Figure 6. ArpC3-deficient synapses are functionally immature. A, Effect of ArpC3 KO on AMPAR/NMDAR IEI ratios in CA1 pyramidal neurons (WT: 0.2 ± 0.2, n = 14; KO: 1.0 ± 0.2, n = 12; p = 0.034). B, Representative traces of pharmacologically isolated AMPAR- and NMDAR-mEPSCs (as indicated) recorded from ArpC3f/f (WT, blue) and ArpC3f/f:Cre+ (KO, red) neurons. C, D, IEI cumulative probability plots for AMPAR-mEPSCs (C) and NMDAR-mEPSCs (D). Insets show median values (see right vertical axes, units are in seconds). AMPAR-mEPSC IEIs were increased in ArpC3 KO versus WT neurons (WT: 0.38 ± 0.13 s, n = 6; KO: 1.87 ± 0.50 s, n = 6; t(10) = 3.54, p = 0.0050); NMDAR-mEPSC IEIs were not significantly different (WT: 1.99 ± 0.38 s, n = 9; KO: 2.02 ± 0.36 s, n = 7; t(14) = 0.043, p = 0.97). E, F, Amplitude cumulative probability plots for AMPAR-mEPSCs (E) and NMDAR-mEPSCs (F). Insets show median values (see right vertical axes, units are in picoamperes). Compared with WT neurons, KO neurons exhibited a left-shifted AMPAR-mEPSC amplitude distribution and reduced median current amplitudes (WT: 13.1 ± 1.3 pA, n = 6; KO: 24.0 ± 3.9 pA, n = 6; t(10) = 3.00, p = 0.013); the NMDAR-mEPSC amplitude distributions and median current amplitudes were comparable between groups (WT: 14.96 ± 0.89 pA, n = 9; KO: 16.43 ± 0.74 pA, n = 7; t(14) = 1.23, p = 0.24). G, Peak-scaled waveform averages of NMDAR-mEPSCs from WT (black, DIV3–5; blue, DIV14–21) and DIV14–21 ArpC3 KO (red) neurons. Scatter plots show that amplitude-weighted decay constants were decreased 69% from DIV 3–5 to DIV 14–21 in WT neurons (DIV 3–5: 85.5 ± 1.1 s, n = 5; DIV 14–21: 50.7 ± 6.5 s, n = 9; t(12) = 3.88, p = 0.0022) but were comparable between WT and KO neurons at DIV 14–21 (KO: 45.1 ± 7.3 s, n = 7; t(14) = 0.58, p = 0.57). H, Waveform averages from minimal stimulation experiments (black, WT and KO failures; blue, WT success; red, KO success) showing selective effects of ArpC3 KO on AMPAR-mEPSC failure rates (WT: 13.5 ± 2.2%, n = 4; KO: 44.0 ± 7.6%, n = 4; t(6) = 4.46, p = 0.0044) compared with NMDAR-mEPSC failures (WT: 39.2 ± 7.2%, n = 5; KO: 30.2 ± 5.5%, n = 5; t(8) = 0.99, p = 0.35). I, Representative traces of AMPAR-mEPSCs recorded from ArpC3f/f (WT, blue), ArpC3f/f:Cre+:FLEx-GFP (KO, red), and ArpC3f/f:Cre+:FLEx-ArpC3 (rescue, green) neurons. J–M, Cumulative probability plots and sample distributions illustrate ArpC3's KO and rescue effects on mEPSC amplitude (ANOVA main effect: F(2,20) = 14.40, p < 0.001) and IEI (ANOVA main effect: F(2,20) = 26.93, p < 0.001). Post hoc comparisons revealed significant differences between WT and KO amplitudes (WT: 9.50 ± 0.69 pA, n = 10; KO: 5.97 ± 0.85 pA, n = 5; p = 0.004) and IEIs (WT: 0.26 ± 0.054 s, n = 10; KO: 1.19 ± 0.11 s, n = 5; p = 0.003). Whereas rescued amplitudes and IEIs were not significantly different from those of WT neurons (rescue amplitude: 9.43 ± 0.45 pA, n = 5; p = 0.99; rescue IEI: 0.32 ± 0.08 s, n = 5; p = 0.92), both values were significantly different from those of KO neurons (KO amplitude, p = 0.02; KO IEI, p = 0.011). Error bars indicate SEM; *p < 0.05; **p < 0.01.