Descending systems direct development of key spinal motor circuits

The formation of mature spinal motor circuits is dependent on both activity dependent and independent mechanisms during postnatal development. During this time, reorganisation and refinement of spinal sensorimotor circuits occurs as supraspinal projections are integrated. However, specific features of postnatal spinal circuit development remain poorly understood. This study provides the first detailed characterisation of rat spinal sensorimotor circuit development in the presence and absence of descending systems. We show that development of proprioceptive afferent (PA) input to motoneurones (MN) and Renshaw cells (RC) is disrupted by thoracic spinal cord transection (TX) at postnatal day 5 (PN5). PN5TX also lead to malformation of GABApre neuron axo-axonic contacts on Ia afferents and the recurrent inhibitory circuit between MN and RC. Using a novel in situ perfused preparation for studying motor control, we show that malformation of these spinal circuits leads to hyperexcitability of the monosynaptic reflex. Our results demonstrate that removing descending input severely disrupts development of spinal circuits and identifies key mechanisms contributing to motor dysfunction in conditions such as cerebral palsy and spinal cord injury

Differential effects of anti-Nogo-A antibody treatment and treadmill training in rats with incomplete spinal cord injury

Locomotor training on treadmills can improve recovery of stepping in spinal cord injured animals and patients. Likewise, lesioned rats treated with antibodies against the myelin associated neurite growth inhibitory protein, Nogo-A, showed increased regeneration, neuronal reorganization and behavioural improvements. A detailed kinematic analysis showed that the hindlimb kinematic patterns that developed in anti-Nogo-A antibody treated versus treadmill trained spinal cord injured rats were significantly different. The synchronous combined treatment group did not show synergistic effects. This lack of synergistic effects could not be explained by an increase in pain perception, sprouting of calcitonin gene-related peptide (CGRP) positive fibres or by interference of locomotor training with anti-Nogo-A antibody induced regeneration and sprouting of descending fibre tracts. The differential mechanisms leading to behavioural recovery during task-specific training and in regeneration or plasticity enhancing therapies have to be taken into account in designing combinatorial therapies so that their potential positive interactive effects can be fully expresse

Training locomotor networks

For a complete adult spinal rat to regain some weight-bearing stepping capability, it appears that a sequence of specific proprioceptive inputs that are similar, but not identical, from step to step must be generated over repetitive step cycles. Furthermore, these cycles must include the activation of specific neural circuits that are intrinsic to the lumbosacral spinal cord segments. For these sensorimotor pathways to be effective in generating stepping, the spinal circuitry must be modulated to an appropriate excitability level. This level of modulation is sustained from supraspinal input in intact, but not spinal, rats. In a series of experiments with complete spinal rats, we have shown that an appropriate level of excitability of the spinal circuitry can be achieved using widely different means. For example, this modulation level can be acquired pharmacologically, via epidural electrical stimulation over specific lumbosacral spinal cord segments, and/or by use-dependent mechanisms such as step or stand training. Evidence as to how each of these treatments can “tune” the spinal circuitry to a “physiological state” that enables it to respond appropriately to proprioceptive input will be presented. We have found that each of these interventions can enable the proprioceptive input to actually control extensive details that define the dynamics of stepping over a range of speeds, loads, and directions. A series of experiments will be described that illustrate sensory control of stepping and standing after a spinal cord injury and the necessity for the “physiological state” of the spinal circuitry to be modulated within a critical window of excitability for this control to be manifested. The present findings have important consequences not only for our understanding of how the motor pattern for stepping is formed, but also for the design of rehabilitation intervention to restore lumbosacral circuit function in humans following a spinal cord injury

Spatiotemporal microvascular changes following contusive spinal cord injury

Microvascular integrity is disrupted following spinal cord injury (SCI) by both primary and secondary insults. Changes to neuronal structures are well documented, but little is known about how the capillaries change and recover following injury. Spatiotemporal morphological information is required to explore potential treatments targeting the microvasculature post-SCI to improve functional recovery. Sprague-Dawley rats were given a T10 moderate/severe (200 kDyn) contusion injury and were perfuse-fixed at days 2, 5, 15, and 45 post-injury. Unbiased stereology following immunohistochemistry in four areas (ventral and dorsal grey and white matter) across seven spinal segments (n = 4 for each group) was used to calculate microvessel density, surface area, and areal density. In intact sham spinal cords, average microvessel density across the thoracic spinal cord was: ventral grey matter: 571 ± 45 mm−2, dorsal grey matter: 484 ± 33 mm−2, ventral white matter: 90 ± 8 mm−2, dorsal white matter: 88 ± 7 mm−2. Post-SCI, acute microvascular disruption was evident, particularly at the injury epicentre, and spreading three spinal segments rostrally and caudally. Damage was most severe in grey matter at the injury epicentre (T10) and T11. Reductions in all morphological parameters (95–99% at day 2 post-SCI) implied vessel regression and/or collapse acutely. Transmission electron microscopy (TEM) revealed disturbed aspects of neurovascular unit fine structure at day 2 post-SCI (n = 2 per group) at T10 and T11. TEM demonstrated a more diffuse and disrupted basement membrane and wider intercellular clefts at day 2, suggesting a more permeable blood spinal cord barrier and microvessel remodelling. Some evidence of angiogenesis was seen during recovery from days 2 to 45, indicated by increased vessel density, surface area, and areal density at day 45. These novel results show that the spinal cord microvasculature is highly adaptive following SCI, even at chronic stages and up to three spinal segments from the injury epicentre. Multiple measures of gross and fine capillary structure from acute to chronic time points provide insight into microvascular remodelling post-SCI. We have identified key vascular treatment targets, namely stabilising damaged capillaries and replacing destroyed vessels, which may be used to improve functional outcomes following SCI in the future

Enhanced Motor Function by Training in Spinal Cord Contused Rats Following Radiation Therapy

Weight-bearing stepping, without supraspinal re-connectivity, can be attained by treadmill training in an animal whose spinal cord has been completely transected at the lower thoracic level. Repair of damaged tissue and of supraspinal connectivity/circuitry following spinal cord injury in rat can be achieved by specific cell elimination with radiation therapy of the lesion site delivered within a critical time window, 2-3 weeks postinjury. Here we examined the effects of training in the repaired spinal cord following clinical radiation therapy. Studies were performed in a severe rat spinal cord contusion injury model, one similar to fracture/crush injuries in humans; the injury was at the lower thoracic level and the training was a combined hindlimb standing and stepping protocol. Radiotherapy, in a similar manner to that reported previously, resulted in a significant level of tissue repair/preservation at the lesion site. Training in the irradiated group, as determined by limb kinematics tests, resulted in functional improvements that were significant for standing and stepping capacity, and yielded a significant direct correlation between standing and stepping performance. In contrast, the training in the unirradiated group resulted in no apparent beneficial effects, and yielded an inverse correlation between standing and stepping performance, e.g., subject with good standing showed poor stepping capacity. Further, without any training, a differential functional change was observed in the irradiated group; standing capacity was significantly inhibited while stepping showed a slight trend of improvement compared with the unirradiated group. These data suggest that following repair by radiation therapy the spinal circuitries which control posture and locomotor were modified, and that the beneficial functional modulation of these circuitries is use dependent. Further, for restoring beneficial motor function following radiotherapy, training seems to be crucial

Ultrastructural synaptic features differ between alpha- and gamma-motoneurons innervating the tibialis anterior muscle in the rat

We investigated the synaptology of retrogradely labeled spinal motoneurons after injection of horseradish peroxidase into the tibialis anterior (TA) muscle of adult rat. In total, 32 TA motoneurons were investigated in the electron microscope and demonstrated a bimodal size distribution with cell diameter peaks at 40 mu m and 20 mu m, likely representing alpha- and gamma-motoneurons, respectively. Both alpha- and gamma-motoneurons were apposed by S- and F-type synaptic boutons, whereas only alpha-motoneurons demonstrated inputs by the large M- and C-type boutons. The proportion of cell body membrane covered by synaptic inputs was surprisingly indistinguishable between alpha-motoneurons (72.2%) and gamma-motoneurons (63.5%). The ratio between the number of F- and S-type boutons in apposition with the motoneuron cell body (F/S ratio) and the ratio between the soma membrane coverage provided by F- and S-type boutons were both significantly higher in alpha- than in gamma-motoneurons. When comparing our data with previous findings in other species, we conclude that rat TA alpha-motoneurons are similar to cat and primate alpha-motoneurons with regard to synaptic terminal morphology, frequency, and distribution. However, rat gamma-motoneurons show a markedly higher total synaptic coverage and frequency than cat gamma-motoneurons, although both species exhibit appositions made by the same synaptic types and similar ratios between inhibitory and excitatory inputs

Spinal stimulation of the upper lumbar spinal cord modulates urethral sphincter activity in rats after spinal cord injury.

After spinal cord injury (SCI), the neurogenic bladder is observed to develop asynchronous bladder and external urethral sphincter (EUS) contractions in a condition known as detrusor-sphincter dyssnergia (DSD). Activation of the EUS spinal controlling center located at the upper lumbar spinal cord may contribute to reduce EUS dyssynergic contractions and decrease urethral resistance during voiding. However, this mechanism has not been well studied. This study aimed at evaluating the effects of epidural stimulation (EpS) over the spinal EUS controlling center (L3) in combination with a serotonergic receptor agonist on EUS relaxation in naive rats and chronic (6-8 wk) T8 SCI rats. Cystometrogram and EUS electromyography (EMG) were obtained before and after the intravenous administration of 5HT-1A receptor agonist and antagonist. The latency, duration, frequency, amplitude, and area under curve of EpS-evoked EUS EMG responses were analyzed. EpS on L3 evoked an inhibition of EUS tonic contraction and an excitation of EUS intermittent bursting/relaxation correlating with urine expulsion in intact rats. Combined with a 5HT-1A receptor agonist, EpS on L3 evoked a similar effect in chronic T8 SCI rats to reduce urethral contraction (resistance). This study examined the effect of facilitating the EUS spinal controlling center to switch between urine storage and voiding phases by using EpS and a serotonergic receptor agonist. This novel approach of applying EpS on the EUS controlling center modulates EUS contraction and relaxation as well as reduces urethral resistance during voiding in chronic SCI rats with DSD

Spinal stimulation of the upper lumbar spinal cord modulates urethral sphincter activity in rats after spinal cord injury.

After spinal cord injury (SCI), the neurogenic bladder is observed to develop asynchronous bladder and external urethral sphincter (EUS) contractions in a condition known as detrusor-sphincter dyssnergia (DSD). Activation of the EUS spinal controlling center located at the upper lumbar spinal cord may contribute to reduce EUS dyssynergic contractions and decrease urethral resistance during voiding. However, this mechanism has not been well studied. This study aimed at evaluating the effects of epidural stimulation (EpS) over the spinal EUS controlling center (L3) in combination with a serotonergic receptor agonist on EUS relaxation in naive rats and chronic (6-8 wk) T8 SCI rats. Cystometrogram and EUS electromyography (EMG) were obtained before and after the intravenous administration of 5HT-1A receptor agonist and antagonist. The latency, duration, frequency, amplitude, and area under curve of EpS-evoked EUS EMG responses were analyzed. EpS on L3 evoked an inhibition of EUS tonic contraction and an excitation of EUS intermittent bursting/relaxation correlating with urine expulsion in intact rats. Combined with a 5HT-1A receptor agonist, EpS on L3 evoked a similar effect in chronic T8 SCI rats to reduce urethral contraction (resistance). This study examined the effect of facilitating the EUS spinal controlling center to switch between urine storage and voiding phases by using EpS and a serotonergic receptor agonist. This novel approach of applying EpS on the EUS controlling center modulates EUS contraction and relaxation as well as reduces urethral resistance during voiding in chronic SCI rats with DSD