The effects of clonidine and yohimbine on locomotion and cutaneous reflexes in the adult chronic spinal cat

The effects on the locomotor pattern of a noradrenergic agonist (clonidine) and an antagonist (yohimbine) were studied in 3 adult chronic spinal cats walking on a treadmill. In the early post-transection period, when the cat walked mainly on the tip of its feet, without supporting its own weight, it was observed that clonidine (150 micrograms/kg) could induce a good bilateral foot placement and intermittent complete weight support. When clonidine was given 1-3 months following the transection, at a time when the spinal cats had a stable and regular locomotor performance, the step length increased markedly, especially at low speeds. This was associated with an increase in the duration of the flexor and extensor bursts, as well as an increase of the angular excursion of all joints. These effects, seen during forward locomotion, were also observed during backward locomotion. In addition, the latter was more easily elicited after clonidine. Yohimbine (1.5-3 mg/kg) partially antagonized these effects. The threshold current needed to elicit a small flexion reflex through wires implanted in the dorsum of the paw was 2-3 times higher after clonidine. Trains of shocks in the animal, standing quietly, did not induce the prolonged late discharges normally found in acute spinal cats. Fast paw shaking, elicited by dipping one paw in water, was abolished by clonidine and reappeared after yohimbine. These results indicate that noradrenergic drugs may influence both spinal locomotion and the excitability of cutaneous reflexes. This class of substances could thus play a useful role in the recovery and/or maintenance of locomotor functions after spinal trauma.     

Recovery of Locomotion after Spinal Cord Hemisection: An X-Ray Study of the Cat Hindlimb

Hemisection of the spinal cord in adult cats is a suitable model to study the mechanisms underlying recovery of motor functions. The initial paresis of the hindlimb is followed by a considerable improvement of locomotor functions of the affected hindlimb. Kinematic analyses of treadmill locomotion were performed from 10 days to 8 months after complete hemisections (right side) of the spinal cord at the thoracolumbar level, using X-ray cinematography for precise measurements of the hindlimb joint angles. The footfall pattern and the electromyogram were recorded. Motor control of both proximal and distal hindlimb joints improved substantially during the 1st postoperative month. However, persistent locomotor deficits were still present several months after hemisection. They could be divided into three groups of symptoms: (1) The gait pattern was disturbed with regard to interlimb coordination. The stance-phase duration of the right hindlimb was shortened. (2) The flexor capacity of the affected hindlimb was reduced, resulting in a slow insufficient flexion of the hip, knee, and ankle during the swing phase. (3) The timing of the flexion–extension events was impaired. The onset of the E1-extension was delayed and the amplitude was reduced. Electromyographic patterns of muscle activity during locomotion of the lesioned side hindlimb differed from the contralateral hindlimb, which served as a control. The results indicate that in spite of a good short-term functional improvement there are long-term locomotor deficits present after spinal cord hemisection.     

Weight-bearing hindlimb stepping in treadmill-exercised adult spinal cats

Hindlimb locomotion on a motor-driven treadmill was studied in 5 cats spinalized at a low thoracic level adults. Six months after surgery, the cats were anesthetized and implanted for electromyographic (EMG) and force recordings in hindlimb muscles. For the last 5 months of the spinalization period, the hindlimbs of each cat were exercised daily for 30 minutes on a treadmill. Data were collected during hindlimb locomotion on a treadmill across the entire range of speeds each cat could accommodate. All trials were filmed (100 frames/s) for kinematic analysis. EMG data were recorded from the soleus (Sol), medial gastrocnemius (MG), tibialis anterior (TA) and extensor digitorum longus (EDL). Forces were recorded in vivo from the Sol and MG tendons. All cats could sustain full weight-bearing stepping without the need for mechanical stimulation of the tail. Although the general stepping pattern of the spinal cats was remarkably similar to that of normal cats, several key differences were identified. Compared to normal cats, the adult spinal cats walked at a lower range of speeds and exhibited a longer swing phase duration. The Sol produced forces and displayed activation periods comparable to those observed in normal cats. The MG of adult spinal cats, however, produced lower forces and had a later onset of activation in comparison to normal cats. Each of the muscles in all spinal cats exhibited tremor during stepping. These results suggest that there were limitations in the activation levels of some hindlimb flexor and extensor muscles during treadmill locomotion. These data further suggest that, in normal cats, accommodation to treadmill speed is accomplished by modulating supraspinal input to the lumbar spinal cord while leaving many of the timing details to be regulated by lumbar spinal networks.     

Treadmill training enhances the recovery of normal stepping patterns in spinal cord contused rats

Treadmill training is known to improve stepping in complete spinal cord injured animals. Few studies have examined whether treadmill training also enhances locomotor recovery in animals following incomplete spinal cord injuries. In the present study, we compared locomotor recovery in trained and untrained rats that received a severe mid-thoracic contusion of the spinal cord. A robotic device was used to train and to test bipedal hindlimb stepping on a treadmill. Training was imposed for 8 weeks. The robotic device supported the weight of the rats and recorded ankle movements in the hindlimbs for movement analyses. Both the trained and untrained rats generated partial weight bearing hindlimb steps after the spinal cord contusion. Dragging during swing was more prevalent in the untrained rats than the trained rats. In addition, only the trained rats performed step cycle trajectories that were similar to normal step cycle trajectories in terms of the trajectory shape and movement velocity characteristics. In contrast, untrained rats executed step cycles that consisted of fast, kick-like movements during forward swing. These findings indicate that spinal cord contused rats can generate partial weight bearing stepping in the absence of treadmill training. The findings also suggest that the effect of treadmill training is to restore normal patterns of hindlimb movements following severe incomplete spinal cord injury in rats.     

Effect of locomotor training in completely spinalized cats previously submitted to a spinal hemisection

After a spinal hemisection in cats, locomotor plasticity occurring at the spinal level can be revealed by performing, several weeks later, a complete spinalization below the first hemisection. Using this paradigm, we recently demonstrated that the hemisection induces durable changes in the symmetry of locomotor kinematics that persist after spinalization. Can this asymmetry be changed again in the spinal state by interventions such as treadmill locomotor training started within a few days after the spinalization? We performed, in 9 adult cats, a spinal hemisection at thoracic level 10 and then a complete spinalization at T13, 3 weeks later. Cats were not treadmill trained during the hemispinal period. After spinalization, 5 of 9 cats were not trained and served as control while 4 of 9 cats were trained on the treadmill for 20 min, 5 d a week for 3 weeks. Using detailed kinematic analyses, we showed that, without training, the asymmetrical state of locomotion induced by the hemisection was retained durably after the subsequent spinalization. By contrast, training cats after spinalization induced a reversal of the left/right asymmetries, suggesting that new plastic changes occurred within the spinal cord through locomotor training. Moreover, training was shown to improve the kinematic parameters and the performance of the hindlimb on the previously hemisected side. These results indicate that spinal locomotor circuits, previously modified by past experience such as required for adaptation to the hemisection, can remarkably respond to subsequent locomotor training and improve bilateral locomotor kinematics, clearly showing the benefits of locomotor training in the spinal state.     

Treadmill training in incomplete spinal cord injured rats

Treadmill training has been shown to accelerate locomotor recovery and to improve weight bearing during treadmill walking in spinal cats. In human patients treadmill training is increasingly used in rehabilitation after incomplete spinal cord injury. In this study we examined training effects in spinal cord injured rats with an incomplete dorsal lesion. Recovery was examined with an open field locomotor score, kinematic analysis on the treadmill, and several functional tests (i.e. foot print evaluation, narrow beam crossing, grid walking, open field exploratory activity). During the course of 5 weeks after the injury, a substantial amount of recovery occurred in the treadmill trained as well as in the untrained rats. If compared to the control lesioned rats, which showed a high level of spontaneous hindlimb movements at 7-14 days post lesion, no additional beneficial effect of a 5-week daily treadmill training on the locomotor outcome could be detected in the trained group. The only change observed was a slightly larger exploratory activity of the trained rats. It is probable that the spared ventral and ventro-lateral fibers allowed spontaneous recovery and 'self-training' to occur to such an extend that systematic treadmill training did not provide additional improvement.     

Excitability level-setting mechanisms in the pons: their behavioral support in decerebrate, reflex standing and freely moving, intact cats

In the acute precollicular-postmammillary decerebrate cat, stimulation of the mesencephalic locomotor region (MLR) induces "controlled locomotion" on a moving treadmill. Stimulation of the dorsal area and of the ventral area of the pons at its midline elicited a long-lasting decrease and an increase in the tone of the hindlimb extensor muscles, respectively. By selecting the stimulus strength according to the stimulus site, it was possible to set the extensor muscle tone level, that is the "background excitability" of the brain stem and the spinal cord. Locomotor effects induced by MLR stimulation were greatly modified by the set level of background excitability. When the background excitability was high, MLR stimulation evoked "spastic" locomotor movement, while "atonic" locomotor movement was evoked when it was low. Furthermore, stimulation of the ventral area alone also evoked "spastic" locomotor movement. During locomotion in intact cats, stimulation of the dorsal area evoked a series of postural changes. Within a few seconds from the beginning of stimulation, the cat ceased to walk, but maintained a standing posture with or without a locomotor figure. With continuation of this stimulation, it squatted and then lay down on the floor in a sequential manner. Stimulation of the ventral area of the pons evoked an almost opposite series of postural changes. Within a few seconds from the beginning of stimulation, the cat changed from a lying to a squatting posture, and then stood, started to walk and continued to walk during the period of stimulation. All these results demonstrate that an increase in extensor muscle tone and activation of the spinal stepping generator are not separate phenomena, and suggest that integration of neuronal mechanisms involved in the setting of the background excitability and in locomotor movement is a prerequisite for successful expression of locomotor behavior both in decerebrate and intact cats.     

Descending pathways eliciting forelimb stepping in the lateral funiculus: Experimental studies with stimulation and lesion of the cervical cord in decerebrate cats

The funicular pathways that elicit forelimb stepping were investigated with stimulation and lesion of the cervical white matter in decerebrate cats with the lower thoracic cord transected. We localized cross-sectional areas where stimulation evoked rhythmic motor-nerve discharges imitating those of stepping (fictive locomotion) in the immobilized animal, and further examined whether or not lesions made in the corresponding areas abolished actual locomotor movements. Stimulation of the C3 lateral funiculus (LF) produced fictive locomotion in the ipsilateral forelimb. The effective areas of stimulation were located separately in the dorsolateral funiculus (DLF) and in the ventrolateral funiculus (VLF), while the VLF was more effective than the DLF. Effective stimuli were pulse trains with a frequency of about 30 Hz, with a rather wide pulse duration of about 0.5 ms. Blocking axonal conduction through the lower thoracic cord by cooling reproducibly facilitated fictive locomotion in both amplitude and frequency. In the lesion experiments, forelimb locomotor movements were elicited spontaneously or by stimulation of the mesencephalic locomotor region (MLR). The locomotor movements were abolished by complete lesions of the C2-C3 LFs on both sides, but these remained when either the DLF or the VLF was intact on one side. These findings together suggested that the descending pathways for the activation of the spinal locomotor network of the single forelimb are located ipsilaterally in the DLF as well as in the VLF. Both the DLF and the VLF pathways can initiate locomotion, while the VLF pathways have a higher potential for its initiation. Lesion experiments further showed that cats can walk with both forelimbs, even though the spinal locomotor network of the single forelimb was deprived of its main descending input by unilateral lesions of the LF. However, when the unilateral lesion extended to the medial part of the LF, the bilateral walking was abolished; the limb on the lesioned side showed only rhythmic extension movements without active flexion movements, which was out of phase with the stepping movements on the intact side. This finding suggested that the medial part of the LF is important for producing flexion movements during the swing phase of stepping.     

Hindlimb loading determines stepping quantity and quality following spinal cord transection

We compared the bipedal hindlimb stepping ability of untrained and trained (step-trained 6 min/day) spinal rats (mid-thoracic spinal cord transection at post-natal day 5) at different levels of body weight support on a treadmill over a 40-day period, starting at 69 days of age. A robotic device provided precise levels of body weight support and recorded hindlimb movement. We assessed stepping ability using: (1) step quantity determined from the measured hindlimb movement, (2) ordinal scales of paw placement, weight-bearing, and limb flexion, and (3) the lowest level of body weight support at which stepping was maintained. Stepping quantity and quality depended strongly on the level of support provided. Stepping ability improved with time, but only at the higher levels of weight-bearing, and independently of training. Increasing limb loading by gradually decreasing body weight support altered the spatiotemporal properties of the steps, resulting in an increase in step length and stance duration and a decrease in swing and step cycle duration. The rats progressively improved their ability to support more load before collapsing from a maximum of about 42 g ( approximately 25% of body weight) at Day 1 to 73 g ( approximately 35% of body weight) at Day 40. We conclude that the level of hindlimb loading provided to a spinally transected rat strongly influences the quantity and quality of stepping. Furthermore, the relationship between stepping ability and loading conditions changes with time after spinal cord transection and is unaltered by small amounts of step training. Finally, load-bearing failure point can be a quantitative measure of locomotor recovery following spinal cord injury, especially for severely impaired animals that cannot step unassisted.     

Initiation and modulation of the locomotor pattern in the adult chronic spinal cat by noradrenergic, serotonergic and dopaminergic drugs

The effects of noradrenergic, serotonergic and dopaminergic drugs, and their interaction were studied in 8 adult spinal cats during the first week following spinalisation and up to 3 months, when the animals had reached a steady state in their locomotor pattern. During the first week, when no episodes of coordinated stepping were observed, injection of the serotonergic precursor (DL-5-HTP) or a dopaminergic agonist (apomorphine) failed to induce locomotion. In contrast, injection of either a noradrenaline precursor (L-DOPA) or an agonist (clonidine) induced locomotion when the hindlimbs were placed on a moving belt. The spinal animal demonstrated a bilateral foot placement on the plantar surface, as well as transient weight support of the hindquarters at a treadmill speed up to 0.80 m/s. The movement pattern and the electromyographic activity resemble those of the intact cat in many aspects. This locomotion-triggering effect of L-DOPA or clonidine was also observed when given after DL-5-HTP or apomorphine. At around 3 months following spinalisation, when the animal showed a stable and regular locomotor pattern, injection of clonidine increased the step cycle duration, resulting in a prolonged flexor and extensor burst duration as the EMG amplitude was unchanged or slightly increased. Injected in the same animal, quipazine, a serotonergic agonist, increased both the duration and the amplitude of flexor and extensor EMGs. In contrast to the serotonergic and the noradrenergic agonists, apomorphine and L-DOPA augmented mainly the flexor activity which could even lead to a sustained flexion when the dose was increased. When combining clonidine to a serotonergic drug, the characteristics of the modulation of the locomotor pattern resulting from each drug were retained. The present results demonstrate that (1) the noradrenergic system is probably the most important system for the initiation of locomotion; (2) the three monoaminergic descending systems (mimicked by the precursor and agonists) can modify rather specifically different aspects of the well established locomotor pattern in the same chronic spinal cat and (3) the effect of monoaminergic drugs are reproducible when given in similar time periods in different chronic spinal cats. The present study provides insight into the role of the noradrenergic, serotonergic and dopaminergic system in the initiation and in the modulation of the locomotion pattern following spinalisation. The above studies also provide a basis to investigate the effects of these drugs in spinal cord-injured patients.