A estimulação epidural do córtex motor (ECM) é eficaz no tratamento da dor neuropática refratária, porém seus mecanismos de ação ainda são incertos. Sabendo que a ECM ativa a via analgésica descendente em ratos, fomos investigar o efeito da ECM sobre os núcleos serotonérgicos descendentes, dorsal da rafe (NDR) e magno da rafe (NMR) e sobre os neurônios da coluna posterior da medula espinhal (CPME). Ratos Wistar, submetidos à ECM, foram avaliados no teste de pressão da pata e seus tecidos foram avaliados frente à imunorreatividade (IR) para Egr-1 (marcador de ativação neuronal), serotonina (5HT) e substância P (SP). A ECM induziu antinocicepção em 62% nos animais, não alterou a ativação do NDR, entretanto ativou o NMR (67%), quando comparado com ratos não estimulados. A ECM aumentou a IR-5HT em 75% no NDR e em 92% no NMR. Na CPME, a ECM inibiu os neurônios nociceptivos (48%), porém não interferiu com a IR-SP. Estes resultados sugerem que a ECM induz analgesia, em parte, via ativação do sistema serotonérgico descendente. / Motor cortex stimulation (MCS) is effective in the treatment of refractory neuropathic pain; however, its mechanisms of action remain unclear. Since the MCS activates the descending pain pathway in rats, we investigated the MCS effect on the descending serotonergic nuclei, dorsal raphe nucleus (DRN) and the magnus raphe nucleus (MRN) and also on the neurons of the dorsal horn of the spinal cord (DHSC). Wistar rats, submitted to MCS, were evaluated by paw pressure test and its tissues were evaluated by immunoreactivity (IR) to Egr-1 (neuronal activation marker), serotonin (5HT) and substance P (SP). MCS induced antinociception by 62% in animals, although did not modify the NDR activation; however, activated the MRN (67%), when compared to control groups. MCS increased the IR-5HT by 75% in the NDR and 92% in the MRN. In the DHSC, MCS inhibited the nociceptive neurons (48%), however did not change the IR-SP. These results suggest that MCS induce antinociception, partly, by the descending serotonergic pathway activation.

A patogenia da hiponatremia decorrente do uso da Fluoxetina em pacientes idosos não está bem definida. Este achado tem sido atribuído a uma secreção inapropriada do Hormônio Antidiurétido (HAD), embora ainda não tenha sido evidenciado um aumento dos níveis plasmático deste hormônio nestes pacientes. Desta forma, este trabalho visa investigar o efeito da Fluoxetina sobre o rim e mais especificamente se ela teria um efeito direto sobre o Ducto Coletor Medular Interno (DCMI) de ratos normais: 1) Estudos in vivo- 10 ratos foram suplementados com aplicações intraperitoniais diárias de 10mg/kg de Fluoxetina. Após 10 dias os animais foram sacrificados e o sangue e os rins foram coletados para posterior análise. 2) Estudos in vitro- a) estudos com a técnica de Imunoblotting para avaliar a expressão da proteína da Aquaporina 2 (AQP2) em ratos suplementados e em suspensão de túbulos de DCMI de 10 ratos normais incubados com 10-7M de Fluoxetina por 30 min. b) estudos com microperfusão de segmentos isolados do nefron- a permeabilidade osmótica à água (Pf, mm./sec) foi determinada em DCMI perfundidos de ratos normais(n=6) com a técnica standart, na presença de10-7M de Fluoxetina. Resultados: 1) Os estudos in vivo mostraram que o peso caiu de 179±1,28g para 158±1,15g (p<0,0001), o Na plasmático caiu de 139,3±0,78mEq/l para 134,9±0,5mEq/l (p<0,0001) e não houve alterações do K e do HAD plasmáticos. 2) Os estudos in vitro-a) a análise densitométrica dos ensaios com imunoblotting mostraram um aumento da expressão da proteína da AQP2 de 40%, ambos, em ratos suplementados (cont-99,6± 5,2 vs Fx 145,6±16,9, p< 0,05) e em frações de membrana da suspensão de túbulos incubados com Fluoxetina (cont-100,0±3,5.vs 143,0±2,0, p<0,01). b) A Fluoxetina aumentou a Pf nos DCMI, na ausência de HAD de 7,24±2,07 para Fx- 15,77±3,25 (p<0,01). Em resumo, nossos dados revelaram que a Fluoxetina determinou uma diminuição do peso corporal e do nível plasmático de Na sem alterar os níveis plasmáticos de K e HAD e ocasionou um aumento da expressão da proteína da AQP2 e um aumento da permeabilidade à água nos DCMI, levando-nos à conclusão de que o efeito direto da Fluoxetina no DCMI, aumentando a reabsorção de água, pode ser responsável pela hiponatremia encontrada nos pacientes idosos com depressão que fazem uso desta droga. / The pathogenesis of the hyponatremia after Fluoxetine(Fx) supply in elderly is not well understood. This event has been attributed to an inappropriate antidiuretic hormone secretion although the vasopressin enhanced plasma level has not been demonstrated yet in these pacients. Thus, this experiment was designed to investigate the effect of Fx on the kidney and more specifically if it would have a direct effect in the inner medullary collecting duct (IMCD) from normal rats: 1) in vivo study- 10 rats were supplied daily with i.p. injections of Fx 10 mg/kg. After five days, the rats were sacrificed and blood and kidneys were collected. 2) in vitro studya) Immunobloting studies for AQP2 protein expression, in IMCD from supplied rats and in IMCD tubules suspension from 10 normal rats incubated with mm./sec) Fx. by 30 min. b) Microperfusion studies- the osmotic water permeability (Pf, mm./sec) was determined in normal rats IMCD(n=6) isolated and perfused by the standart methods.Fx (10-7M) was added to the bath fluid. Results: In vivo study showed that the weight decreased from 179±1.28g to 158±1.5g(p<0.0001); the Na+ plasma level decreased from 139.3±0.78mEq/l to 134.9±0.5mEq/l(p<0.0001), the K+ and the ADH plasma level remained unchanged. In vitro study- a) the densitometric analysis of the immunobloting essays showed an increase in AQP2 protein abundance of about about 40%, both, in supplied rats (cont-99.6± 5.2 vs Fx 145.6±16.9, p< 0.05), and in membrane fraction extracted from IMCD tubule suspension incubated with Fx ( cont-100.0±3.5.vs 143.0±2.0,p<0.01). b) Fx increased the Pf in IMCD in absence of vasopressin from the control period- 7.24±2.07 to Fx- 15.77±3.25 (p<0.01). In summary, our data revealed that, after Fx, weight, and plasma Na+ level decreased, the plasma K+ and ADH levels remained unchanged, whereas the AQP2 protein abundance and the water absorption in IMCD increased, leading us to conclude that the direct effect of Fx in the IMCD, could account for the hyponatremia produced by this drug in depressed elderly.

Les troubles du spectre autistique (TSA) sont définis par l’association de troubles des interactions sociales, de troubles du langage et de comportements stéréotypés et intérêts restreints. Cette entité recouvre en réalité des situations cliniques très hétérogènes, tant par le spectre de sévérité des symptômes que par la variété des comorbidités et signes associés. Si l’étiologie génétique semble prépondérante, les mécanismes moléculaires impliqués semblent complexes et hétérogènes, et les associations génotype-phénotype inconstantes. Une stratégie possible pour décomposer cette hétérogénéité clinique et génétique consiste à s’appuyer sur des endophénotypes, ou phénotypes intermédiaires, pour définir des catégories plus homogènes sur le plan physiopathologique. Parmi les nombreux endophénotypes biologiques décrits dans les TSA, l’augmentation de la sérotonine sanguine est l’un des mieux documentés, mais les mécanismes en sont encore inconnus. Des déficits en mélatonine (qui est un dérivé de la sérotonine) ont également été rapportés par plusieurs études. L’objectif de cette thèse était de caractériser les anomalies de la voie sérotonine-mélatonine dans les TSA, et d’en étudier les mécanismes et les corrélats cliniques. L’exploration exhaustive de la voie sérotonine-mélatonine à partir de prélèvements sanguins dans une grande cohorte de plus de 200 patients avec TSA et leurs apparentés a permis d’estimer la prévalence de l’hypersérotoninémie dans les TSA à 45%, et celle du déficit en mélatonine à environ 60%. Cette étude a mis en évidence des anomalies du catabolisme de la sérotonine ainsi que des anomalies de la synthèse de la mélatonine, et a ainsi permis de proposer des mécanismes biochimiques à ces deux endophénotypes. Les anomalies de synthèse de la mélatonine, qui pourraient s’accompagner d’une augmentation de la N-acétylsérotonine, ont été confirmées sur des échantillons de glandes pinéales et de tractus gastro-intestinal (sources majeures de mélatonine et de sérotonine de l’organisme) issus de patients avec TSA. Les gènes impliqués dans la synthèse de la mélatonine (gènes codant pour les enzymes AANAT et ASMT) ont été étudiés dans les TSA, ainsi que dans des pathologies neurodéveloppementales connexes. Les anomalies de la synthèse de mélatonine pourraient être associées aux troubles du sommeil, fréquents chez les patients avec TSA / Autism Spectrum Disorders (ASD) are defined by three core symptoms: social interaction impairments, language impairments, and stereotyped behavior and restricted interests. Beyond this definition lie extremely diverse clinical situations, in terms of symptoms severity as well as comorbidities and associated features. The aetiology of ASD is considered to be mostly genetic, but the molecular mechanisms involved seem to be complex and heterogeneous, and the genotype-phenotype associations elusive. One possible strategy for decomposing the clinical and genetic complexity is to focus on endophenotypes, or intermediate phenotypes, to define more homogeneous pathophysiological categories. Among many biological endophenotypes reported in ASD, the increase of blood serotonin is well documented but still unexplained. Deficits in melatonin (which chemically derives from serotonin) have also been described. The aim of this work was to characterize the impairments of the serotonin-melatonin pathway in ASD, and to address their mechanisms and clinical correlates. Based on a comprehensive assessment of the serotonin-melatonin pathway from blood samples in a large cohort of 200 patients with ASD and their relatives, the prevalence of hyperserotonemia in ASD was estimated to be 45%, and that of melatonin deficit about 60%. Impairments of serotonin catabolism were shown, as well as impairments of melatonin synthesis, thus providing biochemical mechanisms for both endophenotypes. Abnormal melatonin synthesis, which may involve an increase in N-acetylserotonin, was confirmed on pineal gland and gastro-intestinal tract samples (i.e. the major sources of melatonin and serotonin) from patients with ASD. The genes involved in melatonin synthesis (coding for AANAT and ASMT enzymes) were studied in ASD and in related neurodevelopmental disorders. Abnormal melatonin synthesis may be associated with sleep disorders, frequently observed in patients with ASD

An ideal biosensor is a compact and in-expensive device that is able to readily and rapidly detects different types of analytes with high sensitivity and specificity. The affectability of a biosensing methodology is subject to the limit of nanomaterials to transduce the target binding process to an improved perceptible signal, while the selectivity is accomplished by considering the binding and specificity of certain moieties to their targets. Keeping these requirements in mind we have chosen nanomaterials such as carbon nanotubes (CNTs) and gold nanoparticles (AuNPs) that has catalytic properties combined with their size, shape and configuration dependent chemical and physical properties as essential precursors and signaling components for creation of biosensors with tremendous sensitivity. The primary goal of the research work described in this dissertation is to develop and evaluate novel methods to detect various analytes using nanomaterials, at the same time making an affordable architecture for point-of-care (POC) applications. We report here in chapter 3 a simple and new strategy for preparing disposable, paper-based, porous AuNP/M-SWCNT hybrid thin gold films with high conductivity, rapid electron transfer rates, and excellent electrocatalytic properties to achieve multiple analyte electrochemical detection with a resolution that greatly exceeds that of purchased flat gold slides. We further explored the use of nanomaterial-based paper films in more complex matrices to detect analytes such as NADH, which can act as a biomarker for certain cellular redox imbalances and disease conditions. Carbon nanotubes with their large activated surfaces and edge-plane sites (defects) that are ideal for performing NADH oxidation at low potentials without any help of redox mediators minimizing surface fouling in complex matrices is described in chapter 4. With an instrument-free approach in mind we further focused on a colorimetric platform using split cocaine aptamers and gold nanoparticles (AuNPs) to detect cocaine for on-site applications as described in chapter 5. In chapter 5, the split aptamer sequences were evaluated mainly on three basic criteria, the hybridization efficiency, specificity towards the analyte (cocaine), and the reaction time to observe a distinguishable color change from red to blue. The assay is an enzyme-assisted target recycling (EATR) strategy following the principle that nuclease enzyme recognizes probe–target complexes, cleaving only the probe strand releasing the target for recycling. We have also studied the effect of the number of binding domains with variable chain lengths on either side of the apurinic (AP) site. On the basis of our results, we finally shortlisted the sequence combination with maximum signal enhancement fold which is instrumental in development of colorimetric platform with faster, and specific reaction to observe a distinctive color change in the presence of cocaine.

La neurohormone ocytocine (OT) est de plus en plus étudiée pour son potentiel thérapeutique dans les troubles du comportement social, comme l'autisme, qui sont associés à une dérégulation de plusieurs systèmes de neurotransmission, notamment l'OT et la sérotonine (5-HT). Dans ce cadre, une étape importante afin de développer des médicaments basés sur des mécanismes biologiques est de caractériser les interactions entre l'OT et les autres neurotransmetteurs. La littérature sur les rongeurs montre que la relation entre OT et 5-HT est fortement impliquée dans plusieurs aspects du comportement social. Par ailleurs, nous avons récemment montré chez le sujet sain que le fonctionnement du récepteur 5-HT 1A (5-HT1AR) est modifié suite à l'administration d'OT.neuroJ'ai donc réalisé une première expérience chez des patients autistes en utilisant le scanner TEP avec le radiotraceur [18F]MPPF (spécifique du 5-HT1AR). Aucune différence n'est apparue, à l'état basal, entre 18 patients autistes et 24 sujets contrôles. Par ailleurs, l'OT n'a pas modifié le système 5-HT1AR. Enfin, alors qu'une corrélation entre la densité de 5-HT1AR et le volume de matière grise du striatum a été observé dans le groupe contrôle, cette relation était absente dans le groupe de patients. Ces résultats suggèrent une altération subtile du 5-HT1AR, ne pouvant être détectée qu'au niveau fonctionnel.Parce que le scanner TEP ne permet pas de dire si les changements observés sont dus à une libération de sérotonine ou à une modification directe du récepteur, j'ai réalisé une deuxième expérience chez 3 macaques rhésus, avec le [18F]MPPF et le [11C]DASB (marquant le transporteur de la 5-HT). Par rapport au placebo, l'OT injectée dans le ventricule latéral a significativement augmenté la liaison du [18F]MPPF dans l'amygdale et l'insula tandis que la liaison du [11C]DASB diminuait dans ces mêmes régions. Ainsi, nous pouvons dire que l'OT a provoqué la libération de 5-HT ainsi qu'une modification du 5-HT1AR dans ces régions importantes pour les comportements socio-émotionnels. Une étude par autoradiographie a confirmé cette interprétation.Ces expériences montrent qu'il existe une action régulatrice de l'OT sur la 5-HT chez le primate, mais que ce mécanisme est dérégulé chez les patients avec autisme. Cela ouvre donc la voie à l'investigation de traitements combinés exerçant un effet sur ces deux neurotransmetteurs / The neurohormone oxytocin (OT) is increasingly studied for its therapeutic potential in social disorders, like autism, which are associated with the deregulation of several neurotransmission systems, including OT and serotonin (5-HT). Hence investigating OT’s interactions with other neurotransmitters is a relevant step towards mechanism-based treatments. Studies in rodents demonstrated that the interaction between OT and 5-HT, is critical for several aspects of social behaviour. Moreover, using PET-scan in humans we have recently found that 5-HT 1A receptor (5-HT1AR) function is modified after intra-nasal oxytocin intake. Thus I performed a first experiment in which intra-nasal OT was administered to patients with autism undergoing a [18F]MPPF (a 5-HT1AR radiotracer) PET scanner, in order to study their basal serotonergic system and to look if the oxytocin modulates the 5-HT1AR system. I found no differences of baseline 5-HT1AR concentration between 18 autistic subjects and 24 controls. Critically, in patients, OT did not induce changes on the 5-HT1AR system. Moreover, in controls, there was a correlation between 5-HT1AR and grey matter volume in the striatum, that was not observed in patients. These results suggest a subtle disruption of patients’ serotonergic system, that can only be seen at the functional level. Because PET scan does not tell us if the observed modification is due to a change in 5-HT1AR or 5-HT concentration, I performed a second PET scan experiment on 3 macaque monkeys, using [18F]MPPF and [11C]DASB, that marks the serotonin transporter. Compared to placebo, OT injections in the lateral ventricle significantly reduced [11C]DASB binding potential in right amygdala, insula and hippocampus whereas [18F]MPPF binding potential increased in right amygdala and insula. Thus we reproduced results obtained in healthy humans and extended it by suggesting that OT provokes the release of 5-HT in key limbic regions involved in socio-emotional processing. These results were confirmed with autoradiography.Taken together, these experiments indicate that OT modulates 5-HT release in primates, but this mechanism is disrupted in patients with autism. This opens ways to investigate combined OT/5-HT treatments, especially since FDA approved drugs targeting the two systems are already available for use in patients with autism

La sclérose latérale amyotrophique (SLA) est une maladie neurodégénérative caractérisée par une mort sélective des motoneurones. Les mécanismes impliqués dans cette pathologie sont encore mal connus. Il apparait néanmoins que la SLA est une maladie multifactorielle impliquant différents partenaires tels que les motoneurones, les cellules gliales et musculaires. Le modèle murin SOD1G93A développe un phénotype semblable à celui observé chez les patients SLA. L'étude précise du patron locomoteur des SOD1G93A nous a permis de re-définir la date d'apparition des symptômes à 2 mois d'âge soit 1 un mois avant la date communément admise. Nous corrélons cette donnée fonctionnelle à des modifications histologiques au niveau médullaire, en particulier sur la composante gliale, et musculaire. Sur cette base, nous avons développé deux approches in vivo, l'une physiologique et l'autre pharmacologique. D'une part nous avons caractérisé les effets d'exercices physiques de différentes intensités chez les souris SOD1G93A. Cette étude à mis en évidence le rôle primordial de l'environnement et ne démontre pas d'effet de l'exercice sur la survie des souris SOD1G93A. D'autre part, nous rapportons une augmentation de survie des souris SOD1G93A après traitement chronique à faible dose d'une molécule anti-glutamatergique, la gacyclidine. A forte dose cette molécule semble avoir un effet néfaste.En parallèle, nous avons décrit la répartition anatomique de la sérotonine et d'un de ses récepteurs dans la moelle épinière humaine. Nous observons de grandes similarités topographiques avec les murins et primates, et validons ainsi l'utilisation future de ces modèles animaux dans les pathologies affectant la locomotion tel que la SLA. / Amytrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by a selective death of motoneurons. Pathogenesis and mechanisms of selective vulnerability are not yet fully understood although there is growing evidences that ALS is a complex multi-factorial disease that involves several partners such as neuron, glial and muscle cells. Transgenic mice over-expressing a human mutated form of the gene coding for SOD1 develop a dominantly inherited adult-onset paralytic disorder that mimics human ALS symptoms. The precise description of the SOD1G93A mice locomotor pattern using a gait analysis method allowed us to refine symptoms onset at two months of age. This is one month earlier than described in the literature. We correlate these functional modifications to histological alterations of (1) the glial component of the spinal cord, and (2) muscles. From this referent study, we have then developed and evaluated a physiological and pharmacological in vivo approaches. In our first study, we have characterized the effect of different intensities of physical exercise on SOD1G93A mice. Our study not only demonstrates the crucial role of the environment but also that exercise does not have an impact on the survival of SOD1G93A mice. In the second part of our work, we report an increase in SOD1G93A mice survival when they had been chronically treated with a low dose of gacyclidine, an anti-glutamatergic molecule. At higher dose, this molecule seems to be detrimental.In a parallel study, we have carried out the anatomical description of serotonin and one of its receptor in the adult human spinal cord. We observe topographic similarities with rodents and primates, thus validating their further use as animal models, to study motor pathologies such as ALS.

Interagir avec autrui est crucial pour notre survie. Différentes études ont mis en évidence le rôle de l'ocytocine (OT) dans la sociabilité et l'affiliation. La première partie de notre travail montre qu'il y a un lien entre la concentration d'ocytocine plasmatique et l'ocytocine centrale et que les deux sont corrélées au niveau d'extraversion des individus. L'OT plasmatique corrèle aussi au volume de l'amygdale et de l'hippocampe, deux régions cérébrales importantes pour le comportement social. Nous montrons également que l'administration d'OT entraine une modification (augmentation) de la perception subjective de la sociabilité. L'ensemble de ces résultats suggère que l'OT constitue un biomarqueur de la personnalité sociale et pourrait servir au diagnostic de pathologies sociales comme l'Autisme. Nous avons étudié le fonctionnement central de l'OT et particulièrement son interaction avec la sérotonine (5-HT), impliquée elle aussi dans la régulation du comportement social. Avec la Tomographie par Emission de Positon (TEP) et grâce à un radioligand ([18-F]MPPF) spécifique des récepteurs 5-HT1A de la 5-HT nous montrons que l'administration d'OT entraine, chez le sujet sain une augmentation du potentiel de liaison (BP) du MPPF traduisant une modification de l'activité de la 5 HT au niveau du raphe, de l'amygdale/hippocampe, de l'insula et du cortex orbitofrontal, régions clés pour le traitement du comportement social. Les patients Asperger démontrent une diminution du MPPF BP par rapport aux contrôles au niveau de ces mêmes régions, différence qui s'annule par la prise d'OT. Ces résultats confirment le potentiel thérapeutique de l'OT et ouvrent de nouvelles pistes de recherche visant à intégrer la relation entre les neurotransmetteurs dans les futurs traitements. L'ensemble de ce travail place l'ocytocine au coeur de la physiologie du comportement social et suggère son utilisation tant au niveau du diagnostic que de la prise en charge de l'Autisme / Interacting with others is crucial for human fitness. In the past decade, there has been a growing interest for oxytocin (OXT) and its implication in social behavior. In the first section of this work we show that peripheral and central concentrations of OXT are correlated. Peripheral and central OXT are also correlated with subjects’ extraversion and with the volume of amygdala and hippocampus, two brain regions important for the regulation of social behavior. Interestingly, we show that OXT intake increases the subjective perception of subjects’ sociability. These findings suggest that OXT can be considered a biomarker of social behavior, thus opening the possibility of using this hormone in the screening process of psychiatric disorder like Autism. In a second section, we focused on the central action of OXT and in particular its interaction with another neurotransmitter also essential for social behavior: the serotonin (5-HT). We assessed OXT effect on the central serotoninergic activity in healthy subjects using the Positon Emission Tomography (PET) thanks to a radiotracer ([18-F]MPPF) specific for the 5-HT1A receptors and known to be localised in brain regions important for social processing. Our results show that oxytocin administration increases MPPF binding potential (BP) in raphe nuclei, right amygdala, hippocampus and orbitofrontal cortex. Interestingly, Asperger patients showed a decrease in MPPF BP in these regions compared to controls. This difference disappeared after oxytocin. These results strengthen the role of oxytocin in social behavior and underline the therapeutic potential of this neuromodulator for psychiatric disorders implicating both serotonin and oxytocin dysfunctions

This diploma thesis deals withthe studyof electrochemical behaviorand detection of two structurally different neurotransmitters, dopamine (DA) and serotonin (5-HT), in solutions commonly used for neuron cultivation, namely Neurobasal (NB), NB with phenol red and in phosphate buffer (PB) of a pH close to the physiological value. An electrode based on boron-doped diamondwas used for the study, examiningtwo types of surfaces obtained after oxidation (O-BDD) and mechanical polishing (p-BDD). The results were obtained by two voltametric techniques,namely cyclic and differential pulse voltammetry.The studyrevealedthat DA oxidationis a quasi-reversible process,whereas 5-HT oxidizes irreversibly on O-BDD and p-BDD. Nevertheless, for both neurotransmitters their anodic oxidationonboth BDD surfaces is controlledby diffusion. Due to the passivation of the electrode surface by the oxidation products, anodic reactivation(Eact = +2400 mV, t = 30 s) was first testedto regenerate the O-BDD surface during DA and 5-HT measurements. There was no continuous decrease in DA peak currents on O-BDD and the measuredsignals were characterizedby high repeatabilityin all studiedmedia (sr (Ip) 1.1% in PB of pH 7.0, 1.7% in NB of pH 7.34, 0.9% in NB with phenol red of pH 7.48). In the case of 5-HT, the anodic reactivation was...

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

A polymorphism in the promoter region of the human serotonin transporter (5-HTT)-coding SLC6A4 gene (5-HTTLPR) has been implicated in moderating susceptibility to stress-related psychopathology and to possess regulatory functions on human in vivo 5-HTT availability. However, data on a direct relation between 5-HTTLPR and in vivo 5-HTT availability have been inconsistent. Additional factors such as epigenetic modifications of 5-HTTLPR might contribute to this association. This is of particular interest in the context of obesity, as an association with 5-HTTLPR hypermethylation has previously been reported. Here, we tested the hypothesis that methylation rates of 14 cytosine-phosphate-guanine (CpG) 5-HTTLPR loci, in vivo central 5-HTT availability as measured with [11C]DASB positron emission tomography (PET) and body mass index (BMI) are related in a group of 30 obese (age: 36±10 years, BMI>35 kg/m2) and 14 normal-weight controls (age 36±7 years, BMI<25 kg/m2). No significant association between 5-HTTLPR methylation and BMI overall was found. However, site-specific elevations in 5-HTTLPR methylation rates were significantly associated with lower 5-HTT availability in regions of the prefrontal cortex (PFC) specifically within the obese group when analyzed in isolation. This association was independent of functional 5-HTTLPR allelic variation. In addition, negative correlative data showed that CpG10-associated 5-HTT availability determines levels of reward sensitivity in obesity. Together, our findings suggest that epigenetic mechanisms rather than 5-HTTLPR alone influence in vivo 5-HTT availability, predominantly in regions having a critical role in reward processing, and this might have an impact on the progression of the obese phenotype.:Introduction ............................................................................................................. 3 Homeostatic and hedonic control of food intake...................................................... 3 The obesity epidemic .............................................................................................. 4 The role of 5-HT in energy balance......................................................................... 5 The role of 5-HT in the PFC function....................................................................... 6 The role of the PFC in food intake .......................................................................... 7 The association between central 5-HT transporter (5-HTT) availability and obesity ..................................................................................................................................7 Genetics of obesity ................................................................................................. 8 Epigenetics of obesity ............................................................................................. 9 Objectives and hypothesis of the present work...................................................... 11 Manuscript ..... ....................................................................................................... 12 Summary ............................................................................................................... 20 References ............................................................................................................ 22 Appendices ............................................................................................................I Glossary ................................................................................................................ I Publications ........................................................................................................... IV Selbstständigkeitserklärung................................................................................... V Danksagung .......................................................................................................... VI