Locomotion is one of the most important behaviours and requires interaction between sensors at various levels of the nervous system and the limb muscles of an animal. The basic neural rhythm for locomotion in mammals has been shown to arise from local neural networks residing in the spinal cord and these networks are known as central pattern generators (CPGs). However, during the locomotion, these centres are constantly interacting with the sensory feedback signals coming from muscles, joints and peripheral skin receptors in order to adapt the stepping to varying environmental conditions. Conceptual models of mammalian locomotion have been constructed using mathematical models of locomotor subsystems based on the abundance of neurophysiological evidence obtained primarily in the cat. Several aspects of locomotor control using the cat as an animal model have been investigated employing computer simulations and here we use the same approach to address number of questions or/and hypotheses related to rhythmic locomotion in quadrupeds. Some of the involve questions are, role of mechanical linkage during deafferented walking, finding inherent stabilities/instabilities of muscle-joint interactions during normal walking, estimating phase dependent controlability of muscle action over joints. This thesis presents the basics of a biologically realistic model of mammalian locomotion and summarises methodological approaches in modelling quadruped locomotor subsystems such as CPGs, limb muscles and sensory pathways. In the first appended article, we extensively discuss the construction details of the three-dimensional computer simulator for the study of the hind leg neuro-musculo-skeletal-control system and its interactions during normal walking of the cat. The simulator with the walking model is programmed in Python scripting language with other supported open source libraries such as Open Dynamics Engine (ODE) for simulating body dynamics and OpenGL for three dimensional graphical representation. We have examined the functionality of the simulator and the walking model by simulating deafferented walking. It was possible to obtain a realistic stepping in the hind legs even without sensory feedback to the two controllers (CPGs) for each leg. We conclude that the mechanical linkages between the legs also play a major role in producing alternating gait. The use of simulations of walking in the cat for gaining insights into more complex interactions between the environment and the neuro-muscular-skeletal system is important especially for questions where a direct neurophysiological experiment can not be performed on a real walking animal. For instance, it is experimentally hard to isolate individual mechanisms without disrupting the natural walking pattern. In the second article, we introduce a different approach where we use the walking model to identify what control is necessary to maintain stability in the musculo-skeletal system. We show that the actions of most of the hindlimb muscles over the joints have an inherent stability during stepping, even without the involvement of proprioceptive feedback mechanisms. In addition, we observe that muscles generating movements in the ankle joint of the hind leg must be controlled by neural mechanisms, which may involve supraspinal structures, over the whole step cycle. / QC 20101111

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La communication entre les neurones est fondée sur leur capacité à changer leur patron de décharge pour l’encodage de différents messages. Pour plusieurs fonctions vitales, comme la respiration et la mastication, les neurones doivent pouvoir générer des patrons d’activité répétitifs, et les groupes de neurones responsables de ces décharges rythmiques sont des générateurs de patron central (GPC). En dépit de recherches soutenues, les mécanismes précis qui sous-tendent la rythmogénèse dans les GPCs ne sont pas bien définis. Le plus souvent, la potentielle contribution des astrocytes demeure grandement inexplorée, même si ces cellules sont aujourd’hui connues pour leur implication dans la modulation synaptique neuronale. Pour nos travaux, le noyau sensoriel principal du trijumeau (NVsnpr) a été pris comme modèle à cause de son rôle central dans les mouvements rythmiques de la mastication. Dans ce noyau, des travaux antérieurs ont montré que la décharge en bouffées rythmiques est déclenchée dans les neurones lorsque la concentration de calcium extracellulaire ([Ca2+]e) est artificiellement baissée. Nous fondant sur cette observation, notre première hypothèse a postulé que la baisse de la [Ca2+]e pouvait survenir de façon physiologique en lien avec des stimulations sensorielles pertinentes. Deuxièmement, parce que les astrocytes ont été impliqués dans le tamponnage et l’homéostasie d’ions extracellulaires comme le K+, nous avons postulé que ces cellules pouvaient jouer un rôle équivalent dans le contrôle de la [Ca2+]e. Nos résultats montrent que les astrocytes peuvent réguler la [Ca2+]e et ainsi contrôler la capacité des neurones à changer leur patron de décharge. Premièrement, en stimulant les afférences sensorielles au NVsnpr, nous avons montré que des baisses physiologiques de la [Ca2+]e sont observées en parallèle à l’apparition de bouffées rythmiques neuronales. Deuxièmement, nous avons démontré que les astrocytes répondent aux mêmes stimuli qui induisent l’activité rythmique neuronale, et que leur blocage avec un chélateur de Ca2+ empêche les neurones de générer un patron de décharge en bouffées rythmiques. Cette habilité est rétablie en rajoutant la S100β, une protéine astrocytaire liant le Ca2+, dans le milieu extracellulaire, alors que l’anticorps anti-S100β empêche l’activité rythmique. Ces résultats indiquent que les astrocytes régulent une propriété neuronale fondamentale : la capacité à changer de patron de décharge. Ainsi, les GPCs dépendraient des fonctions intégrées des astrocytes et des neurones. Ces découvertes pourraient avoir des implications transposables à plusieurs autres circuits neuronaux dont la fonction dépend de l’induction d’activité rythmique. / Communication between neurons rests on their capacity to change their firing pattern to encode different messages. For several vital functions, such as respiration and mastication, neurons need to generate a repetitive firing pattern, and the groups of neurons responsible for these rhythmic discharges are called central pattern generator (CPG). Despite intense research in this field, the exact mechanisms underlying rhythmogenesis in CPGs are not completely defined. In most instances, the potential contribution of astrocytes is largely unexplored, even though these cells are now well known to be involved in neuronal synaptic modulation. In our work, the trigeminal main sensory nucleus (NVsnpr) was used as a model owing to its central role in the rhythmic movement of mastication. Previous work have shown that rhythmic bursting discharge is triggered in NVsnpr neurons when extracellular calcium concentration ([Ca2+]e) is artificially decreased. Based on this observation, our first hypothesis postulated that the reduction of [Ca2+]e could also happen physiologically in relation to relevant sensory stimulation. Secondly, because astrocytes have been involved in the buffering and the homeostasis of extracellular ions like potassium, we have postulated that these cells could also play a role in the control of [Ca2+]e. The results presented in this thesis show that astrocytes can regulate [Ca2+]e and thus control the ability of neurons to change their firing pattern. First, we showed that stimulation of sensory afferent fibers to the NVsnpr induced neuronal rhythmic bursting and in parallel reduction of [Ca2+]e . Secondly, we have demonstrated that astrocytes respond to the same sensory stimuli that induce neuronal rhythmic activity, and their blockade with a Ca2+ chelator prevents generation of neuronal rhythmic bursting. This ability is restored by adding S100β, an astrocytic Ca2+-binding protein, to the extracellular space, while the application of an anti- S100β antibody prevents generation of rhythmic activity. These results indicate that astrocytes regulate a fundamental neuronal property: that is the capacity to change their firing pattern. Thus, CPG functions result from integrated neuronal and glial activities. These findings may have broad implications for many other neural networks whose functions depend on the generation of rhythmic activity.

Muscarinic acetylcholine receptors (mAChRs) mediate effects of acetylcholine (ACh) in many systems, including those involved in locomotion. In the stage 37/38 Xenopus laevis tadpole, a well-understood model system of vertebrate locomotion, mAChRs have been found to be located on motor neurons with evidence suggesting that mAChRs are involved in swimming behaviour. The current study aimed to further investigate the role of mAChR-mediated cholinergic transmission by employing extracellular and whole-cell patch clamp recordings to examine the effects of mAChR activation on the properties of different types of neurons in the Xenopus laevis tadpole swimming circuit. It was found that mAChR activation can increase the threshold for initiating swimming by skin stimulation and can lead to the generation of spontaneous motor output in the absence of physical stimuli. These effects were found to be a result of direct inhibition of dorsolateral sensory interneurons of the mechanosensory pathway, direct inhibition of glycinergic inhibitory interneurons in the CPG and a decrease in CPG neuron firing reliability during swimming. The data presented here comprise the first whole-cell patch-clamp investigation into mAChR-mediated cholinergic transmission in the Xenopus laevis tadpole swimming circuit and provide novel evidence that mAChRs modulate the properties of mechanosensory pathway and CPG neurons in this model system of vertebrate locomotion.

A deeper understanding of both movement control and the effects of nociceptor inputs on our motor systems is critical for proper clinical diagnosis of musculo-skeletal dysfunctions and for development of novel rehabilitation schemes. In the jaw system, masticatory movements are produced by a central pattern generator (CPG) located in the brainstem. Considerable efforts have been made in deciphering this neuronal network. The present thesis contributes towards an increasingly detailed understanding of its essential elements, and presents a hypothesis of how deep somatic pain (i.e. muscle pain) may be evoked and interferes with the masticatory CPG circuitry. In Paper I, the expression of c-Fos-like protein was used as a molecular marker to visualize brainstem neurons that were active during induced fictive mastication in the anesthetized and paralyzed rabbit. Our findings provide a previously lacking detailed record of the neuronal populations that form the masticatory motor pattern. Certain cells were located in brainstem areas previously suggested to be involved in the masticatory CPG. However, it was a new finding that neurons in the dorsal part of the trigeminal main sensory nucleus (NVsnpr-d) may belong to this circuitry. Paper II focused on the discovered neurons in NVsnpr in an in vitro slice preparation from young rats.  Intracellular recordings allowed us to define two cell types based on their response to depolarizing current. Microstimulation applied to the trigeminal motor nucleus, its reticular border, the parvocellular reticular formation and the nucleus reticularis pontis caudalis, elicited postsynaptic potentials in 81% of the neurons tested. Responses obtained were predominately excitatory and sensitive to gluta-matergic antagonists DNQX or/and APV. Some inhibitory and biphasic responses were also evoked. Bicuculline methiodide or strychnine blocked the IPSPs indicating that they were mediated by GABAA or glycinergic receptors. About one third of the stimulations activated both types of neurons antidromically. Neurons in NVsnpr-d seem to gather all the conditions that can theoretically account for a role in masticatory rhythm generation. In Paper III, the masticatory model system was used to investigate the possible role of muscle spindle primary afferents in development of persistent musculoskeletal pain. Following intramuscular acidic (pH 4.0) saline injections of rat masseter muscles, in vitro whole cell recordings were done from jaw closing muscle spindle somata located in the trigeminal mesencephalic nucleus (NVmes). Compared to control neurons, the somata of afferents exposed to acid had more hyperpolarized membrane potentials, more hyperpolarized thresholds for firing, high frequency membrane oscillations and ectopic bursting of action potentials. These changes in membrane properties lasted for up to 35 days. Within the same time frame experi-mental animals showed hypersensitivity to touch on the skin covering the injected muscle. Similar saline injections also resulted in a significant increase of activity dependent c-Fos expression in NVmes neurons compared to controls. Immuno-fluorescence and lectin binding studies indicated that small-caliber muscle afferents containing known nociceptor markers (CGRP, SP, P2X3, TRPV1 and IB4) and expressing glutamate receptors are found close to the annulo-spiral endings of the NVmes afferents. Combined, our new observations support the hypothesis that excessive release of glutamate, within muscle spindles due to ectopically evoked antidromic action potentials, could lead to development of persistent musculoskeletal pain by activation and/ or sensitization of adjacent muscle afferent nociceptors.

These studies compare neurotransmitter localization and the behavioral functions of homologous neurons in nudibranch molluscs to determine the types of changes that might underlie the evolution of species-specific behaviors. Serotonin (5-HT) immunohistochemistry in eleven nudibranch species indicated that certain groups of 5 HT-immunoreactive neurons, such as the Cerebral Serotonergic Posterior (CeSP) cluster, are present in all species. However, the locations and numbers of many other 5 HT-immunoreactive neurons were variable. Thus, particular parts of the serotonergic system have changed during the evolution of nudibranchs. To test whether the functions of homologous neurons are phylogenetically variable, comparisons were made in species with divergent behaviors. In Tritonia diomedea, which crawls and also swims via dorsal-ventral body flexions, the CeSP cluster includes the Dorsal Swim Interneurons (DSIs). It was previously shown that the DSIs are members of the swim central pattern generator (CPG); they are rhythmically active during swimming and, along with their neurotransmitter 5-HT, are necessary and sufficient for swimming. It was also known that the DSIs excite efferent neurons used in crawling. DSI homologues, the CeSP-A neurons, were identified in six species that do not exhibit dorsal-ventral swimming. Many physiological characteristics, including excitation of putative crawling neurons were conserved, but the CeSP A neurons were not rhythmically active in any of the six species. In the lateral flexion swimmer, Melibe leonina, the CeSP-A neurons and 5-HT, were sufficient, but not necessary, for swimming. Thus, homologous neurons, and their neurotransmitter, have functionally diverged in species with different behaviors. Homologous neurons in species with similar behaviors also exhibited functional divergence. Like Melibe, Dendronotus iris is a lateral flexion swimmer. Swim interneuron 1 (Si1) is in the Melibe swim CPG. However, its putative homologue in Dendronotus, the Cerebral Posterior ipsilateral Pedal (CPiP) neuron, was not rhythmically active during swim-like motor patterns, but could initiate such a motor pattern. Together, these studies suggest that neurons have changed their functional relationships to neural circuits during the evolution of species-specific behaviors and have functionally diverged even in species that exhibit similar behaviors.

Neuroprosthetics is at the intersection of neuroscience, biomedical engineering, and physics. A biocompatible neuroprosthesis contains artificial neurons exhibiting biophysically plausible dynamics. Hybrid systems analysis could be used to prototype such artificial neurons. Biohybrid systems are composed of artificial and living neurons coupled via real-time computing and dynamic clamp. Model neurons must be thoroughly tested before coupled with a living cell. We use bifurcation theory to identify hazardous regimes of activity that may compromise biocompatibility and to identify control strategies for regimes of activity desirable for functional behavior. We construct real-time artificial neurons for the analysis of hybrid systems and demonstrate a mechanism through which an artificial neuron could maintain duty cycle independent of variations in period.

Neuronal networks produce reliable functional output throughout the lifespan of an animal despite ceaseless molecular turnover and a constantly changing environment. The cellular and molecular mechanisms underlying the ability of these networks to maintain functional stability remain poorly understood. Central pattern generating circuits produce a stable, predictable rhythm, making them ideal candidates for studying mechanisms of activity maintenance. By identifying and characterizing the regulators of activity in small neuronal circuits, we not only obtain a clearer understanding of how neural activity is generated, but also arm ourselves with knowledge that may eventually be used to improve medical care for patients whose normal nervous system activity has been disrupted through trauma or disease. We utilize the pattern-generating pyloric circuit in the crustacean stomatogastric nervous system to investigate the general scientific question: How are specific aspects of rhythmic activity regulated in a small neuronal network? The first aim of this thesis poses this question in the context of a single neuron. We used a single-compartment model neuron database to investigate whether co-regulation of ionic conductances supports the maintenance of spike phase in rhythmically bursting “pacemaker” neurons. The second aim of the project extends the question to a network context. Through a combination of computational and electrophysiology studies, we investigated how the intrinsic membrane conductances of the pacemaker neuron influence its response to synaptic input within the framework of the Phase Resetting Curve (PRC). The third aim of the project further extends the question to a systems-level context. We examined how ambient temperatures affect the stability of the pyloric rhythm in the intact, behaving animal. The results of this work have furthered our understanding of the principles underlying the long-term stability of neuronal network function.

Le mouvement masticatoire est généré et coordonné par un générateur de patron central (GPC) situé au niveau du pont. Plusieurs résultats antérieurs de notre laboratoire soutiennent que le réseau de neurones à l’origine de la rythmogénèse est situé dans le noyau sensoriel principal du nerf trijumeau (NVsnpr). Ces mêmes expériences révèlent que des diminutions de la concentration calcique extracellulaire ([Ca2+]e) tiennent une place importante dans la génération des bouffées de décharges des neurones de cette région. Notre laboratoire tente de vérifier si la contribution des astrocytes à l’homéostasie de la concentration calcique extracellulaire est impliquée dans la genèse du rythme. Cette étude a pour but la caractérisation spatiale du syncytium astrocytaire au sein du NVsnpr dorsal et l’étude de l’effet de la [Ca2+]e sur les propriétés astrocytaires électrophysiologiques et de connectivité. Nous avons utilisés pour ce faire la technique d’enregistrement par patch-clamp sur une préparation en tranche de tronc cérébral de rat. Nous démontrons ici que la diminution de la [Ca2+]e n’affecte pas les propriétés électrophysiologiques astrocytaires, mais induit une augmentation de la taille du syncytium. De plus, nous établissons l’existence au sein du NVsnpr dorsal d’une organisation anatomofonctionnelle du réseau astrocytaire calquée sur l’organisation neuronale. / The masticatory movement is generated and coordinated by a central pattern generator (CPG) located in the pons. Previous results from our laboratory suggest that the neural network responsible for its rythmogenesis is located in the trigeminal main sensory nucleus (NVsnpr). Moreover, results indicate that in this region, decrease in extracellular calcium concentration ([Ca2+]e) plays an important role in genarating burst. One of our laboratory's goal is to assess if the contribution of astrocytes to the extracellular calcium concentration homeostasis is involved in the genesis of the mastication rhythm. With this study, we characterized the astrocyte syncytium within the NVsnpr and measured the effect of [Ca2+]e on the astrocytes electrophysiology and their networks. A patch-clamp recording technique in conjunction with a rat brain stem slice preparation was used. We demonstrate that a decrease in [Ca2+]e does not affect the electrophysiological properties of astrocytes but induces an increase in the size of the syncytium. We also report the existence, within the dorsal NVsnpr, of an anatomofunctional organization between neurons and astrocytes.

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