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. 2016 Nov 9;36(45):11521–11531. doi: 10.1523/JNEUROSCI.1519-16.2016

Figure 2.

Figure 2.

Induction of LTP by group II mGlu receptor activation requires NMDAR activation. A, fEPSPs recorded in the stratum radiatum express LTP only if group II mGlu receptor activation (LY354740, 1 μm, 10 min) occurs concomitantly with repetitive Schaffer collateral stimulation (0.05 Hz). LTP is prevented by blocking NMDARs (D-AP5, 40 μm, 10 min). Insets, Representative traces (averages of six sweeps) before (1) and after (2) drug application. Scale bars, 0.5 mV and 10 ms. B, Quantification of fEPSP slope changes under the conditions presented in A (no stimulation, D-AP5). ****p < 0.0001, *p < 0.05 indicates significantly different from slices treated only with LY354740 (ANOVA followed by Tukey's post hoc test; F(2, 18) = 14.23). C, Whole-cell patch-clamp recordings of responses evoked by Schaffer collateral stimulation show potentiation by activation of group II mGlu receptors during experiments in current-clamp mode, but not in voltage-clamp mode at −70 mV, a potential at which NMDARs are blocked significantly by magnesium. Insets, Representative averaged traces. Scale bars, 10 mV/20 pA and 5 ms. D, Quantification of EPSPs and EPSCs showing LY354740-induced LTP only in current-clamp mode, two-tailed paired Student's t test. E, Pharmacologically isolated NMDA currents (NBQX, 25 μm, 10 min) evoked by Schaffer collateral stimulation at a holding potential of −70 mV with low extracellular magnesium (0.1 mm) are potentiated in response to activation of group II mGlu receptors (LY354740, 1 μm, 10 min). NMDAR-mediated currents are not potentiated if PKC is blocked (bisindolylmaleimide, 1 μm, 20 min). Insets, Representative traces (averages of six sweeps) before (1) and after (2) drug application and after 40 μm D-AP5 (3) to confirm the selective activation of NMDARs. Scale bars, 20 ms and 20 pA. F, Quantification of EPSCs before, during, and after application of LY354740 in control conditions and when PKC is blocked. ****p < 0.0001, *p < 0.05 indicates significantly different from the baseline (ANOVA followed by Tukey's post hoc test; LY35: F(2, 17) = 100.3; LY35 + bisindolylmaleimide; F(2, 15) = 225.7). G, Western blots showing phosphorylation at serine 896 of the GluN1 subunit (GluN1 p-S896) in control conditions after application of 1 μm LY354740 alone or in combination with 1 μm bisindolylmaleimide. The specificity of the primary antibody against GluN1 p-S896 was confirmed by addition of an antibody-blocking peptide. The protein ladder indicates molecular weight markers (M) in kDa. H, Protein quantification reveals increased phosphorylation at GluN1 p-S896 after activation of group II mGlu receptors. This effect is blocked by the PKC antagonist. ****p < 0.0001, *p < 0.05 indicates significantly different from the control (ANOVA followed by Tukey's post hoc test; F(2, 16) = 4.119).