Compensatory adaptation of parallel motor pathways promotes skilled forelimb recovery after spinal cord injury

Abstract

Skilled forelimb patterning is regulated by the corticospinal tract (CST) with support from brainstem regions. When the CST is lesioned, there is a loss of forelimb function; however, if indirect pathways remain intact, rehabilitative training can facilitate recovery. Following spinal cord injury, rehabilitation is thought to enhance the reorganization and plasticity of spared supraspinal-propriospinal circuits, aiding functional recovery. This study focused on the roles of cervical propriospinal interneurons (PNs) and rubrospinal neurons (RNs) in the recovery of reaching and grasping behaviors in rats with bilateral lesions of the CST and dorsal columns at C5. The lesions resulted in a 50% decrease in pellet retrieval, which normalized over four weeks of training. Silencing PNs or RNs after recovery resulted in reduced retrieval success. Notably, silencing both pathways corresponded to greater functional loss, underscoring their parallel contributions to recovery, alongside evidence of CST fiber sprouting in the spinal cord and red nucleus.

Keywords: Biological sciences; Natural sciences; Neuroscience; Systems neuroscience.

Graphical abstract

Figure 1

Inducible inhibitory DREADDs attenuate cortical neuron excitability Fluorescent images of cortical slices with neurons transduced with AAV2-TetOn and HiRet-TRE-hM4Di-mCherry. Example of recorded cortical neuron that expressed hM4Di-mCherry; (A) biocytin, (B) mCherry and (C) merged. Example of recorded cortical neuron (arrowhead) that did not express hM4Di-mCherry; (D) biocytin, (E) mCherry and (F) merged. Recorded neurons showing attenuation upon CNO application in neuronal excitability were hM4Di+/biocytin+. Recorded neurons that showed no CNO-mediated attenuation were hM4Di-/biocytin+. (G) Change in number of spikes measured during electrical excitability of whole-cell patch clamped cortical neurons expressing hM4Di. Two-tailed unpaired t-test was significant: t(7.529, 6); p = 0.0003. (H) Representative electrophysiology traces from a recorded hM4Di+ cortical neuron. Normal cell firing was observed during baseline measurements. CNO application temporarily attenuated cell firing that returned to normal upon washing out cell with ACSF. Data is mean ± SD. N = 2–6. ∗∗∗p < 0.001. Scale bars in (A) and (D) are 50 μm.

Figure 2

Neuronal silencing of C3-C4 PNs and/or RSNs in normal rats does not affect forelimb reaching behavior (A) Silencing of C3/C4 PNs or RSNs was mediated by a 2 viral vector system for doxycycline inducible expression of hM4Di. For this study HiRet-tre-hM4Di-mCherry was injected into laminae VII of the C6-T1 spinal cord where it is preferentially taken up at synapses and retrogradely transported to multiple neuronal populations innervating that region. AAV-TetOn was stereotaxically injected into either the red nucleus, C3/C4 spinal cord or both. Representative images showing labeling of C3 propriospinal neurons (B) or red nucleus (C). Silencing of C3/C4 propriospinal neurons (D), rubrospinal neurons (E), or both (F) in normal rats shows no change in pellet retrieval with the administration of CNO either before or after induction of hM4Di expression with doxycycline. Silencing of both C3/C4 propriospinal neurons and rubrospinal neurons (G) failed to show reductions in hand and digit dexterity before or after CNO administration. Repeated measures one-way ANOVA was found not significant in D) F(4.921, 39.37) = 2.368; p = 0.0578, n = 9; E) F(3.445, 20.67) = 0.9004; p = 0.4695, n = 7; and F) F (4.166, 29.16) = 2.008; p = 0.1172, n = 8 for each group. Data is mean ± SEM. Scale bars in panels B is 250 μm and panel C is 150um.

Figure 3

Recovery of reaching is mediated by C3/C4 propriospinal and rubrospinal neurons after C5 dorsal funiculus lesions (A) Experimental timeline describing training, viral injections, lesion time point, recovery, CNO pre-induction of hM4Di and post induction for silencing of individual populations. (B) Illustration showing lesion area shaded in gray. (C) Representative section showing C5 dorsal funiculus lesion stained by GFAP. (D) Representative section showing loss of PKC-gamma staining of CST in T4 spinal cord section. (E) Illustration showing injection procedure for expressing hM4Di in propriospinal neurons. ((F) Graph showing pellet retrieval scores prior to lesioning (black) and after C5 DC lesions (red). Significant deficits in pellet retrieval were observed 7 days after DC lesions (p > 0.001 any black time point compared to day 52 of trial) with significant recovery back to baseline by day 71 of trial (comparison of day 52 to day 71, p = 0.0003). After induction of hM4Di by administration of doxycycline, CNO induced a decrease of 18% at day 80 (p = 0.0117) and 24.1% at day 82 (p = 0.0021) when compared to vehicle administration. (G) After C5 DC lesions all rats showed persistent deficits in hand, wrist, and digit movements (−7 vs. 52 days; p = 0.0001), which only marginally recovered (52 vs. 71 days; p = 0.3119) and showed no response to CNO treatment. (H) Illustration showing injection procedure for expressing hM4Di within neurons of the red nucleus. (I) Graph showing pellet retrieval scores prior to lesioning (blue) and after lesioning (red) with CNO inducing a 19.3% (p = 0.062) and 20.6% (p = 0.0366) reduction at days 80 and 82, respectively, when compared to vehicle injections. (J) As with panel G, deficits in hand, wrist and digit movements did not resolve over time or change with CNO treatment. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 Data is mean ± SEM, C3/C4 PNs n = 8, RNs n = 6. Scale bars in panels C and D are 500 μm. Also see Figure S2.

Figure 4

Silencing both C3/C4 PNs and RNs together have a combined reduction in the recovery of forelimb reaching (A) Experimental timeline describing training, viral injections, lesion time point, recovery, CNO pre-induction of hM4Di and post induction for simultaneous silencing of both C3/C4 PNs and RNs populations. (B) Illustration showing injections procedure for expressing hM4Di in both propriospinal and rubrospinal neurons and lesion site (black). (C) Graph showing pellet retrieval scores prior to lesioning (blue) and after C5 DC lesions (red). Significant deficits in pellet retrieval were observed 7 days (day 66, E) after DC lesions (p > 0.001 any black time point compared to day 52 of trial) with significant recovery back to baseline by day 71 of trial (comparison of day 52 to day 71, p = 0.001). After doxycycline induction of hM4Di, administration of CNO induced a 32% (p = 0.0127) and 37% (p = 0.043) reduction in pellet retrieval at 94 and 96 days, when compared to vehicle control injections. Repeated measures one-way ANOVA was significant: F (2.739, 19.17) = 12.59, p = 0.0001. Sidak’s post hoc analysis done for multiple comparisons. (D–G) Individual video frames of representative reaching attempts at pellets (arrowheads) at timepoints indicated by letters in panel C. The entire video is in supplemental video. (H) Deficits in hand, wrist and digit movements did not resolve over time or change with CNO treatment. (I) Differences in single pellet retrieval between pre- and post-lesion or vehicle and CNO treatment for each group. (J) T-test comparison between different treatment groups. ∗∗p < 0.01, ∗p < 0.05, ∗∗∗p < 0.001, Data is mean ± SEM, n = 8.Also see Video S1 and Figure S2.

Figure 5

hM4Di+ specifically labels C3-C4 PNs and axon terminals onto motor neurons in the C6-T1 spinal cord (A) Spinal cord C3-C4 sections showing bilateral distribution of hM4Di-mCherry expression in the intermediate gray matter and ChAT+ MNs. (B) Higher magnification of contralateral C3-C4 spinal cord. (C) Higher magnification of ipsilateral C3-C4 spinal cord. No overlapping expression of hM4Di and ChAT was observed in C3-C4. (D) C8 spinal cord section showing hM4Di+ C3-C4 PN axon terminals within the intermediate zone and ventral horn. (E) Higher magnification of contralateral C8 spinal cord showing hM4Di axons crossing the midline toward ChAT+ MNs. (F) Higher magnification of ipsilateral C8 spinal cord showing numerous hM4Di+ axons within gray matter surrounding ChAT+ MNs. (G) Maximum intensity projection (MIP: 20 μm) of confocal z stack showing hM4Di+ C3-C4 axon terminals co-expressing vGlut2 near ChAT+ MNs. (H and I) Orthogonal sectioning showing hM4Di+/vGlut2+ C3-C4 PN axon terminals contacting ChAT+ C8 MN somas. Scale bars in (A) and (D) is 500 μm; (B), (C), and (E) are 200 μm; (F) is 100 um (G) is 10 μm.

Figure 6

hM4Di expressing C3-C4 PN axon terminals in LRN (A) Brainstem section stained for mCherry showing hM4Di expressing axon terminals from C3/C4 propriospinal neurons almost exclusively targeting the LRN (red labeling surrounded by yellow oval), similar area of illustration in Paxinos and Watson 7^th edition, figure 171. (B) Higher magnification image of LRN showing hM4Di+ C3-C4 PN axon terminals. (C) Maximum intensity projection (MIP: 20 μm) confocal image showing hM4Di+/vGlut2+ PN axon terminals around NeuN+ LRN neurons. Orthogonal sectioning of z stack images showing hM4Di+/vGlut2+ PN terminals on NeuN+ LRN neuron (D), (E) somas and (F) dendrite. Red = hM4Di-mCherry, Green = vGlut2, Blue = NeuN. Scale bar in (A) 500 μm, (B) 200 μm and (C) 10 μm. Cu: Cuneate Nucleus; Sp5: spinal trigeminal tract; SC: spinal cord; MdD: Medullary Reticular nucleus, dorsalis; PCRt: Parvicellular reticular nucleus; FN: Facial Nucleus.

Figure 7

CST fiber sprouting in C3-C4 spinal cord and red nucleus post-injury is dependent on rehabilitative training (A) Corticospinal axons extend into the region of the C3-C4 propriospinal neurons. (B and C) Six weeks following C5 dorsal funiculus lesion, animals that underwent rehabilitative behavioral training showed increased sprouting of corticospinal axons into the C3-C4 spinal cord (C), whereas those that did not undergo training showed no sprouting (B). (D) Quantification of thresholded BDA fiber density within the C3-C4 gray matter was statistically significant by one-way ANOVA; F(2, 12) = 75.69; p < 0.0001. Sidak’s post hoc multiple comparison was significant: intact vs. lesion no rehab, p < 0.0001; and lesion with no rehab vs. lesion with rehab; p = 0.0116. (E–G) Corticospinal axons also extend into the red nucleus in intact, non-injured rats. Six weeks following C5 dorsal funiculus lesions with rehabilitative training show extensive sprouting into the red nucleus (G) when compared to a similar group not receiving rehabilitative training (F). (H) Quantification of thresholded BDA fiber density in the red nucleus was statistically significant by one-way ANOVA; F(2, 12) = 10.62; p = 0.0022. Sidak’s posthoc multiple comparison was significant: intact vs. lesion no rehab, p = 0.0079; lesion with no rehab vs. lesion with rehab; p = 0.0058. Data is mean ± SD; n = 3–6. ∗∗p < 0.01, ∗p < 0.05. Scale bars in (A –C & E–G) 100 μm.