Activation of locomotion in adult chronic spinal rats is achieved by transplantation of embryonic raphe cells reinnervating a precise lumbar level.

Traumatic lesions of the spinal cord yield a loss of supraspinal control of voluntary locomotor activity, although the spinal cord contains the necessary circuitry to generate the basic locomotor pattern. In spinal rats, this network, known as central pattern generator (CPG), was shown to be sensitive to serotonergic pharmacological stimulation. In previous works we have shown that embryonic raphe cells transplanted into the sublesional cord of adult rats can reinnervate specific targets, restore the lesion-induced increase in receptor densities of neurotransmitters, promote hindlimb weight support, and trigger a locomotor activity on a treadmill without any other pharmacological treatment or training. With the aim of discriminating whether the action of serotonin on CPG is associated to a specific level of the cord, we have transplanted embryonic raphe cells at two different levels of the sublesional cord (T9 and T11) and then performed analysis of the kinematic and EMG activity synchronously recorded during locomotion. Locomotor performances were correlated to the reinnervated level of the cord and compared to that of intact and transected nontransplanted animals. The movements expressed by T11 transplanted animals correspond to a well defined locomotor pattern comparable to that of the intact animals. On the contrary, T9 transplanted animals developed limited and disorganized movements as those of nontransplanted animals. The correlation of the locomotor performances with the level of reinnervation of the spinal cord suggests that serotonergic reinnervation of the L1-L2 level constitutes a key element in the genesis of this locomotor rhythmic activity. This is the first in vivo demonstration that transplanted embryonic raphe cells reinnervating a specific level of the cord activate a locomotor behavior.

Long-lasting recovery of locomotor function in chronic spinal rat following chronic combined pharmacological stimulation of serotonergic receptors with 8-OHDPAT and quipazine.

In chronic spinal rats, long-term stimulation of 5-HT receptors with quipazine or 8-OHDPAT by means of daily injection, promotes robust locomotor recovery. The question of a possible potentiation between treatments when applied together was addressed. Daily injections of both 8-OHDPAT and quipazine, were performed for a month in spinal animals. Animals were placed on a treadmill and the bipedal hindlimb locomotion was tested. Motor performances (behavioural test) and locomotor parameters (EMG and kinematic) were analysed weekly during the treatment. Furthermore, the locomotor performances were evaluated during two supplemental months following the end of the treatment. Our results suggest that association of both agonists induced long-lasting positive effects on locomotor function. Motor performances were significantly better after combined injection of both drugs than when the agonists were used separately. But, the most significant and new result is that the locomotor scores did not decrease during the weeks that followed the end of the treatment. These results suggests a long-lasting and 5-HT-dependent reorganisation of spinal networks.

An Electromyographic Study of the Hindlimb Locomotor Movements in the Acute Thalamic Rat.

In this paper we have analysed the patterns of muscular activities that underlie hindlimb locomotor movements in the acute thalamic rat. Electromyographic activities of muscles representative of the functional muscle groups of the hindlimbs were recorded bipolarly during locomotion in acute thalamic rats. Locomotor movements occurred spontaneously, but could also be induced by electrical stimulation (0.1 ms pulses; 30 - 70 Hz; 75 - 300 microA) of the lateral hypothalamic area. The two hindlimbs displayed a wide variety of coordination patterns during both types of locomotion. However, alternated coordination of the hindlimbs occurred more frequently during induced than during spontaneous locomotion. Correspondingly, the duration of the spontaneous step cycles had a tendency to be shorter than that of the evoked step cycles, although they had a quite similar range. The patterns of muscular activities within one hindlimb were similar during spontaneous and induced locomotion. During each step cycle, (i) the hip and ankle flexors usually displayed a single burst in alternation with that displayed by the hip, knee and ankle extensors, (ii) a double bursting pattern was sometimes observed in flexors during fast locomotor movements, (iii) within flexors and extensors, muscles were recruited sequentially, and (iv) the activation of other muscles (semitendinosus, rectus femoris, extensor and flexor digitorum longus) consisted of single or double bursting patterns.

Recovery of locomotion following transplantation of monoaminergic neurons in the spinal cord of paraplegic rats.

Severe traumatic lesions of the spinal cord yield a permanent deficit of motricity in adult mammals and specifically a loss of locomotor activity of hindlimbs when the lesion is located at the lower thoracic level. To restore this function, we have developed a paradigm of transplantation in rats based on a transection model of the spinal cord and the subsequent injection at the sublesional level of a suspension of embryonic brainstem monoaminergic neurons which play a key role in the modulation of locomotion. A genuine locomotion was characterized in transplanted animals by electromyographic and electroneurographic recordings. This correlated with a specific reinnervation pattern of targets, where typical synapses were found, and with the normalization of biochemical parameters.

Comparison between ventral spinocerebellar and rubrospinal activities during locomotion in the cat.

During fictive locomotion of the thalamic cat, rhythmic activity related to the efferent discharges in hindlimb nerves was found in rubrospinal neurons (Arshavsky et al., this issue). Since the movements were abolished by curarization, this modulation could not result from rhythmic peripheral inputs and had therefore a central origin. Taking into account the existence of spinal generators, it was suggested that ascending pathways transmit rhythmic activity from these spinal centers to the supraspinal ones. Preliminary results have been obtained for neurons of the ventral spinocerebellar tract (VSCT) recorded during fictive locomotion: (1) their discharge is rhythmically modulated at the periodicity of the locomotor rhythm; (2) their discharge pattern can be complex and variable in relation with the complexity and variability of the pattern of efferent activity in various muscle nerves of the ipsilateral hindlimb; (3) their responses to phasic afferent stimulation of the ipsilateral hindlimb are modulated in parallel with their locomotor-related activity. These results show that VSCT neurons convey information on the central spinal activity during locomotion, and suggest that these neurons contribute to the activity of lumbar-projecting rubrospinal neurons which have similar characteristics.

Median nerve neurotization by peripheral nerve grafts directly implanted into the spinal cord: anatomical, behavioural and electrophysiological evidences of sensorimotor recovery.

Over the years, peripheral nerve grafts, a favorable environment to support axonal elongation, have given rise to increasing interest as a possible solution for promoting spinal cord repair. In the experiments described here, following an avulsion injury of the rat brachial plexus, the median nerve was repaired by a peripheral nerve graft (PN) inserted directly into the dorsal side of the spinal cord. Eight months later the animals were submitted to behavioral tests, electrophysiological and histological studies. Regrowth of axons from both motoneurons and ganglionic neurons was demonstrated following a single superficial dorsal implantation of a PN. Sensorimotor peripheral reinnervation allowed most of the studied animals to recover enough flexor activity for grasping. Reinnervation was achieved even without prior root avulsion suggesting that the presence of a PN is sufficient to induce sprouting in the spinal cord from axotomized and non-axotomized neurons.

Discharges of forelimb spindle primary afferents during locomotor activity in the decorticate cat.

In the acute decorticate cat, the discharge patterns of single spindle primary afferents from various forelimb muscles during locomotion have been examined in relation to the extrafusal activity. In a given muscle under isometric conditions, the activation of spindle primary endings was closely related to that of extrafusal muscle fibers. Since this increase in spindle discharge could occur in the absence of detectable electromyographic activity in the parent muscle, it resulted, at least to some extent from an enhanced activity in gamma efferents. The data are discussed in relation to previous studies of forelimb efferent activity and hindlimb fusimotor control in the same preparation.

Cutaneous fiber groups involved in the inhibition of fictive locomotion in the rabbit.

Decorticate, paralyzed unanaesthetized rabbit preparations can display motoneuron discharges (spontaneous or elicited through various somatic stimulations) which are related to locomotion. These activities are suppressed when manual pressure is exerted on the dorso-lumbar skin. The present study indicates that: (1) locomotor discharges can also be inhibited through repetitive electrical stimulation of one of the skin nerves belonging to dorsal dermatomes (TH5 to L5); (2) other skin nerves, especially those of the limbs, do not present the same inhibitory properties; (3) among the various fiber groups composing these dorsal skin nerves, only A delta are inhibitory; other groups, on the contrary, display moderate (A alpha and A beta) or strong (unmyelinated C fibers) excitatory actions; (4) the inhibitory action of A delta fibers counteracts the excitatory action of C fibers, when both groups are stimulated together. The relationship between this form of motor inhibition and the well known phenomenon of "reflex immobility" is briefly considered.

Fictive locomotion in the adult thalamic rat.

In immobilized adult thalamic rats, electrical stimulation of sites within the lateral hypothalamic area (LHA) or the mesencephalic locomotor region (MLR) were found to elicit fictive locomotor patterns in hindlimb muscle nerves. Significant differences were found between several characteristics (average cycle period, locomotor episode duration, intralimb and interlimb coordination patterns) of the LHA-induced and MLR-induced fictive locomotor activities. These findings support the hypothesis that LHA and MLR play different functional roles during locomotion.

5-HT1A receptors are involved in short- and long-term processes responsible for 5-HT-induced locomotor function recovery in chronic spinal rat.

After thoracic spinal cord transection, a paraplegic syndrome occurs. Previous data showed that an acute administration of a 5-HT2 agonist (quipazine) could promote motor function recovery in spinal rats. However, continuous subdural perfusion of quipazine via an osmotic pump over 1 month proved to be more effective. The present study was designed to investigate the possible involvement of 5-HT1A receptors in such recovery. Motor performances and locomotor parameters were analysed in spinal animals receiving daily, for 1 month, a dose of the 5-HT1A agonist 8-OHDPAT. The results were compared to those obtained in spinal rats receiving either a placebo or quipazine in the same conditions. Using daily injections instead of continuous perfusion of either receptor agonist to spinal animals allowed characterization of short- and long-term consequences of pharmacological stimulation of 5-HT1A and 5-HT2 receptors on motor function recovery. Our data demonstrate that daily injections of a 5-HT1A agonist induce long-term, cumulative, positive effects on motor function recovery, as assessed by the improvement in the walking parameters observed before the 'day-test' injection. This might involve use-dependent processes depending on a chronic and/or repetitive stimulation of the spinal network for locomotion in relation to 5-HT receptor activation. A further improvement in the motor parameters, transiently observed following the injection, suggests a more direct action of 5-HT1A and 5-HT2 receptor activation on spinal neurons involved in motor pattern generation.

Serotonin-induced activation of the network for locomotion in adult spinal rats.

The biogenic amine serotonin has been described in the literature as a powerful modulator of the spinal central pattern generator for locomotion. In the present study, we tested whether administration of serotonin or its agonist quipazine could restore motor activity in a model of paraplegia. One to three weeks after a complete transection of the spinal cord at a low thoracic level, rats were given either intrathecal injections of serotonin (5 mM, 15 microL) or intraperitoneal injections of quipazine (400-600 microg/kg). Both treatments allowed recovery of locomotor activity on a treadmill in response to tail pinching. As compared with the activity elicited before treatment, the locomotor activity produced by spinal animals was characterised by longer locomotor sequences with a larger number of successive steps, better body support, better interlimb coordination, and a higher amplitude of electromyographic bursts. These results suggest that serotonergic drugs could be used for the recovery of motor functions after lesions of the spinal cord.

Interlimb coordination during fictive locomotion in the thalamic cat.

Efferent discharges in muscle nerves of the four limbs were recorded simultaneously during spontaneous fictive locomotion in thalamic cats with the goal of understanding how the central nervous system controls interlimb coordination during stepping. The onset of the bursts of activity in the nerve of a selected flexor muscle in each limb allowed the temporal and the phase relationships between the fictive step cycle of a pair of limbs to be determined. Our main results are the following: 1) the fictive step cycles of the two forelimbs are always strictly alternated whereas the phasing of the step cycles of either the two hindlimbs or pairs of homolateral or diagonal limbs is more variable; 2) the time interval between the onsets of the flexor bursts of one of the two pairs of diagonal limbs is independent of the step cycle duration; 3) distinct patterns of interlimb coordination exist during fictive locomotion; a small number of patterns of coordination involving all four limbs, which correspond to the walking and the trotting gaits in the intact cat, occur very frequently. The results demonstrate that the central nervous system deprived of phasic afferent inputs from the periphery has the capacity to generate most of the patterns of interlimb coordination which occur during real locomotion. They further support the view that the central pattern of interlimb coordination essentially results from diagonal interaction between a forelimb generator for locomotion and a hindlimb one.

Fictive motor activity in rat after 14 days of hindlimb unloading.

The goal of the present study was to examine the effects of chronic hindlimb unloading on fictive motor patterns which can be developed in hindlimb nerves of adult rats. The animals were divided into two groups. The first group was submitted to hindlimb unloading for 2 weeks by tail suspension. The second group served as controls. After this initial phase, the animals of both groups were acutely decorticated, paralysed and electroneurographic efferent activity was recorded from hindlimb muscle nerves under conditions of "fictive locomotion" in order to evaluate variations in central locomotor command. Fictive rhythmic motor episodes were either spontaneous or evoked by electrical stimulation of the mesencephalic locomotor region. Only the second ones were recognised as locomotor-like activities. The motor pattern was not fundamentally affected by unloading except that, after the unloading period, extensor muscle nerves were significantly more frequently activated and their burst durations were increased compared to activity in control animals, despite the fact that the phasic sensory afferent inputs were suppressed. This suggests that unloading induces plastic modifications of the central networks of neurons implicated in the locomotor command. The origin of this extensor hyperactivity is discussed. It is proposed that it could be the consequence of either changes in motoneuronal properties or of an increase in afferent input to motoneurones.

Locomotor recovery in the chronic spinal rat: effects of long-term treatment with a 5-HT2 agonist.

A complete transection of the spinal cord at a low thoracic level induces a paraplegic syndrome that is accompanied by a loss of spinal cord serotonin content. Former experimental data suggest that the central pattern generator for locomotion, located in the lumbar segments of the spinal cord, might be able to generate rhythmic motor outputs (similar to automatic walking under certain circumstances) involving exteroceptive stimulations and activation of serotonergic receptors. In the present study, we investigated the effects of a chronic treatment using a serotonergic agonist, delivered continuously to the sublesionned spinal cord, and its effect on motor function recovery. The data obtained from behavioural, kinematic and electromyographic measurements suggest that the chronic stimulation of 5-HT2 type receptors allows motor function recovery. Behavioural measurements show a clear improvement in motor performances when compared to spinal animals (confirmed by kinematic observations): alternating steps and foot placement is recovered in these animals. However, electromyographic data demonstrate that the pattern of activation of the muscles is only restored partially.

[Can transplantation of neurons facilitate motor recovery in paraplegics? Study of an animal model]. / Les transplantations de neurones peuvent-elles faciliter la récupération motrice chez les paraplégiques? Etude d'un modèle animal.

This review strives forward at least two goals. First, to take from the literature the arguments demonstrating that hindlimbs locomotion is controlled by a spinal network of neurons (the so-called Central Pattern Generator for locomotion--CPG) known to be able to generate locomotor activity independently of the control of supraspinal nervous structures, as it is after thoracic lesions of the spinal cord. The principles of work of the CPG and its intrinsic possibilities to adapt its working are reviewed. Special reference is made to the various ways used during experiments to activate the CPG in spinal animals or clinical practice in paraplegic men: training to walk, electrical stimulations, pharmacological stimulations. Second, to show, from our own results, obtained from the study of an animal model of paraplegia, the adult spinal rat, how it could be possible to take advantage of the autonomy of the CPG, with special reference to its sensibility to monoamines, to obtain locomotor recovery in hindlimbs after section of the thoracic spinal cord, by means of transplantation of noradrenergic and/or serotonergic embryonic neurons in the lumbo-sacral spinal cord. Section of the spinal cord at a thoracic level results in an important locomotor deficit in hindlimbs, likely linked to degeneration of monoaminergic terminals in the lumbar enlargement. In the adult spinal rat, sub-lesional injection of a suspension of embryonic nervous cells, taken from either locus coeruleus or raphe sites, leads to reinnervation of the lumbar enlargement with monoaminergic terminals. Despite the fact that connections with supraspinal structures are not reestablished, transplanted animals recover progressively a posture convenient for locomotion. The hindlimbs, which are in an extended position a few days after the lesion, become progressively flexed and able to support the body weight. This evolution does not appear in spinal but non transplanted animals. But, the main point is that transplanted animals develop, within the few weeks that follow transplantation, a good-quality locomotor activity in hindlimbs which had no equivalent in spinal but non transplanted animals. The reality of a lumbar CPG for locomotion and the efficacy of pharmacological treatments and training to walk, to elicit recovery of stepping, are discussed in man, in connection with the relevance to use transplantation of monoaminergic nervous cells in the spinal cord of paraplegics.

Evidence of rhythmic inhibitory synaptic influences in hindlimb motoneurons during fictive locomotion in the thalamic cat.

Intracellular recordings of various motoneurons of proximal hindlimb muscles were performed on thalamic paralyzed cats, during fictive locomotion that was either spontaneous or evoked by stimulation of the subthalamic region. In motoneurons innervating sartorius (medialis and lateralis), vasti (intermedius, medialis and lateralis) and anterior biceps-semimembranous, one depolarization occurred in each locomotor cycle, alternating with a phase of repolarization that was synchronous with the activation of the antagonistic muscle nerve. This latter phase could be decreased or reversed by intracellular injection of chloride ions or current, revealing the presence of inhibitory inputs onto motoneurons. The pattern of membrane potential variations was more complex in motoneurons of rectus femoris and posterior biceps-semitendinosus muscles, but phases of chloride dependent inhibition were nevertheless identified, mainly during the sartorius nerve activation in the case of rectus femoris, and during the vasti and anterior biceps-semimembranosus nerve activations in the case of posterior biceps-semitendinosus. These inhibitory influences were shown to be controlled by the level of activity in exteroceptive afferents. The characteristics of the excitatory and inhibitory inputs to the hindlimb motoneurons identified here are discussed in relation with the organization of the central pattern generator for locomotion.

Recovery of locomotor activity in the adult chronic spinal rat after sublesional transplantation of embryonic nervous cells: specific role of serotonergic neurons.

Locomotor movements are programmed in a specialised neuronal network that is localised in the central nervous system and referred to as the central pattern generator (CPG) for locomotion. This CPG can be activated by pharmacological agents such as monoamines. The aim of the present study was to try to activate the CPGs by using cells that are supposed to release serotonin locally. Adult chronic spinal rats were injected with embryonic brainstem neurons within the spinal cord under a thoracic transection. This procedure resulted in a monoaminergic reinnervation of the lumbar enlargement. With the help of a specific neurotoxin for noradrenergic neurons (6-hydroxydopamine), it was possible to isolate the serotonergic system. After such transplantation of monoaminergic neurons and even with serotonergic neurons alone, a bilateral, alternating, rhythmic locomotor-like activity recovered in hindlimbs. Furthermore, this locomotor-like activity was clearly facilitated when the re-uptake of serotonin was blocked by zimelidine. Therefore, we conclude that transplanted embryonic serotonergic neurons are able to activate the CPG for locomotion.