Following microsurgical reconstruction of injured peripheral nerves, severed axons are able to undergo spontaneous regeneration. However, the functional result is always unsatisfactory with poor sensory recovery and reduced motor function. One contributing factor is the retrograde neuronal death, which occurs in the dorsal root ganglia (DRG) and in the spinal cord. An additional clinical problem is the loss of nerve tissue that often occurs in the trauma zone and which requires “bridges” to reconnect separated nerve ends. The present thesis investigates the extent of retrograde degeneration in spinal motoneurons and cutaneous and muscular afferent DRG neurons after permanent axotomy and following treatment with N-acetyl-cysteine (NAC). In addition, it examines the survival and growth-promoting effects of nerve reconstructions performed by primary repair and peripheral nerve grafting in combination with NAC treatment. In adult rats, cutaneous sural and muscular medial gastrocnemius DRG neurons and spinal motoneurons were retrogradely labeled with fluorescent tracers from the homonymous transected nerves. Survival of labeled neurons was assessed at different time points after nerve transection, ventral root avulsion and ventral rhizotomy. Axonal regeneration was evaluated using fluorescent tracers after sciatic axotomy and immediate nerve repair. Intraperitoneal or intrathecal treatment with NAC was initiated immediately after nerve injury or was delayed for 1-2 weeks. Counts of labeled gastrocnemius DRG neurons did not reveal any significant retrograde cell death after nerve transection. Sural axotomy induced a delayed loss of DRG cells, which amounted to 43- 48% at 8-24 weeks postoperatively. Proximal transection of the sciatic nerve at 1 week after initial axonal injury did not further increase retrograde DRG degeneration, nor did it affect survival of corresponding motoneurons. In contrast, rhizotomy and ventral root avulsion induced marked 26- 53% cell loss among spinal motoneurons. Primary repair or peripheral nerve grafting supported regeneration of 53-60% of the motoneurons and 47-49% of the muscular gastrocnemius DRG neurons at 13 weeks postoperatively. For the cutaneous sural DRG neurons, primary repair or peripheral nerve grafting increased survival by 19-30% and promoted regeneration of 46-66% of the cells. Regenerating sural and medial gastrocnemius DRG neurons upregulate transcription of peripherin and activating transcription factor 3. The gene expression of the structural neurofilament proteins of high molecular weight was significantly downregulated following injury in both regenerating and non-regenerating sensory neurons. Treatment with NAC was neuroprotective for spinal motoneurons after ventral rhizotomy and avulsion, and sural DRG neurons after sciatic nerve injury. However, combined treatment with nerve graft and NAC had significant additive effect on neuronal survival and also increased the number of sensory neurons regenerating across the graft. In contrast, NAC treatment neither affected the number of regenerating motoneurons nor the number of myelinated axons in the nerve graft and in the distal nerve stump. In summary, the present results demonstrate that cutaneous sural sensory neurons are more sensitive to peripheral nerve injury than muscular gastrocnemius DRG cells. Moreover, the retrograde loss of cutaneous DRG cells taking place despite immediate nerve repair would still limit recovery of cutaneous sensory functions. Experimental data also show that NAC provides a highly significant degree of neuroprotection in animal models of adult nerve injury and could be combined with nerve grafting to further attenuate retrograde neuronal death and to promote functional regeneration.

Spinal cord integrates somatosensory information and generates coordinated motor responses. Temporal integration can be used for discrimination of important stimuli from noise. Here it is shown that temporal integration of somatosensory inputs in sub second time scale is possible without changes of intrinsic properties of motoneurons. The activity of premotor neurons increases during temporal integration and can be a mechanism for short term information storage in spinal cord. Suppression of motor activity after painful somatosensory stimulus is called cutaneous silent period. This motor suppression is well described in humans and used for diagnostics. However it is not known if the suppression of motor activity is due to inhibition of motoneurons or reduction of excitatory drive from premotor neurons. Here it is shown that motoneurons are inhibited during cutaneous silent period. Neural networks of spinal cord not only process somatosensory information but generate locomotion and reflexes too. It is accepted that neural networks controlling front and hind limb movements are located in cervical and lumbar enlargements respectfully. Here it is shown that thoracic segments of spinal cord contribute to hind limb movements as well. It means that neural network generating movements is much more widely distributed than previously thought. / Nugaros smegenys gauna somatosensorinę informaciją, ją integruoja ir generuoja motorinius atsakus. Disertacijoje parodoma, kad somatosensorinių įėjimų viršsekundinė laikinė integracija nugaros smegenų neuronų tinkle vyksta ne dėl motorinių neuronų vidinių savybių kitimo. Laikinės integracijos metu padidėja priešmotorinių neuronų aktyvumas ir tai gali lemti informacijos apie somatosensorinį įėjimą saugojimą. Somatosensorinis tylos periodas – tai motorinio aktyvumo slopinimas po skausmingo stimulo. Jis plačiai aprašytas žmonėse, bei taikomas diagnostikoje. Nepaisant plataus taikymo, somatosensorinio tylos periodo mechanizmai nėra ištirti – nebuvo žinoma ar šis motorinio aktyvumo slopinimas vyksta slopinant motorinius neuronus, ar eliminuojant motorinių neuronų žadinimą. Disertacijoje parodoma, kad somatosensorinio tylos periodo metu motoriniai neuronai yra slopinami. Be somatosensorinės informacijos apdorojimo nugaros smegenų neuronų tinklai užtikrina judėjimo ir refleksų valdymą. Yra priimta, kad priekines ir užpakalines galūnes valdantys neuronų tinklai išsidėstę atitinkamai nugaros smegenų kaklinės ir strėnų sričių išplatėjimuose. Disertacijoje parodoma, kad ir krūtininiai nugaros smegenų segmentai prisideda prie užpakalinių galūnių motorinio aktyvumo generavimo. Tai leidžia manyti, kad neuronų tinklas generuojantis judesius yra išplitęs labiau, nei manyta iki šiol.

Nugaros smegenys gauna somatosensorinę informaciją, ją integruoja ir generuoja motorinius atsakus. Disertacijoje parodoma, kad somatosensorinių įėjimų viršsekundinė laikinė integracija nugaros smegenų neuronų tinkle vyksta ne dėl motorinių neuronų vidinių savybių kitimo. Laikinės integracijos metu padidėja priešmotorinių neuronų aktyvumas ir tai gali lemti informacijos apie somatosensorinį įėjimą saugojimą. Somatosensorinis tylos periodas – tai motorinio aktyvumo slopinimas po skausmingo stimulo. Jis plačiai aprašytas žmonėse, bei taikomas diagnostikoje. Nepaisant plataus taikymo, somatosensorinio tylos periodo mechanizmai nėra ištirti – nebuvo žinoma ar šis motorinio aktyvumo slopinimas vyksta slopinant motorinius neuronus, ar eliminuojant motorinių neuronų žadinimą. Disertacijoje parodoma, kad somatosensorinio tylos periodo metu motoriniai neuronai yra slopinami. Be somatosensorinės informacijos apdorojimo nugaros smegenų neuronų tinklai užtikrina judėjimo ir refleksų valdymą. Yra priimta, kad priekines ir užpakalines galūnes valdantys neuronų tinklai išsidėstę atitinkamai nugaros smegenų kaklinės ir strėnų sričių išplatėjimuose. Disertacijoje parodoma, kad ir krūtininiai nugaros smegenų segmentai prisideda prie užpakalinių galūnių motorinio aktyvumo generavimo. Tai leidžia manyti, kad neuronų tinklas generuojantis judesius yra išplitęs labiau, nei manyta iki šiol. / Spinal cord integrates somatosensory information and generates coordinated motor responses. Temporal integration can be used for discrimination of important stimuli from noise. Here it is shown that temporal integration of somatosensory inputs in sub second time scale is possible without changes of intrinsic properties of motoneurons. The activity of premotor neurons increases during temporal integration and can be a mechanism for short term information storage in spinal cord. Suppression of motor activity after painful somatosensory stimulus is called cutaneous silent period. This motor suppression is well described in humans and used for diagnostics. However it is not known if the suppression of motor activity is due to inhibition of motoneurons or reduction of excitatory drive from premotor neurons. Here it is shown that motoneurons are inhibited during cutaneous silent period. Neural networks of spinal cord not only process somatosensory information but generate locomotion and reflexes too. It is accepted that neural networks controlling front and hind limb movements are located in cervical and lumbar enlargements respectfully. Here it is shown that thoracic segments of spinal cord contribute to hind limb movements as well. It means that neural network generating movements is much more widely distributed than previously thought.

Este trabalho descreve o desenvolvimento de um sistema de simulação de circuitos neuronais, com interface de utilização amigável e arquitetura baseada em web. O sistema é direcionado ao estudo de redes de neurônios da medula espinhal, responsáveis pelo controle motor, sujeitas à ativação por vias superiores e periféricas ou por estímulos elétricos. Sua utilidade está relacionada à criação de hipóteses ou teorias sobre o processamento neuronal realizado no caso são ou patológico, a atividades como a interpretação de resultados de experimentos eletrofisiológicos realizados em humanos e no direcionamento e validação de procedimentos experimentais. Para os propósitos deste projeto, a simulação computacional é o recurso mais indicado a se utilizar, considerando o grande número de variáveis envolvidas e o caráter não-linear dos elementos constituintes. As simulações devem retratar de maneira fidedigna as principais propriedades que caracterizam os núcleos neuronais a se estudar. Essas propriedades estão associadas ao recrutamento de unidades motoras, às relações de entrada-saída dos conjuntos neuronais, à influência das vias aferentes sobre os motoneurônios, ao papel da inibição recorrente e da inibição recíproca, à geração de força e do sinal eletromiográfico, entre outros. A simulação do reflexo H, que é uma técnica muito importante utilizada em estudos neurofisiológicos, está presente neste trabalho. Pretende-se que o sistema de simulação aqui proposto seja uma ferramenta útil para pesquisa e ensino da neurofisiologia do controle motor, provendo subsídios que levem a um melhor entendimento dos circuitos neuronais modelados. / This work describes the development of a simulation system of neuronal circuitry, having a user-friendly interface and based on web architecture. The system is intended for studying spinal cord neuronal networks responsible for muscle control, subjected to descending drive or electrical stimulation. It is potentially useful in many activities, such as the interpretation of electrophysiological experiments conducted with humans, the proposition of hypotheses or theories on neuronal processing. Computer simulation is the most indicated approach to attain the objectives of this project because of the huge number of variables and the non-linear characteristics of the constituting elements. The simulations should mimic in a faithful way the main properties related to the modeled neuronal nuclei. These properties are associated with: i) motor-unit recruitment, ii) neuronal nuclei input-output relations, iii) afferent tract influence on motoneurons, iv) effects of recurrent inhibition and reciprocal inhibition, v) generation of force and electromyogram, and others. The generation of the H-reflex by the Ia-motoneuron pool system, which is an important tool in human neurophysiology, is included in the simulation system. The biological reality obtained with the present simulator and its web-based implementation make it a powerful tool for researchers in neurophysiology.

La sclérose latérale amyotrophique (SLA) est une maladie neurodégénérative incurable, qui touche les motoneurones de la moelle épinière et du cerveau. Elle se manifeste par une faiblesse musculaire qui évolue rapidement vers une paralysie générale, entrainant la mort du patient. Les principes moléculaires conduisant à la dégénérescence sélective des motoneurones demeurent encore mal connus, entravant le développement de nouvelles thérapies. Mon travail de thèse a permis l'identification d'une nouvelle voie de mort spécifique aux motoneurones, qui dépend du récepteur LT-βR et de son ligand LIGHT. De plus, cette voie de mort peut être déclenchée par une cytokine pro-inflammatoire, qui est l'interféron gamma (IFNγ). Nous avons pu montrer des signes d'activation de cette voie de mort chez des souris modèles de la SLA ainsi que dans les tissus de patients atteints de la maladie. En effet, on observe au cours de la maladie une augmentation des niveaux d'IFNγ dans les astrocytes et les motoneurones. Une approche génétique a par la suite permis de démontrer l'implication fonctionnelle de cette voie de mort dans le processus pathologique. / Amyotrophic lateral sclerosis (ALS) is a progressive, fatal neurodegenerative disease affecting primarily motoneurons in the brain and spinal cord. Symptoms of the disease include general muscle weakness, rapidly evolving in an overall paralysis, leading to the death of the patient. The precise mechanisms responsible for the selective vulnerability of motoneurons remain largely unknown, impeding therefore the development of effective therapies. My thesis work led to the discovery of a novel motoneuron selective death pathway dependent on the activation of LT-βR by LIGHT. This death pathway might also be triggered by the pro-inflammatory cytokine interferon gamma (IFNγ). Interestingly, we have documented signs of activation of this pathway in ALS mice and sporadic ALS patients, with IFNγ being upregulated in astrocytes and motoneurons. Furthermore, a genetic approach has provided evidence of the functional involvement of this death pathway in the pathogenic process.

No description available.

No description available.

La sclérose latérale amyotrophique (SLA) est une maladie neurodégénérative due à une dégénérescence des motoneurones. Plus de 40 mutations du gène TARDBP ont été identifiées chez des patients SLA. Les défauts biochimiques de ces mutations étant encore inconnus, les modèles animaux sont présentement la seule mesure possible d’un phénotype. Pour étudier les conséquences physiopathologiques d’une de ces mutations, nous avons développé deux lignées transgéniques de poisson zébré, exprimant le gène humain TARDBP soit de type sauvage, soit avec la mutation G348C liée à la SLA, sous le contrôle d’un promoteur de choc thermique. Ces lignées ont été étudiées sur trois générations, après avoir établi un protocole de choc thermique induisant une expression ubiquitaire du transgène. Les embryons transgéniques de la génération F2 de la lignée exprimant la mutation développent un phénotype moteur suite à un choc thermique de 38.5°C pendant 30 minutes lorsque les embryons sont à 18 heures post-fertilisation. 60% des embryons ont une réponse anormale au toucher. De plus, une réduction de 28% de la longueur de pré-branchement des axones des motoneurones est observée. Ces résultats indiquent que notre lignée exprimant la protéine mutante TDP-43 est un modèle génétique de la SLA prometteur, qui ouvre des perspectives pour la compréhension de la physiopathologie de la maladie et la découverte de molécules thérapeutiques. / Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease due to motoneurons degeneration. More than 40 mutations of the gene TARDBP, coding for the protein TDP-43 have been found in ALS patients. As the biochemical defects of these mutations are not known, in vivo models are currently the only windows onto the pathology. To study the pathophysiological consequences of one of these mutations, we have generated two stable zebrafish transgenic lines, expressing the human gene TARDBP, either the wild-type version, or the G348C mutated version linked to ALS, under the control of a heat shock promotor. These lines were studied for three generation, after establishing a heat shock protocol sufficient to induce a ubiquitous expression of the transgene. The transgenic embryos of the F2 generation of the line expressing the mutant protein develop a motor phenotype after a 38.5°C heat shock for 30 minutes when the embryos are 18 hours post-fertilization. 60% of these embryos have an abnormal touch escaped evoked response, and a 28% reduction of the pre-branching axonal length of the motoneurons axons. These results indicate that our line expressing the mutant TDP-43 protein is a promising genetic model of ALS, opening perspectives for the pathophysiological understanding of the disease, and the discovery of new therapeutics.

LIGHT, un membre de la famille du TNF, déclenche la mort des motoneurones et contribue à la progression de la dégénérescence des motoneurones chez les souris modèles de la sclérose latérale amyotrophique. Étonnement, nous montrons que LIGHT peut également stimuler la croissance axonale in vitro et est nécessaire à la régénération axonale des motoneurones in vivo lors d'une lésion nerveuse périphérique. La compréhension des mécanismes dictant la bivalence de son récepteur mène à l'étude des effets de la régionalisation du signal LIGHT. Les chambres microfluidiques en isolant les axones des corps cellulaires, nous ont permis d'étudier la résultante fonctionnelle d'une stimulation axonale et somatique par LIGHT. Nous montrons que LIGHT active différentiellement la mort, quand il agit au niveau somatique, ou la régénération axonale, quand il stimule l'axone. L'ensemble de ces résultats mène à de nouvelles approches où l'administration de LIGHT au niveau du nerf lésé et du muscle correspondant nous renseignera sur le potentiel thérapeutique de LIGHT lors d'une lésion du système nerveux. / LIGHT, a member of the TNF superfamily, triggers death of motoneurons and contribute to the progression of motoneuron degeneration in an ALS mouse model. Interestingly, we demonstrate that LIGHT can elicit neurite outgrowth in vitro and contribute to axon regeneration in vivo following nerve injury. To better understand mechanisms governing the opposite effects of LIGHT on motoneurons we asked whether a regional activation of LIGHT pathway could trigger these opposite responses. Toward this goal we used microfluidic chambers to isolate the soma from the axon of motoneurons. We shows that LIGHT activates differentially motoneurons death when it acts at the soma and axon outgrowth when it acts at the level of the axon. Together, these results lead us to evaluate new therapeutic approaches to nerve injury.

La Sclérose Latérale Amyotrophique (SLA) est une maladie neurodégénérative au cours de laquelle les motoneurones meurent. Le premier dysfonctionnement des motoneurones est la rétractation de leurs jonctions neuromusculaires. La présence de vacuoles a été décrite dans l’axone et les dendrites des motoneurones avant la dénervation dans les souris SOD1G93A, modèle murin de la maladie. L’origine des vacuoles n’est pas connue. On peut toutefois se demander si elle pourrait résulter d’un stress excitotoxique. L’excitotoxicité pourrait provenir soit d’une hyperexcitabilité intrinsèque du motoneurone, soit d’une hyperexcitation (balance des entrées excitatrices et inhibitrices modifiées au profit d’une plus grande excitation). Or il a été montré que si les motoneurones sont hyperexcitables au stade embryonnaire dans les souris SOD1G93A, seuls les motoneurones résistants à la SLA (type S) sont hyperexcitables à la deuxième semaine postnatale tandis que les motoneurones vulnérables (types FF et FR) deviennent hypoexcitables avant leur dégénérescence chez l’adulte. Nous avons donc étudié les entrées synaptiques reçues par les motoneurones, pour savoir si la balance excitation/inhibition est déplacée et s’ils sont ainsi hyperexcités. Pour cela nous avons réalisé des enregistrements électrophysiologiques de motoneurones lors de la stimulation de circuits pré-moteurs, et des marquages intracellulaires de motoneurones combinés avec des marquages immunohistochimiques des boutons VGlut1, VGlut2 et Vgat. Nous avons montré que l’amplitude des PPSE monosynaptiques Ia était diminuée dans les souris SOD1, les PPSI di- et trisynaptiques étaient moins nombreux et les interneurones inhibiteurs moins excitables. Cette modification des entrées synaptiques n’était pas due à un changement du nombre de synapses. En revanche, les synapses sont particulièrement nombreuses aux domaines dendritiques qui se vacuolisent dans les souris SOD1, suggérant un lien entre l’activité synaptique et la vacuolisation. Des marquages intracellulaires de motoneurones de souris SOD1, montrent que les vacuoles grandissent avec l’évolution de la maladie, suggérant leur implication dans le processus de dégénération. Grâce à des révélations immunohistochimiques, nous avons montré que ces vacuoles apparaissent dans l’espace intermitochondrial lors de la dégénérescence des mitochondries. Le réticulum endoplasmique est également impliqué. Enfin, l’autophagie, mécanisme d’élimination des organites cellulaires, est déficient au moment de l’apparition des vacuoles, expliquant pourquoi elles s’accumulent avec le temps. Ces résultats amènent à reconsidérer l’hypothèse de l’excitotoxicité supposée comme mécanisme à l’origine de la mort des motoneurones. / Glutamate excitotoxicity arising from excessive entry of calcium in the cell, has long been suggested to contribute to the degeneration of motoneurons in Amyotrophic Lateral Sclerosis (ALS). This hypothesis is enhanced by the observation of vacuoles on motoneurones dendritic tree. Such vacuoles were previously observed on neurons under excitotoxic stress. Excitotoxicity may stem from an intrinsic hyperexcitability of the motoneurons or from a shift of the balance of excitatory / inhibitory inputs received by the motoneurons toward more excitation. Thanks to an in vivo preparation that allows us to make intracellular recordings of motoneurons in adult mice, it was shown that spinal motoneurons do not display an intrinsic hyperexcitability just prior to their degeneration in SOD1 G93A mice, the standard model of ALS. Thus, to study excitotoxicity hypothesis, we decided to study dendritic vacuoles and undersand their genesis, and then to study synaptic inputs on motoneurons, to decipher if there is a hyperexcitability. We have shown, with intracellular labelling and immunohistochemistry, that vacuoles grow with age, that they appear in the intermembrane space of mitochondria, and that deficiency in autophagy prevent their elimination. With electrophysiological recordings, we have shown that monosynaptic EPSP amplitude is reduced in SOD1 mice. IPSP were less numerous and inhibitory interneurons were less excitable. These alterations were not due to synapses numbers, however synapses are preferentially localised on dendritic places that vacuolate, suggesting a link between synaptic activity and vacuolation. These results suggest that excitotoxicity might not be the mecanism of motoneuron death.