Dissertations / Theses on the topic 'Embryonic chick spinal cord'

Textbook accounts of vertebrate embryonic development have been based largely upon experiments on amphibian embryos, which have shown that the tissues of the trunk and tail are organised from distinct precursors that existed during gastrulation. In the mouse and chick, however, retrospective clonal analyses and transplantation experiments have demonstrated that the amniote body instead arises progressively from a population of axial precursors that are common to both the neural and mesodermal tissues of the trunk and tail. For this reason, they are known as neuro-mesodermal progenitors (NMps). Detailed studies of NMps have been precluded by their lack of a unique gene expression profile and the technical difficulties associated with isolating them from the embryo. Mouse embryonic stem cells (ESCs) provide the possibility of instead deriving them in vitro. ESCs have been used to model developmental processes, partly through large cellular aggregates known as embryoid bodies. These structures do not, however, resemble the axial organisation of the embryo and they develop in a disordered manner. This thesis presents a novel culture system of small, three-dimensional aggregates of ESCs (gastruloids) that can recreate the events of early post-implantation development, including axial elongation. Gastruloids are the first ESC-based model for axial elongation morphogenesis; this body of work characterises their development and identifies a candidate population of NMps within their elongating tissues. Additionally, this work establishes a xenotransplantation assay for testing the functional properties of in vitro-derived NMp populations in the chicken embryo and applies it to NMps from gastruloid cultures. The results of this assay show that gastruloids are a credible source of NMps in vitro and therefore offer a new experimental means to interrogate their properties. The use of gastruloids to recreate embryonic development has implications for basic research as a synthetic system and for the therapeutic derivation of other embryonic progenitors through bioengineering.

Several recent experiments on developing chick spinal cord have established a time window when the developing spinal cord changes from a permissive to a restrictive environment for regeneration. This time window occurs during embryonic days 13-14 (E13-E14) of chick development. Recent experiments in adult rat, have found two proteins that actively inhibit axonal regeneration. This study has sought possible inhibitory proteins, in chicks, correlating to this temporal change. Proteins continuously present after this change (E14-E20) but not before (E11) were identified. Two-dimensional gel electrophoresis was used for separatation of the proteins. Seven protein spots of interest demonstrated this correlative late-expressing neural protein (LNP) profile. Although the functions of these proteins could not be ascertained in this study, further investigation is warranted.
Science, Faculty of
Zoology, Department of
Graduate

Le but de mon projet de thèse a été d’investiguer le rôle des cellules microgliales in vivo au cours du développement. On s’est particulièrement intéressés au développement de la moelle épinière car ce modèle a été bien caractérisé. Nous avons trouvé que les microglies colonisent la moelle épinière embryonnaire grâce à leur migration et prolifération. A E12. 5 les microglies s’accumulent transitoirement au point d’insertion des ganglions de la racine dorsale et phagocytent les axons apoptotiques des neurones sensoriels. A E13. 5 les microglies interagissent avec les glies radiaires, expriment la galactin-3 et phagocytent les corps apoptotiques de motoneurones (Rigato et al. , 2011). Ces microglies sont capables de proliférer grâce au récepteur P2X7 qui n’est pas couplé à l’hémicanal pannexin-1. Ce récepteur ne contrôle pas l’activation, ce qui indique que pendant le développement embryonnaire de la moelle épinière, la prolifération et l’activation microgliale sont deux processus indépendants (Rigato et al. , 2012). Actuellement nous sommes en train d’analyser le rôle des microglies dans l’apoptose en utilisant des embryons PU. 1-KO, génétiquement dépourvus de microglies. En l’absence de microglies, le nombre de motoneurones et neurones sensoriels qui rentrent en apoptose augmente. Les cellules microgliales semblent donc avoir un rôle protecteur vis à vis de motoneurones, probablement à travers la sécrétion de facteurs de croissance. Ces résultats montrent que les interactions microglies-neurones sont établies très tôt au cours du développement de la moelle épinière et ils ouvrent un nouveau champ de recherche qui permettra de mieux comprendre comment se forment ces interactions
The challenge of my PhD project has been to understand more about embryonic microglial cells in vivo in the developing CNS. I focus my attention to the developing mouse spinal cord as it is a deeply studied and well-characterized model. We found that microglial cells colonize the embryonic spinal cord through migration and proliferation. At E12. 5 microglia transitory accumulate at the insertion point of dorsal root ganglia (DRG) and phagocytose apoptotic axons of sensory neurons. At E13. 5 microglia interact with radial glial cells, express the galactin-3 and phagocytose the apoptotic bodies of dying motoneurons (Rigato et al. , 2001). These ventral microglia at E13. 5 are able to proliferate as they express the purinergic receptor P2X7 that are not coupled to pannexin-1 hemichannel. This receptor strictly controls microglial proliferation but it is not involved in their activation, indicating that during the embryonic development of the spinal cord, microglial proliferation and activation are two independent processes (Rigato et al. , 2012). We are now analysing the putative role of microglia in the developmental cell death process by using PU. 1-KO embryos, genetically devoid of microglial cells. In absence of microglia, the number of motoneurons and sensory neurons that undergo programmed cell death increased. Microglial cells seem to have a protective role towards neurons, probably through the release of some growth factors. These results show that that interactions between microglia and neurons are established very early during spinal cord development and they open a new research field that will permit to better understand how these interactions are formed

During amniote embryogenesis, the segmented pattern characteristic of the vertebral column appears early during development through the sequential formation of multipotent structures called somites. Somites differentiate subsequently into dermomyotome (giving rise later to skin and skeletal muscles) and sclerotome (giving rise to vertebral bone structures and cartilage). In addition, sclerotomes subdivide following their rostro-caudal intrasegmental boundary into an axon growth-permissive region (anterior half) and an axon growth-repulsive region (posterior half). This binary system instructs motor and sensory axon navigation, as well as neural crest cell migration, to ensure that the peripheral nervous system develops without obstruction by the future cartilage and bones of the vertebral column. Repellent cues are expressed in posterior half-sclerotomes in order to exclude navigating axons from “no-go” areas and restrict their growth to specific exit points of the future vertebral column. Interestingly, similar repellent cues (e.g. Eph/Ephrins) are expressed in the adult central nervous system (CNS) and have been shown to control connectivity and plasticity throughout life. Following brain or spinal cord injury, these repellent molecules are upregulated by reactive astrocytes accumulating at the lesion site, and may impede axon regeneration in this region. In this dissertation, I am presenting the results of a differential gene expression analysis of anterior and posterior half-sclerotomes, based on RNA-sequencing data and using the chick embryo as a model organism. This study led to the identification of molecules, previously uncharacterized in this system, that may play a role in adhesive and mechanical properties of somites and in axon guidance and fasciculation. I focused on the functional analysis of one molecule of the posterior half-sclerotome, the extracellular matrix protein Fibulin-2. To look at its role in the segmentation of spinal axons, I used ectopic misexpression in a subset of segments based on somite electroporation. The width of spinal nerve bundle growth was restricted by Fibulin-2 overexpression in posterior and anterior half-sclerotomes, suggesting a role in sharpening/controlling the path of spinal axon growth. In addition, I showed that this could occur via an interaction with the axon growth repellent Semaphorin 3A. Then I looked at the expression of Fibulin-2 in two models of CNS injury: mouse cerebral cortical stab injury and rat dorsal crush spinal cord injury. In both cases, I observed an increase in Fibulin-2 protein level compared to control. I also used primary cultures of rat cortical astrocytes to show that the expression of Fibulin-2 after inflammatory cytokine-induced activation is increased. Finally, I studied a candidate axon growth repellent previously identified in the laboratory. I explored the hypothesis that this repellent molecule is an O-glycosylated, spliced variant form of a known protein. To characterize this repellent molecule, I used RNA-sequencing data from chick embryonic somites and 2D gel electrophoresis of an astrocytic cell line protein extract. Together, these results suggested that the developing vertebral column and the adult CNS share molecular features to control axon growth and plasticity. This type of study could lead to the characterization of molecular systems that regulate axon growth, and to the identification of novel therapeutic targets in brain or spinal cord injury.

This work addresses developmental regulation of neurotransmitter receptors in the vertebrate central nervous system (CNS). Glycine receptors (GlyR) play a major role in inhibitory neurotransmission in the spinal cord. Changes in the types of GlyRs being expressed by embryonic rat spinal cord neurons are examined during development in vitro. Spinal cord neurons are cultured at the fourteenth day of gestation, prior to receiving afferent input. Previous work demonstrated a delayed expression of the adult-type GlyR α subunits. Reverse transcriptase-polymerase chain reaction demonstrates the presence of mRNA for GlyR α2 subunits by these neurons at early times in culture. The presence of GlyR α2 subunits are confirmed by immunofluorescence microscopy with a new α2 subunit specific antibody. These subunits appear by the first day in culture and exhibit a diffuse subcellular distribution. During the course of these experiments, populations of embryonic rats were found to differ in the subtypes of GlyR they expressed at early times during development. The expression of functional GlyRs is investigated in two populations of embryonic rats using whole-cell patch clamp recordings. The GlyR antagonist, strychnine, is used as a tool to distinguish between some forms of the GlyR. The two populations are similar in their onset of responsiveness to glycine and in the ion-dependence of the glycine-induced current. The strychnine-sensitivity of responses to glycine differs between the two populations. Neurons from the first population of rats exhibit a developmentally regulated increase in the sensitivity to strychnine, while the strychnine-sensitivity of responses to glycine by neurons from the second population remains high throughout development in culture. These results suggest that two populations differ in the type of functional GlyR they express during early development in culture. The relatively low sensitivity to strychnine exhibited during the first few days in culture by neurons from the first population of rats cannot be accounted for by changes in sensitivity to glycine or by non-specific cross-activation of γ-aminobutyric acid receptors (GABARs). Neurons from the first population undergo a gradual change from the predominant expression of a strychnine-insensitive GlyR to some form(s) of strychnine-sensitive GlyR.

Les cellules microgliales sont les cellules immunitaires résidentes du système nerveux central (SNC). Elles peuvent déjà être détectées au début du développement embryonnaire du SNC. Dans ce projet de recherche nous avons étudié l'invasion et les caractéristiques phénotypiques des cellules microgliales du cerveau embryonnaire. Nos résultats demontrent que les microglies dans le cortex embryonnaire ont un phénotype de « repos »; elles expriment peu de marqueurs d'activation et n'ont presque aucun canaux K+ à rectification entrante. Pourtant, elles sont très dynamiques comme dans le cerveau adulte. Au cours du développement du plexus choroïde, des microglies activées au phenotype phagocytique s'accumulent à un moment coïncidant avec un pic d'apoptose dans cette structure. La proliferation des microglies dans la moelle épinière embryonnaire dépend de récepteurs P2X7. Nous avons retrouvé les mêmes récepteurs sur les cellules microgliales du cortex. Diverses études ont demontré que les infections et l'activation immunitaire pendant la grossesse donnent un risque accru de développement de maladies neuro-psychiatriques chez les enfants. Puisque les microglies sont les cellules immunitaires du SNC et qu’elles sont présentes au cours du développement embryonnaire, nous avons examiné si elles sont activées après une reaction immunitaire maternelle pendant la grossesse. Nos résultats indiquent qu’il n'y a aucune augmentation de densité ou d’activation des cellules microgliales dans le cerveau embryonnaire après induction d’une réaction immunitaire maternelle
The microglia are the resident immune cells of the central nervous system (CNS). They can be detected from the beginning of the development of the embryonic CNS. In this project we have studied the invasion and phenotypic characteristics of the microglial cells in the embryonic brain. Our results show that embryonic microglia in the cortex have a “resting” phenotype; the express little activation markers and have little to no inward rectifying K+ channels. However, they are very dynamic like observed in the adult brain. During development of the choroid plexus, activated microglia with a phagocytic phenotype accumulate at the moment apoptotic cells are present in this structure. The proliferation of microglial in the embryonic spinal cord depends on P2X7 receptors. We found the same receptors to be present on the microglia in the cortex. Different studies have shown that infections and immune activation during pregnancy increase the risk on neuropsychiatric disorders in the offspring. Since microglia are the immune cells of the CNS and they are present early in development, we studied the effect of maternal inflammation during pregnancy on these cells. Our results indicate that there is no effect on microglia density and activation after maternal immune activation

Traumatic injury to the spinal cord interrupts ascending and descending pathways leading to severe functional deficits of sensory motor and autonomic function which depend on the level and severity of the injury. There are currently no effective therapies for treating such injuries and the adult central nervous system has very limited capacity for repair so that recovery is very limited and functional deficits are usually permanent. Cell transplantation is a potential therapy for spinal cord injury and a range of cell types are being investigated as candidates. Mesenchymal stem cells (MSCs) obtained from bone marrow are one cell type quite extensively studied. When transplanted into animal models of spinal cord injury these cells are reported to affect various aspects of repair and in some cases to improve functional outcome according to behavioural measures. However, the use of these cells has several limitations including the need for an invasive harvesting procedure, variability in cell quality and slow expansion in culture. This project therefore had two main aims: Firstly to investigate whether MSC-like cells closely equivalent to bone marrow derived MSCs could be reliably and consistently differentiated from human embryonic stem cells (hESCs) in order to provide an “off the shelf” cellular therapy product for spinal cord injury and secondly, to transplant such cells into animal models of spinal cord injury in order to, determine whether hESC-derived MSCs replicate or improve on the repair mechanisms reported for bone marrow MSCs.

Persons with spinal cord injury (SCI) suffer life-long consequences including paralysis, loss of involuntary bodily functions, and chronic pain. A subset of SCI patients develop neuropathic pain (NP), a chronic condition resulting from damage to the spinal cord. Hyperexcitability of spinal cord sensory neurons near damaged tissue is believed to underlie SCI-related NP. Although many therapies have been employed clinically to combat SCI-NP, few give satisfactory long-term relief. Transplantation of cells that release GABA, a molecule that inhibits neuronal activity, is being explored as an alternative to current SCI-NP therapies. My experiments made progress toward preclinical modeling of GABA cell therapy for SCI-NP. First, I sought to determine whether quisqualic acid (QUIS)-induced SCI altered responses to tonic pain stimuli or altered GABAergic neural circuitry in rats. Second, I sought to determine whether a combination of genetic and trophic manipulations could promote a GABAergic phenotype in rat embryonic neural precursor cells (NPCs) in an in vitro culture system. The results revealed that QUIS-SCI rats exhibit unusually prolonged nocifensive responses to hind paw formalin injections. There was no significant difference between QUIS-SCI and sham surgery rats in c-Fos immunolabeling of spinal cord sensory neurons after formalin-induced neuronal activity. However, immunohistochemistry revealed substantial decreases in staining for markers of GABA presynaptic vesicles in injured spinal cord tissue. NPCs were enriched for a neuronal phenotype by combining withdrawal of the growth factor FGF-2 from culture media and overexpression of the transcription factor MASH1 in transfected cells. Although glial marker expression was suppressed in NPCs by these manipulations, expression of neuronal markers none the less declined through time. MASH1-overexpressing NPCs exhibited greater clonal expansion and decreased stress-induced PDI expression after FGF-2 withdrawal as compared to naïve. In light of existing data, these results suggest that the QUIS-SCI model may be useful for testing the efficacy of GABAergic NPC transplantation to reduce neuropathic pain. MASH1 overexpression and FGF-2 withdrawal could serve as a first step toward enriching GABA in NPCs for transplantation. Although the mechanism for MASH1 cytoprotection remains unclear, MASH1 may enhance survival of NPCs grafted into the spinal cord. These experiments contributed to the preclinical basis for application of therapeutic GABAergic stem cell transplantation for NP in human SCI patients.

Primary sensory afferent outgrowth within the developing longitudinal pathway of the spinal cord is important for intrasegmental and intersegmental communication that underlies coordination and development of reflexes and contributes to sensory perception. The endogenous mechanisms that regulate primary sensory afferent extension are the primary focus of this dissertation. This dissertation tested the hypothesis that primary sensory afferent extension in the longitudinal pathway is regulated by sphingosine 1-phosphate type 1 receptor (S1P1R) and tyrosine kinase receptor B (TrkB) through a brain derived neurotrophic factor (BDNF) related mechanism. To test this hypothesis we used embryonic day five (E5) chicken embryos, as this is the developmental time point when sensory afferents are growing along the longitudinal axis of the spinal cord but have not yet turned ventrally to make connections with the grey matter of the spinal cord. Chicken embryos were removed from their in ovo environment to allow for labeling of primary afferent neurons in the thoracic 3/4 (T3/4) dorsal root ganglia (DRG). Tissue was then put into culture with or without various pharmacological agents and subsequently assayed for length of growth of the labeled primary afferent axons along the longitudinal axis of the spinal cord. Results showed both BDNF and fingolimod-p, an S1P1R agonist known to increase BDNF mRNA and protein production/secretion in cortical neurons, increased primary axon extension along the longitudinal pathway. Further, fingolimod-p increased BDNF mRNA production in DRG in this system. Conversely, inhibition of BDNF or S1PRs attenuated primary afferent axon extension along the longitudinal pathway. We found BDNF signaling to be required for fingolimod-p's effects as addition of αBDNF attenuated the effects of fingolimod-p on axon outgrowth. TrkB, the high affinity receptor for BDNF, is expressed in chicken DRG during embryonic development. We hypothesized that TrkB activation by BDNF regulates DRG axon extension in the longitudinal pathway through the PLC-γ signaling pathway. We found inhibition of TrkB and/or PLC-γ signaling pathway attenuated DRG axon extension with or without BDNF stimulation. Additional pathways associated with TrkB activation: mitogen activated kinase (MAPK) and phosphoinositide 3-kinase (PI3K) appeared to either have no effect on DRG axon extension or were involved in DRG axon extension through a mechanism that is not related to TrkB. Collectively, these studies suggest an endogenous mechanism for the regulation of DRG axon outgrowth within the longitudinal pathway. With this mechanism, DRG axon outgrowth may be enhanced or attenuated following manipulation of S1P1R, BDNF and/or TrkB. Further, these findings suggest an action through BDNF on CNS axons as a potential therapeutic effect of fingolimod-p, a treatment for relapsing remitting forms of Multiple Sclerosis

Tous les réseaux de neurones immatures génèrent une activité dite « spontanée »qui persiste même en l’absence de toute afférence et est impliquée dans de nombreux processus développementaux. Cette activité apparaît in vitro sous formes de vagues calciques ou électriques pouvant se propager sur de grandes distances et embraser toute la préparation. Toutefois, sa dynamique a été assez peu étudiée jusqu’à présent. En vue de combler quelque peu cette lacune, nous avons utilisé des matrices de microélectrodes (MEA) pour caractériser l’activité rythmique spontanée dans la moelle épinière embryonnaire de souris, sur des préparations aigues et en culture incluant également le tronc cérébral.Les enregistrements MEA produisent des volumes de données très importants qui nécessitent des outils d’analyse performants et adaptés à l’information que l’on souhaite extraire. Nous avons donc développé des méthodes pour la détection, la classification et la cartographie des patrons spatiotemporels d’activité dans les données multicanaux. Notre approche cartographique utilise l’interpolation par splines et est orientée vers la production de cartes multimodales combinant l’activité électrique et des données anatomiques ou biochimiques (marquages). Ces méthodes d’analyse nous ont permis de décrire très précisément l’évolution de l’activité spontanée aux stades précoces (E12.5–E15.5). Nous avons également montré que, à E14.5, l’activité est initiée dans le bulbe, plus précisément dans une région riche en neurones 5-HT, suggérant un nouveau rôle des voies sérotoninergiques descendantes dans la maturation des réseaux spinaux.Enfin, nous avons analysé les mouvements embryonnaires à E14.5 et avons découvert des caractéristiques analogues à la dynamiques spatiotemporelle des activités intraspinales
Immature neural networks generate a peculiar type of activity that persists even in the absence of electrical inputs and was termed for this reason “endogenous”or “spontaneous”. This activity is ubiquitous and was found involved in a wide range of developmental events. In vitro, it can be observed as calcium or electrical waves propagating over great distances, often invading the whole preparation,but its dynamics remain poorly described. In order to somewhat fill this gap,we used multielectrode arrays (MEAs) to characterise the spontaneous rhythmic activity in the mouse developing spinal cord, in both acute and cultured isolated hindbrain-spinal cord preparations.To extract relevant information from the massive amounts of data yielded by MEA recordings, adapted analysis tools are needed. Thus, we have developedmethods for the detection, classification and mapping of spatiotemporal patternsof activity in multichannel data. Our mapping approach is based on the thin plates pline interpolation and includes the possibility to combine maps of activity with anatomical or stained data for multimodal imaging.These methods allowed us to analyse in great detail the evolution of spontaneousactivity at early stages (E12.5–E15.5). In addition, we have localised theinitiation site of E14.5 activity in the medulla and shown that it matches a densemidline population of serotoninergic neurons, suggesting a new role for 5-HTpathways in the maturation of spinal networks. Finally, we have recorded andtracked spontaneous limb movements of E14.5 embryos and found that features of motility were consistent with patterns of spinal activity

Les réseaux vasculaires et nerveux présentent des similitudes frappantes (points de branchements, superposition, voies afférentes/efférentes, …) et tous deux interagissent lors du développement ou dans le cadre de pathologies.Dans un premier projet, nous avons voulu déterminer si un facteur pro-angiogénique, c'est-à-dire induisant la formation de nouveaux vaisseaux, peut avoir un effet direct sur le réseau neuronal. Des études menées in vitro ou in vivo chez l’adulte, ont montré une implication directe du Vascular Endothelial Growth Factor (VEGF) sur le système nerveux (survie, prolifération neuronale, croissance axonale, …). Nous avons cherché à savoir si ce facteur a un effet sur le développement ou l’activité des réseaux neuronaux lors de la vie embryonnaire alors que les systèmes vasculaires et nerveux se mettent progressivement en place. Avec une approche électrophysiologique, nous avons focalisé notre attention sur les motoneurones de la moelle épinière de souris entre les stades E13,5 et P0. Nos résultats montrent que le VEGF augmente de façon significative la fréquence des activités synaptiques liées à la libération de GABA et de Glycine pendant une fenêtre temporelle correspondant à la mise en place de ces mêmes activités (E13,5 et E15,5). Cet effet modulateur met en évidence un nouveau rôle du VEGF dans la maturation fonctionnelle des réseaux neuronaux et ouvre de nouvelles perspectives dans l’étude des neurodégénérescences précoces. Dans un second projet, nous nous sommes intéressés au glioblastome, cancer cérébral très invasif. Nous montrons que l’inhibition d’IRE1 (Inositol Requiring-Enzyme 1, senseur du stress du réticulum endoplasmique) dans un modèle d’implantation orthotopique chez la souris induit la formation de tumeurs plus petites, moins vascularisées et plus dispersées avec un meilleur pronostic de survie. Nous observons aussi des altérations du microenvironnement tumoral (matrice extracellulaire, réaction astrocytaire) avec des modifications de l’expression de nombreux facteurs de croissance dont le TGFß
The nervous and the vascular systems share similarities (branching points, afferent/efferent parts …) and are closely connected during development and pathology.In the first part of this project, we questioned whether the pro-angiogenic key factor VEGF (Vascular Endothelial Growth Factor), which promotes new blood vessels formation, can directly interact with neural networks while nervous and vascular systems are developing. In the present study, using an electrophysiological approach, we focused on the effect of VEGF on embryonic spinal lumbar motoneurons (MNs). Our results demonstrate that VEGF increases the frequency of the GABA/glycinergic events at early developmental stages (E13.5 and E15.5) but not at the perinatal stage E17.5. Our data highlight a new role for VEGF which can control both the maturation of the vascular and neuronal networks and may likely be involved in early MNs degeneration.In the second part, we focused on glioblastoma, the most agressive form of brain cancer. Our results show that inhibition of IRE1 (Inositol Requiring-Enzyme 1, stress sensor of endoplasmic reticulum) leads to formation of smaller, less vascularized, more invasive tumors with a better prognosis. We also observe that tumoral microenvironnement is altered (reactive astrogliosis, extracellular matrix) and expression of several growth factors like TGFß is modified

L'etude in vitro de cellules neuronales obtenues a partir de cellules primaires de moelle epiniere d'embryons de rat de 14 jours a permis de montrer l'influence, sur la croissance des neurites, de plusieurs facteurs extracellulaires. Les resultats montrent que les neurones spinaux sont capables de repondre a ces differents facteurs par induction de plusieurs formes de croissance neuritique. Les cellules non neuronales jouent un role dans la regulation de ces differentes formes de croissance des neurites

The embryonic central nervous system (CNS) is more plastic than adult CNS, and the early embryonic brain and spinal cord has been suggested to recover more readily from a severe injury. The following studies were designed to determine the stages of chicken (Gallus domesticus) embryonic development during which descending brainstem-spinal tracts maintain their capacity for anatomical and functional repair after complete thoracic spinal cord transection. Brainstem-spinal neurons begin to project their axons through the spinal cord at approximately embryonic day (E)4, and these projections are essentially complete to the lumbar level by E12. Disruption of these brainstem-spinal pathways was produced by complete thoracic spinal cord transections at different developmental ages from E3 through E14. Control sham operations were conducted in parallel. The post-operative embryonic recovery period varied from 5 to 8 days. Following recovery, the extent of anatomical repair was assessed by injecting a fluorescent dye into the lumbar spinal cord caudal to the transection site. Brainstem tissue sections were subsequently examined for the presence of retrogradely labelled brainstem-spinal neurons. Anatomical results indicated similar distributions of retrogradely labelled neurons within the brainstem of both sham transected controls and embryos transected prior to E13 (Hasan et al., 1991, 1992). The neuroanatomical recovery shown by chick embryos can be attributed either to axonal regeneration of previously severed axons or to the subsequent development of new axonal projections from later developing neurons. In order to address this issue, the embryonic lumbar spinal cord was injected before and after thoracic transection with two different retrograde tract tracing fluorescent dyes. Co-localization of both labels within the same brainstem-spinal neuron would be indicative of regeneration of previously axotomized projections rather than the subsequent development of new axonal projections. Findings indicated that there were double-labelled brainstem—spinal neurons after a transection prior to E13 and the number of double-labelled brainstem-spinal neurons decreased after an E13-E15 transection. In addition, at each subsequent stage of development from E1O-E12, a higher ratio of double-labelled brainstem-spinal neurons (indicating regeneration of previously severed axons) to the number of cell bodies labelled with the second fluorescent tracer alone (indicating possible subsequent development) was observed. This would suggest that during successive stages of development, regeneration of previously axotomized fibers increasingly contributes to the observed anatomical and functional recovery after thoracic cord transections prior to E13 (Hasan et al., 1992). Functional recovery was assessed by focal electrical stimulation of identified brainstem locomotor regions in transected or sham-transected E18-E20 embryos. Leg muscle electromyographic (EMG) recordings were used to monitor brainstem stimulated locomotor activity (Valenzuela et al., 1990; Hasan et al., 1991, 1992). Functional repair was evident among E18-E20 embryos that had had their spinal cords transected prior to E13 and this brainstem evoked locomotion was indistinguishable from brainstem-evoked locomotion in control (sham-transected or untransected) embryos. In addition, voluntary open-field locomotion and brainstem evoked locomotion in hatchling chicks transected prior to E13 was indistinguishable from that observed in control hatchlings, indicating that complete functional recovery had occurred. Embryos and hatchling chicks transected on or after E13 showed reduced functional repair abilities (Hasan et al., 1991, 1992.

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.