any loss during sample work-up. The internal standard corrects for losses due to degradation, extraction efficiency, binding to silica present in the glass tubes, gas phase equilibrium and ionization efficiencies. The concentration of 2-AG was normalized to amounts of DCN protein to avoid the errors introduced by handling small amounts of tissue per DCN slice (typically below 1 mg).

Experiments with KO mice

Imaging and biochemical experiments were blinded (Fig. 4, 8). Electrophysiological experiments were partially blinded: initial experiments were not blinded (approximately half n), but all subsequent experiments were blinded (Fig. 11). As we did not observe any differences in the two data sets, we have included all these experiments.

Drugs and KO mice

SR95531, NBQX disodium salt, QX-314, AM251, TTX, WIN55, 212-2 mesylate (WIN), were purchased from Ascent Scientific. ZnCl₂, strychnine, BAPTA, GDP-β-S, tricine, and tetrahydrolipstatin (THL) were purchased from Sigma-Aldrich. The 2-AG, 1-AG and ²H₈ 2-AG standards were purchased from Cayman Chemical (Ann Arbor, MI). U73122 was purchased from Tocris Cookson. Fura-2AM and Newport Green were purchased from TefLabs and Invitrogen, respectively. GPR39/mZnR KO mice were kindly provided by D. Moechars from Janssen, Pharmaceutical companies of Johnson & Johnson. ZnT3 KO mice were purchased from Jackson.

Zn²⁺ Decreases Probability of Release via Activation of Endocannabinoid Signaling

To investigate the effects of Zn²⁺ on parallel fiber excitatory postsynaptic current (EPSC) strength, we obtained whole-cell recordings from DCN principal neurons (fusiform cells). Bath application of Zn²⁺ (100 μM, 5 min) significantly decreased EPSC amplitude (Fig.1A). The inhibitory effect of Zn²⁺ on EPSC strength was reversed upon Zn²⁺ removal, suggesting that extracellular Zn²⁺ mediates a short-lasting decrease in synaptic strength (Fig.1A). The observed Zn²⁺-induced decrease in synaptic strength is due to pre- and or postsynaptic mechanisms. To investigate whether the effects of Zn²⁺ on synaptic strength are mediated by presynaptic mechanisms, we used two assays that are sensitive to changes in presynaptic neurotransmitter release (Pr): paired-pulse ratio (PPR) and coefficient of variation (CV) analysis (Faber and Korn, 1991; Tsien and Malinow, 1991). Paired-pulse facilitation, an increased second response to two stimuli applied in rapid succession, is thought to reflect an increase in Pr. The coefficient of variation (CV) is the standard deviation of the EPSC amplitudes normalized to the mean amplitude and varies oppositely with quantal content; the inverse square, CV^-2, is directly proportional to quantal content (quantal content = n p, where n = number of release sites and p = Pr). Application of Zn²⁺ increased PPR and decreased 1/CV² both of which indicate a decrease in Pr (Fig. 1 B, C). Baclofen (2-5 μM), which is known to inhibit synaptic strength via a presynaptic mechanism (Dutar and Nicoll, 1988), caused a similar increase in PPR and a similar reduction in 1/CV², indicating that PPR and CV assays are highly sensitive to changes in Pr (Fig. 1B, C). Conversely, 0.5 μM NBQX (2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione), which reduces synaptic strength by partially blocking postsynaptic glutamate receptors, left PPR and 1/CV² unaffected, thus confirming that PPR and CV assays are not sensitive to changes in synaptic strength that are mediated by postsynaptic manipulations (Fig.1B, C). Consistent with these results, application of Zn²⁺ decreased miniature EPSC (mEPSC) frequency without affecting mEPSC amplitude (Fig. 1D). Taken together, these findings show that the Zn²⁺-induced EPSC depression is mediated by a decrease in Pr.

Given the role of endocannabinoid retrograde signaling in determining the Pr of DCN parallel fiber synapses (Tzounopoulos et al., 2007; Tzounopoulos and Kraus, 2009; Sedlacek et al., 2011; Zhao et al., 2011; Zhao and Tzounopoulos, 2011), we tested whether endocannabinoids are involved in Zn²⁺-mediated modulation of Pr. We found that the specific cannabinoid receptor 1 (CB1R) antagonist AM-251 (1μM) prevented Zn²⁺-mediated EPSC depression and the concomitant changes in PPR and 1/CV² (Fig. 2A-C). Bath application of AM-251 (1 μM) did not affect baseline synaptic transmission, indicating lack of tonic activation of CB1Rs and lack of tonic endocannabinoid signaling in modulating baseline Pr (Fig. 2D). These results suggest that Zn²⁺- dependent endocannabinoid signaling mediates a Pr reduction, thus revealing an unexpected link between Zn²⁺ and endocannabinoid signaling.

Endocannabinoid modulation of Pr has both postsynaptic (endocannabinoid synthesis and release) and presynaptic (activation of cannabinoid receptors, CB1Rs) components (Alger, 2012). Therefore, we investigated whether Zn²⁺-dependent modulation of Pr is mediated through direct presynaptic activation or allosteric modulation of CB1Rs, or through initiation of a postsynaptic signaling cascade leading to endocannabinoid synthesis and release. Intracellular application of the calcium chelator BAPTA (10 mM) in fusiform cells blocked Zn²⁺-induced depression of EPSCs and the concomitant changes in PPR and 1/CV², indicating that an increase in postsynaptic Ca²⁺ is necessary for Zn²⁺-mediated modulation of Pr (Fig. 3A-C). While BAPTA also chelates intracellular Zn²⁺ (Hyrc et al., 2000), subsequent experiments using the extracellular zinc chelator tricine (e.g. see Figs. 4, 9 and 11) are consistent with a lack of dependence of our proposed mechanism on an intracellular Zn²⁺ increase. In addition, loading of fusiform cells with GDP-β-S (0.5 mM), a nonhydrolyzable analog of GDP, blocked the effects of Zn²⁺ on EPSC amplitude, PPR and 1/CV², suggesting that postsynaptic activation of G-protein-coupled receptors (GPCRs) mediate Zn²⁺-induced activation of endocannabinoid signaling (Fig. 3A-C; see also figure legend for control experiments on the lack of effects on GDP-β-S on baseline synaptic transmission). Given that endocannabinoid synthesis from the postsynaptic neuron can be induced by elevations in intracellular Ca²⁺, by activation of GPCRs, or by concerted activation of GPCRs and elevations in intracellular Ca²⁺ (Wilson and Nicoll, 2001; Kim et al., 2002; Zhao and Tzounopoulos, 2011), our results indicate that Zn²⁺ likely promotes endocannabinoid production through a postsynaptic activation of GPCRs and a rise in intracellular Ca²⁺.

Zn²⁺-Induced Decrease in Pr Is Mediated by a mZnR-dependent Increase of 2-AG Synthesis

Since endocannabinoid production is tightly linked to G_q-coupled receptors in the DCN (Zhao and Tzounopoulos, 2011), and because the previously orphan G_q-coupled receptor 39 (GPR39) has emerged as a putative metabotropic Zn²⁺-sensing receptor (mZnR) (Besser et al., 2009), we evaluated whether mZnR activation is involved in the Zn²⁺-triggered promotion of endocannabinoid production in the DCN. As activation of mZnRs is thought to lead to Zn²⁺-mediated increases in intracellular Ca²⁺ in the hippocampus and in cortical neurons (Besser et al., 2009; Saadi et al., 2012; but see Evstratova and Toth, 2011), we determined whether synaptically released Zn²⁺ leads to an mZnR-mediated increase in intracellular Ca²⁺ in the DCN. We measured intracellular Ca²⁺ responses in the fusiform cell layer of DCN slices from wild type (WT) and from genetically modified mice lacking GPR39 (Moechars et al., 2006) (mZnR KO mice). While parallel fiber stimulation (100 Hz, 1s) caused robust intracellular Ca²⁺ responses in slices from mZnR WT mice, measurements from littermate mZnR KO mice displayed significantly reduced Ca²⁺ responses (Fig. 4A). To determine whether reduced Ca²⁺ responses in mZnR KO mice are due to the lack of Zn²⁺-triggered, mZnR mediated signaling, we measured the sensitivity of the Ca²⁺ responses to tricine: an extracellular Zn²⁺ chelator (Paoletti et al., 2009). Application of 10 mM tricine reduced Ca²⁺ responses in WT mice and left Ca²⁺ responses unaffected in mZnR KO mice; tricine also reduced Ca²⁺ responses in WT mice to levels similar to those observed in mZnR KO mice (Fig. 4A). Because tricine only reduces Ca²⁺ responses in WT mice, but not in KO mice, we conclude that the observed differences in Ca²⁺ responses are due to Zn²⁺-mediated, mZnR-dependent signaling.

To further support that vesicular Zn²⁺ triggers Zn²⁺-mediated signaling via mZnR, we used a mouse genetic model lacking the transporter that loads Zn²⁺ into synaptic vesicles (ZnT3 KO) mice (Cole et al., 1999). ZnT3 KO mice lack vesicular Zn²⁺ staining and vesicular Zn²⁺ release (Cole et al., 1999; Qian and Noebels, 2005; Chorin et al., 2011). As expected, while ZnT3 WT mice displayed DCN vesicular Zn²⁺ release after parallel fiber stimulation, DCN vesicular Zn²⁺ release was undetectable in ZnT3 KO mice (Fig. 4B-C). In response to parallel fiber stimulation, ZnT3 KO mice displayed reduced Ca²⁺ responses when compared to WT mice; in addition, tricine did not affect Ca²⁺ responses in ZnT3 KO mice (Fig. 4A). These results indicate that a significant proportion of the Ca²⁺ response following stimulation of the parallel fibers requires both vesicular Zn²⁺ and the mZnR. While Fura-2 can also detect changes in intracellular Zn²⁺, previous studies have established that activation of mZnRs by extracellular Zn²⁺ leads to a PLC-dependent increase in intracellular Ca²⁺ that is not associated with Zn²⁺ influx (Besser et al., 2009; Chorin et al., 2011; Saadi et al., 2012). Finally, mZnR KO mice displayed normal DCN vesicular Zn²⁺ release (Fig. 4B-C). Together these findings suggest that mZnRs are functionally expressed in the DCN neurons and participate in Zn²⁺-triggered increases in intracellular Ca²⁺ responses.

Given the dependence of the Zn²⁺-induced reduction of Pr on postsynaptic Ca²⁺ increases and GPCRs activation (Fig. 3A), as well as the mZnR-mediated increase in Ca²⁺ responses in DCN neurons (Fig. 4A), we tested whether mZnRs are necessary for the Zn²⁺-induced reduction of synaptic strength. We found that Zn²⁺-mediated modulation of EPSC amplitude, PPR and 1/CV² were absent in mZnR KO mice (Fig. 5A-C). In contrast, littermate WT mice displayed reversible Zn²⁺-induced reductions on EPSC amplitude and Pr (Fig. 5A-C). PPR levels were not different between WT mZnR and mZnR KO mice, suggesting that the absence of mZnR does not affect baseline Pr (Fig. 5B, p=0.22). Importantly, mEPSC analysis revealed that basal quantal properties -- mini EPSC frequency and amplitude -- were not different between mZnR WT and KO mice (Fig. 6A, B). Moreover, mEPSC rise and decay times were not different between WT and KO mice (Fig. 6 C, D). These results indicate that the lack of effect of Zn²⁺ on reducing synaptic strength and Pr in mZnR KO mice is not due to differences in basal synaptic properties between WT and KO mice. The lack of Zn²⁺-mediated changes in Pr in mZnR KO mice was not caused by non-functional endocannabinoid signaling in mZnR KO mice. This was determined by the fact that depolarization-induced suppression of excitation (DSE; DSE is induced by postsynaptic depolarization leading to rises in post synaptic Ca²⁺ levels that, in turn, trigger endocannabinoid synthesis and Pr reduction) was not different between mZnR WT and KO mice (Fig. 6E). DSE in mZnR KOs was blocked by AM-251, confirming that it is mediated by CB1Rs (n=5, not shown). These results reveal that baseline Pr and depolarization-induced endocannabinoid signaling are intact in mZnR KO mice. Finally, application of 2μM WIN-55, 212-2 (WIN, a CB1R agonist) caused a quantitatively and kinetically similar decrease in EPSC amplitude in WT and KO mice (Fig. 6F), suggesting that CB1R function is not altered between mZnR WT and mZnR KO mice. Because Zn²⁺-induced modulation of Pr and the Zn²⁺-induced promotion of endocannabinoid signaling are absent in mZnR KO mice, we conclude that mZnRs are necessary for the Zn²⁺-induced reduction of Pr, this Pr reduction is likely occurring via the activation of postsynaptic endocannabinoid synthesis.

To determine the specific endocannabinoid that leads to mZnR mediated endocannabinoid signaling and subsequent Pr reduction, we used a combined electrophysiological and biochemical approach. 2-arachidonoylglycerol (2-AG), the endocannabinoid that mediates retrograde signaling in the DCN, is synthesized following phospholipase-C (PLC) activation and the cleavage of diacylglycerol by a diacylglycerol lipase (DGL) (Zhao et al., 2009). To determine whether Zn²⁺-induced changes in Pr are mediated through the 2-AG biosynthetic pathway, we first investigated the effect of pharmacological blockade of PLC and DGL on Zn²⁺-mediated Pr modulation. We observed that incubation of slices with either a PLC inhibitor (5 μM U73122), or a DGL inhibitor (10 μM tetrahydrolipstatin, THL) blocked Zn²⁺-induced depression of EPSC amplitude and the concomitant Zn²⁺-induced changes in PPR and 1/CV² (Fig. 7A-C). Moreover, bath application of both these inhibitors did not affect baseline synaptic transmission, or CV of evoked EPSCs, indicating that their inhibitory effect on Zn²⁺-induced Pr reduction is not due to a non-specific change of baseline Pr that occludes or blocks further changes in Pr by Zn²⁺ (Fig. 7D-F). Taken together, these results suggest that application of Zn²⁺ leads to a PLC-dependent production of 2-AG.

Next, we employed isotopic dilution liquid chromatography/tandem mass spectrometry (LC/MS/MS) to quantify 2-AG levels and their dependence on Zn²⁺. The two different isomers of AG (2-AG and 1-AG) were chromatographically resolved and base peak separations were achieved (Fig. 8A). Consistent with previous reports (Zhang et al., 2010), the isotopically labeled 2-AG-d8 had a 0.12 min shorter retention time (Fig. 8A). The formation of endogenous 2-AG was confirmed by coelution with internal standard (Fig. 8A) and by high resolution MS and MS/MS data obtained on the 2-AG peak, where a similar fragmentation pattern was observed between the standard and the DCN tissue samples (Fig. 8B, C). In agreement with our hypothesis on the enhancing effect of Zn²⁺ on the 2-AG metabolic pathway (Fig. 7), HPLC-mass spectrometry analysis showed that DCN 2-AG levels increased significantly after Zn²⁺ (100 μM for 5 minutes) application (Fig. 8D). Moreover, Zn²⁺-mediated increases in 2-AG levels were abolished when slices were either pretreated with the PLC inhibitor (U73122, 5 μM for 30 min prior to Zn²⁺ application) or in mZnR KO mice (Fig. 8D). These results indicate that exogenous Zn²⁺ leads to increased 2-AG synthesis via a PLC-dependent pathway. Given that 2-AG synthesizing enzymes have been localized in the dendrites of DCN fusiform cells (Zhao et al., 2009), our results suggest that activation of mZnR leads to the activation of postsynaptic PLC and DGL leading to enhanced postsynaptic 2-AG production.

Endogenous Synaptic Zn²⁺ Levels Decrease Pr via mZnR and Endocannabinoid Signaling

Next, we investigated whether endogenous, synaptically released Zn²⁺ reduces Pr via promotion of endocannabinoid signaling. Brief bursts of presynaptic activity can promote the synthesis and release of endocannabinoids from postsynaptic cells, leading to presynaptic CB1 receptors activation that, in turn, suppress neurotransmitter release (Brown et al., 2003). This process leads to endocannabinoid-mediated short-term plasticity: short-term depression in this case. Therefore, to determine whether endogenous Zn²⁺ triggers endocannabinoid signaling, we investigated the role of synaptically-released Zn²⁺ in short-term plasticity. We measured parallel fiber-evoked EPSCs at low frequencies (0.3 Hz) before and after a high frequency stimulus train (100 Hz, 1sec). This stimulus train produced short-term facilitation in EPSC amplitude (termed in figures STF; Fig. 9A1). Bath application of the extracellular Zn²⁺ chelator tricine after the initial stimulus train -- in the same cell -- significantly increased the magnitude of short-term facilitation induced by an identical stimulus train (Fig. 9A1, A3). These results suggest that chelation of synaptically released Zn²⁺ during the high frequency train enhanced the magnitude of induced short-term facilitation. Brief trains of presynaptic activity -- apart from promoting synthesis and release of endocannabinoids -- trigger a build-up of calcium in presynaptic boutons resulting in forms of short-term plasticity (Zucker and Regehr, 2002), such as augmentation and post-tetanic potentiation (PTP). In many synapses, the combined effect of PTP and endocannabinoid signaling determines the size and the sign of short-term plasticity (Beierlein et al., 2007). Therefore, one possible interpretation of the effect of tricine in enhancing the amount of short-term facilitation is that the chelation of Zn²⁺ blocks Zn²⁺-mediated endocannabinoid signaling and short-term depression, thereby leading to enhanced short-term facilitation. This hypothesis predicts that blockade of endocannabinoid signaling should increase the amount of short-term facilitation and occlude the enhancing effect of tricine. Consistent with this hypothesis, application of AM-251 increased short-term facilitation and occluded the enhancement of short-term facilitation produced by Zn²⁺ chelation (Fig. 9A2, A3). These results suggest that endogenous, synaptically-released Zn²⁺ modulates short-term facilitation by enhancing endocannabinoid signaling.

If chelation of synaptic Zn²⁺ enhances short-term facilitation (Fig. 9A) by affecting the balance between opposing forms of modulation of Pr, we expect that synaptic Zn²⁺ would modulate the threshold for inducing short-term facilitation. To test this hypothesis, we determined the effect of zinc chelation on threshold conditions under which presynaptic trains of action potentials fail to induce short-term plasticity. We varied the frequency of presynaptic bursts and investigated the effect of tricine on the resulting short-term plasticity. We found that although a 30 Hz, 1 sec stimulation did not affect synaptic strength, application of tricine -- under identical conditions -- resulted in significant short-term facilitation (Fig. 9B1, B3). Moreover, application of AM-251 alone under these conditions also revealed short-term facilitation and, importantly, occluded the enhancing effect of tricine (Fig. 9B2, B3). Taken together, these results suggest that the release of endogenous Zn²⁺ during bursts of presynaptic activity promotes endocannabinoid signaling and shifts the balance of opposing short-term plasticity mechanisms towards reduction -- or decreased enhancement -- of synaptic strength.

Because PTP depends on baseline Pr (Zucker and Regehr, 2002) potential effects of tricine on baseline Pr would affect the amount of observed short-term facilitation. However, tricine did not affect baseline EPSC strength, PPR or CV, thus suggesting that its effects on short-term plasticity are not due to changes of baseline Pr (Fig. 10A-C). Moreover, these observations indicate that any release of synaptic Zn²⁺ during baseline synaptic transmission is not capable of promoting endocannabinoid signaling; presynaptic trains of action potentials are needed for Zn²⁺-induced promotion of endocannabinoid signaling. Taken together, these findings (Figs 9-10) reveal a previously unknown role for synaptic Zn²⁺ in setting the threshold for short-term plasticity via enhancement of endocannabinoid signaling.

Given that exogenous Zn²⁺ triggers endocannabinoid synthesis is dependent on mZnRs (Figs. 2-8), we hypothesized that endogenous Zn²⁺-mediated reduction of short-term facilitation (Fig. 9) is also dependent on mZnRs. If mZnRs are necessary for the Zn²⁺-induced endocannabinoid synthesis, then we expect that short-term facilitation should be increased in mZnR KO mice; the enhancing effect of tricine on short-term facilitation should also be absent in mZnR KO mice. In agreement with our hypotheses, short-term facilitation in response to 100 Hz stimulation was enhanced in mZnR KO mice (Fig. 11 A1, A3). Moreover, chelation of synaptically-released Zn²⁺ by tricine did not increase the magnitude of short-term facilitation in mZnR KO mice, suggesting that the lack of the zinc receptor -- similarly to application of AM-251 in control mice in Fig. 9 -- occludes the effect of tricine (Fig. 11A2, A3). Since depolarization-induced endocannabinoid signaling is intact in mZnR KO and baseline Pr is not affected by the absence of mZnR (Fig. 6), these results suggest that the enhancing effect of synaptic Zn²⁺ on short-term plasticity is dependent on mZnR activation of endocannabinoid signaling.

To determine the effect of elimination of synaptic Zn²⁺ release on endocannabinoid signaling and short-term plasticity, we also used ZnT3 KO mice, which do not display any DCN vesicular Zn²⁺ release (Fig. 4B,C). Given that Zn²⁺-chelation experiments with tricine suggest that the release of vesicular Zn²⁺ decreases short-term facilitation levels (Fig. 9), ZnT3 KO mice are expected to show more robust short-term facilitation than WT littermate mice. Moreover, we expected that the enhancing effect of tricine on short-term facilitation would be occluded in ZnT3 KO mice. Indeed, comparison of short-term facilitation levels between ZnT3 WT and ZnT3 KO littermates revealed increased levels of short-term facilitation (100 Hz stimulation) in ZnT3 KO mice (Fig. 11B1, B3). Additionally, the enhancing effect of tricine was occluded in ZnT3 KO mice (Fig. 11 B2, B3). Together these results suggest that endogenous, synaptically-released Zn²⁺ mediates the depressing effects in short-term plasticity observed in fusiform cells.

During milder presynaptic stimulation (30 Hz, 1sec) synaptic Zn²⁺ increased the threshold for short-term facilitation (Fig. 9B1-B3). Based on this finding, we hypothesized that the threshold for inducing short-term facilitation would be lower in mZnR KO and in ZnT3 KO mice. To test this hypothesis we investigated the amount of short-term facilitation after a 30 Hz 1sec presynaptic stimulus. In accordance with our hypothesis, while mZnR WT and ZnT3 WT mice revealed minimal levels of short-term facilitation, mZnR KO and ZnT3 KO mice revealed significantly enhanced levels of short-term facilitation (Fig. 11C1-C5). The observed effects in ZnT3KO mice are not due to changes in either, basal quantal properties, baseline Pr, or in deficient endocannabinoid signaling in ZnT3 KO mice, as WT and KO mice displayed similar mEPSC frequency, amplitude, rise time, decay time, similar levels of baseline PPR, and similar depolarization-induced endocannabinoid signaling (Fig 12 A-F). Taken together, our physiological, pharmacological, genetic and biochemical results show that release of endogenous, vesicular Zn²⁺ reduces neurotransmitter release via mZnR-dependent, Gq-coupled receptor activation of 2-AG synthesis. While a G_q-coupled receptor promotion of endocannabinoid synthesis has been well established during the last decade, our results reveal a surprising link between synaptically released Zn²⁺ and retrograde endocannabinoid signaling, thus providing a significant advance towards the elucidation of the role of Zn²⁺ in synaptic physiology.

The Role of Zn²⁺ and mZnR in Modulating Synaptic Transmission

Our studies provide the first evidence for inhibitory effects of Zn²⁺-mediated signaling on Pr. We demonstrate a synaptic Zn²⁺-mediated negative feedback loop that reduces Pr at DCN parallel fiber synapses through a signaling pathway linking Zn²⁺ release to endocannabinoid synthesis. Bath application of exogenous Zn²⁺ as well as release of endogenous Zn²⁺ caused a reversible reduction of Pr. This inhibitory effect of Zn²⁺ is opposite to the effect that has been observed in mossy fiber synapses (Li et al., 2001; Huang et al., 2008), where exogenous Zn²⁺ application leads to LTP of synaptic response via presynaptic increases in Pr (Huang et al., 2008; Pan et al., 2011). Given that mZnRs are likely present in mossy fiber synapses (Besser et al., 2009; Chorin et al., 2011), the lack of the inhibitory effect of Zn²⁺ on Pr is likely due to the absence of endocannabinoid signaling in mossy fiber synapses (Hofmann et al., 2008). Our results suggest that the synaptic effects of Zn²⁺ are synapse-specific and, similar to other neuromodulatory systems, Zn²⁺ -mediated effects depend on the molecular composition of specific synapses.

While previous studies have shown that Zn²⁺ is released in an activity and Ca²⁺-dependent manner (Qian and Noebels, 2005, 2006), there has a been a controversy whether synaptic Zn²⁺ is released or whether Zn²⁺ creates a layer in the extracellular space that maps onto the synaptic Zn²⁺ stained region (Zn²⁺ veneer theory; (Kay, 2003; Kay and Toth, 2008). Indeed, the amount of synaptic Zn²⁺ that reaches the postsynaptic membrane during synaptic stimulation remains unknown with estimates from ~0 or no Zn²⁺ release (Kay, 2003; Kay and Toth, 2008), to more than 100 μM (Vogt et al., 2000). Here, we focused on determining whether synaptically-released (endogenous) Zn²⁺ -- regardless of the actual concentration reached in synaptic cleft -- was sufficient to trigger endocannabinoid signaling. The results presented in Figs 9 and 11 show that endogenous Zn²⁺ reduces Pr via endocannabinoid signaling in the DCN. Thus, our pharmacological and genetic manipulations support a ZnT3/mZnR–dependent, endocannabinoid-mediated decrease of Pr as a result of release of endogenous levels of Zn²⁺ in response to brief stimulus trains.

While recent results have indicated that synaptically released Zn²⁺ induced by a single action potential blocks NMDAR responses (Pan et al., 2011), our results show that mZnR-dependent triggering of endocannabinoid signaling requires trains of presynaptic action potentials. The requirement for trains of presynaptic action potentials is consistent with the recent proposal that Zn²⁺-containing synaptic mossy fiber vesicles populate the reserve -- not the readily released -- pool (Lavoie et al., 2011). Although the detailed physiological conditions leading to mZnR-mediated signaling remain undetermined, our findings suggest that, in response to a brief 30 Hz train, endogenous Zn²⁺ levels set the threshold for eliciting short-term plasticity (Fig. 9 B1 and Fig. 11C). While in vivo recordings from DCN granule cells have not been obtained, sensory stimulation can produce bursts of spikes in cerebellar granule cells at very high firing frequencies (more than 200 Hz) (Chadderton et al., 2004). Given the strong parallels between DCN and cerebellar granule cells (Oertel and Young, 2004), the brief 30 Hz stimulation that we used is expected to be well within the physiological range of parallel fiber activity, thus suggesting that Zn²⁺-mediated endocannabinoid pathway can be engaged by physiologically relevant stimuli.

Our results demonstrate that GPR39 (mZnR) is necessary for Zn²⁺-mediated endocannabinoid synthesis; it is likely that synaptically released Zn²⁺ mediates its effects on endocannabinoid signaling and Pr via direct activation of GPR39 (mZnR, Figure 13). GPR39, a previously orphan G-protein coupled receptor was initially proposed to mediate obestatin signaling and to regulate food intake (Zhang et al., 2005). However, the absence of GPR39 in the hypothalamus (Jackson et al., 2006) and the identification of Zn²⁺, rather than obestatin, as its putative endogenous ligand (Yasuda et al., 2007) have raised questions regarding the physiological role of GPR39. Recent studies have suggested that synaptically released Zn²⁺ triggers metabotropic signaling via mZnR in the hippocampus (Besser et al., 2009), enhancing the activity of the potassium chloride co-transporter 2 in CA3 pyramidal neurons (Chorin et al., 2011). We show that during synaptic activation of excitatory terminals, similar to group 1 mGluRs (Maejima et al., 2001; Varma et al., 2001; Kushmerick et al., 2004), the mZnR is necessary for 2-AG synthesis and thus, contributes to the overall signaling profile of excitatory synaptic transmission that is tightly coupled to postsynaptic endocannabinoid synthesis. Because the 2-AG synthesizing enzymes -- DGLα and DGLβ — have been localized to the dendrites of DCN fusiform cells (Zhao et al., 2009), our electrophysiological, imaging and biochemical results are consistent with a postsynaptic localization of mZnRs.

The Role of Zn²⁺ in Brain Function

The allosteric modulation of NMDA and glycine receptors by Zn²⁺ has suggested roles in learning, and motor control respectively (Hirzel et al., 2006; Adlard et al., 2010). Synaptically released Zn²⁺ has also been associated with Alzheimer’s disease and pain processing (Deshpande et al., 2009; Duce et al., 2010; Nozaki et al., 2011). Although initial studies did not uncover cognitive deficits in ZnT3KO mice (Cole et al., 2001), recent evidence reveals impaired fear memory (Martel et al., 2010), impaired spatial memory and impaired behavior dependent on hippocampus and perirhinal cortex (Martel et al., 2011), accelerated aging-related decline of spatial memory (Adlard et al., 2010), as well as impairments in spatial working memory and contextual discrimination in mice that lack vesicular Zn²⁺ (Sindreu et al., 2011). Our studies, by demonstrating a Zn²⁺-mediated negative feedback loop that reduces Pr at central auditory synapses -- through a specific pathway linking mZnR activation, endocannabinoid synthesis and Pr inhibition -- pave the way for novel in-vivo and behavioral experimental paradigms aimed at further defining the role of Zn²⁺ on brain function.

In sensory systems, the levels of synaptic Zn²⁺ in somatosensory cortex can be rapidly and dynamically regulated by sensory experience, although the physiological consequences of these changes have not been determined (Brown and Dyck, 2003; Nakashima and Dyck, 2009, 2010). Although it has been known for 25 years that DCN parallel fibers contain very large amounts of synaptic Zn²⁺ (Frederickson et al., 1988), the physiological role of this metal in modulating auditory circuits has not been previously evaluated. Based on our observations, we propose that synaptic Zn²⁺ -- by providing a negative feedback loop that reduces synaptic strength -- may decrease the responsiveness of the cochlear nucleus to acoustic stimuli: thus, synaptic Zn²⁺ release is expected to reduce the gain of input-output transformations between the auditory nerve and the cochlear nucleus. This is consistent with the proposed inhibitory role of Zn²⁺ in visual and auditory processing centers (Hirzel et al., 2006; Chappell et al., 2008), thus supporting the notion that Zn²⁺ is an inhibitory neuromodulator in sensory systems.

In conclusion, our results reveal the existence of a cellular pathway linking synaptically released Zn²⁺ and endocannabinoids: a previously unrecognized connection between two fundamental neuromodulatory systems, leading to activity-dependent inhibition of synaptic strength.