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In the present study 5-hydroxytryptamine (5-HT) 1A receptor knockout mice (KO), mice overexpressing the 5-HT1A receptor (OE), and wild-type (WT) mice were used to investigate the influence of 5-HT1A receptor on the development of the serotonergic system in the brain, from the embryonic day 12.5 to the postnatal day 15.5. Neither the absence nor the overexpression of 5-HT1A receptor influenced the development and differentiation of serotonergic neurons in the raphe area of the mouse brain. However, a delay in the initial development of the serotonergic projections to the mesencephalic tegmentum, cerebral cortex and hypothalamus was observed in both transgenic mice lines. The brain levels of 5-HT and 5-hydroxyindoleacetic acid were significantly higher in both transgenic mice lines during the late embryonic and early postnatal periods as compared to WT mice. An increase in the turnover of 5-HT was not observed before the early postnatal period. Both the absence and the overexpression of 5-HT1A receptor delayed the development of the dopaminergic system of the mesencephalic tegmentum in the early embryonic period. In OE mice the postnatal development of the noradrenergic system appeared to be exaggerated. The immunoreactivity for the neurotrophic protein S100ß was higher in the cerebral cortex, striatum and hippocampus of OE mice as compare to WT and KO mice. The expression of synaptic proteins, such as synapatobrevin and synaptotagmin was reduced in KO and OE mice during the early embryonic period. This reduction may be linked to the delayed development of the serotonergic projections and the dopaminergic system. In addition, no influence of 5-HT1A receptor mutations on the myelination of the brain was observed. Zusammenfassung In der vorliegenden Arbeit wurden die 5-Hydroxytryptamin (5-HT)1A Rezeptor Knockout (KO), überexprimierenden (ÜE) Mäuse und die Wild-Typ (WT) Mäuse, in den Entwicklungsperioden vom embryonalen Tag 12,5 bis postnatalen Tag 15,5 untersucht, um weitere Informationen über den Einfluss vom 5-HT1A Rezeptor auf die Entwicklung des serotonergen Systems im Gehirn zu erhalten. Sowohl das Fehlen des 5-HT1A Rezeptors als auch dessen Überexpression hatten zwar keinen Einfluss auf die Entwicklung und Differenzierung der serotonergen Neurone in den Raphe Regionen, verzögerte aber die erste Entwicklung der serotonergen Innervierungen im mesencephalen Tegmentum, Hypothalamus und cerebralen Cortex. In den späten embryonalen und insbesondere frühpostnatalen Perioden waren die 5-HT- und 5-HIAA-Spiegel bei KO und ÜE Mäusen im Vergleich zu WT Mäusen signifikant erhöht. Eine Erhöhung des 5-HT Turnovers wurde erst in der frühpostnatalen Periode beobachtet. Auch die Entwicklung des dopaminergen Systems im Mesencephalon war in der frühen embryonalen Periode sowohl bei KO als auch bei ÜE Mäusen verlangsamt. Die Überexpression des 5-HT1A Rezeptors begünstigte möglicherweise die postnatale Entwicklung des noradrenergen Systems. Bei ÜE Mäusen war die Immunreaktivität des neurotrophen Proteins S100? im cerebralen Cortex, Hippocampus und Striatum stärker als bei WT und KO Mäusen. Die Expression der synaptischen Proteine wie Synaptobrevin und Synaptotagmin war sowohl bei KO als auch bei ÜE Mäusen in der frühen embryonalen Periode verzögert. Dies könnte mit der verzögerten Entwicklung der serotonergen Projektionen und des dopaminergen Systems in Zusammenhang stehen. Darüber hinaus hatten transgene Veränderungen am 5-HT1A Rezeptor keinen Einfluss auf die Myelinisierung im Gehirn der Maus. Schlagwörter: serotonerges System, Entwicklung des Gehirns, 5-HT1A Rezeptor, transgene Mäuse, dopaminerges System, noradrenerges System, S100ß, Synaptisches Protein, Myelinisierung / In the present study 5-hydroxytryptamine (5-HT) 1A receptor knockout mice (KO), mice overexpressing the 5-HT1A receptor (OE), and wild-type (WT) mice were used to investigate the influence of 5-HT1A receptor on the development of the serotonergic system in the brain, from the embryonic day 12.5 to the postnatal day 15.5. Neither the absence nor the overexpression of 5-HT1A receptor influenced the development and differentiation of serotonergic neurons in the raphe area of the mouse brain. However, a delay in the initial development of the serotonergic projections to the mesencephalic tegmentum, cerebral cortex and hypothalamus was observed in both transgenic mice lines. The brain levels of 5-HT and 5-hydroxyindoleacetic acid were significantly higher in both transgenic mice lines during the late embryonic and early postnatal periods as compared to WT mice. An increase in the turnover of 5-HT was not observed before the early postnatal period. Both the absence and the overexpression of 5-HT1A receptor delayed the development of the dopaminergic system of the mesencephalic tegmentum in the early embryonic period. In OE mice the postnatal development of the noradrenergic system appeared to be exaggerated. The immunoreactivity for the neurotrophic protein S100ß was higher in the cerebral cortex, striatum and hippocampus of OE mice as compare to WT and KO mice. The expression of synaptic proteins, such as synapatobrevin and synaptotagmin was reduced in KO and OE mice during the early embryonic period. This reduction may be linked to the delayed development of the serotonergic projections and the dopaminergic system. In addition, no influence of 5-HT1A receptor mutations on the myelination of the brain was observed.

Submitted by PPG Pediatria e Sa?de da Crian?a (pediatria-pg@pucrs.br) on 2018-05-17T17:25:42Z No. of bitstreams: 1 TESE Thiago Viola Final.pdf: 3221820 bytes, checksum: 98e6a1c6936e1a12973e6fe2d7d12ce5 (MD5) / Approved for entry into archive by Caroline Xavier (caroline.xavier@pucrs.br) on 2018-05-30T16:45:08Z (GMT) No. of bitstreams: 1 TESE Thiago Viola Final.pdf: 3221820 bytes, checksum: 98e6a1c6936e1a12973e6fe2d7d12ce5 (MD5) / Made available in DSpace on 2018-05-30T16:49:19Z (GMT). No. of bitstreams: 1 TESE Thiago Viola Final.pdf: 3221820 bytes, checksum: 98e6a1c6936e1a12973e6fe2d7d12ce5 (MD5) Previous issue date: 2018-03-12 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Introduction: Child development in adverse environments and conditions, such as with the lack of economic resources or with parental care deprivation, is considered a major risk factor for neurological and psychiatric diseases. Altered cognitive processing is thought to mediate this relationship, however, the neurobiological mechanisms underlying the effects of early adverse experiences on cognition have not yet been fully revealed. Evidence indicates that dopaminergic neurotransmission and the corticotrophinergic system have important functions in the neurobiology of decision-making and risk assessment, which are cognitive processes associated with the functionality of the cerebral cortex. Similarly, working memory is another cognitive domain that underlies cortical activity, and some studies indicate that alterations in neuroimmunologic signaling may contribute to the decline of these higher order cognitive functions. Objectives: To investigate the effects of impoverished housing conditions during early life on risk assessment processing and its associated cortical neurobiological and epigenetic mechanisms in C57BL/6 adolescent mice. In addition, we investigated the effects maternal care deprivation during early life, and the effects of systemic activation of the toll-type receptor (TLR)-3 on working memory performance, and its associated cortical neurobiological mechanisms in male BALB/c mice. Methods: Two studies with rodent experimental models were proposed. The first study used a model of impoverished housing from the postnatal day (P) 2 to P9. During adolescence, risk assessment was investigated using a behavioral paradigm that explores the conflict between two biologically relevant stimuli: the motivation to consume a sweet and highly palatable solution while being threatened by predatory olfactory cues. The expression of dopaminergic (Drd1, Drd2) and corticotrophinergic (Cfr, Crfr1) genes in the medial prefrontal cortex (mPFC) were investigated by real-time PCR. The accumulation of histone marks (H3K9me3, H3R2me2s) were assessed at the promoter region of genes associated with behavioral outcomes. In addition, plasma corticosterone levels were assessed by ELISA. In the second study, a rodent model of maternal care deprivation from P2 to P15 was applied. During adolescence, animals were injected with a TLR-3 agonist, which is a viral receptor implicated with inflammatory signaling, and then tested in a working memory task. The expression of pro-inflammatory genes (Nfkb1, Il6 and Tnf-?) and the receptor itself (Tlr3), were performed in the mPFC by real-time PCR. Results: In the first study, we found increased anxiety-like behavior, increased HPA axis response to stress and impaired RA processing in female adolescent mice, with no effect in males. These sex-specific effects were associated with increased Crfr1 mRNA expression in the medial prefrontal cortex (mPFC), which correlated with an increase in the occupancy of the histone mark H3R2me2s, a histone modification known to be involved in transcriptional activation and epigenetic priming, within the promoter of the Crfr1 gene. In the second study, we found that systemic administration of a TLR-3 agonist can modulate and exacerbate early life stress induced working memory impairments, and that higher gene expression levels of Nfkb1 in the mPFC was associated a lower working memory performance. Conclusions: The findings of the first study indicated a deleterious effect of impoverished housing exposure on risk assessment processing in females, which could be detrimental for cognitive performance in potentially dangerous situations, and suggest that the epigenetic priming of the Crfr1 gene may represent a critical factor mediating the relationship between early life stress and altered cognitive processing later in life in females. Finally, the findings of the second study demonstrated that the systemic activation of TLR-3 can induce working memory impairments, revealing an important mediating role of the neuroinflammatory signalling in the cerebral cortex associated with the cognitive changes resulting from maternal care deprivation exposure during early in life. / Introdu??o: O desenvolvimento infantil em ambientes e condi??es adversas, como frente a escassez de recursos econ?micos ou de cuidado parental, ? considerado fator de risco para doen?as neurol?gicas e psiqui?tricas. Altera??es em processos cognitivos parecem mediar esta rela??o, contudo, os mecanismos neurobiol?gicos adjacentes aos efeitos de experi?ncias adversas precoces sobre a cogni??o ainda n?o foram completamente revelados. Evid?ncias apontam que a neurotransmiss?o dopamin?rgica e o sistema corticotrofin?rgico possuem importantes fun??es na neurobiologia da tomada de decis?o e avalia??o do risco, que s?o processos cognitivos associados a funcionalidade do c?rtex cerebral. Similarmente, a mem?ria de trabalho ? outro dom?nio cognitivo que envolve atividade cortical, e alguns estudos apontam que altera??es na sinaliza??o neuroimunol?gica podem contribuir para o decl?nio destas fun??es cognitivas superiores. Objetivos: Investigar o efeito da exposi??o a moradia empobrecida na inf?ncia sobre o processamento cognitivo de avalia??o do risco e mecanismos neurobiol?gicos e epigen?ticos corticais associados em camundongos adolescentes da linhagem C57BL/6. Al?m disso, investigar o efeito da priva??o do cuidado materno na inf?ncia e da ativa??o sist?mica do receptor do tipo toll (TLR)-3 sobre a mem?ria de trabalho e mecanismos neurobiol?gicos corticais associados em camundongos machos adolescentes da linhagem BALB/c. M?todos: Foram propostos dois estudos com modelos experimentais murinos. O primeiro estudo utilizou um modelo de moradia empobrecida do dia p?s-natal (P) 2 ao P9. Quando os animais encontravam-se no per?odo da adolesc?ncia, o processamento de avalia??o do risco foi investigado por uma tarefa que explora um conflito entre dois est?mulos biologicamente fundamentais na vida de um roedor, a motiva??o de consumir uma solu??o doce e altamente palat?vel (leite condensado) tendo que se expor a pistas olfativas de um predador natural, o coiote. Os n?veis de express?o de genes dopamin?rgicos (Drd1, Drd2) e corticotrofin?rgicos (Cfr, Crfr1) no c?rtex medial pr?-frontal (mPFC) foram investigados por PCR em tempo real. Os n?veis de altera??es de histonas (H3K9me3, H3R2me2s) foram avaliados na regi?o promotora de genes associados aos desfechos comportamentais. Adicionalmente, os n?veis de corticosterona plasm?tica foram avaliados por ELISA. No segundo estudo, o modelo de adversidade utilizado foi o de priva??o do cuidado materno do P2 ao P15. Similarmente, quando os animais encontravam-se no per?odo da adolesc?ncia, ocorreu a administra??o sist?mica de um agonista de TLR-3, um receptor viral relacionado a sinaliza??o inflamat?ria, e posteriormente os animais foram testados em uma tarefa de mem?ria de trabalho. Os n?veis de express?o g?nica de genes pr?-inflamat?rios (Nfkb1, Il6 e Tnf-?) e do pr?prio receptor (Tlr3), foram avaliados no mPFC por PCR em tempo real. Resultados: no primeiro estudo, observou-se um aumento de comportamentos do tipo ansioso, maior responsividade do eixo Hipot?lamo-Pituit?ria-Adrenal (HPA) e uma diminui??o do processamento de avalia??o do risco nas f?meas expostas a moradia empobrecida, ao passo que n?o ocorreram altera??es nos animais machos. A diminui??o de avalia??o do risco foi associada a um aumento na express?o de Crfr1 no mPFC, o que se correlacionou com um aumento dos n?veis de H3R2me2s na regi?o promotora deste gene. No segundo estudo, observou-se que a ativa??o sist?mica de TLR-3 exacerbou os preju?zos de mem?ria de trabalho decorrentes da exposi??o a priva??o do cuidado materno, e este efeito correlacionou-se aos n?veis de express?o de Nfkb1 no mPFC. Conclus?es: os achados do estudo 1 indicam um efeito delet?rio da exposi??o a moradia prec?ria na inf?ncia sobre o processamento de avalia??o do risco em f?meas, revelando um preju?zo espec?fico referente ao engajamento cognitivo frente a situa??es potencialmente perigosas. Al?m disso, evidenciou-se um efeito a n?vel epigen?tico de regula??o da express?o cortical de Crfr1, indicando um importante papel deste gene sobre a rela??o entre pobreza na inf?ncia e altera??es cognitivas em f?meas adolescentes. Por fim, os achados do estudo 2 demonstraram que a ativa??o sist?mica do TLR-3 pode exacerbar os preju?zos de mem?ria de trabalho induzidos pelo estresse precoce, revelando um papel mediador da sinaliza??o neuroinflamat?ria no c?rtex cerebral relacionada as altera??es cognitivas decorrentes da exposi??o a priva??o do cuidado materno.

La dopamine est un neurotransmetteur important dans le fonctionnement des ganglions de la base, région du cerveau impliquée dans la fonction motrice et l’apprentissage. Un dérèglement de la dynamique de la dopamine peut être à l’origine de différentes pathologies neurologiques, telles que la maladie de Parkinson et le trouble de déficit de l’attention avec ou sans hyperactivité (TDAH). La lévodopa, un précurseur de la dopamine, est utilisée pour réduire les symptômes associés à la maladie de Parkinson, sans action directe sur ses causes. La lévodopa est très efficace au début de la maladie, mais la durée de son effet ainsi que son index thérapeutique diminuent avec la progression de la dénervation induite par la maladie. Ces changements compliquent considérablement l’optimisation des régimes posologiques. Le méthylphénidate, quant à lui, est administré pour réduire les symptômes du TDAH et agit entre autres en bloquant la recapture de la dopamine. Bien que les données confirment une certaine implication de la dopamine dans le TDAH, son étiologie exacte demeure inconnue. Peu d’études ont cerné l’effet de la lévodopa sur le système dopaminergique des ganglions de la base et son évolution avec la progression de la maladie. Aussi, bien que le TDAH ait suscité beaucoup d’intérêt, rares sont les études quantitatives de nature mécanistiques sur le sujet. L’approche de modélisation mathématique utilisée dans cette thèse s’inscrit dans un effort global visant l’optimisation de la lévodopa et du méthylphénidate, appuyé par l’élucidation des mécanismes impliqués dans la maladie de Parkinson et dans le TDAH. En adoptant une approche de pharmacologie quantitative des systèmes (QSP), nous avons développé un modèle intégratif du système dopaminergique des ganglions de la base, avec l’objectif d’élucider les mécanismes impliqués, d’évaluer l’impact de la dopamine chez dessujets souffrant de Parkinson ou de TDAH, et recevant ou non un traitement, et enfin de guider objectivement l’exercice d’optimisation des régimes posologiques. À notre connaissance, c’est le premier cadre unificateur de modélisation qui s’adresse à ces deux pathologies. Le modèle développé dans cette thèse est composé de trois sous-modèles : le premier décrit la pharmacocinétique du médicament concerné, soit la lévodopa ou le méthylphénidate ; le deuxième exprime mathématiquement les différents mécanismes impliqués dans la dynamique de la dopamine ; le troisième représente la complexité de la neurotransmission dans les ganglions de la base. Avec des adaptations appropriées, nous avons appliqué ce même modèle au contexte de la maladie de Parkinson et au TDAH, ainsi qu’à leurs thérapies respectives. Pour représenter physiologiquement la maladie de Parkinson, nous avons intégré dans le modèle l’évolution de la perte neuronale ainsi que les différents mécanismes de compensation qui en résultent. La fréquence de tapotement des doigts est utilisée comme mesure clinique de la bradykinésie, définie comme le ralentissement des mouvements chez les patients parkinsoniens. Le modèle développé se base sur les connaissances actuelles de la pathophysiologie et pharmacologie du Parkinson, assurant ainsi sa validité en comparaison à des observations expérimentales et cliniques. Ensuite, à l’aide de ce modèle, les relations non-linéaires entre la concentration plasmatique de lévodopa, la concentration en dopamine dans le cerveau et la réponse à une tâche motrice sont étudiées. Le rétrécissement de l’index thérapeutique de la lévodopa au cours de la progression de la maladie dû à ces non-linéarités est investigué. Enfin, pour assurer l’aspect translationnel de notre approche, nous avons développé une application web à laquelle ce modèle a été intégré. Cette application sert de preuve de concept à un outil facilitant l’optimisation et l’individualisation des régimes posologiques. Pour l’étude du TDAH, nous avons adapté le modèle du système dopaminergique en y intégrant la libération tonique et phasique de la dopamine, cette dernière se produisant durant une tâche d’apprentissage par renforcement. Des individus virtuels ont été créés avec et sans déséquilibre du ratio tonique/phasique de la dopamine. En simulant une tâche de réponse à des stimuli dans un contexte de déséquilibre de la dopamine, le modèle nous a permis d’observer des symptômes similiaires à ceux de patients réels souffrant de TDAH. Finalement, la réponse au méthylphénidate résultant de l’inhibition de la recapture de la dopamine, à travers différents scénarios d’apprentissage a aussi été étudiée. Le développement d’une métrique nous a permis de différencier les répondants des non-répondants, et ainsi de mettre en évidence l’implication possible d’un apprentissage excessif chez les nonrépondants. Une meilleure compréhension de la réponse au méthylphénidate permettrait d’éviter la surmédication chez les non-répondants et d’aider les cliniciens dans leur pratique. Malgré la complexité du système dopaminergique et des traitements associés, cette thèse est un pas en avant dans la compréhension des mécanismes sous-jacents et de leur implication dans la thérapie. Ces avancées ont été réalisées en adoptant une approche de pharmacologie quantitative des systèmes, associée à une modélisation neurocomputationnelle du domaine du génie électrique, et complétée par un aspect de transfert au chevet du patient. Ce n’est qu’en transcendant ainsi les frontières disciplinaires qu’une visée aussi globale et intégrative est possible, afin de faire face aux défis multidimensionnels du système de la santé. / Dopamine is an important neurotransmitter of the basal ganglia, a region of the brain involved in motor function and learning. Disruption of dopamine dynamics can cause various neurological conditions, such as Parkinson’s disease and attention deficit hyperactivity disorder (ADHD). Levodopa, a dopamine precursor, is used to reduce the symptoms associated with Parkinson’s disease, without directly alleviating its causes. Levodopa is very effective in the early stages of the disease, but its effect duration along with its therapeutic index decrease with disease-induced denervation. These modifications further challenge determination of optimal dosing regimens of levodopa. In the case of ADHD, methylphenidate is administered to reduce its symptoms by, among other things, blocking dopamine recapture. Although evidence supports involvement of dopamine in ADHD, its exact etiology remains unknown. Few studies have investigated the effect of levodopa on the basal ganglia dopaminergic system and how it evolves with disease progression. Also, although ADHD has received a lot of interest, few quantitative studies of a mechanistic nature have been conducted on the subject. The mathematical modeling approach used in this thesis is part of an overall effort to optimize levodopa and methylphenidate, supported by the elucidation of the mechanisms involved in Parkinson’s disease and ADHD. Using a quantitative systems pharmacology (QSP) approach, we have developed an integrative model of the basal ganglia dopaminergic system, with the objective of elucidating the mechanisms involved, assessing the impact of dopamine in subjects with Parkinson’s or ADHD, with and without treatment, and objectively guiding the dosing regimens optimization. To the best of our knowledge, this is the first unifying modeling framework that addresses at the same time these two pathologies and their therapies. The model developed in this thesis includes three sub-models: the first one describes the drug pharmacokinetics, either levodopa or methylphenidate; the second one translates mathematically the different mechanisms involved in the dopamine dynamics; the third one is a computational representation of the complexity of neurotransmission in the basal ganglia. With appropriate adaptations, we have applied this same model to the context of Parkinson’s disease and ADHD, as well as to their respective pharmacotherapies. In order to physiologically represent Parkinson’s disease, we have integrated the denervation process in the model as well as the resulting compensation mechanisms. The finger tapping frequency is used as a clinical endpoint of bradykinesia, defined as the slowing of movements. The developed model is based on up-to-date knowledge of the pathophysiology and pharmacology of Parkinson’s disease, thus ensuring its validity in comparison with experimental and clinical observations. Using this model, the non-linear relationships between plasma levodopa concentration, dopamine concentration in the brain and response to a motor task were studied. The narrowing of levodopa therapeutic index during the progression of the disease due to these non-linearities was investigated. Finally, to ensure the translational aspect of our approach, we developed a web application in which this model was integrated. This application serves as a proof of concept for a tool aimed to facilitate the optimization and individualization of dosing regimens. For the study of ADHD, we adapted the developed model by integrating tonic and phasic dopamine release, the latter occurring during a reinforcement learning task. Virtual individuals were created with and without dopamine imbalance in the tonic/phasic ratio. By simulating a stimulus-response task, we observe ADHD-like symptoms among virtual patients with dopamine imbalance. Finally, the response to methylphenidate resulting from dopamine recapture inhibition, through different learning scenarios, was also studied. The development of a metric allowed us to differentiate responders from non-responders, and thus to highlight the possible implication of excessive learning in non-responders. A better understanding of methylphenidate response would help avoid overmedication in non-responders and assist clinicians in their practice. Despite the complexity of the dopaminergic system and its associated therapies, this thesis is a step forward in understanding the underlying mechanisms and their involvement in pharmacotherapy. These advances were achieved by adopting a quantitative systems pharmacology approach, combined with neurocomputational modeling borrowed from the electrical engineering field, and complemented by a translational bedside aspect. It is only by transcending disciplinary boundaries and adopting such an integrative approach that this ultimate goal of having a real impact on the multifaceted health system is possible.