Context-Dependent Modulation of Excitatory Synaptic Strength by Synaptically Released Zinc

Abstract

Synaptically released zinc inhibits baseline excitatory neurotransmission; however, the role of this neuromodulator on short-term plasticity during different levels of synaptic activity remains largely unknown. This lack of knowledge prevents our understanding of information transfer across zinc-releasing synapses, including 50% of excitatory synapses in cortical areas. We used in vitro electrophysiology in mouse brain slices and discovered that the effects of zinc on excitatory postsynaptic current (EPSC) amplitudes are context-dependent. At lower frequencies of activity, synaptically released zinc reduces EPSC amplitudes. In contrast, at higher stimulation frequencies and vesicular release probability (Pr), zinc inhibits EPSC amplitudes during the first few stimuli but leads to enhanced steady-state EPSC amplitudes during subsequent stimuli. This paradoxical enhancement is due to zinc-dependent potentiation of synaptic facilitation via the recruitment of endocannabinoid signaling. Together, these findings demonstrate that synaptically released zinc is a modulator of excitatory short-term plasticity, which shapes information transfer among excitatory synapses.

Keywords: ZnT3; auditory brainstem; auditory synapses; short-term plasticity; synaptic zinc.

Figure 1.

Frequency- and history-dependent effects of synaptically released zinc on AMPA EPSCs. A–C, Representative traces of cartwheel cell AMPA EPSCs evoked by 20-pulse train stimulation of parallel fiber at 5 Hz (A), 20 Hz (B), and 50 Hz (C middle panel), before (grey) and after 100 µM ZX1 (blue); (C side panels) same as in upper panel but zoomed on the first 2 pulses (left) and the last 5 pulses (right). For improved visualization, responses after ZX1 are slightly shifted to the right. D, Summary graph showing the ratio of AMPA EPSCs in ZX1 to that in control at each pulse at 5 Hz (maroon), 20 Hz (orange), and 50 Hz (blue). E, Summary graph comparing the ratio of AMPA EPSCs in ZX1 to that in control for the first two and last five pulses in the 20-pulse train at 5 Hz (maroon, n = 10, p = 0.82), 20 Hz (orange, n = 10, p = 0.63), and 50 Hz (blue, n = 10, p < 0.01). Values represent mean ± SEM. Detailed values and statistical tests: D, E, ZX_1–2/control_1–2 vs. ZX_16–20/control_16–20: 5 Hz: 1.51 ± 0.07 vs. 1.55 ± 0.08, n = 10, p = 0.82, t = 0.24, df = 9; 20 Hz: 1.47 ± 0.09 vs. 1.41 ± 0.06, n = 10, p = 0.63, t = 0.49, df = 9; 50 Hz: 1.44 ± 0.07 vs. 0.86 ± 0.08, n = 10, p < 0.01, t = 5.45, df = 9; paired t-tests; ZX_1–2/control_1–2: 5 Hz vs. 20 Hz, p = 0.65, t = 0.46, df = 9; 5 Hz vs. 50 Hz, p = 0.38, t = 0.92, df = 9; 20 Hz vs. 50 Hz, p = 0.68, t = 0.42, df = 9; ZX_16–20/control_16–20: 5 Hz vs. 20 Hz, p = 0.09, t = 1.87, df = 9; 5 Hz vs. 50 Hz, p < 0.01, t = 5.09, df = 9; 20 Hz vs 50 Hz, p < 0.01, t = 4.98, df = 9; paired t-tests; AMPA EPSC amplitudes_16–20: control: 50 Hz: 1.00 ± 0.00 vs. ZX1: 0.86 ± 0.08, p = 0.042, t = 2.36, df = 9; one-sample t-test.

Figure 2.

ZnT3-dependent, synaptically released zinc enhances EPSC_SS in high Pr. A, B, Upper panels, representative traces of AMPA EPSCs evoked by 50-Hz 20-pulse train in 1.2 mm (gray) and 2.4 mm (color) external calcium in control (A) and ZX1 (B). Lower panels, summary graphs showing average AMPA EPSC amplitudes during the train in 1.2 mm (gray) and 2.4 mm (color) external calcium in control (A) and ZX1 (B; EPSC_16–20 amplitude: control: 1.2 vs. 2.4 mm: n = 7, p< 0.01; ZX1: 1.2 vs. 2.4 mm: n = 7, p = 0.09). C, D, Upper panels, representative traces of AMPA EPSCs evoked by 50-Hz 20-pulse train in 1.2 mm (black) and 2.4 mm (gray or green) external calcium in ZnT3WT (C) and ZnT3KO mice (D). Lower panels, summary graphs showing average AMPA EPSC amplitudes during the train in 1.2 mm (black) and 2.4 mm (gray or green) external calcium in ZnT3WT (C) and ZnT3KO mice (D; ZnT3 WT: 1.2 vs. 2.4 mm: n = 5, p < 0.01; ZnT3KO: 1.2 vs. 2.4 mm: n = 5, p = 0.24). Values represent mean ± SEM. Detailed values and statistical tests. A, B, lower panels: EPSC_16–20 amplitude: control: 1.2 mm calcium: 1109 pA ± 90 pA vs. 2.4 mm calcium: 1605 pA ± 147 pA, n = 7, p < 0.01, F = 40.89, DFn = 1, DFd = 60; ZX1: 1.2 mm calcium: 1158 pA ± 75 pA vs. 2.4 mm calcium: 1301 pA ± 116 pA, n = 7, p = 0.09, F = 2.9, DFn = 1, DFd = 60; two-way ANOVA. C, D, lower panels: EPSC_16–20 amplitude. ZnT3WT: 1.2 mm calcium: 996 pA ± 95 pA vs. 2.4 mm calcium: 1564 pA ± 135 pA, n = 5, p < 0.01, F = 58.51, DFn = 1, DFd = 40; ZnT3KO: 1.2 mm calcium: 750 pA ± 185 pA vs. 2.4 mm calcium: 876 pA ± 156 pA, n = 5, p = 0.24, F = 1.40, DFn = 1, DFd = 40; two-way ANOVA.

Figure 3.

Endocannabinoid signaling is necessary for the zinc-mediated enhancement of EPSC_SS. A, Upper panel, representative traces of AMPA EPSCs, evoked by 50-Hz 20-pulse train, in the presence of 1 µm AM-251 before (gray) and after ZX1 (orange). Lower panel, summary graph showing average ratios of AMPA EPSCs in the presence of ZX1 to that in control during the train (ZX_1–2/control_1–2 vs. ZX_16–20/control_16–20: 50 Hz: n = 5, p = 0.13). B, Upper panel, representative traces of AMPA EPSCs evoked by 50-Hz 20-pulse train in 1.2 mm (gray) and 2.4 mm (orange) external calcium in the presence of AM-251. Lower panel, summary graph showing average AMPA EPSC amplitudes during the train in 1.2 mm (gray) and 2.4 mm (orange) external calcium in the presence of AM-251. (EPSC_16–20 amplitude in AM-251: 1.2 vs. 2.4 mm calcium, n = 5, p = 0. 09). Values represent mean ± SEM. Detailed values and statistical tests: A, lower panel: ZX_1–2/control_1–2 vs. ZX_16–20/control_16–20: 50 Hz: control: 1.47 ± 0.06 vs. ZX1: 1.36 ± 0.08, n = 5, p = 0.13, t = 1.4, df = 9; paired t-test. B, lower panel: EPSC_16–20 amplitude: AM-251: 1.2 mm calcium: 805 pA ± 97 pA vs. 2.4 mm calcium: 921 pA ± 80 pA, n = 5, p = 0.19, F = 1.7, DFn = 1, DFd = 40; two-way ANOVA.

Figure 4.

AMPAR saturation or desensitization do not contribute to the zinc-mediated enhancement of EPSC_SS. A, C, Upper panels, representative traces of AMPA EPSCs before (gray) and after (color) bath application of 0.5–1 mm kynurenic acid (A) and 100 µm cyclothiazide (C). Lower panels, summary graphs showing normalized AMPA EPSC amplitude (A) and decay time constant (C), before (gray) and after (color) 0.5–1 mm kynurenic acid (A) and 100 µm cyclothiazide (C; AMPA EPSC amplitude: control vs. kynurenic acid, n = 5, p < 0.01; AMPA EPSC decay time constant: control vs. cyclothiazide, n = 5, p = 0.04). B, D, Upper panels, representative traces of AMPA EPSCs evoked by 50-Hz 20-pulse train in 1.2 mm (gray) and 2.4 mm (color) external calcium, in 0.5–1 mm kynurenic acid and ZX1 (B) or in 100 µm cyclothiazide and ZX1 (D). Lower panels, summary graphs showing average AMPA EPSC amplitudes during the train, in kynurenic acid and ZX1 (B), or in cyclothiazide and ZX1 (D). (EPSC_16–20 amplitude: kynurenic acid: 1.2 vs. 2.4 mm calcium, n = 5, p = 0.13; cyclothiazide: 1.2 vs. 2.4 mm calcium, n = 5, p = 0.06.) Values represent mean ± SEM. Detailed values and statistical tests: A, lower panel: AMPA EPSC amplitude in the presence of kynurenic acid: 59.63% ± 8.5% of baseline, n = 5, p < 0.01, t = 4.74, df = 4; one-sample t-test. C, lower panel: AMPA EPSC decay time constant: control vs. cyclothiazide: 5.99 ± 0.81 vs. 11.32 ± 2.15, n = 5, p = 0.04; paired t-test, t = 2.95, df = 4. B, lower panel: EPSC_16–20 amplitude: kynurenic acid: 1.2 mm calcium: 718 pA ± 94 pA vs. 2.4 mm calcium: 812 pA ± 96 pA, n = 5, p = 0.13, F = 2.37, DFn = 1, DFd = 40; two-way ANOVA. D, lower panel: cyclothiazide: 1.2 mm calcium: 122 pA ± 32 pA vs. 2.4 mm calcium: 186 pA ± 41 pA, n = 5, p = 0.06, F = 3.6, DFn = 1, DFd = 40; two-way ANOVA.

Figure 5.

Enhanced zinc-mediated synaptic facilitation depends on endocannabinoid signaling. A, B, Upper panels, representative traces of AMPA EPSCs evoked by 50-Hz 20-pulse train before (gray) and after ZX1 application (color), in the absence (A) or presence (B) of AM-251. Lower panels, summary graphs showing average AMPA EPSC amplitudes during the train, normalized to EPSC amplitude elicited by first pulse, before (A and B, gray) and after (A and B, color) ZX1 application, in the absence (A), or presence (B) of AM-251 (ratio of the amplitude of 16–20 EPSC to first EPSC at 50 Hz: in the absence of AM-251: control vs. ZX1, n = 10, p < 0.01; in the presence of AM-251: control vs. ZX1, n = 5, p = 0.32). Values represent mean ± SEM. Detailed values and statistical tests: Lower panels: ratio of the amplitude of 16–20 EPSC to first EPSC at 50 Hz: in the absence of AM-251: control: 2.29 ± 0.25 vs. ZX1: 1.32 ± 0.15, n = 10, p < 0.01, F = 52.81, DFn = 1, DFd = 90; in the presence of AM-251: control: 1.19 ± 0.15 vs. ZX1: 1.04 ± 0.09, n = 5, p = 0.32, F = 1.0, DFn = 1, DFd = 40; two-way ANOVA.

Figure 6.

AMPA EPSC facilitation by synaptically released zinc is frequency, history, and Pr dependent. A, B, Upper panels, representative traces of AMPA EPSCs evoked by 20-pulse train at 5 Hz (A) and 20 Hz (B), before (gray) and after (color) 100 µM ZX1, in 2.4 mm external calcium. Lower panels, summary graphs showing average AMPA EPSC amplitudes during the 20-pulse train, normalized to EPSC amplitude of the first pulse, before (gray) and after (color) ZX1 application at 5 Hz (A) and 20 Hz (B), in 2.4 mm external calcium (ratio of the amplitude of 16–20 EPSC to first EPSC: 2.4 mm calcium: 5 Hz: control vs. ZX1, n = 5, p = 0.58; 20 Hz: control vs. ZX1, n = 5, p = 0.09). C–E, Upper panels, representative traces of AMPA EPSCs evoked by 20-pulse train at 5 Hz (C), 20 Hz (D), and 50 Hz (E), before (gray) and after (color) 100 µM ZX1, in 1.2 mm external calcium. Lower panels, summary graphs showing average AMPA EPSC amplitudes during the 20-pulse train, normalized to EPSC amplitude of the first pulse, before (gray) and after (color) ZX1 application at 5 Hz (C), 20 Hz (D), and 50 Hz (E) in 1.2 mm external calcium (ratio of the amplitude of 16–20 EPSC to first EPSC: 1.2 mm calcium: 5 Hz: control vs. ZX1, n = 5, p = 0.34; 20 Hz: control vs. ZX1, n = 5, p = 0.45; 50 Hz vs. ZX1, n = 10, p < 0.01). F, G, Upper panels, representative traces of AMPA EPSCs evoked by 50-Hz 20-pulse train, before (black) and after (color) 100 µm ZX1, in ZnT3WT (F) and ZnT3KO (G) mice. Lower panels, summary graphs showing average AMPA EPSC amplitudes during the 50-Hz 20-pulse train, normalized to EPSC amplitude of the first pulse, before (black) and after (color) ZX1 application, in ZnT3WT (F) and ZnT3KO (G) mice (ratio of the amplitude of 16–20 EPSC to first EPSC: ZnT3WT mice: 50 Hz control vs. ZX1, n = 6, p < 0.01; ZnT3KO mice: 50 Hz: control vs. ZX1, n = 5, p = 0.85). H, Upper panel, representative traces of peak-scaled AMPA EPSCs evoked by 50-Hz 20-pulse train in ZnT3WT (black) and ZnT3KO mice (green). Lower panel, summary graphs showing average AMPA EPSC amplitudes during the 50-Hz 20 pulse train, normalized to EPSC amplitude of the first pulse in control conditions, in ZnT3WT (black) and ZnT3KO (color) mice (ratio of the amplitude of 20 EPSC to first EPSC: ZnT3WT vs. ZnT3KO: control: 50 Hz, n = 5, p < 0.01). Values represent mean ± SEM. Detailed values and statistical tests: A, B, lower panels: ratio of the amplitude of 16–20 EPSC to first EPSC: 2.4 mm calcium: 5 Hz: control: 1.40 ± 0.12 vs. ZX1: 1.34 ± 0.15, n = 5, p = 0.58, F = 0.31, DFn = 1, DFd = 40; 20 Hz: control: 2.03 ± 0.09 vs. ZX1: 1.83 ± 0.14, n = 5, p = 0.09, F = 3.0, DFn = 1, DFd = 40; two-way ANOVA. C–E, lower panels: ratio of the amplitude of 16–20 EPSC to first EPSC: 1.2 mm calcium: 5 Hz: control: 1.54 ± 0.14 vs. ZX1: 1.61 ± 0.11, n = 5, p = 0.34, F = 0.9, DFn = 1, DFd = 40; 20 Hz: control: 2.78 ± 0.29 vs. ZX1: 2.94 ± 0.34, n = 5, p = 0.45, F = 0.56, DFn = 1, DFd = 40; 50 Hz: 4.44 ± 0.47 vs. ZX1: 3.14 ± 0.37, n = 10, p < 0.01, F = 22.59, DFn = 1, DFd = 90; two-way ANOVA. F, G, lower panels: ratio of the amplitude of 16–20 EPSC to first EPSC: ZnT3WT mice: 50 Hz: 5.580 ± 0.39 vs. ZX1: 3.96 ± 0.55, n = 6, p < 0.01, F = 28.37, DFn = 1, DFd = 50; ZnT3KO mice: control: 50 Hz: 1.71 ± 0.27 vs. ZX1: 1.74 ± 0.33, n = 5, p = 0.85, F = 0.04, DFn = 1, DFd = 40; two-way ANOVA. H, lower panel: ratio of the amplitude of 16–20 EPSC to first EPSC: ZnTWT vs. ZnT3KO: control: 50 Hz: 5.58 ± 0.39 vs. 1.71 ± 0.27, n = 5, p < 0.01, t = 8.15, df = 8; unpaired t-test.

Figure 7.

The postsynaptic inhibitory effect of zinc on AMPA EPSC does not contribute to the zinc-mediated enhancement of synaptic facilitation. A, Representative traces of AMPA EPSCs, evoked by 50-Hz 20-pulse train in the presence of 50 nm WIN 55 212-2, before (gray) and after (maroon) ZX1. B, Summary graph showing average AMPA EPSC amplitudes during the train, normalized to EPSC amplitude elicited by the first pulse, before (gray) and after (maroon) ZX1 (ratio of the amplitude of 16–20 EPSC to first EPSC at 50 Hz: control vs. ZX1, n = 5, p = 0.18). C, Summary graph showing average ratios of AMPA EPSCs in the presence of ZX1 to that in control during the train (ZX_1–2/control_1–2 vs. ZX_16–20/control_16–20, n = 5, p = 0.23). Values represent mean ± SEM. Detailed values and statistical tests: B, Ratio of the amplitude of 16–20 EPSC to first EPSC at 50 Hz: control: 3.63 ± 0.35 vs. ZX1: 3.23 ± 0.22, n = 5, p = 0.18, F = 1.84, DFn = 1, DFd = 40; two-way ANOVA; C, ZX_1–2/control_1–2 vs. ZX_16–20/control_16–20: 1.40 ± 0.04 vs. 1.35 ± 0.05, n = 5, p = 0.23, t = 1.39, df = 4, paired t-test.