Fig. 4: Adult IRSp53 re-expression rescues NMDA/AMPA ratios in IRSp53-cKO cortical neurons.

a Experimental scheme showing injection of PHP.eB-IRSp53 or control (PHP.eB-EGFP) into the retro-orbital sinus of WT or IRSp53-cKO mice at postnatal week 8 and electrophysiological measurements (evoked and miniature EPSCs) at postnatal week ~12. b Examples of layer 5 pyramidal neurons in the prelimbic region of the mPFC, where NMDAR/AMPAR-EPSCs or mEPSCs were measured, from WT mice injected with PHP.eB-EGFP into the retro-orbital sinus at postnatal week 8 and visualized under fluorescence microscopy at postnatal week 12 without additional EGFP staining. The EGFP signals in the neurons were enhanced to clearly indicate the neurons analyzed by patch-clamp recordings. c Sample traces of evoked NMDAR- and AMPAR-EPSCs from EGFP/IRSp53-expressing WT/IRSp53-cKO mice. d PHP.eB-IRSp53 injection normalizes the ratio of NMDAR-EPSCs and AMPAR-EPSCs in mPFC layer 5 pyramidal neurons of adult IRSp53-cKO mice. Note that IRSp53 overexpression in WT mice increases the NMDA/AMPA ratio (n = 10 neurons from 6 mice [WT-EGFP], 10, 4 [WT-IRSp53],17, 6 [IRSp53-cKO-EGFP], and 14, 4 [IRSp53-cKO-IRSp53], two-way ANOVA with Sidak’s test). e, f IRSp53 overexpression in WT neurons increases silent synapses, whereas IRSp53-re-expression in IRSp53-cKO neurons does not affect silent synapses (n = 11, 4 [WT-EGFP], 7, 2 [WT-IRSp53], 9, 3 [IRSp53-cKO-EGFP], and 10, 3 [IRSp53-cKO-IRSp53], two-way ANOVA with Sidak’s test, #p < 0.05 [Mann-Whitney test]). g, h Increased paired-pulse facilitation (PPF) in IRSp53-overexpressing WT mPFC layer 5 pyramidal neurons. WT mice were infected with PHP.eB-IRSp53, or PHP.eB-EGFP (control), at 8 weeks, and EGFP-positive neurons were used to measure PPF at 12 weeks (n = 11, 2 [WT-EGFP], 8, 2 [WT-IRSp53], two-way ANOVA with Sidak’s test). i, j Decreased PPF in IRSp53-re-expressing IRSp53-cKO mPFC layer 5 pyramidal neurons. IRSp53-cKO mice (Emx1) were infected with PHP.eB-IRSp53, or PHP.eB-EGFP (control), at 8 weeks, and EGFP-positive neurons were used to measure PPF at 12 weeks (n = 11, 2 [cKO-EGFP], 7, 2 [cKO-IRSp53], two-way ANOVA with Sidak’s test). Significance values are indicated as *p < 0.05, **p < 0.01, ***p < 0.001, or ns (not significant, p > 0.05). Error bars represent the standard errors of means.