The spinal cord contains many descending and ascending longitudinal tracts whose development appears to be controlled by distinct guidance systems. We identified a population of dorsal spinal neurons marked by coexpression of the transcription factor Zic2 and the guidance receptor EphA4. Zic2+;EphA4+ neurons are surrounded by mechanosensory terminals, suggesting innervation by mechanoreceptor afferents. Their axons form an ipsilateral ascending pathway that develops during embryogenesis and projects within the ventral aspect of the dorsal funiculus, the same location as the descending corticospinal tract (CST), which develops postnatally. Interestingly, the same guidance mechanism, namely, ephrinB3-induced EphA4 forward signaling, is required for the guidance of both ascending and descending axon tracts. Our analysis of conditional EphA4 mutant mice also revealed that the development of the dorsal funiculus occurs independently of EphA4 expression in descending CST axons and is linked to the distribution of Zic2+;EphA4+ spinal neurons and the formation of the ascending pathway.

We previously showed that electrical stimulation of motor cortex (M1) after unilateral pyramidotomy in the rat increased corticospinal tract (CST) axon length, strengthened spinal connections, and restored forelimb function. Here, we tested: (i) if M1 stimulation only increases spinal axon length or if it also promotes connections to brain stem forelimb control centers, especially magnocellular red nucleus; and (ii) if stimulation-induced increase in axon length depends on whether pyramidotomy denervated the structure. After unilateral pyramidotomy, we electrically stimulated the forelimb area of intact M1, to activate the intact CST and other corticofugal pathways, for 10 days. We anterogradely labeled stimulated M1 and measured axon length using stereology. Stimulation increased axon length in both the spinal cord and magnocellular red nucleus, even though the spinal cord is denervated by pyramidotomy and the red nucleus is not. Stimulation also promoted outgrowth in the cuneate and parvocellular red nuclei. In the spinal cord, electrical stimulation caused increased axon length ipsilateral, but not contralateral, to stimulation. Thus, stimulation promoted outgrowth preferentially to the sparsely corticospinal-innervated and impaired side. Outgrowth resulted in greater axon density in the ipsilateral dorsal horn and intermediate zone, resembling the contralateral termination pattern. Importantly, as in spinal cord, increase in axon length in brain stem also was preferentially directed towards areas less densely innervated by the stimulated system. Thus, M1 electrical stimulation promotes increases in corticofugal axon length to multiple M1 targets. We propose the axon length change was driven by competition into an adaptive pattern resembling lost connections.

The corticospinal tract (CST) has dense contralateral and sparse ipsilateral spinal cord projections that converge with proprioceptive afferents on common spinal targets. Previous studies in adult rats indicate that the loss of dense contralateral spinal CST connections after unilateral pyramidal tract section (PTx), which models CST loss after stroke or spinal cord injury, leads to outgrowth from the spared side into the affected, ipsilateral, spinal cord. The reaction of proprioceptive afferents after this CST injury, however, is not known. Knowledge of proprioceptive afferent responses after loss of the CST could inform mechanisms of maladaptive plasticity in spinal sensorimotor circuits after injury. Here, we hypothesize that the loss of the contralateral CST results in a reactive increase in muscle afferents from the impaired limb and enhancement of their physiological actions within the cervical spinal cord. We found that 10 d after PTx, proprioceptive afferents sprout into cervical gray matter regions denervated by the loss of CST terminations. Furthermore, VGlut1-positive boutons, indicative of group 1A afferent terminals, increased on motoneurons. PTx also produced an increase in microglial density within the gray matter regions where CST terminations were lost. These anatomical changes were paralleled by reduction in frequency-dependent depression of the H-reflex, suggesting hyperreflexia. Our data demonstrate for the first time that selective CST injury induces maladaptive afferent fiber plasticity remote from the lesion. Our findings suggest a novel structural reaction of proprioceptive afferents to the loss of CST terminations and provide insight into mechanisms underlying spasticity.

Selective dorsal rhizotomy (SDR) consists of microsurgical partial deafferentation of sensory nerve roots (L1-S2). It is primarily used today in decreasing spasticity in young cerebral palsy (CP) patients. Intraoperative monitoring (IOM) is an essential part of the surgical decision-making process, aimed at improving functional results. The role played by SDR-IOM is examined, while realizing that connections between complex EMG responses to nerve-root stimulation and a patient's individual motor ability remain to be clarified.

Neural activity helps construct neural circuits during development and this function is leveraged by neuromodulation protocols to promote connectivity and repair in maturity. Neuromodulation targeting the motor cortex (MCX) strengthens connections for evoking muscle contraction (MEPs). Mechanisms include promoting local MCX and corticospinal tract (CST) synaptic efficacy and also axon terminal structural changes.

To restore motor control after spinal cord injury requires reconnecting the brain with spinal motor circuits below the lesion. A bridge around the injury is an important alternative to promoting axon regeneration through the injury. Previously, we reported a novel motor bridge in rats. The thirteenth thoracic nerve was detached from the muscle it innervates and the cut end implanted caudally into the lumbar gray matter where motor bridge axons regenerate. In this study, we first determined that regenerating bridge axons project to spinal motor circuits. Stable projections were present in ventral motor laminae of the cord, including putative synapses directly on motoneurons, 2 months after insertion in the intact cord. At this time, earlier-forming dorsal horn projections were mostly eliminated. Regenerating axons were effective in evoking leg motor activity as early as 2 weeks. We next determined that bridge axons could regenerate caudal to a chronic injury. We hemisected the spinal cord at L2 and inserted the bridge nerve 1 month later at L5 and found ventral laminae projections similar to those in intact animals, including onto motoneurons directly. Finally, we determined that the bridge circuit could be activated by neural pathways rostral to its origin. For spinally hemisected animals, we electrically stimulated the rostral spinal cord and recorded evoked potentials from the bridge and, in turn, motor responses in the sciatic nerve. Our findings suggests that bridge motoneurons could be used by descending motor pathways as premotor interneurons to transmit neural signals to bypass a chronic spinal injury.

Spared corticospinal tract (CST) and proprioceptive afferent (PA) axons sprout after injury and contribute to rewiring spinal circuits, affecting motor recovery. Loss of CST connections post-injury results in corticospinal signal loss and associated reduction in spinal activity. We investigated the role of activity loss and injury on CST and PA sprouting. To understand activity-dependence after injury, we compared CST and PA sprouting after motor cortex (MCX) inactivation, produced by chronic MCX muscimol microinfusion, with sprouting after a CST lesion produced by pyramidal tract section (PTx). Activity suppression, which does not produce a lesion, is sufficient to trigger CST axon outgrowth from the active side to cross the midline and to enter the inactivated side of the spinal cord, to the same extent as PTx. Activity loss was insufficient to drive significant CST gray matter axon elongation, an effect of PTx. Activity suppression triggered presynaptic site formation, but less than PTx. Activity loss triggered PA sprouting, as PTx. To understand injury-dependent sprouting further, we blocked microglial activation and associated inflammation after PTX by chronic minocycline administration after PTx. Minocycline inhibited myelin debris phagocytosis contralateral to PTx and abolished CST axon elongation, formation of presynaptic sites, and PA sprouting, but not CST axon outgrowth from the active side to cross the midline. Our findings suggest sprouting after injury has a strong activity dependence and that microglial activation after injury supports axonal elongation and presynaptic site formation. Combining spinal activity support and inflammation control is potentially more effective in promoting functional restoration than either alone.

Motor recovery after spinal cord injury is limited due to sparse descending pathway axons caudal to the injury. Rehabilitation is the primary treatment for paralysis in humans with SCI, but only produces modest functional recovery. Here, we determined if dual epidural motor cortex (M1) intermittent theta burst stimulation (iTBS) and cathodal transcutaneous spinal direct stimulation (tsDCS) enhances the efficacy of rehabilitation in improving motor function after cervical SCI. iTBS produces CST axon sprouting and tsDCS enhances M1-evoked spinal activity and muscle contractions after SCI. Rats were trained to perform the horizontal ladder task. Animals received a moderate midline C4 contusion, producing bilateral forelimb impairments. After 2 weeks, animals either received 10 days of iTBS+tsDCS or no stimulation; subsequently, all animals received 6 weeks of daily rehabilitation on the horizontal ladder task. Lesion size was not different in the two animal groups. Rehabilitation alone improved performance by a 22% reduction in skilled locomotion error rate, whereas stimulation+rehabilitation was markedly more effective (52%), and restored error rate to pre-injury levels. Stimulation+rehabilitation significantly increased CST axon length caudal to the injury and the amount of ventral horn label was positively correlated with functional improvement. The stimulation+rehabilitation group had significantly less proprioceptive afferent terminal labelling in the intermediate zone and fewer synapses on motoneurons . Afferent fiber terminal labeling was negatively correlated with motor recovery. Thus, the dual neuromodulation protocol promotes adaptive plasticity in corticospinal and proprioceptive afferents networks after contusion SCI, leading to enhanced rehabilitation efficacy and recovery of skilled locomotion.

Injury to the supraspinal motor systems, especially the corticospinal tract, leads to movement impairments. In addition to direct disruption of descending motor pathways, spinal motor circuits that are distant to and not directly damaged by the lesion undergo remodeling that contributes significantly to the impairments. Knowing which spinal circuits are remodeled and the underlying mechanisms are critical for understanding the functional changes in the motor pathway and for developing repair strategies. Here, we target spinal premotor cholinergic interneurons (IN) that directly modulate motoneuron excitability via their cholinergic C-bouton terminals. Using a model of unilateral medullary corticospinal tract lesion in male rats, we found transneuronal downregulation of the premotor cholinergic pathway. Phagocytic microglial cells were upregulated in parallel with cholinergic pathway downregulation and both were blocked by minocycline, a microglia activation inhibitor. Additionally, we found a transient increase in interneuronal complement protein C1q expression that preceded cell loss. 3D reconstructions showed ongoing phagocytosis of C1q-expressing cholinergic INs by microglia 3 d after injury, which was complete by 10 d after injury. Unilateral motor cortex inactivation using the GABAA receptor agonist muscimol replicated the changes detected at 3 d after lesion, indicating activity dependence. The neuronal loss after the lesion was rescued by increasing spinal activity using cathodal trans-spinal direct current stimulation. Our finding of activity-dependent modulation of cholinergic premotor INs after CST injury provides the mechanistic insight that maintaining activity, possibly during a critical period, helps to protect distant motor circuits from further damage and, as a result, may improve motor functional recovery and rehabilitation.SIGNIFICANCE STATEMENT Supraspinal injury to the motor system disrupts descending motor pathways, leading to movement impairments. Whether and how intrinsic spinal circuits are remodeled after a brain injury is unclear. Using a rat model of unilateral corticospinal tract lesion in the medulla, we show activity-dependent, transneuronal downregulation of the spinal premotor cholinergic system, which is mediated by microglial phagocytosis, possibly involving a rapid and transient increase in neuronal C1q before neuronal loss. Spinal cord neuromodulation after injury to augment spinal activity rescued the premotor cholinergic system. Our findings provide the mechanistic insight that maintaining activity, possibly during an early critical period, could protect distant motor circuits from further damage mediated by microglia and interneuronal complement protein and improve motor functional outcomes.

The strength of connections between motor cortex (MCX) and muscle can be augmented with a variety of stimulation protocols. Augmenting MCX-to-muscle connection strength by neuromodulation may be a way to enhance the intact motor system's capacity for acquiring motor skills and promote function after injury to strengthen spared connections. But this enhancement must be maintained for functional improvements.

Evidence suggests that motor experience plays a role in shaping development of the corticospinal system and voluntary motor control, which is a key motor function of the system. Here we used a mouse model with conditional forebrain deletion of the gene for EphA4 (Emx1-Cre:EphA4tm2Kldr), which regulates development of the laterality of corticospinal tract (CST). We combined study of Emx1-Cre:EphA4tm2Kldr with unilateral forelimb constraint during development to expand our understanding of experience-dependent CST development from both basic and translational perspectives. This mouse develops dense ipsilateral CST projections, a bilateral motor cortex motor representation, and bilateral motor phenotypes. Together these phenotypes can be used as readouts of corticospinal system organization and function and the changes brought about by experience. The Emx1-Cre:EphA4tm2Kldr mouse shares features with the common developmental disorder cerebral palsy: bilateral voluntary motor impairments and bilateral CST miswiring. Emx1-Cre:EphA4tm2Kldr mice with typical motor experiences during development display the bilateral phenotype of "mirror" reaching, because of a strongly bilateral motor cortex motor representation and a bilateral CST. By contrast, Emx1-Cre:EphA4tm2Kldr mice that experienced unilateral forelimb constraint from P1 to P30 and tested at maturity had a more contralateral motor cortex motor representation in each hemisphere; more lateralized CST projections; and substantially more lateralized/independent reaching movements. Changes in CST organization and function in this model can be explained by reduced synaptic competition of the CST from the side without developmental forelimb motor experiences. Using this model we show that unilateral constraint largely abrogated the effects of the genetic mutation on CST projections and thus demonstrates how robust and persistent experience-dependent development can be for the establishment of corticospinal system connections and voluntary control. Further, our findings inform the mechanisms of and strategies for developing behavioral therapies to treat bilateral movement impairments and CST miswiring in cerebral palsy.

Cervical spinal cord injury (SCI) impairs arm and hand function largely by interrupting descending tracts. Most SCI spare some axons at the lesion, including the corticospinal tract (CST), which is critical for voluntary movement. We targeted descending motor connections with paired electrical stimulation of motor cortex and cervical spinal cord in the rat. We sought to replicate the previously published effects of intermittent theta burst stimulation of forelimb motor cortex combined with trans-spinal direct current stimulation placed on the skin over the neck to target the cervical enlargement. We hypothesized that paired stimulation would improve performance in skilled walking and food manipulation (IBB) tasks. Rats received a moderate C4 spinal cord contusion injury (200 kDynes), which ablates the main CST. They were randomized to receive paired stimulation for 10 consecutive days starting 11 days after injury, or no stimulation. Behavior was assessed weekly from weeks 4-7 after injury, and then CST axons were traced. Rats with paired cortical and spinal stimulation achieved significantly better forelimb motor function recovery, as measured by fewer stepping errors on the horizontal ladder task (34 ± 9% in stimulation group vs. 51 ± 18% in control, p = .013) and higher scores on the food manipulation task (IBB, 0-9 score; 7.2 ± 0.8 in stimulated rats vs. 5.2 ± 2.6 in controls, p = .025). The effect size for both tasks was large (Cohen's d = 1.0 and 0.92, respectively). The CST axon length in the cervical spinal cord did not differ significantly between the groups, but there was denser and broader ipsilateral axons distribution distal to the spinal cord injury. The large behavioral effect and replication in an independent laboratory validate this approach, which will be trialed in cats before being tested in people using non-invasive methods.

Spinal reflexes reorganize in cerebral palsy (CP), producing hyperreflexia and spasticity. CP is more common among male infants, and gender might also influence brain and spinal-cord reorganization. This retrospective study investigated the frequency of higher-graded EMG responses elicited by electrical nerve-root stimulation during selective dorsal rhizotomy (SDR), prior to partial nerve- root deafferentation, considering not only segmental level and body side, but also gender.

Early postnatal mammals, including human babies, can perform only basic motor tasks. The acquisition of skilled behaviors occurs later, requiring anatomical changes in neural circuitry to support the development of coordinated activation or suppression of functionally related muscle groups. How this circuit reorganization occurs during postnatal development remains poorly understood. Here we explore the connectivity between corticospinal (CS) neurons in the motor cortex and muscles in mice. Using trans-synaptic viral and electrophysiological assays, we identify the early postnatal reorganization of CS circuitry for antagonistic muscle pairs. We further show that this synaptic rearrangement requires the activity-dependent, non-apoptotic Bax/Bak-caspase signaling cascade. Adult Bax/Bak mutant mice exhibit aberrant co-activation of antagonistic muscle pairs and skilled grasping deficits but normal reaching and retrieval behaviors. Our findings reveal key cellular and molecular mechanisms driving postnatal motor circuit reorganization and the resulting impacts on muscle activation patterns and the execution of skilled movements.

No abstract available

The motor cortex represents muscle and joint control and projects to spinal cord interneurons and-in many primates, including humans-motoneurons, via the corticospinal tract (CST). To examine these spinal CST anatomical mechanisms, we determined if motor cortex sites controlling individual forelimb joints project differentially to distinct cervical spinal cord territories, defined regionally and by the locations of putative last-order interneurons that were transneuronally labeled by intramuscular injection of pseudorabies virus. Motor cortex joint-specific sites were identified using intracortical-microstimulation. CST segmental termination fields from joint-specific sites, determined using anterograde tracers, comprised a high density core of terminations that was consistent between animals and a surrounding lower density projection that was more variable. Core terminations from shoulder, elbow, and wrist control sites overlapped in the medial dorsal horn and intermediate zone at C5/C6 but were separated at C7/C8. Shoulder sites preferentially terminated dorsally, in the dorsal horn; wrist/digit sites, more ventrally in the intermediate zone; and elbow sites, medially in the dorsal horn and intermediate zone. Pseudorabies virus injected in shoulder, elbow, or wrist muscles labeled overlapping populations of predominantly muscle-specific putative premotor interneurons, at a survival time for disynaptic transfer from muscle. At C5/C6, CST core projections from all joint zones were located medial to regions of densely labeled last-order interneurons, irrespective of injected muscle. At C7/C8 wrist CST core projections overlapped the densest interneuron territory, which was located in the lateral intermediate zone. In contrast, elbow CST core projections were located medial to the densest interneuron territories, and shoulder CST core projections were located dorsally and only partially overlapped the densest interneuron territory. Our findings show a surprising fractionation of CST terminations in the caudal cervical enlargement that may be organized to engage different spinal premotor circuits for distal and proximal joint control.