In the spinal cord, an intricate neural network generates and coordinates the patterning of limb movements during locomotion. This network, known as the locomotor central pattern generator (CPG), comprises of various cell populations that together orchestrate the output of motor neurons. Identification of CPG neurons through their specific gene expression is a valuable tool that can provide considerable insight to the character, intrinsic properties and role of a population, which represents a step toward understanding locomotor circuit function and correlating neural activity to behaviour. We selectively targeted two inhibitory CPG populations to investigate their molecular characteristics, circuitry and functional role; Renshaw cells (RCs) marked by their specific expression of the cholinergic nicotinic receptor α2 (Chrna2) and a subset of the dI6 population derived by their selective expression of the Doublesex and mab-3 related transcription factor 3 (Dmrt3). We found that RCs have hyperpolarisation-activated cation (Ih) and small calcium-activated potassium (ISK) modulatory currents that differentially regulate their excitation and firing properties, which influence the instantaneous feedback to motor neurons through the recurrent inhibition circuit. Due to previous difficulties isolating RCs from the surrounding locomotor circuits, their functional role remains poorly defined. For the first time, we selectively silenced RC inhibition and found that all aspects of motor behaviour, including coordination and gait were normal. The deletion of RC signalling instead altered the electrical and synaptic properties of the recurrent inhibitory circuit, suggesting that developmental plasticity compensates for the loss of RC inhibition. We reveal Dmrt3 neurons comprise a population of glycinergic inhibitory, spike-frequency adapting commissural interneurons active during locomotion. Conditional silencing of the Dmrt3 population resulted in considerable gait abnormalities in the neonatal and adult mouse. This manifested as an uncoordinated CPG output in vitro, impaired limb coordination in pups and increased fore- and hindlimb synchrony in adults that was exacerbated at faster locomotor speeds. Dmrt3 mediated inhibition subsequently impacts locomotion and suggests the Dmrt3 population contribute to coordinating speed dependent left-right limb alternation. This thesis provides cellular, circuit and behavioural insights into the Renshaw cell and Dmrt3 populations and enhances our knowledge regarding their probable function within the locomotor CPG.

Locomotion is an innate behaviour that is controlled by different areas of the central nervous system, which allow for effectiveness of movement. The spinal cord is an important centre involved in the generation and maintenance of rhythmic patterns of locomotor activity such as walking and running. Interneurons throughout the ventral horn of the spinal cord form the locomotor central pattern generator (CPG) circuit, which produces rhythmic activity responsible for hindlimb movement. Motoneurons within the lumbar region of the spinal cord innervate the leg muscles to convey rhythmic CPG output to drive appropriate muscle contractions. Intrinsic modulators, such as acetylcholine acting via M2 and M3 muscarinic receptors, regulate CPG circuitry to allow for flexibility of motor output. Using electrophysiology and genetic techniques, this work characterized the receptors involved in cholinergic modulation of locomotor networks and the role and mechanism of action of a subpopulation of genetically identified cholinergic interneurons in the lumbar region of the neonatal mouse spinal cord. Firstly, the effects of M2 and M3 muscarinic receptors on the output of the lumbar locomotor network were characterised. Experiments in which fictive locomotor output was recorded from the ventral roots of isolated spinal cord preparations revealed that M3 muscarinic receptors are important in stabilizing the locomotor rhythm while M2 muscarinic receptor activation seems to increase the irregularity of the locomotor frequency whilst increasing the strength of the motor output. This work then explored the cellular mechanisms through which M2 and M3 muscarinic receptors modulate motoneuron output. M2 and M3 receptor activation exhibited contrasting effects on motoneuron function suggesting that there is a fine balance between the activation of these two receptor subtypes. M2 receptor activation induces an outward current and decreases synaptic drive to motoneurons while M3 receptors are responsible for an inward current and increase in synaptic inputs to motoneurons. Despite the different effects of M2 and M3 receptor activation on synaptic drive and subthreshold properties of MNs, both M2 and M3 receptors are required for muscarine-induced increase in motoneuron output. CPG networks therefore appear to be subject to balanced cholinergic modulation mediated by M2 and M3 receptors, with the M2 subtype also being important for regulating the intensity of motor output. Next, using Designer Receptor Exclusively Activated by Designer Drug (DREADD) technology, the impact of the activation or inhibition of a genetically identified group of cholinergic spinal interneurons that express the Paired-like homeodomain 2 (Pitx2) transcription factor was explored. Stimulation of these interneurons increased motoneuron output through the activation of M2 muscarinic receptors and subsequent modulation of Kv2.1 channels. Inhibition of Pitx2+ interneurons during fictive locomotion decreased the amplitude of locomotor bursting. Genetic ablation of these cells confirmed that Pitx2+ interneurons increase the strength of locomotor output by activating M2 muscarinic receptors. Overall, this work provides new insights into the receptors and mechanisms involved in intraspinal cholinergic modulation. Furthermore, this study provides direct evidence of the mechanism through which Pitx2+ interneurons regulate motor output. This work is not only important for advancing understanding of locomotor networks that control hindlimb locomotion, but also for dysfunction and diseases where the cholinergic system is impaired such as Spinal Cord Injury and Amyotrophic Lateral Sclerosis.

Neural circuit flexibility is fundamental to the production of adaptable behaviors. Invertebrate models offer relatively simple networks consisting of large, identifiable neurons that are useful for investigating the electrophysiological properties that contribute to circuit output. In particular, central pattern generating circuits within the crustacean stomatogastric nervous system have been well characterized with regard to their synaptic connectivities, cellular properties, and response to modulatory influences. Monoaminergic modulation is essential for the production of adaptable circuit output in most species. Monoamines, such as dopamine and serotonin, signal via metabotropic receptors, which activate intracellular signaling cascades. Many of the neuronal and network targets of monoaminergic modulation in the crustacean stomatogastric nervous system are known, but nothing is known of the signal transduction cascades that mediate the biophysical response. This work represents a thorough characterization of monoaminergic receptors in the crustacean stomatogastric nervous system. We took advantage of the close phylogenetic relationship between crustaceans and insects to clone monoaminergic receptors from the spiny lobster. Using a novel database mining strategy, we were able to identify several uncharacterized monoaminergic receptors in the Panulirus interruptus genome. We cloned one serotonin (5-HT2βPan) and three dopamine receptors (D1αPan, D1βPan, and D2αPan), and characterized them with regard to G protein coupling and signal transduction cascades. We used a heterologous expression system to show that G protein couplings and signaling properties of monoaminergic receptors are strongly conserved among vertebrate and invertebrate species. This work further shows that DAR-G protein couplings in the stomatogastric nervous system are unique for a given receptor subtype, and receptors can couple to multiple signaling pathways, similar to their mammalian homologs. Custom made antibodies were used to localize monoamine receptors in the stomatogastric ganglion, and in identified neurons. Pyloric neurons show unique receptor expression profiles, which supports the idea of receptor expression as an underlying mechanism for cell-type specific effects of a given modulator. Receptors are localized to the synaptic neuropil, but are not expressed in the membrane of large diameter processes or the soma. The localization of dopamine receptors in identified pyloric neurons suggests that they may respond to synaptic, paracrine or neurohormonal dopamine signals. This work also supports the idea that different types of signals can be generated by a single receptor.

This dissertation creates practical methods for Poincaré return mappings of individual and networked neuron models. Elliptic bursting models are found in numerous biological systems, including the external Globus Pallidus (GPe) section of the brain; the focus for studies of epileptic seizures and Parkinson's disease. However, the bifurcation structure for changes in dynamics remains incomplete. This dissertation develops computer-assisted Poincaré ́maps for mathematical and biologically relevant elliptic bursting neuron models and central pattern generators (CPGs). The first method, used for individual neurons, offers the advantage of an entire family of computationally smooth and complete mappings, which can explain all of the systems dynamical transitions. A complete bifurcation analysis was performed detailing the mechanisms for the transitions from tonic spiking to quiescence in elliptic bursters. A previously unknown, unstable torus bifurcation was found to give rise to small amplitude oscillations. The focus of the dissertation shifts from individual neuron models to small networks of neuron models, particularly 3-cell CPGs. A CPG is a small network which is able to produce specific phasic relationships between the cells. The output rhythms represent a number of biologically observable actions, i.e. walking or running gates. A 2-dimensional map is derived from the CPGs phase-lags. The cells are endogenously bursting neuron models mutually coupled with reciprocal inhibitory connections using the fast threshold synaptic paradigm. The mappings generate clear explanations for rhythmic outcomes, as well as basins of attraction for specific rhythms and possible mechanisms for switching between rhythms.

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(工学) / 甲第18946号 / 工博第3988号 / 新制||工||1614 / 31897 / 京都大学大学院工学研究科航空宇宙工学専攻 / (主査)教授 泉田 啓, 教授 藤本 健治, 教授 松野 文俊 / 学位規則第4条第1項該当

A multifunctional central pattern generator (CPG) can produce bursting polyrhythms that determine locomotive activity in an animal: for example, swimming and crawling in a leech. Each rhythm corresponds to a specific attractor of the CPG. We employ a Hodgkin-Huxley type model of a bursting leech heart interneuron, and connect three such neurons by fast inhibitory synapses to form a ring. This network motif exhibits multistable co-existing bursting rhythms. The problem of determining rhythmic outcomes is reduced to an analysis of fixed points of Poincare mappings and their attractor basins, in a phase plane defined by the interneurons' phase differences along bursting orbits. Using computer assisted analysis, we examine stability, bifurcations of attractors, and transformations of their basins in the phase plane. These structures determine the global bursting rhythms emitted by the CPG. By varying the coupling synaptic strength, we examine the dynamics and patterns produced by inhibitory networks.

Nerve cells are organized into complex networks that comprise the building blocks of our nervous system. Neurons communicate by transmitting messenger molecules released from synaptic vesicles. Alterations in neuronal circuitry and synaptic signaling contribute to a wide range of neurological conditions, often with consequences for movement. Intrinsic neuronal networks in the spinal cord serve to coordinate vital rhythmic motor functions. In spite of extensive efforts to address the organization of these neural circuits, much remains to be revealed regarding the identity and function of specific interneuron cell types and how neuromodulation tune network activity. In this thesis, two novel genes initially identified as markers for spinal neuronal populations were investigated: Slc10a4 and Dmrt3. The orphan transporter SLC10A4 was found to be expressed on synaptic vesicles of the cholinergic system, including motor neurons, as well as in the monoaminergic system, including dopaminergic, serotonergic and noradrenergic nuclei. Thus, it constitutes a novel molecular denominator shared by these classic neuromodulatory systems. SLC10A4 was found to influence vesicular transport of dopamine and affect neuronal release and reuptake efficiency in the striatum. Mice lacking Slc10a4 displayed impaired monoamine homeostasis and were hypersensitive to the drugs amphetamine and tranylcypromine. These findings demonstrate that SLC10A4 is capable of modulating the modulatory systems of the brain with potential clinical relevance for neurological and mental disorders. The transcription factor encoded by Dmrt3 was found to be expressed in a population of inhibitory commissural interneurons originating from the dorsal interneuron 6 (dI6) domain in the spinal cord. In parallel, a genome-wide association study revealed that a non-sense mutation in horse DMRT3 is permissive for the ability to perform pace among other alternate gaits. Further analysis of Dmrt3 null mutant mice showed that Dmrt3 has a central role for spinal neuronal network development with consequences for locomotor behavior. The dI6 class has been suggested to take part in motor circuits but remains one of the least studied classes due to lack of molecular markers. To further investigate the Dmrt3-derived neurons, and the dI6 population in general, a Dmrt3Cre mouse line was generated which allowed for characterization on the molecular, cellular and  behavioral level. It was found that Dmrt3 neurons synapse onto motor neurons, receive extensive synaptic inputs from various neuronal sources and are rhythmically active during fictive locomotion. Furthermore, silencing of Dmrt3 neurons in Dmrt3Cre;Viaatlx/lx mice led to impaired motor coordination and alterations in gait, together demonstrating the importance of this neuronal population in the control of movement.

Locomotion is an integral part of a whole range of animal behaviours. The basic rhythm for locomotion in vertebrates has been shown to arise from local networks residing in the spinal cord and these networks are known as central pattern generators (CPG). 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 or swimming to varying environmental conditions. Conceptual models of vertebrate locomotion have been constructed using mathematical models of locomotor subsystems based on the neurophysiological evidence obtained primarily in the cat and the salamander, an amphibian with a sprawling posture. Such models provide opportunity for studying the key elements in the transition from aquatic to terrestrial locomotion. Several aspects of locomotor control using the cat or the salamander as an animal model have been investigated employing computer simulations and here we use the same approach to address a number of questions or/and hypotheses related to rhythmic locomotion in quadrupeds. Some of the involved questions are, the role of mechanical linkage during deafferented walking, finding inherent stabilities/instabilities of muscle-joint interactions during normal walking and estimating phase dependent controlability of muscle action over joints. Also we investigate limb and body coordination for different gaits, use of side-stepping in front limbs for turning and the role of sensory feedback in gait generation and transitions in salamanders.      This thesis presents the basics of the biologically realistic models of cat and salamander locomotion and summarizes computational methods in modeling quadruped locomotor subsystems such as CPG, limb muscles and sensory pathways. In the case of cat hind limb, we conclude that the mechanical linkages between the legs play a major role in producing the alternating gait. In another experiment we use the model to identify open-loop linear transfer functions between muscle activations and joint angles while ongoing locomotion. We hypothesize that the musculo-skeletal system for locomotion in animals, at least in cats, operates under critically damped condition.      The 3D model of the salamander is successfully used to mimic locomotion on level ground and in water. We compare the walking gait with the trotting gait in simulations. We also found that for turning, the use of side-stepping alone or in combination with trunk bending is more effective than the use of trunk bending alone. The same model together with a more realistic CPG composed of spiking neurons was used to investigate the role of sensory feedback in gait generation and transition. We found that the proprioceptive sensory inputs are essential in obtaining the walking gait, whereas the trotting gait is more under central (CPG) influence compared to that of the peripheral or sensory feedback.      This thesis work sheds light on understanding the neural control mechanisms behind vertebrate locomotion. Additionally, both neuro-mechanical models can be used for further investigations in finding new control algorithms which give robust, adaptive, efficient and realistic stepping in each leg, which would be advantageous since it can be implemented on a controller of a quadruped-robotic device. / This work is Funded by Swedish International Development cooperation Agency (SIDA). QC 20111110

Thesis (Ph. D.)--Miami University, Dept. of Zoology, 2005. / Title from second page of PDF document. Includes bibliographical references (p. 100-108).

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Tecnologia, Departamento de Engenharia Elétrica, 2014. / Submitted by Ana Cristina Barbosa da Silva (annabds@hotmail.com) on 2014-11-25T17:23:31Z No. of bitstreams: 1 2014_RafaelCortesdePaiva.pdf: 7660330 bytes, checksum: eaad53db8e1c76edec638a3e30ee5f3e (MD5) / Approved for entry into archive by Raquel Viana(raquelviana@bce.unb.br) on 2014-11-25T17:58:53Z (GMT) No. of bitstreams: 1 2014_RafaelCortesdePaiva.pdf: 7660330 bytes, checksum: eaad53db8e1c76edec638a3e30ee5f3e (MD5) / Made available in DSpace on 2014-11-25T17:58:54Z (GMT). No. of bitstreams: 1 2014_RafaelCortesdePaiva.pdf: 7660330 bytes, checksum: eaad53db8e1c76edec638a3e30ee5f3e (MD5) / Nesse trabalho foi realizado o estudo de técnicas bio-inspiradas para gerar a marcha de um robô bípede. Foi utilizado o conceito de CPG, Central Pattern Generator (CPG), que é uma rede neural capaz de produzir respostas rítmicas. Elas foram modeladas como osciladores acoplados chamados de osciladores neurais. Para tanto foram utilizados alguns modelos de osciladores, o modelo de Matsuoka, o modelo de Kuramoto e o modelo de Kuramoto com acoplamento entre a dinâmica do oscilador e a dinâmica da marcha. Foram usados dois modelos de robôs, o Bioloid e o NAO. Para otimizar os parâmetros dos osciladores foram utilizados o Algoritmo Genético (AG), o Particle Swarm Optimization (PSO) e o Nondominated sorting Genetic Algorithm II (NSGA-II). Foi utilizada uma função de custo que através de determinadas condições tem como objetivo obter uma marcha eficiente. No NSGA-II, além dessa função de custo, foi utilizada outra função de custo que considera o trabalho realizado pelo robô. Além disso, também foi utilizada a aprendizagem por reforço para treinar um controlador que corrige a postura do robô durante a marcha. Foi possível propor um framework para obter os parâmetros dos osciladores e através dele obter uma marcha estável em ambas as plataformas. Também foi possível propor um framework utilizando aprendizagem por reforço para treinar um controlador para corrigir a postura do robô com a marcha sendo gerado pelo oscilador de Kuramoto com acoplamento. O objetivo do algoritmo foi minimizar a velocidade do ângulo de arfagem do corpo do robô, dessa forma, a variação do ângulo de arfagem também foi minimizada consequentemente. Além disso, o robô andou mais “cautelosamente” para poder manter a postura e dessa forma percorreu uma distância menor do que se estivesse sem o controlador. ______________________________________________________________________________ ABSTRACT / This document describes computational optimized bipedal robot gait generators. Thegaits are applied by a neural oscillator, composed of coupled central pattern generators(CPG), which are neural networks capable of producing rhythmic output. The models ofthe oscillators used were the Matsuoka model, Kuramoto model and Kura moto model withcoupling between the dynamics of the oscillator and dynamics of the gait. Two bipedalrobots, a NAO and a Bioloid, were used. The neural oscillators were optimized with threealgorithms, a Genetic Algorithm (GA), Particle Swarm Optimization (PSO) and Nondominatedsorting Genetic Algorithm II (NSGA-II). It was used a fitness function that has theobjective to obtain an efficient gait through some conditions. In NSGA-II, besides this fitnessfunction, another one was used that has the objective to minimize the work done by therobot. Additionally, reinforcement learning techniques were used to train a controller thatcorrects the robots gait posture. It was proposed a framework to obtain the parameters of theoscillators used and obtain efficient gaits in both robots. Also, it was proposed a frameworkusing reinforcement learning to train a controller to correct the robots gait posture. The objective of the algorithm was to minimize the pitch angular velocity, consequently the pitchangle standard deviation was minimized. Additionally, the robot moved with more “caution” and walked less compared with the walk without the posture controller.