PMC

Activation of hM4di in the NRM fails to alter skilled locomotion after sham lesion. A–D, Skilled locomotion assessment of ngr1^+/+ (n = 10) and ngr1^−/− (n = 10) mice using grid-walking analysis for 5 weeks starting 3 weeks after AAV-mediated transfection of the NRM with hM4di showed that the percentage of missed steps made by all four limbs was unaltered before or at anytime after sham lesion after treatment with vehicle or CNO. Error bars indicate SEM.

Bilateral pyramidotomy does not alter raphespinal terminal distribution. A, A schematic shows the location of the NRM and the bilateral spinal innervation of the RPST. B, Bar chart shows average density of 5HT+ axons per mm² (±SEM) in cervical spinal ventral horn in bPyX and sham-lesioned ngr1^+/+ and ngr1^−/− mice. C–J, Photomicrographs show 5-HT+ raphespinal axons terminating in the C6/C7 spinal ventral horn contralateral (C–F) and ipsilateral (G–J) to a unilateral BDA injection into the red nucleus in ngr1^+/+ (C, G, E, I) and ngr1^−/− (D, H, F, J) mice after sham (C, D, G, H; n = 9 for ngr1^+/+; n = 8 for ngr1^−/−) or bPyX (E, F, I, J; n = 11 per genotype). There was no significant difference in the density of 5-HT+ raphespinal axons in the ipsilateral (G–J) or contralateral (C–F) ventral horn between ngr1^+/+ (C, G, E, I) and ngr1^−/− mice (D, H, F, J) after bPyX or sham lesion (B). Scale bar, 500 μm.

Bilateral pyramidotomy-induced rubral sprouting drives spontaneous recovery of function after complete corticospinal tract lesion. A–E, Summary schematics show the origin and termination pattern of the CST (green lines), RST (from the red nucleus; red lines), and the RPST (from the nucleus raphe magnus; blue lines) in the brainstem (A) and spinal cord (D) of adult intact mice. bPyX (B, C) results in spinal degeneration of the CST and retraction of CST terminals from the spinal cord in both ngr1^+/+ (E) and plasticity-sensitized ngr1^−/− (F) mice. Lesion-induced de novo connections (red stippled lines) formed between the red nucleus and premotor brainstem nuclei (B; basilar pontine nuclei and nucleus raphe magnus) and motor neurons in the spinal cord (E) after complete ablation of the CST in adult ngr1^+/+ mice initiates spontaneous functional recovery. bPyX-induced sprouting of rubral connections in the brainstem (C) and spinal cord (F) is enhanced in ngr1^−/− mice, significantly elevating spontaneous recovery of function.

Bilateral pyramidotomy increases plasticity of intact rubrofugal terminals in the basilar pontine nuclei. A, A schematic shows the location of the BPNs, the site of unilateral BDA injection into the red nucleus, and the innervation pattern of rubropontine axons (red stippled line) into the BPNs. B, Bar chart shows average density of BDA+ rubropontine axons per mm² (±SEM) in BPNs in bPyX and sham-lesioned ngr1^+/+ and ngr1^−/− mice. C–J, Photomicrographs show BDA+ rubropontine axons terminating in the BPNs contralateral (C–F) and ipsilateral (G–J) to the unilateral BDA injection into the red nucleus in ngr1^+/+ (C, G, E, I) and ngr1^−/− (D, H, F, J) mice after sham (C, D, G, H) or bPyX (E, F, I, J) lesion. There was no significant difference in the density of BDA+ rubropontine axons in the ipsilateral (G, H) or contralateral (C, D) BPNs between ngr1^+/+ (n = 9) and ngr1^−/− (n = 8) mice after sham lesion. The density of BDA+ rubropontine axons was significantly elevated in both the contralateral and ipsilateral BPNs 6 weeks after bPyX in ngr1^+/+ and ngr1^−/− mice compared to sham-lesioned ngr1^+/+ and ngr1^−/− mice (B; ^#p < 0.005, ANOVA; compare C, G and E, I; compare D, H and F, J). Lesion-induced enhanced plasticity of BDA+ rubropontine axon terminals was significantly augmented in ngr1^−/− compared to ngr1^+/+ mice in both contralateral and ipsilateral BPNs 6 weeks after bPyX (B; *p < 0.005, ANOVA; compare E, F; compare I, J). Scale bar, 100 μm.

Bilateral pyramidotomy significantly increases rubrofugal innervation of the nucleus raphe magnus. A, A schematic shows the relative location of the red nuclei and the NRM, the site of the unilateral BDA injection, and the location of the de novo rubro–raphe circuit (red stippled line). B–M, Photomicrographs show BDA+ rubro-raphe axons (B, E, H, K) in close apposition to 5-HT+ NRM neurons (C, F, I, L) and overlay (D, G, J, M; red, BDA+ axons; green, 5-HT+ NRM neurons) 6 weeks after bPyX (H–M; n = 11 per genotype) or sham lesion (B–G; n = 9 for ngr1^+/+; n = 8 for ngr1^−/−) in ngr1^+/+ (B–D, H–J) and ngr1^−/− (E–G, K–M) mice. There was no significant difference in the density of BDA+ rubro–raphe axons in the NRM between ngr1^+/+ and ngr1^−/− mice after sham lesion (N; data are shown as the average density of BDA+ axons per square millimeter ± SEM; compare D, G). The density of BDA+ rubro–raphe axons was significantly elevated in the NRM 6 weeks after bPyX in ngr1^+/+ and ngr1^−/− mice compared to sham-lesioned ngr1^+/+ and ngr1^−/− mice (N; ^#p < 0.005, ANOVA; compare D, J; compare G, M). Lesion-induced enhanced plasticity of BDA+ rubro–raphe axon terminals was significantly augmented in ngr1^−/− compared to ngr1^+/+ mice in the NRM 6 weeks after bPyX (N; *p < 0.005, ANOVA; compare E, F; compare I, J). Scale bar, 100 μm.

Bilateral pyramidotomy results in a transient deficit in skilled forelimb function. A–D, Skilled forelimb motor function was assessed using the tape removal task and analyzed using repeated-measures ANOVA with a Bonferroni correction for multiple comparisons (A, B) with post hoc ANOVA (A–D). Data are shown as the average time taken in seconds to remove the tape from each forelimb within each group ± SEM. The time taken to remove the tape was significantly increased by the left forelimb (A, ^#F_(3,20) = 11.872, p < 0.005, post hoc; *p < 0.005) and the right forelimb (B, ^#F_(3,20) = 9.403, p < 0.005, post hoc; *p < 0.005) in ngr1^+/+ mice after bPyX (A, B, black lines; n = 7) compared to sham-lesioned controls (black stippled lines; n = 6). Lesion-induced deficits failed to reach significance in the time taken to remove the tape by the left forelimb (A; NSD, p = 0.09) or right forelimb (B; NSD, p = 0.061) in ngr1^−/− mice after bPyX (red lines; n = 7) compared to sham-lesioned (red stippled lines; n = 6) controls. There was no significant difference in the time taken to remove the tape between bPyX ngr1^+/+ and bPyX ngr1^−/− mice in either their left forelimb (A; NSD, p = 0.387) or right forelimb (B; NSD, p = 0.44). Assessment of the effect of lesion over time within each group showed that both ngr1^+/+ and ngr1^−/− mice displayed a significant lesion induced increase in the time taken to remove the tape after bPyX (*p < 0.05) in both left (C) and right forelimbs (D). Both ngr1^+/+ and ngr1^−/− demonstrated spontaneous restitution of function back to prelesion baseline levels by day 21 in the left forelimb (C) and by day 14 in the right forelimb (D).

Bilateral pyramidotomy does not influence nonskilled locomotor function. A, A schematic shows the location of the bPyX and degeneration of the CST. B–E, Photomicrographs of caudal brainstem and C7 spinal cord immunostained for PKCγ show the intact corticospinal tract in the medullary pyramids (B, green) and the spinal ventral dorsal column projection (D, white) of ngr1^+/+ mice 4 weeks after sham lesion and its absence 4 weeks after bPyX (C, E). Intense GFAP immunoreactivity delineates the extent of the lesion and confirms its confinement to the pyramidal tract (C). F, G, Assessment of nonskilled locomotor function with footprint analysis revealed that neither ngr1^+/+ (n = 9, black bars) nor ngr1^−/− (n = 9) mice displayed a functional deficit 4 d (F) or 35 d (G) after bPyX in forelimb and hindlimb stride length or forelimb and hindlimb base of support compared to sham-lesioned ngr1^+/+ (n = 7) and ngr1^−/− (n = 8) controls. Gait parameters described in F are schematized in F1. Data are displayed as average distance in gait parameter as a percentage of sham ± SEM. F1–F4, G1–G4, Examination of footprints (red ink applied to forelimbs and blank ink to hindlimbs) from ngr1^+/+ (F1, F2) and ngr1^−/− mice (F3, F4) 4 d after bPyX (F2, F4) or sham lesion (F1, F3), and ngr1^+/+ (G1, G2) and ngr1^−/− mice (G3, G4) 35 d after bPyX (G2, G4) or sham lesion (G1, G3) shows no difference in stride length or base of support parameters (described in F1). Scale bar, 500 μm.

Bilateral pyramidotomy results in significant and sustained functional deficits in skilled locomotion. A–H, Skilled locomotion was assessed by counting the number of footfalls made during 3 min of grid walking on a 2.5 × 2.5 cm mesh grid and analyzed using repeated-measures ANOVA with a Bonferroni 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. A, B, E, F, Both ngr1^+/+ (black lines; n = 11) and ngr1^−/− (red lines; n = 12) mice showed a significant increase in the percentage of missed steps made by the left forelimbs (A; ^#F_(3,32) = 297.555, p < 0.005), the right forelimbs (B; ^#F_(3,32) = 210.306, p < 0.005), the left hindlimbs (E; ^#F_(3,32) = 265.443, p < 0.005), and the right hindlimbs (F; ^#F_(3,32) = 319.533, p < 0.005) compared with sham-lesioned ngr1^+/+ (n = 9) and ngr1^−/− (n = 9) controls. Additionally, ngr1^−/− mice recovered significant function of all limbs compared to ngr1^+/+ mice after bPyX. ^#p < 0.005 with post hoc ANOVA; *p < 0.005. C, D, G, H, Assessment of the effect of lesion over time within each group showed that both ngr1^+/+ and ngr1^−/− mice showed a significant lesion-induced increase in the percentage of missed steps made by all limbs after bPyX that was sustained throughout the testing period. Ngr1^+/+ demonstrated significant spontaneous functional recovery in all limbs by 14 dpl compared to the maximum deficits observed at 2 dpl. However, ngr1^−/− showed accelerated spontaneous functional recovery, as by day 7 after lesion they had already made significantly fewer errors compared to the maximum deficits observed at 2 dpl. *p < 0.005.

Bilateral pyramidotomy increases plasticity of the intact rubrospinal tract. A, A schematic shows the location of the red nuclei, the location of the BDA injection, and the unilateral projection of the rubrospinal tract in the lateral funiculus of the spinal cord and its termination pattern at every spinal segment. B, Photomicrograph shows a coronal section of brain (with corresponding plate from mouse atlas) through the red nucleus 6 weeks after a 75 nl stereotaxic microinjection of BDA (BDA, red; DAPI, blue). The discrete immunofluorescence confirms the fidelity of targeting the red nuclei specifically. C, D, Bar charts show average density of BDA+ RST axons per mm² (±SEM) in the cervical spinal cord (C) and PnC (D) in bPyX and sham-lesioned ngr1^+/+ and ngr1^−/− mice. E–H, Photomicrographs show BDA+ RST axons (with map of corresponding spinal segment) in the DLF and terminals in intermediate laminae in coronal sections of C7 spinal cord of ngr1^+/+ (E, G) and ngr1^−/− (F, H) mice after bPyX (G, H) and sham lesion (E, F). There was no significant difference in the density of BDA+ RST axons in the dorsal or ventral halves (E; delineated in line with the central canal) of the spinal cord at cervical segments C6–C8 between ngr1^+/+ and ngr1^−/− mice after sham lesion (C; compare photomicrographs E, F). The density of BDA+ RST axons was significantly elevated in both the dorsal and ventral halves of the spinal cord 6 weeks after bPyX in ngr1^+/+ (n = 11) and ngr1^−/− (n = 11) mice compared to sham-lesioned ngr1^+/+ (n = 9) and ngr1^−/− (n = 9) mice (C; ^#p < 0.005, ANOVA, compare E, G; compare F, H). Lesion-induced enhanced plasticity of BDA+ RST axon terminals was significantly augmented in ngr1^−/− compared to ngr1^+/+ mice in both dorsal and ventral halves of the spinal cord 6 weeks after bPyX (C; *p < 0.005, ANOVA, compare G, H). Assessment of BDA+ axon density in the pontine reticular nucleus revealed no significant difference in BDA tracing efficiency between genotypes after bPyX or sham lesion (D; data are shown as density of BDA+ axons per square millimeter ± SEM). Scale bar, 100 μm.

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 (), 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 (). Consistent with , 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.

Activation of the inhibitory DREADD receptor hM4di abrogates activity-induced c-fos expression in NRM neurons. A, D, A schematic shows the relative location of the red nuclei, NRM, and rubro–raphe axons (red stippled line) and the location of AAV2-CAG-hSyn-hM4di-HA-IRES-mCitrine injection into the NRM (A) and its relative location to the LVNs (D). B, Intraperitoneal injection of CNO activates hM4di and silences NRM-projecting raphespinal axons (outlined in blue). The time line for experiments described in – is schematized in C. Ngr1^+/+ (n = 36) and ngr1^−/− (n = 36) mice received a single 75 nl stereotaxic injection of high-titer (1 × 10¹³ pfu/ml) AAV-hM4di into the NRM. After 3 weeks, mice were split into two groups: a red group, to anatomically assess the expression of hM4di and the molecular activation status of NRM neurons expressing hM4di (n = 12 per genotype), and a blue group (n = 24 per genotype), to assess the functional impact of transiently silencing NRM neurons after bPyX or sham lesion. Unlesioned ngr1^+/+ and ngr1^−/− mice in the red group underwent behavioral assessment using the grid-walking task on day 0 (3 weeks after AAV injection), 30 min after an intraperitoneal injection of either vehicle (n = 6 per genotype) or CNO (n = 6 per genotype), and a second behavioral assessment 24 h later. E, There was no significant difference in the percentage of missed steps made by naive ngr1^+/+ or ngr1^−/− mice after CNO compared to the vehicle treatment or 24 h after drug washout. F–W, Photomicrographs show coronal sections of brainstem stained with antibodies to c-fos (white and red) and 5-HT (green; arrowheads denote colocalization). Few c-fos+ nuclei were observed in the NRM or LVNs of naive ngr1^+/+ (F–H) and ngr1^−/− (I–K) mice. Significantly more c-fos+ nuclei were observed in 5-HT+ neurons after 3 min of running on the rotarod in both ngr1^+/+ (L, M) and ngr1^−/− (O, P) mice compared to naive controls (X; *p < 0.005, ANOVA). CNO treatment significantly reduced the number of c-fos+5-HT+ NRM neurons in ngr1^+/+ (R, S) and ngr1^−/− (O, P) mice compared to nave controls (X; *p < 0.005, ANOVA). Naive ngr1^+/+ (H) and ngr1^−/− (K) mice that were not run on the rotarod showed little c-fos induction in the LVNs (Y). Significantly more c-fos+ nuclei were observed in the LVNs in both ngr1^+/+ (N) and ngr1^−/− (Q) mice 60 min after running on the rotarod (Y; *p < 0.005, ANOVA). Delivery of CNO failed to significantly alter the number of c-fos+ LVNs in either ngr1^+/+ (T) or ngr1^−/− (W) mice compared to vehicle-treated controls (Y). Data are shown as the average percentage of missed steps during 3 min of grid walking ± SEM (E), as the average of the percentage of c-fos+5-HT+ neurons ± SEM (X), and the average number of c-fos+ neurons in the LVNs ± SEM (Y). Scale bars, 100 μm.