Fig 2: Effects of PCDH7 overexpression in hippocampal slices. (A) Representative images of hippocampal CA1 pyramidal neurons overexpressing either GFP or PCDH7 + GFP in cultured hippocampal slices after biolistic transfection for 1 ~ 4 days. Images were taken from basal dendrites. Scale bar 10 µm. (B) Sample traces and quantification of evoked NMDAR mediated EPSCs from paired transfected (with either GFP or PCDH7) and neighboring untransfected hippocampal CA1 pyramidal neurons in organotypic brain slice cultures. Each black circle represents current amplitude of one pair of neurons. Blue circle represents mean amplitude ± SEM. Recordings were performed 3–4 days after transfection. 3 independent experiments, n = 11–14 pairs of neurons. **p < 0.01, Paired t test. (C) and (D) Representative images of hippocampal CA1 pyramidal neurons expressing c-terminal GFP tagged PCDH7 (PCDH7-GFP) with either DsRed (C) or c-terminal tdTomato tagged SHANK3 (SHANK3-tdTomato) (D). Both whole cell (top) and zoomed in basal dendrites (bottom) are shown. Scale bar 10 µm.

Fig 3: The ACh-M1R-PKC cascade activates PAK signaling in striatum/accumbal slices ex vivo.A, B Striatal/accumbal slices were pretreated with the M1R-specific antagonist VU0255035 (20 µM) or the PKC inhibitor GF109203X (10 µM) for 90 min and were then stimulated with carbachol for 5 min. NR1 phosphorylation, PAK autophosphorylation, and LIMK phosphorylation were quantified by immunoblotting over four independent experiments. One-way ANOVA followed by Tukey’s test, **p < 0.01, ***p < 0.001. C Striatal/accumbal slices were pretreated with the group I-specific PAK inhibitor FRAX486 (20 µM) for 6 h and were then treated with carbachol (1 mM) for 5 min. NR1 phosphorylation, PAK autophosphorylation, LIMK phosphorylation, and cofilin phosphorylation were quantified by immunoblotting over four independent experiments. The error bars represent the mean ± SEM. One-way ANOVA followed by Tukey’s test, n.s. not significant, **p < 0.01, ****p < 0.0001.

Fig 4: AMPA receptors have altered glycosylation state and are mislocalised in the cytoplasm in Frrs1 mice. (A) Post-translational glycosylation state is altered in Frrs1l-/-, demonstrated by differential digestion with glycosylation-sensitive enzymes compared with wild-type littermates. Frrs1l-/- show greater sensitivity of GLUA2 and GLUA4 digestion with ENDO-H (P<0.01); ~18% of wild-type GLUA2 and GLUA4 is digested compared with ~65% and 45%, respectively, in Frrs1l-/-. (B) Localisation of AMPA receptors is altered, with increased amount of AMPA receptor in the cytoplasm rather than the synapse in Frrs1l-/-. GLUA1 proportions in the cytoplasm and the synapse were not altered. (C) Overall levels of GRIN1 (NMDA receptor marker) in the post-synaptic fraction were unchanged between Frrs1l-/- and wild-type controls. Data analysed using Student’s t-test (n=3 Frrs1l-/-, n=3 Frrs1l+/+). Percentage of total GLUA2 and GLUA4 data are mean±s.d. ns, non-significant; *P<0.05, ****P<0.0001.

Fig 5: Electric foot shock activates PAK through M1Rs in accumbal D2R-MSNs.A VU0255035 was administered i.p. 25 min before the electric foot shock (0.4 mA, 2.0 s). n = 10. Error bars indicate mean ± SEM. Kruskal–Wallis test, followed by Dunn’s multiple comparisons test, *p < 0.05, **p < 0.01. B FRAX486 was administered s.c. 6 h before the electric foot shock (0.4 mA, 2.0 s). n = 8. Error bars indicate mean ± SEM. Kruskal–Wallis test, followed by Dunn’s multiple comparisons test, n.s. not significant, **p < 0.01. C VU0255035 was administered (40 mg/kg, i.p.) before the electric foot shock (0.4 mA, 2.0 s). Ten minutes after electric foot shock, NR1 phosphorylation and PAK autophosphorylation were quantified by immunoblotting. n = 7. Error bars indicate mean ± SEM. One-way ANOVA followed by Tukey’s test, ***p < 0.001, ****p < 0.0001. D Drd1a-tdTomato/Drd2-mVenus double transgenic mice were administered with VU0255035 (40 mg/kg, i.p.) before the electric foot shock. Ten minutes after electric foot shock, immunohistochemical analysis was performed. Immunofluorescence staining using an anti-pPAK1 (S144) antibody, an anti-GFP antibody, and TO-PRO3 is shown. Arrows indicate pPAK1 (S144)-positive D2R-MSNs. Scale bar, 10 µm. E The percentage of D1R-MSNs or D2R-MSNs among pPAK1 (S144)-positive cells after electric foot shock. Error bars indicate the mean ± SEM of three independent experiments. Student’s t test, **p < 0.01. F Quantification of the pPAK1 (S144)-positive D2R-MSNs in the NAc core. Error bars indicate the mean ± SEM of three independent experiments. One-way ANOVA followed by Tukey’s test, ****p < 0.0001.