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<p>At birth the gastrointestinal (GI) tract is rapidly colonized by microbial organisms which exhibit considerable fluctuations in composition across the first two years of life. During this period, the enteric nervous system (ENS) continues to undergo significant structural and functional changes. In the present study, we investigated whether exposure to intestinal microbiota influences the postnatal development of the ENS. We focused our investigations on dopaminergic neurons as they are among the latest populations of neurons to differentiate during enteric development. The myenteric plexus of specific pathogen-free (SPF) and germ-free (GF) mice were examined in whole-mount preparations of the small and large intestine at three time-points: postnatal day 1 (P1), P7, and P28. The density of dopaminergic neurons did not differ significantly between SPF and GF mice in any region of the intestine examined at P1. However, at P7, GF mice had significantly fewer myenteric dopaminergic neurons in the ileum than did SPF mice, and this difference was maintained at P28.</p> <p>The proportion of enteric dopaminergic neurons has been shown to be dependent upon the availability of serotonin. In the GI tract, serotonin can be of neuronal or enterochromaffin (EC) cell origin. We therefore tested the hypothesis that reductions in myenteric dopaminergic neuron densities in the ileum of GF mice were secondary to changes in enteric serotonergic neuron densities or EC cell frequencies. Neither serotonergic neurons nor EC cell numbers were affected by GF status during the postnatal period. The reduction in dopaminergic neurons seen in GF mice must therefore be attributable to a mechanism that has yet to be determined.</p> <p>These findings are consistent with the notion that enteric microbiota can influence the development of late-born neuronal populations. The reduced proportion of dopaminergic neurons in the ileum of GF mice at P7 and P28 may contribute to the previously described altered motility patterns in postnatal GF mice.</p> / Master of Science (MSc)

Mucosal inflammation in conditions ranging from infective acute enteritis or colitis to inflammatory bowel disease (IBD) is accompanied by alteration in enterochromaffin (EC) cell numbers and serotonin (5-hydroxytryptamine; 5-HT) content in the gut. Previously we had shown that CD4+ T cells, via production of T helper (Th)2 cytokines, regulate EC cell biology in the Trichuris muris-infectious colitis model. I further examined the mechanisms of immuno-endocrine interactions in the context of intestinal inflammation. In chapter 3, utilizing human EC cell line and Trichuris muris-mouse model of infectious colitis we identified a critical role of interleukin (IL)-13, a key Th2 cytokine, in increasing EC cell numbers, tryptophan hydroxylase (TPH)1 expression (rate-limiting enzyme of mucosal 5-HT bio-synthesis), and 5-HT production. In chapter 4, we show that IL-13 driven intestinal inflammation is critically dependent on increased 5-HT production using dextran sulfate sodium (DSS) and dinitrobenzene sulphonic acid (DNBS) models of colitis. In DSS-induced colitis, we were the first to identify the increased production of IL-13 and its pathogenic role as IL-13 knockout (IL-13-KO) mice had less severe inflammation compared to wild-type, which was exacerbated following replenishment of 5-HT in IL-13-KO mice. In chapter 5, biopsy examination revealed, higher mucosal IL-13 expression accompanied inflammation in Crohn's disease (CD), which was additionally associated with increased TPH1, 5-HT receptor (5-HTR)3A, 5-HTR7 and decreased 5-HT transporter (5-HTT) expressions. Moreover, CD patients had elevated plasma and platelet-poor plasma 5-HT levels compared to healthy controls (HCs). Furthermore, 5-HTT polymorphism associated genotypes causing inefficiency in 5-HT re-uptake were more common in our patient cohort than HCs. The findings included in this thesis further emphasize the role of immuno-endocrine interactions in intestinal inflammation, which may be a step toward a better diagnosis or management or even a cure for a disease that is of growing concern, and in understanding IBD pathogenesis. / Dissertation / Doctor of Philosophy (PhD) / The gut produces most of the serotonin found in our body, where it regulates many normal functions. A group of special cells, named enterochromaffin cells, produces nearly all of the serotonin in the gut. In diseases of the gut, especially ones that involve inflammation resulting in symptoms like abdominal pain, diarrhea and bleeding, the number of these cells and serotonin concentration are different from that in the normal gut. I found that these changes are controlled by a particular protein produced by immune cells, called interleukin-13, and alteration in serotonin levels, in turn, contributes to the inflammatory process. Our laboratory experiments with cells and animals establish this connection between interleukin-13 and serotonin in gut inflammation. We further confirm this association between interleukin-13 and serotonin in human inflammatory bowel disease. Moreover, we identify a potential genetic cause of these changes in serotonin concentrations which may ultimately result in inflammatory bowel disease.

Les troubles anxieux comptent parmi les troubles psychiatriques les plus courants dans le monde, les femmes étant presque deux fois plus susceptibles que les hommes de recevoir un diagnostic de trouble d’anxiété au cours de leur vie. Les neurones sérotoninergiques (5-HT) du raphé médian sont fortement impliqués dans la régulation de l’humeur et de l’anxiété, mais les substrats neuronaux sous-tendant les différences liées au sexe dans l’anxiété sont encore largement méconnus. L'hippocampe ventral (HPv), une région qui a été décrite comme un modulateur majeur de l'anxiété, entre autres grâce à ses communications oscillatoires avec d'autres zones cérébrales, reçoit des afférences denses de 5-HT des noyaux du raphé. Des résultats préliminaires obtenus par notre laboratoire montrent que l’activation optogénétique des neurones 5-HT du raphé qui projettent à l’HPv influence le niveau d’anxiété des souris femelles, mais pas des mâles. En se basant sur ces résultats, l’objectif de mon projet est d’explorer les causes de ce dimorphisme sexuel de la voie raphé-HPv dans l’anxiété. J’analyserai l’expression du marqueur d’activation c-Fos après un test d’anxiété, avec ou sans activation optogénétique de la voie 5-HT raphé-HPv. Notre hypothèse est qu’il existe une différence mâle-femelle dans l’excitabilité des neurones 5-HT projetant à l’HPv. Les résultats obtenus permettront de mettre en lumière i) l’expression de c-Fos dans les neurones 5-HT qui projettent à l’HPv en conditions basales (eYFP) chez les mâles et femelles et ii) la différence dans l’expression de c-Fos après activation optogénétique de notre population d’intérêt chez les mâles et femelles. Nous démontrons que l’activation optogénétique de la voie 5-HT du raphé projetant à l’HPv augmente les comportements anxieux, seulement pour les souris femelles. Nous démontrons aussi que l’activation de cette projection diminue l’activité locomotrice. Par ailleurs, les comportements anxieux semblent activer différemment les sous-régions du raphé en fonction du sexe. Ce travail contribue à une meilleure compréhension des mécanismes sous-jacents au rôle de la voie 5-HT du raphé projetant à l’HPv dans la modulation différentielle des comportements d’anxiété selon le sexe. / Anxiety disorders rank among the most common psychiatric conditions worldwide, with women being nearly twice as likely as men to receive a diagnosis of an anxiety disorder during their lifetime. Serotonergic neurons (5-HT) in the median raphe are heavily involved in regulating mood and anxiety, but the neuronal substrates underlying sex-related differences in anxiety are still largely unknown. The ventral hippocampus (vHP), a region described as a major modulator of anxiety, including through oscillatory communication with other brain areas, receives dense inputs of 5-HT from the raphe nuclei. Preliminary results from our laboratory indicate that optogenetic activation of 5-HT neurons projecting to the vHP influences the level of anxiety in female mice but not in males. Building upon these findings, the aim of my project is to explore the causes of this sexual dimorphism in the raphe-vHP pathway related to anxiety. I will analyze the expression of the c-Fos activation marker after an anxiety test, with or without optogenetic activation of the raphe-vHP 5-HT pathway. Our hypothesis is that there is a male-female difference in the excitability of 5-HT neurons projecting to the vHP. The results obtained will shed light on i) c-Fos expression in 5-HT neurons projecting to the vHP under baseline conditions (eYFP) in males and females, and ii) the difference in c-Fos expression after optogenetic activation of our population of interest in males and females. We demonstrate that optogenetic activation of the raphe-vHP 5-HT pathway increases anxiety behaviors, only in female mice. We also show that activation of this projection decreases locomotor activity. Furthermore, anxiety behaviors appear to activate different subregions of the raphe depending on sex. This work contributes to a better understanding of the underlying mechanisms of the role of the raphe-vHP 5-HT pathway in the differential modulation of anxiety behaviors based on sex.

Obesity is a physiological consequence of dysregulated energy homeostasis. Energy homeostasis depends on energy intake and energy expenditure. Factors controlling the development of different adipose tissue deposits in the body and their distinct metabolic phenotypes are of considerable interest from both an agricultural and biomedical perspective. Following the literature review, the first chapter was devoted to studies designed to bridge the neural-adipose interface in understanding the relationship between appetite regulation and adipose tissue deposition in chickens, using chickens selected for low or high juvenile body weight as a model. Appetite regulation in the brain, particularly the hypothalamus, is the main factor governing food intake. Neuropeptide Y (NPY), known as a potent orexigenic factor, also promotes energy storage in fat in mammals and thus has a dual role in promoting energy intake via appetite regulation in the brain and energy storage/expenditure via direct effects on adipose tissue function. There have been no reports of the effects of NPY on adipose tissue function in any avian species. By exposing chicken preadipocytes to different concentration of NPY, we found that NPY enhances both proliferation and differentiation and thus appears to play a major role in chicken adipogenesis, an effect that has not yet been reported, to our knowledge. In the body weight selected chicken lines, we found that NPY and receptor sub-type expression was elevated in the abdominal fat of chickens from the high body weight chicken line and expression of these genes displayed heterosis in the reciprocal crosses of the parental lines as compared to both the high and low body weight selected lines. Intriguingly, expression of those same genes was greater in the low weight than high weight chickens in the hypothalamus. Hypothalamic transcriptomic profiling revealed that genes involved in serotonergic and dopaminergic systems may also play an important role in both appetite regulation and insulin-regulated energy homeostasis in the body weight chicken lines. Intracerebroventricular injection of serotonin in broiler chicks was associated with a dose and time dependent reduction in food intake that was coupled with the activation of the ventromedial hypothalamus and arcuate nucleus, as determined by c-fos immunoreactivity. The remainder of this dissertation project describes the effects of knocking down expression of a recently discovered transcription factor, ZBED6, on mouse preadipocyte proliferation and differentiation. The dissertation ends with a study using diet-induced porcine prepubertal obesity as a model to examine differences in adipokine gene expression between different fat depots from pigs that consumed diets that differed in carbohydrate composition. Overall, we conclude that both NPY and monoamines such as serotonin and dopamine are of importance in the regulation of energy balance in chickens. Moreover, we propose that NPY is a factor that mediates hypothalamus and adipose tissue crosstalk in chickens. An understanding of this system may provide a new avenue for the treatment of obesity and associated disease complications by re-orchestrating the neuronal outputs or adiposity inputs. This information may also be of value in developing strategies to improve feed conversion and meat yield in commercial broilers. / Ph. D.

Flera miljoner människor världen över är idag drabbade av depression vilket skapar ett stort lidande. Depression leder även många gånger till självmord. Känt är att tarmmikrobiotan är en viktig del i sjukdomsförloppet [MB1] och att kunna karakterisera tarmmikrobiota som är av psykobiotisk karaktär blir därför viktigt i ett behandlande syfte men också för att kunna ställa diagnos mer objektivt. Syftet med den här litteraturstudien är att genom artikelgranskning i PubMed undersöka vad senaste forskningen säger om tarmmikrobiotans roll i tryptofanmetabolismen med efterföljande effekt på depression. Skiljer sig tarmmikrobiotan och tryptofanmetabolismen mellan individer med och utan depression och kan tarmmikrobiotan och tryptofanmetaboliter användas som biomarkörer för att diagnostisera depression? Resultatet visar att tarmmikrobiotan och vissa tryptofanmetaboliter skiljer sig signifikant åt mellan friska individer och individer med depression. Studiens slutsats är att tarmmikrobiota och tryptofans metaboliter skulle kunna användas som biomarkörer för att kunna diagnostisera depression mer objektivt. Dock förekommer flera olika potentiella confounders som bör tas i beaktning. / Several million people worldwide are today affected by depression, which creates a lot of suffering. Depression also often leads to suicide. It is known that the gut microbiota is an important part of the course of the disease and being able to characterize gut microbiota that is of a psychobiotic nature therefore becomes important for a treatment purpose but also to be able to make a diagnosis more objectively. The aim of this literature study is to examine, through article review in PubMed, what the latest research says about the role of gut microbiota in tryptophan metabolism with subsequent effect on depression. Do gut microbiota and tryptophan metabolism differ between individuals with and without depression and can gut microbiota and tryptophan metabolites be used as biomarkers to diagnose depression? The results show that the gut microbiota and certain tryptophan metabolites differ significantly between healthy individuals and individuals with depression. The study's conclusion is that gut microbiota and tryptophan metabolites could be used as biomarkers to diagnose depression more objectively. However, there are several different potential confounders that should be taken into account.

The purpose of this study was the investigation of several transport systems expressed at the BBB. The identification and functional characterization of such transport systems is essential to provide a basis for strategies to regulate drug disposition into the brain. Immortalized rat brain endothelial cells (RBE4 cells) have been used in this study as an in vitro model of the BBB. The present study has shown that the RBE4 cells are a suitable model of the BBB for transporter studies. These cells do express the amino acid transport systems L and y+, which are known to be present at the BBB. The uptake of L-tryptophan, a neutral amino acid transported by system L, exhibited a half saturation constant (Kt) of 31 µM and a maximal velocity rate (Vmax) of about 1 nmol/mg/min in RBE4 cells. The kinetic constants of the L-arginine uptake, representing system y+ transport activity, into RBE4 cells were determined with a Kt value of about 55 µM and a Vmax of 0.56 nmol/mg/min. Furthermore the expression of two sodium dependent transporters, the 5-HT transporter (SERT) and the organic cation/carnitine transporter OCTN2, was shown at the RBE4 cells. Uptake studies with radiolabeled 5-HT exhibited a saturable, sodium dependent transport at RBE4 cells with a Kt value of about 0.40 µM and a Vmax of about 52 fmol/mg/min. L-carnitine and TEA (tetraethylammonium) are known to be transported by the OCTN2 transporter. The uptake of L-carnitine into RBE4 cells was shown to be sodium dependent and saturable with a Kt value of 54 µM and a maximal velocity of about 3.6 pmol/mg/min. In contrast, the organic cation TEA follows a sodium independent uptake mechanism at RBE4 cells. Also a sodium independent choline uptake into the cells was discovered but the molecular identity remained unknown. This saturable choline transport exhibited a Kt value of about 22 µM and a maximal velocity of about 52 pmol/mg/min.

Cloninger’s Psychobiological Theory of Personality proposes four temperament dimensions, each underpinned by a different neurotransmitter system. The serotonergic system is purportedly linked to Harm Avoidance (HA). The aim of this study was to explore the relationship between HA and serotonin in migraine without aura (MO). A second aim was to explore the personality profile of MO patients. Sixty-six participants completed an online questionnaire and donated blood samples. Results indicated no significant association between HA and serotonin and a significant relationship between MO and HA. This study indicates that both Cloninger’s Psychobiological Theory of Personality and the Tridimensional Personality Questionnaire used for its assessment have value in South African personality research. In addition, the findings of the study reveal support for personality influences on the processes involved in migraine. This not only produces worthwhile avenues of research but also an alternative perspective for clinical practice. / Psychology / M.A. (Psychology (Research Consultation))

Major depressive disorder (MDD) is a mood disorder majorly responsible for disability and mortality worldwide. With a lifetime prevalence of 15-20%, it is the main cause of functional impairment in Western societies as well as the fourth most debilitating illness in the world. Although the pathophysiology of MDD is not yet fully understood, some evidence that suggest the presence of a neuroanatomical deficiency have given rise to the theory of a specific imbalance in the monoamine neurotransmitters noradrenaline (NA) and/or serotonin (5-HT) levels in the brain. Overall, the various classes of antidepressant agents that have been developed to increase monoamine levels on the basis of this proposal have been successful. However, facts relating to prevalent escalation in the illness and recurring episodes of depression point towards a need to enhance clinical treatment. Most conventional antidepressants such as selective serotonin reuptake inhibitors (SSRI) and selective serotonin and noradrenaline inhibitors (SNRI) pose problems in symptomatic improvement. These include therapeutic lag, safety and tolerability issues, making more than 30% patients with MDD unable to reach adequate relief. In this respect, the action mechanism has moved beyond conventional SSRI and lead to the introduction of vilazodone, a novel antidepressant with an additional 5-HT1A partial agonist profile argued to be of potential benefit for a greater efficacy, faster onset of action and better tolerability. Using secondary data, this project aimed to evaluate the role of vilazodone as a SPARI-drug in the overall clinical treatment of MDD as well as its potential in addressing some of the most common obstacles in antidepressant treatment. Study results proved vilazodone’s efficacy to be superior to placebo. Patients across all studies showed significant improvement in depressive symptoms measured in MADRS and HAMD17. Vilazodone was also shown to be generally safe and tolerable but was not positively distinguished from placebo with regards to adverse effects. An overall, meaningful improvement in depressive symptoms was demonstrated in vilazodone, which reinforces its merit as an important treatment option for patients with MDD.

The purpose of this study was the investigation of several transport systems expressed at the BBB. The identification and functional characterization of such transport systems is essential to provide a basis for strategies to regulate drug disposition into the brain. Immortalized rat brain endothelial cells (RBE4 cells) have been used in this study as an in vitro model of the BBB. The present study has shown that the RBE4 cells are a suitable model of the BBB for transporter studies. These cells do express the amino acid transport systems L and y+, which are known to be present at the BBB. The uptake of L-tryptophan, a neutral amino acid transported by system L, exhibited a half saturation constant (Kt) of 31 µM and a maximal velocity rate (Vmax) of about 1 nmol/mg/min in RBE4 cells. The kinetic constants of the L-arginine uptake, representing system y+ transport activity, into RBE4 cells were determined with a Kt value of about 55 µM and a Vmax of 0.56 nmol/mg/min. Furthermore the expression of two sodium dependent transporters, the 5-HT transporter (SERT) and the organic cation/carnitine transporter OCTN2, was shown at the RBE4 cells. Uptake studies with radiolabeled 5-HT exhibited a saturable, sodium dependent transport at RBE4 cells with a Kt value of about 0.40 µM and a Vmax of about 52 fmol/mg/min. L-carnitine and TEA (tetraethylammonium) are known to be transported by the OCTN2 transporter. The uptake of L-carnitine into RBE4 cells was shown to be sodium dependent and saturable with a Kt value of 54 µM and a maximal velocity of about 3.6 pmol/mg/min. In contrast, the organic cation TEA follows a sodium independent uptake mechanism at RBE4 cells. Also a sodium independent choline uptake into the cells was discovered but the molecular identity remained unknown. This saturable choline transport exhibited a Kt value of about 22 µM and a maximal velocity of about 52 pmol/mg/min.