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Clues about the organization of spinal networks responsible for rhythmic motor behaviors have come from the examination of reflex circuitry, lesioning studies, and single-cell recordings. Recently, more attention has been paid to extracellularly recorded multiunit signals thought to represent the general activity of local cellular potentials. Focusing on the gross localization of spinal locomotor networks, we used multiunit signals of the lumbar cord to classify the activation and organization of those networks. We employed power spectral analysis to compare multiunit power across rhythmic conditions and locations and to infer patterns of activation based on coherence and phase measures. We found greater multiunit power in midlumbar segments during stepping, supportive of previous lesioning studies isolating rhythm-generating capabilities to these segments. We also found much greater multiunit power during the flexion phase of stepping than during the extension phase for all lumbar segments. Greater multiunit power at flexion indicates increased neural activity during this phase and is suggestive of previously reported asymmetries between flexor- and extensor-related interneuronal populations of the spinal rhythm-generating network. Finally, the multiunit power showed no phase lag at coherent frequencies throughout the lumbar enlargement indicative of a longitudinal standing wave of neural activation. Our results suggest that the multiunit activity may be representative of the spinal rhythm-generating activity that is distributed in a rostrocaudal gradient. Additionally, our results indicate that this multiunit activity may operate as a flexor-dominant standing wave of activation that is synchronized throughout the rostrocaudal extent of the lumbar enlargement. NEW & NOTEWORTHY We report on the power spectral analysis of multiunit activity (MUA) of lumbar spinal interneurons during a locomotor task. In line with prior studies, we found evidence of greater power at the frequency of locomotion in high lumbar segments and during the flexion phase. Our results also confirm prior observations from our laboratory that the rhythmically active MUA behaves as a longitudinal standing wave of neural activation that is flexor dominant.

Cutaneous feedback from feet is involved in regulation of muscle activity during locomotion, and the lack of this feedback results in motor deficits. We tested the hypothesis that locomotor changes caused by local unilateral anesthesia of paw pads in the cat could be reduced/reversed by electrical stimulation of cutaneous and proprioceptive afferents in the distal tibial nerve during stance. Several split-belt conditions were investigated in four adult female cats. In addition, we investigated the effects of similar distal tibial nerve stimulation on overground walking of one male cat that had a transtibial, bone-anchored prosthesis for 29 months and, thus, had no cutaneous/proprioceptive feedback from the foot. In all treadmill conditions, cats walked with intact cutaneous feedback (control), with right fore- and hindpaw pads anesthetized by lidocaine injections, and with a combination of anesthesia and electrical stimulation of the ipsilateral distal tibial nerve during the stance phase at 1.2× threshold of afferent activation. Electrical stimulation of the distal tibial nerve during the stance phase of walking with anesthetized ipsilateral paw pads reversed or significantly reduced the effects of paw pad anesthesia on several kinematic variables, including lateral center of mass shift, cycle and swing durations, and duty factor. We also found that stimulation of the residual distal tibial nerve in the prosthetic hindlimb often had different effects on kinematics compared with stimulation of the intact hindlimb with paw anesthetized. We suggest that stimulation of cutaneous and proprioceptive afferents in the distal tibial nerve provides functionally meaningful motion-dependent sensory feedback, and stimulation responses depend on limb conditions.

Neuromodulatory therapies for spinal cord injury (SCI) such as electrical epidural stimulation (EES) are increasingly effective at improving patient outcomes. These improvements are thought to be due, at least in part, to plasticity in neuronal circuits. Precisely which circuits are influenced and which afferent classes are most effective in stimulating change remain important open questions. Genetic tools, such as Designer Receptors Exclusively Activated by Designer Drugs (DREADDs), support targeted and reversible neuromodulation as well as histological characterization of manipulated neurons. We therefore transduced and activated lumbar large diameter peripheral afferents with excitatory (hM3Dq) DREADDs, in a manner analogous to EES, in a rat hemisection model, to begin to trace plasticity and observe concomitant locomotor changes. Chronic DREADDs activation, coupled with thrice weekly treadmill training, was observed to increase afferent fluorescent labeling within motor pools and Clarke's column when compared to control animals. This plasticity may underlie kinematic differences that we observed across stages of recovery, including an increased and less variable hindquarters height in DREADDs animals, shorter step durations, a more flexed ankle joint early in recovery, a less variable ankle joint angle in swing phase, but a more variable hip joint angle. Withdrawal of DREADDs agonist, clozapine- N -oxide (CNO) left these kinematic differences largely unaffected; suggesting that DREADDs activation is not necessary for them later in recovery. However, we observed an intermittent "buckling" phenomenon in DREADDs animals without CNO activation, that did not occur with CNO re-administration. Future studies could use more refined genetic targeted of specific afferent classes, and utilize muscle recordings to find where afferent modulation is most influential in altering motor output., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Eisdorfer, Sobotka-Briner, Schramfield, Moukarzel, Chen, Campion, Smit, Rauscher, Lemay, Smith and Spence.)

Spinal interneurons play a critical role in motor output. A given interneuron may receive convergent input from several different sensory modalities and descending centers and relay this information to just as many targets. Therefore, there is a critical need to quantify populations of spinal interneurons simultaneously. Here, we quantify the functional connectivity of spinal neurons through the concurrent recording of populations of lumbar interneurons and hindlimb motor units in the in vivo cat model during activation of either the ipsilateral sural nerve or contralateral tibial nerve. Two microelectrode arrays were placed into lamina VII, one at L3 and a second at L6/7, while an electrode array was placed on the surface of the exposed muscle. Stimulation of tibial and sural nerves elicited similar changes in the discharge rate of both interneurons and motor units. However, these same neurons showed highly significant differences in prevalence and magnitude of correlated activity underlying these two forms of afferent drive. Activation of the ipsilateral sural nerve resulted in highly correlated activity, particularly at the caudal array. In contrast, the contralateral tibial nerve resulted in less, but more widespread correlated activity at both arrays. These data suggest that the ipsilateral sural nerve has dense projections onto caudal lumbar spinal neurons, while contralateral tibial nerve has a sparse pattern of projections., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor declared a past collaboration with one of the author ML., (Copyright © 2022 Zaback, Tiwari, Krupka, Marchionne, Negro, Lemay and Thompson.)

We explored the relationship between population interneuronal network activation and motor output in the adult, in vivo, air-stepping, spinal cat. By simultaneously measuring the activity of large numbers of spinal interneurons, we explored ensembles of coherently firing interneurons and their relation to motor output. In addition, the networks were analyzed in relation to their spatial distribution along the lumbar enlargement for evidence of localized groups driving particular phases of the locomotor step cycle. We simultaneously recorded hindlimb EMG activity during stepping and extracellular signals from 128 channels across two polytrodes inserted within lamina V-VII of two separate lumbar segments. Results indicated that spinal interneurons participate in one of two ensembles that are highly correlated with the flexor or the extensor muscle bursts during stepping. Interestingly, less than half of the isolated single units were significantly unimodally tuned during the step cycle whereas >97% of the single units of the ensembles were significantly correlated with muscle activity. These results show the importance of population scale analysis in neural studies of behavior as there is a much greater correlation between muscle activity and ensemble firing than between muscle activity and individual neurons. Finally, we show that there is no correlation between interneurons' rostrocaudal locations within the lumbar enlargement and their preferred phase of firing or ensemble participation. These findings indicate that spinal interneurons of lamina V-VII encoding for different phases of the locomotor cycle are spread throughout the lumbar enlargement in the adult spinal cord. NEW & NOTEWORTHY We report on the ensemble organization of interneuronal activity in the spinal cord during locomotor movements and show that lumbar intermediate zone interneurons organize in two groups related to the two major phases of walking: stance and swing. Ensemble organization is also shown to better correlate with muscular output than single-cell activity, although ensemble membership does not appear to be somatotopically organized within the spinal cord.

Objective: H-Reflex is a test that is carried out to measure the relative excitability of reflex pathways. Although reliable, conventional methods consist of performing many small steps, which requires a high level of attentiveness, and thus can carry an elevated risk of human error, despite proper training. Equipment that is available to perform those tests with different levels of automation are typically proprietary, inextensible by the user, and expensive. Here we present a novel MATLAB application that can accurately and reliably perform automated H-Reflex measurements, test the stimulating electrodes, and carry out typical subsequent analyses., Methods: This application is a Graphical User Interface that works with inexpensive equipment and offers many important features such as measuring electrode impedance in-situ, automating lengthy measurements like recruitment curves and frequency response trials, standardizing electric stimulation properties, automatic exporting of digital data and metadata, and immediately analyzing acquired data with single-click events., Results: Our new method was validated against conventional H-Reflex measurement methods with 2 anesthetized rats. The difference between acquired data using both methods was negligible (mean difference=0.0038; std=0.0121). Our app also detected electrode impedance with high accuracy (94%)., Conclusion: The method presented here allows reliable and efficient automated H-reflex measurements and can accurately analyze the collected data., Significance: The features provided by our app can speed up data collection and reduce human error, and unlike conventional methods, allow the user to analyze data immediately after the record. This can result in higher research quality and give broader access to the technique.

Delivery of neurotrophins to the spinal injury site via cellular transplants or viral vectors administration has been shown to promote recovery of locomotion in the absence of locomotor training in adult spinalized animals. These delivery methods involved risks of secondary injury to the cord and do not allow for precise and controlled dosing making them unsuitable for clinical applications. The present study was aimed at evaluating the locomotor recovery efficacy and safety of the neurotrophin BDNF delivered intrathecally to the lumbar locomotor centers using an implantable and programmable infusion mini-pump. Results showed that BDNF treated spinal cats recovered weight-bearing plantar stepping at all velocities tested (0.3-0.8 m/s). Spinal cats treated with saline did not recover stepping ability, especially at higher velocities, and dragged their hind paws on the treadmill. Histological evaluation showed minimal catheter associated trauma and tissue inflammation, underlining that intrathecal delivery by an implantable/programmable pump is a safe and effective method for delivery of a controlled BDNF dosage; it poses minimal risks to the cord and is clinically translational.

Spinal cord injury (SCI) often results in life-long sensorimotor impairment. Spontaneous recovery from SCI is limited, as supraspinal fibers cannot spontaneously regenerate to form functional networks below the level of injury. Despite this, animal models and humans exhibit many motor behaviors indicative of recovery when electrical stimulation is applied epidurally to the dorsal aspect of the lumbar spinal cord. In 1976, epidural stimulation was introduced to alleviate spasticity in Multiple Sclerosis. Since then, epidural electrical stimulation (EES) has been demonstrated to improve voluntary mobility across the knee and/or ankle in several SCI patients, highlighting its utility in enhancing motor activation. The mechanisms that EES induces to drive these improvements in sensorimotor function remain largely unknown. In this review, we discuss several sensorimotor plasticity mechanisms that we hypothesize may enable epidural stimulation to promote recovery, including changes in local lumbar circuitry, propriospinal interneurons, and the internal model. Finally, we discuss genetic tools for afferent modulation as an emerging method to facilitate the search for the mechanisms of action., (Copyright © 2020 Eisdorfer, Smit, Keefe, Lemay, Smith and Spence.)

Sensorimotor training providing motion-dependent somatosensory feedback to spinal locomotor networks restores treadmill weight-bearing stepping on flat surfaces in spinal cats. In this study, we examined if locomotor ability on flat surfaces transfers to sloped surfaces and the contribution of length-dependent sensory feedback from lateral gastrocnemius (LG) and soleus (Sol) to locomotor recovery after spinal transection and locomotor training. We compared kinematics and muscle activity at different slopes (±10° and ±25°) in spinalized cats ( n = 8) trained to walk on a flat treadmill. Half of those animals had their right hindlimb LG/Sol nerve cut and reattached before spinal transection and locomotor training, a procedure called muscle self-reinnervation that leads to elimination of autogenic monosynaptic length feedback in spinally intact animals. All spinal animals trained on a flat surface were able to walk on slopes with minimal differences in walking kinematics and muscle activity between animals with/without LG/Sol self-reinnervation. We found minimal changes in kinematics and muscle activity at lower slopes (±10°), indicating that walking patterns obtained on flat surfaces are robust enough to accommodate low slopes. Contrary to results in spinal intact animals, force responses to muscle stretch largely returned in both SELF-REINNERVATED muscles for the trained spinalized animals. Overall, our results indicate that the locomotor patterns acquired with training on a level surface transfer to walking on low slopes and that spinalization may allow the recovery of autogenic monosynaptic length feedback following muscle self-reinnervation. NEW & NOTEWORTHY Spinal locomotor networks locomotor trained on a flat surface can adapt the locomotor output to slope walking, up to ±25° of slope, even with total absence of supraspinal CONTROL. Autogenic length feedback (stretch reflex) shows signs of recovery in spinalized animals, contrary to results in spinally intact animals.

Objective: We aimed to refine electroneurogram techniques for monitoring hypogastric nerve activity during bladder filling, and then examined nerve activity in normal intact versus acutely decentralized bladders., Methods: Effects of electrical stimulation of hypogastric nerves or lumbar ventral roots on detrusor pressure were examined, as were effects of isoflurane versus propofol anesthetics on hypogastric nerve stimulation evoked pressure. Hypogastric nerve activity was then recorded using custom-made bipolar cuff electrodes during bladder filling before and after its transection between the spinal cord and electrode to eliminate efferent nerve signals., Results: Electrical stimulation of hypogastric nerves evoked low amplitude detrusor pressures that did not differ between the two anesthetics. Upper lumbar (L2) ventral root stimulation evoked detrusor pressures were suppressed, yet not eliminated, after transection of hypogastric nerves and all spinal roots below L5. Afferent and efferent hypogastric nerve activity did not change with bladder filling in neuronally intact bladders yet decreased in decentralized bladders. No change in afferent activity was observed during bladder filling in either intact or decentralized bladders., Conclusions: These findings indicate that a more complete decentralized bladder model should include transection of lumbosacral spinal roots innervating the bladder as well as hypogastric nerves. These refined electroneurogram recording methods may be suitable for evaluating the effectiveness of nerve transfer surgeries for bladder reinnervation by monitoring sensory activity in the transferred nerve., Competing Interests: The authors have declared that no competing interests exist.

Adult cats show limited spontaneous locomotor capabilities following spinal transection, but recover treadmill stepping with body-weight-supported training. Delivery of neurotrophic factors such as brain-derived neurotrophic factor (BDNF) and neurotrophic factor 3 (NT-3) can substitute for body-weight-supported training, and promotes a similar recovery in a shorter period of time. Autologous cell grafts would negate the need for the immunosuppressive agents currently used with most grafts, but have not shown functional benefits in incomplete spinal cord injury models and have never been tested in complete transection or chronic injury models. In this study, we explored the effects of autologous fibroblasts, prepared from the individual cats and modified to produce BDNF and NT-3, on the recovery of locomotion in acute, sub-chronic and chronic full-transection models of spinal injury. Fourteen female cats underwent complete spinal transection at T11/T12. Cats were separated into four groups: sham graft at the time of injury, and BDNF and NT-3 producing autologous fibroblasts grafted at the time of injury, 2 weeks after injury, or 6 weeks after injury. Kinematics were recorded 3 and 5 weeks after cell graft. Additional kinematic recordings were taken for some cats until 12 weeks post-graft. Eleven of 12 cats with neurotrophin-producing grafts recovered plantar weight-bearing stepping at treadmill speeds from 0.3 to 0.8 m/sec within 5 weeks of grafting, whereas control cats recovered poor quality stepping at low speeds only (≤ 0.4 m/sec). Further, kinematic measures in cats with grafts were closer to pre-transection values than those for controls, and recovery was maintained up to 12 weeks post-grafting. Our results show that not only are autologous neurotrophin-producing grafts effective at promoting recovery of locomotion, but that delayed delivery of neurotrophins does not diminish the therapeutic effect, and may improve outcome.

Body-weight supported locomotor training (BWST) promotes recovery of load-bearing stepping in lower mammals, but its efficacy in individuals with a spinal cord injury (SCI) is limited and highly dependent on injury severity. While animal models with complete spinal transections recover stepping with step-training, motor complete SCI individuals do not, despite similarly intensive training. In this review, we examine the significant differences between humans and animal models that may explain this discrepancy in the results obtained with BWST. We also summarize the known effects of SCI and locomotor training on the muscular, motoneuronal, interneuronal, and supraspinal systems in human and non-human models of SCI and address the potential causes for failure to translate to the clinic. The evidence points to a deficiency in neuronal activation as the mechanism of failure, rather than muscular insufficiency. While motoneuronal and interneuronal systems cannot be directly probed in humans, the changes brought upon by step-training in SCI animal models suggest a beneficial re-organization of the systems' responsiveness to descending and afferent feedback that support locomotor recovery. The literature on partial lesions in humans and animal models clearly demonstrate a greater dependency on supraspinal input to the lumbar cord in humans than in non-human mammals for locomotion. Recent results with epidural stimulation that activates the lumbar interneuronal networks and/or increases the overall excitability of the locomotor centers suggest that these centers are much more dependent on the supraspinal tonic drive in humans. Sensory feedback shapes the locomotor output in animal models but does not appear to be sufficient to drive it in humans.

Background: The measurement of ground reaction forces (GRFs) in animals trained to locomote on a treadmill after spinal cord injury (SCI) could prove valuable for evaluating training outcomes; however, quantitative measures of the GRFs in spinal felines are limited., New Method: A split belt treadmill was designed and constructed to measure the GRFs of feline hindlimbs during stepping. The treadmill consists of two independent treadmill assemblies, each mounted on a force plate. The design allows measurements of the vertical (F z ), fore-aft (F y ) and mediolateral (F x ) ground-reaction forces for both hindlimbs while the forelimbs are resting on a platform., Results: Static and dynamic noise tests revealed little to no noise at frequencies below 6Hz. Validation of the force plate measurements with a hand-held force sensor force showed good agreement between the two force readings. Peak normalized (to body mass) vertical GRFs for intact cats were 4.89±0.85N/kg for the left hindlimb and 4.79±0.97N/kg for the right. In comparison, trained spinalized cats peak normalized vertical GRFs were 2.20±0.94N/kg for the left hindlimb and 2.85±0.99N/kg for the right., Comparison With Other Existing Methods: Previous methods of measuring GRFs used stationary single force plates or treadmill mounted to single force plate. Using independent treadmills for each hindlimb allows measurement of the individual hindlimb's GRFs in spinalized cats following body-weight supported treadmill training., Conclusions: The split belt force treadmill enables the simultaneous recording of ground-reaction forces for both hindlimbs in cats prior to spinalization, and following spinalization and body-weight-supported treadmill training (BWST)., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Nuclear exclusion of the transcriptional regulators and potent oncoproteins, YAP/TAZ, is considered necessary for adult tissue homeostasis. Here we show that nuclear YAP/TAZ are essential regulators of peripheral nerve development and myelin maintenance. To proliferate, developing Schwann cells (SCs) require YAP/TAZ to enter S-phase and, without them, fail to generate sufficient SCs for timely axon sorting. To differentiate, SCs require YAP/TAZ to upregulate Krox20 and, without them, completely fail to myelinate, resulting in severe peripheral neuropathy. Remarkably, in adulthood, nuclear YAP/TAZ are selectively expressed by myelinating SCs, and conditional ablation results in severe peripheral demyelination and mouse death. YAP/TAZ regulate both developmental and adult myelination by driving TEAD1 to activate Krox20. Therefore, YAP/TAZ are crucial for SCs to myelinate developing nerve and to maintain myelinated nerve in adulthood. Our study also provides a new insight into the role of nuclear YAP/TAZ in homeostatic maintenance of an adult tissue., Competing Interests: The authors declare that no competing interests exist.

While it is well established that the axons of adult neurons have a lower capacity for regrowth, some regeneration of certain CNS populations after spinal cord injury (SCI) is possible if their axons are provided with a permissive substrate, such as an injured peripheral nerve. While some axons readily regenerate into a peripheral nerve graft (PNG), these axons almost always stall at the distal interface and fail to reinnervate spinal cord tissue. Treatment of the glial scar at the distal graft interface with chondroitinase ABC (ChABC) can improve regeneration, but most regenerated axons need further stimulation to extend beyond the interface. Previous studies demonstrate that pharmacologically inhibiting kinesin-5, a motor protein best known for its essential role in mitosis but also expressed in neurons, with the pharmacological agent monastrol increases axon growth on inhibitory substrates in vitro. We sought to determine if monastrol treatment after an SCI improves functional axon regeneration. Animals received complete thoracic level 7 (T7) transections and PNGs and were treated intrathecally with ChABC and either monastrol or DMSO vehicle. We found that combining ChABC with monastrol significantly enhanced axon regeneration. However, there were no further improvements in function or enhanced c-Fos induction upon stimulation of spinal cord rostral to the transection. This indicates that monastrol improves ChABC-mediated axon regeneration but that further treatments are needed to enhance the integration of these regrown axons., (Copyright © 2014. Published by Elsevier Inc.)

Background. Transplants of cellular grafts expressing a combination of 2 neurotrophic factors, brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) have been shown to promote and enhance locomotor recovery in untrained spinalized cats. Based on the time course of recovery and the absence of axonal growth through the transplants, we hypothesized that recovery was due to neurotrophin-mediated plasticity within the existing locomotor circuitry of the lumbar cord. Since BDNF and NT-3 have different effects on axonal sprouting and synaptic connectivity/strengthening, it becomes important to ascertain the contribution of each individual neurotrophins to recovery. Objective. We studied whether BDNF or NT-3 only producing cellular grafts would be equally effective at restoring locomotion in untrained spinal cats. Methods. Rat fibroblasts secreting one of the 2 neurotrophins were grafted into the T12 spinal transection site of adult cats. Four cats in each group (BDNF alone or NT-3 alone) were evaluated. Locomotor recovery was tested on a treadmill at 3 and 5 weeks post-transection/grafting. Results. Animals in both groups were capable of plantar weight-bearing stepping at speed up to 0.8 m/s as early as 3 weeks and locomotor capabilities were similar at 3 and 5 weeks for both types of graft. Conclusions. Even without locomotor training, either BDNF or NT-3 only producing grafts promote locomotor recovery in complete spinal animals. More clinically applicable delivery methods need to be developed., (© The Author(s) 2014.)

Although axons lose some of their intrinsic capacity for growth after their developmental period, some axons retain the potential for regrowth after injury. When provided with a growth-promoting substrate such as a peripheral nerve graft (PNG), severed axons regenerate into and through the graft; however, they stop when they reach the glial scar at the distal graft-host interface that is rich with inhibitory chondroitin sulfate proteoglycans. We previously showed that treatment of a spinal cord injury site with chondroitinase (ChABC) allows axons within the graft to traverse the scar and reinnervate spinal cord, where they form functional synapses. While this improvement in outgrowth was significant, it still represented only a small percentage (<20%) of axons compared to the total number of axons that regenerated into the PNG. Here we tested whether providing exogenous brain-derived neurotrophic factor (BDNF) via lentivirus in tissue distal to the PNG would augment regeneration beyond a ChABC-treated glial interface. We found that ChABC treatment alone promoted axonal regeneration but combining ChABC with BDNF-lentivirus did not increase the number of axons that regenerated back into spinal cord. Combining BDNF with ChABC did increase the number of spinal cord neurons that were trans-synaptically activated during electrical stimulation of the graft, as indicated by c-Fos expression, suggesting that BDNF overexpression improved the functional significance of axons that did reinnervate distal spinal cord tissue., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The high clinical relevance of models of incomplete cervical spinal cord injury (SCI) creates a need to address the spontaneous neuroplasticity that underlies changes in functional activity that occur over time after SCI. There is accumulating evidence supporting long projecting propriospinal neurons as suitable targets for therapeutic intervention after SCI, but focus has remained primarily oriented toward study of descending pathways. Long ascending axons from propriospinal neurons at lower thoracic and lumbar levels that form inter-enlargement pathways are involved in forelimb-hindlimb coordination during locomotion and are capable of modulating cervical motor output. We used non-invasive magnetic stimulation to assess how a unilateral cervical (C5) spinal contusion might affect transmission in intact, long ascending propriospinal pathways, and influence spinal cord plasticity. Our results show that transmission is facilitated in this pathway on the ipsilesional side as early as 1 week post-SCI. We also probed for descending magnetic motor evoked potentials (MMEPs) and found them absent or greatly reduced on the ipsilesional side as expected. The frequency-dependent depression (FDD) of the H-reflex recorded from the forelimb triceps brachii was bilaterally decreased although H(max)/M(max) was increased only on the ipsilesional side. Behaviorally, stepping recovered, but there were deficits in forelimb-hindlimb coordination as detected by BBB and CatWalk measures. Importantly, epicenter sparing correlated to the amplitude of the MMEPs and locomotor recovery but it was not significantly associated with the inter-enlargement or segmental H-reflex. In summary, our results indicate that complex plasticity occurs after a C5 hemicontusion injury, leading to differential changes in ascending vs. descending pathways, ipsi- vs. contralesional sides even though the lesion was unilateral as well as cervical vs. lumbar local spinal networks.

We compared the activity profiles and synergies of spinal motoneurons recorded during fictive locomotion evoked in immobilized decerebrate cat preparations by midbrain stimulation to the activity profiles and synergies of the corresponding hindlimb muscles obtained during forward level walking in cats. The fictive locomotion data were collected in the Spinal Cord Research Centre, University of Manitoba, and provided by Dr. David McCrea; the real locomotion data were obtained in the laboratories of M. A. Lemay and B. I. Prilutsky. Scatterplot representation and minimum spanning tree clustering algorithm were used to identify the possible motoneuronal and muscle synergies operating during both fictive and real locomotion. We found a close similarity between the activity profiles and synergies of motoneurons innervating one-joint muscles during fictive locomotion and the profiles and synergies of the corresponding muscles during real locomotion. However, the activity patterns of proximal nerves controlling two-joint muscles, such as posterior biceps and semitendinosus (PBSt) and rectus femoris (RF), were not uniform in fictive locomotion preparations and differed from the activity profiles of the corresponding two-joint muscles recorded during forward level walking. Moreover, the activity profiles of these nerves and the corresponding muscles were unique and could not be included in the synergies identified in fictive and real locomotion. We suggest that afferent feedback is involved in the regulation of locomotion via motoneuronal synergies controlled by the spinal central pattern generator (CPG) but may also directly affect the activity of motoneuronal pools serving two-joint muscles (e.g., PBSt and RF). These findings provide important insights into the organization of the spinal CPG in mammals, the motoneuronal and muscle synergies engaged during locomotion, and their afferent control.

The lumbar spinal cord circuitry can autonomously generate locomotion, but it remains to be determined which types of neurons constitute the locomotor generator and how their population activity is organized spatially in the mammalian spinal cord. In this study, we investigated the spatiotemporal dynamics of the spinal interneuronal population activity in the intermediate zone of the adult mammalian cord. Segmental interneuronal population activity was examined via multiunit activity (MUA) during air-stepping initiated by perineal stimulation in subchronic spinal cats. In contrast to single-unit activity, MUA provides a continuous measure of neuronal activity within a ∼100-μm volume around the recording electrode. MUA was recorded during air-stepping, along with hindlimb muscle activity, from segments L3 to L7 with two multichannel electrode arrays placed into the left and right hemicord intermediate zones (lamina V-VII). The phasic modulation and spatial organization of MUA dynamics were examined in relation to the locomotor cycle. Our results show that segmental population activity is modulated with respect to the ipsilateral step cycle during air-stepping, with maximal activity occurring near the ipsilateral swing to stance transition period. The phase difference between the population activity within the left and right hemicords was also found to correlate to the left-right alternation of the step cycle. Furthermore, examination of MUA throughout the rostrocaudal extent showed no differences in population dynamics between segmental levels, suggesting that the spinal interneurons targeted in this study may operate as part of a distributed "clock" mechanism rather than a rostrocaudal oscillation as seen with motoneuronal activity.

Sensory feedback plays a crucial role in the control of locomotion and in the recovery of function after spinal cord injury. Investigations in reduced preparations have shown that the locomotor cycle can be modified through the activation of afferent feedback at various phases of the gait cycle. We investigated the effect of phase-dependent electrical stimulation of a cutaneous afferent nerve on the locomotor pattern of trained spinal cord-injured cats. Animals were first implanted with chronic nerve cuffs on the sural and sciatic nerves and electromyographic electrodes in different hindlimb muscles. Cats were then transected at T12 and trained daily to locomote on a treadmill. We found that electrical stimulation of the sural nerve can enhance the ongoing flexion phase, producing higher (+129%) and longer (+17.4%) swing phases of gait even at very low threshold of stimulation. Sural nerve stimulation can also terminate an ongoing extension and initiate a flexion phase. A higher prevalence of early switching to the flexion phase was observed at higher stimulation levels and if stimulation was applied in the late stance phase. All flexor muscles were activated by the stimulation. These results suggest that electrical stimulation of the sural nerve may be used to increase the magnitude of the swing phase and control the timing of its onset after spinal cord injury and locomotor training.

Transplantation of neural progenitor cells (NPC) is a promising therapeutic strategy for replacing neurons lost after spinal cord injury, but significant challenges remain regarding neuronal integration and functional connectivity. Here we tested the ability of graft-derived neurons to reestablish connectivity by forming neuronal relays between injured dorsal column (DC) sensory axons and the denervated dorsal column nuclei (DCN). A mixed population of neuronal and glial restricted precursors (NRP/GRP) derived from the embryonic spinal cord of alkaline phosphatase (AP) transgenic rats were grafted acutely into a DC lesion at C1. One week later, BDNF-expressing lentivirus was injected into the DCN to guide graft axons to the intended target. Six weeks later, we observed anterogradely traced sensory axons regenerating into the graft and robust growth of graft-derived AP-positive axons along the neurotrophin gradient into the DCN. Immunoelectron microscopy revealed excitatory synaptic connections between regenerating host axons and graft-derived neurons at C1 as well as between graft axons and DCN neurons in the brainstem. Functional analysis by stimulus-evoked c-Fos expression and electrophysiological recording showed that host axons formed active synapses with graft neurons at the injury site with the signal propagating by graft axons to the DCN. We observed reproducible electrophysiological activity at the DCN with a temporal delay predicted by our relay model. These findings provide the first evidence for the ability of NPC to form a neuronal relay by extending active axons across the injured spinal cord to the intended target establishing a critical step for neural repair with stem cells.

Spinal locomotor circuits are intrinsically capable of driving a variety of behaviors such as stepping, scratching, and swimming. Based on an observed rostrocaudal wave of activity in the motoneuronal firing during locomotor tasks, the traveling-wave hypothesis proposes that spinal interneuronal firing follows a similar rostrocaudal pattern of activation, suggesting the presence of spatially organized interneuronal modules within the spinal motor system. In this study, we examined if the spatial organization of the lumbar interneuronal activity patterns during locomotor activity in the adult mammalian spinal cord was consistent with a traveling-wave organizational scheme. The activity of spinal interneurons within the lumbar intermediate zone was examined during air-stepping in subchronic spinal cats. The preferred phase of interneuronal activity during a step cycle was determined using circular statistics. We found that the preferred phases of lumbar interneurons from both sides of the cord were evenly distributed over the entire step cycle with no indication of functional groupings. However, when units were subcategorized according to spinal hemicords, the preferred phases of units on each side largely fell around the period of extensor muscle activity on each side. In addition, there was no correlation between the preferred phases of units and their rostrocaudal locations along the spinal cord with preferred phases corresponding to both flexion and extension phases of the step cycle found at every rostrocaudal level of the cord. These results are consistent with the hypothesis that interneurons operate as part of a longitudinally distributed network rather than a rostrocaudally organized traveling-wave network.

Activity-based therapies such as passive bicycling and step-training on a treadmill contribute to motor recovery after spinal cord injury (SCI), leading to a greater number of steps performed, improved gait kinematics, recovery of phase-dependent modulation of spinal reflexes, and prevention of decrease in muscle mass. Both tasks consist of alternating movements that rhythmically stretch and shorten hindlimb muscles. However, the paralyzed hindlimbs are passively moved by a motorized apparatus during bike-training, whereas locomotor movements during step-training are generated by spinal networks triggered by afferent feedback. Our objective was to compare the task-dependent effect of bike- and step-training after SCI on physiological measures of spinal cord plasticity in relation to changes in levels of neurotrophic factors. Thirty adult female Sprague-Dawley rats underwent complete spinal transection at a low thoracic level (T12). The rats were assigned to one of three groups: bike-training, step-training, or no training. The exercise regimen consisted of 15 min/d, 5 days/week, for 4 weeks, beginning 5 days after SCI. During a terminal experiment, H-reflexes were recorded from interosseus foot muscles following stimulation of the tibial nerve at 0.3, 5, or 10 Hz. The animals were sacrificed and the spinal cords were harvested for Western blot analysis of the expression of neurotrophic factors in the lumbar spinal cord. We provide evidence that bike- and step-training significantly increase the levels of brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and NT-4 in the lumbar enlargement of SCI rats, whereas only step-training increased glial cell-derived neurotrophic factor (GDNF) levels. An increase in neurotrophic factor protein levels that positively correlated with the recovery of H-reflex frequency-dependent depression suggests a role for neurotrophic factors in reflex normalization.

Peripheral nerve grafts (PNG) into the rat spinal cord support axon regeneration after acute or chronic injury, with synaptic reconnection across the lesion site and some level of behavioral recovery. Here, we grafted a peripheral nerve into the injured spinal cord of cats as a preclinical treatment approach to promote regeneration for eventual translational use. Adult female cats received a partial hemisection lesion at the cervical level (C7) and immediate apposition of an autologous tibial nerve segment to the lesion site. Five weeks later, a dorsal quadrant lesion was performed caudally (T1), the lesion site treated with chondroitinase ABC 2 days later to digest growth inhibiting extracellular matrix molecules, and the distal end of the PNG apposed to the injury site. After 4-20 weeks, the grafts survived in 10/12 animals with several thousand myelinated axons present in each graft. The distal end of 9/10 grafts was well apposed to the spinal cord and numerous axons extended beyond the lesion site. Intraspinal stimulation evoked compound action potentials in the graft with an appropriate latency illustrating normal axonal conduction of the regenerated axons. Although stimulation of the PNG failed to elicit responses in the spinal cord distal to the lesion site, the presence of c-Fos immunoreactive neurons close to the distal apposition site indicates that regenerated axons formed functional synapses with host neurons. This study demonstrates the successful application of a nerve grafting approach to promote regeneration after spinal cord injury in a non-rodent, large animal model., (Copyright 2010 Elsevier Inc. All rights reserved.)

A simple neuromechanical model has been developed that describes a spinal central pattern generator (CPG) controlling the locomotor movement of a single-joint limb via activation of two antagonist (flexor and extensor) muscles. The limb performs rhythmic movements under control of the muscular, gravitational and ground reaction forces. Muscle afferents provide length-dependent (types Ia and II) and force-dependent (type Ib from the extensor) feedback to the CPG. We show that afferent feedback adjusts CPG operation to the kinematics and dynamics of the limb providing stable "locomotion." Increasing the supraspinal drive to the CPG increases locomotion speed by reducing the duration of stance phase. We show that such asymmetric, extensor-dominated control of locomotor speed (with relatively constant swing duration) is provided by afferent feedback independent of the asymmetric rhythmic pattern generated by the CPG alone (in "fictive locomotion" conditions). Finally, we demonstrate the possibility of reestablishing stable locomotion after removal of the supraspinal drive (associated with spinal cord injury) by increasing the weights of afferent inputs to the CPG, which is thought to occur following locomotor training.

The H-reflex habituates at relatively low frequency (10 Hz) stimulation in the intact spinal cord, but loss of descending inhibition resulting from spinal cord transection reduces this habituation. There is a return towards a normal pattern of low-frequency habituation in the reflex activity with cycling exercise of the affected hind limbs. This implies that repetitive passive stretching of the muscles in spinalized animals and the accompanying stimulation of large (Group I and II) proprioceptive fibers has modulatory effects on spinal cord reflexes after injury. To test this hypothesis, we induced pyridoxine neurotoxicity that preferentially affects large dorsal root ganglia neurons in intact and spinalized rats. Pyridoxine or saline injections were given twice daily (IP) for 6 weeks and half of the spinalized animals were subjected to cycling exercise during that period. After 6 weeks, the tibial nerve was stimulated electrically and recordings of M and H waves were made from interosseous muscles of the hind paw. Results show that pyridoxine treatment completely eliminated the H-reflex in spinal intact animals. In contrast, transection paired with pyridoxine treatment resulted in a reduction of the frequency-dependent habituation of the H-reflex that was not affected by exercise. These results indicate that normal Group I and II afferent input is critical to achieve exercise-based reversal of hyper-reflexia of the H-reflex after spinal cord injury.

Because there currently is no treatment for spinal cord injury, most patients are living with long-standing injuries. Therefore, strategies aimed at promoting restoration of function to the chronically injured spinal cord have high therapeutic value. For successful regeneration, long-injured axons must overcome their poor intrinsic growth potential as well as the inhibitory environment of the glial scar established around the lesion site. Acutely injured axons that regenerate into growth-permissive peripheral nerve grafts (PNGs) reenter host tissue to mediate functional recovery if the distal graft-host interface is treated with chondroitinase ABC (ChABC) to cleave inhibitory chondroitin sulfate proteoglycans in the scar matrix. To determine whether a similar strategy is effective for a chronic injury, we combined grafting of a peripheral nerve into a highly relevant, chronic, cervical contusion site with ChABC treatment of the glial scar and glial cell line-derived neurotrophic factor (GDNF) stimulation of long-injured axons. We tested this combination in two grafting paradigms: (1) a peripheral nerve that was grafted to span a chronic injury site or (2) a PNG that bridged a chronic contusion site with a second, more distal injury site. Unlike GDNF-PBS treatment, GDNF-ChABC treatment facilitated axons to exit the PNG into host tissue and promoted some functional recovery. Electrical stimulation of axons in the peripheral nerve bridge induced c-Fos expression in host neurons, indicative of synaptic contact by regenerating fibers. Thus, our data demonstrate, for the first time, that administering ChABC to a distal graft interface allows for functional axonal regeneration by chronically injured neurons.

Current evidence suggests that significant morphological changes occur in nerve-muscle connections caudal to spinal cord injury (SCI). To determine whether neuromuscular junction (NMJ) function is compromised after SCI, we investigated the contribution of NMJ failure to hindlimb muscle fatigue in control and spinalized adult rats. Repetitive supramaximal nerve stimulation was applied to two muscle-nerve preparations: medial gastrocnemius (MG)-tibial and tibialis anterior (TA)-peroneal. NMJ transmission failure was evident in control and SCI animals after repetitive stimulation. At 2 wk post-SCI, NMJ transmission failure was greater in SCI animals compared with controls, but the difference was not significant (P = 0.205 for the MG and P = 0.053 for the TA). At 6 wk post-SCI, there was a significant but small difference in NMJ transmission failure for the TA between control and spinal animals. These results demonstrate that, although there may be a mild decrement in NMJ function, NMJ transmission remains largely intact for supramaximal nerve stimulation.

We measured the forces produced at the cat's hindpaw by microstimulation of the lumbar spinal cord and the movements resulting from those forces. We also measured the forces and movements produced by co- and sequential activation of two intraspinal sites. Isometric force responses were measured at nine limb configurations with the paw attached to a force transducer. The active forces elicited at different limb configurations were summarized as patterns representing the sagittal plane component of the forces produced at the paw throughout the workspace. The force patterns divided into the same distinct types found with the femur fixed. The responses during simultaneous activation of two spinal sites always resembled the response for activation of one of the two sites, i.e., winner-take-all, and we did not observe vectorial summation of the forces produced by activation of each site individually as reported in chronic spinal animals. The movements produced by activation of each of the sites were consistent with the force orientations, and different movements could be created by varying the sequence of activation of individual sites. Our results highlight the absence of a vectorial summation phenomenon during intraspinal microstimulation in decerebrate animals, and the preservation during movement of the orientation of isometric forces.

Chronic spinal cats with neurotrophin-secreting fibroblasts (NTF) transplants recover locomotor function. To ascertain possible mechanisms, intraspinal microstimulation was used to examine the lumbar spinal cord motor output of four groups of chronic spinal cats: untrained cats with unmodified-fibroblasts graft (Op-control) or NTF graft and locomotor-trained cats with unmodified-fibroblasts graft (Trained) or NTF graft (Combination). Forces generated via intraspinal microstimulation at different hindlimb positions were recorded and interpolated, generating representations of force patterns at the paw. Electromyographs (EMGs) of hindlimb muscles, medial gastrocnemius, tibialis anterior, vastus lateralis, and biceps femoris posterior, were also collected to examine relationships between activated muscles and force pattern types. The same four force pattern types obtained in spinal-intact cats were found in chronic spinal cats. Proportions of force patterns in spinal cats differed significantly from those in intact cats, but no significant differences in proportions were observed among individual spinal groups (Op-control, NTF, Trained, and Combination). However, the proportions of force patterns differed significantly between trained (Trained and Combination) and untrained groups (Op-control and NTF). Thus the frequency of expression of some response types was modified by injury and to a lesser extent by training. Force pattern laminar distribution differed in spinal cats compared with intact, with more responses obtained dorsally (0-1,000 microm) and fewer ventrally (3,200-5,200 microm). EMG analysis demonstrated that muscle activity highly predicted some force pattern types and was independent of hindlimb position. We conclude that spinal motor output modularity is preserved after injury.

In spinal cats, locomotor recovery without rehabilitation is limited, but weight-bearing stepping returns with treadmill training. We studied whether neurotrophins administered to the injury site also restores locomotion in untrained spinal cats and whether combining both neurotrophins and training further improves recovery. Ordinary rat fibroblasts or a mixture of fibroblasts secreting brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) (Fb-NTF) were grafted into T12 spinal transection sites. Cats with each type of transplant were divided into two groups: one receiving daily training and the other receiving no training. As expected, trained cats with/without neurotrophin-producing transplants could step on the treadmill. Untrained cats without neurotrophin-producing transplants could not locomote. However, untrained cats with neurotrophin-secreting transplants performed plantar weight-bearing stepping at speeds up to 0.8 m/s as early as 2 wk after transection. Locomotor capability and stance lengths in these animals were similar to those in animals receiving training alone, suggesting that administration of BDNF/NT-3 was equivalent to treadmill training in restoring locomotion in chronically spinalized cats. Cats receiving both interventions showed the greatest improvement in step length. Anatomical evaluation indicated that all transections were complete and that axons did not enter the cord caudal to the graft. Thus BDNF/NT-3 secreting fibroblasts were equivalent to training in their ability to engage the locomotor circuitry in chronic spinal cats. Furthermore, the rapid time-course of recovery and the absence of axonal growth through the transplants indicate that the restorative mechanisms were not related to supraspinal axonal growth. Finally, the results show that transplants beneficial in rodents are applicable to larger mammals.

Fibrillation potentials and positive sharp waves (spontaneous potentials) are the electrophysiological hallmark of denervated skeletal muscle, and their detection by intramuscular electromyography (EMG) is the clinical gold standard for diagnosing denervated skeletal muscle. Surprisingly, spontaneous potentials have been described following human and experimental spinal cord injury (SCI) in muscles innervated by spinal cord segments distal to the level of direct spinal injury. To determine whether electrophysiological abnormalities are improved by two therapeutic interventions for experimental SCI, neurotrophic factors and exercise training, we studied four representative hindlimb muscles in adult domestic short-hair cats following complete transection of the spinal cord at T11-T12. In untreated cats, electrophysiological abnormalities persisted unchanged for 12 weeks postinjury, the longest duration studied. In contrast, fibrillations and positive sharp waves largely resolved in animals that underwent weight-supported treadmill training or received grafts containing fibroblasts genetically modified to express brain-derived neurotrophic factor and neurotrophin-3. These findings suggest that neurotrophins and activity play an important role in the poorly understood phenomenon of fibrillations distal to SCI.

We used a musculoskeletal model of the cat hindlimb to compare the patterns of endpoint forces generated by all possible combination of 12 hindlimb muscles under three different muscle activation rules: homogeneous activation of muscles based on uniform activation levels, homogeneous activation of muscles based on uniform (normalized) force production, and activation based on the topography of spinal motoneuron pools. Force patterns were compared with the patterns obtained experimentally by microstimulation of the lumbar spinal cord in spinal intact cats. Magnitude and orientation of the force patterns were compared, as well as the proportion of the types found, and the proportions of patterns exhibiting points of zero force (equilibrium points). The force patterns obtained with the homogenous activation and motoneuron topography models were quite similar to those measured experimentally, with the differences being larger for the patterns from the normalized endpoint forces model. Differences in the proportions of types of force patterns between the three models and the experimental results were significant for each model. Both homogeneous activation and normalized endpoint force models produced similar proportions of equilibrium points as found experimentally. The results suggest that muscle biomechanics play an important role in limiting the number of endpoint force pattern types, and that muscle combinations activated at similar levels reproduced best the experimental results obtained with intraspinal microstimulation.

In experimental decerebration of mammals, the cerebral cortex and thalamus are surgically or otherwise inactivated under traditional (pharmacologic) general anesthesia. Once the effects of the pharmacologic anesthesia have dissipated, the animal remains alive, but there is neither pain sensation nor consciousness. Because the Animal Welfare Act and its regulations recognize drugs as the only means to alleviate pain, it is unclear whether a decerebrate animal should be placed in U.S. Department of Agriculture (USDA) pain and distress category D (pain or distress alleviated by drugs) or E (unalleviated pain or distress). We present a rationale for including decerebrate animals in USDA category D. We also provide a general review of decerebration and suggestions for institutional animal care and use committees having to evaluate decerebration protocols.

Spontaneous potentials in skeletal muscle distal to human spinal cord injury (SCI) have been reported in the literature. Two animal models of SCI were studied for the presence of similar potentials. Six rats and two cats with surgical transections of the thoracic spinal cord were followed for 4-6 weeks with serial electromyography. As a control for the effects of anesthesia and serial testing, three intact rats were anesthetized and tested weekly for 4 weeks. In rats with spinal cord transection, spontaneous potentials emerged 4-7 days after surgery and persisted for the duration of the study (28-32 days). Spontaneous potentials were absent in controls at all timepoints. In cats, spontaneous potentials were observed 8 days postinjury and gradually diminished, starting at 2 weeks. Spontaneous potentials therefore occur after SCI in animals as well as in humans. The utilization of animal models will facilitate the understanding of alterations that occur distal to spinal cord lesions and affect the function of lower motor neurons, leading to peripheral denervation after SCI.

We studied the forces produced at the cat's hindpaw by microstimulation of the ipsi- and contralateral lumbar spinal cord in spinal intact alpha-chloralose anesthetized (n = 3) or decerebrate (n = 3) animals. Isometric force and EMG responses were measured at 9-12 limb configurations, with the paw attached to a force transducer and with the hip and femur fixed. The active forces elicited at different limb configurations were summarized as force fields representing the sagittal plane component of the forces produced at the paw throughout the workspace. The forces varied in amplitude over time but the orientations were stable, and the pattern of an active force field was invariant through time. The active force fields divided into four distinct types, and a few of the fields showed convergence to an equilibrium point. The fields were generally produced by coactivation of the hindlimb muscles. In addition, some of the fields were consistent with known spinal reflexes and the stimulation sites producing them were in laminae where the interneurons associated with those reflexes are known to be located. Muscle activation produced by intraspinal stimulation, as assessed by intramuscular EMG activity, was modified with limb configuration, suggesting that the responses were not fixed, but were modified by position-dependent sensory feedback. The force responses may represent basic outputs of the spinal circuitry and may be related to similar spinal primitives found in the frog and rat.