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. 2021 Feb 24;118(9):e2019194118. doi: 10.1073/pnas.2019194118

Fig. 3.

Fig. 3.

Np65 deletion reduces AMPAR-mediated transmission. (A) Western blot for Np65 protein expression in primary neuronal cultures infected with Nptn sgRNA-expressing lentivirus and untransfected control (n = 3 replicates; ***P < 0.001). (B) Western blot results of the protein expression of Np65, GluA1, GluA2, NR2B, PSD-95, and Gapdh in Np65-knockout (KO) and untransfected wild-type (WT) neurons. Representative blots and bar graphs of the normalized protein expression levels are shown (mean ± SEM; n = 3 experiments; **P < 0.01; ***P < 0.001). (C and E) Scatterplots show the amplitudes of (C) AMPAR-EPSCs and (E) NMDAR-EPSCs for single pairs (open circles) of untransfected (control) cells and cells transfected with CRISPR_Nptn; the filled circle represents mean ± SEM amplitude. Insets show sample traces from control (gray) and transfected cells (green). The bar graphs to the right of the scatterplots show the mean ± SEM amplitude of (C) AMPAR-EPSC (control: 168 ± 11.9; CRISPR_Nptn: 120 ± 11.0; n = 32 pairs; five mice; ***P < 0.001) and (E) NMDAR-EPSCs (control: 64.5 ± 5.79; CRISPR_Nptn: 56.8 ± 4.57; n = 17 pairs; three mice; P = 0.4586; ns: not significant), (Scale bars, 25 pA, 25 ms.) (D) Representative traces of AMPAR-mediated currents from outside out patches from CRISPR_Nptn-transfected (green) and control (gray) neurons. (Scale bar, 200 pA, 500 ms.) The summary bar graph shows the mean ± SEM amplitude (control: 612 ± 51.5, n = 14; CRISPR_Nptn: 425 ± 47.5, n = 14; three mice; **P < 0.01). (F) Representative traces of paired-pulse ratio (PPR) from neurons transfected with CRISPR_Nptn (green) and control (gray). The bar graph to the right of the scatterplots shows the mean ± SEM of PPR (control: 2 ± 0.06; CRISPR_Nptn: 2 ± 0.07; n = 22 pairs; four mice; P = 0.9493; ns: not significant). (Scale bar, 25 pA, 25 ms.) (G) Representative traces of mEPSCs recorded from CA1 neurons electroporated with CRISPR_Nptn (green) and from untransfected control (gray) neurons. (Scale bars, 5 pA, 1 s.) Cumulative distributions of the mEPSC amplitude and interevent intervals are shown. The bar graphs show the mean ± SEM of mEPSC amplitude (control: 11.64 ± 0.47; CRISPR_Nptn: 9.9 ± 0.49; n = 10 pairs; three mice; *P < 0.05) and frequency (control: 0.22 ± 0.02; CRISPR_Nptn: 0.21 ± 0.01; n = 10 pairs; three mice; P = 0.92; ns: not significant).