Two inhibitory neuronal classes govern acquisition and recall of spinal sensorimotor adaptation.

Spinal circuits are central to movement adaptation, yet the mechanisms within the spinal cord responsible for acquiring and retaining behavior upon experience remain unclear. Using a simple conditioning paradigm, we found that dorsal inhibitory neurons are indispensable for adapting protective limb-withdrawal behavior by regulating the transmission of a specific set of somatosensory information to enhance the saliency of conditioning cues associated with limb position. By contrast, maintaining previously acquired motor adaptation required the ventral inhibitory Renshaw cells. Manipulating Renshaw cells does not affect the adaptation itself but flexibly alters the expression of adaptive behavior. These findings identify a circuit basis involving two distinct populations of spinal inhibitory neurons, which enables lasting sensorimotor adaptation independently from the brain.

Small leucine-rich proteoglycans inhibit CNS regeneration by modifying the structural and mechanical properties of the lesion environment.

Extracellular matrix (ECM) deposition after central nervous system (CNS) injury leads to inhibitory scarring in humans and other mammals, whereas it facilitates axon regeneration in the zebrafish. However, the molecular basis of these different fates is not understood. Here, we identify small leucine-rich proteoglycans (SLRPs) as a contributing factor to regeneration failure in mammals. We demonstrate that the SLRPs chondroadherin, fibromodulin, lumican, and prolargin are enriched in rodent and human but not zebrafish CNS lesions. Targeting SLRPs to the zebrafish injury ECM inhibits axon regeneration and functional recovery. Mechanistically, we find that SLRPs confer mechano-structural properties to the lesion environment that are adverse to axon growth. Our study reveals SLRPs as inhibitory ECM factors that impair axon regeneration by modifying tissue mechanics and structure, and identifies their enrichment as a feature of human brain and spinal cord lesions. These findings imply that SLRPs may be targets for therapeutic strategies to promote CNS regeneration.

Met/HGFR triggers detrimental reactive microglia in TBI.

The complexity of signaling events and cellular responses unfolding in neuronal, glial, and immune cells upon traumatic brain injury (TBI) constitutes an obstacle in elucidating pathophysiological links and targets for intervention. We use array phosphoproteomics in a murine mild blunt TBI to reconstruct the temporal dynamics of tyrosine-kinase signaling in TBI and then scrutinize the large-scale effects of perturbation of Met/HGFR, VEGFR1, and Btk signaling by small molecules. We show Met/HGFR as a selective modifier of early microglial response and that Met/HGFR blockade prevents the induction of microglial inflammatory mediators, of reactive microglia morphology, and TBI-associated responses in neurons and vasculature. Both acute and prolonged Met/HGFR inhibition ameliorate neuronal survival and motor recovery. Early elevation of HGF itself in the cerebrospinal fluid of TBI patients suggests that this mechanism has translational value in human subjects. Our findings identify Met/HGFR as a modulator of early neuroinflammation in TBI with promising translational potential.

Neurotransmitter phenotype switching by spinal excitatory interneurons regulates locomotor recovery after spinal cord injury.

Severe spinal cord injury in adults leads to irreversible paralysis below the lesion. However, adult rodents that received a complete thoracic lesion just after birth demonstrate proficient hindlimb locomotion without input from the brain. How the spinal cord achieves such striking plasticity remains unknown. In this study, we found that adult spinal cord injury prompts neurotransmitter switching of spatially defined excitatory interneurons to an inhibitory phenotype, promoting inhibition at synapses contacting motor neurons. In contrast, neonatal spinal cord injury maintains the excitatory phenotype of glutamatergic interneurons and causes synaptic sprouting to facilitate excitation. Furthermore, genetic manipulation to mimic the inhibitory phenotype observed in excitatory interneurons after adult spinal cord injury abrogates autonomous locomotor functionality in neonatally injured mice. In comparison, attenuating this inhibitory phenotype improves locomotor capacity after adult injury. Together, these data demonstrate that neurotransmitter phenotype of defined excitatory interneurons steers locomotor recovery after spinal cord injury.

Proprioception: Bottom-up directive for motor recovery after spinal cord injury.

Proprioceptive feedback provides movement-matched sensory information essential for motor control and recovery after spinal cord injury. While it is understood that the fundamental contribution of proprioceptive feedback circuits in locomotor recovery is to activate the local spinal cord interneurons and motor neurons in a context-dependent manner, the precise mechanisms by which proprioception enables motor recovery after a spinal cord injury remain elusive. Furthermore, how proprioception contributes to motor learning mechanisms intrinsic to spinal cord networks and gives rise to motor recovery is currently unknown. This review discusses the existence of motor learning mechanisms intrinsic to spinal cord circuits and circuit-level insights on how proprioception might contribute to spinal cord plasticity, adaptability, and learning, in addition to the logic in which proprioception helps to establish an internal motor command to execute motor output using spared circuits after a spinal cord injury.

Functional Local Proprioceptive Feedback Circuits Initiate and Maintain Locomotor Recovery after Spinal Cord Injury.

Somatosensory feedback from proprioceptive afferents (PAs) is essential for locomotor recovery after spinal cord injury. To determine where or when proprioception is required for locomotor recovery after injury, we established an intersectional genetic model for PA ablation with spatial and temporal confinement. We found that complete or spatially restricted PA ablation in intact mice differentially affects locomotor performance. Following incomplete spinal cord injury, PA ablation below but not above the lesion severely restricts locomotor recovery and descending circuit reorganization. Furthermore, ablation of PAs after behavioral recovery permanently reverts functional improvements, demonstrating their essential role for maintaining regained locomotor function despite the presence of reorganized descending circuits. In parallel to recovery, PAs undergo reorganization of activity-dependent synaptic connectivity to specific local spinal targets. Our study reveals that PAs interacting with local spinal circuits serve as a continued driving force to initiate and maintain locomotor output after injury.

Long-Distance Descending Spinal Neurons Ensure Quadrupedal Locomotor Stability.

Locomotion is an essential animal behavior used for translocation. The spinal cord acts as key executing center, but how it coordinates many body parts located across distance remains poorly understood. Here we employed mouse genetic and viral approaches to reveal organizational principles of long-projecting spinal circuits and their role in quadrupedal locomotion. Using neurotransmitter identity, developmental origin, and projection patterns as criteria, we uncover that spinal segments controlling forelimbs and hindlimbs are bidirectionally connected by symmetrically organized direct synaptic pathways that encompass multiple genetically tractable neuronal subpopulations. We demonstrate that selective ablation of descending spinal neurons linking cervical to lumbar segments impairs coherent locomotion, by reducing postural stability and speed during exploratory locomotion, as well as perturbing interlimb coordination during reinforced high-speed stepping. Together, our results implicate a highly organized long-distance projection system of spinal origin in the control of postural body stabilization and reliability during quadrupedal locomotion.

Multisensory Signaling Shapes Vestibulo-Motor Circuit Specificity.

The ability to continuously adjust posture and balance is necessary for reliable motor behavior. Vestibular and proprioceptive systems influence postural adjustments during movement by signaling functionally complementary sensory information. Using viral tracing and mouse genetics, we reveal two patterns of synaptic specificity between brainstem vestibular neurons and spinal motor neurons, established through distinct mechanisms. First, vestibular input targets preferentially extensor over flexor motor pools, a pattern established by developmental refinement in part controlled by vestibular signaling. Second, vestibular input targets slow-twitch over fast motor neuron subtypes within extensor pools, while proprioceptors exhibit inversely correlated connectivity profiles. Genetic manipulations affecting the functionality of proprioceptive feedback circuits lead to adjustments in vestibular input to motor neuron subtypes counterbalancing the imposed changes, without changing the sparse vestibular input to flexor pools. Thus, two sensory signaling systems interact to establish complementary synaptic input patterns to the final site of motor output processing.

Muscle spindle feedback directs locomotor recovery and circuit reorganization after spinal cord injury.

Spinal cord injuries alter motor function by disconnecting neural circuits above and below the lesion, rendering sensory inputs a primary source of direct external drive to neuronal networks caudal to the injury. Here, we studied mice lacking functional muscle spindle feedback to determine the role of this sensory channel in gait control and locomotor recovery after spinal cord injury. High-resolution kinematic analysis of intact mutant mice revealed proficient execution in basic locomotor tasks but poor performance in a precision task. After injury, wild-type mice spontaneously recovered basic locomotor function, whereas mice with deficient muscle spindle feedback failed to regain control over the hindlimb on the lesioned side. Virus-mediated tracing demonstrated that mutant mice exhibit defective rearrangements of descending circuits projecting to deprived spinal segments during recovery. Our findings reveal an essential role for muscle spindle feedback in directing basic locomotor recovery and facilitating circuit reorganization after spinal cord injury.

Axon regeneration can facilitate or suppress hindlimb function after olfactory ensheathing glia transplantation.

Reports based primarily on anatomical evidence suggest that olfactory ensheathing glia (OEG) transplantation promotes axon regeneration across a complete spinal cord transection in adult rats. Based on functional, electrophysiological, and anatomical assessments, we found that OEG promoted axon regeneration across a complete spinal cord transection and that this regeneration altered motor responses over time. At 7 months after transection, 70% of OEG-treated rats showed motor-evoked potentials in hindlimb muscles after transcranial electric stimulation. Furthermore, a complete spinal cord retransection performed 8 months after injury demonstrated that this axon regeneration suppressed locomotor performance and decreased the hypersensitive hindlimb withdrawal response to mechanical stimulation. OEG transplantation alone promoted reorganization of lumbosacral locomotor networks and, when combined with long-term training, enhanced some stepping measures. These novel findings demonstrate that OEG promote regeneration of mature axons across a complete transection and reorganization of spinal circuitry, both of which contribute to sensorimotor function.

Further evidence of olfactory ensheathing glia facilitating axonal regeneration after a complete spinal cord transection.

Spinal Wistar Hannover rats injected with olfactory ensheathing glia (OEG) have been shown to recover some bipedal stepping and climbing abilities. Given the intrinsic ability of the spinal cord to regain stepping with pharmacological agents or epidural stimulation after a complete mid-thoracic transection, we asked if functional recovery after OEG injections is due to changes in the caudal stump or facilitation of functional regeneration of axons across the transection site. OEG were injected rostral and caudal to the transection site immediately after transection. Robotically assisted step training in the presence of intrathecal injections of a 5-HT(2A) receptor agonist (quipazine) was used to facilitate recovery of stepping. Bipedal stepping as well as climbing abilities were tested over a 6-month period post-transection to determine any improvement in hindlimb functional due to OEG injections and/or step training. The ability for OEG to facilitate regeneration was analyzed electrophysiologically by transcranially stimulating the brainstem and recording motor evoked potentials (MEP) with chronically implanted intramuscular EMG electrodes in the soleus and tibalis anterior with and without intrathecal injections of noradrenergic, serotonergic, and glycinergic receptor antagonists. Analyses confirmed that along with improved stepping ability and increased use of the hindlimbs during climbing, only OEG rats showed recovery of MEP. In addition the MEP signals were eliminated after a re-transection of the spinal cord rostral to the original transection and were modified in the presence of receptor antagonists. These data indicate that improved hindlimb function after a complete transection was coupled with OEG-facilitated functional regeneration of axons. This article is part of a Special Issue entitled: Understanding olfactory ensheathing glia and their prospect for nervous system repair.

Noradrenergic innervation of the rat spinal cord caudal to a complete spinal cord transection: effects of olfactory ensheathing glia.

Transplantation of olfactory bulb-derived olfactory ensheathing glia (OEG) combined with step training improves hindlimb locomotion in adult rats with a complete spinal cord transection. Spinal cord injury studies use the presence of noradrenergic (NA) axons caudal to the injury site as evidence of axonal regeneration and we previously found more NA axons just caudal to the transection in OEG- than media-injected spinal rats. We therefore hypothesized that OEG transplantation promotes descending coeruleospinal regeneration that contributes to the recovery of hindlimb locomotion. Now we report that NA axons are present throughout the caudal stump of both media- and OEG-injected spinal rats and they enter the spinal cord from the periphery via dorsal and ventral roots and along large penetrating blood vessels. These results indicate that the presence of NA fibers in the caudal spinal cord is not a reliable indicator of coeruleospinal regeneration. We then asked if NA axons appose cholinergic neurons associated with motor functions, i.e., central canal cluster and partition cells (active during fictive locomotion) and somatic motor neurons (SMNs). We found more NA varicosities adjacent to central canal cluster cells, partition cells, and SMNs in the lumbar enlargement of OEG- than media-injected rats. As non-synaptic release of NA is common in the spinal cord, more associations between NA varicosities and motor-associated cholinergic neurons in the lumbar spinal cord may contribute to the improved treadmill stepping observed in OEG-injected spinal rats. This effect could be mediated through direct association with SMNs and/or indirectly via cholinergic interneurons.

Serotonergic innervation of the caudal spinal stump in rats after complete spinal transection: effect of olfactory ensheathing glia.

Spinal cord injury studies use the presence of serotonin (5-HT)-immunoreactive axons caudal to the injury site as evidence of axonal regeneration. As olfactory ensheathing glia (OEG) transplantation improves hindlimb locomotion in adult rats with complete spinal cord transection, we hypothesized that more 5-HT-positive axons would be found in the caudal stump of OEG- than media-injected rats. Previously we found 5-HT-immunolabeled axons that spanned the transection site only in OEG-injected rats but detected labeled axons just caudal to the lesion in both media- and OEG-injected rats. Now we report that many 5-HT-labeled axons are present throughout the caudal stump of both media- and OEG-injected rats. We found occasional 5-HT-positive interneurons that are one likely source of 5-HT-labeled axons. These results imply that the presence of 5-HT-labeled fibers in the caudal stump is not a reliable indicator of regeneration. We then asked if 5-HT-positive axons appose cholinergic neurons associated with motor functions: central canal cluster and partition cells (active during fictive locomotion) and somatic motor neurons (SMNs). We found more 5-HT-positive varicosities in lamina X adjacent to central canal cluster cells in lumbar and sacral segments of OEG- than media-injected rats. SMNs and partition cells are less frequently apposed. As nonsynaptic release of 5-HT is common in the spinal cord, an increase in 5-HT-positive varicosities along motor-associated cholinergic neurons may contribute to the locomotor improvement observed in OEG-injected spinal rats. Furthermore, serotonin located within the caudal stump may activate lumbosacral locomotor networks.

OEG implantation and step training enhance hindlimb-stepping ability in adult spinal transected rats.

Numerous treatment strategies for spinal cord injury seek to maximize recovery of function and two strategies that show substantial promise are olfactory bulb-derived olfactory ensheathing glia (OEG) transplantation and treadmill step training. In this study we re-examined the issue of the effectiveness of OEG implantation but used objective, quantitative measures of motor performance to test if there is a complementary effect of long-term step training and olfactory bulb-derived OEG implantation. We studied complete mid-thoracic spinal cord transected adult female rats and compared four experimental groups: media-untrained, media-trained, OEG-untrained and OEG-trained. To assess the extent of hindlimb locomotor recovery at 4 and 7 months post-transection we used three quantitative measures of stepping ability: plantar stepping performance until failure, joint movement shape and movement frequency compared to sham controls. OEG transplantation alone significantly increased the number of plantar steps performed at 7 months post-transection, while training alone had no effect at either time point. Only OEG-injected rats plantar placed their hindpaws for more than two steps by the 7-month endpoint of the study. OEG transplantation combined with training resulted in the highest percentage of spinal rats per group that plantar stepped, and was the only group to significantly improve its stepping abilities between the 4- and 7-month evaluations. Additionally, OEG transplantation promoted tissue sparing at the transection site, regeneration of noradrenergic axons and serotonergic axons spanning the injury site. Interestingly, the caudal stump of media- and OEG-injected rats contained a similar density of serotonergic axons and occasional serotonin-labelled interneurons. These data demonstrate that olfactory bulb-derived OEG transplantation improves hindlimb stepping in paraplegic rats and further suggest that task-specific training may enhance this OEG effect.