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

Injury induced neuroplasticity and cell specific targeting of the lumbar enlargement for gene therapy.

This dissertation is an examination of spinal cord injury induced neuroplasticity and tests whether noninvasive gene therapy can successfully target neurons in the lumbar spinal cord. It begins with an overview of neural control of locomotion and a brief summary of therapeutics that are used and/or in development for treating spinal anatomically characterize s subset of neurons in the spinal cord, long ascending propriospinal neurons, that are involved in interlimb coordination. Characterization of these neurons allows for subsequent evaluation of their potential involvement in injury induced neuroplasticity. This dissertation is divided into five chapters, covering spinal cord injury and therapeutics. Chapter One gives background on locomotor control, propriospinal neurons, spinal cord injury, and therapeutics. Chapter Two develops and characterizes viral tracing methods for spinal cord anatomy. Chapter Three then uses these methods to characterize long ascending propriospinal neurons and evaluate their involvement in injury induced plasticity. Chapter Four then focuses on the development of noninvasive delivery of gene transfer to the lumbar enlargement. This involves optimizing focused ultrasound and intravenous microbubble delivery to focally and transiently permeabilize the blood spinal cord barrier of the lumbar spinal cord. This optimization then allows for successful gene transfer in neurons in the lumbar spinal cord following intravenous delivery of viral vector. Lasty, Chapter Five discusses the implications for all of these findings and how these findings have contributed to our understanding spinal cord anatomy and injury, and how the proof-of-concept in Chapter 4 provides a promising new avenue for spinal cord injury therapeutics

Reversible silencing of spinal neurons unmasks a left-right coordination continuum.

This dissertation is focused on dissecting the functional role of two anatomically-defined pathways in the adult rat spinal cord. A TetOn dual virus system was used to selectively and reversibly induce enhanced tetanus neurotoxin expression in L2 neurons that project to L5 (L2-L5) or C6 (long ascending propriospinal neurons, LAPNs). Results focus on the changes observed during overground locomotion. The dissertation is divided into four chapters. Chapter One is a focused introduction to locomotion, including its broad description, the central mechanisms of its expression, how genetic-based approaches defined these mechanisms, and the limitations in these approaches. It concludes with details of the silencing paradigm used here and a summary of the main findings. Chapter Two describes the functional consequences of silencing L2-L5 interneurons. The focus is on selective disruption of hindlimb coordination during overground locomotion, revealing a continuum from walk to hop. These changes are independent of speed, step frequency, and other spatiotemporal features of gait. Left-right alternation was restored during swimming and stereotypic exploration, suggesting a task-specific role. Silencing L2-L5 interneurons partially uncoupled the hindlimbs, allowing spontaneous shifts in coordination on a step-by-step basis. It is proposed this pathway distributes temporal information for left-right hindlimb alternation, securing effective coordination in a context-dependent manner. Chapter Three focuses on the consequences of silencing LAPNs.Three patterns of interlimb coupling are disrupted: left-right forelimb, left-right hindlimb, and contralateral hindlimb-forelimb coordination. Observed again was a context-dependent continuum from walk-to-hop, irrespective of step frequency, speed, and the salient features that define locomotion. However, instead of spontaneous shifts in coordination as observed from L2-L5 interneuron silencing, the breadth of coupling patterns expressed were maintained on a step-by-step basis. It is proposed that this ascending, inter-enlargement pathway distributes temporal information required for left-right alternation at the shoulder and pelvic girdles in a context-dependent manner. Collectively, these data suggest that L2-L5 interneurons and LAPNs are key pathways that distribute left-right patterning information throughout the neuraxis. The functional role(s) of these pathways are exquisitely gated to the context at hand, suggesting that the locomotor circuitry undergoes functional reorganization thereby endowing or masking the silencing-induced disruptions to interlimb coordination

Kainic acid-induced lumbar spinal cord damage leads to coordination deficits for the examination of cellular replacement therapies

Damage to the spinal cord can result in life-long deficits including locomotion. Most spinal cord injury cellular therapies focus on the regeneration of long-distance white matter descending motor tracts. However, locomotion is a complex task involving excitatory and inhibitory circuitry also in the spinal gray matter. The aim of this study was to create a discrete, gray matter lesion in the lumbar spinal cord to investigate the role of spinal interneurons in this region, as well as to establish a model that can evaluate proof-of principle targeted cell replacement therapies. A kainic acid lesion was modified to damage intermediate gray matter (laminae V-VII) in the lumbar spinal enlargement (spinal L2-L4) in 10-week-old female Fischer rats. A thorough, tailored behavioral evaluation revealed deficits in gross hindlimb function, skilled walking, coordination, balance and gait two-weeks post-injury. These deficits strongly correlated with structural deficits in the rostro-caudal axis. Machine-learning quantification confirmed interneuronal damage to laminae V-VII in spinal L2-L4 correlates with hindlimb dysfunction. White matter area and lower motoneuron numbers at lesion epicenters did not correlate with behavioral deficits. Animals do not regain lost sensorimotor function three months after injury, indicating that natural recovery mechanisms of the spinal cord cannot compensate for loss of laminae V-VII neurons. Together, this established model is ideal to evaluate cellular transplantation therapies that replace these lost neuronal pools vital to sensorimotor function. Rat spinal cells taken from embryos age 14 contain a mix of neural stem cells and neural precursor cells and have shown potential to aid functional recovery. Studies have shown that they survive and differentiate into gray matter interneurons both in vitro and in vivo, despite being transplanted into white matter lesions. Using the newly established KA-model, E14 spinal graft survival in a lumbar gray matter lesion could be evaluated for future proof-of-principle, targeted cell replacement experiments. Histological analysis revealed that grafted cells survive well both in the intact and KA-lesioned lumbar spinal cord without additional growth factors. Furthermore, grafts differentiate into neuronal NeuN+ cells both in the white and the gray matter. While future experiments will need to adjust injection parameters as well as evaluate graft differentiation and functional benefits, this work lays the groundwork to assess the potential cellular replacement has to restore lost function after a lumbar spinal cord injury

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

Activity dependent processes guide remodeling after traumatic spinal cord injury

Spinal cord injury causes a complete life change for patients and their social environment with severe economic, societal, and health implications. So far, there is no cure and only very limited treatment for patients suffering from spinal cord injury. Although central axons fail to regrow successfully after injury, incomplete spinal cord injury is accompanied by spontaneous but limited functional recovery. This recovery is attributed to compensatory neuroanatomical plasticity, where fibers remodel distal to the injury to establish new connections, so called neuronal detour circuits. Whilst various detour circuits in rodents are anatomically characterized in detail, the mechanisms of remodeling are not completely understood. Activity dependent processes, such as N-methyl-D-aspartate receptor (NMDAR) signaling, cyclic AMP response element-binding (CREB) transcription, and neuronal activity itself, play a crucial role in the formation of neural circuits during embryogenesis. We analyzed the role of these processes in the cervical spinal cord, where the corticospinal tract has been demonstrated to sprout and contact novel target neurons after traumatic spinal cord injury. More specifically, we perturbed NMDAR signaling, (ii) suppressed CREB transcription, and (iii) silenced neurons with designer receptor exclusively activated by designer drugs (DREADDs). Inhibiting NMDAR and CREB function in the cervical area during detour circuit formation both resulted in anatomically aberrant remodeling of the corticospinal tract. However, cervical circuitry remained unaltered when activity dependent processes were manipulated before spinal cord injury and after a mature detour circuit had already been established. These experiments demonstrate that spinal cord injury transiently opens a critical window of activity dependent plasticity, enabling detour circuit formation. Furthermore, I argue that target selection during detour circuit formation after spinal cord injury is based on the neuron’s relative level of activity. Firstly, this interpretation is based on our observation that global cervical silencing had no measurable effects whilst selective silencing of specific neuronal populations created an anatomically and functionally defective detour circuit. Secondly, within this defective detour circuit, the degree of neuronal silencing negatively correlated with the likelihood of that neuron to be contacted by corticospinal tract collaterals. With the help of a detailed literature analysis, I demonstrate similarities in plasticity between the developing CNS, the adult intact, and injured CNS and propose future experiments. Taken together, this thesis illustrates that activity dependent processes guide spontaneous detour circuit formation after spinal cord injury

Inducing Neural Plasticity After Spinal Cord Injury To Recover Impaired Voluntary Movement

Spinal cord injury (SCI) is often an incapacitating neural injury most commonly caused by a traumatic blow to the spine. A SCI causes damage to the axons that carry sensory and motor signals between the brain and spinal cord, and in turn, the rest of the body. Depending on the severity and location of a SCI, many corticospinal axons and other descending motor pathways can remain intact. Moderate spontaneous functional recovery occurs in patients and animal models following incomplete SCI. This recovery is linked to changes occurring via the remaining pathways and throughout the entire nervous system, which is generally referred to as neuronal plasticity. It has been shown that plasticity can be induced via electrical stimulation of the brain and spinal cord targeting specific descending pathways, which can further improve impaired motor function. Most importantly, it has been shown that activity dependent stimulation (ADS), which is based on mechanisms of spike timing-dependent plasticity, can strengthen remaining pathways and promote functional recovery in various preclinical injury models of the central nervous system. The purpose of this dissertation was to determine if precisely-timed stimulation of the spinal cord triggered by the firing of neurons in the hindlimb motor cortex would result in potentiation of corticospinal connections as well as enhance hindlimb motor recovery after spinal cord contusion. In order to achieve this, we needed to determine the optimal neurophysiological conditions which would allow activity dependent stimulation (ADS) to facilitate enhanced communication between the cerebral cortex and spinal cord motor neurons. Thus, this dissertation project investigated three specific aims. The first study determined the effects of a contusive spinal cord injury on spinal motor neuron activity, corticospinal coupling, and conduction time in rats. It was discovered that spinal cord responses could still be evoked after spinal cord contusion, most likely via the cortico-reticulo-spinal pathway. The second study determined the optimal spike-stimulus delay for increasing synaptic efficacy in descending motor pathways using an ADS paradigm in an acute, anesthetized rat model of SCI. It was discovered that bouts of ADS conditioning can increase synaptic efficacy in intact descending motor pathways, as measured by cortically evoked activity in the spinal cord, after SCI. The third study determined whether spike-triggered intraspinal microstimulation (ISMS), using optimized spike-stimulus delays, results in improved motor performance in an ambulatory rat model of SCI. It was determined that ADS therapy can enhance the behavioral recovery of locomotor function after spinal cord injury. The results from this study indicate that activity-dependent stimulation is an effective treatment for behavioral recovery following a moderate spinal cord contusion in the rodent. The implications of these results have the potential to lead to a novel treatment for a variety of neurological disease and disorders

Axon regeneration and circuit reorganization after complete spinal cord injury

First human trials involving neuroprosthetic rehabilitation demonstrated recently that significant functional benefits can be achieved with lumbosacral neuromodulation and reorganized spared projections. However, complete spinal cord injuries (SCI) wholly isolate infralesional circuits from any supraspinal control, leading to irreversible motor deficits that neuroprosthetics still fails to address. As axons fail to regrow across the lesion site, neuroregenerative research after SCI has been dogmatically focusing attention on minimizing the environmental inhibitions to axon regeneration. In the present work, we demonstrate that spontaneous axon regeneration failure can be reversed, and that propriospinal axons are able to grow across complete non-neural lesion cores when the required facilitators are provided. We identified three mechanisms needed for propriospinal regrowth : i) enhancing neuronal intrinsic growth capacities using viral technologies, ii) remodeling the lesion core with growth factors, in order to densify permissive substrates, and iii) guiding axons chemically across the SCI site. Propriospinal neurons - that coordinate different spinal segments in healthy conditions - are known to support recovery of locomotion in incomplete models of spinal cord injury. However, restoring a robust descending bridge of propriospinal axons does not by itself promote any functional benefit. We hypothesize that a lack of somatosensory feedback to the motor cortex (M1), together with insufficient descending motor control, may prevent coherent exchange of information between lumbosacral and cortical motor centers. Therefore, we explored the neuroregenerative potential of reticulospinal axons - which are projected from the brainstem to the spinal cord - as a second relay for the descending motor cortical command. Together with axon guidance and lesion remodeling, our viral manipulation of intrinsic growth programs induced limited yet significant reticulospinal regeneration into the lesion core. In order to enhance this reticulospinal regrowth, we explored activity-dependent mechanisms of neurite growth control. As we observed that activity does not recruit growth programs after SCI, we revealed a possible divergence between the mechanisms that underly reticulospinal axon regrowth and neurite sprouting. In the mean time, we are developing a somatosensory interface that aims at restoring sensory feedback to the motor cortex after complete SCI. Based on hindlimb electromyographic activity, we linked locomotion to optogenetic neuromodulation of the motor thalamus, and were able to evoke responses in M1 by selectively activating thalamocortical projections, both before and after complete spinal lesion. Such technology enables a direct recruitment of thalamic nuclei that gate motor learning and motor planning at a cortical level. Coming experiments will test wether a strategy combining propriospinal regeneration with locomotion-dependent thalamocortical modulation is sufficient to restore voluntary stepping after complete SCI

Mechanisms and novel therapies in cervical spinal cord injury.

PhDRecent epidemiological data indicate that more than half of SCI patients have injuries of the cervical spine. There is no satisfactory treatment for these injuries either in the acute or the chronic phase. Docosahexaenoic acid (DHA) is an omega-3 polyunsaturated fatty acid that is essential in brain development and has structural and signalling roles. Acute DHA administration has been shown to improve neurological functional recovery following injury in rodent thoracic spinal cord injury (SCI) animal models. In this thesis, we characterized a cervical SCI model comprising a hemisection lesion applied at the C4-5 level of the rat spinal cord, and tested the effects of an acute treatment with 250 nmol/kg DHA delivered intravenously 30 minutes after injury. The acute intravenous bolus of DHA not only increased the number of neuronal cells spared at three weeks following injury but also resulted in robust sprouting of uninjured corticospinal and serotonergic fibres. Next, we used a mouse pyramidotomy model to confirm that this robust sprouting was not species or injury model specific. We demonstrated that the number of V2a interneurons contacted by collateral corticospinal sprouting fibres is positively correlated with skilled motor recovery. To address the mechanism behind the neuroplasticity-promoting effect of DHA, we investigated the expression of miR-21 and phosphatase and tensin homolog (PTEN) in cortical neurons and raphe nuclei after DHA treatment. We found that DHA significantly up-regulates miR-21 and down-regulates PTEN in corticospinal neurons one day after SCI. Downregulation of PTEN by DHA was also seen in dorsal root ganglion (DRG) neuron 3 cultures and was accompanied by increased neurite outgrowth. Lastly, we investigated whether DHA treatment combined with specific-task rehabilitation maximized the recovery of skilled forelimb function following cervical SCI. The rats receiving combined therapy achieved greater skilled forelimb functional recovery compared to DHA treatment or rehabilitation only. In summary, this study shows that DHA has therapeutic potential in cervical SCI and provides evidence that DHA could exert its beneficial effects in SCI via enhancement of neuroplasticityMemorial Hospital, Taiwan for funding this research (grant number CMRPG3A105