Evolutionary robotics and neuroscience

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Development and plasticity of locomotor circuits in the zebrafish spinal cord

A fundamental goal in neurobiology is to understand the development and organization of neural circuits that drive behavior. In the embryonic spinal cord, the first motor activity is a slow coiling of the trunk that is sensory-independent and therefore appears to be centrally driven. Embryos later become responsive to sensory stimuli and eventually locomote, behaviors that are shaped by the integration of central patterns and sensory feedback. In this thesis I used a simple vertebrate model, the zebrafish, to investigate in three manners how developing spinal networks control these earliest locomotor behaviors. For the first part of this thesis, I characterized the rapid transition of the spinal cord from a purely electrical circuit to a hybrid network that relies on both chemical and electrical synapses. Using genetics, lesions and pharmacology we identified a transient embryonic behavior preceding swimming, termed double coiling. I used electrophysiology to reveal that spinal motoneurons had glutamate-dependent activity patterns that correlated with double coiling as did a population of descending ipsilateral glutamatergic interneurons that also innervated motoneurons at this time. This work (Knogler et al., Journal of Neuroscience, 2014) suggests that double coiling is a discrete step in the transition of the motor network from an electrically coupled circuit that can only produce simple coils to a spinal network driven by descending chemical neurotransmission that can generate more complex behaviors. In the second part of my thesis, I studied how spinal networks filter sensory information during self-generated movement. In the zebrafish embryo, mechanosensitive sensory neurons fire in response to light touch and excite downstream commissural glutamatergic interneurons to produce a flexion response, but spontaneous coiling does not trigger this reflex. I performed electrophysiological recordings to show that these interneurons received glycinergic inputs during spontaneous fictive coiling that prevented them from firing action potentials. Glycinergic inhibition specifically of these interneurons and not other spinal neurons was due to the expression of a unique glycine receptor subtype that enhanced the inhibitory current. This work (Knogler & Drapeau, Frontiers in Neural Circuits, 2014) suggests that glycinergic signaling onto sensory interneurons acts as a corollary discharge signal for reflex inhibition during movement. v In the final part of my thesis I describe work begun during my masters and completed during my doctoral degree studying how homeostatic plasticity is expressed in vivo at central synapses following chronic changes in network activity. I performed whole-cell recordings from spinal motoneurons to show that excitatory synaptic strength scaled up in response to decreased network activity, in accordance with previous in vitro studies. At the network level, I showed that homeostatic plasticity mechanisms were not necessary to maintain the timing of spinal circuits driving behavior, which appeared to be hardwired in the developing zebrafish. This study (Knogler et al., Journal of Neuroscience, 2010) provided for the first time important in vivo results showing that synaptic patterning is less plastic than synaptic strength during development in the intact animal. In conclusion, the findings presented in this thesis contribute widely to our understanding of the neural circuits underlying simple motor behaviors in the vertebrate spinal cord.Un objectif important en neurobiologie est de comprendre le développement et l'organisation des circuits neuronaux qui entrainent les comportements. Chez l'embryon, la première activité motrice est une lente contraction spontanée qui est entrainée par l'activité intrinsèque des circuits spinaux. Ensuite, les embryons deviennent sensibles aux stimulations sensorielles et ils peuvent éventuellement nager, comportements qui sont façonnées par l'intégration de l'activité intrinsèque et le rétrocontrôle sensoriel. Pour cette thèse, j'ai utilisé un modèle vertébré simple, le poisson zèbre, afin d'étudier en trois temps comment les réseaux spinaux se développent et contrôlent les comportements locomoteurs embryonnaires. Pour la première partie de cette thèse j'ai caractérisé la transition rapide de la moelle épinière d'un circuit entièrement électrique à un réseau hybride qui utilise à la fois des synapses chimiques et électriques. Nos expériences ont révélé un comportement embryonnaire transitoire qui précède la natation et qu'on appelle « double coiling ». J'ai démontré que les motoneurones spinaux présentaient une activité dépendante du glutamate corrélée avec le « double coiling » comme l'a fait une population d'interneurones glutamatergiques ipsilatéraux qui innervent les motoneurones à cet âge. Ce travail (Knogler et al., Journal of Neuroscience, 2014) suggère que le « double coiling » est une étape distincte dans la transition du réseau moteur à partir d'un circuit électrique très simple à un réseau spinal entrainé par la neurotransmission chimique pour générer des comportements plus complexes. Pour la seconde partie de ma thèse, j'ai étudié comment les réseaux spinaux filtrent l'information sensorielle de mouvements auto-générés. Chez l'embryon, les neurones sensoriels mécanosensibles sont activés par un léger toucher et ils excitent en aval des interneurones sensoriels pour produire une réponse de flexion. Par contre, les contractions spontanées ne déclenchent pas ce réflexe même si les neurones sensoriels sont toujours activés. J'ai démontré que les interneurones sensoriels reçoivent des entrées glycinergiques pendant les contractions spontanées fictives qui les empêchaient de générer des potentiels d'action. L'inhibition glycinergique de ces interneurones, mais pas des autres neurones spinaux, est due à l'expression d'un sous-type de récepteur glycinergique unique qui augmente iii le courant inhibiteur. Ce travail (Knogler & Drapeau, Frontiers in Neural Circuits, 2014) suggère que la signalisation glycinergique chez les interneurones sensoriels agit comme un signal de décharge corolaire pour l'inhibition des réflexes pendant les mouvements auto- générés. Dans la dernière partie de ma thèse, je décris le travail commencé à la maîtrise et terminé au doctorat qui montre comment la plasticité homéostatique est exprimée in vivo aux synapses centrales à la suite des changements chroniques de l'activité du réseau. J'ai démontré que l'efficacité synaptique excitatrice de neurones moteurs spinaux est augmentée à la suite d’une diminution de l'activité du réseau, en accord avec des études in vitro précédentes. Par contre, au niveau du réseau j'ai démontré que la plasticité homéostatique n'était pas nécessaire pour maintenir la rythmicité des circuits spinaux qui entrainent les comportements embryonnaires. Cette étude (Knogler et al., Journal of Neuroscience, 2010) a révélé pour la première fois que l'organisation du circuit est moins plastique que l'efficacité synaptique au cours du développement chez l'embryon. En conclusion, les résultats présentés dans cette thèse contribuent à notre compréhension des circuits neuronaux de la moelle épinière qui sous-tendent les comportements moteurs simples de l'embryon

Inverse modulation of motor neuron cellular and synaptic properties can maintain the same motor output

Although often examined in isolation, a single neuromodulator typically has multiple cellular and synaptic effects. Here, we have examined the interaction of the cellular and synaptic effects of 5-HT in the lamprey spinal cord. 5-HT reduces the amplitude of glutamatergic synaptic inputs and the slow post-spike afterhyperpolarization (sAHP) in motor neurons. We examined the interaction between these effects using ventral root activity evoked by stimulation of the spinal cord. While 5-HT reduced excitatory glutamatergic synaptic inputs in motor neurons to approximately 60% of control, ventral root activity was not significantly affected. The reduction of the sAHP by 5-HT increased motor neuron excitability by reducing spike frequency adaptation, an effect that could in principle have opposed the reduction of the excitatory synaptic input. Support for this was sought by reducing the amplitude of the sAHP by applying the toxin apamin before 5-HT application. In these experiments, 5-HT reduced the ventral root response, presumably because the reduction of the synaptic input now dominated. This was supported by computer simulations that showed that the motor output could be maintained over a wide range of synaptic input values if they were matched by changes in postsynaptic excitability. The effects of 5-HT on ventral root responses were altered by spinal cord lesions: 5-HT significantly increased ventral root responses in animals that recovered good locomotor function, consistent with a lesion-induced reduction in the synaptic effects of 5-HT, which thus biases its effects to the increase in motor neuron excitability.Non

Principles Governing Locomotion in Vertebrates: Lessons From Zebrafish

Locomotor behaviors are critical for survival and enable animals to navigate their environment, find food and evade predators. The circuits in the brain and spinal cord that initiate and maintain such different modes of locomotion in vertebrates have been studied in numerous species for over a century. In recent decades, the zebrafish has emerged as one of the main model systems for the study of locomotion, owing to its experimental amenability, and work in zebrafish has revealed numerous new insights into locomotor circuit function. Here, we review the literature that has led to our current understanding of the neural circuits controlling swimming and escape in zebrafish. We highlight recent studies that have enriched our comprehension of key topics, such as the interactions between premotor excitatory interneurons (INs) and motoneurons (MNs), supraspinal and spinal circuits that coordinate escape maneuvers, and developmental changes in overall circuit composition. We also discuss roles for neuromodulators and sensory inputs in modifying the relative strengths of constituent circuit components to provide flexibility in zebrafish behavior, allowing the animal to accommodate changes in the environment. We aim to provide a coherent framework for understanding the circuitry in the brain and spinal cord of zebrafish that allows the animal to flexibly transition between different speeds, and modes, of locomotion

Control of Turning Behaviors by Spinal Projection Neurons in the Larval Zebrafish

This thesis aims to examine how hindbrain spinal projection neurons (SPNs), namely RoV3, MiV1 and MiV2 control tail undulations during turning behaviors. I find that phototaxic turns differ from forward swims by an increased tail bend and a prolonged cycle period during the first undulation, while the later undulations are largely identical. Interestingly, laser ablation of RoV3, MiV1 and MiV2 neurons specifically affects the first undulation cycle by reducing the tail bend and the cycle period. Thus fish without the SPNs mainly perform forward swims in response to the turn-inducing phototaxic cues. These results suggest that the descending motor command that generates turns in larval zebrafish are composed of two pathways: one generates symmetric tail undulations, and the other, mediated by RoV3, MiV1 and MiV2 neurons, provides a brief and biased effect that modulates the first cycle of tail movement. Furthermore, fish whose unilateral SPNs are ablated are unable to perform turns toward the ablated side during the phototaxis, the optomotor response, the dark-flash response, and spontaneous swims, indicating the universal role of the SPNs in controlling visually-induced and spontaneous turns. Simultaneous two-photon calcium imaging and motor nerve recording in paralyzed fish show that RoV3, MiV2 and most MiV1 neurons on the turning side are active during turns, and that these activities are linearly correlated to the vigor of the intended turns. However, some MiV1 neurons are broadly tuned for all swimming directions. Computer simulations suggest that unilateral descending innervations to a specific type of spinal interneurons, namely commissural inhibitory interneurons, can generate a two-fold increase in the spinal network’s cycle period. This suggests that the SPNs could potentially innervate two types of spinal interneurons, namely $CoBL_{gly}$ and CoLo, in order to control the rhythm during turns. An additional chapter of this thesis examines the ontogeny of operant and classical learning behaviors in zebrafish. Using strategically positioned visual cues paired with electroshocks, I find that both learning behaviors are expressed reliably around week 3, and reach adult performance levels at week 6. These memories are behaviorally expressed in adults for 6 hours and retrievable for 12 hours

In Vitro, In Vivo, and In Silico Studies of Reticulospinal Circuits and Generalized Arousal

Generalized arousal (GA) is a fundamental force in the nervous system that alerts an individual to abrupt changes in its environment. A state of high GA is operationally defined by increases in an animal’s a.) locomotor output, b.) responsiveness to sensory stimuli, and c.) emotional reactivity. Previous studies have identified the nucleus gigantocellularis (NGC), a small group of large-bodied neurons in the hindbrain reticular formation, as a potential neuronal substrate for GA. These neurons are responsive to a wide range of sensory modalities and have diverse projections that target both forebrain areas and motor effectors directly within the spinal cord, thereby facilitating rapid responses to sensory stimulation. Here, we used three different approaches to study the role of GA in driving and modulating mammalian motor activity: in silico modeling of GA circuits, in vitro culture of a reticulospinal circuit, and in vivo behavioral assays of circadian transitions in GA. In our in silico study, we constructed a variety of computational models of the generalized arousal circuit and asked how modifying specific aspects of the NGC and its connectivity would influence the responsiveness of motor effectors in the circuit to arousing sensory stimuli. These models reveal that an NGC with a homogeneous microstructure that integrates all inputs equally and bifurcating projections that simultaneously target limbic and spinal areas is most effective at transducing an arousing sensory signal

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

Determining how stable network oscillations arise from neuronal and synaptic mechanisms

Many animal behaviors involve the generation of rhythmic patterns and movements. These rhythmic patterns are commonly mediated by neural networks that produce an oscillatory activity pattern, where different neurons maintain a relative phase relationship. This thesis examines the relationships between the cellular and synaptic properties that give rise to stable activity in the form of phase maintenance, across different frequencies in a well-suited model system, the pyloric network of the crab Cancer borealis. The pyloric network has endogenously oscillating ‘pacemaker’ neurons that inhibit ‘follower’ neurons, which in turn feed back onto the pacemaker neurons. The focus of this thesis was to determine the methods by which phase maintenance is achieved in an oscillatory network. This thesis examines the idea that phase maintenance occurs through the actions of intrinsic properties of isolated neurons or through the dynamics of their synaptic connections or both. A combination of pharmacological and electrophysiological techniques a used to show how identified membrane properties and short-term synaptic plasticity are involved with phase maintenance over a range of biologically relevant oscillation frequencies. To examine whether network stability is due to the characteristic stable activity of the identified pyloric neuron types, the hypothesis that phase maintenance is an inherent property of synaptically-isolated individual neurons in the pyloric network was first tested. A set of parameters were determined (frequency-dependent activity profile) to define the response of each isolated pyloric neuron to sinusoidal input at different frequencies. The parameters that define the activity profile are: burst onset phase, burst end phase, resonance frequency and intra-burst spike frequency. Each pyloric neuron type was found to possess a unique activity profile, indicating that the individual neuron types are tuned to produce a particular activity pattern at different frequencies depending on their role in the network. To elucidate the biophysical properties underlying the frequency-dependent activity profiles of the neurons, the hyperpolarization activated current (Ih) was measured and found to possess frequency-dependent properties. This implies that Ih has a different influence on the activity phase of pyloric neurons at different frequencies. Additionally, it was found that the Ih contribution to the burst onset phase depends on the neuron type: in the pacemaker group neurons (PD) it had no influence on the burst onset phase at any frequency whereas in follower neurons it acted to advance the onset phase in one neuron type (LP) and, paradoxically, to delay it in a different neuron type (PY). The results from this part of the study provided evidence that stability is due in part to the intrinsic neuronal properties but that these intrinsic properties do not fully explain network stability. To address the contribution of pyloric synapses to network stability, the mechanisms by which synapses promote phase maintenance were investigated. An artificial synapse that mimicked the feedforward PD to LP synapse, was used so that the synaptic parameters could be varied in a controlled manner in order to examine the influence of the properties of this synapse on the postsynaptic LP neuron. It was found that a static synapse with fixed parameters (such as strength and peak phase) across frequencies cannot result in a constant activity phase in the LP neuron. However, if the synaptic strength decreases and the peak phase is delayed as a function of frequency, the LP neuron can maintain a constant activity phase across a large range of frequencies. These dynamic changes in the strength and peak phase of the PD to LP synapse are consistent with the short-term plasticity properties previously reported for this synapse. In the pyloric network, the follower neuron LP provides the sole transmitter-mediated feedback to the pacemaker neurons. To understand the role of this synapse in network stability, this synapse was blocked and replaced by an artificial synapse using the dynamic clamp technique. Different parameters of the artificial synapse, including strength, peak phase, duration and onset phase were found to affect the pyloric cycle period. The most effective parameters that influence cycle period were the synaptic duration and its onset phase. Overall this study demonstrated that both the intrinsic properties of individual neurons and the dynamic properties of the synapses are essential in producing stable activity phases in this oscillatory network. The insight obtained from this thesis can provide a general understanding of the contribution of intrinsic properties to neuronal activity phase and how short-term synaptic dynamics can act to promote phase maintenance in oscillatory networks

Role of endocannabinoid system and acid sensing ion channels on spinal locomotor circuits during physiological and pathological conditions

Background: Mammalian spinal cord can generate well-coordinated locomotor activity called fictive locomotion in the absence of any higher brain center input or of rhythmic sensory feedback. This activity clearly provides evidence for the central pattern generator (CPG) that produces the locomotor rhythm. Such CPG consists of glutamatergic excitatory and glycinergic and GABAergic inhibitory interneuronal connections that finally excite or inhibit the motoneuronal pools. Many factors including fast (ion channels) and slow (modulatory G-protein coupled receptors; GPCRs) processes control these neuronal pools to act rhythmically. These factors are perturbed following spinal cord injury (SCI) in the early and late phases. The present study addresses the role of a few modulatory processes, namely acid sensing ion channels (ASICs) and cannabinoid 1 receptors (CB1Rs) at both initial and the late phases of injury.Objectives: Recent evidence has shown that the deletion of ASICs slows down the progression of disease in ischemic conditions, whereas the same protocol increases seizure severity. CB1R activation or deletion also results in neuroprotective or toxic mechanisms. In order to understand the importance of ASICs and CB1Rs in the spinal locomotor circuits, it is crucial to analyze them in physiological and pathological conditions. To investigate this issue, both organotypic slice culture and an in vitro rat spinal cord model were used. With the latter, fictive locomotion can be recorded from the ventral roots of the lumbar region for a time window of 24 h. Network parameters like synaptic transmission, fictive locomotion and disinhibited bursting provide information to explain the physiological modifications and pathological severity after excitotoxicity caused by transient kainate (KA; glutamate analog) application. Drugs that modulate CB1Rs and ASICs may supply evidence for the role of these processes in fictive locomotion.Results and conclusion: Our results show that the CB1R activation or block for 24 h diminished the locomotor rhythm. In particular, CB1R pharmacological block completely depressed both dorsal root (DR) and chemically evoked fictive locomotion. This depression was amplified following KA treatment. Furthermore, a limited neuroprotection was observed after CB1R agonists (anandamide; AEA or 2-arachidanoyl glycerol; 2AG) and an endogenous cannabinoid uptake inhibitor. These results allow us to propose the innate activity of CB1R (that is well preserved) to be important after KA mediated excitotoxicity, while any neuroprotective role might come in later phases after injury. A low concentration of KA that can induce a borderline injury elicited rapid glutamate release combined with proton discharge (acidification) in the organotypic SCI model. In response to this challenge, the ASIC subtypes (1a, 1b, 2a and 3) mRNA levels were found to be elevated after 24 h. Both neuronal numbers and network activity were highly depressed after application of ASIC pharmacological blockers that intensified the consequences of KA treatment. These results indicate that moderate acidification might be beneficial for the recovery (or limitation) of KA mediated excitotoxicity. Hence, this study demonstrates that both ASICs and CB1Rs activity are important in the early phase of experimental SCI in vitro. Their pharmacological modulation can outline future strategies for neuroprotection

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