Re-Establishment of Cortical Motor Output Maps and Spontaneous Functional Recovery via Spared Dorsolaterally Projecting Corticospinal Neurons after Dorsal Column Spinal Cord Injury in Adult Mice

Abstract

Motor cortical plasticity contributes to spontaneous recovery after incomplete spinal cord injury (SCI), but the pathways underlying this remain poorly understood. We performed optogenetic mapping of motor cortex in channelrhodopsin-2 expressing mice to assess the capacity of the cortex to re-establish motor output longitudinally after a C3/C4 dorsal column SCI that bilaterally ablated the dorsal corticospinal tract (CST) containing ∼96% of corticospinal fibers but spared ∼3% of CST fibers that project via the dorsolateral funiculus. Optogenetic mapping revealed extensive early deficits, but eventual reestablishment of motor cortical output maps to the limbs at the same latency as preoperatively by 4 weeks after injury. Analysis of skilled locomotion on the horizontal ladder revealed early deficits followed by partial spontaneous recovery by 6 weeks after injury. To dissociate between the contributions of injured dorsal projecting versus spared dorsolateral projecting corticospinal neurons, we established a transient silencing approach to inactivate spared dorsolaterally projecting corticospinal neurons specifically by injecting adeno-associated virus (AAV)-expressing Cre-dependent DREADD (designer receptor exclusively activated by designer drug) receptor hM4Di in sensorimotor cortex and AAV-expressing Cre in C7/C8 dorsolateral funiculus. Transient silencing uninjured dorsolaterally projecting corticospinal neurons via activation of the inhibitory DREADD receptor hM4Di abrogated spontaneous recovery and resulted in a greater change in skilled locomotion than in control uninjured mice using the same silencing approach. These data demonstrate the pivotal role of a minor dorsolateral corticospinal pathway in mediating spontaneous recovery after SCI and support a focus on spared corticospinal neurons as a target for therapy.

Significance statement: Spontaneous recovery can occur after incomplete spinal cord injury (SCI), but the pathways underlying this remain poorly understood. We performed optogenetic mapping of motor cortex after a cervical SCI that interrupts most corticospinal transmission but results in partial recovery on a horizontal ladder task of sensorimotor function. We demonstrate that the motor cortex can reestablish output to the limbs longitudinally. To dissociate the roles of injured and uninjured corticospinal neurons in mediating recovery, we transiently silenced the minor dorsolateral corticospinal pathway spared by our injury. This abrogated spontaneous recovery and resulted in a greater change in skilled locomotion than in uninjured mice using the same approach. Therefore, uninjured corticospinal neurons substantiate remarkable motor cortical plasticity and partial recovery after SCI.

Keywords: corticospinal; motor cortex; optogenetics; plasticity; recovery; spinal cord injury.

Copyright © 2016 the authors 0270-6474/16/364081-13$15.00/0.

Figures

Figure 1.

Optogenetic and pharmacogenetic dissection of motor cortical plasticity underlying recovery after SCI. A, Cross-section through cervical spinal cord in a Thy1-ChR2 mouse immunostained for PKCγ (red), YFP (green), and BDA (pink) with euthanasia 2 weeks after BDA injections into sensorimotor cortex. B, Close-up of the dorsal column showing BDA+ corticospinal axons comprising the dCST. C, Close-up of the dorsolateral funiculus showing BDA+ corticospinal axons comprising the dlCST and their branches into gray matter. D, Close-up of the ventral column showing sparse BDA+ immunoreactivity. E, Schematic of descending motor corticofugal pathways involved in distal limb control. The motor cortex projects directly to the spinal cord via the dCST (∼96%), dlCST (∼3%), and vCST (∼1%), but also provides major excitatory input to the red nucleus (RN) and reticular formation (RF), which descend the rubrospinal tract and reticulospinal tract, respectively. After C3/C4 SCI, the dCST is bilaterally interrupted, but other motor corticofugal pathways remain intact. F, Horizontal ladder task of sensorimotor dysfunction and recovery after C3/C4 dorsal column SCI. Arrows denote errors of the forelimb and/or hindlimb. G, Longitudinal optogenetic motor mapping through intact skull before and after SCI. Anesthetized, head-fixed mice are placed with their left FL and HL suspended from the ground to allow free movement. Laser motion sensors direct at targets on the limbs record movements evoked by optogenetic cortical stimulation. Mapping is repeated in the same animal at multiple time points after SCI. A chronic cranial window preparation allows optical access through completely intact skull. The window is directed over the right cortical hemisphere and centered around bregma (yellow square) rostro-caudally. A 12 × 14 array of cortical points with 300 μm spacing is stimulated in semirandom order by a fixed ∼100-μm-diameter 473 nm laser (10 ms pulse). After three repetitions of stimulation, a map of average evoked forelimb and hindlimb movements are assembled and scaled based on amplitude. Inset brain atlas was adapted from the Allen Brain Atlas and indicates approximate location of motor mapping area within the red outline. Bright green area is an approximate area of primary motor cortex. Scale bars: A, 500 μm; B–D, 50 μm.

Figure 2.

Partial spontaneous recovery and sustained deficits in skilled locomotion after C3/C4 dorsal column SCI. A, Left, Transverse (cross-) sections of cervical spinal cord rostral to a dorsal column lesion immunostained for YFP and PKCγ showing the presence of the dorsal corticospinal tract. YFP labels neurons diffusely in both gray and white matter and extensively labels the dCST in Thy1-ChR2/YFP mice. Middle, Transverse sections of cervical spinal cord at the lesion epicenter immunostained for PKCy and YFP. The lesion includes the dorsal column and the dCST bilaterally. Right, Transverse sections of cervical spinal cord caudal to the SCI immunostained for PKCy and YFP. B, C, Close-up on dorsal column for PKCy and YFP rostral (B) and caudal (C) to injury. Note the absence of the dCST caudal to injury. C–G, Horizontal ladder error percentage (errors/steps * 100) for the left forelimb (D), right forelimb (E), left hindlimb (F), and right hindlimb (G) analyzed using two-way ANOVA with a Holm–Sidak's multiple-comparisons test. The left and right FLs have significantly higher error percentages up to 56 d after injury versus preinjury. ***p < 0.001, **p < 0.01, *p < 0.05 versus preoperation. The left and right FLs have significantly lower error percentages at 42 and 56 d after injury versus 7 d after injury. ##p < 0.01, #p < 0.05 versus 7 d after injury. The left and right HLs completely recover horizontal ladder placement by 7–14 d after injury. ***p < 0.001, **p < 0.01, *p < 0.05 versus preoperation. n = 16 SCI mice and n = 8 sham-operated mice. Error bars indicate SEM. Scale bars: A, 500 μm; B, 50 μm.

Figure 3.

Effect of acute SCI on motor maps. A, Average upsampled ChR2-stimulated motor maps (100 × 100 μm pixels) normalized to their respective mean and aligned based on the location of bregma (yellow square). Average FL and HL motor maps before injury (Ai) and immediately after C3/C4 dorsal column SCI (Aii) (<1 h after injury; n = 8). Inset brain atlas was adapted from the Allen Brain Atlas and indicates approximate location of motor mapping area within the red outline. Bright green area is an approximate area of primary motor cortex. B, Position of center of gravity of FL and HL maps with respect to bregma. FL is displaced caudally from 0.30 ± 0.14 mm rostral to bregma preoperatively to 0.35 ± 0.07 mm caudal to bregma acutely. C, FL and HL motor amplitude are reduced after acute SCI. D, FL and HL map area defined by the number of cortical sites capable of generating FL and HL movement, respectively, are reduced after acute SCI. E, Latencies of optogenetic stimulation to FL and HL movement are increased after acute SCI. n = 8 injured mice. *p < 0.05, **p < 0.01, ***p < 0.001, preinjury versus acute injory. Error bars indicate SEM.

Figure 4.

Spontaneous motor map reestablishment after C3/C4 dorsal column SCI. A, Baseline averaged ChR2-stimulated motor maps of the FL and HL at multiple time points before and after SCI. Bregma is denoted by the yellow square in each panel. B, FL motor map area, defined by the number of cortical sites from which movement could be generated is lower acutely and 3, 7, and 14 d after SCI. C, FL motor output, defined as the average maximal displacement at the 9 pixels encompassing the center of gravity, is significantly lower acutely and at 3 and 7 d after SCI. D, Latency to FL movement is longer acutely and at 3, 7, and 14 d after SCI. E, The FL map shifts caudally after SCI acutely and at 3 d after SCI. F, HL motor map area is lower acutely and at 3 d after SCI. G, HL motor output is lower acutely and at 3 and 7 d after SCI. H, Latency to HL movement is longer acutely and at 3 and 7 d after SCI. I, HL map does not shift in center position after SCI. n = 8 injured and n = 8 sham mice. *p < 0.05, **p < 0.01, ***p < 0.001 between groups. Error bars indicate SEM.

Figure 5.

Specific targeting of DREADD receptor hM4Di to spared dorsolaterally projecting corticospinal neurons. A–C, Schematic of targeting uninjured dorsolaterally projecting corticospinal neurons. A, Corticospinal neurons originating in layer V motor cortex project their axons via the dCST and dlCST. B, At 4 weeks after injury, an AAV-expressing Cre-dependent hM4Di (AAV1-hSyn-dio-hM4D(Gi)-mCherry) was administered to sensorimotor cortex and an AAV-expressing Cre (AAV1-Cre) was administered to the dorsolateral funiculus at C7/C8, such that only spared dorsolaterally projecting corticospinal neurons are transduced by both viruses in injured animals. C, After CNO administration, only corticospinal neurons expressing both viruses are silenced for 1–2 h (red); dCST neurons axotomized by the dorsal column SCI are not silenced. D, Histological verification of mCherry+ dual-transduced corticospinal axons at C2 spinal cord rostral to the injury. After SCI, mCherry+ corticospinal axons can be observed in the dorsolateral funiculus, but not in the dorsal column. After sham operation, mCherry+ corticospinal axons can be observed in both the dorsolateral funiculus and in the dorsal column. E, F, Quantification of mCherry+ axon numbers in the dorsolateral funiculus and dorsal column in injured and sham-operated mice. E, There is no difference in the number of mCherry+ axons found in the dorsolateral funiculus between SCI and sham-operated mice. F, There are significantly more mCherry+ axons in the dorsal column in sham-operated mice versus SCI mice. G, There are no mCherry+ axons in the ventral funiculi. *p < 0.05. Scale bars in D are 20 μm. Error bars indicate SEM.

Figure 6.

Activation of hM4Di in spared dorsolaterally projecting corticospinal neurons abrogates spontaneous recovery on the horizontal ladder task after C3/C4 dorsal column SCI. A, Experimental schedule. AAV injections were made at 4 weeks after injury and DREADD receptor behavioral experiments at 55–58 d after injury by administering CNO on day 56 and 58. B, Timeline of left FL error percentage in injured and sham-operated mice with AAV1-hSyn-dio-hM4D(Gi)-mCherry + AAV1-iCre injections (hm4Di Cre+) or just AAV1-hSyn-dio-hM4D(Gi)-mCherry for control (hm4Di Cre−). HM4Di Cre− SCI mice have lower error percentage at 55–58 d after injury versus 7 d after injury. HM4Di Cre+ SCI mice have lower error percentage at 55 and 57 d after injury versus 7 d after injury, but not at 56 and 58 d after injury, the 2 d when CNO was administered. C, Left FL error percentages after vehicle administration and CNO administration for each group (calculated as an average of scores on 55 and 57 d and 56 and 58 d, respectively). HM4Di SCI Cre+ mice have a higher error percentage after CNO administration versus vehicle administration and versus sham hM4Di Cre+ mice after CNO administration. hM4DI sham-operated mice have a higher error percentage after CNO administration versus vehicle administration. D, Left FL delta error percentage calculated as the absolute difference between CNO error percentage and vehicle error percentage. hM4Di SCI Cre+ mice have a higher delta error percentage than hM4Di Sham Cre+ mice. E, Timeline of right FL error percentage in injured and sham-operated mice as in B. All four groups have a lower error percentage at 55–58 d after injury versus 7 d after injury. F, Right FL error percentages after vehicle administration and CNO administration for each group. hM4Di SCI Cre+ mice trend toward a higher error percentage after CNO administration versus vehicle administration (p = 0.06). G, Right FL delta error percentage. ***p < 0.001, **p < 0.01, *p < 0.05. Error bars indicate SEM.

Figure 7.

Sensorimotor cortical analysis of mCherry and c-fos expression. A, Photomicrographs of coronal sections of sensorimotor cortex with antibodies to mCherry (red) and c-fos (light blue) in addition to fluoro-Nissl staining (dark blue) to illuminate the cortical layers. MCherry is confined to layer V, consistent with specific transduction of corticospinal neurons. A′, Close-up images of mCherry+ neurons from A showing mCherry (red) and c-fos (light blue). Arrows denote c-fos negative mCherry+ corticospinal neurons, and arrowheads denote c-fos+ mCherry+ corticospinal neurons. B, Quantification of total mCherry+ neurons in sensorimotor cortex. There are significantly more mCherry+ neurons in sham-operated mice (with dual transduction of dlCST and dCST) than in C3/C4 dorsal column SCI mice (with transduction confined to dlCST). C, Quantification of percentage of c-fos+ nuclei in mCherry+ corticospinal neurons after administration of CNO to activate hM4Di or vehicle for control. **p < 0.01, *p < 0.05. Scale bars: A, 50 μm; A′, 25 μm. Error bars indicate SEM.

Figure 8.

Model of corticospinal plasticity after cervical SCI. A, In the uninjured adult mouse, ChR2 stimulation of layer V motor cortex evokes FL and HL movement principally via the dCST and there is baseline skilled locomotion on the horizontal ladder task. B, After C3/C4 dorsal column SCI, the dCST is interrupted (gray dashed line), resulting in depressed cortical motor output and a deficit in skilled locomotion. C, Chronically after C3/C4 SCI, cortical motor output is reestablished and there is partial recovery in skilled locomotion, likely in part mediated by dlCST neurons (green). D, When hM4Di is activated in dCST and dlCST neurons (red), there is a small but statistically significant change in skilled locomotion. E, When hM4DI is activated in dlCST neurons chronically in C3/C4 dorsal column SCI mice (red), there is an abrogation of spontaneous recovery and a greater deficit in skilled locomotion than in uninjured hM4Di activated mice as in IV despite similar dlCST silencing and no dCST silencing, representing a shift in function from the injured major dCST pathway to the uninjured minor dlCST pathway.