Figure 6.
G1 mGluR-dependent Z-LTD reduces presynaptic zinc release and zinc inhibition of NMDARs. A, Left, Time course of AMPAR EPSC amplitude before and after HFS, and NMDAR EPSC amplitude before and after subsequent ZX1 application (blue); similar time course in control experiments (without HFS, red); and similar time course with HFS in the presence of LY367385 (100 μM) and MPEP (4 μM) (green). After obtaining a stable baseline of AMPAR EPSCs, HFS was delivered; then DNQX (20 μM) was applied. NMDAR EPSCs were then recorded at 40 mV, normalized to the baseline NMDAR EPSC amplitude before ZX1 application. The switch from AMPAR to NMDAR EPSC time course, and the renormalization of EPSC amplitude are indicated by a gap and restart of timing in the x axis. In controls, ZX1 potentiated NMDAR EPSCs (n = 5, *p = 0.027, paired t test). After HFS, ZX1 did not potentiate NMDAR EPSCs (n = 6, not significant, p = 0.16, paired t test). After HFS + LY367385/MPEP, ZX1 potentiated NMDAR EPSCs (n = 5, *p = 0.038, paired t test). Right, Example NMDAR EPSCs before and after ZX1 application. B, Average ZX1 potentiation (% increase from baseline) during the last 5 min of ZX1 application (minutes 11-15). Control: n = 5; HFS: n = 6; HFS + LY367385, MPEP: n = 5. HFS reduced ZX1 potentiation compared with control (*p = 0.012); this reduction was blocked by LY367385 and MPEP (*p = 0.015). One-way ANOVA/Bonferroni. C, Dose–response of NMDAR EPSCs (% baseline) for increasing concentrations of ifenprodil, in controls (red) and after HFS (blue). Control: n = 3-5 per concentration; HFS: n = 3 or 4 per concentration. D, IC50 of ifenprodil, from dose–responses in C. Not significant, p = 0.97, comparison of fits, extra sum-of-squares F test. E, Top left, Representative stimulus-evoked (50 pulses, 100 Hz) zinc-mediated fluorescent signals using LZ9 (2 μM), before and after HFS. Dotted white line outlines the edge of the DCN slice. White triangle represents the stimulating electrode. Yellow square represents the ROI. Top right, Time course of representative ratiometric fluorescent signals, before and after HFS. Arrow indicates the time of stimulation. HFS significantly reduced stimulus-evoked zinc fluorescence (maximum ΔR/R) (n = 6, *p = 0.0002, paired t test). Bottom, Same as above, but in the presence of LY367385 (100 μM) and MPEP (4 μM). F, Average stimulus-evoked zinc fluorescence (maximum ΔR/R) after HFS, normalized to baseline before HFS. Compared with HFS alone (n = 6), LY367385/MPEP (n = 6) significantly attenuated the HFS-induced reduction of zinc fluorescence (*p = 0.0005, unpaired t test).