Figure 5. Riluzole blocks GSK3-induced excitability.

(a,b) Representative model recordings (1 s) from nighttime cell with I_NaP at WT (a) or GSK3-KI (b) level shows that increasing I_NaP alone is sufficient to induce spontaneous activity in a silent cell. Scale bar, 20 mV, 200 ms. Grey line indicates RMP (−63.4 mV). (c) Percentage of silent versus spiking neurons seen in SCN network model at night with WT or GSK3-KI levels of I_NaP before and after I_NaP blockade with riluzole. (d) Box plot of early-night spontaneous AP frequencies of SCN neurons from WT (grey) or GSK3-KI (red) mice treated with vehicle (DMSO, 0.01%) or riluzole (10 μM), showing 10th and 90th percentiles (whiskers), 25th and 75th percentiles (box borders), median (centre line) and outliers (symbols). Scheirer–Ray–Hare Kruskal–Wallis test, main effect of genotype, H₍₁₎=7.009, P<0.001; main effect of treatment, H₍₁₎=8.089, P<0.001 and interaction, H₍₁₎=4.834, P<0.001, post hoc asymptotic significance, P<0.001. (e) Representative cell-attached loose-patch traces (5 s) from each group. Scale bar, 20 mV, 1 s. (f) Quantification of silent versus non-silent cells for each group in d and e. Three-way loglinear analysis, three-way interaction, χ²₍₁₎=25.852, P<0.001. Follow-up χ² tests revealed that GSK3-KI cells were significantly more likely to be spiking than WT vehicle-treated cells (χ²₍₁₎=32.428, P<0.001). Blocking I_NaP with riluzole increased the proportion of silent cells in GSK3-KI slices (χ²₍₁₎=44.735, P<0.001) up to that of WT levels (χ²₍₁₎=0.430, P=0.313), whereas riluzole had no effect on the proportion of silent cells in WT slices (χ²₍₁₎=0.034, P=0.5). For d and f: WT and WT+riluzole, n=60 cells, 2 animals; GSK3-KI, n=84 cells, three animals; GSK3-KI+riluzole, n=86 cells, three animals.