Immunohistochemical studies were conducted to assess the subunits of ionotropic and metabotropic glutamate receptor present in the rostral ventrolateral medulla (RVLM) of the rat. Double labeling the medullary sections with polyclonal GluR1, GluR2/3, GluR4, NMDAR1, NMDAR2A/B, mGluR1α, and mGluR2/3 antiserum and monoclonal tyrosine hydroxylase (TH) antiserum revealed nearly all TH immunoreactive (irTH) cells and many TH-negative neurons were immunoreactive to GluR2/3 (irGluR2/3), NMDAR1 (irNMDAR1), and NMDAR2A/B (irNMDAR2A/B). A few RVLM neurons were immunoreactive to GluR1 (irGluR1) and GluR4 (irGluR4), but they were generally TH-negative. Immunoreactivity to mGluR1α (irmGluR1α) appeared to be localized exclusively to fiber-like elements in the RVLM area. Our results show that neurons in the RVLM, including irTH, are endowed mainly with GluR2/3 and NMDAR1 or NMDAR2A/B ionotropic receptor subunits, and that irmGluR1α splice variant appears to be located on nerve fibers ramifying within the RVLM. Moreover, TH-negative neurons in the RVLM appear to bear similar subunits of ionotropic glutamate receptors.

No description available.

No description available.

No description available.

La méthylation de l’ADN est une modification majeure du génome des eucaryotes permettant de moduler l’expression génique et contrôler le développement des mammifères. La protéine Mecp2 (Methyl CpG binding protein 2), dont le gène est situé sur le chromosome X, appartient à la famille des protéines de liaison à l’ADN méthylé. Sur la base de sa structure et de ses interactions Mecp2 a été décrit comme un répresseur de l’expression des gènes. A l’heure actuelle, son implication en tant qu’activateur de la transcription et organisateur de la structure chromatinienne lui confère un rôle plus global dans la régulation de l’épigénome. Des mutations de MECP2 conduisent à des troubles neurologiques dont le principal est le syndrome de Rett (RTT). Cette pathologie dominante liée à l’X affecte principalement les jeunes filles (incidence: 1/15000 naissances). Même si les causes précises du phénotype RTT ne sont pas connues, le profil d’expression de Mecp2 est en lien avec la synaptogenèse, la maturation et la maintenance des réseaux neuronaux. A mon arrivée en thèse l’équipe qui m’a accueilli venait d’identifier des déficits neuronaux, affectant notamment les groupes catécholaminergiques bulbaires et périphériques, à l’origine de troubles respiratoires chez un modèle murin de cette pathologie. Mon travail de thèse a permis de caractériser l’évolution postnatale des déficits moteurs et physiologiques affectant la souris Mecp2-déficiente. L’étude de structures catécholaminergiques d’intérêt telles que la Substantia Nigra et le Locus Coeruleus a révélé que les neurones dopaminergiques et noradrénergiques centraux ont un métabolisme affecté. Le nombre de neurones immunomarqués apparait significativement réduit dans ces groupes ce qui résulterait d’une perte progressive du phénotype « catécholaminergique », en l’absence de mort cellulaire. Nos données suggèrent que ces atteintes constituent un corrélat neuropathologique aux troubles comportementaux observés chez les souris Mecp2-déficientes. Ainsi certains troubles moteurs ont pu être améliorés, à l’aide d’un agent pharmacologique pro-dopaminergique, la L-Dopa. En relation avec les déficits en Bdnf (Brain-derived neurotrophic factor) décrits chez les patientes et les souris Mecp2-déficientes, nous avons identifié qu’une modification du dosage de Mecp2 induit une dérégulation de gènes (Htt, Hap1) codant des protéines impliquées dans le transport intracellulaire des vésicules de Bdnf. Nos travaux nous permettent de postuler que chez la souris Mecp2-déficiente, une altération de la dynamique de transport des vésicules chargées en Bdnf pourrait exacerber le déficit d’expression de cette neurotrophine. Notre traitement des souris Mecp2-déficientes par la cystéamine, une molécule capable d’agir sur les contenus, la libération et la sécrétion du Bdnf permet d’augmenter la survie des animaux et de réduire leurs troubles moteurs. Nos résultats montrent que les déficiences en Mecp2 entrainent des déficits de transport axonal du Bdnf qui s’ajoutent aux déficits de production du Bdnf. Par ailleurs, avec l’utilisation d’agents pharmacologiques agissant sur ce transport, nous offrons de nouvelles perspectives thérapeutiques. / DNA methylation is the major modification of eukaryotic genomes and plays an essential role in mammalian development. The protein Mecp2 (Methyl CpG binding protein 2), encoded by a gene located on the X chromosome, belongs to the ‘Methyl Binding domain’ protein family. Based on its structure and its interactions Mecp2 has historically been described as a repressor of expression for many genes. Currently, its involvement as an activator of transcription and its role in chromatin architecture suggests that it could be a global regulator of the epigenome. Mutations in MECP2 lead to neurological disorders, among which Rett syndrome (RTT). This dominant X-linked pathology mainly affects girls (incidence: 1/15000 live births). Although the precise causes of the RTT phenotype are unknown, the pattern of Mecp2 expression is related to synaptogenesis, maturation and neuromaintenance. Before my integration in the ‘Human Neurogenetics’ team, this group identified neural deficits, affecting brainstem and peripheral catecholaminergic cell groups, causing respiratory disturbances in a mouse model of this disease. My thesis work enabled the characterization of the postnatal physiological and motor deficits affecting the Mecp2-deficient mice. The study of catecholaminergic structures of interest such as the substantia nigra pars compacta and the locus coeruleus has revealed that the central noradrenergic and dopaminergic neurons are affected in their metabolism. The number of immunolabelled neurons of these groups appears significantly reduced and would result in a gradual loss of the mature ‘catecholaminergic’ phenotype, in the absence of cell death. Our data suggest that these defects are a neuropathological correlate for behavioral disorders observed in Mecp2-deficient mice. Some motor deficits have been improved, with L-Dopa, a pro-dopaminergic drug. In relation with Bdnf (Brain-derived neurotrophic factor) reduction described in patients and Mecp2-deficient mice, we identified that a change in the dosage of Mecp2 deregulates genes (Htt, Hap1) encoding proteins involved in the intracellular transport of Bdnf. Our work allows to postulate that in the Mecp2-deficient neurons, an altered dynamics of Bdnf vesicles transport could exacerbate the deficit of expression of this neurotrophin. Our treatment of Mecp2-deficient mice with cysteamine, a molecule able to increase Bdnf contents and enhancing its release and secretion, increased the survival of the animals and reduced their motor defects. Our results show that the Mecp2-deficiencies lead to alteration in the axonal transport of Bdnf in addition to deficits in Bdnf production. In addition, by the use of pharmacological agents that affect this transport, we offer new therapeutic perspectives.

Introdução: A exposição crônica a diferentes tipos de dieta altera o metabolismo hipotalâmico e mesolímbico, podendo causar alterações no comportamento alimentar do indivíduo. O BDNF, fator de crescimento neuronal, pode atuar na modulação do comportamento alimentar tanto em vias hedônicas quanto homeostáticas. O objetivo do estudo foi investigar como o BDNF atua na modulação do comportamento alimentar em vias homeostáticas e hedônicas em ratas fêmeas com diferentes perfis metabólicos. Materiais e métodos: Ratas Wistar fêmeas adultas randomizadas por peso foram divididas em: dieta controle (C) contendo 22% de proteína e 4% de lipídios; dieta hipoproteica (LP) 8% de proteína ou dieta hiperlipídica (HF) 45% de lipídios, ad libitum, por 5 semanas, sendo o consumo medido a cada 72 horas e o peso semanalmente. O trabalho foi dividido em duas partes. Na primeira parte, após as 5 semanas de dieta os animais ficaram em jejum por 4 horas e foram expostos ao alimento doce (Froot Loops®), previamente pesado, por 1 hora, a fim de verificar o consumo de alimento palatável em ratas com diferentes perfis metabólicos. Imediatamente após coletou-se sangue e cérebro, assim como, o peso da gordura abdominal foi mensurado. Na segunda parte do estudo, após as 5 semanas de dieta, os animais ficaram durante 7 dias no BioDAQ®, um sistema computadorizado de análise do comportamento alimentar, para avaliar o consumo da dieta habitual em ratas com diferentes perfis metabólicos. O consumo foi mensurado através de mordidas (diferença de 0,1 g na balança) e refeições (conjunto de porções por um tempo igual ou menor a 15 min, porções são mordidas ininterruptas). No dia 10 foi realizado o teste de preferência alimentar no qual o animal poderia escolher entre a dieta habitual (dieta que eles receberam por 5 semanas) ou a dieta hipersacarídica (HP) (contendo 34% lipídios, 30,2% carboidratos, 14% proteínas, 20% sacarose), com duração de 20 horas. Após 1 semana, foi coletado sangue, cérebro e mensurada a gordura abdominal. Foi realizado western blotting para tirosina hidroxilase (TH) e fosfo- tirosina hidroxilase (pTH) no núcleo accumbens, STAT3 e fosfo-STAT3 (pSTAT3) no hipotálamo. Adicionalmente, foi mensurado BDNF no soro, no núcleo do trato solitário (NTS), área tegmentar ventral (VTA) e glicemia no soro. Resultados: Nas 5 semanas de tratamento, em relação ao ganho de peso dos animais não houve diferenças significativas entre os grupos, não houve interação, apenas apresentaram efeito do tempo (p< 0,001). A gordura abdominal foi maior nas ratas HF (p= 0,002) e LP (p= 0,023) em relação aos controles. Os animais que receberam dieta HF comeram menos gramas de Froot Loops® do que os animais controle (p= 0,003). Durante a habituação ao BioDAQ® não houve diferença significativa no consumo de dieta entre os grupos. Na análise do comportamento alimentar no BioDAQ®, comparando os grupos controle versus hiperlídica (C x HF), animais HF tiveram o tamanho da refeição (g) (p=0,049), número de porções (p= p< 0,001), tamanho da refeição ciclo escuro (p= 0,002), número de porções ciclo escuro (p < 0,001) menor do que os controles e tamanho da porção ciclo escuro (p=0,006) maior do que os controles. Na análise do comportamento alimentar, comparando os grupos controle versus hipoproteica (C x LP) foram encontradas diferenças significativas nos seguintes parâmetros: média do tamanho da porção (g) (p= 0,004), média do tamanho da porção no ciclo claro (g) (p= 0,042), média do tamanho da refeição no ciclo escuro (g) (p= 0,035), média do tamanho da porção no ciclo escuro (g) (p= 0,006). Em todos os parâmetros os animais LP tiveram uma média maior do que os animais controle. No teste de preferência alimentar, os animais HF apresentaram uma média inferior aos animais controle nos seguintes parâmetros: consumo de dieta hipersacarídica (Kcal) (p= 0,034), número de refeições de dieta hipersacarídica (p= 0,016), número de porções de dieta hipersacarídica (p= 0,001). Os animais HF apresentaram médias superiores aos animais C em relação ao tamanho da porção de dieta hipersacarídica (g) (p= 0,023), PMI (intervalo entre refeições) total de dieta hipersacarídica (p= 0,022), saciedade total de dieta hipersacarídica (p= 0,008). Durante o teste de preferência alimentar comparando-se controle versus hipoproteica (C x LP), os animais (LP) apresentaram o consumo de dieta hipersacarídica (kcal) (p= 0,030) e o número de porções de dieta hipersacarídica (p= 0,003) menor que os animais controles. Os animais LP apresentaram saciedade total de dieta hipersacarídica (p= 0,046) maior do que os controles. Não houve diferenças significativas nos níveis de glicemia no soro entre os grupos. Os animais HF apresentaram as médias de pSTAT3 maior do que os controles (p= 0,013) e fosfo-tirosina hidroxilase menor do que os controles (p= 0,05). Os animais LP apresentaram STAT3 menor do que os animais controle (p= 0,035) e tiveram resultados de BDNF próximos da significância (p=0,053), tendo apresentado uma média inferior aos animais controle. Conclusões: A exposição aos diferentes tipos de dieta muda os padrões de alimentação bem como a estrutura corpórea. Tanto animais expostos a dieta HF quanto a LP apresentam alterações compatíveis com um estado de pré-resistência à leptina. O BDNF parece modular as vias homeostáticas e hedônicas do comportamento alimentar, no entanto mais estudos são necessários para entendermos melhor esses mecanismos. / Introduction: The chronic exposure to different types of diet modifies the hypothalamic and mesolimbic metabolism, what can lead to changes in the individual feeding behavior. BDNF, a neuronal growing factor, can act modulating feeding behavior in hedonic as well as in homeostatic pathways. The aim of this study was to investigate how BDNF acts modulating feeding behavior regarding hedonic and homeostatic pathways in female rats with different metabolic profiles. Materials and methods: Female adult Wistar rats randomized by weight were divided in: control diet (C) with 22% of protein and 4% of lipids; low-protein diet (LP) with 8% of protein or high fat diet (HF) with 45% of lipids, ad libitum, for 5 weeks, with the diet consumption verified every 72 hours and the weight verified weekly. The study was divided in two parts. In the first part, after 5 weeks receiving diet, animals were submitted to 4 hours of fasting and then were exposed to sweet food (Froot Loops®), previously weighted, for 1 hour to verify the palatable food consumption in rats with different metabolic profiles. Immediately after, blood and brain were collected and the abdominal fat weight was verified. In the second part of the study, after 5 weeks receiving diet, the animals were submitted to the BioDAQÒ, a computer system of feeding behavior analyses, to evaluate the habitual diet consumption in rats with different metabolic profiles for 7 days. The consumption was measured through bites (0.1g of difference in the scale) and meals (a group of portions for a period of time less or equal than 15min, portions are uninterrupted bites). On day 10 the food preference test was done and the animals could choose between the habitual diet (the one they have received for 5 weeks) and the high sucrose palatable diet (HP) (with 34% of lipids, 30.2% of carbohydrates, 14% of protein, 20% of sucrose) for a period of 20 hours. After one week, blood and brain were collected and abdominal fat was measured. Western blotting was used to verify the content of tyrosine hydroxylase (TH) and phospho-tyrosine hydroxylase (pTH) in the nucleus accumbens, STAT3 and phospho-STAT3 (pSTAT3) in the hypothalamus. Additionally, BDNF was measured in the serum, in the solitary tract nucleus (NTS) and in the ventral tegmental area (VTA) and glycemia was also verified. Results: Considering the 5 weeks of treatment regarding animals weight gain there was no significant difference between groups and no interaction, only an effect of time (p<0.001). Abdominal fat was higher in HF (p=0.002) and LP (p=0.023) rats comparing to controls. Animals that received HF diet ate less grams of Froot Loopsâ than controls (p=0.003). During the habituation to the BioDAQÒ no significant difference was seen in the consumption of the diet between the groups. When analyzing feeding behavior in the BioDAQÒ, comparing control against high fat groups (C x HF), HF animals had a decrease in the size of the meal (g) (p=0.049), number of portion (p<0.001), meal size in the dark phase (p=0.002), number of portions in the dark phase (p<0.001) compared to controls while the size of the portion in the dark phase (p=0.006) was bigger than controls. Analyzing feeding behavior comparing control against low-protein groups (C x LP) a significant difference was observed in the following parameters: average of the size of the portion (g) (p=0.004), average of the size of the portion in the light phase (g) (p=0.042), average of the size of the meal in the dark phase (g) (p=0.035), average of the size of the portion in the dark phase (g) (p=0.006). LP animals showed a higher average than controls in all parameters. In the food preference test, HF animals showed a lower average than controls in the following parameters: consumption of high sucrose palatable diet (Kcal) (p=0.034), number of meals of high sucrose palatable diet (p=0.016), number of portions of high sucrose palatable diet (p=0.001). HF animals showed higher averages than controls regarding the size of the portion of the high sucrose palatable diet (g) (p=0.023), total PMI (post interval meal) of high sucrose palatable diet (p=0.022) and total satiety of high sucrose palatable diet (p=0.008). In the food preference test comparing controls against low-protein groups (C x LP), LP animals showed a lower consumption of high sucrose palatable diet (Kcal) (p=0.030) and a lower number of portions of high sucrose palatable diet (p=0.003) than control animals. LP animals showed total satiety of high sucrose palatable diet (p=0.046) bigger than controls. There was no significant difference in the glycemia levels between groups. HF animals had pSTAT3 averages higher than controls (p=0.013) and phospho-tyrosine hydroxylase lower than controls (p=0.05). LP animals showed STAT3 lower than control animals (p=0.035) and had BDNF results close to the significance (p=0.053), showing a lower average than control animals. Conclusion: The exposure to different types of diets changes the feeding patterns as well as the body structure. Animals exposed to HF diet as well as those exposed to LP diet showed changes related to a leptin pre-resistant state. BDNF seems to modulate homeostatic and hedonic feeding behavior pathways, however more studies are necessary to a better understanding of the mechanisms.

Studies have indicated that approximately 30% of people with heart disease experience major depressive disorder (MDD). Despite strong clinical evidence of a link between the two diseases, the neurobiological processes involved in the relationship are poorly understood. A growing number of studies are revealing similar neuroanatomical and neurochemical abnormalities resulting from both depression and heart disease. The locus coeruleus (LC) is a group of neurons in the pons that synthesize and release norepinephrine, and that is known to play a significant role in depression pathobiology. For example, there is evidence that tyrosine hydroxylase (TH) is elevated in the LC in depression. In addition, there is evidence that the LC plays a role in cardiovascular autonomic regulation. The hippocampus is another region that exhibits abnormalities in both depression and heart disease. In this study, the levels of TH in the hippocampus and LC were examined in the guinea pig pressure-overload model of heart disease. TH levels were also measured in the pressure-overload model treated with vagal nerve stimulation, a new investigational therapeutic intervention in heart disease. This study found that there were no changes in TH levels in the LC or the hippocampus of the pressure-overload model or in the pressure-overload model treated with vagal nerve stimulation.

La tyrosine hydroxylase (TH) est l'enzyme clé de la biosynthèse des catécholamines, classe fondamentale de neurotransmetteurs regroupant la dopamine, la noradrénaline et l'adrénaline. Une expression atypique des catécholamines pourrait conduire à de nombreuses complications neuropsychiatriques. L'étude des facteurs qui régulent le gène de la TH est donc déterminante.<br /><br />Ce travail a pour objectif l'isolement de facteurs de transcription potentiellement impliqués dans l'expression cellulaire restreinte du gène TH de rat. Dans ce but, nous avons développé une stratégie de simple-hybride dans la levure en utilisant la boîte E et les sites octamérique/heptamérique du promoteur TH comme cibles. Deux banques d'ADN complémentaire différentes ont été criblées, issues respectivement de cerveau de rat adulte et de mésencéphale embryonnaire. Ces criblages ont conduit à l'identification de 3 facteurs de transcription candidats : rITF2, un facteur bHLH ubiquitaire déjà décrit pour son aptitude à interagir avec la boîte E du promoteur TH, Lhx9, une protéine LIM à homéodomaine, et ZENON, une nouvelle protéine à doigts de zinc spécifiquement exprimée dans les neurones. La caractérisation de ces 3 facteurs a révélé des propriétés tout à fait intéressantes. Ils sont capables d'interagir spécifiquement avec le promoteur TH, et modulent la transcription du gène TH dans des lignées cellulaires. Leurs patrons d'expression sont complexes et s'inscrivent dans une dynamique développementale très particulière, mais ne sont pas spécifiques du système catécholaminergique. Pour comprendre le rôle de ces facteurs dans l'expression cellulaire restreinte de la TH, nous avons mené une étude de transgenèse des sites cibles utilisés en simple-hybride. La boîte E joue un rôle subtil dans le système catécholaminergique périphérique, illustrant la complexité des mécanismes conduisant à l'expression cellulaire restreinte de la TH. <br /><br />Ce travail souligne les difficultés rencontrées pour isoler des facteurs de transcription impliqués dans des processus d'expression cellulaire restreinte et définit une nouvelle famille de protéines à doigts de zinc, dont ZENON, le premier membre, semble lié au phénotype neuronal. L'identification et l'étude d'autres protéines appartenant à cette famille pourraient donc s'avérer capitales pour la compréhension des mécanismes impliqués dans la mise en place et le maintien des caractères neuronaux au cours du développement.

Parkinson’s disease (PD) is a common neurodegenerative disorder that is characterized by the degeneration of dopaminergic (DA) neurons in the substantia nigra and motor deficits. Although the majority of PD cases are sporadic, several genetic defects in rare familial cases have been identified. Animal models of these genetic defects have been created and have provided unique insights into the molecular mechanisms of the pathogenesis of PD. However, the etiology of PD is still not well understood. Here, taking advantage of the unique features offered by zebrafish, I characterized the functions of PINK1 (PTEN-induced kinase 1) gene, which is associated with recessive familial PD, in the development and survival of DA neurons. In zebrafish, antisense morpholino knockdown of pink1 did not cause a large loss of DA neurons in the ventral diencephalon (vDC), but the patterning of these neurons and their projections were perturbed. The pink1 morphants also showed impaired response to touch stimuli and reduced swimming behaviour. Moreover, the pink1 knockdown caused a significant reduction in the number of mitochondria, as well as mitochondrial morphological defects such as smaller size or loss of cristae, thus affecting mitochondrial function. These results suggest that zebrafish pink1 plays conserved important roles in the development of DA neurons and in the mitochondrial morphology and function. To better follow DA neurons after injury or administration of toxins, I generated a transgenic zebrafish line, Tg(dat:EGFP), in which the green fluorescent protein (GFP) is expressed under the control of cis-regulatory elements of dopamine transporter (dat). In Tg(dat:EGFP) fish, all major groups of DA neurons are correctly labeled with GFP, especially the ones in the vDC, which are analogous to the ascending midbrain DA neurons in mammals. In addition, we observed that the DA neurons in the vDC could partially be replaced after severe laser cell ablation. This suggests that zebrafish may have the unique capacity of regenerating DA neurons after injury. Taken together, my studies suggested that zebrafish could be a useful alternative animal model for the study of the molecular mechanisms underlying PD and for the screening of potential therapeutic compounds for PD.

Parkinson’s disease (PD) is a common neurodegenerative disorder that is characterized by the degeneration of dopaminergic (DA) neurons in the substantia nigra and motor deficits. Although the majority of PD cases are sporadic, several genetic defects in rare familial cases have been identified. Animal models of these genetic defects have been created and have provided unique insights into the molecular mechanisms of the pathogenesis of PD. However, the etiology of PD is still not well understood. Here, taking advantage of the unique features offered by zebrafish, I characterized the functions of PINK1 (PTEN-induced kinase 1) gene, which is associated with recessive familial PD, in the development and survival of DA neurons. In zebrafish, antisense morpholino knockdown of pink1 did not cause a large loss of DA neurons in the ventral diencephalon (vDC), but the patterning of these neurons and their projections were perturbed. The pink1 morphants also showed impaired response to touch stimuli and reduced swimming behaviour. Moreover, the pink1 knockdown caused a significant reduction in the number of mitochondria, as well as mitochondrial morphological defects such as smaller size or loss of cristae, thus affecting mitochondrial function. These results suggest that zebrafish pink1 plays conserved important roles in the development of DA neurons and in the mitochondrial morphology and function. To better follow DA neurons after injury or administration of toxins, I generated a transgenic zebrafish line, Tg(dat:EGFP), in which the green fluorescent protein (GFP) is expressed under the control of cis-regulatory elements of dopamine transporter (dat). In Tg(dat:EGFP) fish, all major groups of DA neurons are correctly labeled with GFP, especially the ones in the vDC, which are analogous to the ascending midbrain DA neurons in mammals. In addition, we observed that the DA neurons in the vDC could partially be replaced after severe laser cell ablation. This suggests that zebrafish may have the unique capacity of regenerating DA neurons after injury. Taken together, my studies suggested that zebrafish could be a useful alternative animal model for the study of the molecular mechanisms underlying PD and for the screening of potential therapeutic compounds for PD.