Fig 2: PC-Specific Absence of SHISA6 Leads to Reduced PF to PC Excitatory Synaptic Input(A) Sample traces of evoked EPSCs generated by stimulating afferent PFs with increasing stimulation strengths. Evoked EPSCs were consistently of lower amplitude in the Shisa6L7 KO, whereas the PPF ratio was unaffected (control, 21–25 cells/6 animals; Shisa6L7 KO, 25–27 cells/7 animals).(B) Whole-cell patch clamp recording configuration of mEPSCs including raw traces. Top panels: mEPSC rise times and mEPSC decay times are significantly faster in the Shisa6L7 KO in comparison to controls (control, 30 cells/4 animals; ShisaL7 KO, 31 cells/4 animals). Lower panels: mEPSCs in Shisa6L7 KO PCs occur at a significantly lower frequency, but with comparable median amplitude (control, 30 cells/4 animals; KO, 31 cells/4 animals).(C) With the addition of FSK, we increase the GluA3-containing AMPAR conductance. Top panels: both the rise and decay time are significantly different between control and Shisa6L7 KO PCs, replicating the mEPSC trials without FSK. Lower panels: the mEPSC frequency is still largely reduced in the absence of SHISA6, but now a median amplitude difference is also revealed in the presence of FSK (control, 14 cells/3 animals; KO, 13 cells/3 animals).Data are represented as mean ± SEM; *p < 0.05, **p < 0.01. See also Figure S3 for CF and sIPSC data.

Fig 3: Rabbit anti-AMPAR GluR3B immunogenicity response. (A) ELISA of protein A-purified AMPAR (GluR3) Aabs against the GluR3 immunisation peptide or an irrelevant peptide; n = 3 technical replicates. (B) Western blot of mouse whole brain lysate probed with a commercial anti-AMPAR antibody (cAMPAR), anti-AMPAR Aabs, a class-specific negative control rIgG (naïve) or secondary antibody only (negative control). Representative blots from n = 3 technical replicates. (C) Immunocytochemical staining of fixed primary cortical neuron on cultures. Cells (DIV8) were stained with anti-AMPAR Aabs (red), anti-ßIII tubulin (green), GFAP (white) and nuclei counterstained with DAPI (blue). Examples of labelling of hippocampal neurons with anti-AMPAR Aabs (red) is indicated by the white arrows. Scale bars = 20 µm. Representative images from n = 3 technical replicates.