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. 2024 Jan 23;5(2):101389. doi: 10.1016/j.xcrm.2023.101389

Figure 6.

Figure 6

AUT00206 modulates Kv3 current, enhances CGC excitability, and improves motor function and seizure susceptibility of H/+ mice

(A‒C) HEK293T cells co-transfected with Kv3.1-WT and Kv3.1-Arg320His variant at a 1:1 ratio. Shown in (A) are families of currents generated in response to voltage steps from −40 to +40 mV in increments of 5 mV from a holding potential of −80 mV. (B) Overlaid currents generated in response to voltage steps to −10 mV (indicated by a star [★] in A) under control conditions (light blue), after application of 10 and 30 μM AUT00206 (dark blue), and after washout (black; overlaid with control trace). AUT00206 (AUT6) produces a leftward shift in the conductance-voltage (G-V) plot and increased current/conductance at relevant voltages (C). DMSO, n = 13; 10 μM, n = 12; 30 μM, n = 12; 100 μM, n = 9; washout, n = 6.

(D) Representative traces of CGC firing from 6-month-old mice before/after bath application of 100 μM AUT6.

(E) Current-frequency (I-f) curves show increased excitability of CGCs from both +/+ and H/+ mice with application of AUT6. +/+ mice, n = 20; H/+ mice, n = 20.

(F) Representative traces of action potentials of PV-INs in response to repetitive stimulation at 40 Hz before/after bath application of 100 μM AUT6.

(G) Summary plot of PV-IN firing fidelity in response to 40 and 80 Hz repetitive stimulation before and after bath application of 100 μM AUT6. 40 Hz, n = 8; 80 Hz, n = 8.

(H) A single i.p. injection of 30 mg/kg AUT6 improves performance on the rotarod. H/+ mice: vehicle, n = 9; AUT6, n = 15. +/+ mice: vehicle, n = 12; AUT6, n = 16. Each mouse received 4 test sessions on the rotarod. Shown here is the average of the 4 test sessions.

(I) A single i.p. injection of 30 mg/kg AUT6 improves performance on the ledge test. H/+ mice: vehicle, n = 7; AUT6, n = 10. +/+ mice: vehicle, n = 7; AUT6, n = 11.

(J) Acute i.p. injections of 30 mg/kg AUT6 delivered 45–60 min prior to PTZ administration attenuates kindling epileptogenesis in H/+ mice. Note that the two lines, +/+ vehicle (gray) and +/+ AUT6 (black), are completely superimposed. H/+ mice: vehicle, n = 6; AUT6, n = 10. +/+ mice: vehicle, n = 7; AUT6, n = 11.

(K) Continuous video EEG detected baseline spontaneous seizures (2 or more seizures in 72 h) in 6 out of 19 mice.

(L) Summary of average seizure frequency per day during baseline monitoring (vehicle, without AUT6) and drug delivery phase (with AUT6) in the 6 mice with spontaneous seizures. See also Figures S7–S11 and Table S1. Data are mean ± SEM.

Statistical analyses: two-way ANOVA with Šídák’s multiple comparisons test (E), paired t test (G and L), unpaired t test (H and I), and Mantel-Cox test (J). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.001. Exact p values and experimental n can be found in Table S1.