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.

Alveolar and cystic echinococcosis, caused by Echinococcus multilocularis and Echinococcus granulosus respectively, are severe zoonotic diseases with limited treatment options. The sole curative treatment is the surgical removal of the complete parasite material. Due to late diagnosis, chemotherapeutic treatment often is the only treatment option. Treatment is based on benzimidazoles, which merely act parasitostatic and often display strong side effects. Therefore, new therapeutic drugs are urgently needed. Evolutionarily conserved signalling pathways are known to be involved in hostparasite cross-communication, parasite development and survival. Moreover, they represent potential targets for chemotherapeutic drugs. In this context the roles of the serotonin- and cAMP-signalling pathways in Echinococcus were studied. Genes encoding serotonin receptors, a serotonin transporter and enzymes involved in serotonin biosynthesis could be identified in the E. multilocularis and E. granulosus genomes indicating that these parasites are capable of synthesizing and perceiving serotonin signals. Also the influence of exogenous serotonin on parasite development was studied. Serotonin significantly increased metacestode vesicle formation from primary cells and re-differentiation of protoscoleces. Inhibition of serotonin transport with citalopram significantly reduced metacestode vesicle formation from primary cells and caused death of protoscoleces and metacestodes. Furthermore, it could be shown that serotonin increased phosphorylation of protein kinase A substrates. Taken together, these results show that serotonin and serotonin transport are essential for Echinococcus development and survival. Consequently, components of the serotonin pathway represent potential drug targets. In this work the cAMP-signalling pathway was researched with focus on G-protein coupled receptors and adenylate cyclases. 76 G-protein coupled receptors, including members of all major families were identified in the E. multilocularis genome. Four genes homologous to adenylate cyclase IX were identified in the E. multilocularis genome and three in the E. granulosus genome. While glucagon caused no significant effects, the adenylate cyclase activator forskolin and the adenylate cyclase inhibitor 2’, 5’ didesoxyadenosine influenced metacestode vesicle formation from primary cells, re-differentiation of protoscoleces and survival of metacestodes. It was further shown that forskolin increases phosphorylation of protein kinase A substrates, indicating that forskolin activates the cAMP-pathway also in cestodes. These results indicate that the cAMP signalling pathway plays an important role in Echinococcus development and survival. To complement this work, the influence of different media and additives on E. granulosus protoscoleces was investigated. Anaerobic conditions and the presence of FBS prolonged protoscolex survival while different media influenced protoscolex activation and development. Taken together, this work provided important insights into developmental processes in Echinococcus and potential drug targets for echinococcosis chemotherapy. / Alveoläre und zystische Echinokokkose, hervorgerufen durch Echinococcus multilocularis und Echinococcus granulosus, sind schwere zoonotische Erkrankungen mit eingeschränkten Behandlungsmöglichkeiten. Die einzig kurative Therapie besteht in der chirurgischen Entfernung des gesammten Parasitenmaterials. Aufgrund später Diagnosestellung stellt Chemotherapie oft die einzige Behandlungsmöglichkeit dar. Die derzeitige Therapie basiert auf Benzimidazolen, welche nur parasitostatisch wirken und oft schwere Nebenwirkungen hervorrufen. Neue Medikamente werden daher dringend benötigt. Evolutionär konservierte Signalwege sind bekanntermaßen an Wirt-Parasit Kreuzkommunikation, Parasitenentwicklung und deren Überleben beteiligt. Darüber hinaus stellen sie auch mögliche Angriffspunkte für Chemotherapeutika dar. In diesem Zusammenhang wurden die Rollen des Serotonin- und des cAMP-Signalwegs in Echinococcus untersucht. Gene für Serotoninrezeptoren, einen Serotonintransporter und für Enzyme, die in der Serotoninsynthese involviert sind, konnten in den E. multilocularis und E. granulosus Genomen identifiziert werden, was darauf schließen lässt, dass diese Parasiten in der Lage sind, Serotonin selbst herzustellen und zu sensieren. Des Weiteren wurde der Einfluss von exogenem Serotonin auf die Parasitenentwicklung untersucht. Serotonin förderte die Bildung von Metazestodenvesikeln aus Primärzellen und die Rückdifferenzierung von Protoskolizes signifikant. Die Hemmung des Serotonintransports mit Citalopram reduzierte die Bildung von Metazestodenvesikeln aus Primärzellen signifikant und führte zum Absterben von Protoskolizes undMetazestoden. Des Weiteren konnte gezeigt werden, dass Serotonin die Posphorylierung von Proteinkinase A Substraten erhöht. Zusammengefasst zeigen diese Ergebnisse, dass Serotonin und Serotonintransport essentiell f¨ur die Entwicklung und das Überleben von Echinococcus sind. Folglich stellen Komponenten des Serotoninsignalwegs potentielle Angriffspunkte für Medikamente dar. In dieser Arbeit wurde der cAMP-Signalweg mit Schwerpunkt auf G-Protein gekoppelte Rezeptoren und Adenylatzyklasen untersucht. 76 G-Protein gekoppelte Rezeptoren, inclusive Mitglieder aller Hauptfamilien, wurden im E. multilocularis-Genom identifiziert. Vier Homologe zur Adenylatzyklase IX wurden im E. multilocularis- Genom und drei im E. granulosus-Genom identifiziert. Während Glukagon keine signifikanten Effekte hervorrief, beeinflussten der Adenylatzyklase-Aktivator Forskolin und der Adenylatzyklase-Inhibitor 2’, 5’-Didesoxyadenosin die Bildung von Metazestodenvesikeln aus Primärzellen, die Rückdifferenzierung von Protoskolizes und das Überleben vonMetazestoden. Zudem wurde gezeigt, dass Forskolin die Phosphorylierung von Proteinkinase A-Substraten erhöht. Dies bestätigt, dass Forskolin den cAMP-Signalweg aktiviert. Diese Ergebnisse legen nahe, dass der cAMP-Signalweg eine wichtige Rolle in der Entwicklung und dem Überleben von Echinococcus spielt. Um diese Arbeit zu vervollständigen, wurde der Einfluss von verschiedenen Medien und Zusätzen auf E. granulosus Protoskolizes untersucht. Anaerobe Bedingungen und die Anwesenheit von FBS verlängerten das Überleben von Protoskolizes, während verschiedene Medien die Aktivierung und die Entwicklung von Protoskolizes beeinflussten. Insgesamt gibt diese Arbeit wichtige Einblicke in Entwicklungsprozesse von Echinococcus und zeigt potentielle Angriffspunkte für Medikamente auf.

Aberrations in brain serotonin (5-HT) neurotransmission have been implicated in psychiatric disorders including anxiety, depression and deficits in learning and memory. Many of these disorders are treated with drugs which promote the availability of 5-HT in the synapse. Selective serotonin uptake inhibitors (SSRIs) are known to stimulate the production of new neurons in the hippocampus (HPC) by increasing synaptic concentration of serotonin (5-HT). However, it is not clear which of the 5-HT receptors are involved in behavioral improvements and enhanced neurogenesis. The current study aimed to investigate the effects of 5HT2A agonists psilocybin and 251-NBMeO and the 5HT2A/C antagonist ketanserin on neurogenesis and hippocampal-dependent learning. Agonists and an antagonist to the 5-HT2A receptor produced alterations in hippocampal neurogenesis and trace fear conditioning. Future studies should examine the temporal effects of acute and chronic psilocybin administration on hippocampal-dependent learning and neurogenesis.

No description available.

Exercise has been shown to cause an increase in the concentration of brain serotonin (5-hydroxytryptamine, 5-HT) in humans and experimental animals. The increase in brain serotonin coincides with the onset of fatigue and is referred to as "central fatigue". Experiments in humans and animals involving serotonin receptor agonists have demonstrated reductions in exercise performance by simulating the exercise-induced increase in endogenous serotonin. Conversely, the administration of serotonin receptor antagonists has been shown to extend exercise performance in experimental animals, but not in humans. Although the relationship between the concentration of brain serotonin and exercise performance is well described in the literature, the precise effect of central fatigue on muscle function per se is unclear. Furthermore, there appear to be differences in serotonergic function between trained and untrained cohorts. However, it is not clear whether the differences are due to a training adaptation or if the differences are inherent in the individual. In addition, the time course of these adaptations and the mechanisms of adaptation are not known. The initial purpose of this thesis was to determine whether six weeks of endurance exercise training had any effect on central serotonin receptor sensitivity in Wistar rats. The rats ran on a treadmill 4 times per week with 2 exercise tests of endurance performance per week. Receptor sensitivity was determined indirectly, at the end of each training week, by the reduction in endurance performance, under the influence of a 5-HT1a agonist, (m-Chlorophenylpiperazine, m-CPP). Improved tolerance to the fatiguing effects of the serotonin agonist would suggest desensitisation of central serotonin receptors, probably 5-HT1a receptors. Two groups of controls were used to examine, i) the effect of the injection per se on exercise performance and ii) changes in serotonin receptor sensitivity associated with maturation, in the absence of any exercise training. In the training group, undrugged exercise performance significantly improved by 47% after 6 weeks of training (mean ± SEM, 4518 ± 729 s vs. 6640 ± 903 s, p=0.01). Drugged exercise performance also increased significantly from week 1 to week 6 (306 ± 69 s to 712 ± 192 s, p=0.004). Control group results indicated that the dose of m-CPP alone caused fatigue during exercise tests and that maturation was not responsible for any decrease in receptor sensitivity. Endurance training appears to stimulate an adaptive response to the fatiguing effects of increased brain serotonin, which may enhance endurance exercise performance. The purpose of the second set of experiments described in this thesis was to investigate changes in serotonin receptor sensitivity in response to exercise training in human subjects. Twelve male volunteers completed 30 minutes of stationary cycling at 70% of VO2peak, on 3 days per week, for 9 weeks. Serotonin receptor sensitivity was assessed indirectly by measuring the prolactin response to a serotonin receptor agonist (buspirone hydrochloride), using a placebo controlled, blind cross-over design. A sedentary group of control subjects were also recruited to control for possible seasonal variations in serotonin receptor sensitivity. Endurance capacity was also assessed as time to exhaustion while cycling at 60% of VO2peak. The exercise training caused a significant increase in aerobic power (VO2peak, 3.1±0.16 to 3.6±0.15 L.m-1, p< 0.05) and endurance capacity (93±8 to 168±11 min, p<0.05), but there was no change (p>0.05) in the prolactin response to a serotonin agonist. However, 25% of the subjects in the training group demonstrated a decrease in receptor sensitivity, as indicated by a decrease in prolactin response. These results suggest that while the exercise training caused an increase in aerobic power and endurance capacity, there was no measurable change in 5-HT receptor sensitivity. In addition, it is possible that changes in receptor sensitivity may take longer to occur, the training stimulus used in the present investigation was inadequate or that changes occurred in other 5-HT receptor subtypes that were not assessed by the present methodology. The third set of experiments described here, investigated the changes in neuromuscular function under the influence of a serotonin receptor agonist (buspirone hydrochloride). Subjects were administered the agonist or a placebo in a blind cross over design. Measures of neuromuscular function included reaction time (RT), hand eye coordination (HEC), isometric neuromuscular control (INC), maximal voluntary isometric contractile force (MVIC-F), isometric muscular endurance capacity (IMEC) and various electromyographic (EMG) indices of fatigue in biceps brachii. A preliminary experiment was conducted to determine a drug dose that did not cause sedation of the research subjects. The agonist caused a significant (p<0.05) decrease in MVIC-F, INC and IMEC. There was a non significant (p = 0.08) decrease in EMG amplitude during the MVIC-F trial with the agonist, compared to the effect of the placebo. The median EMG frequency during the IMEC test was also significantly less with the agonist, when compared to the placebo effect. There was a decline in RT and HEC, although this was not significant. These findings indicate that a serotonin receptor agonist causes a decrease in neuromuscular function during isometric muscle contractions. The decrements in muscle function reported in this study may help to explain previous reports of an association between increased brain serotonin concentration and a reduction in endurance performance. Although the present study does not exclude the possibility that an increase in brain serotonin does cause fatigue by affecting organs peripheral to the brain, it provides evidence of fatigue within the central nervous system. Further examination of the effect of a serotonin agonist on muscle function during non-isometric muscle contractions is warranted.

Previous research indicates that serotonin (5-HT) enhances the acquisition of stress-induced changes in behavior; although it is unclear which serotonin receptors mediate this enhancement. 5-HT2 receptors are potential candidates because activation at these receptors is associated with increased fear and anxiety. In this study we investigated whether pharmacological treatments targeting 5-HT2A and 5-HT2C receptors modulated the acquisition and expression of conditioned defeat. Conditioned defeat is a social defeat model in Syrian hamsters (Mesocricetus auratus) that is characterized by increased submissive and defensive behavior and a loss of territorial aggression following social defeat. In experiment 1, we injected the 5-HT2C receptor agonist mCPP (0.3, 1.0, or 3.0 mg/kg) or vehicle prior to social defeat and tested subjects for conditioned defeat behavior in a social interaction test 24 hours later. In experiment 2, subjects received a social defeat, and 24 hours later we injected mCPP (0.3, 1.0, or 3.0 mg/kg) or vehicle prior to a social interaction test. We found that injection of mCPP increased the expression, but not acquisition, of conditioned defeat. In experiment 3, we injected the 5-HT2A receptor antagonist MDL 11,939 (0.5 or 2.0 mg/kg) or vehicle prior to a social defeat and tested subjects for conditioned defeat behavior. In experiment 4, subjects received a social defeat, and 24 hours later we injected MDL 11,939 (0.5 or 2.0 mg/kg) or vehicle prior to a social interaction test. We found that injection of MDL 11,939 significantly decreased the acquisition, but not expression, of conditioned defeat. These data suggest that pharmacological activation of 5-HT2C receptors enhances the expression of conditioned defeat, while pharmacological blockade of 5-HT2A receptors impairs the acquisition of conditioned defeat. These data extend other studies indicating that 5-HT signaling at 5-HT2A receptors facilitate memories for aversive events and 5-HT signaling at 5-HT2C receptors enhance stress-induced anxiety.

Recent approaches in depression therapeutics include triple reuptake inhibitors, drugs that target three monoamine systems. Using in vivo electrophysiological and microdialysis techniques, the effects of 2- and 14-day treatments of escitalopram, nomifensine and the co-administration of these two drugs (TRI) were examined in male Sprague-Dawley rats. Short- and long-term TRI administration decreased NE firing and had no effect on DA neurons. Normal 5-HT firing rates were maintained after 2-day TRI administration compared to the robust inhibitory action of selective serotonin reuptake inhibitors (SSRIs). Escitalopram treatment enhanced the tonic activation of the 5-HT1A receptors given the increase in firing observed following WAY100635 administration. Nomifensine treatment enhanced tonic activation of the α2–adrenoceptors following idazoxan administration. TRI treatment caused a robust increase in extracellular DA levels that was in part mediated by a serotonergic contribution. Therapeutic effects of the drugs examined in this study may be due to the enhancement of 5-HT, NE and/or DA neurotransmission.

Antidepressants are generally believed to exert their clinical efficacy by enhancing 5-HT transmission. Interestingly, sustained administration of selective serotonin (5-HT) reuptake inhibitors (SSRIs) strongly suppresses in the first few days the firing activity of 5-HT neurons in the dorsal raphe nucleus (DRN), thereby severely hampering the increase of 5-HT in target regions. Remarkably, the firing activity of 5-HT neurons gradually recovers over the time course of treatment and this recovery is believed to be accounted for by the desensitization of 5-HT1A somatodendritic autoreceptors. Here, we sought to investigate whether additional mechanisms might contribute to the dynamic regulation of excitability of 5-HT neurons during the course of SSRI treatments. Borrowing from the well-described homeostatic strengthening of glutamatergic synapses onto cortical pyramidal neurons following prolonged periods of inactivity, we hypothesized that a similar homeostatic-like regulation of synaptic strength might be operant on 5-HT cells during an SSRI treatment. To test this possibility, we used whole-cell electrophysiological recordings on acute midbrain slices to monitor glutamatergic synapses onto 5-HT neurons. We found that a two-day treatment with the SSRI citalopram induced a robust reduction in both the amplitude and frequency of AMPAR-mediated mEPSCs. We also show that this depression in synaptic strength, induced by an SSRI, is transient since excitatory drive onto 5-HT neurons was enhanced by 7 days of treatments. Altogether, these results document a dynamic regulation of glutamatergic synaptic transmission during the time course of a prolonged treatment with an SSRI. Further elucidation of the cellular and molecular mechanisms driving this synaptic plasticity might identify novel pharmacological target to shorten the delay of antidepressant action.

Osteocytes are believed to be the mechanosensory cells that detect and respond to mechanical loading. Physiological loading by oscillatory fluid flow (OFF) activates osteocytes to increase intracellular calcium concentration and release prostaglandin E2 (PGE2). Osteocytes are also sensitive to chemical stimulations such as serotonin, which can also increase PGE2 release. However, it is unclear whether mechanical stimulation can influence osteocyte sensitivity towards serotonin. In this thesis, MLO-Y4 osteocyte-like cells were subjected to serotonin with or without precondition by OFF, the responses of intracellular calcium and PGE2 release were measured. Serotonin increased intracellular calcium and PGE2 release in osteocytes. The effects were significantly reduced by OFF precondition, suggesting mechanical precondition by OFF can reduce osteocyte sensitivity towards serotonin. In terms of mechanisms, OFF- and serotonin-induced calcium responses depended on intra- and extracellular calcium stores. ATP was found to partially mediate OFF modulation of serotonin-induced PGE2 release but not calcium.

Osteocytes are believed to be the mechanosensory cells that detect and respond to mechanical loading. Physiological loading by oscillatory fluid flow (OFF) activates osteocytes to increase intracellular calcium concentration and release prostaglandin E2 (PGE2). Osteocytes are also sensitive to chemical stimulations such as serotonin, which can also increase PGE2 release. However, it is unclear whether mechanical stimulation can influence osteocyte sensitivity towards serotonin. In this thesis, MLO-Y4 osteocyte-like cells were subjected to serotonin with or without precondition by OFF, the responses of intracellular calcium and PGE2 release were measured. Serotonin increased intracellular calcium and PGE2 release in osteocytes. The effects were significantly reduced by OFF precondition, suggesting mechanical precondition by OFF can reduce osteocyte sensitivity towards serotonin. In terms of mechanisms, OFF- and serotonin-induced calcium responses depended on intra- and extracellular calcium stores. ATP was found to partially mediate OFF modulation of serotonin-induced PGE2 release but not calcium.