AMPA receptor inhibition by synaptically released zinc

Abstract

The vast amount of fast excitatory neurotransmission in the mammalian central nervous system is mediated by AMPA-subtype glutamate receptors (AMPARs). As a result, AMPAR-mediated synaptic transmission is implicated in nearly all aspects of brain development, function, and plasticity. Despite the central role of AMPARs in neurobiology, the fine-tuning of synaptic AMPA responses by endogenous modulators remains poorly understood. Here we provide evidence that endogenous zinc, released by single presynaptic action potentials, inhibits synaptic AMPA currents in the dorsal cochlear nucleus (DCN) and hippocampus. Exposure to loud sound reduces presynaptic zinc levels in the DCN and abolishes zinc inhibition, implicating zinc in experience-dependent AMPAR synaptic plasticity. Our results establish zinc as an activity-dependent, endogenous modulator of AMPARs that tunes fast excitatory neurotransmission and plasticity in glutamatergic synapses.

Keywords: AMPA receptors; ZnT3; auditory; synaptic plasticity; zinc.

Fig. 1.

Synaptic ZnT3-dependent zinc inhibits AMPAR EPSCs in DCN parallel fiber synapses via a postsynaptic mechanism. (A) Schematic of the experimental setup for electrophysiological experiments in cartwheel cells. In this figure, AMPAR EPSCs were recorded from cartwheel cells and evoked by parallel fiber stimulation (PF EPSCs). (B) Representative PF EPSCs before and after ZX1 application. (C) Time course of PF EPSC amplitude before and after ZX1 application (PF EPSC amplitude, 15–20 min after ZX1 application: 140.22 ± 7.66% of baseline, n = 7, P < 0.01). (D) Representative PF EPSCs in response to two stimuli 20 ms apart: before and after ZX1 application. (E) Summary graph of paired-pulse ratio (n = 7, P = 0.36 for control vs. ZX1). (F) Summary graph of normalized 1/CV² (n = 7, P = 0.01 for second pulse vs. first pulse; n = 7, P = 0.95 for control first pulse vs. ZX1 first pulse). (G) Representative PF EPSCs in control, after tricine, and after tricine and ZX1 application. (H) Time course of PF AMPAR EPSC amplitude before, after tricine, and after tricine and ZX1 application (PF EPSC amplitude: 15–20 min after tricine application: 100.07 ± 3.35% of baseline, n = 5, P = 0.97; 15–20 min after tricine and ZX1 application: 146.51 ± 7.20% of baseline, n = 5, P < 0.01). (I) Representative PF EPSCs from ZnT3WT and ZnT3KO mice before and after ZX1 application. (J) Time course of PF EPSC amplitude from ZnT3WT and ZnT3KO mice before and after ZX1 application (PF EPSC amplitude, 15–20 min after ZX1 application: ZnT3WT: 147.02 ± 8.82% of baseline, n = 5, P < 0.01; ZnT3KO: 94.69 ± 6.85% of baseline, n = 5, P = 0.16; ZnT3WT vs. ZnT3KO: P < 0.01). Values represent mean ± SEM. For details of statistical tests and detailed values shown in main figures, see SI Materials and Methods.

Fig. S1.

(A) Representative PF AMPAR EPSCs before and after ZX1 application in the presence of 1 μM of AM-251. (B) Summary of PF AMPAR EPSC amplitude,15–20 min after ZX1 application in the following: control: 140.22 ± 7.66% of baseline; AM-251: 148.97 ± 3.35% of baseline, n = 5, P = 0.32, Student unpaired t test. (C and D) Summary graph of PPR (C) and normalized 1/CV² (D) of CWC PF EPSCs. (PPR: control: 2.06 ± 0.25; tricine: 2.23 ± 0.19; ZX1: 2.19 ± 0.20; control vs. tricine: P = 0.89, n = 5; control vs. ZX1: P = 0.68, n = 5; tricine vs. ZX1: P = 0.91, n = 5; 1/CV² normalized to control first pulse: control second pulse: 2.53 ± 0.24, P < 0.01, n = 5; tricine first pulse: 1.02 ± 0.10, P = 0.99, n = 5; ZX1 first pulse: 0.95 ± 0.11, P = 0.74, n = 5, one-way ANOVA, post hoc Tukey.) Values represent mean ± SEM.

Fig. S2.

(A–C) Summary graph of PPR (A), normalized 1/CV² (B), and CV (C) of CWC PF EPSCs from ZnT3WT and ZnT3KO mice (PPR: ZnT3WT: Control: 2.47 ± 0.11; ZX1: 2.43 ± 0.16; P = 0.75, n = 5; ZnT3KO: Control: 2.32 ± 0.17; ZX1: 2.29 ± 0.13; P = 0.78, n = 5, Student paired t test; Control PPR: ZnT3WT vs. ZnT3KO; P = 0.48, n = 5, Student unpaired t test; 1/CV² normalized and compared with control first pulse: ZnT3WT: control second pulse: 2.35 ± 0.32, P < 0.01, n = 5; ZX1 first pulse: 1.15 ± 0.15, P = 0.99, n = 5; ZnT3KO: control second pulse: 2.20 ± 0.23, P < 0.01, n = 5; ZX1 first pulse: 0.93 ± 0.15, P = 0.99, n = 5, one-way ANOVA, post hoc Tukey; CV of pulse 1: ZnT3WT: 0.26 ± 0.04; ZnT3KO: 0.28 ± 0.12; P = 0.87, Student unpaired t test). Values represent mean ± SEM.

Fig. 2.

Inhibition of AMPAR EPSCs by synaptic zinc is dependent on evoked, action potential-driven release of zinc from presynaptic terminals. (A) Schematic of the location of glutamate uncaging. (B) Representative AMPAR currents in response to glutamate uncaging before and after ZX1 application. (C) Time course of amplitude of AMPAR uncaging currents before and after ZX1 application (AMPAR current amplitude, 15–20 min after ZX1 application: 98.46 ± 6.27% of baseline, n = 5, P = 0.84). (D) Representative traces of spontaneous AMPAR EPSCs (sEPSCs) before and ZX1 application. (E) Time course of the mean sEPSC amplitude before and after ZX1 application (mean sEPSC amplitude, 15–20 min after ZX1 application: 104.34 ± 8.78% of baseline, n = 5, P = 0.56). (F–I) Cumulative probability plot of sEPSC amplitude (F), frequency (G), rise time (H), and decay time (I) before and after ZX1 application (mean sEPSC amplitude: n = 5, P = 0.24 for control vs. ZX1; mean sEPSC frequency: n = 5, P = 0.72 for control vs. ZX1; mean sEPSC rise time: n = 5, P = 0.25 for control vs. ZX1; mean sEPSC decay time: n = 5, P = 0.10 for control vs. ZX1).

Fig. 3.

Zinc-mediated inhibition of AMPAR EPSCs is input specific in DCN synapses and occurs in hippocampal synapses. (A, Left) Brightfield image of a DCN slice showing the molecular and deep layer of the DCN where parallel fiber (PF) and auditory nerve (AN) inputs reside, respectively. (Center) DA-ZP1, a cell-permeable fluorescent zinc sensor reveals zinc-mediated fluorescence in the molecular but not deep layer of a DCN slice from a WT mouse. (Right) Absence of DA-ZP1 fluorescence in a DCN slice from a ZnT3KO mouse. (B) Illustration of two-pathway imaging experiments with stimulating electrodes placed in the molecular and deep layer of the DCN. (C) In response to a 100-Hz, 1-s stimulation in the molecular layer, ZP1-6COOH, a membrane-impermeable fluorescent zinc sensor reveals evoked zinc signals in the molecular but not in the deep layer of the DCN. No fluorescence is evoked by identical electrical stimulation in the deep layer. (D) Representative ZP1-6COOH fluorescent responses in response to a 100-Hz, 1-s electrical stimulation. (E) Schematic of the experimental setup for two-pathway electrophysiological experiments in fusiform cells. (F) Representative traces from two-pathway experiment showing, in response to paired-pulse stimulation, PF EPSCs, and AN EPSCs recorded from the same fusiform cell. (G) Representative PF and AN EPSCs, recorded from the same fusiform cell as shown in F, before and after ZX1 application. (H) Time course of PF and AN EPSC amplitude before and after ZX1 application (AMPAR EPSC amplitude, 10–15 min after ZX1 application: PF EPSC: 151.09 ± 7.05% of baseline, n = 3, P < 0.01; AN EPSC: 100.01 ± 1.66% of baseline, n = 3, P = 0.79; PF EPSC vs. AN EPSC: P < 0.01). (I) Schematic of the experimental setup for experiments in the hippocampus, including stimulation of Schaffer collaterals (SC) and recording from CA1 neurons (J) Representative SC CA1 EPSCs from ZnT3WT and ZnT3KO mice before and after ZX1 application. (K) Time course of ZnT3WT and ZnT3KO SC CA1 EPSC amplitude before and after ZX1 application (AMPAR EPSC amplitude, 15–20 min after ZX1 application: ZnT3WT: 146.71 ± 5.66% of baseline; n = 5, P < 0.01; ZnT3nKO: 92.23 ± 9.20% of baseline; n = 5, P = 0.18; ZnT3WT vs. ZnT3KO: P < 0.01).

Fig. S3.

(A) Summary graph of PPR of fusiform cell PF and AN EPSCs from two-pathway experiments. (PF EPSCs: control: 1.88 ± 0.475; ZX1: 1.85 ± 0.334, P = 0.97, n = 3; AN EPSCs: control: 1.18 ± 0.13; ZX1: 1.17 ± 0.03, P = 0.94, n = 3, Student paired t test.) (B–D) Summary graph of PPR (B), normalized 1/CV² (C), and CV (D) of SC EPSCS from CA1 hippocampal neurons form ZnT3WT and ZnT3KO mice (PPR: ZnT3WT: control: 1.89 ± 0.28; ZX1: 2.03 ± 0.19, P = 0.18, n = 5; ZnT3KO: control: 1.82 ± 0.20; ZX1: 1.85 ± 0.29, P = 0.35, n = 5, Student paired t test; Control PPR: ZnT3WT vs. ZnT3KO; P = 0.84, n = 5, Student unpaired t test; 1/CV² normalized and compared with control first pulse: ZnT3WT: control second pulse: 2.19 ± 0.17, P < 0.01, n = 5; ZX1 first pulse: 1.11 ± 0.26, P = 0.97, n = 5; ZnT3KO: control second pulse: 2.16 ± 0.17, P < 0.01, n = 5; ZX1 first pulse: 1.14 ± 0.18, P = 0.79, n = 5, one-way ANOVA, post hoc Tukey; CV of pulse 1: ZnT3WT: 0.32 ± 0.08; ZnT3KO: 0.27 ± 0.07; P = 0.65, Student unpaired t test). (E) Representative hippocampal fEPSPs recorded in stratum radiatum before and after ZX1 application. (F) Time course of initial slope of fEPSPs and fiber volley amplitude before and after ZX1 application (initial slope of fEPSPs: 20–25 min after ZX1 application: 148.24 ± 3.47% of baseline, n = 5, P < 0.01, Student paired t test; fiber volley amplitude: 20–25 min after ZX1 application: -4.09 ± 1.52% of baseline, Student paired t test, n = 5, P = 0.10). (G, Left) Representative traces showing granule cell response to current injections before and after ZX1 application. (Right) Summary graph of granule cell action potential threshold before and after ZX1 application (control: 48.46 ± 4.41 vs. ZX1: 45.71 ± 4.94, n = 4, P = 0.68, Wilcoxon rank sum test). (H) Summary graph of granule cell firing frequency as a function of injected current amplitude (F-I curve) before and after ZX1 application (5 pA: control: 0.83 ± 0.83, ZX1: 0.83 ± 0.83, n = 4, P = 0.84; 15 pA: control: 10.04 ± 1.60, ZX1: 10.78 ± 1.58, n = 4, P = 0.88; 25 pA: control: 34.23 ± 4.17, ZX1: 35.94 ± 5.31, n = 4, P = 0.68, Wilcoxon rank sum test). Values represent mean ± SEM.

Fig. 4.

Plasticity of AMPAR EPSCs by sound-evoked reduction of presynaptic zinc levels in DCN parallel fiber synapses. (A) Representative PF EPSCs from sham- and noise-exposed mice before and after ZX1 application. (B) Time course of PF EPSC amplitude from sham- and noise-exposed mice before and after ZX1 application (PF EPSC amplitude: sham-exposed: 145.55 ± 6.87% of baseline, n = 5, P < 0.01; noise-exposed: 96.98 ± 4.85% of baseline, n = 5, P = 0.83; sham- vs. noise-exposed: P < 0.01). (C) Representative zinc-mediated fluorescent signals in sham- and noise-exposed mice at increasing concentrations of DA-ZP1. (D) Summary graph of fluorescence intensity at different concentrations of DA-ZP1 (fluorescence intensity in arbitrary units: sham- vs. noise-exposed, n = 5, P = 0.02 for 0.25 µM; P = 0.15 for 0.5 µM; P < 0.01 for 0.75 µM; P = 0.03 for 1 µM). (E, Left) Representative evoked, zinc-mediated fluorescent signals in sham- and noise-exposed mice in response to increasing number of pulses at 100 Hz. (Right) Time course of representative ratiometric fluorescent signals. (F) Summary graph of normalized extracellular zinc concentrations. Concentrations from noise-exposed mice are normalized to sham-exposed average concentrations.

Fig. S4.

(A–C) Representative traces of ABR traces in response to sound clicks presented at different intensities (dB) levels from a sham-exposed, exposed ear (A), noise-exposed, exposed ear (B), and noise-exposed animal, unexposed, contralateral ear (C). I-V represent the different waves of the ABR. (D) Summary graph of ABR thresholds from sham-exposed, ipsilateral ears and noise-exposed, contralateral ears (sham-exposed: 34 ± 5.47 dB, n = 5; noise-exposed contralateral ear: 61.8 ± 8.72 dB, n = 5, P < 0.01, Student unpaired t test). ABR thresholds for noise-exposed animals, exposed ears were above 90 dB were not detected with our system. (E and F) Summary of PPR (E) and CV (F) of fusiform cell PF EPSCs from sham- and noise-exposed mice. (PPR: sham-exposed: 2.14 ± 0.17, noise-exposed: 2.45 ± 0.27; P = 0.35, n = 5, Student unpaired t test; CV: sham-exposed: 0.26 ± 0.04, noise-exposed: 0.20 ± 0.03; P = 0.26, Student unpaired t test.) (G and H) Summary graph of PPR (G) and CV (H) of AN EPSCs from sham- and noise-exposed mice (PPR: sham-exposed: 0.93 ± 0.07, noise-exposed: 0.98 ± 0.04, P = 0.42, n = 5, Student unpaired t test; CV: sham-exposed: 0.18 ± 0.02; noise-exposed: 0.33 ± 0.03, P < 0.01, Student unpaired t test). Values represent mean ± SEM.