Figure 8: Double KO of SALM3 and SALM4 normalizes excitatory synapse numbers in both Salm4^−/− and Salm3^−/− neurons.

(a) Comparison of SALM3 and SALM4 protein expression patterns in sagittal brain sections by X-gal staining of Salm3^+/− and Salm4^+/− single KO slices (5 weeks). Note that the hippocampal CA3 and CA1 signals are stronger than those in the dentate gyrus. Scale bar, 1 mm. (b) Salm3^−/−; Salm4^−/− CA1 pyramidal neurons display mEPSC frequencies comparable to those of WT neurons (P18–21), indicative of normalization in both in Salm4^−/− and Salm3^−/− neurons, although such effects are weaker in Salm3^−/− neurons. Note that the double KO has no effect on mEPSC amplitudes. n=12 cells (four mice) for WT, 11 (three mice) for Salm3^−/−, 12 (three mice) for Salm4^−/− and 13 (four mice) for Salm3^−/−; Salm4^−/−, *P<0.05, **P<0.01, ***P<0.001, ns, not significant, ANOVA with Bonferroni’ multiple comparison test; ^#P<0.05, Student’s t-test. (c) The double KO does not rescue the increased mIPSC frequency in Salm4^−/− CA1 pyramidal neurons. n=15 (four mice) for WT, 13 (three mice) for Salm3^−/−, 12 (three mice) for Salm4^−/− and 12 (four mice) for Salm3^−/−; Salm4^−/−, *P<0.05, ns, not significant, ANOVA with Bonferroni’ multiple comparison test. (d) The increased PSD density in the Salm4^−/− hippocampus is normalized by the double KO, as shown by the EM analysis of the hippocampal CA1 stratum radiatum region (P20). Normal and perforated PSDs are indicated by arrows and arrowheads, respectively. Scale bar, 500 nm. n=3 mice for WT, Salm4^−/−, and Salm3^−/−;Salm4^−/−, *P<0.05, **P<0.01, ns, not significant (P=0.1551 for PSD thickness and 0.3996 for perforated spines), ANOVA. (e) The increased density of symmetric, electron-dense synaptic structures in the Salm4^−/− hippocampus is not normalized by the double KO, as shown by the EM analysis of the hippocampal CA1 stratum radiatum region (P20). GABAergic terminals (asterisks) are indicated by EM immunogold staining for GABA. Postsynaptic dendrites (d) are also indicated. Scale bar, 500 nm. n=3 mice for WT, Salm4^−/−, and Salm3^−/−; Salm4^−/−, ***P<0.001, ns, not significant, ANOVA.