Genetic identification of spinal interneurons that coordinate left-right locomotor activity necessary for walking movements

The sequential stepping of left and right limbs is a fundamental motor behavior that underlies walking movements. This relatively simple locomotor behavior is generated by the rhythmic activity of motor neurons under the control of spinal neural networks known as central pattern generators (CPGs) that comprise multiple interneuron cell types. Little, however, is known about the identity and contribution of defined interneuronal populations to mammalian locomotor behaviors. We show a discrete subset of commissural spinal interneurons, whose fate is controlled by the activity of the homeobox gene Dbx1, has a critical role in controlling the left-right alternation of motor neurons innervating hindlimb muscles. Dbx1 mutant mice lacking these ventral interneurons exhibit an increased incidence of cobursting between left and right flexor/extensor motor neurons during drug-induced locomotion. Together, these findings identify Dbx1-dependent interneurons as key components of the spinal locomotor circuits that control stepping movements in mammals.     

Possible contributions of CPG activity to the control of rhythmic human arm movement

There is extensive modulation of cutaneous and H-reflexes during rhythmic leg movement in humans. Mechanisms controlling reflex modulation (e.g., phase- and task-dependent modulation, and reflex reversal) during leg movements have been ascribed to the activity of spinal central pattern generating (CPG) networks and peripheral feedback. Our working hypothesis has been that neural mechanisms (i.e., CPGs) controlling rhythmic movement are conserved between the human lumbar and cervical spinal cord. Thus reflex modulation during rhythmic arm movement should be similar to that for rhythmic leg movement. This hypothesis has been tested by studying the regulation of reflexes in arm muscles during rhythmic arm cycling and treadmill walking. This paper reviews recent studies that have revealed that reflexes in arm muscles show modulation within the movement cycle (e.g., phase-dependency and reflex reversal) and between static and rhythmic motor tasks (e.g., task-dependency). It is concluded that reflexes are modulated similarly during rhythmic movement of the upper and lower limbs, suggesting similar motor control mechanisms. One notable exception to this pattern is a failure of contralateral arm movement to modulate reflex amplitude, which contrasts directly with observations from the leg. Overall, the data support the hypothesis that CPG activity contributes to the neural control of rhythmic arm movement.     

Probing spinal circuits controlling walking in mammals

Locomotion in mammals is a complex motor act that involves the activation of a large number of muscles in a well-coordinated pattern. Understanding the network organization of the intrinsic spinal networks that control the locomotion, the central pattern generators, has been a challenge to neuroscientists. However, experiments using the isolated rodent spinal cord and combining electrophysiology and molecular genetics to dissect the locomotor network have started to shed new light on the network structure. In the present review, we will discuss findings that have revealed the role of designated populations of neurons for the key network functions including coordinating muscle activity and generating rhythmic activity. These findings are summarized in proposed organizational principles for the mammalian segmental CPG.     

Functional organization of locomotor interneurons in the ventral lumbar spinal cord of the newborn rat

Although the mammalian locomotor CPG has been localized to the lumbar spinal cord, the functional-anatomical organization of flexor and extensor interneurons has not been characterized. Here, we tested the hypothesis that flexor and extensor interneuronal networks for walking are physically segregated in the lumbar spinal cord. For this purpose, we performed optical recordings and lesion experiments from a horizontally sectioned lumbar spinal cord isolated from neonate rats. This ventral hemi spinal cord preparation produces well-organized fictive locomotion when superfused with 5-HT/NMDA. The dorsal surface of the preparation was visualized using the Ca(2+) indicator fluo-4 AM, while simultaneously monitoring motor output at ventral roots L2 and L5. Using calcium imaging, we provided a general mapping view of the interneurons that maintained a stable phase relationship with motor output. We showed that the dorsal surface of L1 segment contains a higher density of locomotor rhythmic cells than the other segments. Moreover, L1 segment lesioning induced the most important changes in the locomotor activity in comparison with lesions at the T13 or L2 segments. However, no lesions led to selective disruption of either flexor or extensor output. In addition, this study found no evidence of functional parcellation of locomotor interneurons into flexor and extensor pools at the dorsal-ventral midline of the lumbar spinal cord of the rat.     

Hindlimb dominance during primate high-speed locomotion

Quadrupedal locomotion was mechanically studied for four species of primates, the chimpanzee, the rhesus macaque, the tufted capuchin, and the ring-tailed lemur, from low to high speeds of about two to ten times the anterior trunk length per second. A wide variety of locomotor patterns was observed during the high-speed locomotion of these primates. Positive correlations were observed between the peak magnitude of foot force components and speed. The differentiation of the foot force between the forelimb and the hindlimb did not largely change with a change of speed for each species. The vertical component and the accelerating component for the rhesus macaque were relatively large in the forelimb from low- to high-speed locomotion. The rhesus macaque, which habitually locomotes on the ground, differed in the quadrupedal locomotion from the other relatively arboreal primates, for which the hindlimb was clearly dominant in their dynamic force-producing distribution between the forelimbs and the hindlimbs. The previously reported locomotor difference, which was indicated among primates from the foot force pattern between the forelimb and the hindlimb during walking, also applied to high-speed locomotion.     

Activation of interlimb interactions increases the motor output in legs of healthy subjects: Study under the conditions of arm and leg unloading

The effect of arm movements and movements of individual arm joints on the electrophysiological and kinematic characteristics of voluntary and vibration-triggered stepping-like leg movements was studied under the conditions of horizontal support of the upper and lower limbs. The horizontal support of arms provided a significant increase in the rate of activation of locomotor automatism by noninvasive impact on tonic sensory inputs. The addition of active arm movements during involuntary stepping-like leg movements led to an increase in the EMG activity of hip muscles and was accompanied by an increase in the amplitude of hip and shin movements. The movement of the shoulder joints led to an increase in the activity of hip muscles and was accompanied by an increase in the amplitude of hip and shin movements. Passive arm movements had the same effect on induced leg movements. The movement of the shoulder joints led to an increase in the activity of hip muscles and an increase in the amplitude of movements of knee and hip joints. At the same time, the movement of forearms and wrists had a similar facilitating effect on the physiological and kinematic characteristics of rhythmic stepping-like movements, but influenced the distal segments of legs to a greater extent. Under the conditions of subthreshold vibration of leg muscles, voluntary arm movements led to activation of involuntary rhythmic stepping movements. During voluntary leg movements, the addition of arm movements had a significantly smaller impact on the parameters of rhythmic stepping than during involuntary leg movements. Thus, the simultaneous movements of the upper and lower limbs are an effective method of activation of neural networks connecting the rhythm generators of arms and legs. Under the conditions of arm and leg unloading, the interactions between the cervical and lumbosacral segments of the spinal cord seem to play the major role in the impact of arm movements on the patterns of leg movements. The described methods of activation of interlimb interactions can be used in the rehabilitation of post-stroke patients and patients with spinal cord injuries, Parkinson’s disease, and other neurological diseases.     

Electromyography of back muscles during quadrupedal and bipedal walking in primates

Despite the extensive electromyographic research that has addressed limb muscle function during primate quadrupedalism, the role of the back muscles in this locomotor behavior has remained undocumented. We report here the results of an electromyographic (EMG) analysis of three intrinsic back muscles (multifidus, longissimus, and iliocostalis) in the baboon (Papio anubis), chimpanzee (Pan troglodytes), and orangutan (Pongo pygmaeus) during quadrupedal walking. The recruitment patterns of these three back muscles are compared to those reported for the same muscles during nonprimate quadrupedalism. In addition, the function of the back muscles during quadrupedalism and bipedalism in the two hominoids is compared. Results indicate that the back muscles restrict trunk movements during quadrupedalism by contracting with the touchdown of one or both feet, with more consistent activity associated with touchdown of the contralateral foot. Moreover, despite reported differences in their gait preferences and forelimb muscle EMG patterns, primates and nonprimate mammals recruit their back muscles in an essentially similar fashion during quadrupedal walking. These quadrupedal EMG patterns also resemble those reported for chimpanzees, gibbons and humans (but not orangutans) walking bipedally. The fundamental similarity in back muscle function across species and locomotor behaviors is consistent with other data pointing to conservatism in the evolution of the neural control of tetrapod limb movement, but does not preclude the suggestion (based on forelimb muscle EMG and spinal lesion studies) that some aspects of primate neural circuitry are unique. © 1994 Wiley-Liss, Inc.     

Development and functional organization of spinal locomotor circuits

The coordination and timing of muscle activities during rhythmic movements, like walking and swimming, are generated by intrinsic spinal motor circuits. Such locomotor networks are operational early in development and are found in all vertebrates. This review outlines and compares recent advances that have revealed the developmental and functional organization of these fundamental spinal motor networks in limbed and non-limbed animals. The comparison will highlight common principles and divergence in the organization of the spinal locomotor network structure in these different species as well as point to unresolved issues regarding the assembly and functioning of these networks.     

Sensory-evoked turning locomotion in red-eared turtles: kinematic analysis and electromyography

We examined the limb kinematics and motor patterns that underlie sensory-evoked turning locomotion in red-eared turtles. Intact animals were held by a band-clamp in a water-filled tank. Turn-swimming was evoked by slowly rotating turtles to the right or left via a motor connected to the shaft of the band-clamp. Animals executed sustained forward turn-swimming against the direction of the imposed rotation. We recorded video of turn-swimming and computer-analyzed the limb and head movements. In a subset of turtles, we also recorded electromyograms from identified limb muscles. Turning exhibited a stereotyped pattern of (1) coordinated forward swimming in the hindlimb and forelimb on the outer side of the turn, (2) back-paddling in the hindlimb on the inner side, (3) a nearly stationary, “braking” forelimb on the inner side, and (4) neck bending toward the direction of the turn. Reversing the rotation caused animals to switch the direction of their turns and the asymmetric pattern of right and left limb activities. Preliminary evidence suggested that vestibular inputs were sufficient to drive the behavior. Sensory-evoked turning may provide a useful experimental platform to examine the brainstem commands and spinal neural networks that underlie the activation and switching of different locomotor forms.     

Hox genes and the regulation of movement in Drosophila

Many animals show regionally specialized patterns of movement along the body axis. In vertebrates, spinal networks regulate locomotion, while the brainstem controls movements of respiration and feeding. Similarly, amongst invertebrates diversification of appendages along the body axis is tied to the performance of characteristically different movements such as those required for feeding, locomotion, and respiration. Such movements require locally specialized networks of nerves and muscles. Here we use the regionally differentiated movements of larval crawling in Drosophila to investigate how the formation of a locally specialized locomotor network is genetically determined. By loss and gain of function experiments we show that particular Hox gene functions are necessary and sufficient to dictate the formation of a neuromuscular network that orchestrates the movements of peristaltic locomotion.     

CTCF regulates ataxin-7 expression through promotion of a convergently transcribed, antisense noncoding RNA

Neural networks in the spinal cord control two basic features of locomotor movements: rhythm generation and pattern generation. Rhythm generation is generally considered to be dependent on glutamatergic excitatory neurons. Pattern generation involves neural circuits controlling left-right alternation, which has been described in great detail, and flexor-extensor alternation, which remains poorly understood. Here, we use a mouse model in which glutamatergic neurotransmission has been ablated in the locomotor region of the spinal cord. The isolated in?vitro spinal cord from these mice produces locomotor-like activity-when stimulated with neuroactive substances-with prominent flexor-extensor alternation. Under these conditions, unlike in control mice, networks of inhibitory interneurons generate the rhythmic activity. In the absence of glutamatergic synaptic transmission, the flexor-extensor alternation appears to be generated by Ia inhibitory interneurons, which mediate reciprocal inhibition from muscle proprioceptors to antagonist motor neurons. Our study defines a minimal inhibitory network that is needed to produce flexor-extensor alternation during locomotion.     

Decoding Hindlimb Movement for a Brain Machine Interface after a Complete Spinal Transection

Stereotypical locomotor movements can be made without input from the brain after a complete spinal transection. However, the restoration of functional gait requires descending modulation of spinal circuits to independently control the movement of each limb. To evaluate whether a brain-machine interface (BMI) could be used to regain conscious control over the hindlimb, rats were trained to press a pedal and the encoding of hindlimb movement was assessed using a BMI paradigm. Off-line, information encoded by neurons in the hindlimb sensorimotor cortex was assessed. Next neural population functions, or weighted representations of the neuronal activity, were used to replace the hindlimb movement as a trigger for reward in real-time (on-line decoding) in three conditions: while the animal could still press the pedal, after the pedal was removed and after a complete spinal transection. A novel representation of the motor program was learned when the animals used neural control to achieve water reward (e.g. more information was conveyed faster). After complete spinal transection, the ability of these neurons to convey information was reduced by more than 40%. However, this BMI representation was relearned over time despite a persistent reduction in the neuronal firing rate during the task. Therefore, neural control is a general feature of the motor cortex, not restricted to forelimb movements, and can be regained after spinal injury.     

The effects of long-standing limb loss on anatomical reorganization of the somatosensory afferents in the brainstem and spinal cord

We examined the terminations of sensory afferents in the brainstem and spinal cord of squirrel monkeys and prosimian galagos 4-8 years after a therapeutic forelimb or hindlimb amputation within 2 months of birth. In each animal, the distributions of labeled sensory afferent terminations from remaining body parts proximal to the limb stump were much more extensive than in normal animals. These sprouted afferents extended into the portions of the dorsal horn of the spinal cord as well as the cuneate and external cuneate nuclei of the brainstem (forelimb amputees) or spinal Clarke's column (hindlimb amputee) related to the amputated limb. Such reorganization in sensory afferents along with reorganization of the motor efferents to muscles (Wu and Kaas, J Neurosci 19 : 7679-7697, 1999, Neuron 28 : 967-978, 2000) may provide a basis for mislocated phantom sensations of missing forelimb movements accompanying actual shoulder movements during cortical stimulation or movement imagery in patients with amputations.     

The effects of long-standing limb loss on anatomical reorganization of the somatosensory afferents in the brainstem and spinal cord

We examined the terminations of sensory afferents in the brainstem and spinal cord of squirrel monkeys and prosimian galagos 4-8 years after a therapeutic forelimb or hindlimb amputation within 2 months of birth. In each animal, the distributions of labeled sensory afferent terminations from remaining body parts proximal to the limb stump were much more extensive than in normal animals. These sprouted afferents extended into the portions of the dorsal horn of the spinal cord as well as the cuneate and external cuneate nuclei of the brainstem (forelimb amputees) or spinal Clarke's column (hindlimb amputee) related to the amputated limb. Such reorganization in sensory afferents along with reorganization of the motor efferents to muscles (Wu and Kaas, J Neurosci 19: 7679-7697, 1999, Neuron 28: 967-978, 2000) may provide a basis for mislocated phantom sensations of missing forelimb movements accompanying actual shoulder movements during cortical stimulation or movement imagery in patients with amputations.     

Computation of trunk equilibrium and stability in free flexion-extension movements at different velocities

Velocity of movement has been suggested as a risk factor for low-back disorders. The effect of changes in velocity during unconstrained flexion-extension movements on muscle activations, spinal loads, base reaction forces and system stability was computed. In vivo measurements of kinematics and ground reaction forces were initially carried out on young asymptomatic subjects. The collected kinematics of three subjects representing maximum, mean and minimum lumbar rotations were subsequently used in the kinematics-driven model to compute results during the entire movements at three different velocities. Estimated spinal loads and muscle forces were significantly larger in fastest pace as compared to slower ones indicating the effect of inertial forces. Spinal stability was improved in larger trunk flexion angles and fastest movement. Partial or full flexion relaxation of global extensor muscles occurred only in slower movements. Some local lumbar muscles, especially in subjects with larger lumbar flexion and at slower paces, also demonstrated flexion relaxation. Results confirmed the crucial role of movement velocity on spinal biomechanics. Predictions also demonstrated the important role on response of the magnitude of peak lumbar rotation and its temporal variation.     

A Brain-Machine-Muscle Interface for Restoring Hindlimb Locomotion after Complete Spinal Transection in Rats

A brain-machine interface (BMI) is a neuroprosthetic device that can restore motor function of individuals with paralysis. Although the feasibility of BMI control of upper-limb neuroprostheses has been demonstrated, a BMI for the restoration of lower-limb motor functions has not yet been developed. The objective of this study was to determine if gait-related information can be captured from neural activity recorded from the primary motor cortex of rats, and if this neural information can be used to stimulate paralysed hindlimb muscles after complete spinal cord transection. Neural activity was recorded from the hindlimb area of the primary motor cortex of six female Sprague Dawley rats during treadmill locomotion before and after mid-thoracic transection. Before spinal transection there was a strong association between neural activity and the step cycle. This association decreased after spinal transection. However, the locomotive state (standing vs. walking) could still be successfully decoded from neural recordings made after spinal transection. A novel BMI device was developed that processed this neural information in real-time and used it to control electrical stimulation of paralysed hindlimb muscles. This system was able to elicit hindlimb muscle contractions that mimicked forelimb stepping. We propose this lower-limb BMI as a future neuroprosthesis for human paraplegics.     

Neuronal firing activity in the lateral vestibular nucleus during locomotion in the guinea pig

The effects of spontaneous locomotor activity on neuronal background firing in the lateral vestibular nucleus was investigated during experiments on decerebrate guinea pigs. The onset of rhythmic muscular activity in the extramities was found to produce a rise in the rate of such discharges, which increased from 10–15 to 100 spikes/sec in most neurons. A higher rate occurred as activity began in the ipsilateral forelimb extensor muscles (the stage corresponding to the end of the swing phase and start of the stance phase in the locomotor cycle). The alterations noted in vestibular neuronal activity during locomotion are thought to ensure the background of high anti-graveity muscle tonus against which rhythmic limb movements take place.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 23, No. 5, pp. 536–541, September–October, 1991.     

Thoracic Hemisection in Rats Results in Initial Recovery Followed by a Late Decrement in Locomotor Movements,with Changes in Coordination Correlated with Serotonergic Innervation of the Ventral Horn

Lateral thoracic hemisection of the rodent spinal cord is a popular model of spinal cord injury, in which the effects of various treatments, designed to encourage locomotor recovery, are tested. Nevertheless, there are still inconsistencies in the literature concerning the details of spontaneous locomotor recovery after such lesions, and there is a lack of data concerning the quality of locomotion over a long time span after the lesion. In this study, we aimed to address some of these issues. In our experiments, locomotor recovery was assessed using EMG and CatWalk recordings and analysis. Our results showed that after hemisection there was paralysis in both hindlimbs, followed by a substantial recovery of locomotor movements, but even at the peak of recovery, which occurred about 4 weeks after the lesion, some deficits of locomotion remained present. The parameters that were abnormal included abduction, interlimb coordination and speed of locomotion. Locomotor performance was stable for several weeks, but about 3–4 months after hemisection secondary locomotor impairment was observed with changes in parameters, such as speed of locomotion, interlimb coordination, base of hindlimb support, hindlimb abduction and relative foot print distance. Histological analysis of serotonergic innervation at the lumbar ventral horn below hemisection revealed a limited restoration of serotonergic fibers on the ipsilateral side of the spinal cord, while on the contralateral side of the spinal cord it returned to normal. In addition, the length of these fibers on both sides of the spinal cord correlated with inter- and intralimb coordination. In contrast to data reported in the literature, our results show there is not full locomotor recovery after spinal cord hemisection. Secondary deterioration of certain locomotor functions occurs with time in hemisected rats, and locomotor recovery appears partly associated with reinnervation of spinal circuitry by serotonergic fibers.     

Brachially innervated ectopic hindlimbs in the chick embryo. I. Limb motility and motor system anatomy during the development of embryonic behavior

The functional status of brachially innervated hindlimbs, produced by transplanting hindlimb buds of chick embryos in place of forelimb buds, was quantified by analyzing the number and temporal distribution of spontaneous limb movements. Brachially innervated hindlimbs exhibited normal motility until E10 but thereafter became significantly less active than normal limbs and the limb movements were more randomly distributed. Contrary to the findings with axolotls and frogs, functional interaction between brachial motoneurons and hindlimb muscles cannot be sustained in the chick embryo. Dysfunction is first detectable at E10 and progresses to near total immobility by E20 and is associated with joint ankylosis and muscular atrophy. Although brachially innervated hindlimbs were virtually immobile by the time of hatching (E21), they produced strong movements in response to electrical stimulation of their spinal nerves, suggesting a central rather than peripheral defect in the motor system. The extent of motoneuron death in the brachial spinal cord was not significantly altered by the substitution of the forelimb bud with the hindlimb bud, but the timing of motoneuron loss was appropriate for the lumbar rather than brachial spinal cord, indicating that the rate of motoneuron death was dictated by the limb. Measurements of nuclear area indicated that motoneuron size was normal during the motoneuron death period (E6-E10) but the nuclei of motoneurons innervating grafted hindlimbs subsequently became significantly larger than those of normal brachial motoneurons. Although the muscle mass of the grafted hindlimb at E18 was significantly less than that of the normal hindlimb (and similar to that of a normal forelimb), electronmicroscopic examination of the grafted hindlimbs and brachial spinal cords of E20 embryos revealed normal myofiber and neuromuscular junction ultrastructure and a small increase in the number of axosomatic synapses on cross-sections of motoneurons innervating grafted hindlimbs compared to motoneurons innervating normal forelimbs. The anatomical data indicate that, rather than being associated with degenerative changes, the motor system of the brachial hindlimb of late-stage embryos is intact, but inactive. © 1993 John Wiley & Sons, Inc.     

Kinematics of different types of locomotor movements in the deafferented rat

The kinematics of rat hindlimb movements were assessed and compared pre- and post-deafferentation during swimming, forelimb treadmill locomotion plus hindlimb swimming motion, and walking using all four limbs. All types of locomotion were characterized by an increase in the frequency of locomotor rhythm and reduced amplitude of motion at the hindlimb joints following deafferentation. The reduced change observed in the angle of the coxofemoral joint, indicative of a horizontal component in locomotor motion, was mainly brought about by less marked extension. This would confirm evidence indicating that increased load on the extremities, with its ensuing naturally-occurring afferent outflow, is accompanied by a reduced locomotor motion rate and a rise in the amplitude of the latter due to intensified extension of the limb. The increased forward carriage of the hind limb seen during the transition to four-legged locomotion persisted after deafferentation; this may be considered a sign of coordination amongst the limbs. Deafferentation led to a reduction in the MEG of muscle activity, which was found to be lowest in swimming and highest during walking. The role of the afferent inflow in shaping different types of locomotor motion is evaluated.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 19, No. 4, pp. 520–525, July–August, 1987.