Speed-dependent modulation of the locomotor behavior in adult Mice reveals attractor and transitional gaits

Locomotion results from an interplay between biomechanical constraints of the muscles attached to the skeleton and the neuronal circuits controlling and coordinating muscle activities. Quadrupeds exhibit a wide range of locomotor gaits. Given our advances in the genetic identification of spinal and supraspinal circuits important to locomotion in the mouse, it is now important to get a better understanding of the full repertoire of gaits in the freely walking mouse. To assess this range, young adult C57BL/6J mice were trained to walk and run on a treadmill at different locomotor speeds. Instead of using the classical paradigm defining gaits according to their footfall pattern, we combined the inter-limb coupling and the duty cycle of the stance phase, thus identifying several types of gaits: lateral walk, trot, out-of-phase walk, rotary gallop, transverse gallop, hop, half-bound, and full-bound. Out-of-phase walk, trot, and full-bound were robust and appeared to function as attractor gaits (i.e., a state to which the network flows and stabilizes) at low, intermediate, and high speeds respectively. In contrast, lateral walk, hop, transverse gallop, rotary gallop, and half-bound were more transient and therefore considered transitional gaits (i.e., a labile state of the network from which it flows to the attractor state). Surprisingly, lateral walk was less frequently observed. Using graph analysis, we demonstrated that transitions between gaits were predictable, not random. In summary, the wild-type mouse exhibits a wider repertoire of locomotor gaits than expected. Future locomotor studies should benefit from this paradigm in assessing transgenic mice or wild-type mice with neurotraumatic injury or neurodegenerative disease affecting gait

Analysis of basic motor behaviors in quadrupeds

Ability to perform locomotion in different directions and maintain upright body posture is crucial for normal life. At present, mice, which allows employing genetic approaches, are widely used in studying the locomotor system. In these investigations different experimental setups are used to evoke locomotion. First aim of the present study was to compare kinematics of forward (FW) and backward (BW) locomotion performed in different environmental conditions (i.e. in a tunnel, on a treadmill and on an air-ball). On all set-ups, average speed, step amplitude and swing duration during BW locomotion were significantly smaller compared to those observed during FW locomotion. The extent of rostro-caudal paw trajectory in relation to the hip projection to the surface (HP) strongly depended on hip height. With high hip height, the trajectory was symmetrical in relation to HP (middle steps). When hip was low, steps were either displaced rostrally (anterior steps) or caudally (posterior steps) in relation to HP. During FW locomotion, predominantly anterior and posterior steps were observed, respectively, on the treadmill and air-ball, while all three stepping forms were observed in the tunnel. We observed only anterior steps during BW locomotion. Intralimb coordination depended on the form of stepping. Second aim of the present study was to reveal the role of two populations of commissural interneurons (V0V and V0D CINs) in control of a number of basic motor behaviours (BW locomotion, scratching, righting, and postural corrections). For this purpose two types of knockout mice (Vglut2Cre;Dbx1DTA mice and Hoxb8Cre;Dbx1DTA mice with only V0V and all V0 CINs ablated, respectively) as well as wild-type littermates were used. Our results suggest that the functional effect of excitatory V0V CINs during BW locomotion and scratching is inhibitory, and that execution of scratching involves active inhibition of the contralateral scratching CPG mediated by V0V CINs. By contrast, V0D CINs are elements of spinal postural network, generating postural corrections. Finally, both V0D and V0V CINs contribute to generation of righting behavior. Thus, our study shows the differential contribution of V0 neuron subpopulations in generation of diverse motor acts. Single steps in different directions are used for control of balance or body configuration. However, our knowledge about neural mechanisms responsible for their generation is limited. The third aim of the present study was to characterize postural response to disturbance of basic body configuration caused by forward, backward or outward displacement of the hindlimb. In intact rabbits, displacement of the hindlimb in any direction caused a postural response consisting of two components. First, a lateral trunk movement towards the supporting (contralateral) hindlimb was performed, and then a corrective step in the direction opposite to the direction of the initial limb displacement was executed. These two components were generated by different mechanisms activated in a strict order by sensory information from the deviated limb signalling distortion of the limb/limb-trunk configuration. We have shown that the integrity of the forebrain was not critical for generation of this postural response. We proposed a hypothesis about operation of mechanisms generating the postural response characterized in the present study

Functional contribution of the mesencephalic locomotor region to locomotion

Parce qu'il est naturel et facile de marcher, il peut sembler que cet acte soit produit aussi facilement qu'il est accompli. Au contraire, la locomotion nécessite une interaction neurale complexe entre les neurones supraspinaux, spinaux et périphériques pour obtenir une locomotion fluide et adaptée à l'environnement. La région locomotrice mésencéphalique (MLR) est un centre locomoteur supraspinal situé dans le tronc cérébral qui a notamment pour rôle d'initier la locomotion et d'induire une transition entre les allures locomotrices. Cependant, bien que cette région ait initialement été identifiée comme le noyau cunéiforme (CnF), un groupe de neurones glutamatergiques, et le noyau pédonculopontin (PPN), un groupe de neurones glutamatergiques et cholinergiques, son corrélat anatomique est encore un sujet de débat. Et alors qu'il a été prouvé que, que ce soit lors d’une stimulation de la MLR ou pour augmenter la vitesse locomotrice, la plupart des quadrupèdes présentent un large éventail d'allures locomotrices allant de la marche, au trot, jusqu’au galop, la gamme exacte des allures locomotrices chez la souris est encore inconnue. Ici, en utilisant l'analyse cinématique, nous avons d'abord décidé d'identifier d’évaluer les allures locomotrices des souris C57BL / 6. Sur la base de la symétrie de la démarche et du couplage inter-membres, nous avons identifié et caractérisé 8 allures utilisées à travers un continuum de fréquences locomotrices allant de la marche au trot puis galopant avec différents sous-types d'allures allant du plus lent au plus rapide. Certaines allures sont apparues comme attractrices d’autres sont apparues comme transitionnelles. En utilisant une analyse graphique, nous avons également démontré que les transitions entre les allures n'étaient pas aléatoires mais entièrement prévisibles. Nous avons ensuite décidé d'analyser et de caractériser les contributions fonctionnelles des populations neuronales de CnF et PPN au contrôle locomoteur. En utilisant des souris transgéniques exprimant une opsine répondant à la lumière dans les neurones glutamatergiques (Glut) ou cholinergiques (CHAT), nous avons photostimulé (ou photo-inhibé) les neurones glutamatergiques du CnF ou du PPN ou les neurones cholinergiques du PPN. Nous avons découvert que les neurones glutamatergiques du CnF initient et modulent l’allure locomotrice et accélèrent le rythme, tandis que les neurones glutamatergiques et cholinergiques du PPN le ralentissent. En initiant, modulant et en accélérant la locomotion, notre étude identifie et caractérise des populations neuronales distinctes de la MLR. Définir et décrire en profondeur la MLR semble d’autant plus urgent qu’elle est devenue récemment une cible pour traiter les symptômes survenant après une lésion de la moelle épinière ou liés à la maladie de Parkinson.Because it is natural and easy to walk, it could seem that this act is produced as easily as it is accomplished. On the contrary, locomotion requires an intricate and complex neural interaction between the supraspinal, spinal and peripheric neurons to obtain a locomotion that is smooth and adapted to the environment. The Mesencephalic Locomotor Region (MLR) is a supraspinal brainstem locomotor center that has the particular role of initiating locomotion and inducing a transition between locomotor gaits. However, although this region was initially identified as the cuneiform nucleus (CnF), a cluster of glutamatergic neurons, and the pedunculopontine nucleus (PPN), a cluster of glutamatergic and cholinergic neurons, its anatomical correlate is still a matter of debate. And while it is proven that, either under MLR stimulation or in order to increase locomotor speed, most quadrupeds exhibit a wide range of locomotor gaits from walk, to trot, to gallop, the exact range of locomotor gaits in the mouse is still unknown. Here, using kinematic analysis we first decided to identify to assess locomotor gaits C57BL/6 mice. Based on the symmetry of the gait and the inter-limb coupling, we identified and characterized 8 gaits during locomotion displayed through a continuum of locomotor frequencies, ranging from walk to trot and then to gallop with various sub-types of gaits at the slowest and highest speeds that appeared as attractors or transitional gaits. Using graph analysis, we also demonstrated that transitions between gaits were not random but entirely predictable. Then we decided to analyze and characterize the functional contributions of the CnF and PPN’s neuronal populations to locomotor control. Using transgenic mice expressing opsin in either glutamatergic (Glut) or cholinergic (CHAT) neurons, we photostimulated (or photoinhibited) glutamatergic neurons of the CnF or PPN or cholinergic neurons of the PPN. We discovered that glutamatergic CnF neurons initiate and modulate the locomotor pattern, and accelerate the rhythm, while glutamatergic and cholinergic PPN neurons decelerate it. By initiating, modulating, and accelerating locomotion, our study identifies and characterizes distinct neuronal populations of the MLR. Describing and defining thoroughly the MLR seems all the more urgent since it has recently become a target for spinal cord injury and Parkinson’s disease treatment

Afferent information modulates spinal network activity in vitro and in preclinical animal models

Primary afferents are responsible for the transmission of peripheral sensory information to the spinal cord. Spinal circuits involved in sensory processing and in motor activity are directly modulated by incoming input conveyed by afferent fibres. Current neurorehabilitation exploits primary afferent information to induce plastic changes within lesioned spinal circuitries. Plasticity and neuromodulation promoted by activity-based interventions are suggested to support both the functional recovery of locomotion and pain relief in subjects with sensorimotor disorders. The present study was aimed at assessing spinal modifications mediated by afferent information. At the beginning of my PhD project, I adopted a simplified in vitro model of isolated spinal cord from the newborn rat. In this preparation, dorsal root (DR) fibres were repetitively activated by delivering trains of electrical stimuli. Responses of dorsal sensory-related and ventral motor-related circuits were assessed by extracellular recordings. I demonstrated that electrostimulation protocols able to activate the spinal CPG for locomotion, induced primary afferent hyperexcitability, as well. Thus, evidence of incoming signals in modulating spinal circuits was provided. Furthermore, a robust sensorimotor interplay was reported to take place within the spinal cord. I further investigated hyperexcitability conditions in a new in vivo model of peripheral neuropathic pain. Adult rats underwent a surgical procedure where the common peroneal nerve was crushed using a calibrated nerve clamp (modified spared nerve injury, mSNI). Thus, primary afferents of the common peroneal nerve were activated through the application of a noxious compression, which presumably elicited ectopic activity constitutively generated in the periphery. One week after surgery, animals were classified into two groups, with (mSNI+) and without (mSNI-) tactile hypersensitivity, based on behavioral tests assessing paw withdrawal threshold. Interestingly, the efficiency of the mSNI in inducing tactile hypersensitivity was halved with respect to the classical SNI model. Moreover, mSNI animals with tactile hypersensitivity (mSNI+) showed an extensive neuroinflammation within the dorsal horn, with activated microglia and astrocytes being significantly increased with respect to mSNI animals without tactile hypersensitivity (mSNI-) and to sham-operated animals. Lastly, RGS4 (regulator of G protein signaling 4) was reported to be enhanced in lumbar dorsal root ganglia (DRGs) and dorsal horn ipsilaterally to the lesion in mSNI+ animals. Thus, a new molecular marker was demonstrated to be involved in tactile hypersensitivity in our preclinical model of mSNI. Lastly, we developed a novel in vitro model of newborn rat, where hindlimbs were functionally connected to a partially dissected spinal cord and passively-driven by a robotic device (Bipedal Induced Kinetic Exercise, BIKE). I aimed at studying whether spinal activity was influenced by afferent signals evoked during passive cycling. I first demonstrated that BIKE could actually evoke an afferent feedback from the periphery. Then, I determined that spinal circuitries were differentially affected by training sessions of different duration. On one side, a short exercise session could not directly activate the locomotor CPG, but was able to transiently facilitate an electrically-induced locomotor-like activity. Moreover, no changes in reflex or spontaneous activity of dorsal and ventral networks were promoted by a short training. On the other side, a long BIKE session caused a loss in facilitation of spinal locomotor networks and a depression in the area of motor reflexes. Furthermore, activity in dorsal circuits was long-term enhanced, with a significant increase in both electrically-evoked and spontaneous antidromic discharges. Thus, the persistence of training-mediated effects was different, with spinal locomotor circuits being only transiently modulated, whereas dorsal activity being strongly and stably enhanced. Motoneurons were also affected by a prolonged training, showing a reduction in membrane resistance and an increase in the frequency of post-synaptic currents (PSCs), with both fast- and slow-decaying synaptic inputs being augmented. Changes in synaptic transmission onto the motoneuron were suggested to be responsible for network effects mediated by passive training. In conclusion, I demonstrated that afferent information might induce changes within the spinal cord, involving both neuronal and glial cells. In particular, spinal networks are affected by incoming peripheral signals, which mediate synaptic, cellular and molecular modifications. Moreover, a strong interplay between dorsal and ventral spinal circuits was also reported. A full comprehension of basic mechanisms underlying sensory-mediated spinal plasticity and bidirectional interactions between functionally different spinal networks might lead to the development of neurorehabilitation strategies which simultaneously promote locomotor recovery and pain relief

Studies on the pharmacology of locomotion in adult chronic spinal cat

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal

Lbx1-expressing cells lacking the repellent EphA4 receptor are involved in axonal midline crossing in the spinal cord and evoke a minor gait defect

Most limbed animals, including mice and human beings, show alternating hindlimb movement mediated by neuronal circuits in the spinal cord. However, when the repulsive EphA4 receptor expressed by subsets of spinal neurons is mutated, hindlimbs lose their typical left-right alternating pattern and as a consequence, mice exhibit a hopping gait. EphA4 tyrosine kinase receptor binds to its ligand ephrinB3 at the midline of the spinal cord resulting in axonal growth cone collapse. Therefore, in wild type mice, EphA4-expressing axons are prevented from crossing the midline towards the contralateral side. In full EphA4-/- mutants, it has been suggested that the hopping gait is caused by an increased number of axons derived from excitatory neurons crossing the spinal midline (Kullander 2003, Restrepo 2011). However, it remained unclear, which subpopulations of spinal interneurons are misguided towards the contralateral side and are involved in the observed hopping gait phenotype. Hence, we aim to determine the cellular origin contributing to axon misguidance and hopping gait in EphA4-/- mutant mice, by influencing the balance between excitation and inhibition across the spinal midline. Among 11 main neuron populations in the spinal cord, the interneurons derived from the progenitor domain territory dorsal dI4-6 and marked by the transcription factor Lbx1, were targeted in this study. Here, we investigated the premotor interneuron distribution of motor neurons targeting specific hindlimb muscles in EphA4 mutant mice by means of monosynaptic rabies tracing technique. We also assessed the gait behavior on a treadmill in the conditional EphA4 mutant mice, whose EphA4 receptor was deleted in Lbx1-expressing neurons. We found that a deletion of EphA4 in Lbx1-positive neurons resulted in aberrant axon guidance of dorsal neurons across the spinal midline and minor gait defects such as a hopping gait at low velocities on the treadmill and a reduced swing time during alternating gait. Moreover, 3-week old conditional EphA4 mutants perfomed a slight aberrant hopping gait at higher velocities compared to adults. Therefore, Lbx1-expressing interneurons appear to be partially responsible for the phenotypes observed in full EphA4 mutant mice. In conclusion, we show that the EphA4 receptor plays an important role in preventing axons of Lbx1-expressing interneurons from crossing the spinal midline. Further, EphA4-expression in Lbx1-positive neurons is essential to conserve a complete alternating gait. Lbx1-expressing neurons might be one component of several cell types contributing to the locomotor CPG. Moreover, we also found that deletion of the EphA4 receptor in all inhibitory neurons of conditional EphA4flox/-vGATCre/+ mutant mice caused a partial hopping gait demonstrating that proper axon guidance of inhibitory neurons beside excitatory neurons is important to maintain alternating gait. Finally, although alpha2 chimaerin was shown to be an EphA4 downstream effector and full alpha2 chimaerin mutant mice exhibited a hopping gait (Beg 2007; Wegmeyer 2007), we found no anatomical and gait behavioral defects in the conditional alpha2 chimaerin mutant mice, lacking alpha2 chimaerin in Lbx1-positive cells. In addition, full alpha2 chimaerin-/- mutants displayed significantly decreased synchronous hindlimb movement compared to the full EphA4-/- mutant. These findings suggests that deletion of a single EphA4 effector has less effect on the anatomical and gait behavioral phenotypes than it was observed for the EphA4 receptor itself

The role of inter-enlargement propriospinal neurons in locomotion following spinal cord injury.

The focus of this dissertation is to explore the functional role of two anatomically-defined pathways in the adult rat spinal cord before and after spinal cord injury (SCI). To do this, a TetOn dual virus system was used to selectively and reversibly silence neurons with cell bodies at spinal segment L2 and projections to spinal segment C6 (long ascending propriospinal neurons, LAPNs) and neurons that originate in the C6 spinal segment and terminate at L2 spinal segment (long descending propriospinal neurons, LDPNs). This dissertation is divided into five chapters. Chapter One provides background information regarding spinal cord injury, locomotion, and a brief introduction to propriospinal neurons. Chapter Two details the functional consequences of silencing LAPNs and LDPNs in uninjured animals, with specific regard to sensory context during overground locomotion. Chapter Three describes the consequences of silencing LAPNs following a mild/moderate spinal cord contusion injury. Spinal cord injury (SCI) fundamentally affects the ability to maintain patterned weight-supported stepping. Chapter Four focuses on the functional outcomes of silencing the reciprocal descending inter-enlargement pathway, LDPNs, after mild/moderate spinal cord contusion injury. Finally, Chapter Five compares the differential roles of LAPNs and LDPNs in left-right coordination prior to injury, especially in a sensory context-dependent manner. A section of this chapter is devoted to a recap of injured data for both LAPN and LDPN silencing post-injury and attempts to place this work in context with other studies whose focus is on propriospinal pathways after SCI

Functional organization of cutaneous reflex pathways during locomotion and reorganization following peripheral nerve and/or spinal cord lesions

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal