Figure 10. Activation of hM4di in the NRM abrogates spontaneous recovery of skilled locomotor function after bilateral pyramidotomy. A–H, The effects of transiently silencing the NRM via activation of hM4di on skilled locomotion after bPyX was assessed by counting the number of footfalls made by forelimbs (A, B) and hindlimbs (E, F) during 3 min of grid walking on a 2.5 × 2.5 cm mesh grid and analyzed using repeated-measures ANOVA with Bonferroni's correction for multiple comparisons (A, B, E, F) with post hoc ANOVA (A–H). Data are shown as the average number of missed steps expressed as a percentage of total steps made over 3 min ± SEM. As in (Fig. 3), both ngr1+/+ (n = 14) and ngr1−/− (n = 14) mice made significantly more errors with their left forelimbs (#F(3,29) = 1802.362, p < 0.0005), right forelimbs (#F(3,29) = 1960.634, p < 0.0005), left hindlimbs (#F(3,29) = 2202.006, p < 0.0005), and right hindlimbs (#F(3,29) = 1959.227, p < 0.0005) after bPyX compared to sham-lesioned control ngr1+/+ (n = 10) and ngr1−/− (n = 10) mice (Fig. 9). Consistent with Figure 3, ngr1+/+ mice demonstrated significant spontaneous functional recovery in all four limbs by 14 dpl, and ngr1−/− mice by 7 dpl, compared to maximum deficits at 4 dpl (C, D, G, H; *p < 0.005). Intraperitoneal CNO significantly attenuated spontaneous recovery made by both ngr1+/+ and ngr1−/− mice in left forelimbs (A; #F(3, 32) = 62.569, p < 0.0005), right forelimbs (B; #F(3, 32) = 65.452, p < 0.0005), left hindlimbs (E; #F(3, 32) = 104.047, p < 0.0005), and right hindlimbs (F; #F(3, 32) = 95.395, p < 0.0005). For ngr1+/+ mice, compare black lines and black dots for intraperitoneal vehicle with black lines and blue dots for intraperitoneal CNO (black arrow heads; post hoc ANOVA, p < 0.005). For ngr1−/− mice, compare red lines and red dots for intraperitoneal vehicle with red lines and blue dots for intraperitoneal CNO (red arrow heads; post hoc ANOVA, p < 0.005). Post hoc analyses showed that intraperitoneal CNO nullified spontaneous recovery observed in ngr1+/+ mice up to 35 dpl in the left forelimb (C), and up to 21 dpl in the right forelimb (D) and left (G) and right (H) hindlimbs. Intraperitoneal CNO also nullified spontaneous recovery in ngr1−/− mice up to 28 dpl in the left forelimb (C) and up to 21 dpl in the right forelimb (D) and left (G) and right (H) hindlimbs. *p < 0.005. Although CNO treatment abrogated spontaneous functional recovery in both genotypes, ngr1−/− mice recovered significantly better than ngr1+/+ mice after bPyX after both vehicle (A, B, E, F, black asterisk #p < 0.0005, post hoc ANOVA; *p < 0.005, compare black lines and black spots with red lines and blue spots) and CNO (A, B, E, F, blue asterisk; #p < 0.05, post hoc ANOVA; *p < 0.05, compare black lines and blue spots with red lines and blue spots) treatment.