La corne dorsale de la moelle épinière (CDME) est un site d’intégration et de modulation de l’information somatosensorielle. Les laminae profondes de la CDME jouent un rôle important dans la modulation des informations nociceptives. Notre objectif a été de caractériser les effets de la NA sur la transmission synaptique des laminae profondes de la CDME. Nous montrons que la NA facilite la transmission synaptique inhibitrice dans les laminae III-V de la CDME. Ce phénomène met en jeu l’activation d’adrénocepteurs alpha1, alpha2, et bêta et nécessite une communication interlaminaire intacte entre les laminae III-IV et V. L’inhibition du métabolisme glial produit les mêmes effets qu’une section mécanique entre les laminae IV et V. Une interaction entre les cellules gliales et les neurones des laminae profondes la CDME semble donc indispensable à l’effet facilitateur de la NA. / The dorsal horn of the spinal cord (DH) is an important site of integration and modulation of somatosensory information and deep laminae of the DH play an important role in the modulation of nociceptive information in the neuronal network of the spinal cord.Our aim was to characterize the effects of NA on synaptic transmission in deep laminae of the DH.We show that NA facilitates inhibitory synaptic transmission in laminae III-IV of the DH. This phenomenon involves the activation of alpha1, alpha2, and beta adrenoceptors and requires intact interlaminar communications between laminae III-IV and V. Glial cell metabolism inhibition has the same consequences as a mechanical section between laminae IV and V. These results indicate that an interaction between glial cell and deep laminae neurons of the DH seems essential for the facilitatory effect of NA on inhibitory synaptic communications in laminae III-IV of the DH.

Le bulbe olfactif est le siège d'une neurogenèse adulte pouvant jouer un rôle dans les apprentissages olfactifs. Nous nous sommes intéressés à son rôle dans un type d'apprentissage olfactif : l'apprentissage perceptif. Tout d'abord, l'enrichissement permet d'améliorer la discrimination d'odeurs proches : c'est l'apprentissage perceptif. En étudiant les effets de la suppression de la neurogenèse pendant l'enrichissement nous avons montré que la neurogenèse était nécessaire à l'apprentissage perceptif en modulant l'inhibition des cellules mitrales par les cellules granulaires. Ensuite, nous avons étudié le rôle de la noradrénaline dans cet apprentissage. La noradrénaline est nécessaire à l'apprentissage perceptif et suffisante pour en mimer les effets. De plus, nous avons montré que la neurogenèse bulbaire était essentielle à l'action de la noradrénaline pour permettre l'apprentissage perceptif. Enfin, nous nous sommes intéressés à l'effet du vieillissement sur l'apprentissage perceptif. Nous avons trouvé qu'il existait un défaut d'apprentissage perceptif chez la souris âgée en lien avec une diminution de la neurogenèse. En revanche, une stimulation noradrénergique permet de restaurer l'apprentissage perceptif sans modulation de la neurogenèse bulbaire suggérant l'existence de mécanismes compensatoires. L'apprentissage perceptif est sous-tendu par la neurogenèse bulbaire, via le système noradrénergique, pour permettre une hausse d'inhibition des cellules mitrales par les cellules granulaires améliorant la discrimination des odeurs proches. Avec l'âge, l'apprentissage perceptif peut être restauré suggérant une plasticité toujours présente dans un système olfactif vieillissant / The olfactory bulb is the target of a well described adult neurogenesis which has been involved in different kinds of learning. We focused on the role of adult neurogenesis on olfactory perceptual learning which consists on an improvement of olfactory discrimination after odor enrichment. We found that experience-driven improvement in olfactory discrimination (perceptual learning) requires the addition of newborn neurons in the olfactory bulb. More specifically, we showed that adult-born neurons are required for perceptual learning by modulating the inhibition of mitral cells by granule cells. Then, we studied the role of noradrenaline on perceptual learning. Direct manipulation of noradrenergic transmission significantly effect on adult-born neuron survival and perceptual learning. Finally, we investigated the effect of aging on perceptual learning. We found that perceptual learning was impaired by aging in line with an alteration of neurogenesis. However, noradrenergic stimulation restores perceptual learning without modulating neurogenesis suggesting compensatory mechanisms. Neural mechanisms underlying perceptual learning involve neurogenesis and noradrenergic system to allow an increase of mitral cell inhibition thanks to the granule cells leading to an improvement of odor discrimination. During aging, perceptual learning can be restored suggesting that the olfactory system is still plastic

BACKGROUND: Obesity is a leading health burden of the 21st century. Alterations of the individual endocrine stress response and the monoamine system may pathophysiologically contribute to the obesity pandemic and its metabolic and mental complications. OBJECTIVES: (i) to measure hypothalamic-pituitary-adrenal (HPA) axis responsiveness and its relation to serum concentrations of the arginine-vasopressin (AVP) surrogate copeptin in subjects with obesity (OB) compared to non-obesity controls (NOC), (ii) to test whether HPA axis responsiveness and copeptin are related to central noradrenaline (NA) transporter (NAT) availability, (iii) to assess brain serotonin transporter (SERT) binding potentials in OB compared to NOC. METHODS: 40 subjects with obesity (BMI > 35kg/m2) were compared to 25 non-obesity controls, matched for age and sex. (i) All individuals underwent the combined dexamethasone/corticotropin releasing hormone (dex/CRH) test. Plasma ACTH and cortisol curve parameters were derived, and copeptin was assessed in the 1500h sample. (ii) Positron emission tomography (PET) was applied in 10 OB and 10 NOC using the NAT-selective radiotracer S,S-[11C]O-methylreboxetine (MRB) and associated to curve indicators derived from the dex/CRH test as well as to copeptin. (iii) PET using the SERT selective radiotracer [11C] DASB was performed in 30 OB and 15 NOC for intergroup comparison. RESULTS: (i) OB subjects showed an increased HPA axis responsiveness as measured by cortisol concentrations after CRH stimulation. Correspondingly, the AVP surrogate copeptin was higher in OB along with being significantly associated to HPA axis reactivity. OB subjects had a higher adrenal sensitivity as measured by a lower ACTH/cortisol ratio. (ii) In NOC, the HPA response was related to NAT availability of the amygdala and the orbitofrontal cortex while in OB, this association was located in the hypothalamus. (iii) There were no differences in SERT availability between OB and NOC, but a higher inter-regional SERT connectivity was observed in OB. CONCLUSION: This work supports the notion of an increased endocrine stress response in human obesity, pointing to interacting alterations of the HPA and neurohypophyseal axes. Normally, these stress axes seem to be linked to prefrontal-limbic NA signaling, whereas a loss of this association in favor of a hypothalamic-centered relation is observed in OB. The SERT network pattern is more closely inter-related in OB, albeit central SERT concentrations per se do not differ between OB and NOC.:ABBREVIATIONS 4 LIST OF FIGURES 5 I. BIBLIOGRAPHIC DESCRIPTION 6 II. INTRODUCTION 7 2.1 Obesity as a global health burden 7 2.2 Neurobiology of stress 8 2.3 Stress and obesity 8 2.4 Neuroendocrine correlates of the stress response – The hypothalamic pituitary-adrenaland neurohypophyseal axes 9 2.4.1 Anatomy of the hypothalamic-pituitary-adrenal and neurohypophyseal axes 10 2.4.2 The role of CRH, ACTH and cortisol in the context of metabolism and obesity 11 2.4.3 The role of AVP in the context of metabolism and obesity 12 2.4.4 Measuring HPA axis responsiveness by means of the combined dexamethasonecorticotropin-releasing hormone (dex/CRH) test 12 2.4.5 Measuring AVP secretion by its equally-released surrogate copeptin 14 2.5 The noradrenergic system in the context of obesity and stress axis modulation 14 2.5.1 NA and its influence on feeding behavior16 2.5.2 The association of the noradrenergic system with the HPA and neurohypophyseal axes 16 2.5.3 Monoamine transporters as regulators of neurotransmitter signaling 17 2.5.4 Noradrenaline transporter imaging 18 2.6 The serotonergic system in obesity 19 2.6.1 Role of serotonin in the context of feeding behavior and metabolism 20 2.6.3 5-HTT imaging 21 2.7 Objectives and hypotheses 22 2.8 Study design 23 III. RESULTS 24 3. 1 Post-dexamethasone serum copeptin corresponds to HPA axis responsiveness in human obesity 24 3. 2 Central noradrenaline transporter availability is linked with HPA axis responsiveness and copeptin in human obesity and non-obese controls 34 3. 3 Central serotonin transporter availability in highly obese individuals compared with nonobese controls: A [11C] DASB positron emission tomography study 46 IV. SUMMARY 56 4.1 Subjects with obesity show an enhanced HPA axis responsiveness which correlates to serum concentrations of the AVP surrogate copeptin and abdominal fat distribution 56 4.2 HPA axis responsiveness and copeptin concentrations are differentially related to central NAT availability in subjects with obesity compared to non-obesity controls 58 4.3 Central serotonin transporter availability does not significantly differ in subjects with obesity compared to their non-obesity counterparts 59 4.4 Future direction 61 V. References 62 VI. APPENDICES 79 6.1 Curriculum vitae 79 6.2 Publications 81 6.3 Scientific contribution of the doctoral candidate to the publications 82 6.4 Declaration of the independent writing of this thesis 83 6.5 Acknowledgements 84

During differentiation stem cells are exposed to a range of microenvironmental chemical and physical cues. In this study, human multipotent progenitor cells (hMLPCs) were differentiated from umbilical cord into oligodendrocytes and astrocytes. Chemical cues were represented by a novel defined differentiation medium containing the neurotransmitter norepinephrine (NE). In traditional 2 dimensional (2D) conditions, the hMLPCs differentiated into oligodendrocyte precursors, but did not progress further. However, in a constructed 3 dimensional (3D) environment, the hMLPCs differentiated into committed oligodendrocytes that expressed MBP. When co-cultured with rat embryonic hippocampal neurons (EHNs), hMLPCs developed in astrocytes or oligodendrocytes, based on presence of growth factors in the differentiation medium. In co-culture, physical cues provided by axons were essential for complete differentiation of both astrocytes and oligodendrocytes. This study presents a novel method of obtaining glia from human MLPCs that could eliminate many of the difficulties associated with their differentiation from embryonic stem cells. In addition, it reveals the complex interplay between physical cues and biomolecules on stem cell differentiation.

O estresse parece ser um dos principais fatores responsáveis pela predisposição do indivíduo à depressão. Estudos prévios de nosso laboratório mostraram que a inativação aguda da transmissão sináptica no núcleo leito da estria terminal (NLET) provoca efeitos tipo-antidepressivos em animais submetidos ao modelo animal do nado forçado, indicando que sua ativação durante o estresse facilita o desenvolvimento de consequências comportamentais relacionadas à neurobiologia da depressão. Contudo, os neurotransmissores envolvidos na mediação de tais efeitos permanecem desconhecidos. Sabe-se que a neurotransmissão noradrenérgica no NLET é abundante e tem participação importante na regulação de processos emocionais relacionados à resposta ao estresse. Assim, o presente trabalho investigou o envolvimento da neurotransmissão noradrenérgica existente no núcleo leito da estria terminal (NLET) no desenvolvimento das consequências comportamentais do estresse relacionadas à neurobiologia da depressão, através da administração local de antagonistas noradrenérgicos em animais submetidos ao modelo animal do nado forçado. Os resultados mostraram que a administração local de WB4101 (antagonista ?1) (10 e 15 nmol), CGP20712 (antagonista ?1) (5 e 10 nmol) e ICI118,551 (antagonista ?2) (5 nmol) reduziu o tempo de imobilidade de animais submetidos ao teste do nado forçado. Além disso, a administração local das mesmas doses dessas drogas em grupos independentes de animais submetidos ao teste do campo aberto não causou alteração na atividade locomotora dos animais, descartando um possível efeito inespecífico sobre a atividade locomotora. Os resultados sugerem, portanto, o envolvimento da neurotransmissão noradrenérgica no NLET, através da ativação de receptores noradrenérgicos ?1, ?1 e ?2, na mediação das consequências comportamentais do estresse e nos mecanismos neurais envolvidos na neurobiologia da depressão. / Stress seems to be a major factor responsible for the individual\'s predisposition to depression. Previous studies from our laboratory have shown that acute inactivation of synaptic transmission within the bed nucleus of the stria terminalis (NLET) with cobalt chloride causes antidepressant-like effects in rats submitted to the forced swimming test, suggesting that NLET activation during stress facilitates the development of the behavioral consequences related to the neurobiology of depression. However, the neurotransmitters involved in mediating these effects remain unknown. It is known that the noradrenergic neurotransmission in NLET is abundant and plays an important role in regulating emotional processes related to stress response. Thus, the present study investigated the involvement of the noradrenergic neurotransmission in the bed nucleus of the stria terminalis (NLET) in the development of the behavioral consequences of stress related to the neurobiology of depression by local administration of noradrenergic antagonists in rats submitted to the forced swimming animal model. The results showed that the local administration of WB4101 (?1-antagonist) (10 and 15 nmol), CGP20712 (?1-antagonist) (5 and 10 nmol) and ICI118, 551 (?2-antagonist) (5 nmol) reduced the immobility time of animals submitted to the forced swimming test, an antidepressant-like effect. Furthermore, local administration of the same doses of these drugs in independent groups of animals submitted to the open field test did not change their locomotor activity, discarding a possible unspecific effect on locomotor activity. The results, therefore, suggest the involvement of the noradrenergic neurotransmission within the NLET, through the activation of noradrenergic receptors ?1, ?1 and ?2, in mediating the behavioral consequences of stress and the neural mechanisms involved in the neurobiology of depression.

RESUMO FORTALEZA, E. A. T. Função da neurotransmissão noradrenérgica do núcleo medial da amígdala na modulação do sistema cardiovascular em ratos. 2013. 153p. Tese (Doutorado) Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, 2013. O núcleo medial da amígdala (NMA) está envolvido no controle de uma variedade de processos fisiológicos e comportamentais, dentre eles, a regulação do sistema cardiovascular durante situações aversivas. Além disso, existem evidências indicando a presença de receptores noradrenérgicos no NMA e que os níveis de noradrenalina (NA) no NMA aumentam quando animais são submetidos ao modelo de estresse por restrição (ER). Portanto, o presente estudo foi dividido em duas partes: 1) avaliamos os efeitos da microinjeção local de NA no NMA de ratos, bem como os receptores locais e os mecanismos periféricos envolvidos na sua mediação e 2) investigamos o papel desempenhado pela neurotransmissão noradrenérgica do NMA sobre as respostas cardiovasculares causadas pelo ER. A primeira parte dos nossos resultados indicou que doses crescentes de NA (3, 9, 27 ou 45nmol/100nL) microinjetadas no NMA causavam respostas pressoras e bradicardicas de maneira dose-dependente em ratos não anestesiados. Os efeitos da NA no NMA foram abolidos pelo pré-tratamento local com antagonista de receptores 2-adrenérgicos (RX821002, 10 nmol/100nL), porém não foram alterados pelo pré-tratamento com antagonista de receptores 1adrenérgicos (WB4101, 10 nmol/100nL). A magnitude da resposta pressora à NA no NMA foi aumentada pelo pré-tratamento intravenoso (i.v.) com o bloqueador ganglionar pentolínio (5mg/kg) e foi significativamente reduzida pelo pré-tratamento i.v. com antagonista dos receptores vasopressinérgicos do tipo V1 dTyr(CH2)5(Me)AVP (50 g/Kg) sugerindo que o mecanismo periférico envolvido nestas respostas cardiovasculares é a liberação de vasopressina na circulação sanguínea. Como estruturas finais participando das respostas cardiovasculares desencadeadas pela microinjeção de NA no NMA, foi observado o possível envolvimento dos núcleos paraventricular (NPV) e supra-óptico (NSO) do hipotálamo, que sintetizam vasopressina. O bloqueio de sinapses, tanto no NPV quanto no NSO, com cloreto de cobalto (CoCl2, 1mM/100nL), reduziu as respostas cardiovasculares desencadeadas pela microinjeção de NA no NMA, sugerindo que ambos os núcleos são responsáveis pela liberação de vasopressina em resposta à NA no NMA. Na segunda parte deste estudo, os resultados mostraram que a microinjeção bilateral de WB4101, antagonista seletivo dos receptores 1- adrenérgicos (10, 15 e 20nmol/100nL) no NMA 10 min antes do ER, reduziu a resposta taquicárdica evocada pelo ER, de maneira dose-dependente. Em contraste, a microinjeção bilateral de RX821002, antagonista seletivo dos receptores 2- adrenérgicos (10, 15 e 20nmol/100nL) no NMA 10 min antes do ER, aumentou a resposta taquicárdica evocada pelo ER de maneira dose-dependente. Ambos os tratamentos não causaram alterações na resposta pressora observada nos animais submetidos ao ER. Estes resultados sugerem que receptores 1- e 2-adrenérgicos no NMA desempenham papel facilitatório e inibitório, respectivamente, na resposta taquicárdica durante o ER. Além disso, a microinjeção bilateral de propranolol, antagonista não seletivo dos receptores -adrenérgicos (10, 15 e 20nmol/100nL), no MNA 10 min antes do ER, causou aumento na resposta taquicárdica apenas quando os animais foram pré-tratados com a dose de 15 nmol, sem efeito significativo sobre a resposta pressora observada durante o ER. Este resultado indica que receptores -adrenérgicos no NMA desempenham um papel inibitório sobre a frequência cardíaca durante o ER. Uma vez que foi observada a participação dos receptores - adrenérgicos, realizamos o pré-tratamento com os antagonistas seletivos dos receptores 1- e 2- adrenérgicos, CGP 20712 e ICI 118,551, respectivamente. A microinjeção bilateral de ICI 118,551 (10, 15 e 20nmol/100nL) no NMA aumentou as respostas taquicárdicas ao ER após a microinjeção das doses de 15 e 20 nmol. Esse efeito foi semelhante ao observado após o tratamento prévio com propranolol, na dose de 15 nmol, sem efeito significativo sobre a resposta da pressora durante o ER. Porém, o pré-tratamento com o CGP 20712 (10, 15 e 20 nmol/100 nL) causou um efeito oposto sobre a resposta taquicárdica evocada pelo ER, ocorrendo uma redução da resposta taquicárdica que foi observada apenas após a dose de 20 nmol no NMA, sem efeito significativo sobre a resposta pressora evocada pelo ER. Esses resultados sugerem que os receptores 2-adrenérgicos desempenham influência inibitória sobre a resposta taquicárdica, ao passo que, os receptores 1-adrenérgicos desempenham influência facilitatória na resposta taquicárdica evocada pelo ER. / ABSTRACT FORTALEZA, E. A. T. Noradrenergic neurotransmission function of the medial nucleus of the amygdala in the modulation of the cardiovascular system in rats. 2013. 153p. Thesis (Doctoral) School of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, 2013. The medial amygdaloid nucleus (MeA) regulates a variety of physiological and behavioral processes, as well as regulation of the cardiovascular system during aversive situations. Moreover, there is evidence indicating that noradrenergic receptors are present into the MeA and that the levels of noradrenaline (NA) in this nucleus increases when animals are submitted to acute restraint model. Therefore, this study was divided into two parts: 1) we tested the effects of local NA microinjection into the MeA on cardiovascular responses in unaesthetized rats. Moreover, we describe the type of adrenoceptors activated and the peripheral mechanisms involved in the cardiovascular responses and 2) we investigated the involvement of MeA adrenoceptors in the modulation of cardiovascular responses that are observed during acute restraint. The first part of our results indicated that increasing doses of NA (3, 9, 27 or 45 nmol/ 100nL) microinjected into the MeA of unanesthetized rats caused dose-related pressor and bradycardiac responses. The cardiovascular effects of NA were abolished by MeA local pretreatment with 10 nmol/ 100nL of the specific 2-adrenoceptor antagonist RX821002, but were not affected by local pretreatment with 10 nmol/100nL of the specific 1-adrenoceptor antagonist WB4101. The magnitude of pressor response evoked by NA microinjected into the MeA was potentiated by intravenous pretreatment with the ganglion blocker pentolinium (5 mg/kg, i.v.) and blocked by intravenous pretreatment with the selective V1 vasopressin antagonist dTyr (CH2)5(Me) AVP (50 g/Kg, i.v.). Our results suggest that microinjection of NA into the MeA of unanesthetized rats activates local 2- adrenoceptors, evoking pressor and bradycardiac responses, which are mediated by vasopressin release in the blood circulation. The vasopressin is synthesized by magnocellular cells of paraventricular (PVN) and supraoptic nucleus (SON) of the hypothalamus. Thus, it was investigated the role of these hypothalamic nucleus on the cardiovascular pathway activated by the microinjection of NA into the MeA. Pressor and bradycardiac responses to NA (27nmol/100nL) were blocked when cobalt chloride (CoCl2, 1mM/100nL) was microinjected into the PVN or SON, thus indicating that both hypothalamic nucleus are responsible for the mediation of the cardiovascular responses evoked by NA microinjection into the MeA. Our results suggest that pressor and bradycardic responses caused by NA microinjection into the MeA are mediated by magnocellular neurons in the PVN and SON. In the second part of this study Male Wistar rats received bilateral microinjections of the selective 1-adrenoceptor antagonist WB4101 (10, 15, and 20 nmoL/100nL) or the selective 2-adrenoceptor antagonist RX821002 (10, 15, and 20 nmoL/nL) into the MeA, 10 min before the exposure to acute restraint. The injection of WB4101 into the MeA reduced the restraint-evoked tachycardia. In contrast, the injection of RX821002 increased the tachycardia. Both drugs had no effect on the BP increase observed during the acute restraint. Our findings indicate that 1 and 2-adrenoceptors in the MeA play different roles in the modulation of the HR increase evoked by restraint stress in rats. Results suggest that 1-adrenoceptors and 2-adrenoceptors respectively mediate the MeA facilitatory and inhibitory influences on restraint-related HR responses. Moreover, we investigated the involvement of -adrenoceptors in the MeA in cardiovascular responses evoked in rats submitted to an acute restraint stress. We first pretreated Wistar rats with the nonselective -adrenoceptor antagonist propranolol, microinjected bilaterally into the MeA (10, 15, and 20 nmol/100nL) 10 min before exposure to acute restraint. The pretreatment with propranolol did not affect the BP increase evoked by restraint. However, it increased the tachycardiac response caused by acute restraint when animals were pretreated with a dose of 15 nmol, without a significant effect on the BP response. This result indicates that -adrenoceptors in the MeA have an inhibitory influence on restraintevoked HR changes. Pretreatment with the selective 2- adrenoceptor antagonist ICI 118,551 (10, 15, and 20 nmol/100 nL) significantly increased the restraint evoked tachycardiac response after the doses of 15 and 20 nmol, an effect that was similar to that observed after the pretreatment with propranolol at the dose of 15 nmol, without a significant effect on the BP response. Pretreatment of the MeA with the selective 1-adrenoceptor antagonist CGP 20712 (10, 15, and 20 nmol/100nL) caused an opposite effect on the HR response, and a significant decrease in the restraint-evoked tachycardia was observed only after the dose of 20nmol, without a significant effect on the BP response. Because propranolol is an equipotent antagonist of both 1 and 2-adrenoceptors, and opposite effects were observed after the treatment with the higher doses of the selective antagonists ICI 118,551 and CGP 20712, the narrow window in the dose-response to propranolol could be explained by a functional antagonism resulting from the simultaneous inhibition of 1 and 2-adrenoceptors by the treatment with propranolol.

A perimenopausa representa a transição da vida reprodutiva para não reprodutiva. É geralmente caracterizada por alterações neuroendócrinas, metabólicas e comportamentais, um possível resultado da depleção folicular ovariana e consequente redução do número de folículos ovarianos. É o período em que as mulheres podem apresentar maior susceptibilidade a manifestar transtornos afetivos e de ansiedade. A exposição de roedores ao resíduo químico 4-diepóxido de vinilciclohexeno (VCD) é um modelo bem estabelecido para estudos sobre perimenopausa, pois o VCD acelera o processo natural de atresia folicular. Embora as concentrações plasmáticas de estradiol estejam normais ou elevadas durante a perimenopausa, a terapia com estradiol pode ser benéfica para mulheres sintomáticas na perimenopausa. Portanto, o objetivo do presente trabalho foi investigar se a depleção folicular gradativa acelerada pelo VCD resulta em alterações na neuroquímica de ratas fêmeas em núcleos cerebrais que controlam o humor, além de avaliar se o estradiol seria capaz de reverter as possíveis alterações. Ratas da linhagem Wistar (28 dias pós-natal) receberam diariamente, durante 15 dias consecutivos, injeções subcutâneas de VCD (160 mg / kg) ou óleo de milho (O). Aproximadamente 55 dias após a primeira injeção, cápsulas de silastic contendo 17?-estradiol (E) ou O foram inseridas subcutaneamente (Grupos O+O; VCD+O; VCD+E). Cerca de 21 dias após o implante das cápsulas, as ratas dos grupos O+O e VCD+O foram decapitadas na manhã do diestro, enquanto que as do grupo VCD+E foram decapitadas exatamente 21 dias após o implante das cápsulas contendo estradiol, entre 0900 h e 1100 h. O sangue foi colhido para avaliação das concentrações plasmáticas de estradiol e progesterona por radioimunoensaio (RIE). Os cérebros foram removidos para microdissecção do hipocampo, amígdala, Locus coeruleus (LC) e Núcleo Dorsal da Rafe (NDR), para posterior análise dos níveis de RNAm para os receptores de progesterona (PR) e estradiol do tipo beta (ER?) por meio de RT/PCR. Este experimento foi replicado para remoção do hipocampo e amígdala para dosagem dos conteúdos de noradrenalina (NA) e serotonina (5-HT) por meio de cromatografia líquida de alta performance, seguida de detecção eletroquímica (HPLC/ED). Outro conjunto de ratas submetidas às mesmas condições10 experimentais foi perfundido para imunohistoquímica para TPH no NDR e TH no LC. Como esperado, na periestropausa (grupo VCD+O) as concentrações plasmáticas de estradiol não foram diferentes daquelas das ratas controles (O+O). As concentrações plasmáticas de progesterona na periestropausa foram menores que as do grupo controle, o que foi revertido pelo estradiol. No LC, a expressão de PR na periestropausa foi igual à das ratas controles, enquanto a expressão do ER? foi menor; a terapia com estradiol não modificou a expressão de nenhum destes receptores. A densidade de neurônios noradrenérgicos (TH+) no LC não foi alterada nem pela depleção folicular nem pela terapia estrogênica. Na periestropausa, o conteúdo de NA foi menor na amígdala, mas não no hipocampo, e o estradiol não alterou este conteúdo em nenhuma das áreas. No NDR, a expressão de PR e de ER? nas ratas na periestropausa foi menor que nas ratas controles; o estradiol preveniu o declínio da expressão de ER?, mas não de PR. O NDR foi analisado separadamente por toda a extensão rostro-caudal em 3 níveis anatômicos: rostral, médio e caudal, cada um dividido em 3 sub-regiões: lateral, dorsal e ventral. O número de neurônios serotonérgicos (TPH+) no NDR foi menor na periestropausa, e o estradiol foi capaz de reverter esse efeito, atuando principalmente na região caudal. A expressão gênica de PR não foi alterada nem pela depleção folicular nem pela terapia estrogênica tanto na amígdala como no hipocampo. A expressão de ER? também não foi diferente na periestropausa, quando comparada ao grupo controle, mas o estradiol aumentou esta expressão no hipocampo. Tanto na amígdala como no hipocampo houve redução no conteúdo de 5-HT na periestropausa e estradiol foi capaz de reestabelecer os níveis deste neurotransmissor aos valores controles apenas no hipocampo. Estes dados elucidam, pelo menos em parte os mecanismos do efeito positivo da terapia estrogênica nos sintomas de mulheres normoestrogênicas na perimenopausa. Estes efeitos parecem não envolver de forma importante o sistema noradrenérgico central, mas resultar do aumento da biossíntese de progesterona periférica em associação com a regulação positiva de ER? no NDR e hipocampo, que parece potencializar a via serotonérgica NDR/HPC. Portanto, o desenvolvimento de novas terapias que ativem os ER? pode ser uma alternativa para obter os efeitos positivos da ação do estradiol, eliminando os efeitos colaterais das terapias de estradiol que normalmente resultam da ativação do ER?. / Perimenopause represents the transition from reproductive to non-reproductive life. It is usually characterized by neuroendocrine, metabolic and behavioural changes, which result from a follicular depletion and reduced number of ovarian follicles. During this period, women are more likely to express mood disorders and anxiety. The exposure of animals to diepoxide 4-vinylcyclohexene (VCD) is a well-established experimental model for perimenopause studies, as VCD induces loss of ovarian small follicles (primary and primordial) in mice and rats by accelerating the natural process of atresia. Although estrogens levels are normal or even high during perimenopause, estrogen therapy can be beneficial for symptomatic perimenopausal women. The aim of this study was to investigate whether gradual follicular depletion induced by VCD results in changes in the neurochemistry of female rats in brain nuclei that control mood and the role of estradiol on these changes. Female rats (28 days) were daily injected with VCD or corn oil (O) for 15 days. Around 55 days after the first injection, pellets of 17?-estradiol (E) or O were inserted s.c (Groups O+O; VCD+O; VCD+E). Around 21 days after, rats O+O and VCD+O were decapitated between 0900 h and 1100 of diestrus while rats VCD+E were decapitated exactly 21 days after the onset of E therapy. Another set of rats followed the same experimental design and were perfused for TH and TPH immunohistochemistry in Locus coeruleus (LC) and Dorsal Raphe Nuclei (DRN), respectively. Blood was collected for estradiol and progesterone measurement by radioimmunoassay (RIA). The brains were removed from decapitated rats to punch out LC, DRN, hippocampus and amygdala to analyse the expression of mRNA for ER? and PR by RT/PCR. This experiment was replicated to punch out the hippocampus and amygdala for the determination of noradrenaline (NE) and serotonin (5-HT) contents by High Performance Liquid Chromatography, followed by Electrochemical Detection (HPLC/ED). As expected, plasma concentrations of estradiol were not different from those of control rats (O + O). Plasma concentrations of progesterone in the periestropause were lower than those in the control group, which was reversed by estradiol. In the LC, the PR expression in the periestropause was similar to that of the control rats, whereas the ER? expression was lower; estradiol therapy did not modify the expression of any of these receptors. The12 density of noradrenergic (TH +) neurons in LC was not altered by either follicular depletion or estrogen therapy. In periestropause, NA content was lower in the amygdala, but not in the hippocampus, and estradiol did not alter this content in any of the areas. In NDR, the expression of PR and ER? in periestropausal rats was lower than in controls; estradiol prevented the decrease of ER? expression, but not PR. The NDR was analyzed separately for the entire rostrocaudal axis in three anatomical levels: rostral, middle and caudal, each divided into three sub-regions: lateral, dorsal and ventral. The number of serotonergic neurons (TPH +) in NDR was lower in the periestropause, and estradiol was able to reverse this effect, acting mainly in the caudal region. PR gene expression was not altered by either follicular depletion or estrogen therapy in either the amygdala or the hippocampus. ER? expression was also no different in periestropause compared to the control group, but estradiol increased this expression in the hippocampus. Both in the amygdala and in the hippocampus there was a reduction in 5-HT content in the periestropause, and estradiol was able to reestablish the levels of this neurotransmitter at the control values only in the hippocampus. These data elucidate, at least in part, the mechanisms of the positive effect of estrogen therapy on the symptoms of normoestrogenic women in perimenopause. These effects do not appear to significantly involve the central noradrenergic system but result from increased peripheral progesterone biosynthesis in association with positive regulation of ER? in the NDR and hippocampus, which appears to potentiate the serotonergic NDR/HPC pathway. Therefore, the development of new therapies that activate ER? may be an alternative to obtain the positive effects of the estradiol action, eliminating the side effects of the estradiol therapies that normally result from the activation of ER?.

O estresse parece ser um dos principais fatores responsáveis pela predisposição do indivíduo à depressão. Estudos prévios de nosso laboratório mostraram que a inativação aguda da transmissão sináptica no núcleo leito da estria terminal (NLET) provoca efeitos tipo-antidepressivos em animais submetidos ao modelo animal do nado forçado, indicando que sua ativação durante o estresse facilita o desenvolvimento de consequências comportamentais relacionadas à neurobiologia da depressão. Contudo, os neurotransmissores envolvidos na mediação de tais efeitos permanecem desconhecidos. Sabe-se que a neurotransmissão noradrenérgica no NLET é abundante e tem participação importante na regulação de processos emocionais relacionados à resposta ao estresse. Assim, o presente trabalho investigou o envolvimento da neurotransmissão noradrenérgica existente no núcleo leito da estria terminal (NLET) no desenvolvimento das consequências comportamentais do estresse relacionadas à neurobiologia da depressão, através da administração local de antagonistas noradrenérgicos em animais submetidos ao modelo animal do nado forçado. Os resultados mostraram que a administração local de WB4101 (antagonista ?1) (10 e 15 nmol), CGP20712 (antagonista ?1) (5 e 10 nmol) e ICI118,551 (antagonista ?2) (5 nmol) reduziu o tempo de imobilidade de animais submetidos ao teste do nado forçado. Além disso, a administração local das mesmas doses dessas drogas em grupos independentes de animais submetidos ao teste do campo aberto não causou alteração na atividade locomotora dos animais, descartando um possível efeito inespecífico sobre a atividade locomotora. Os resultados sugerem, portanto, o envolvimento da neurotransmissão noradrenérgica no NLET, através da ativação de receptores noradrenérgicos ?1, ?1 e ?2, na mediação das consequências comportamentais do estresse e nos mecanismos neurais envolvidos na neurobiologia da depressão. / Stress seems to be a major factor responsible for the individual\'s predisposition to depression. Previous studies from our laboratory have shown that acute inactivation of synaptic transmission within the bed nucleus of the stria terminalis (NLET) with cobalt chloride causes antidepressant-like effects in rats submitted to the forced swimming test, suggesting that NLET activation during stress facilitates the development of the behavioral consequences related to the neurobiology of depression. However, the neurotransmitters involved in mediating these effects remain unknown. It is known that the noradrenergic neurotransmission in NLET is abundant and plays an important role in regulating emotional processes related to stress response. Thus, the present study investigated the involvement of the noradrenergic neurotransmission in the bed nucleus of the stria terminalis (NLET) in the development of the behavioral consequences of stress related to the neurobiology of depression by local administration of noradrenergic antagonists in rats submitted to the forced swimming animal model. The results showed that the local administration of WB4101 (?1-antagonist) (10 and 15 nmol), CGP20712 (?1-antagonist) (5 and 10 nmol) and ICI118, 551 (?2-antagonist) (5 nmol) reduced the immobility time of animals submitted to the forced swimming test, an antidepressant-like effect. Furthermore, local administration of the same doses of these drugs in independent groups of animals submitted to the open field test did not change their locomotor activity, discarding a possible unspecific effect on locomotor activity. The results, therefore, suggest the involvement of the noradrenergic neurotransmission within the NLET, through the activation of noradrenergic receptors ?1, ?1 and ?2, in mediating the behavioral consequences of stress and the neural mechanisms involved in the neurobiology of depression.

Les troubles de l’initiation du mouvement de la maladie de Parkinson sont invalidants et sans solution thérapeutique satisfaisante. Dans ce travail, nous nous intéressons à deux catégories de symptômes, classiquement explorées de façon indépendante : d’un côté, la lenteur à l’initiation du mouvement ou akinésie, et de l’autre, la difficulté à retenir l’initiation d’un mouvement ou impulsivité. En nous appuyant sur des avancées théoriques et méthodologiques récentes, nous tentons de revisiter ces troubles de l’initiation du mouvement en termes de dysfonctionnements exécutifs dans la maladie de Parkinson.Dans ce but, nous avons : 1) recherché les liens entre marqueurs cliniques (akinésie et impulsivité) et troubles du contrôle inhibiteur ; 2) identifié les dysfonctionnements cérébraux associés au moyen de méthodes électroencéphalographiques innovantes permettant de localiser les sources cérébrales et d’observer la dynamique des processus cérébraux ; et 3) sondé l’origine neurochimique de cette fonction à l’aide d’un protocole pharmacologique.Nos résultats suggèrent que l’impulsivité et l’akinésie sont les deux faces d’une même pièce, aux origines d’un dysfonctionnement du contrôle inhibiteur proactif non sélectif. Ils indiquent également que ce contrôle exécutif est sous-tendu par l’activité du noyau sous-thalamique et d’un réseau cortical médian comprenant le précuneus et l’aire motrice supplémentaire. Un rôle du système noradrénergique a également été mis en évidence à l’origine de ces troubles exécutifs. Ces travaux ouvrent donc la voie à de nouvelles perspectives thérapeutiques pour la maladie de Parkinson / Movement initiation disorders in Parkinson’s disease are multifaceted, are debilitating and have no satisfying therapeutic option. On the one hand, slowness and difficulties initiating voluntary movements contribute to akinesia, a cardinal symptom of the disease which is usually considered to be motor in origin and which is not fully alleviated by current medication. On the other hand, difficulties refraining voluntary movements contribute to impulsivity, a frequent side effect of current dopaminergic and neurosurgical therapies. Here, based on systematic analyses of the clinical neuroimaging literature, we suggest that these opposite forms of movement initiation disorders might be executive, not purely motor, in origin.To empirically test this hypothesis, we: 1) related clinical markers of the disease (akinesia and impulsivity) to behavioral indexes of inhibitory control impairment; 2) identified associated cerebral dysfunctions by means of advanced electroencephalographic (EEG) methods; and 3) investigated the neurochemical origin of these dysfunctions by combining pharmacological and cerebral stimulations with EEG recordings.Results suggest that impulsivity and akinesia are the two sides of the same coin. Indeed, they were found to be associated with opposite inhibitory control disorders, and dysfunctions of subthalamic cortical loops involving medial executive areas. Importantly, it was also found that restoring normal behavior and normal activity within these loops by means of deep brain stimulation depends on the level of noradrenergic tonus, opening the way for new therapeutic approaches for Parkinson’s disease

L'inhibition de réponse joue un rôle majeur dans le contrôle cognitif. Mais les relations entre activité cérébrale et comportement sont particulièrement difficiles à appréhender puisque la fonction est précisément destinée à prévenir tout comportement observable… Dans une première partie, nous proposons une revue de questions mettant en évidence la nécessité d'utiliser des méthodes psychophysiques adaptées. Nous proposons également une analyse systémique des travaux utilisant l'électroencéphalographie, méthode largement privilégiée pour sonder la dynamique des mécanismes inhibiteurs. Dans une seconde partie, nous mettons en œuvre ces propositions dans le cadre d'une étude combinée EEG/IRMf. Grâce à des méthodes avancées de traitement du signal EEG, nous suggérons la mise en jeu de mécanismes automatiques, non sélectifs, d'inhibition de réponse dès lors que le contexte événementiel est incertain. Il reposerait sur la pré-activation d'une circuiterie auto-inhibitrice dans le complexe moteur supplémentaire, implémenté par un large réseau médial frontopariétal. Dans les troisième et quatrième parties, nous montrons le rôle majeur de cette fonction dans d'autres processus cognitifs et illustrons les conséquences cliniques de son dysfonctionnement. Nous montrons en particulier que l'impulsivité n'est pas la seule expression des troubles du contrôle inhibiteur. La difficulté à initier une action constituerait, dans la maladie de Parkinson, une conséquence directe de ce dysfonctionnement. L'implication d'une dérégulation du système noradrénergique et de l'activité du noyau sous-thalamique dans l'occurrence du trouble est suggérée, et les perspectives thérapeutiques discutées / Response inhibition plays a major role in cognitive control. Understanding these mechanisms is essential because their dysfunctions may cause symptoms in many psychiatric and neurological diseases. The relationship between brain activity and behavior are particularly difficult to understand because the function of interest is specifically intended to prevent any observable behavior… In the first part, we propose a review of issues highlighting the need for a new theoretical model and adapted psychophysical methods. We also provide systematic analysis of studies using electroencephalography, widely preferred method for probing the dynamics of inhibitory mechanisms. Convergently, the review calls for revisiting both acquisition methods and processing methods of EEG signal used in conventional studies. In the second part, we implement these proposals by combining advanced treatment methods of EEG signal (blind source separation, analysis test by test, spectral analysis in the sources, location) and comparing these results with the fMRI data. We reveal the involvement of automatic and nonselective inhibitory mechanisms, when the context is uncertain. A large medial frontoparietal network would ensure the implementation. The involvement of an automatic behavior (when the environment becomes predictable) would require a top-down control that deactivates in advance the excitability of the self-inhibitory circuitry of the SMC. In the third and fourth sections, we show the key role of this function in other cognitive processes and illustrate the clinical consequences of its dysfunction. We show in particular that the difficulty in initiating an action (akinesia) would, in Parkinson’s disease, a direct consequence of this dysfunction. The involvement of the noradrenergic system and the activity of the subthalamic nucleus in the occurrence of the disorder are suggested, and therapeutic perspectives discussed