Reconstruction and Simulation of Neocortical Microcircuitry.

We present a first-draft digital reconstruction of the microcircuitry of somatosensory cortex of juvenile rat. The reconstruction uses cellular and synaptic organizing principles to algorithmically reconstruct detailed anatomy and physiology from sparse experimental data. An objective anatomical method defines a neocortical volume of 0.29 ± 0.01 mm(3) containing ~31,000 neurons, and patch-clamp studies identify 55 layer-specific morphological and 207 morpho-electrical neuron subtypes. When digitally reconstructed neurons are positioned in the volume and synapse formation is restricted to biological bouton densities and numbers of synapses per connection, their overlapping arbors form ~8 million connections with ~37 million synapses. Simulations reproduce an array of in vitro and in vivo experiments without parameter tuning. Additionally, we find a spectrum of network states with a sharp transition from synchronous to asynchronous activity, modulated by physiological mechanisms. The spectrum of network states, dynamically reconfigured around this transition, supports diverse information processing strategies. PAPERCLIP: VIDEO ABSTRACT.

A neuroanatomical basis for electroacupuncture to drive the vagal-adrenal axis.

Somatosensory autonomic reflexes allow electroacupuncture stimulation (ES) to modulate body physiology at distant sites1-6 (for example, suppressing severe systemic inflammation6-9). Since the 1970s, an emerging organizational rule about these reflexes has been the presence of body-region specificity1-6. For example, ES at the hindlimb ST36 acupoint but not the abdominal ST25 acupoint can drive the vagal-adrenal anti-inflammatory axis in mice10,11. The neuroanatomical basis of this somatotopic organization is, however, unknown. Here we show that PROKR2Cre-marked sensory neurons, which innervate the deep hindlimb fascia (for example, the periosteum) but not abdominal fascia (for example, the peritoneum), are crucial for driving the vagal-adrenal axis. Low-intensity ES at the ST36 site in mice with ablated PROKR2Cre-marked sensory neurons failed to activate hindbrain vagal efferent neurons or to drive catecholamine release from adrenal glands. As a result, ES no longer suppressed systemic inflammation induced by bacterial endotoxins. By contrast, spinal sympathetic reflexes evoked by high-intensity ES at both ST25 and ST36 sites were unaffected. We also show that optogenetic stimulation of PROKR2Cre-marked nerve terminals through the ST36 site is sufficient to drive the vagal-adrenal axis but not sympathetic reflexes. Furthermore, the distribution patterns of PROKR2Cre nerve fibres can retrospectively predict body regions at which low-intensity ES will or will not effectively produce anti-inflammatory effects. Our studies provide a neuroanatomical basis for the selectivity and specificity of acupoints in driving specific autonomic pathways.

Patterns of spinal sensory-motor connectivity prescribed by a dorsoventral positional template.

Sensory-motor circuits in the spinal cord are constructed with a fine specificity that coordinates motor behavior, but the mechanisms that direct sensory connections with their motor neuron partners remain unclear. The dorsoventral settling position of motor pools in the spinal cord is known to match the distal-to-proximal position of their muscle targets in the limb, but the significance of invariant motor neuron positioning is unknown. An analysis of sensory-motor connectivity patterns in FoxP1 mutant mice, where motor neuron position has been scrambled, shows that the final pattern of sensory-motor connections is initiated by the projection of sensory axons to discrete dorsoventral domains of the spinal cord without regard for motor neuron subtype or, indeed, the presence of motor neurons. By implication, the clustering and dorsoventral settling position of motor neuron pools serve as a determinant of the pattern of sensory input specificity and thus motor coordination.

Changes in intra- and interlimb reflexes from hindlimb cutaneous afferents after staggered thoracic lateral hemisections during locomotion in cats.

When the foot dorsum contacts an obstacle during locomotion, cutaneous afferents signal central circuits to coordinate muscle activity in the four limbs. Spinal cord injury disrupts these interactions, impairing balance and interlimb coordination. We evoked cutaneous reflexes by electrically stimulating left and right superficial peroneal nerves before and after two thoracic lateral hemisections placed on opposite sides of the cord at 9- to 13-week interval in seven adult cats (4 males and 3 females). We recorded reflex responses in ten hindlimb and five forelimb muscles bilaterally. After the first (right T5-T6) and second (left T10-T11) hemisections, coordination of the fore- and hindlimbs was altered and/or became less consistent. After the second hemisection, cats required balance assistance to perform quadrupedal locomotion. Short-latency reflex responses in homonymous and crossed hindlimb muscles largely remained unaffected after staggered hemisections. However, mid- and long-latency homonymous and crossed responses in both hindlimbs occurred less frequently after staggered hemisections. In forelimb muscles, homolateral and diagonal mid- and long-latency response occurrence significantly decreased after the first and second hemisections. In all four limbs, however, when present, short-, mid- and long-latency responses maintained their phase-dependent modulation. We also observed reduced durations of short-latency inhibitory homonymous responses in left hindlimb extensors early after the first hemisection and delayed short-latency responses in the right ipsilesional hindlimb after the first hemisection. Therefore, changes in cutaneous reflex responses correlated with impaired balance/stability and interlimb coordination during locomotion after spinal cord injury. Restoring reflex transmission could be used as a biomarker to facilitate locomotor recovery. KEY POINTS: Cutaneous afferent inputs coordinate muscle activity in the four limbs during locomotion when the foot dorsum contacts an obstacle. Thoracic spinal cord injury disrupts communication between spinal locomotor centres located at cervical and lumbar levels, impairing balance and limb coordination. We investigated cutaneous reflexes during quadrupedal locomotion by electrically stimulating the superficial peroneal nerve bilaterally, before and after staggered lateral thoracic hemisections of the spinal cord in cats. We showed a loss/reduction of mid- and long-latency responses in all four limbs after staggered hemisections, which correlated with altered coordination of the fore- and hindlimbs and impaired balance. Targeting cutaneous reflex pathways projecting to the four limbs could help develop therapeutic approaches aimed at restoring transmission in ascending and descending spinal pathways.

Coordination amongst quadriceps muscles suggests neural regulation of internal joint stresses, not simplification of task performance.

Many studies have demonstrated covariation between muscle activations during behavior, suggesting that muscles are not controlled independently. According to one common proposal, this covariation reflects simplification of task performance by the nervous system so that muscles with similar contributions to task variables are controlled together. Alternatively, this covariation might reflect regulation of low-level aspects of movements that are common across tasks, such as stresses within joints. We examined these issues by analyzing covariation patterns in quadriceps muscle activity during locomotion in rats. The three monoarticular quadriceps muscles (vastus medialis [VM], vastus lateralis [VL], and vastus intermedius [VI]) produce knee extension and so have identical contributions to task performance; the biarticular rectus femoris (RF) produces an additional hip flexion. Consistent with the proposal that muscle covariation is related to similarity of muscle actions on task variables, we found that the covariation between VM and VL was stronger than their covariations with RF. However, covariation between VM and VL was also stronger than their covariations with VI. Since all vastii have identical actions on task variables, this finding suggests that covariation between muscle activity is not solely driven by simplification of overt task performance. Instead, the preferentially strong covariation between VM and VL is consistent with the control of internal joint stresses: Since VM and VL produce opposing mediolateral forces on the patella, the high positive correlation between their activation minimizes the net mediolateral patellar force. These results provide important insights into the interpretation of muscle covariations and their role in movement control.

In Vivo Calcium Imaging Visualizes Incision-Induced Primary Afferent Sensitization and Its Amelioration by Capsaicin Pretreatment.

Previous studies have shown that infiltration of capsaicin into the surgical site can prevent incision-induced spontaneous pain like behaviors and heat hyperalgesia. In the present study, we aimed to monitor primary sensory neuron Ca2+ activity in the intact dorsal root ganglia (DRG) using Pirt-GCaMP3 male and female mice pretreated with capsaicin or vehicle before the plantar incision. Intraplantar injection of capsaicin (0.05%) significantly attenuated spontaneous pain, mechanical, and heat hypersensitivity after plantar incision. The Ca2+ response in in vivo DRG and in in situ spinal cord was significantly enhanced in the ipsilateral side compared with contralateral side or naive control. Primary sensory nerve fiber length was significantly decreased in the incision skin area in capsaicin-pretreated animals detected by immunohistochemistry and placental alkaline phosphatase (PLAP) staining. Thus, capsaicin pretreatment attenuates incisional pain by suppressing Ca2+ response because of degeneration of primary sensory nerve fibers in the skin.SIGNIFICANCE STATEMENT Postoperative surgery pain is a major health and economic problem worldwide with ∼235 million major surgical procedures annually. Approximately 50% of these patients report uncontrolled or poorly controlled postoperative pain. However, mechanistic studies of postoperative surgery pain in primary sensory neurons have been limited to in vitro models or small numbers of neurons. Using an innovative, distinctive, and interdisciplinary in vivo populational dorsal root ganglia (DRG) imaging (>1800 neurons/DRG) approach, we revealed increased DRG neuronal Ca2+ activity from postoperative pain mouse model. This indicates widespread DRG primary sensory neuron plasticity. Increased neuronal Ca2+ activity occurs among various sizes of neurons but mostly in small-diameter and medium-diameter nociceptors. Capsaicin pretreatment as a therapeutic option significantly attenuates Ca2+ activity and postoperative pain.

Asymmetric and transient properties of reciprocal activity of antagonists during the paw-shake response in the cat.

Mutually inhibitory populations of neurons, half-center oscillators (HCOs), are commonly involved in the dynamics of the central pattern generators (CPGs) driving various rhythmic movements. Previously, we developed a multifunctional, multistable symmetric HCO model which produced slow locomotor-like and fast paw-shake-like activity patterns. Here, we describe asymmetric features of paw-shake responses in a symmetric HCO model and test these predictions experimentally. We considered bursting properties of the two model half-centers during transient paw-shake-like responses to short perturbations during locomotor-like activity. We found that when a current pulse was applied during the spiking phase of one half-center, let's call it #1, the consecutive burst durations (BDs) of that half-center increased throughout the paw-shake response, while BDs of the other half-center, let's call it #2, only changed slightly. In contrast, the consecutive interburst intervals (IBIs) of half-center #1 changed little, while IBIs of half-center #2 increased. We demonstrated that this asymmetry between the half-centers depends on the phase of the locomotor-like rhythm at which the perturbation was applied. We suggest that the fast transient response reflects functional asymmetries of slow processes that underly the locomotor-like pattern; e.g., asymmetric levels of inactivation across the two half-centers for a slowly inactivating inward current. We compared model results with those of in-vivo paw-shake responses evoked in locomoting cats and found similar asymmetries. Electromyographic (EMG) BDs of anterior hindlimb muscles with flexor-related activity increased in consecutive paw-shake cycles, while BD of posterior muscles with extensor-related activity did not change, and vice versa for IBIs of anterior flexors and posterior extensors. We conclude that EMG activity patterns during paw-shaking are consistent with the proposed mechanism producing transient paw-shake-like bursting patterns found in our multistable HCO model. We suggest that the described asymmetry of paw-shaking responses could implicate a multifunctional CPG controlling both locomotion and paw-shaking.

Evidence That the Central Nervous System Can Induce a Modification at the Neuromuscular Junction That Contributes to the Maintenance of a Behavioral Response.

Neurons within the spinal cord are sensitive to environmental relations and can bring about a behavioral modification without input from the brain. For example, rats that have undergone a thoracic (T2) transection can learn to maintain a hind leg in a flexed position to minimize exposure to a noxious electrical stimulation (shock). Inactivating neurons within the spinal cord with lidocaine, or cutting communication between the spinal cord and the periphery (sciatic transection), eliminates the capacity to learn, which implies that it depends on spinal neurons. Here we show that these manipulations have no effect on the maintenance of the learned response, which implicates a peripheral process. EMG showed that learning augments the muscular response evoked by motoneuron output and that this effect survives a sciatic transection. Quantitative fluorescent imaging revealed that training brings about an increase in the area and intensity of ACh receptor labeling at the neuromuscular junction (NMJ). It is hypothesized that efferent motoneuron output, in conjunction with electrical stimulation of the tibialis anterior muscle, strengthens the connection at the NMJ in a Hebbian manner. Supporting this, paired stimulation of the efferent nerve and tibialis anterior generated an increase in flexion duration and augmented the evoked electrical response without input from the spinal cord. Evidence is presented that glutamatergic signaling contributes to plasticity at the NMJ. Labeling for vesicular glutamate transporter is evident at the motor endplate. Intramuscular application of an NMDAR antagonist blocked the acquisition/maintenance of the learned response and the strengthening of the evoked electrical response.SIGNIFICANCE STATEMENT The neuromuscular junction (NMJ) is designed to faithfully elicit a muscular contraction in response to neural input. From this perspective, encoding environmental relations (learning) and the maintenance of a behavioral modification over time (memory) are assumed to reflect only modifications upstream from the NMJ, within the CNS. The current results challenge this view. Rats were trained to maintain a hind leg in a flexed position to avoid noxious stimulation. As expected, treatments that inhibit activity within the CNS, or disrupt peripheral communication, prevented learning. These manipulations did not affect the maintenance of the acquired response. The results imply that a peripheral modification at the NMJ contributes to the maintenance of the learned response.

Neonatal Injury Evokes Persistent Deficits in Dynorphin Inhibitory Circuits within the Adult Mouse Superficial Dorsal Horn.

Neonatal tissue damage induces long-term deficits in inhibitory synaptic transmission within the spinal superficial dorsal horn (SDH) that include a reduction in primary afferent-evoked, feedforward inhibition onto adult projection neurons. However, the subpopulations of mature GABAergic interneurons which are compromised by early-life injury have yet to be identified. The present research illuminates the persistent effects of neonatal surgical injury on the function of inhibitory SDH interneurons derived from the prodynorphin (DYN) lineage, a population that synapses directly onto lamina I spinoparabrachial neurons and is known to suppress mechanical pain and itch in adults. The results demonstrate that hindpaw incision at postnatal day 3 (P3) significantly decreased the strength of primary afferent-evoked glutamatergic drive onto DYN neurons within the adult mouse SDH while increasing the appearance of afferent-evoked inhibition onto the same population. Neonatal injury also dampened the intrinsic membrane excitability of mature DYN neurons, and reduced their action potential discharge in response to sensory input, compared with naive littermate controls. Furthermore, P3 incision decreased the efficacy of inhibitory DYN synapses onto adult spinoparabrachial neurons, which reflected a prolonged reduction in the probability of GABA release. Collectively, the data suggest that early-life tissue damage may persistently constrain the ability of spinal DYN interneurons to limit ascending nociceptive transmission to the adult brain. This is predicted to contribute to the loss of feedforward inhibition onto mature projection neurons, and the "priming" of nociceptive circuits in the developing spinal cord, following injuries during the neonatal period.SIGNIFICANCE STATEMENT Neonatal injury has lasting effects on pain processing in the adult CNS, including a reduction in feedforward inhibition onto ascending projection neurons in the spinal dorsal horn. While it is clear that spinal GABAergic interneurons are comprised of multiple subpopulations that play distinct roles in somatosensation, the identity of those interneurons which are compromised by tissue damage during early life remains unknown. Here we document persistent deficits in spinal inhibitory circuits involving dynorphin-lineage interneurons previously implicated in gating mechanical pain and itch. Notably, neonatal injury reduced the strength of dynorphin-lineage inhibitory synapses onto mature lamina I spinoparabrachial neurons, a major output of the spinal nociceptive network, which could contribute to the priming of pain pathways by early tissue damage.

Poststroke Impairment and Recovery Are Predicted by Task-Specific Regionalization of Injury.

Lesion size and location affect the magnitude of impairment and recovery following stroke, but the precise relationship between these variables and functional outcome is unknown. Herein, we systematically varied the size of strokes in motor cortex and surrounding regions to assess effects on impairment and recovery of function. Female Sprague Dawley rats (N = 64) were evaluated for skilled reaching, spontaneous limb use, and limb placement over a 7 week period after stroke. Exploration and reaching were also tested in a free ranging, more naturalistic, environment. MRI voxel-based analysis of injury volume and its likelihood of including the caudal forelimb area (CFA), rostral forelimb area (RFA), hindlimb (HL) cortex (based on intracranial microstimulation), or their bordering regions were related to both impairment and recovery. Severity of impairment on each task was best predicted by injury in unique regions: impaired reaching, by damage in voxels encompassing CFA/RFA; hindlimb placement, by damage in HL; and spontaneous forelimb use, by damage in CFA. An entirely different set of voxels predicted recovery of function: damage lateral to RFA reduced recovery of reaching, damage medial to HL reduced recovery of hindlimb placing, and damage lateral to CFA reduced recovery of spontaneous limb use. Precise lesion location is an important, but heretofore relatively neglected, prognostic factor in both preclinical and clinical stroke studies, especially those using region-specific therapies, such as transcranial magnetic stimulation.SIGNIFICANCE STATEMENT By estimating lesion location relative to cortical motor representations, we established the relationship between individualized lesion location, and functional impairment and recovery in reaching/grasping, spontaneous limb use, and hindlimb placement during walking. We confirmed that stroke results in impairments to specific motor domains linked to the damaged cortical subregion and that damage encroaching on adjacent regions reduces the ability to recover from initial lesion-induced impairments. Each motor domain encompasses unique brain regions that are most associated with recovery and likely represent targets where beneficial reorganization is taking place. Future clinical trials should use individualized therapies (e.g., transcranial magnetic stimulation, intracerebral stem/progenitor cells) that consider precise lesion location and the specific functional impairments of each subject since these variables can markedly affect therapeutic efficacy.

Formation of a novel supraspinal-spinal connectome that relearns the same motor task after complete paralysis.

Having observed that electrical spinal cord stimulation and training enabled four patients with paraplegia with motor complete paralysis to regain voluntary leg movement, the underlying mechanisms involved in forming the newly established supraspinal-spinal functional connectivity have become of great interest. van den Brand et al. (Science 336: 1182-1185, 2012) subsequently, demonstrated the recovery, in response to spinal electro-neuromodulation and locomotor training, of voluntary stepping of the lower limbs in rats that received a lesion that is assumed to eliminate all long-descending cortical axons that project to lumbosacral segments. Here, we used a similar spinal lesion in rats to eliminate long-descending axons to determine whether a novel, trained motor behavior triggered by a unique auditory cue learned before a spinal lesion, could recover after the lesion. Hindlimb stepping recovered 1 mo after the spinal injury, but only after 2 mo, the novel and unique audio-triggered behavior was recovered, meaning that not only was a novel connectivity formed but also further evidence suggested that this highly unique behavioral response was independent of the recovery of the circuitry that generated stepping. The unique features of the newly formed supraspinal-spinal connections that mediated the recovery of the trained behavior is consistent with a guidance mechanism(s) that are highly use dependent.NEW & NOTEWORTHY Electrical spinal cord stimulation has enabled patients with paraplegia to regain voluntary leg movement, and so the underlying mechanisms involved in this recovery are of great interest. Here, we demonstrate in rodents the recovery of trained motor behavior after a spinal lesion. Rodents were trained to kick their right hindlimb in response to an auditory cue. This behavior recovered 2 mo after the paralyzing spinal cord injury but only with the assistance of electrical spinal cord stimulation.

Spleen tyrosine kinase facilitates neutrophil activation and worsens long-term neurologic deficits after spinal cord injury.

BACKGROUND: Spinal cord injury elicits widespread inflammation that can exacerbate long-term neurologic deficits. Neutrophils are the most abundant immune cell type to invade the spinal cord in the early acute phase after injury, however, their role in secondary pathogenesis and functional recovery remains unclear. We have previously shown that neutrophil functional responses during inflammation are augmented by spleen tyrosine kinase, Syk, a prominent intracellular signaling enzyme. In this study, we evaluated the contribution of Syk towards neutrophil function and long-term neurologic deficits after spinal cord injury. METHODS: Contusive spinal cord injury was performed at thoracic vertebra level 9 in mice with conditional deletion of Syk in neutrophils (Sykf/fMRP8-Cre). Hindlimb locomotor recovery was evaluated using an open-field test for 35 days following spinal cord injury. Long-term white matter sparing was assessed using eriochrome cyanide staining. Blood-spinal cord barrier disruption was evaluated by immunoblotting. Neutrophil infiltration, activation, effector functions, and cell death were determined by flow cytometry. Cytokine and chemokine expression in neutrophils was assessed using a gene array. RESULTS: Syk deficiency in neutrophils improved long-term functional recovery after spinal cord injury, but did not promote long-term white matter sparing. Neutrophil activation, cytokine expression, and cell death in the acutely injured spinal cord were attenuated by the genetic loss of Syk while neutrophil infiltration and effector functions were not affected. Acute blood-spinal cord barrier disruption was also unaffected by Syk deficiency in neutrophils. CONCLUSIONS: Syk facilitates specific neutrophil functional responses to spinal cord injury including activation, cytokine expression, and cell death. Long-term neurologic deficits are exacerbated by Syk signaling in neutrophils independent of acute blood-spinal cord barrier disruption and long-term white matter sparing. These findings implicate Syk in pathogenic neutrophil activities that worsen long-term functional recovery after spinal cord injury.

Cutaneous inputs from perineal region facilitate spinal locomotor activity and modulate cutaneous reflexes from the foot in spinal cats.

It is well known that mechanically stimulating the perineal region potently facilitates hindlimb locomotion and weight support in mammals with a spinal transection (spinal mammals). However, how perineal stimulation mediates this excitatory effect is poorly understood. We evaluated the effect of mechanically stimulating (vibration or pinch) the perineal region on ipsilateral (9-14 ms onset) and contralateral (14-18 ms onset) short-latency cutaneous reflex responses evoked by electrically stimulating the superficial peroneal or distal tibial nerve in seven adult spinal cats where hindlimb movement was restrained. Cutaneous reflexes were evoked before, during, and after mechanical stimulation of the perineal region. We found that vibration or pinch of the perineal region effectively triggered rhythmic activity, ipsilateral and contralateral to nerve stimulation. When electrically stimulating nerves, adding perineal stimulation modulated rhythmic activity by decreasing cycle and burst durations and by increasing the amplitude of flexors and extensors. Perineal stimulation also disrupted the timing of the ipsilateral rhythm, which had been entrained by nerve stimulation. Mechanically stimulating the perineal region decreased ipsilateral and contralateral short-latency reflex responses evoked by cutaneous inputs, a phenomenon we observed in muscles crossing different joints and located in different limbs. The results suggest that the excitatory effect of perineal stimulation on locomotion and weight support is mediated by increasing the excitability of central pattern-generating circuitry and not by increasing excitatory inputs from cutaneous afferents of the foot. Our results are consistent with a state-dependent modulation of reflexes by spinal interneuronal circuits.

Establishment of a Sheep Model for Hind Limb Peripheral Nerve Injury: Common Peroneal Nerve.

Thousands of people worldwide suffer from peripheral nerve injuries and must deal daily with the resulting physiological and functional deficits. Recent advances in this field are still insufficient to guarantee adequate outcomes, and the development of new and compelling therapeutic options require the use of valid preclinical models that effectively replicate the characteristics and challenges associated with these injuries in humans. In this study, we established a sheep model for common peroneal nerve injuries that can be applied in preclinical research with the advantages associated with the use of large animal models. The anatomy of the common peroneal nerve and topographically related nerves, the functional consequences of its injury and a neurological examination directed at this nerve have been described. Furthermore, the surgical protocol for accessing the common peroneal nerve, the induction of different types of nerve damage and the application of possible therapeutic options were described. Finally, a preliminary morphological and stereological study was carried out to establish control values for the healthy common peroneal nerves regarding this animal model and to identify preliminary differences between therapeutic methods. This study allowed to define the described lateral incision as the best to access the common peroneal nerve, besides establishing 12 and 24 weeks as the minimum periods to study lesions of axonotmesis and neurotmesis, respectively, in this specie. The post-mortem evaluation of the harvested nerves allowed to register stereological values for healthy common peroneal nerves to be used as controls in future studies, and to establish preliminary values associated with the therapeutic performance of the different applied options, although limited by a small sample size, thus requiring further validation studies. Finally, this study demonstrated that the sheep is a valid model of peripheral nerve injury to be used in pre-clinical and translational works and to evaluate the efficacy and safety of nerve injury therapeutic options before its clinical application in humans and veterinary patients.

Adaptation to slope in locomotor-trained spinal cats with intact and self-reinnervated lateral gastrocnemius and soleus muscles.

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.

Enhancement by TNF-α of TTX-resistant NaV current in muscle sensory neurons after femoral artery occlusion.

Femoral artery occlusion in rats has been used to study human peripheral artery disease (PAD). Using this animal model, a recent study suggests that increases in levels of tumor necrosis factor-α (TNF-α) and its receptor lead to exaggerated responses of sympathetic nervous activity and arterial blood pressure as metabolically sensitive muscle afferents are activated. Note that voltage-dependent Na+ subtype NaV1.8 channels (NaV1.8) are predominately present in chemically sensitive thin fiber sensory nerves. The purpose of this study was to examine the role played by TNF-α in regulating activity of NaV1.8 currents in muscle dorsal root ganglion (DRG) neurons of rats with PAD induced by femoral artery occlusion. DRG neurons from control and occluded limbs of rats were labeled by injecting the fluorescent tracer DiI into the hindlimb muscles 5 days before the experiments. A voltage patch-clamp mode was used to examine TTX-resistant (TTX-R) NaV currents. Results were as follows: 72 h of femoral artery occlusion increased peak amplitude of TTX-R [1,922 ± 139 pA in occlusion (n = 11 DRG neurons) vs. 1,178 ± 39 pA in control (n = 10), means ± SE; P < 0.001 between the 2 groups] and NaV1.8 currents [1,461 ± 116 pA in occlusion (n = 11) and 766 ± 48 pA in control (n = 10); P < 0.001 between groups] in muscle DRG neurons. TNF-α exposure amplified TTX-R and NaV1.8 currents in DRG neurons of occluded muscles in a dose-dependent manner. Notably, the amplification of TTX-R and NaV1.8 currents induced by TNF-α was attenuated in DRG neurons with preincubation with respective inhibitors of the intracellular signaling pathways p38-MAPK, JNK, and ERK. In conclusion, our data suggest that NaV1.8 is engaged in the role of TNF-α in amplifying muscle afferent inputs as the hindlimb muscles are ischemic; p38-MAPK, JNK, and ERK pathways are likely necessary to mediate the effects of TNF-α.

Noradrenergic Components of Locomotor Recovery Induced by Intraspinal Grafting of the Embryonic Brainstem in Adult Paraplegic Rats.

Intraspinal grafting of serotonergic (5-HT) neurons was shown to restore plantar stepping in paraplegic rats. Here we asked whether neurons of other phenotypes contribute to the recovery. The experiments were performed on adult rats after spinal cord total transection. Grafts were injected into the sub-lesional spinal cord. Two months later, locomotor performance was tested with electromyographic recordings from hindlimb muscles. The role of noradrenergic (NA) innervation was investigated during locomotor performance of spinal grafted and non-grafted rats using intraperitoneal application of α2 adrenergic receptor agonist (clonidine) or antagonist (yohimbine). Morphological analysis of the host spinal cords demonstrated the presence of tyrosine hydroxylase positive (NA) neurons in addition to 5-HT neurons. 5-HT fibers innervated caudal spinal cord areas in the dorsal and ventral horns, central canal, and intermediolateral zone, while the NA fiber distribution was limited to the central canal and intermediolateral zone. 5-HT and NA neurons were surrounded by each other's axons. Locomotor abilities of the spinal grafted rats, but not in control spinal rats, were facilitated by yohimbine and suppressed by clonidine. Thus, noradrenergic innervation, in addition to 5-HT innervation, plays a potent role in hindlimb movement enhanced by intraspinal grafting of brainstem embryonic tissue in paraplegic rats.

A Spinal Mechanism Related to Left-Right Symmetry Reduces Cutaneous Reflex Modulation Independently of Speed During Split-Belt Locomotion.

Task- and phase-dependent reflex modulation during locomotion is well established, but we do not know the signals driving this modulation. To determine whether signals related to left-right symmetry of the locomotor pattern modulate cutaneous reflexes, we stimulated the superficial peroneal nerve in five intact female cats and in four spinal-transected cats (spinal cats, two males and two females) during split-belt locomotion at different left-right speeds. We compared cutaneous reflexes evoked in three ipsilateral and two contralateral hindlimb muscles during split-belt locomotion with those evoked during tied-belt (equal left-right speeds) locomotion at matched speeds of the slow and fast limbs. Our results showed similar phase-dependent modulation of cutaneous reflexes during tied-belt and split-belt locomotion in intact and spinal cats. During tied-belt locomotion in intact cats, an increase in speed significantly increased reflex modulation from minimum to maximum values, whereas in spinal cats, we observed a significant decrease. However, in all muscles of intact and spinal cats, split-belt locomotion significantly reduced reflex modulation compared with tied-belt locomotion independently of which limb was stepping on the slow or fast belt. Additionally, reflex modulation correlated more with spatial left-right symmetry, as opposed to a temporal one, in intact and spinal cats. Our results indicate that signals related to left-right symmetry reduce cutaneous reflex modulation independently of speed via a spinal mechanism. We propose that asymmetric sensory feedback from the left and right legs alters the state of the spinal network, thereby reducing cutaneous reflexes to prevent inputs from destabilizing a potentially unstable pattern.SIGNIFICANCE STATEMENT When we contact an obstacle during walking, receptors in the skin send signals to the CNS to alter the trajectory of the leg to maintain balance. This response, or reflex, is different when the leg is in the air and when it is contacting the ground. The reflex also differs when we walk at different speeds. Here, we investigated this reflex when the left and right legs were walking at different speeds on a split-belt treadmill in cats. We show that the reflex is smaller during split-belt locomotion compared with when both legs are walking at equal speeds. We propose that the spinal locomotor network controlling walking reduces the reflex response to optimize balance when gait is unstable.

Ipsilesional Motor Cortex Plasticity Participates in Spontaneous Hindlimb Recovery after Lateral Hemisection of the Thoracic Spinal Cord in the Rat.

After an incomplete spinal cord injury (SCI) spontaneous motor recovery can occur in mammals, but the underlying neural substrates remain poorly understood. The motor cortex is crucial for skilled motor learning and the voluntary control of movement and is known to reorganize after cortical injury to promote recovery. Motor cortex plasticity has also been shown to parallel the recovery of forelimb function after cervical SCI, but whether cortical plasticity participates in hindlimb recovery after SCI remains unresolved. Using intracortical microstimulation (ICMS) mapping, behavioral and cortical inactivation techniques in the female Long-Evans rat, we evaluated the spontaneous cortical mechanisms of hindlimb motor recovery 1-5 weeks after lateral hemisection of the thoracic (T8) spinal cord that ablated the crossed corticospinal tract (CST) from the contralesional motor cortex while sparing the majority of the CST from the ipsilesional motor cortex. Hemisection initially impaired hindlimb motor function bilaterally but significant recovery occurred during the first 3 weeks. ICMS revealed time-dependent changes in motor cortex organization, characterized by a chronic abolishment of hindlimb motor representation in the contralesional motor cortex and the development of transient bilateral hindlimb representation in the ipsilesional motor cortex 3 weeks after hemisection, when significant behavioral recovery occurred. Consistently, reversible inactivation of the ipsilesional, but not the contralesional motor cortex, during skilled ladder walking 3 weeks after hemisection reinstated deficits in both hindlimbs. These findings indicate that the ipsilesional motor cortex transiently reorganizes after lateral hemisection of the thoracic spinal cord to support recovery of hindlimb motor function.SIGNIFICANCE STATEMENT Partial motor recovery can occur after an incomplete spinal cord injury and is hypothesized to result from the reorganization of spared descending motor pathways. The motor cortex is crucial for the control of voluntary movement and contains topographical movement representations (motor maps) that are highly plastic. We examined the organization of hindlimb motor maps bilaterally after a lateral hemisection of the spinal cord to show that while motor maps are abolished in the deefferented cortex, the spared ipsilesional cortex transiently reorganizes to gain a representation of the affected hindlimb after injury that relates to recovery. This finding demonstrates that plasticity in the ipsilesional motor cortex at early time points after spinal cord hemisection is initially important to support motor recovery.

Spinal Angulation: A Limitation of the Fetal Lamb Model of Myelomeningocele.

INTRODUCTION: The surgically induced fetal lamb model is the most commonly used large animal model of myelomeningocele (MMC) but is subject to variation due to surgical technique during defect creation. MATERIAL AND METHODS: Thirty-one fetal lambs underwent creation of the MMC defect, followed by defect repair with either an extracellular matrix (ECM) patch (n = 10) or ECM seeded with placental mesenchymal stromal cells (n = 21). Postnatal hindlimb function was assessed using the Sheep Locomotor Rating (SLR) scale. Postmortem magnetic resonance imaging of the lumbar spine was used to measure the level and degree of spinal angulation, as well as cross-sectional area of remaining vertebral bone. RESULTS: Median level of angulation was between the 2nd and 3rd lumbar vertebrae, with a median angle of 24.3 degrees (interquartile range 16.2-35.3). There was a negative correlation between angulation degree and SLR (r = -0.44, p = 0.013). Degree of angulation also negatively correlated with the normalized cross-sectional area of remaining vertebral bone (r = -0.75, p < 0.0001). DISCUSSION: Surgical creation of fetal MMC leads to varying severity of spinal angulation in the ovine model, which affects postnatal functional outcomes. Postnatal assessment of spinal angulation aids in standardization of the surgical model of fetal MMC repair.