Interaction between Ephrins and mGlu5 metabotropic glutamate receptors in the induction of long-term synaptic depression in the hippocampus

Abstract

We applied the group-I metabotropic glutamate (mGlu) receptor agonist, 3,5-dihydroxyphenylglycine (DHPG), to neonatal or adult rat hippocampal slices at concentrations (10 microM) that induced a short-term depression (STD) of excitatory synaptic transmission at the Schaffer collateral/CA1 synapses. DHPG-induced STD was entirely mediated by the activation of mGlu5 receptors because it was abrogated by the mGlu5 receptor antagonist, MPEP [2-methyl-6-(phenylethynyl)pyridine], but not by the mGlu1 receptor antagonist, CPCCOEt [7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester]. Knowing that ephrin-Bs functionally interact with group-I mGlu receptors (Calò et al., 2005), we examined whether pharmacological activation of ephrin-Bs could affect DHPG-induced STD. We activated ephrin-Bs using their cognate receptor, EphB1, under the form of a preclustered EphB1/Fc chimera. Addition of clustered EphB1/Fc alone to the slices induced a small but nondecremental depression of excitatory synaptic transmission, which differed from the depression induced by 10 microM DHPG. Surprisingly, EphB1/Fc-induced synaptic depression was abolished by MPEP (but not by CPCCOEt) suggesting that it required the endogenous activation of mGlu5 receptors. In addition, coapplication of DHPG and EphB1/Fc, resulted in a large and nondecremental long-term depression. The effect of clustered EphB1/Fc was specific because it was not mimicked by unclustered EphB1/Fc or clustered EphA1/Fc. These findings raise the intriguing possibility that changes in synaptic efficacy mediated by mGlu5 receptors are under the control of the ephrin/Eph receptor system, and that the neuronal actions of ephrins can be targeted by drugs that attenuate mGlu5 receptor signaling.

Figure 1.

Pharmacological activation of mGlu5 receptors and ephrin-Bs depresses synaptic transmission at the Schaffer collateral/CA1 synapses in adult rat hippocampal slices. Pooled data normalized to the baseline, illustrating the effects of DHPG (A), clustered EphB1/Fc (B), DHPG + MPEP (C), DHPG + CPCCOEt (D), clustered EphB1/Fc + MPEP (E), clustered EphB1/Fc + CPCCOEt (F), clustered EphB1/Fc + DHPG (G), and clustered EphB1/Fc + DHPG + MPEP (H) on the slope of fEPSP are shown. Values are means ± SEM. Statistical analysis was performed by one-way ANOVA followed by Tukey's PLSD. In A (n = 5), values corresponding to the peak of synaptic depression (40–60 min) were significantly different from baseline values (p < 0.05); in B (n = 6), all values following the termination of Eph/Fc exposure were significantly different from baseline values (p > 0.05) except the value at time 0; in C (n = 3), none of the values with DHPG + MPEP differed from baseline values; in D (n = 3), peak values of synaptic depression (40–50 min) were significantly different from baseline values (p < 0.05); in E (n = 3), none of the values with EphB1/Fc + MPEP differed from baseline values; in F (n = 5), peak values of synaptic depression (40–60 min) were significantly different from the two initial baseline values; in G, (n = 6) note a robust nondecremental LTD induced by coapplication of EphB1/Fc and DHPG. DHPG was applied for 20 min following a 60 min exposure to EphB1/Fc. All values recorded at the end of DHPG exposure were significantly different from values recorded during exposure to EphB1/Fc alone (p < 0.05). Note in H (n = 5) that the synergism between EphB1/Fc and DHPG was abolished when DHPG was applied in the presence of MPEP. Representative traces illustrating fEPSP before, during, and after drug applications are shown in all figures. Traces are averages of four consecutive responses at the indicated time points.

Figure 2.

Same as in Figure 1, but in hippocampal slices from rats at postnatal days 7–9. In A and D (n = 6 and 4, respectively), values corresponding to the peak of synaptic depression (40–60 min) were significantly different from baseline values (p < 0.05); in B (n = 5), values following the termination of Eph/Fc exposure were significantly different from baseline values (p < 0.05); in F (n = 5), values from 50 to 60 min were significantly different from baseline values (p < 0.05); in C and E (n = 4 and 5, respectively), none of the values with DHPG + MPEP or clustered EphB1/Fc + MPEP differed from baseline values; in G (n = 5), we found the same synergism between EphB1/Fc and DHPG observed in adult hippocampal slices (see Fig. 1G). All values recorded at the end of DHPG exposure were significantly different from values recorded during exposure to EphB1/Fc alone (p < 0.05). Again, the synergism between EphB1/Fc and DHPG was abolished when DHPG was applied in the presence of MPEP (n = 5). Pool data from a different set of experiments with hippocampal slices from rats at postnatal days 7–9 are shown in I–M. In I (DHPG; n = 5) and K (unclustered EphB1/Fc + DHPG; n = 5), values corresponding to the peak of synaptic depression (from 40 to 80 min in I and from 79 to 110 min in K) were significantly different from baseline values (p < 0.05); the lack of effect of unclustered EphB1/Fc on synaptic transmission is shown in J (n = 6); in L (n = 6), clustered EphA1/Fc is shown to induce a long-lasting increase in synaptic transmission. Values at times >40 min are significantly different from baseline values; in M (n = 5), DHPG is still able to reduce synaptic transmission in the presence of clustered EphA1/Fc. Representative traces illustrating fEPSP are shown in all figures. Traces are averages of four consecutive responses at the indicated time points.

Figure 3.

A, mGlu5 receptors coimmunoprecipitate with ephrin-B2 in hippocampal protein extracts. From the left side: (1) expression of mGlu5 receptors in the hippocampus of adult wild-type or mGlu5 knock-out mice; (2) expression of mGlu5 receptors, ephrin-B2, and KCC2 in protein extracts from the hippocampus of 7- to 9-d-old rats used for immunoprecipitation (input); and (3) expression of mGlu5 receptors in ephrin-B2 immunoprecipitates, but not in KCC2 immunoprecipitates. IP, immunoprecipitates; WB, Western blotting. B, C, Stimulation of PI hydrolysis in neonate hippocampal slices challenged with DHPG (10 μm) in the absence or presence of CPCCOEt (10 μm) or MPEP (1 μm) or in the absence or presence of EphB1/Fc with or without MPEP is shown in B and C, respectively. Data are means ± SEM of 5–6 determinations. p < 0.05 [(one-way ANOVA + Tukey's PLSD) vs the respective values obtained in the absence of DHPG (*) or vs values obtained with DHPG alone (#)]. Tyrosine phosphorylation in ephrin-B2 immunoprecipitates from neonate hippocampal slices challenged with clustered EphB1/Fc, DHPG, or MPEP applied alone or in combination is shown in D. Data were normalized by the levels of total ephrin-B2 in immunoprecipitates. Densitometric values are means ± SEM of 4–5 determinations. *p < 0.05 (one-way ANOVA + PLSD vs control values).

Figure 4.

A, ERK1/2 phosphorylation in neonate hippocampal slices challenged with DHPG and clustered EphB1/Fc applied alone or in combination. Note that DHPG induced a slight a concentration-dependent activation of the MAPK pathway, whereas clustered EphB1/Fc was inactive. Values are means ± SEM of four determinations. p < 0.05 (one-way ANOVA + Tukey's PLSD) vs controls (*) or 10 μm DHPG (#). The effect of DHPG, clustered EphB1/Fc, and DHPG + clustered EphB1/Fc in neonate hippocampal slices treated with the MEK inhibitor, UO126, is shown in B–D, respectively (values are means + SEM of 4–5 determinations).

Figure 5.

A, Akt phosphorylation in neonate hippocampal slices challenged with DHPG and clustered EphB1/Fc applied alone or in combination. Note that neither DHPG nor clustered EphB1/Fc activated the PI-3-K pathway. The effect of DHPG, clustered EphB1/Fc, and DHPG + clustered EphB1/Fc in neonate hippocampal slices treated with the PI-3-K inhibitor, LY294002, is shown in B–D, respectively. Vales are means ± SEM. In B (n = 4), values from 40 to 80 min were significantly different from baseline values p < 0.05 (one-way ANOVA + Tukey's PLSD); in C (n = 5), values >70 min were significantly different from baseline values (p < 0.05); in D, the synergism between clustered EphB1/Fc and DHPG persists in the presence of LY294002. Values from 100 to 140 min were significantly different from values obtained with clustered EphB1/Fc alone (p < 0.05) (all points in the presence of LY294002).