Figure 4. Reduced intrinsic excitability and abnormal spontaneous firing activity of cerebellar PCs in ARF4+/−/ARF5−/− mice. A, Representative traces of AP firing in response to smaller (left; 400 pA injection) and larger (right; 800 pA injection) depolarizing current injections (500 ms) in WT (gray) and ARF4+/−/ARF5−/− (black) PCs. B, The average number of evoked APs plotted against the injected current amplitudes in WT (gray; n = 33) and ARF4+/−/ARF5−/− (black; n = 32) PCs. The data were collected from more than six mice in each condition. A repeated-measures two-way ANOVA indicates a significant interaction (genotype × injected current; F(19,1197) = 3.42; p < 0.0001). Multiple-comparison tests with Holm–Sidak's method show significant differences between WT and ARF4+/−/ARF5−/− at injected current strength indicated by: *1p = 0.0249, *2p = 0.0221, *3p = 0.0176, *4p = 0.0121, *5p = 0.0111. C, D, Respective pooled data of spike-train duration (ms) and interspike intervals (ms) at smaller 400 pA injection and larger 800 pA current injection. Spike-train duration was defined as the time difference between the first and the last AP events at each current injection. The interspike interval was measured as the mean of intervals between adjacent spike events at each current injection. *p < 0.005 between WT and ARF4+/−/ARF5−/− mice at 800 pA injection, Student's t test with multiple test correction. E, Pooled data of AP amplitudes at smaller 400 pA injection (left) and larger 800 pA current injection (right). Insets show the 15th AP waveforms (gray, WT; black, ARF4+/−/ARF5−/−) aligned with the AP threshold time points at which their differentiated waveform (dV/dt) exceeded 20 mV/ms. Horizontal and vertical scale bars in insets correspond to 0.5 ms and 20 mV, respectively. AP amplitude was measured from the threshold potential to the positive peak of each AP. Multiple-comparison tests show significant differences between WT and ARF4+/−/ARF5−/− from the 8th to 15th AP amplitudes (*p < 0.05) at 800 pA larger current injection, but not at 400 pA smaller current injection. F, Percentage of spontaneously active (i.e., spiking) PCs out of all the recorded PCs. Fischer's exact test indicates that the fraction of spontaneously active PCs is significantly smaller in ARF4+/−/ARF5−/− mice compared with WT (*p = 0.0101). G, Representative traces of spontaneously fired APs in WT (left, top, gray) and ARF4+/−/ARF5−/− (left, bottom, black) PCs. Right, Mean spontaneous spike rates in WT (gray) and ARF4+/−/ARF5−/− (black) PCs. *p = 0.0273, Student's t test.