No description available.

Transcription factors of the helix-loop-helix (HLH) gene family are widespread in the animal kingdom. Among them, members of HLH subfamilies such as ASCL, Neurogenin, NeuroD, COE, Atonal, Oligo, NSCL, Hairy/E(spl) and Hey (here referred to as neural HLH genes) have been shown to be fundamental for the development of the nervous system. They are expressed at different time periods of neuronal differentiation, from the specification of ectoderm towards a neural lineage, to the ultimate differentiation of neurons. Few HLH genes have been identified in the lamprey; however, considering the wide diversity of HLH gene subfamilies in metazoans, including vertebrates, it is very likely that lampreys possess a large repertoire of HLH genes in their genome. In the present study, the identification of several HLH genes in the lamprey genome, as well as the isolation and expression of different lamprey neural HLH genes is reported. As expected, a wide repertoire of HLH genes was identified in the sea lamprey (Petromyzon marinus) genome. On the other hand, the identification and expression analysis of different neural HLH genes of the ASCL, Neurogenin, COE and Hairy/E(spl) in the brook lamprey Lampetra planeri showed an overall conservation with other vertebrates, both at the sequence and expression pattern levels. In addition, novel features of the lamprey nervous system are revealed, such as the identification of possible new sensory cranial placodes in pharyngeal arches. Furthermore, these genes can serve as molecular markers for different cranial placodes and dorsal root ganglia (DRG), and their expression also highlights the presence of a ventricular zone in the brain and spinal cord, along with a complementary marginal zone. Finally, with the use of a Notch pathway inhibitor in developing L. planeri embryos, the regulation of expression of the isolated genes by the Notch signaling pathway was shown to be generally conserved between lampreys and gnathostomes in the spinal cord. This functional study also revealed that the lamprey spinal cord likely presents an independent developmental programme from the brain. All together, the present study shows that the analysis of neural HLH genes represents an excellent tool to understand the lamprey nervous system.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Diversos estudos demonstram que o estresse infantil, incluindo situações de perda dos pais, negligência e abusos, representa um forte fator de risco para o desenvolvimento de transtornos de ansiedade, sendo de especial interesse para este trabalho, o transtorno do pânico. Modelos de estresse neonatal em animais de laboratório, que se baseiam na ruptura da relação mãe-filhote, como a separação materna e a privação materna, têm sido amplamente utilizados para avaliar as consequências desse estressor sobre a expressão de comportamentos defensivos associados à ansiedade na vida adulta. No entanto, pouco se sabe sobre seus efeitos em modelos animais de ataques de pânico, mais especificamente aqueles que associam esta condição emocional à resposta defensiva de fuga em animais. Diante disso, o objetivo inicial do presente trabalho foi o de estender as investigações dos efeitos do estresse neonatal sobre o comportamento de fuga de ratos adultos (após 60 dias de nascimento) observado no labirinto em T elevado (LTE), pela estimulação elétrica da substância cinzenta periaquedutal (SCPD) e durante a exposição a um ambiente em hipóxia (7% O2). Para efeitos comparativos, esses animais também foram testados em modelos animais associados à ansiedade generalizada e a depressão. Observamos que ratos Wistar submetidos à separação materna (3h/dia, do 2º ao 21º dia pós-nascimento) não diferiram de animais controles nos parâmetros comportamentais analisados nos modelos de pânico (fuga no LTE e pela estimulação elétrica da SCPD), nos de ansiedade (resposta de esquiva no LTE e o beber punido no teste de conflito de Vogel) ou no de depressão (tempo gasto em imobilidade no teste do nado forçado). Já em ratos privados da mãe (por 24h no 11º dia pós- nascimento), embora este estressor não tenha alterada a resposta de fuga no LTE, ele aumentou a expressão deste comportamento durante a exposição à hipóxia, sugestivo de um efeito panicogênico. Ainda empregando a privação materna, observamos que a administração intraperitoneal de um inibidor da síntese de serotonina, a pclorofenilalanina metil éster (p-CPA - 100mg/Kg/dia, por 4 dias antes dos testes comportamentais) facilitou a expressão do comportamento de fuga durante o teste da hipóxia nos animais controle, de maneira semelhante ao efeito obtido somente com a privação materna. Porém este tratamento não potencializou a fuga promovida pela privação materna. Já os níveis plasmáticos de corticosterona foram aumentados pela exposição à hipóxia, independentemente dos animais terem sido previamente privados da mãe ou terem recebido o pCPA antes do teste. Por fim, também observamos, através de uma análise por Western Blotting, que nem a privação materna ou a exposição à hipóxia altera a concentração de receptores serotonérgicos do tipo 5-HT1A na SCPD ou na amígdala. Em suma, nossos resultados mostram que a privação materna promove uma facilitação da resposta de fuga na hipóxia, sugerindo uma relação entre esse estresse neonatal e o desencadeamento de ataques de pânico de um subtipo específico, o pânico respiratório. Contudo, no que diz respeito ao envolvimento da neurotransmissão serotonérgica, mais estudos são necessários para entender sua participação nessa resposta. / Early life stress (ELS), including parental loss due to death, neglect or abuse, represents a major risk factor for the late development of psychiatric disorders, such as anxiety disorders. Animal models of ELS that are based on the disruption of mother-infant relationship, such as the repeated maternal separation or maternal deprivation, have been extensively used for the investigation of the longterm effects of these stressors on the expression of defensive behaviors associated with anxiety. However, little is known about their effects on animal models of panic attacks, more specifically in those that associate this emotional condition with escape behavior in animals. Therefore, the aim of the present study was to extend the investigation on the long-term consequences of neonatal stress on the escape response of adult rats (60 days after birth) evoked by the elevated T maze (ETM), electrical stimulation of the dorsal periaqueductal gray (DPAG) or hypoxia (7% O2). For comparative reasons, these animals were also tested in animal models of anxiety and depression. The results showed that the repeated maternal separation of male Wistar (3 hours/day from day 2 to 21 after birth) did not affect the behavioral indexes measured in the panic (escape in the ETM or after DPAG electrical stimulation), anxiety (ETM inhibitory avoidance or punished licking in the Vogel conflict test) or depression (time in immobility in the forced swimming test) models. On the other hand, rats submitted to maternal deprivation (24 hs in the 11th day after birth), although not differing from the control animals on escape expression in the ETM, showed a pronounced escape response during hypoxia, indicating a panicogenic-like response. Also, using this maternal deprivation protocol, we observed that systemic administration of a 5-HT synthesis inhibitor, p-chlrophenilalanine metylester (p-CPA - 100mg/Kg/day, for 4 days before the behavioral tests), facilitated escape expression during hypoxia in non-deprived animals to a level observed in non-pharmacologically treated deprived animals. We also observed that plasma corticosterone levels were increased 30 minutes after hypoxia exposure, independently of the previous condition of the animals (deprivation or drug treatment). Finally, we observed that the number of 5-HT1A receptors in the DPAG or amygdala, measured by Western Blotting, was not affect by previous maternal deprivation or exposure to hypoxia. Taken together, our results show that a single maternal deprivation episode facilitates the expression of escape behavior during hypoxia, suggesting a relationship between this ELS with the observation of a specific subtype of panic attack, the respiratory panic. Further studies are required in order to clarify the 5- HT involvement on these responses.

Extensos dados na literatura têm assinalado a importância da serotonina (5-HT) na modulação de comportamentos de medo e ansiedade em roedores, a grande maioria utilizando ratos machos como sujeitos experimentais. No presente trabalho, foi analisada a influência da neurotransmissão 5-HT periférica e central sobre a expressão da resposta de medo condicionado e incondicionado de ratos machos e fêmeas, previamente selecionados pelo fenótipo como animais de baixa (BA) ou de alta (AA) reatividade emocional. Para este fim foi utilizado o teste do sobressalto potencializado pelo medo (SPM). A influência global e central de 5-HT foi avaliada pela utilização da administração sistêmica ou intraventricular aguda do inibidor irreversível da enzima triptofano hidroxilase PCPA (p-clorofenilalanina) nas doses de 200 mg/mL (i.p.) e 200 µg/5 µL (i.v.), ou através de infusões locais de 5-HT (10 nmol/0.2 µL) ou do agonista seletivo de receptores 5-HT1A 8-hidroxi-2-(di-n-propilamino)-tetralina (8-OH-DPAT) na dose de 0,3 µg/0.2 µL, diretamente em áreas encefálicas conhecidas por sua influência na modulação do medo e ansiedade a saber: o córtex pré-limbico (CPL), o núcleo basolateral da amígdala (BLA), e a substância cinzenta periaqueductal dorsal (SCPd) do mesencéfalo. As variáveis dependentes registradas foram a amplitude e a latência das respostas incondicionadas e condicionadas de sobressalto e a amplitude do SPM. / Extensive data in the literature have signaled the importance of serotonin (5-HT) on the modulation of fear and anxiety-like behaviors in rodents. In the present study, we have analyzed the influence of peripheral and central 5-HT neurotransmission on the expression of the conditioned and unconditioned fear, and on the fear-potentiated startle in male and female rats previously selected as low- (LA) or high-anxiety (HA). For this purpose, we used the fear-potentiated startle (FPS) test. The global and central influence of 5-HT was evaluated by using the acute systemic or intraventricular administration of the irreversible tryptophan hydroxylase inhibitor PCPA (p-chlorophenylalanine - 200 mg/mL i.p., or 200 µg/5 µL i.v.). Local effects were evaluated through local infusions of 5-HT itself (10 nmol/0.2 µL) or the selective 5-HT1A receptors agonist 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT 0.3 µg/0.2 µL) in the prelimbic cortex (PrL), basolateral amygdala (BLA) or the dorsal periaqueductal gray (DPAG). These brain regions were chosen for the present study based on their great importance in the modulation and expression of conditioned and unconditioned fear. Dependent variables recorded were the amplitude and latency of unconditioned and conditioned fear, and fear-potentiated startle (FPS).

La douleur neuropathique est due à une lésion ou une pathologie du système nerveux somatosensoriel. La prégabaline, un anticonvulsivant, et la duloxétine, un antidépresseur, sont des traitements de référence, efficaces chez un tiers des patients. Mieux comprendre leurs mécanismes d’action est crucial pour améliorer leur tolérance et leur efficacité. En utilisant un modèle murin de douleur neuropathique périphérique, nous montrons que : 1) la prégabaline, dont l’action est indépendante du système opioïdergique, agit sur la composante neuroimmunitaire périphérique de la douleur ; 2) la duloxétine agit via deux mécanismes indépendants, l’un central (contrôles descendants) pour un traitement aigu et l’autre périphérique (ganglion rachidien) pour un traitement chronique. Dans ce cas, l’analyse transcriptomique met en évidence une inhibition de l’inflammation neurogène. La comparaison des taux plasmatiques de duloxétine chez l’homme et chez la souris suggère une action périphérique chez l’homme. / Neuropathic pain is caused by a lesion or a disease of the somatosensory nervous system. Pregabalin, an anticonvulsant, and duloxetine, an antidepressant, are the standard treatments, effective in one-third of patients. A better understanding of their mechanisms of action is a crucial point to improve their tolerance and efficiency. By using a murine model of peripheral neuropathy, we have shown that : 1) pregabalin, whose effect is independent from the opioid system, acts on the peripheral neuroimmune component of pain ; 2) duloxetine acts via two independent mechanisms, one central (descending controls) for an acute treatment and the other peripheral (dorsal root ganglia) for a chronic treatment. In this case, transcriptomic analysis hightlights an inhibition of the neurogenic inflammation. Comparison of duloxetine plasmatic levels in humans and mice suggests a peripheral action in humans.

Diversos estudos demonstram que o estresse infantil, incluindo situações de perda dos pais, negligência e abusos, representa um forte fator de risco para o desenvolvimento de transtornos de ansiedade, sendo de especial interesse para este trabalho, o transtorno do pânico. Modelos de estresse neonatal em animais de laboratório, que se baseiam na ruptura da relação mãe-filhote, como a separação materna e a privação materna, têm sido amplamente utilizados para avaliar as consequências desse estressor sobre a expressão de comportamentos defensivos associados à ansiedade na vida adulta. No entanto, pouco se sabe sobre seus efeitos em modelos animais de ataques de pânico, mais especificamente aqueles que associam esta condição emocional à resposta defensiva de fuga em animais. Diante disso, o objetivo inicial do presente trabalho foi o de estender as investigações dos efeitos do estresse neonatal sobre o comportamento de fuga de ratos adultos (após 60 dias de nascimento) observado no labirinto em T elevado (LTE), pela estimulação elétrica da substância cinzenta periaquedutal (SCPD) e durante a exposição a um ambiente em hipóxia (7% O2). Para efeitos comparativos, esses animais também foram testados em modelos animais associados à ansiedade generalizada e a depressão. Observamos que ratos Wistar submetidos à separação materna (3h/dia, do 2º ao 21º dia pós-nascimento) não diferiram de animais controles nos parâmetros comportamentais analisados nos modelos de pânico (fuga no LTE e pela estimulação elétrica da SCPD), nos de ansiedade (resposta de esquiva no LTE e o beber punido no teste de conflito de Vogel) ou no de depressão (tempo gasto em imobilidade no teste do nado forçado). Já em ratos privados da mãe (por 24h no 11º dia pós- nascimento), embora este estressor não tenha alterada a resposta de fuga no LTE, ele aumentou a expressão deste comportamento durante a exposição à hipóxia, sugestivo de um efeito panicogênico. Ainda empregando a privação materna, observamos que a administração intraperitoneal de um inibidor da síntese de serotonina, a pclorofenilalanina metil éster (p-CPA - 100mg/Kg/dia, por 4 dias antes dos testes comportamentais) facilitou a expressão do comportamento de fuga durante o teste da hipóxia nos animais controle, de maneira semelhante ao efeito obtido somente com a privação materna. Porém este tratamento não potencializou a fuga promovida pela privação materna. Já os níveis plasmáticos de corticosterona foram aumentados pela exposição à hipóxia, independentemente dos animais terem sido previamente privados da mãe ou terem recebido o pCPA antes do teste. Por fim, também observamos, através de uma análise por Western Blotting, que nem a privação materna ou a exposição à hipóxia altera a concentração de receptores serotonérgicos do tipo 5-HT1A na SCPD ou na amígdala. Em suma, nossos resultados mostram que a privação materna promove uma facilitação da resposta de fuga na hipóxia, sugerindo uma relação entre esse estresse neonatal e o desencadeamento de ataques de pânico de um subtipo específico, o pânico respiratório. Contudo, no que diz respeito ao envolvimento da neurotransmissão serotonérgica, mais estudos são necessários para entender sua participação nessa resposta. / Early life stress (ELS), including parental loss due to death, neglect or abuse, represents a major risk factor for the late development of psychiatric disorders, such as anxiety disorders. Animal models of ELS that are based on the disruption of mother-infant relationship, such as the repeated maternal separation or maternal deprivation, have been extensively used for the investigation of the longterm effects of these stressors on the expression of defensive behaviors associated with anxiety. However, little is known about their effects on animal models of panic attacks, more specifically in those that associate this emotional condition with escape behavior in animals. Therefore, the aim of the present study was to extend the investigation on the long-term consequences of neonatal stress on the escape response of adult rats (60 days after birth) evoked by the elevated T maze (ETM), electrical stimulation of the dorsal periaqueductal gray (DPAG) or hypoxia (7% O2). For comparative reasons, these animals were also tested in animal models of anxiety and depression. The results showed that the repeated maternal separation of male Wistar (3 hours/day from day 2 to 21 after birth) did not affect the behavioral indexes measured in the panic (escape in the ETM or after DPAG electrical stimulation), anxiety (ETM inhibitory avoidance or punished licking in the Vogel conflict test) or depression (time in immobility in the forced swimming test) models. On the other hand, rats submitted to maternal deprivation (24 hs in the 11th day after birth), although not differing from the control animals on escape expression in the ETM, showed a pronounced escape response during hypoxia, indicating a panicogenic-like response. Also, using this maternal deprivation protocol, we observed that systemic administration of a 5-HT synthesis inhibitor, p-chlrophenilalanine metylester (p-CPA - 100mg/Kg/day, for 4 days before the behavioral tests), facilitated escape expression during hypoxia in non-deprived animals to a level observed in non-pharmacologically treated deprived animals. We also observed that plasma corticosterone levels were increased 30 minutes after hypoxia exposure, independently of the previous condition of the animals (deprivation or drug treatment). Finally, we observed that the number of 5-HT1A receptors in the DPAG or amygdala, measured by Western Blotting, was not affect by previous maternal deprivation or exposure to hypoxia. Taken together, our results show that a single maternal deprivation episode facilitates the expression of escape behavior during hypoxia, suggesting a relationship between this ELS with the observation of a specific subtype of panic attack, the respiratory panic. Further studies are required in order to clarify the 5- HT involvement on these responses.

Les glucocorticoïdes (GC) sont des hormones stéroïdes synthétisées par les glandes surrénales. La production de ces GC est une des réponses de l’organisme pour rétablir l’homéostasie grâce à différentes actions comprenant des effets centraux sur le comportement et la douleur. C’est ce dernier qui a fait l’objet de mes travaux dans le cadre de cette thèse.Les afférences sensorielles primaires véhiculent les informations de la périphérie dans les cornes dorsales de la moelle épinière. Ces informations qui peuvent être nociceptives sont modulées par un réseau de neurones spinal avant d’être transmises et intégrées. Nous avons montré que les GC sont impliqués dans la maturation et le fonctionnement de la transmission inhibitrice faisant intervenir le neurotransmetteur GABA. Dans les cornes dorsales, cette inhibition est cruciale pour limiter les mécanismes de transmission de l’information nociceptive. / Glucocorticoids (GC) are steroid hormones synthesized in adrenals following HPA axis activation. GC production is a response of the organism to alleviate homeostasis perturbations through different actions. One of them involves central neuronal modulation of behavior and pain perception.Primary afferents convey peripheral sensory information in the dorsal horns of the spinal cord. This information can be nociceptive and are modulated by a spinal neuronal network before being transmitted and integrated. We showed that GC are implied in the maturation and functioning of the inhibitory transmission involving GABA neurotransmitter. In the dorsal horns this inhibitory transmission is of major importance, limiting the processing of nociceptive information.

Doctor of Philosophy(PhD) / Two recently identified transient receptor potential (TRP) channels, TRPM8 and TRPA1, have been proposed to play an important role in mammalian cool and cold peripheral sensory transduction. When expressed in cell-lines the cloned TRPM8 and TRPA1 receptors have distinct pharmacological and temperature response characteristics. Although these receptors are also transported to the central terminals of primary afferents, little is known about their centrally mediated actions. In this thesis, I use an in vitro electrophysiological approach to investigate the dorsal horn processing of cool afferent modalities and the role of TRP ion channels. The results of this thesis provide further information on thermal processing, indicate direction for further research and suggest possible therapeutic targets for the management of abnormal cold sensory processing. Initial experiments demonstrate that the cooling agents and known TRPM8 and TRPA1 agonists, menthol and icilin, inhibit primary afferent evoked excitatory postsynaptic currents (EPSCs) in rat spinal cord dorsal horn neurons. In addition, temperature reduction, menthol and icilin increase the frequency of miniature EPSCs without affecting amplitude distribution or kinetics. Little or no direct postsynaptic effect on dorsal horn neurons, GABAergic or glycinergic transmission was found. In combination, these observations demonstrate that temperature reduction, menthol and icilin act presynaptically to increase the probability of glutamate release from primary afferent fibres. Further examination of the changes in glutamatergic synaptic transmission induced by temperature reduction, menthol and icilin reveals a subset of neurons sensitive to innocuous cool (< 29 oC) and low concentrations of icilin (3-10 µM) which closely match the temperature activation and pharmacological profile of TRPM8. In addition, the majority of lamina I and II neurons displayed characteristics partly consistent with TRPA1-activation, including a concentration-dependent response to icilin and blockade by ruthenium red. The present experiments did not allow thermal characterisation of these TRPA1-like responses. Together these observations indicate that the effects of menthol and icilin on glutamatergic synaptic transmission in the superficial dorsal horn are mediated by TRPM8 and possibly by TRPA1. Examination of the anatomical location of neurons activated by temperature reduction, menthol, icilin and capsaicin allowed the central termination pattern of thermoreceptive primary afferent fibres with specific TRP-like response characteristics to be determined. TRPM8-like presynaptic activation was confined to a subpopulation of neurons located in lamina I and outer lamina II, while the majority of neurons throughout laminae I and II received inputs sensitive to menthol, high concentrations of icilin and capsaicin. These findings suggest that innocuous cool sensation projects to a specific subpopulation of superficial dorsal horn neurons unlike other modalities (mediated by TRPV1, possibly TRPA1 and other receptors), which non-selectively engage circuits within the entire superficial dorsal horn. No morphological specificity was identified for recovered neurons after electrophysiological characterisation. Finally, mu-opioids were shown to inhibit basal glutamatergic synaptic transmission as well as menthol- and icilin-induced transmission in the superficial dorsal horn. Of particular interest, delta-opioids selectively inhibited icilin-induced synaptic transmission within the same location. The selective effect of delta-opioids suggests a possible role in modulating receptors activated by icilin (TRPM8 and TRPA1). Overall, this thesis provides further evidence that TRPM8 is responsible for the transduction of innocuous cold sensation in mammals and is a potential therapeutic target in humans with cold hyperaesthesia secondary to abnormal thermal processing. The use of delta-opioid agonists warrants further investigation in cold hypersensitivity states and potentially other forms of pain.

Peripheral nerve trauma is a common cause of considerable functional morbidity, and healthcare expenditure. Particularly in the ~15% of injuries unsuitable for primary repair, standard clinical management results in inadequate sensory restitution in the majority of cases, despite the rigorous application of complex microsurgical techniques. This can largely be explained by the failure of surgical management to adequately address the neurobiological hurdles to optimal regeneration. Most significant of these is the extensive sensory neuronal death that follows injury, and which is accompanied by a reduction in the regenerative potential of axotomised neurons, and in the supportive capacity of the Schwann cell population if nerve repair is delayed. The present study aimed to accurately delineate the timecourse of neuronal death, in order to identify a therapeutic window during which clinically applicable neuroprotective strategies might be adopted. It then proceeded to investigate means to increase the regenerative capacity of chronically axotomised neurons, and to augment the Schwann cells’ ability to promote that regenerative effort. Unilateral sciatic nerve transection in the rat was the model used, initially assessing neuronal death within the L4&amp;5 dorsal root ganglia by a combination of morphology, TdT uptake nick-end labelling (TUNEL), and statistically unbiased estimation of neuronal loss using the stereological optical disector technique. Having identified 2 weeks, and 2 months post-axotomy as the most biologically relevant timepoints to study, the effect upon neuronal death of systemic treatment with acetyl-L-carnitine (ALCAR 10, or 50mg/kg/day) or N-acetyl-cysteine (NAC 30, or 150mg/kg/day) was determined. A model of secondary nerve repair was then adopted; either 2 or 4 months after unilateral sciatic nerve division, 1cm gap repairs were performed using either reversed isografts, or poly-3-hydroxybutyrate (PHB) conduits containing an alginate-fibronectin hydrogel. Six weeks later nerve regeneration and the Schwann cell population were quantified by digital image analysis of frozen section immunohistochemistry. Sensory neuronal death begins within 24 hours of injury, but takes 1 week to translate into significant neuronal loss. The rate of neuronal death peaks 2 weeks after injury, and neuronal loss is essentially complete by 2 months post-axotomy. Nerve repair is incompletely neuroprotective, but the earlier it is performed the greater the benefit. Two clinically safe pharmaceutical agents, ALCAR &amp; NAC, were found to virtually eliminate sensory neuronal death after peripheral nerve transection. ALCAR also enhanced nerve regeneration independently of its neuroprotective role. Plain PHB conduits were found to be technically simple to use, and supported some regeneration, but were not adequate in themselves. Leukaemia inhibitory factor enhanced nerve regeneration, though cultured autologous Schwann cells (SC’s) were somewhat more effective. Both were relatively more efficacious after a 4 month delay in nerve repair. The most profuse regeneration was found with recombinant glial growth factor (rhGGF-2) in repairs performed 2 months after axotomy, with results that were arguably better than were obtained with nerve grafts. A similar conclusion can be drawn from the result found using both rhGGF-2 and SC’s in PHB conduits 4 months after axotomy. In summary, these findings reinforce the significance of sensory neuronal death in peripheral nerve trauma, and the possibility of its` limitation by early nerve repair. Two agents for the adjuvant therapy of such injuries were identified, that can virtually eliminate neuronal death, and enhance regeneration. Elements in the creation of a bioartificial nerve conduit to replace, or surpass autologous nerve graft for secondary nerve repair are presented.