Spinal Locomotor Circuits Research Articles

The Multifunctional Mesencephalic Locomotor Region

In 1966, Shik, Severin and Orlovskii discovered that electrical stimulation of a region at the junction between the midbrain and hindbrain elicited controlled walking and running in the cat. The region was named Mesencephalic Locomotor Region (MLR). Since then, this locomotor center was shown to control locomotion in various vertebrate species, including the lamprey, salamander, stingray, rat, guinea-pig, rabbit or monkey. In human subjects asked to imagine they are walking, there is an increased activity in brainstem nuclei corresponding to the MLR (i.e. pedunculopontine, cuneiform and subcuneiform nuclei). Clinicians are now stimulating (deep brain stimulation) structures considered to be part of the MLR to alleviate locomotor symptoms of patients with Parkinson's disease. However, the anatomical constituents of the MLR still remain a matter of debate, especially relative to the pedunculopontine, cuneiform and subcuneiform nuclei. Furthermore, recent studies in lampreys have revealed that the MLR is more complex than a simple relay in a serial descending pathway activating the spinal locomotor circuits. It has multiple functions. Our goal is to review the current knowledge relative to the anatomical constituents of the MLR, and its physiological role, from lamprey to man. We will discuss these results in the context of the recent clinical studies involving stimulation of the MLR in patients with Parkinson's disease.

Ephrin-B2 expression in the proprioceptive sensory system

Recent studies have shown that ephrin-B2 on sensory afferent fibers from the dorsal root ganglia (DRG) controls transmission of pain sensation to the spinal cord. We examined ephrin-B2 expression in mouse DRG and spinal cord using an ephrin-B2/ß-galactosidase chimeric allele. We found that ephrin-B2 is expressed exclusively in proprioceptive neurons and fibers in neonates, while expression in lamina III and IV of the adult spinal cord was observed in addition to that in the deeper laminae. We confirmed that ephrin-B2 protein causes co-clustering of EphB2 and glutamate receptors in spinal cord neurons. Our data are consistent with a role for ephrin-B2 in transmission of positional information to the CNS, and thus suggest a role in synaptic plasticity of spinal cord locomotor circuits that are known to be sensitive to proprioceptive sensory input after spinal cord injury.

Hindbrain V2a Neurons in the Excitation of Spinal Locomotor Circuits during Zebrafish Swimming

During locomotion in vertebrates, reticulospinal neurons in the hindbrain play critical roles in providing descending excitation to the spinal cord locomotor systems. However, despite the fact that many genes that are used to classify the neuronal identities of neurons in the hindbrain have been identified, the molecular identity of the reticulospinal neurons that are critically involved in locomotor drive is not well understood. Chx10-expressing neurons (V2a neurons) are ipsilaterally projecting glutamatergic neurons in the spinal cord and the hindbrain. Many of the V2a neurons in the hindbrain are known to project to the spinal cord in zebrafish, making hindbrain V2a neurons a prime candidate in descending locomotor drive. We investigated the roles of hindbrain V2a neurons using optogenetic and electrophysiological approaches. The forced activation of hindbrain V2a neurons using channelrhodopsin efficiently evoked swimming, whereas the forced inactivation of them using Archearhodopsin3 or Halorhodpsin reliably stopped ongoing swimming. Electrophysiological recordings of two populations of hindbrain reticulospinal V2a neurons showed that they were active during swimming. One population of neurons, small V2a neurons in the caudal hindbrain, fired with low rhythmicity, whereas the other population of neurons, large reticulospinal V2a neurons, called MiV1 neurons, fired more rhythmically. These results indicated that hindbrain reticulospinal V2a neurons play critical roles in providing excitation to the spinal locomotor circuits during swimming by providing both tonic and phasic inputs to the circuits.

Decoding the mechanisms of gait generation in salamanders by combining neurobiology, modeling and robotics

Vertebrate animals exhibit impressive locomotor skills. These locomotor skills are due to the complex interactions between the environment, the musculo-skeletal system and the central nervous system, in particular the spinal locomotor circuits. We are interested in decoding these interactions in the salamander, a key animal from an evolutionary point of view. It exhibits both swimming and stepping gaits and is faced with the problem of producing efficient propulsive forces using the same musculo-skeletal system in two environments with significant physical differences in density, viscosity and gravitational load. Yet its nervous system remains comparatively simple. Our approach is based on a combination of neurophysiological experiments, numerical modeling at different levels of abstraction, and robotic validation using an amphibious salamander-like robot. This article reviews the current state of our knowledge on salamander locomotion control, and presents how our approach has allowed us to obtain a first conceptual model of the salamander spinal locomotor networks. The model suggests that the salamander locomotor circuit can be seen as a lamprey-like circuit controlling axial movements of the trunk and tail, extended by specialized oscillatory centers controlling limb movements. The interplay between the two types of circuits determines the mode of locomotion under the influence of sensory feedback and descending drive, with stepping gaits at low drive, and swimming at high drive.

Coapplication of noisy patterned electrical stimuli and NMDA plus serotonin facilitates fictive locomotion in the rat spinal cord

A new stimulating protocol [fictive locomotion-induced stimulation (FListim)], consisting of intrinsically variable weak waveforms applied to a single dorsal root is very effective (though not optimal as it eventually wanes away) in activating the locomotor program of the isolated rat spinal cord. The present study explored whether combination of FListim with low doses of pharmacological agents that raise network excitability might further improve the functional outcome, using this in vitro model. FListim was applied together with N-methyl-d-aspartate (NMDA) + serotonin, while fictive locomotion (FL) was electrophysiologically recorded from lumbar ventral roots. Superimposing FListim on FL evoked by these neurochemicals persistently accelerated locomotor-like cycles to a set periodicity and modulated cycle amplitude depending on FListim rate. Trains of stereotyped rectangular pulses failed to replicate this phenomenon. The GABA(B) agonist baclofen dose dependently inhibited, in a reversible fashion, FL evoked by either FListim or square pulses. Sustained episodes of FL emerged when FListim was delivered, at an intensity subthreshold for FL, in conjunction with subthreshold pharmacological stimulation. Such an effect was, however, not found when high potassium solution instead of NMDA + serotonin was used. These results suggest that the combined action of subthreshold FListim (e.g., via epidural stimulation) and neurochemicals should be tested in vivo to improve locomotor rehabilitation after injury. In fact, reactivation of spinal locomotor circuits by conventional electrical stimulation of afferent fibers is difficult, while pharmacological activation of spinal networks is clinically impracticable due to concurrent unwanted effects. We speculate that associating subthreshold chemical and electrical inputs might decrease side effects when attempting to evoke human locomotor patterns.

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.

Influence of spinal reflexes on the locomotor pattern after spinal cord injury

In complete spinal cord injured (cSCI) subjects a shift from dominant early (60–120ms latency) to dominant late (120–450ms latency) spinal reflex (SR) components occurs over time after injury. This shift is assumed to reflect a spinal neuronal dysfunction below the level of a spinal lesion. The neuronal pathways of SR are suggested to be closely connected with spinal locomotor circuits. The aim of this study was to explore the influence of the two SR components on the electromyographic (EMG) pattern induced by assisted locomotion in cSCI subjects. Leg muscle EMG activity was analysed during assisted locomotion in both healthy and motor cSCI subjects. SR were evoked by non-noxious tibial nerve stimulation during mid-stance phase of the gait cycle. Early and late SR components had a differential influence on the locomotor pattern. In healthy and cSCI subjects with a dominant early SR component the locomotor EMG pattern was modulated in the form of a short increase in leg flexors activity in the stance phase (tibialis anterior, biceps femoris). In contrast, in chronic cSCI subjects with a dominant late SR component no activation in biceps femoris but a long-lasting activation of tibialis anterior and rectus femoris muscles during the stance phase was evoked. It is concluded that the same tibial nerve stimuli activated two different neuronal pathways, resulting in divergent interactions with spinal locomotor circuitries. It is proposed that the two SR components have different physiological roles during locomotion.

Controlling Specific Locomotor Behaviors through Multidimensional Monoaminergic Modulation of Spinal Circuitries

Descending monoaminergic inputs markedly influence spinal locomotor circuits, but the functional relationships between specific receptors and the control of walking behavior remain poorly understood. To identify these interactions, we manipulated serotonergic, dopaminergic, and noradrenergic neural pathways pharmacologically during locomotion enabled by electrical spinal cord stimulation in adult spinal rats in vivo. Using advanced neurobiomechanical recordings and multidimensional statistical procedures, we reveal that each monoaminergic receptor modulates a broad but distinct spectrum of kinematic, kinetic, and EMG characteristics, which we expressed into receptor-specific functional maps. We then exploited this catalog of monoaminergic tuning functions to devise optimal pharmacological combinations to encourage locomotion in paralyzed rats. We found that, in most cases, receptor-specific modulatory influences summed near algebraically when stimulating multiple pathways concurrently. Capitalizing on these predictive interactions, we elaborated a multidimensional monoaminergic intervention that restored coordinated hindlimb locomotion with normal levels of weight bearing and partial equilibrium maintenance in spinal rats. These findings provide new perspectives on the functions of and interactions between spinal monoaminergic receptor systems in producing stepping, and define a framework to tailor pharmacotherapies for improving neurological functions after CNS disorders.

Initiation of Locomotion in Adult Zebrafish

Motor behavior is generated by specific neural circuits. Those producing locomotion are located in the spinal cord, and their activation depends on descending inputs from the brain or on sensory inputs. In this study, we have used an in vitro brainstem-spinal cord preparation from adult zebrafish to localize a region where stimulation of descending inputs can induce sustained locomotor activity. We show that a brief stimulation of descending inputs at the junction between the brainstem and spinal cord induces long-lasting swimming activity. The swimming frequencies induced are remarkably similar to those observed in freely moving adult fish, arguing that the induced locomotor episode is highly physiological. The motor pattern is mediated by activation of ionotropic glutamate and glycine receptors in the spinal cord and is not the result of synaptic interactions between neurons at the site of the stimulation in the brainstem. We also compared the activity of motoneurons during locomotor activity induced by electrical stimulation of descending inputs and by exogenously applied NMDA. Prolonged NMDA application changes the shape of the synaptic drive and action potentials in motoneurons. When escape activity occurs, the swimming activity in the intact zebrafish was interrupted and some of the motoneurons involved became inhibited in vitro. Thus, the descending inputs seem to act as a switch to turn on the activity of the spinal locomotor network in the caudal spinal cord. We propose that recurrent synaptic activity within the spinal locomotor circuits can transform a brief input into a well coordinated and long-lasting swimming pattern.

Prolonged electrical stimulation over hip flexors increases locomotor output in human SCI

Objectives The objective of this study was to determine whether enhanced feedback from thigh afferents improves locomotor output in human spinal cord injury (SCI). Methods The effects of afferent feedback originating from the upper thigh muscles on locomotion was examined using electrical stimulation in 10 subjects with incomplete SCI and three neurologically intact controls during robotic-assisted treadmill walking. Electrical stimulation consisted of 20 pulses at 30 Hz, applied bilaterally to the skin of the medial thigh, approximately over the sartorius muscle. The stimulation was applied at four different phases of the gait cycle. Torque responses of hip and knee joints and electromyograms of both legs were recorded during baseline with no stimulation, stimulation, and post-stimulation. Results During stimulation, enhanced hip and knee extension and flexion torque responses were observed during the stance and swing phases, respectively, for all four different stimulation conditions. Larger hip extension torque was observed when the stimulation was applied during the stance phase and the transition from stance to swing. Conclusions Enhanced afferent feedback produced by electrical stimulation may increase the excitability of the spinal cord locomotor circuits in human SCI. Significance Results from this study emphasize the contribution of sensory information from thigh muscles, particularly the sartorius muscle afferents, to locomotor control in human SCI during treadmill walking.

Preferred locomotor phase of activity of lumbar interneurons during air-stepping in subchronic spinal cats

Spinal locomotor circuits are intrinsically capable of driving a variety of behaviors such as stepping, scratching, and swimming. Based on an observed rostrocaudal wave of activity in the motoneuronal firing during locomotor tasks, the traveling-wave hypothesis proposes that spinal interneuronal firing follows a similar rostrocaudal pattern of activation, suggesting the presence of spatially organized interneuronal modules within the spinal motor system. In this study, we examined if the spatial organization of the lumbar interneuronal activity patterns during locomotor activity in the adult mammalian spinal cord was consistent with a traveling-wave organizational scheme. The activity of spinal interneurons within the lumbar intermediate zone was examined during air-stepping in subchronic spinal cats. The preferred phase of interneuronal activity during a step cycle was determined using circular statistics. We found that the preferred phases of lumbar interneurons from both sides of the cord were evenly distributed over the entire step cycle with no indication of functional groupings. However, when units were subcategorized according to spinal hemicords, the preferred phases of units on each side largely fell around the period of extensor muscle activity on each side. In addition, there was no correlation between the preferred phases of units and their rostrocaudal locations along the spinal cord with preferred phases corresponding to both flexion and extension phases of the step cycle found at every rostrocaudal level of the cord. These results are consistent with the hypothesis that interneurons operate as part of a longitudinally distributed network rather than a rostrocaudally organized traveling-wave network.

Brainstem Regions Affecting Minimum Alveolar Concentration and Movement Pattern during Isoflurane Anesthesia

Spinal transection or selective delivery of volatile anesthetics to the spinal cord reduces minimum alveolar concentration (MAC), whereas precollicular decerebration does not. The authors sought to determine which brainstem regions influence anesthetic requirements and movement responses with isoflurane. Movement (biceps femoris electromyogram) and MAC were measured in adult rats before and after decerebration at the precollicular, mid-collicular, pontine or medullary level, or decerebellation. Additional experiments assessed the effects of lidocaine inactivation of the mesencephalic locomotor region on MAC and the effects of isoflurane on nociceptive neuronal responses in this region. Transections placed at the level of the mid-colliculus, rostral pons, and pontomedullary junction significantly reduced MAC by approximately 10, 40, and 45%, respectively. MAC was decreased 9% after mid-medullary transections that were placed caudal to the nucleus raphe magnus but rostral to the dorsal reticular nucleus; however, only weak, single movements occurred. Caudal medullary transections at the obex decreased MAC by 60%. Bilateral inactivation of the mesencephalic locomotor region with lidocaine caused a reversible, 32% decrease in MAC and reduced the number and amplitude of movements at sub-MAC isoflurane concentrations. Neuronal responses of mesencephalic locomotor region neurons to supramaximal noxious tail clamp were reduced by 87% by 1.2 MAC isoflurane. The authors conclude that the mesencephalic locomotor region influences anesthetic requirements and promotes repetitive movement with sub-MAC isoflurane by facilitating ventral spinal locomotor circuits, where anesthetics seem to exert their key immobilizing effects. However, net brainstem influences on MAC seem to result from interaction among descending nociceptive and locomotor modulatory pathways.

Endogenous extracellular serotonin modulates the spinal locomotor network of the neonatal mouse

Serotonin (5-HT) can potently activate and modulate spinal locomotor circuits in a variety of species. Many of these findings have been obtained by applying serotonin exogenously to the isolated spinal cord of in vitro preparations, which has the drawback of indiscriminately activating extrasynaptic receptors and neurons. To investigate the role of endogenously released serotonin in modulating locomotor networks, the selective serotonin reuptake inhibitor citalopram was used. Fictive locomotion was elicited by either electrical stimulation of the brainstem or the sacral 4 (S4) dorsal root. The addition of 20 microm of citalopram caudal to thoracic segment 5 (T5) had an overall inhibitory effect on the lumbar central pattern generator (CPG). Left-right and flexor-extensor coupling were significantly decreased, and there was also a phase shift in the flexor-extensor relationship. In addition, there was a significant decrease in burst amplitude. These effects were observed during both afferent and brainstem evoked fictive locomotion. When citalopram was added in the presence of 5-HT(1A) and 5-HT(1B) antagonists, the inhibitory effects were largely reversed. The remaining excitatory effects were mediated by 5-HT(7) and 5-HT(2) receptors. These results suggest that endogenous 5-HT release can modulate locomotor-like activity early in neonatal development.

Opposing Aminergic Modulation of Distinct Spinal Locomotor Circuits and Their Functional Coupling during Amphibian Metamorphosis

The biogenic amines serotonin (5-HT) and noradrenaline (NA) are well known modulators of central pattern-generating networks responsible for vertebrate locomotion. Here we have explored monoaminergic modulation of the spinal circuits that generate two distinct modes of locomotion in the metamorphosing frog Xenopus laevis. At metamorphic climax when propulsion is achieved by undulatory larval tail movements and/or by kicking of the newly developed adult hindlimbs, the underlying motor networks remain spontaneously active in vitro, producing either separate fast axial and slow appendicular rhythms or a single combined rhythm that drives coordinated tail-based and limb-based swimming in vivo. In isolated spinal cords already expressing distinct axial and limb rhythms, bath-applied 5-HT induced coupled network activity through an opposite slowing of axial rhythmicity (by increasing motoneuron burst and cycle durations) and an acceleration of limb rhythmicity (by decreasing burst and cycle durations). In contrast, in preparations spontaneously expressing coordinated fictive locomotion, exogenous NA caused a dissociation of spinal activity into separate faster axial and slower appendicular rhythms by decreasing and increasing burst and cycle durations, respectively. Moreover, in preparations from premetamorphic and postmetamorphic animals that express exclusively axial-based or limb-based locomotion, 5-HT and NA modified the developmentally independent rhythms in a similar manner to the amines' opposing effects on the coexisting circuits at metamorphic climax. Thus, by exerting differential modulatory actions on one network that are opposite to their influences on a second adjacent circuit, these two amines are able to precisely regulate the functional relationship between different rhythmogenic networks in a developing vertebrate's spinal cord.

Development and function of spinal locomotor circuits in zebrafish

Development and function of spinal locomotor circuits in zebrafish

Roles of inhibitory interneurons in the spinal locomotor circuit of developing rodents

Roles of inhibitory interneurons in the spinal locomotor circuit of developing rodents

Spinal control of locomotion – from cat to man

For a large number of vertebrate species it is now indisputable that spinal networks have the capability of generating the basic locomotor rhythm. The aim of this review is to summarize the evidence for spinal pattern generators in cats and primates, including man and its interaction with sensory signals from the limbs. For all species the sensory feed-back from the moving limb is very important to achieve effective locomotor behaviour by adapting to the environment and compensating for unexpected postural disturbances. Sensory regulation of stepping can occur via reflex pathways to motoneurones (by-passing the locomotor rhythm generators) or by acting on the spinal locomotor networks themselves. The sensory feed-back serves to control the timing of the different phases in the step cycle, to shape the pattern of muscle activity, to contribute to the excitatory drive of the motoneurones and to the long-term adaptation of the locomotor activity. In this review we discuss the spinal locomotor circuits and the sensory feed-back in animals (mainly the cat) and human subjects. Special emphasis is given to work that has been of importance for the development of new rehabilitation paradigms following spinal cord injury.

Development and function of spinal locomotor circuits in zebrafish

Development and function of spinal locomotor circuits in zebrafish

Alx, a Zebrafish Homolog ofChx10, Marks Ipsilateral Descending Excitatory Interneurons That Participate in the Regulation of Spinal Locomotor Circuits

Recent molecular genetic studies suggest that the expression of transcription factors in the developing spinal cord helps determine the morphological and physiological properties of neurons. Using the zebrafish preparation, we have examined the properties of neurons marked by alx, a zebrafish homolog of mammalian Chx10. We performed morphological and physiological studies using transgenic zebrafish expressing fluorescent reporter constructs in cells that had at any time point expressed alx (alx neurons). Our data reveal that zebrafish alx neurons are all ipsilateral descending neurons that are positive for vesicular glutamate transporter 2, suggesting that they are glutamatergic excitatory interneurons. Patch recordings show that earlier-born neurons are active during stronger movements such as escapes and fast swimming (strong movement class), whereas later-born ones are involved in sustained weak swimming (weak movement class). Paired recordings between alx neurons and motoneurons show that neurons of the strong movement class make frequent monosynaptic excitatory connections onto motoneurons. Thus, neurons of this class are likely premotor interneurons that regulate motoneuron activity during escapes and fast swimming. We also show the existence of a monosynaptic connection between an alx neuron of the weak movement class and a motoneuron. Collectively, our data demonstrate that alx marks ipsilateral descending neurons that are involved in the regulation of motoneuron activity during forms of locomotion, such as escape and swimming.

Cholinergic Input Is Required during Embryonic Development to Mediate Proper Assembly of Spinal Locomotor Circuits

Rhythmic limb movements are controlled by pattern-generating neurons within the ventral spinal cord, but little is known about how these locomotor circuits are assembled during development. At early stages of embryogenesis, motor neurons are spontaneously active, releasing acetylcholine that triggers the depolarization of adjacent cells in the spinal cord. To investigate whether acetylcholine-driven activity is required for assembly of the central pattern-generating (CPG) circuit, we studied mice lacking the choline acetyltransferase (ChAT) enzyme. Our studies show that a rhythmically active spinal circuit forms in ChAT mutants, but the duration of each cycle period is elongated, and right-left and flexor-extensor coordination are abnormal. In contrast, blocking acetylcholine receptors after the locomotor network is wired does not affect right-left or flexor-extensor coordination. These findings suggest that the cholinergic neurotransmitter pathway is involved in configuring the CPG during a transient period of development.