Thesis (M. S.)--Wake Forest University Graduate School of Arts and Sciences, Molecular Genetics Program, May 2003. / Kent E. Vrana, advisor. Includes curriculum vita. Includes bibliographical references.

In helminths, including the free-living nematode, Caenorhabditis elegans, serotonin (5-HT) acts as an important neuroactive agent and is associated with carbohydrate metabolism, glucouse utilization, motility, feeding and reproductive behaviour. In mammals and other organisms, 5-HT is synthesized through the action of tryptophan hydroxylase (TPH). TPH is the rate limiting enzyme in the biosynthesis of 5-HT, and as such sets the pace for the formation of 5-HT. TPH is a member of a family of enzymes that hydroxylate aromatic amino acids and have an absolute requirement for the pterin cofactor, tetrahydrobiopterin (BH4). It is unknown if this same enzyme catalyzes the synthesis of 5-HT in C. elegans and other helminths. / Based on sequence information from the C. elegans Genome Data Base and RT-PCR, we have cloned a full-length C. elegans TPH cDNA (CeTPH) that shows high homology to mammalian TPH. The predicted coding sequence of CeTPH was subcloned into the prokaryotic expression vector, pET-15b, and the resulting construct was introduced into E. coli (BL21 DE3 pLys strain) for IPTG-inducible expression of CeTPH protein. Results show that CeTPH expressed in E. coli has TPH activity and also shows an absolute requirement for the cofactor, BH4, just as shown previously for the mammalian enzyme. It has been well established that 5-HT is present and is biologically active in the tissues of C. elegans. By way of characterizing furthur CeTPH, we examined the localization of TPH in whole mounts of C. elegans by immunofluoresence using a polyclonal antibody against TPH. / Taken together, the results of this thesis characterize at the structural, functional and in situ levels one of the most primitive forms of TPH enzyme ever cloned.

No description available.

Serotonin (5-HT) containing neuroepithelial cells (NECs) are O2 sensitive chemoreceptors found throughout the skin of larval zebrafish (Danio rerio). Zebrafish larvae are sensitive to changes in ambient PO2 as early as 2 days post fertilisation (dpf) and hyperventilate in response to hypoxia beginning at 3 dpf. Tryptophan hydroxylase (tph) is the rate-limiting enzyme in 5-HT synthesis; three tph paralogs are present in zebrafish (tph1a, tph1b and tph2). Although 5-HT has been implicated as a key neurotransmitter mediating hypoxic hyperventilation, it has not been possible to discern the role of 5-HT specifically contained within the NECs in promoting hypoxic hyperventilation. The purpose of this study was to determine the role of NEC 5-HT in regulating the hypoxic ventilatory response in larval zebrafish. It was hypothesised that 5-HT is a key neurotransmitter released from NECs which contributes to hypoxic hyperventilation. Immunohistochemistry was used to determine the distribution of tph paralogs and their role in 5-HT production in NECs. Tph1a was present in NECs and nerves innervating NECs. Exposure to the non-selective tph inhibitor, para-chlorophenylalanine (pCPA), or translational gene knockdown of tph1a, diminished 5-HT expression within NECs. Exposure to acute hypoxia (PO2 = 30 mmHg) revealed a blunted hypoxic ventilatory response (reduced breathing frequency) in fish exhibiting depleted 5-HT in NECs. The hypoxic hyperventilatory response was rescued with application of 5-HT. The results of these experiments demonstrate that tph1a is responsible for 5-HT production in NECs of larval zebrafish, and that 5-HT released from NECs is involved in establishing their hypoxic hyperventilatory response.

Disruptions in brain serotonin (5-hydroxytryptamine, 5-HT) signaling pathways have been associated with etiology and pathogenesis of various neuropsychiatric disorders, but specific neural mechanisms of 5-HT function are yet to be fully elucidated. Tryptophan hydroxylase 2 (TPH2) is the rate-limiting enzyme for brain 5-HT synthesis. Therefore, in this study a tamoxifen (Tam)-inducible cre-mediated conditional gene (Tph2) knockout in adult mouse brain (Tph2icKO) has been established to decipher the specific role of brain 5-HT in the regulation of behavior in adulthood. Immunohistochemistry and high-performance liquid chromatography (HPLC) were used first to test the efficacy of Tam-inducible inactivation of Tph2 and consequential reduction of 5-HT in adult mouse brain. Tam treatment resulted in ≥90% reduction in the number of 5-HT immuno-reactive cells in the anterior raphe nuclei. HPLC revealed a significant reduction in concentration of 5-HT and its metabolite 5-hydroxyindole acetic acid (5-HIAA) in selected brain regions of Tph2icKO, indicating the effectiveness of the protocol used. Second, standard behavioral tests were used to assess whether reduced brain 5-HT concentrations could alter anxiety-, fear- and depressive-like behavior in mice. No altered anxiety- and depressive-like behaviors were observed in Tph2icKO compared to control mice (Tph2CON) in all indices measured, but Tph2icKO mice exhibited intense and sustained freezing during context-dependent fear memory retrieval. Tph2icKO mice also exhibited locomotor hyperactivity in the aversive environments, such as the open field, and consumed more food and fluid than Tph2CON mice. Lastly, the combined effect of maternal separation (MS) stress and adult brain 5-HT depletion on behavior was assessed in male and female mice. Here, MS stress, 5-HT depletion and their interaction elicited anxiety-like behavior in a sex-dependent manner. MS reduced exploratory behavior in both male and female mice. Reduced 5-HT enhanced anxiety in female, but not in male mice. Furthermore, expression of genes related to the 5-HT system and emotionality (Tph2, Htr1a, Htr2a, Maoa and Avpr1a) was assessed by performing a quantitative real-time PCR. In Tph2icKO mice there was a reduction in expression of Tph2 in the raphe nuclei of both male and female mice. Interaction between MS stress and 5-HT deficiency was detected showing increased Htr2a and Maoa expression in raphe and hippocampus respectively of female mice. In male mice, MS stress and 5-HT depletion interaction effects reduced Avpr1a expression in raphe, while the expression of Htr1a, Htr2a and Maoa was differentially altered by 5-HT depletion and MS in various brain regions. / Unterbrechungen der Serotonin-Stoffwechselwege (5-Hydroxytryptamin, 5-HT) im Gehirn wurden mit der Ätiologie und der Pathogenese von verschiedenen neuropsychiatrischen Erkrankungen assoziiert, wobei die neuronalen Mechanismen der 5-HT Funktionen noch vollständig entschlüsselt werden müssen. Die Tryptophan-Hydroxylase 2 (TPH2) ist das limitierende Enzym für die 5-HT Synthese im Gehirn, weshalb der durch Tamoxifen (Tam) induzierbare, cre-vermittelte Tph2 Gen-Knockout (Tph2icKO) im adulten Mausgehirn möglicherweise helfen könnte die spezifische Rolle von 5-HT im Gehirn in der Regulation von adultem Verhalten zu entschlüsseln. Zuerst wurden Hochleistungsflüssigkeitschromatographie (HPLC) und Immunhistochemische Analysen durchgeführt um die Effizienz der Tam induzierten Inaktivierung des Tph2 und die daraus folgende Reduktion von 5-HT im Gehirn zu überprüfen. Die Behandlung mit Tam resultierte in einer ≥86% Reduktion der Anzahl von 5-HT immunoreaktiven Zellen in der anterioren Raphe im Gehirn. Die HPLC zeigte eine signifikante Reduktion der 5-HT Konzentration und dessen Stoffwechselprodukts 5-Hydroxyindolylessigsäure (5-HIAA) in ausgewählten Gehirn regionen von Tph2icKO, was auf die Effektivität des benutzten Protokolls hindeutet. Danach wurden standarisierte Verhaltens tests durchgeführt um festzustellen, ob eine reduzierte 5-HT Konzentrationen im Gehirn zu einer Veränderung in der Angstreaktion, Depression und im Furchtverhalten der Mäuse führt. Bei allen Tests konnte sowohl in den Tph2icKO-Mäusen als auch in den Kontrolltieren kein offensichtliches angstbezogenes und depressionsähnliches Verhalten festgestellt werden, wobei die Tph2icKO-Mäuse intensive und anhaltende Furcht im Kontext „dependent fear retrieval“ zeigten. Tph2icKO-Mäuse zeigten zudem lokomotorische Hyperaktivität und konsumierten mehr Futter und Flüssigkeit als die Kontrolltiere. Zuletzt wurde der kombinierte Effekt von Stress durch mütterliche Trennung (MS) und adulter 5-HT Reduktion im Gehirn auf das Verhalten von männlichen und weiblichen Mäusen untersucht. Wieder rief nicht der depressionsähnliche Phänotyp, sondernder Stress durch die mütterliche Trennung (MS) und 5-HT Verarmung und deren Interaktion ein angstähnliches Verhalten in Abhängigkeit vom Geschlecht hervor. Reduziertes 5-HT vergrößerte die Angst in weiblichen, aber nicht in männlichen Mäusen. Stress durch mütterliche Trennung (MS) reduzierte das explorative Verhalten sowohl in Männchen als auch in Weibchen. Die Expression von Genen, welche im Bezug zum 5-HT System stehen (Tph2, Htr1a, Htr2a, Maoa und Avpr1a) wurden mit Hilfe von quantitativer Real-Time PCR untersucht. Die Tam Behandlung reduzierte dasTph2 Level in der Raphe bei beiden Geschlechtern signifikant. In weiblichen Mäusen steigertedie Interaktion zwischen Stress durch mütterliche Trennung (MS) und 5-HT Verarmung das Htr2a und Maoa Expressions level in der Raphe und im Hippokampus. In männlichen Mäusen reduzierte die Interaktion von Stress durch mütterliche Trennung (MS) und 5-HT Reduktion die Avpr1a Expression in der Raphe. Die Expression von Htr1a, Htr2a und Maoa wurde in verschiedenen Gehirn regionen unterschiedlich von Tam und Mütterliche Trennung MS verändert. In der Amygdala wurde nur ein MS Effekt auf die Tph2 Expression in den Mäusen sichtbar.

No description available.

Serotonin is a very important neuromodulatory agent that affects many physiological and behavioral responses of both vertebrates and invertebrates. In helminths, especially parasitic ones, not much is known about the biosynthesis and mode of action of serotonin or any of the related biogenic amine neurotransmitters, such as catecholamines (dopamine and noradrenaline). In this study, we cloned two full length cDNAs from Schistosoma mansoni encoding tryptophan hydroxylase (TPH) and tyrosine hydroxylase (TH). TPH and TH catalyze the rate limiting steps in the biosynthesis of serotonin and catecholamines, respectively. Both enzymes were expressed in Escherichia coli and the purified proteins were shown to have TPH and TH activities. This indicates that S. mansoni, and possibly other parasitic helminths, may be capable of synthesizing serotonin and catecholamines endogenously. In the second part of our studies, we looked at the mode of action of serotonin in helminths, in particular the molecular properties of serotonergic G protein-coupled receptors (GPCR). We cloned two helminth GPCRs, one from the free living nematode Caenorhabditis elegans and the second from S. mansoni. The C. elegans receptor (5-HT2Ce) was shown to encode a functional serotonin receptor with structural and signaling properties similar to those of mammalian 5-HT2 receptors. However, its agonist I antagonist binding profile differed from previously characterized serotonin receptors. The cloned S. mansoni receptor (SmGPCRx) was found to represent a new structural class of receptor, which shared about the same level of amino acid sequence homology with various biogenic amines receptors, such as serotonin, catecholamines, and octopamine receptors. Additional sequence analysis and immunolocalization studies confirmed that SmGPCRx possesses structural characteristics of a GPCR. SmGPCRx is the first GPCR ever cloned from a parasitic flatworm. Taken together, these studies mark an important first step to

Serotonin (5-Hydroxytryptamin, 5-HT) ist ein wichtiger Neurotransmitter im Zentralnervensystem (ZNS). Seine Biosynthese erfolgt unter Beteiligung des Enzyms Tryptophanhydroxylase 2 (TPH2). Polymorphismen im TPH2 Gen beim Menschen sind Risikofaktoren bei der Entstehung von Depressionen und Angstverhalten. Die gängigsten Antidepressiva und Anxiolytika wirken auf das Serotonin System. Unklar ist, ob das komplette oder teilweise Fehlen von Serotonin im Gehirn zu Entwicklungsstörungen und neurochemischen oder psychologischen Veränderungen führt. In dieser Arbeit werden Mauslinien mit unterschiedlichen TPH2 Aktivitäten im ZNS verglichen und der Einfluss verringerter 5-HT Konzentrationen auf Entwicklung und Verhalten der Tiere untersucht. Zentrales Serotonin ist nur für die postnatale Entwicklung notwendig. Das verzögerte Wachstum von Tph2-/- Tieren ist nicht auf eine Störung der Hypothalamus-Hypophysen-Nebennieren-Achse oder auf metabolische Veränderungen zurückzuführen, sondern kann aus verringerter Vokalisation im Ultraschallbereich resultieren. Tph2-/- Mäuse wurden mit generierten Mausmodellen mit niedriger TPH2 Aktivität vergleichen. Die Ergebnisse zeigen, dass 20% weniger zentrales Serotonin nicht ausreichen, um Depression oder Angst-Verhalten herbeizuführen. Möglicherweise greifen kompensatorische Mechanismen wie ein verringerter Serotoninmetabolismus oder eine gesteigerte 5-HT1A-Rezeptorsensitivität. Der komplette Verlust von Serotonin im Gehirn führt zu einem starken depressiven und weniger ängstlich Verhalten, mit erhöhter Aggression - ohne Veränderung in Aktivität, Geruchsinn, Gedächnis und adulter Neurogenese. Fluoxetine Behandlung von Tph2-defizienten Mäusen zeigte einen Serotonin-unabhängigen Effekt dieses Antodepressivums auf Angst-Verhalten und Depression. Fluoxeine reduzieren den Serotoningehalt im Gehirn von Mäusen mit geringen TPH2-Aktivität, was zeigt, dass TPH-Aktivität die Effizienz von Serotonin beeinflussenAntidepressiva bestimmen, / Serotonin (5-HT) is a major neurotransmitter in the brain biosynthesis of which is initiated by tryptophan hydroxylase 2 (TPH2). Polymorphisms in the TPH2 gene are suggested as risk factors associated with depression and anxiety in humans. Furthermore, the most frequently prescribed antidepressants and anxiolytics target the serotonergic system. However, the question whether a complete ablation or partial reduction in brain serotonin leads to the developmental, neurochemical, or psychological abnormalities remains unresolved. In this study, I took advantage of mouse lines with various degree of decrease in TPH2 activity in order to dissect the impact of 5-HT loss on development, brain neurochemistry and behavior. Using Tph2-deficient mice I showed that central serotonin is essential for normal postnatal, but not prenatal development. Growth retardation of Tph2-/- mice was not a result of a disruption of the hypothalamo-pituitary-adrenal axis, metabolic abnormalities, or impaired thermoregulation, but could result from reduced ultrasonic vocalization. I tested Tph2-/- mice along with other newly generated mouse models with partial TPH2 reduction, and showed that 20% reduction in central serotonin is not enough to cause changes in anxiety- and depression-like behaviors most likely due to compensatory mechanisms including reduced serotonin metabolism and increased 5-HT1A receptor sensitivity. However, complete loss of central serotonin leads to a depression-like phenotype, reduced anxiety-like behavior, and exaggerated aggression, but no differences in activity, olfaction, memory, and adult neurogenesis. Fluoxetine treatment of Tph2-/- mice revealed serotonin-independent action of this antidepressant on anxiety- and depression-like behavior. Furthermore, fluoxetine drastically reduced the brain 5-HT content in mice with low TPH2 activity indicating that TPH2 activity may determine the efficiency of antidepressants targeting the serotonergic system.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 6 uppsatser.

No description available.