Vesicular zinc promotes presynaptic and inhibits postsynaptic long-term potentiation of mossy fiber-CA3 synapse

Abstract

The presence of zinc in glutamatergic synaptic vesicles of excitatory neurons of mammalian cerebral cortex suggests that zinc might regulate plasticity of synapses formed by these neurons. Long-term potentiation (LTP) is a form of synaptic plasticity that may underlie learning and memory. We tested the hypothesis that zinc within vesicles of mossy fibers (mf) contributes to mf-LTP, a classical form of presynaptic LTP. We synthesized an extracellular zinc chelator with selectivity and kinetic properties suitable for study of the large transient of zinc in the synaptic cleft induced by mf stimulation. We found that vesicular zinc is required for presynaptic mf-LTP. Unexpectedly, vesicular zinc also inhibits a form of postsynaptic mf-LTP. Because the mf-CA3 synapse provides a major source of excitatory input to the hippocampus, regulating its efficacy by these dual actions, vesicular zinc is critical to proper function of hippocampal circuitry in health and disease.

Figure 1

Synthesis and X-ray structures of ZX1 and its zinc complex. (A) Using the intramolecular pyridinium salt 1 as a synthetic precursor for installing the DPA unit, ZX1 was prepared by reductive amination with 2-sulfonated aniline. (B) X-ray structure of ZX1 (left), displaying the two orientations of the disordered pyridine ring refined at 50:50 occupancy. Hydrogen atoms, except those on N2 and N3, are omitted for clarity; X-ray structure of ZX1-Zn(II)(OAc) (right). Hydrogen atoms are omitted for clarity.

Figure 2

Zinc-binding and fluorescence quenching properties of ZX1. (A) The experimental potentiometric equilibrium curves for free ZX1 (blue) and ZX1/Zn(II) (1:1) complex (magenta) as a function of added titrant (0.1 M NaOH). (B) Fluorescence quenching of a 1.67 μM solution of ZP3-Zn(II) (1:1) by zinc chelators in pH 7.0 buffer (50 mM PIPES, 100 mM KCl). ZX1 (magenta) binds zinc more rapid than CaEDTA (blue). (C) ZX1 (100 μM), but not CaEDTA (7.5 mM), inhibits the NMDA receptor mediated EPSC (I_NMDA) evoked in CA3 pyramids by mf stimulation (0.033 Hz). Pharmacologically isolated synaptic I_NMDA were recorded in CA3 pyramids at a holding potential of + 30 mv. Representative traces from ZX1 (left) and CaEDTA (right) reflect 5 minute epochs immediately before (1) and between 5 and 10 minutes after (2) application of each chelator to the bath (denoted by horizontal line). ZX1 produced a 40% ± 13% increase of I_NMDA (n = 6 cells, p = 0.02, paired t-test) whereas CaEDTA produced no significant increase (3% ± 17%, n = 5 cells, p> 0.05).

Figure 3

ZX1 inhibits induction of LTP at MF-CA3 synapse. Top left: Field EPSPs (fEPSPs) were recorded from hippocampal slices acutely isolated from WT mice (P28-P42) and the effects of ZX1 were examined on LTP of mf-CA3 synapse. Representative traces from WT (left) and ZX1 (right) represent baseline (1) and 30 min after HFS (2). Arrow denotes the timing of application of HFS and arrowheads denote the baseline fEPSP (1) and fEPSP after HFS (2). Horizontal line denotes timing of application of ZX1 to bath. Top right: Reduction of LTP induced by HFS is plotted as function of increasing concentrations of ZX1. Plot is based upon the following results: compared to baseline, vehicle 149 ± 9 (n = slices from 10 mice); ZX1 50 μM 142 ± 3% (n = 4); ZX1 100 μM 124 ± 3% (n = 7); ZX1 200 μM 120 ± 5% (n = 8). Middle and bottom panels: Whole cell recordings of CA3 pyramids were performed in hippocampal slices acutely isolated from WT mice (P21–P29) and the effects of ZX1 (100 μM) were examined on LTP and PPF of mf-CA3 synapse following HFS of the mossy fibers. Percent potentiation induced by HFS (middle left and right): vehicle 188 ± 16 (n = 8); ZX1 100 μM 131 ± 21% (n = 9). PPF before and after HFS (bottom left and right): vehicle 2.8 ± 0.5 and 1.3 ± 0.1 respectively, (paired t-test, p = 0.04); ZX1 3.1 ± 0.8 and 2.7 ± 0.7 respectively, (paired t-test, p = 0.2). Representative traces from Vehicle (left) and ZX1 (right) before and after HFS. Arrows denote administration of HFS. Horizontal lines in right middle and bottom panels denote timing of application of ZX1 to bath.

Figure 4

Divergent effects of HFS on LTP and PPF of mf-CA3 synapse in slices of WT and ZnT3 −/− mice. Whole cell recordings of CA3 pyramids were performed in hippocam-pal slices acutely isolated from WT or ZnT3 −/− mice and the effects on LTP and PPF of mf-CA3 synapse were examined following HFS of the mossy fibers. Percent LTP induced by HFS: WT 167 ± 14% (n = 17, p = 0.0001) (left top); ZnT3 −/− 180 ± 15% (n = 14, p = 0.0001) (right top). PPF before and 10–30 min after HFS: WT 3.1 ± 0.3 and 2.1 ± 0.2 (paired t-test, p = 0.0002); ZnT3 −/− 2.7± 0.3 and 2.6 ± 0.2 (paired t-test, p = 0.5). Arrows denote the time of the application of HFS.

Figure 5

Analysis of mEPSCs accompanying mf-CA3 LTP. mEPSCs accompanying mf-CA3 LTP were examined in slices of WT and ZnT3 −/− mice. Top panels present results from representative slices before (basal) and approximately 15 min after HFS. Middle and bottom panels present cumulative probability of event frequency and amplitude respectively with insets displaying mean ± SEM. In WT, HFS increased the frequency without change of amplitude of mEPSCs (left middle and bottom panels respectively) as evident in significant increase of event frequency (mean ± SEM) from 3.2 ± 0.5 Hz recorded in 10 min epoch prior to HFS compared to 4.2 ± 0.6 Hz recorded 10–30 min after HFS (n=15, paired t-test, p=0.04). In ZnT3 −/−, HFS decreased the event frequency and increased the event amplitude of mEPSCs (right middle and bottom panels respectively) as evident in a decrease of frequency from 5.3 ± 0.7 Hz to 3.0 ± 0.6 Hz (mean ± SEM, n=6, paired t-test, p = 0.05); the amplitude increased from 28 ± 4.3 pA to 34.7 ± 4.9 pA after HFS (n=6, paired t-test, p=0.02).

Figure 6

Effects of dialyzing CA3 pyramids with BAPTA on mf-CA3 LTP in ZnT3 −/− and WT mice. In WT in ACSF (top), the percent potentiation was similar when CA3 pyramids were dialyzed with BAPTA 171 ± 17% (n=7) compared to vehicle 180 ± 22 % (n=8) (Student’s t-test, p = 0.8). In ZnT3 −/− in ACSF (middle), the percent potentiation was reduced when CA3 pyramids were dialyzed with BAPTA 123 ± 11% (n=6) compared to vehicle 166 ± 16% (n = 12) (Student’s t-test, p = 0.009). The amount of potentiation in BAPTA dialyzed cells was significantly greater in WT compared to ZnT3 −/− animals (t-test, p = 0.04). In WT with ZX1 (100 μM) in the bath (bottom), the percent potentiation was eliminated when CA3 pyramids were dialyzed with BAPTA 82 ± 7% (n = 5) compared to vehicle 134 ± 20% (n = 9) (t-test, p = 0.04); note that data of vehicle dialyzed CA3 pyramids with ZX1 in bath are reprinted from experiment presented in Figure 3 (right middle). Arrows denote the time of application of HFS and arrowheads denote the baseline EPSC (1) and the EPSC after HFS (2) for BAPTA experiments.

Figure 7

ZX1 disinhibits mf-LTP in rim1α null mutant mice. In rim1α null mutant mice, HFS of the mf did not induce LTP as detected in field potential recordings in presence of ACSF (a small nonsignificant decrease of fEPSP of 93 ± 11%, n = 4 when measured 50–60 min after compared to the 10 min immediately preceding HFS) (left panel). By contrast, with ZX1 (100 μM) in the bath, HFS of the mf induced LTP in slices from rim1α null mutant mice (an increase of fEPSP of 151 ± 14%, n = 4, p = 0.016, vehicle vs ZX1) (right panel); this mf-LTP was not accompanied by a reduction of paired pulse facilitation (bottom right).

Figure 8

Model proposing how vesicular zinc promotes increased glutamate P_r underlying presynaptic mf-CA3 LTP. Invasion of mossy terminal by a high frequency train of action potentials results in calcium influx and fusion of synaptic vesicles with the terminal plasma membrane (1), resulting in release of zinc into synaptic cleft. The synaptically released zinc reenters the same or nearby presynaptic terminal through a voltage gated calcium channel (2). The local elevation of zinc concentration within the terminal activates a src family kinase (3) which phosphorylates and promotes activation of TrkB, one consequence of which is the phosphorylation and activation of PLCγ1 (4). PLCγ1 in turn catalyzes cleavage of phosphatidyl inositol bisphosphate (PIP₂, leading to formation of diacyl glycerol (DAG) and inositol 3,4,5 phosphate (IP₃) (5). IP₃ binds to and triggers release of calcium from endoplasmic reticulum (6) resulting in activation of calcium-calmodulin adenylate cyclase (Ca-CaM AC) (7), formation of cAMP and activation of protein kinase A (8). Subsequent steps include interaction of the synaptic vesicle associated protein, Rab3A, and its partner in the active zone, Rim1α (9), the net result being an increased glutamate P_r (10). This proposal is based in part upon the finding that zinc is capable of activating TrkB and its downstream signaling independently of brain-derived neurotrophic factor (BDNF) (Huang et al., 2008; Huang and McNamara, 2010). Inhibition of TrkB kinase prevents induction of mf-LTP (Huang et al, 2008) as well as the accompanying reduction of PPF (unpublished). Preventing TrkB activation of PLCγ1 signaling inhibits induction of mf-CA3 LTP (He et al., 2010) and the accompanying reduction of PPF (unpublished). Because activation of PLCγ_γ1 results in formation of IP3 and release of calcium from the endoplasmic reticulum, this model is consistent with the inhibition of mf-CA3 LTP by presynaptic calcium chelation with EGTA (Tong et al., 1996) and by genetic or pharmacological inhibition of α1E – containing calcium channels (Breustedt et al., 2003; Dietrich et al., 2003). This model is also consistent with evidence that TrkB activation promotes transmitter release from presynaptic terminals (Jovanovic et al. 2000; Tyler et al., 2002; Lohof et al., 1993). The requirements of the synaptic vesicle protein, Rab3a, and its interacting partner, Rim1α, for induction of mf-CA3 LTP (Castillo et al., 1997; Castillo et al., 2002) suggest that TrkB-activated PLC_γ1 signaling somehow interacts with these molecular components of the release apparatus to promote increased P_r and LTP. A requirement for Rab3a in the BDNF-mediated increase of mEPSC frequency in hippocampal neurons supports this suggestion (Alder et al., 2005).