INTRODUÇÃO: Testes provocativos com drogas serotonérgicas não apresentaram resultados consensuais em pacientes com TOC, sugerindo que a atividade serotonérgica seja heterogênea neste transtorno. É possível que a disfunção serotonérgica se relacione à resistência ao tratamento com inibidores de recaptura de serotonina (IRS) neste transtorno. O objetivo deste estudo foi comparar a atividade serotonérgica em pacientes com TOC resistente e responsivo ao tratamento com IRS e voluntários normais através de teste provocativo com citalopram. MÉTODOS: Foram estudados 30 pacientes com TOC resistente a IRS (RT), 30 pacientes com TOC respondedor (RP) e 30 voluntários sem transtorno mental (CN) pareados por sexo e idade. Cada indivíduo recebeu 20 mg de citalopram intravenoso. Foram dosados: prolactina, cortisol, hormônio de crescimento no plasma e serotonina em plasma rico em plaquetas nos seguintes tempos: -20, 0, 20, 40, 60, 80, 100, 120, 140 e 160 minutos. Como medidas da variação hormonal foram comparadas a variação percentual máxima (delta%) e a área sob a curva da variação. RESULTADOS: O citalopram não induziu sintomas ansiosos ou piora dos sintomas obsessivo-compulsivos nos pacientes. Também não houve mudanças significativas na concentração periférica de serotonina e na concentração de GH. A droga induziu um pico de secreção de prolactina no grupo controle (delta%=65,76 ± 105,1) maior que nos grupos RT e RP (RT delta%=17,41 ± 31,06; RP delta%=15,87 ± 31,71; p<0,05 em relação ao grupo controle). A secreção de cortisol mostrou-se atenuada apenas no grupo RT: (RT delta%=20,98 ± 58,14; RP delta%= 47,69 ± 66,94; CN delta%= 63,58 ± 88,4; p<0,05). CONCLUSÕES: Tanto pacientes resistentes como respondedores ao tratamento com IRS apresentaram resposta atenuada de prolactina ao citalopram em comparação a voluntários saudáveis, mas apenas pacientes resistentes apresentaram também resposta atenuada de cortisol, sugerindo maior disfunção serotonérgica neste grupo. / INTRODUCTION: Serotonergic pharmacological challenge tests have failed to produce consensual results in patients with OCD diagnosis, suggesting a heterogeneous 5-HT activity in this disorder. It is possible that serotonergic dysfunction is related with inadequate response to treatment with serotonin reuptake inhibitors (SRI). The aim of this study was to compare the neuroendocrine response to a serotonergic challenge in serotonin reuptake inhibitors treatment resistant and responders OCD patients and healthy volunteers. Methods: Thirty OCD resistant patients, 30 responders patients and 30 healthy volunteers, age and sex matched, were included. Each subject received 20 mg of intravenous citalopram. Prolactin, cortisol, growth hormone and serotonin were determined at the following times after the onset of citalopram infusion: -20, 0, 20, 40, 60, 80, 100, 120, 140 and 160 minutes. The maximal percentage variation (delta%) and area under the curve were compared as measures of hormonal variation. RESULTS: Citalopram has not induced any worsening of anxious or obsessive-compulsive symptoms among patients. No significant changes were observed either at platelet rich plasma serotonin concentration or at plasma growth hormone concentration. Citalopram has induced an increase in prolactin secretion in the control group (delta%=65.76 ± 105.1) while RT and RP groups showed blunted prolactin response (RT delta%=17.41 ± 31.06; RP delta%=15.87 ± 31.71; p<0.05). The cortisol response to citalopram was attenuated only in the RT group: (RT delta%=20.98 ± 58.14; RP delta%= 47.69 ± 66.94; CN delta%= 63.58 ±88.4; p<0.05). CONCLUSIONS: We concluded that either treatment resistant as responders patients have blunted prolactin response to citalopram, but only resistant patients also show an attenuated cortisol response, suggesting a more disrupted central serotonergic transmission in this group.

Die Tryptophanhydroxylase (TPH) katalysiert den geschwindigkeitsbestimmenden Schritt der Synthese des wichtigen Neurotransmitters Serotonin. Kürzlich wurde ein zweites TPH-Isozym, die TPH2, entdeckt. Es stellte sich heraus, dass dieses Isozym für die Serotoninsynthese im Zentralnervensystem verantwortlich ist, wohingegen die TPH1 lediglich der Ausgangspunkt der Serotoninsynthese in den peripheren Geweben ist. Da Störungen im Serotoninstoffwechsel mit einer Vielzahl von psychiatrischen Erkrankungen in Verbindung gebracht werden, rückt nun die als neuronales Enzym identifizierte TPH2 in den Fokus der Forschung. In dieser Arbeit konnte gezeigt werden, dass TPH1 und 2 sich nicht nur in ihren Expressionsorten sondern auch in ihren grundlegenden biochemischen Eigenschaften voneinander unterscheiden. Die TPH1 stellte sich als aktiveres der beiden Enzyme heraus. Der N- und C-Terminus der TPH2 konnten als auf die Aktivität des Enzyms inhibierend bzw. aktivierend wirkende Strukturen identifiziert werden und stellen damit interessante Angriffspunkte für die pharmakologische Beeinflussung dar, wobei der N-Terminus als TPH2-spezifische Struktur eine gezielte Beeinflussung des serotonergen Systems im Zentralnervensystem ohne Auswirkungen auf das periphere System ermöglichen würde. In weiteren Projekten konnte die Existenz von mindestens zwei, die Enzymaktivität nicht beeinflussenden Proteinkinase A-Phosphorylierungsstellen in der TPH2 nachgewiesen werden, es konnte ein auf einem fluorometrischen Prinzip basierender High-Throughput-Assay zur Bestimmung der TPH-Aktivität entwickelt werden, Tubulin beta2A wurde als Interaktionpartner der TPH2 identifiziert, die Auswirkungen eines in vitro aktivitätssenkenden Tph2-SNPs auf die Serotoninlevel und das Verhalten verschiedener Mausstämme konnte durch die Generierung und Untersuchung von congenen Mäusen als unbedeutend eingestuft werden und die Expression von TPH1-mRNA wurde als Marker für endometriale Karzinome identifiziert. / Tryptophan hydroxylase (TPH) catalyzes the rate limiting step of the synthesis of the important neurotransmitter serotonin. Recently a new TPH isoenzyme, TPH2, was discovered. It turned out that this isoenzyme is responsible for the serotonin synthesis within the central nervous system, whereas the TPH1 is merely the starting point of serotonin synthesis in peripheral tissues. Since dysfunction in the metabolism of serotonin is related to a large number of psychiatric diseases, the neuronal TPH2 moved into the centre of interest. As a basis for the pharmacological manipulation of the central nervous serotonergic system, without influencing the periphery, the identification of differences between the two isoenzymes is essential. In this thesis it was shown that TPH1 and 2 not only differ in their expression sites but also in their basic biochemical characteristics. TPH1 turned out to be the more active enzyme. Furthermore it was shown that the N- and C-termini of TPH2 have an inhibitory respectively activating influence on the enzymatic activity. Therefore they became interesting targets for pharmacological interference, whereas the N-terminus as a TPH2 specific structure would facilitate the manipulation of the central nervous serotonergic system without exerting influence on the peripheral system. In further projects the existence of at least two protein kinase A phosphorylation sites could be verified, whereas the phosphorylation doesn’t seem to have any influence on the enzymatic activity, a high throughput assay for determination of TPH activity, based on a fluorometric principle, was developed, Tubulin beta2A was identified as a TPH2 interaction partner, the effect of a SNP in the Tph2 gene that decreases the TPH2 activity in vitro on the serotonin level and the behaviour of different mouse strains could be rated as insignificant by the generation of congenic mice und the expression of TPH1 mRNA was identified as a marker for endometrial cancer.

Die schlafendokrine Regulation wird massgeblich beeinflusst durch Hirnareale, die auch für die Pathophysiologie psychiatrischer, und insbesondere affektiver Störungen, eine wichtige Rolle spielen. Dazu zählen neben dem Hypothalamus, und insbesondere dem paraventrikulären Kerngebiet (PVN), auch limbische Areale wie der Hippokampus und die Amygdala, der präfrontale Kortex sowie die afferenten Kerngebiete im Hirnstamm, insbesondere der Locus coeruleus (LC) und die Raphe Kerne. Der Schlaf und die nächtliche Hormonsekretion können als dynamische Prozesse begriffen werden, die gekennzeichnet sind durch eine fein abgestimmte Abfolge neuronaler Aktivitäten in bestimmten Kerngebieten im Verlauf einer Nacht. Somit ermöglichen polysomnographische und endokrine Untersuchungen Aussagen über mögliche Funktionsänderungen im Zusammenspiel dieser verschiedenen Kerngebiete und leisten daher einen wichtigen Beitrag für die (neuro-) psychiatrische Forschung. In der folgenden Arbeit haben wir insbesondere die dynamischen Aspekte der Schlafarchitektur und die nächtliche Hormonsekretion als Parameter verwendet, um verschiedene Einflussfaktoren auf die schlafendokrine Regulation zu untersuchen. Dabei war es mein Ziel, neben der Diagnose Major Depression auch eine Interaktion zwischen Geschlecht, Alter und Symptomatologie der Depression zu untersuchen. Wir haben dargestellt, dass das Geschlecht einen massgeblichen Einfluss auf diejenigen Schlafparameter ausübt, die bisher als charakteristische Merkmale einer Major Depression angesehen wurden. Weiterhin konnten wir zeigen, dass das Alter (bzw. die Menopause bei Frauen) die schlafendokrinen Veränderungen im Rahmen einer Major Depression geschlechtsspezifisch moduliert. Auch beobachteten wir, dass die klinischen Merkmale einer Major Depression durch geschlechtsspezifische Faktoren beeinflusst werden, wie auch neuere Studien nahelegen. Insbesondere stellen wir die Hypothese auf, dass sogenannte atypische Symptome der Depression bei jüngeren Frauen gehäuft vorkommen und in einer Unteraktivität der HPA Achse (vielleicht als Ausdruck einer genetischen Untererregbarkeit), der afferenten, aktivierenden Serotoninneurone und der Amygdala Kerne begründet liegen. An dieser Konstellation könnte auch eine chronische, aber moderate inflammatorische Immunreaktion beteiligt sein. Im Gegensatz dazu weisen Patienten mit nicht atypischer Depression Zeichen einer Überaktivität der HPA Achse und der Amygdala Kerne auf, die sich im Sinne eines Teufelskreises gegenseitig verstärken und afferente katecholaminerge Neurone einbeziehen können. Die Möglichkeit, dass eine durch inflammatorische Zytokine hervorgerufene Sensibilisierung der HPA Aktivität zu dieser Entwicklung beiträgt scheint plausibel und sollte in zukünftigen Studien überprüft werden. Die unmittelbare Relevanz dieser Überlegungen und Befunde wird unterstrichen durch geschlechtsspezifische Behandlungserfolge bei Patienten mit Major Depression und durch unsere Befunde einer geschlechtsspezifischen Beeinflussung der schlafendokrinen Regulation durch Peptidhormone. Schliesslich haben wir in einer Gruppe von Patientinnen mit Multipler Sklerose (MS) gezeigt, dass unabhängig von den Symptomen einer Major Depression, die hochdosierte Gabe von Glukokortikoiden depressions-typische Veränderungen der schlafendokrinen Regulation induzieren kann. Da diese Patientinnen neben der akuten Glukokortikoidgabe auch eine akute inflammatorische Reaktion (akuter MS Schub) aufwiesen, stellt sich die Frage nach einer Interaktion zwischen der inflammatorischen Reaktion und der Hypothalamus-Hypophysen-Nebennierenrinden (HPA) Achse. Einerseits können immunologische Prozesse den Schlaf beeinflussen, und andererseits gibt es zunehmend Hinweise für einen Zusammenhang zwischen affektiven Symptomen und Immunreaktionen. Da das Geschlecht Immunreaktionen wesentlich beeinflusst, könnten die geschlechtsspezifischen Unterschiede hinsichtlich der klinischen Symptome und der schlafendokrinen Veränderungen im Rahmen einer Major Depression Ausdruck unterschiedlicher zugrundeliegender immunologischer Mechanismen sein. Unterschiede in der Pathophysiologie depressiver Störungen eröffnen die Möglichkeit, die Therapie depressiver Patienten spezifischer auf die zugrundeliegenden Pathomechanismen auszurichten. Dadurch könnten erstens die Behandlung optimiert und zweitens neue spezifischere Behandlungsstrategien für Patienten mit affektiven Störungen entwickelt werden. / The sleep-endocrine regulation is critically influenced by brain areas, which also play an important role for the pathophysiology of psychiatric, and particularly affective disorders. These include the hypothalamus, and particularly the paraventricular nucleus (PVN), but also limbic areas such as the hippocampus and the amygdala, the prefrontal cortex as well as afferent brainstem nuclei, in particular the locus coeruleus (LC) and the raphe nuclei. Sleep and nocturnal hormone secretion can be viewed as dynamic processes, which are characterised by a fine-tuned pattern of neuronal activity in certain brain nuclei in the course of a night. Thus, polysomnographic and endocrine investigations give us insight into possible functional changes in the interplay of these different nuclei and hence provide an important contribution to the field of (neuro-) psychiatric research. In the following piece of work we have used in particular parameters which highlight the dynamic aspects of the sleep-architecture and hormone secretion to examine the various modulators of sleep-endocrine regulation. My aim was to investigate, besides the effect of the diagnosis of major depression, also the interaction of gender, ageing and clinical features of major depression. We have demonstrated that gender has a critical influence on sleep-endocrine changes, which so far have been considered typical of major depression. Also, we could show that ageing (respectively the menopause in women) affects sleep-endocrine changes in patients with major depression in a gender-specific manner. Furthermore, we noted that the clinical features of major depression are also influenced by gender-specific factors, as suggested by recent studies. In particular, we put forward the hypothesis that so-called atypical features of depression are common in young female patients and reflect hypoactivity of the HPA axis (possibly due to a genetic hyposensitivity to stimulation), the afferent activating serotonin neurones and the amygdala nuclei. A chronic but moderate inflammatory immune reaction could also play a role in this situation. Conversely, patients with non-atypical depression show signs of overactivity of the HPA axis and the amygdala nuclei, which can be mutually reinforced, leading to a vicious circle and which can include afferent catecholaminergic neurones. The possibility that priming of the HPA axis with inflammatory cytokines contributes to this development seems plausible and should be examined in future studies. The immediate relevance of these considerations and data is highlighted by gender-specific treatment responses among patients with major depression and our own data demonstrating gender-specific modulation of sleep-endocrine regulation by peptide hormones. Finally, we have shown in a group of female patients with multiple sclerosis (MS) that high doses of glucocorticoids can induce depression-like changes of sleep-endocrine regulation, independently of clinical symptoms of depression. As these patients were treated with high doses of glucocorticoids and suffered from an acute inflammatory reaction (acute MS relapse), the question arises about an interaction between the inflammatory reaction and the hypothalamo-pituitary-adrenocortical (HPA) axis. On the one hand immunological processes can affect sleep and on the other hand there is a growing number of studies suggesting an association between affective symptoms and immune reactions. As gender critically influences immune reactions, the gender-specific differences in clinical features and sleep-endocrine changes in patients with major depression could reflect underlying differences in immunological mechanisms. Differences regarding the pathophysiology of major depression open up the possibility to adjust treatments more specifically to the underlying pathophysiology. Thereby, one could firstly optimise the therapy and secondly develop new and more specific treatment strategies for patients with affective disorders.

Serotonin ist ein Neurotransmitter im zentralen Nervensystem (ZNS), die eine Vielzahl von Funktionen in der menschlichen Physiologie hat. Serotonergen Neuronen in der Raphe-Kerne des Gehirns Unser Ziel war die Leitung der Differenzierung von embryonalen Stammzellen (ES-Zellen) eingeengt und pluripotenten Stammzellen (iPS) Zellen in eine angereicherte Population von Serotonin-produzierenden Zellen, neuartige Gene, die wesentlich für die Entwicklung zu identifizieren und die Funktion des serotonergen Systems. Zu diesem Zweck haben wir differenzierten ES-Zellen in Serotonin-produzierenden Neuronen. Verwendung von RNA zu verschiedenen Zeitpunkten im Verlauf der ES-Zelldifferenzierung wir Gene spezifisch in serotonergen Linie von Affymetrix Genarray angereichert identifiziert isoliert. Um Kandidatengene bewerten wir neu programmiert Maus und Ratte embryonale Fibroblasten zu iPS-Zellen und anschließend differenziert sie serotonergen Neuronen. Wir haben uns für Cacna2d1, für eine alpha2 / delta-Untereinheit von spannungsabhängigen Calciumkanäle als prominentesten Kandidaten unter diesen Genen kodiert. Zur Analyse der Rolle des Proteins Cacna2d1 wir verwendet Cacna2d1 Knockout-Mäusen und Morpholino-Knockdown im Zebrafisch. Wir versäumt, direkte Beteiligung der Cacna2d1 mit serotonergen Systems sehen. Allerdings Immunfärbung für Cacna2d1 in Zebrafisch zeigte zeitabhängige Muster während der frühen Entwicklung. Cacna2d1 Expression wurde in seitlichen Mittellinie Stamm gesehen; vermutlich in Neuromasten Zellen. Übereinstimmend mit ihrer Charakterisierung als Neuromasten werden diese Cacna2d1-positiven Zellen in Richtung der Schwanz der Migration. Darüber hinaus zeigte Zebrafisch gestörten Migrationsverhalten der Neuromasten nach Morpholino-Knockdown von Cacna2d1. So ist diese Studie stellte klar, dass Cacna2d1 ist für Zebrafisch Seitenlinie Entwicklung aber keinen Einfluss auf die Einrichtung des serotonergen Systems. / Serotonin is a neurotransmitter in the central nervous system (CNS), which has a wide range of functions in human physiology. Serotonergic neurons are concentrated in the raphe nuclei of the brain We aimed at directing the differentiation of embryonic stem (ES) cells and induced pluripotent stem (iPS) cells into an enriched population of serotonin producing cells to identify novel genes that are essential for the development and function of serotonergic system. To this purpose we differentiated ES cells into serotonin producing neurons. Using RNA isolated at different time points during the course of ES cell differentiation we identified genes specifically enriched in the serotonergic lineage by Affymetrix gene array. To evaluate candidate genes we reprogrammed mouse and rat embryonic fibroblast to iPS cells and subsequently differentiated them to serotonergic neurons. We selected Cacna2d1, coding for an alpha2/delta subunit of voltage dependent calcium channels as a most prominent candidate among these genes. To analyse the role of the Cacna2d1 protein we used Cacna2d1 knockout mice and morpholino-knockdown in zebrafish. We failed to see direct involvement of Cacna2d1 with serotonergic system. However immunostaining for Cacna2d1 in zebrafish revealed time-dependent pattern during early development. Cacna2d1 expression was seen in lateral midline trunk; presumably in neuromast cells. Concordantly with their characterization as neuromasts, these Cacna2d1-positive cells are migrating towards the tail. Moreover, zebrafish showed disturbed migration behaviours of neuromasts after morpholino-knockdown of Cacna2d1. Thus, this study clarified that Cacna2d1 is essential for zebrafish lateral line development but does not affect the establishment of the serotonergic system.

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.

INTRODUÇÃO: Testes provocativos com drogas serotonérgicas não apresentaram resultados consensuais em pacientes com TOC, sugerindo que a atividade serotonérgica seja heterogênea neste transtorno. É possível que a disfunção serotonérgica se relacione à resistência ao tratamento com inibidores de recaptura de serotonina (IRS) neste transtorno. O objetivo deste estudo foi comparar a atividade serotonérgica em pacientes com TOC resistente e responsivo ao tratamento com IRS e voluntários normais através de teste provocativo com citalopram. MÉTODOS: Foram estudados 30 pacientes com TOC resistente a IRS (RT), 30 pacientes com TOC respondedor (RP) e 30 voluntários sem transtorno mental (CN) pareados por sexo e idade. Cada indivíduo recebeu 20 mg de citalopram intravenoso. Foram dosados: prolactina, cortisol, hormônio de crescimento no plasma e serotonina em plasma rico em plaquetas nos seguintes tempos: -20, 0, 20, 40, 60, 80, 100, 120, 140 e 160 minutos. Como medidas da variação hormonal foram comparadas a variação percentual máxima (delta%) e a área sob a curva da variação. RESULTADOS: O citalopram não induziu sintomas ansiosos ou piora dos sintomas obsessivo-compulsivos nos pacientes. Também não houve mudanças significativas na concentração periférica de serotonina e na concentração de GH. A droga induziu um pico de secreção de prolactina no grupo controle (delta%=65,76 ± 105,1) maior que nos grupos RT e RP (RT delta%=17,41 ± 31,06; RP delta%=15,87 ± 31,71; p<0,05 em relação ao grupo controle). A secreção de cortisol mostrou-se atenuada apenas no grupo RT: (RT delta%=20,98 ± 58,14; RP delta%= 47,69 ± 66,94; CN delta%= 63,58 ± 88,4; p<0,05). CONCLUSÕES: Tanto pacientes resistentes como respondedores ao tratamento com IRS apresentaram resposta atenuada de prolactina ao citalopram em comparação a voluntários saudáveis, mas apenas pacientes resistentes apresentaram também resposta atenuada de cortisol, sugerindo maior disfunção serotonérgica neste grupo. / INTRODUCTION: Serotonergic pharmacological challenge tests have failed to produce consensual results in patients with OCD diagnosis, suggesting a heterogeneous 5-HT activity in this disorder. It is possible that serotonergic dysfunction is related with inadequate response to treatment with serotonin reuptake inhibitors (SRI). The aim of this study was to compare the neuroendocrine response to a serotonergic challenge in serotonin reuptake inhibitors treatment resistant and responders OCD patients and healthy volunteers. Methods: Thirty OCD resistant patients, 30 responders patients and 30 healthy volunteers, age and sex matched, were included. Each subject received 20 mg of intravenous citalopram. Prolactin, cortisol, growth hormone and serotonin were determined at the following times after the onset of citalopram infusion: -20, 0, 20, 40, 60, 80, 100, 120, 140 and 160 minutes. The maximal percentage variation (delta%) and area under the curve were compared as measures of hormonal variation. RESULTS: Citalopram has not induced any worsening of anxious or obsessive-compulsive symptoms among patients. No significant changes were observed either at platelet rich plasma serotonin concentration or at plasma growth hormone concentration. Citalopram has induced an increase in prolactin secretion in the control group (delta%=65.76 ± 105.1) while RT and RP groups showed blunted prolactin response (RT delta%=17.41 ± 31.06; RP delta%=15.87 ± 31.71; p<0.05). The cortisol response to citalopram was attenuated only in the RT group: (RT delta%=20.98 ± 58.14; RP delta%= 47.69 ± 66.94; CN delta%= 63.58 ±88.4; p<0.05). CONCLUSIONS: We concluded that either treatment resistant as responders patients have blunted prolactin response to citalopram, but only resistant patients also show an attenuated cortisol response, suggesting a more disrupted central serotonergic transmission in this group.

A epilepsia é a condição crônica mais prevalente dentre as doenças neurológicas graves, associada com taxas significativas de morbidade e mortalidade. Pacientes com epilepsia farmacorresistente possuem uma taxa de mortalidade 2 a 3 vezes maior que indivíduos sem epilepsia. A epilepsia do lobo temporal (ELT) é a principal causa de epilepsia farmacorresistente nos adultos e também o protótipo da epilepsia cirurgicamente tratável, portanto, com maior acessibilidade para estudo de possíveis mecanismos epileptogênicos. Esclerose hipocampal (EH) é o achado neuropatológico mais comum em pacientes com ELT. Evidências, baseadas em experimentos animais e estudos em humanos, sugerem que as vias serotoninérgicas desempenham um importante papel na epileptogênese. Pacientes com ELT, causadas pela EH, apresentam maior prevalência de transtornos do humor e psicose contribuindo para uma pior qualidade de vida, com consequente impacto negativo nas respostas terapêuticas farmacológicas e cirúrgicas. Além disso, alguns estudos sugerem a existência de um mecanismo patogênico operante comum entre estas condições. Portanto, esta é um importante variável para análise. O objetivo desse estudo foi correlacionar as variáveis clínicas da epilepsia e a presença de transtornos psiquiátricos coexistentes com a concentração de serotonina (5-HT), a densidade dos receptores serotoninérgicos e a densidade do transportador serotoninérgico (5-HTT) no hipocampo dos pacientes com ELT-EH, que foram submetidos à cirurgia devido à presença de epilepsia farmacorresistente. Foram avaliadas amostras de 44 hipocampos de pacientes cirurgicamente tratados para ELT-EH. A concentração de 5-HT foi avaliada por cromatografia líquida de alta eficiência (HPLC) com detecção por fluorescência. 5-HTT e os receptores serotoninérgicos 5-HT1A, 5-HT2A, 5-HT6 e 5-HT7 foram avaliados por Western Blot. Níveis mais baixos de concentração de 5-HT estiveram associados com a presença de crises TCG (Wilcoxon-Mann-Whitney; p = 0.019). A densidade aumentada do receptor 5-HT1A esteve associada com maior duração da epilepsia (coeficiente de correlação de Spearman: p = 0.040) e a densidade diminuída do receptor 5-HT6 esteve associado com presença de EME (Wilcoxon-Mann-Whitney: p = 0.0027). A densidade dos receptores 5-HT2A e 5-HT7 e do 5-HTT não estiveram associadas com variáveis clínicas da epilepsia. A concentração de 5-HT, a densidade dos receptores e a densidade de 5-HTT não estiveram associadas à presença dos transtornos psiquiátricos neste grupo de pacientes. Nossos achados sugerem que as vias serotoninérgicas estão associadas com mecanismos de epileptogênese. Não foi evidenciado associações entre as vias serotoninérgicas e a presença de comorbidades psiquiátricas neste grupo de pacientes com ELT-EH / Epilepsy is the most prevalent neurological condition and it is associated with significative morbidities and mortalities rates. Patients with refractory epilepsy have a 2- to 3-fold higher mortality rate than people without epilepsy. Temporal lobe epilepsy (TLE) is the most common form of adult drug-resistant epilepsy. It is also the prototype of a surgically treatable epilepsy and because of that it is the most accessible for studies focused in epileptogenesis. Hippocampal sclerosis (HS) is the most common neuropathological finding in patients with TLE. Evidences from experimental, clinical and image studies suggest that the serotonergic system play an important role in epileptogenesis. Patients with TLE-HS have a higher prevalence of mood disorder and psychosis contributing for a worse quality of life with and consequent negative impact in pharmacological and surgical responses. Furthermore, studies suggest a common pathogenic mechanism operant in both conditions. Therefore, this is an important analytical variable. The objective of this study was to correlate clinical variables of epilepsy and the presence of psychiatric disease with serotonin (5-HT) concentration, serotonergic receptor density and serotonergic transporter (5-HTT) density in the hippocampus of TLE-HS patients submitted for surgery due to refractory epilepsy. It was analyzed 44 hippocampal tissue samples from surgical treated TLE-HS patients. 5-HT concentration was assessed by high pressure liquid chromatography (HPLC) with fluorescence detection. 5-HTT and serotonergic receptors 5-HT1A, 5-HT2A, 5-HT6 and 5- HT7 were assessed by Western Blotting. Lower levels of 5-HT concentration were associated with the presence of generalized tonic-clonic seizures (Wilcoxon-Mann-Whitney; p = 0.019). A higher 5-HT1A receptor density was associated with longer epilepsy duration (Spearman correlation coefficient: p = 0.040). Lower 5-HT6 receptor density was associated with the presence of status epilepticus (Wilcoxon-Mann-Whitney: p = 0.0027). 5-HT2A receptor, 5- HT7 receptor and 5-HTT densities were not associated with clinical variables of epilepsy. 5-HT concentration, serotonergic receptors and transporter densities were not associated with the presence of psychiatric disease in this group of patients. Our findings suggest that the serotonergic pathways are associated with epileptogenesis mechanisms. It was not evidenced any association between serotonergic pathways and psychiatric comorbidities in this group of TLE-HS patients

Serotonin (5-hydroxytryptamine, 5-HT) is a neurotransmitter involved in the embryonic neural development and adult neurogenesis. But the effects of 5-HT on stem cells are not fully known. In this study, the effects and underlying signal pathways of 5- HT on proliferation and neural differentiation of mouse embryonic stem (ES) cells, neural progenitor (NP) cell line C 17.2 and embryonic neural stem (NS) cells were explored. Molecular analysis, immunostaining and western blotting revealed that NP/NB cells expressed the rate-limiting enzyme tryptophan hydroxylase (TPH) and produced endogenous 5-HT. While mouse ES cells showed no expression of TPH. Quantitative PCR demonstrated that ES cells and NPINS cells expressed majority of 5-HT receptor sUbtypes. In serum free propagation culture, WST1, BrdU incorporation and neural colony forming cell assay demonstrated that 5-HT enhanced proliferation of ES cells and NPINS cells in a dose-dependent manner. Tryptophan hydroxylase (TPH) inhibitor para-chlorophenylalanine (PCPA) which can inhibit biosynthesis of endogenous 5-HT decreased viability of mouse NP/NS cells. Mouse ES cells derived embryoid bodies (EB) and NS/NP cells were subjected to neural induction in serum-free medium with and without 5-HT or PCPA. On day 8 of EB cultures, immunofluorescence staining displayed a less percentage of SSEA-1+ cells derived from cultures supplemented with 5-HT. Nestin positivity are comparable. Quantitative PCR analysis suggested that supplement of 5-HT in EB culture inhibit neural differentiation of ES cells and induce mesodermal commitment. On day 21 of ES cells neural induction, compared to cultures without 5-HT treatment, a significantly less number of ß-tubulin III+ neurons, GEAP+ astrocytes and GaIC+ oligodendrocytes were noted in 5-HT -supplemented cultures. For NS/NP cells, the inhibitory effects of 5-HT on neuronal and oligodendrocytic commitment were also observed. And the application of PCPA exerted a promoting effect on neural differentiation of NS cells. Manipulating 5-HT level can affect the expression level of key genes which involved in 5-HT metabolism. ES and NS/NP cells treated with 5-HT showed decreased production of endogenous reactive oxygen species (ROS). 5-HT demonstrated a significant anti-apoptotic effect on NP cells and this antiapoptotic effect may be mediated by up-regulated expression of anti-apoptotic gene Bel- 2. Whole genome cDNA microarray analysis and quantitative RT-PCR revealed that notch signal pathway was involved in mediating the biological effects of 5-HT. Western blotting further confirmed that 5-HT treatment up-regulated the protein level of NICD and notch downstream effectors Hes-l and Hes-5. Finally, the therapeutic effects of ES cell-derived neural cells were testified in a mouse model of global ischemia. Two weeks post-transplantation, BrdU labeled ES cell-derived neural cells survived and migrated throughout brain parenchyma. A majority of transplanted cells remained nestin positive. The cognitive functions of cell transplanted groups showed significant recovery compared with untransplanted arms, but no significant difference was observed between transplanted groups treated with and without 5-HT. Taken together, data of this study indicated 5-HT play an important role in neural development and ES cell-derived neural cells might be applicable in the treatment of stroke. / Li, Jin. / "November 2011." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 195-241). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstracts in English. / ACKNOWLEDGEMENTS --- p.i / LIST OF PUBLICATIONS --- p.ii / ABSTRACT --- p.iii / ABSTRACT [in Chinese] --- p.v / TABLE OF CONTENT --- p.vi / LISTS OF FLOWCHARTS --- p.xii / LISTS OF FIGURES --- p.xiii / LIST OF TABLES --- p.xvi / LIST OF EQUIPMENTS --- p.xvii / LIST OF ABBREVATIONS --- p.xvii / Chapter Chapter1 --- Introduction --- p.1 / Chapter 1.1 --- Central nervous system disorder --- p.1 / Chapter 1.1.1 --- Stroke --- p.1 / Chapter 1.1.2 --- Spinal cord injuries --- p.4 / Chapter 1.1.3 --- Parkinson's disease --- p.6 / Chapter 1.1.4 --- Amyotrophic Lateral Sclerosis --- p.8 / Chapter 1.2 --- Stem cell therapy --- p.10 / Chapter 1.2.1 --- General considerations in stem cell therapy --- p.11 / Chapter 1.2.2 --- Stem cell therapy for stroke --- p.11 / Chapter 1.2.3 --- Stem cell therapy for spinal cord injury --- p.15 / Chapter 1.2.4 --- Stem cell therapy for Parkinson's disease --- p.16 / Chapter 1.2.5 --- Stem cell therapy for ALS --- p.18 / Chapter 1.3 --- Stem cells --- p.20 / Chapter 1.3.1 --- Embryonic stem cells --- p.21 / Chapter 1.3.1.1 --- Derivation and characterization --- p.21 / Chapter 1.3.1.2 --- Biology of ES cells --- p.21 / Chapter 1.3.1.2.1 --- Pluripotency of ES cells --- p.21 / Chapter 1.3.1.2.2 --- Differentiation of ES cells to multiple lineages --- p.24 / Chapter 1.3.1.2.2.1 --- Ectodermal differentiation --- p.25 / Chapter 1.3.1.2.2.2 --- Mesodermal differentiation --- p.27 / Chapter 1.3.1.2.2.3 --- Endodermal differentiation --- p.28 / Chapter 1.3.2 --- Neural stem cells --- p.30 / Chapter 1.3.2.1 --- Derivation and characterization --- p.30 / Chapter 1.3.2.2 --- Biology of NS cells --- p.32 / Chapter 1.3.3 --- Induced pluripotent stem cells --- p.34 / Chapter 1.3.4 --- Mesenchymal stem cells --- p.35 / Chapter 1.4 --- Serotonin (5-HT) --- p.36 / Chapter 1.4.1 --- Distribution --- p.37 / Chapter 1.4.2 --- Metabolism --- p.37 / Chapter 1.4.3 --- Biological effects of 5-HT --- p.38 / Chapter 1.4.4 --- Serotonin receptor subtypes and receptor signal transduction pathways --- p.40 / Chapter Chapter2 --- Aim --- p.43 / Chapter 2.1 --- Hypothesis and study objectives --- p.43 / Chapter Chapter3 --- Materials and Methods --- p.49 / Chapter 3.1 --- Chemicals and Reagents --- p.49 / Chapter 3.1.1 --- Cell culture --- p.49 / Chapter 3.1.2 --- Serotonin, serotonin receptor subtypes specific agonists/antagonists and drugs that regulate serotonin metabolism --- p.51 / Chapter 3.1.3 --- Cell proliferation assay --- p.52 / Chapter 3.1.4 --- Cell apoptosis assay --- p.52 / Chapter 3.1.5 --- Immunohistochemistry and staining --- p.52 / Chapter 3.1.6 --- Western blotting --- p.55 / Chapter 3.1.7 --- Molecular biology --- p.56 / Chapter 3.1.8 --- Whole genome cDNA micro array --- p.58 / Chapter 3.1.9 --- MAO activity assay --- p.58 / Chapter 3.1.10 --- Endogenous ROS production assay --- p.58 / Chapter 3.2 --- Consumable --- p.58 / Chapter 3.3 --- Cells --- p.60 / Chapter 3.3.1 --- Feeder cell --- p.60 / Chapter 3.3.1.1 --- Mouse embryonic fibroblasts --- p.60 / Chapter 3.3.2 --- ES cells --- p.61 / Chapter 3.3.2.1 --- ES cell D3 --- p.61 / Chapter 3.3.2.2 --- ES cell-E14TG2a --- p.61 / Chapter 3.3.3 --- NS cells --- p.61 / Chapter 3.3.3.1 --- Neural progenitor cells line C172 --- p.61 / Chapter 3.3.3.2 --- Mouse embryonic neural stem cells --- p.61 / Chapter 3.4 --- In-house prepared solutions --- p.62 / Chapter 3.4.1 --- Stock solution ofInsulin, Transferrin, Selentine (ITS) Supplement --- p.63 / Chapter 3.4.2 --- Gelatin solution 01% --- p.62 / Chapter 3.4.3 --- Paraformaldehyde solution 4% (PFA) --- p.62 / Chapter 3.4.4 --- Tritox X-lOO solution 03% --- p.63 / Chapter 3.4.5 --- Popidium iodide solution 1 ug/ml (PI) --- p.63 / Chapter 3.4.6 --- Poly-L-ornithine solution --- p.63 / Chapter 3.4.7 --- Laminin solution --- p.64 / Chapter 3.4.7 --- MEF Maintenance medium --- p.64 / Chapter 3.4.9 --- Cryopreservation Media for MEF and C172 (2X) --- p.64 / Chapter 3.4.10 --- Cryopreservation Media for mouse ES cell (2X) --- p.65 / Chapter 3.4.11 --- Cryopreservation Media for mouse NS cell (2X) --- p.65 / Chapter 3.4.12 --- Serum based maintenance medium for C172 --- p.65 / Chapter 3.4.13 --- Serum free maintenance medium for C172 --- p.66 / Chapter 3.4.14 --- Serum-based propagation medium for ES cells --- p.66 / Chapter 3.4.15 --- Serum-free propagation medium forES cells --- p.67 / Chapter 3.4.16 --- Serum-free induction medium for ES cells --- p.67 / Chapter 3.4.16.1 --- Serum-free induction medium I --- p.67 / Chapter 3.4.16.2 --- Serum-free induction medium II --- p.68 / Chapter 3.4.16.3 --- Serum-free induction medium III --- p.68 / Chapter 3.4.17 --- Tris-HCl (1 M), pH 74 --- p.68 / Chapter 3.4.18 --- Tris-HCl (1 M), pH 87 --- p.69 / Chapter 3.4.19 --- Tris-HCI (1 M), pH 69 --- p.69 / Chapter 3.4.20 --- APS 10% (wt/vol) --- p.69 / Chapter 3.4.21 --- Protease inhibitor (10X) --- p.70 / Chapter 3.4.22 --- RIPA --- p.70 / Chapter 3.4.23 --- Resolving buffer (8X) --- p.70 / Chapter 3.4.24 --- Stacking buffer (4X) --- p.71 / Chapter 3.4.25 --- Protein running buffer (lOX) --- p.71 / Chapter 3.4.26 --- Transfer buffer (10X) --- p.72 / Chapter 3.4.27 --- Transfer buffer (IX) --- p.72 / Chapter 3.4.28 --- Blocking buffer (lOX) --- p.72 / Chapter 3.4.29 --- TBS (10X) --- p.73 / Chapter 3.4.30 --- TBS-T (IX) --- p.73 / Chapter 3.4.31 --- Stacking gel --- p.73 / Chapter 3.4.32 --- Resolving gel --- p.74 / Chapter 3.5 --- Methods --- p.75 / Chapter 3.5.1 --- Cell culture --- p.75 / Chapter 3.5.1.1 --- Preparation of acid washed cover slips --- p.75 / Chapter 3.5.1.2 --- Preparation of gelatinized culture wares --- p.75 / Chapter 3.5.1.3 --- Poly-L-omithine and laminin coating --- p.76 / Chapter 3.5.1.4 --- Thawing cryopreserved cells --- p.76 / Chapter 3.5.1.5 --- Passage of culture --- p.77 / Chapter 3.5.1.5 --- 6 Cell count --- p.78 / Chapter 3.5.1.7 --- Cytospin --- p.78 / Chapter 3.5.1.8 --- Trypan blue dye exclusion test --- p.78 / Chapter 3.5.1.9 --- Cryopreservation --- p.79 / Chapter 3.5.1.10 --- Derivation and culture of mouse embryonic fibroblasts (MEF) --- p.79 / Chapter 3.5.1.11 --- Propagation of ES cells in serum-based/free medium --- p.81 / Chapter 3.5.1.12 --- Neural differentiation ofES cells --- p.83 / Chapter 3.5.1.13 --- Propagation ofNP cell C172 in serum-based or serum-free medium --- p.84 / Chapter 3.5.1.14 --- Neural differentiation ofC172 --- p.85 / Chapter 3.5.1.15 --- Derivation and propagation of embryonic NS cells --- p.85 / Chapter 3.5.1.13 --- Neural differentiation of embryonic NS cells --- p.86 / Chapter 3.5.1.17 --- BrdU labeling of the ES cells derived products --- p.87 / Chapter 3.5.2 --- Cell proliferation assay --- p.87 / Chapter 3.5.2.1 --- Cell morphology --- p.87 / Chapter 3.5.2.2 --- WST-1 assay --- p.88 / Chapter 3.5.2.3 --- BrdU incorporation assay --- p.88 / Chapter 3.5.2.4 --- NCFC assay --- p.89 / Chapter 3.5.3 --- Conventional and quantitative RT-PCR --- p.89 / Chapter 3.5.3.1 --- RNA extraction --- p.89 / Chapter 3.5.3.2 --- RNA quantitation --- p.90 / Chapter 3.5.3.3 --- Reverse Transcription ofthe First Strand complementary DNA --- p.90 / Chapter 3.5.3.4 --- Polymerase chain reaction --- p.91 / Chapter 3.5.3.5 --- RNA Integrity Check --- p.91 / Chapter 3.5.3.6 --- Electrophoresis and visualization of gene products --- p.91 / Chapter 3.5.3.7 --- Real-time quantitative PCR --- p.92 / Chapter 3.5.4 --- Microarray --- p.94 / Chapter 3.5.5 --- Immunofluoresent staining --- p.94 / Chapter 3.5.6 --- Western blot --- p.95 / Chapter 3.5.6.1 --- Harvesting samples --- p.95 / Chapter 3.5.6.2 --- Protein extraction --- p.96 / Chapter 3.5.6.3 --- Protein quantification --- p.96 / Chapter 3.5.6.4 --- SDS-PAGE --- p.97 / Chapter 3.5.6.5 --- Wet transfer of protein to PVDF membrane --- p.97 / Chapter 3.5.6.6 --- Blocking the membrane --- p.97 / Chapter 3.5.6.7 --- Immunoblotting --- p.97 / Chapter 3.5.6.8 --- Signal detection --- p.98 / Chapter 3.5.7 --- Cell apoptosis assay --- p.98 / Chapter 3.5.7.1 --- ANNEXINV-FITC apoptosis detection --- p.98 / Chapter 3.5.7.2 --- TUNEL --- p.99 / Chapter 3.5.8 --- Endogenous ROS assay --- p.100 / Chapter 3.5.9 --- In vivo studies --- p.101 / Chapter 3.5.9.1 --- Induction of cerebral ischemia in mice --- p.101 / Chapter 3.5.9.2 --- Transplantation --- p.101 / Chapter 3.5.9.3 --- Assessment of learning ability and memory --- p.102 / Chapter 3.5.10 --- Histological analysis --- p.103 / Chapter 3.5.10.1 --- Animal sacrifice for brain harvest --- p.103 / Chapter 3.5.10.2 --- Cryosectioning --- p.103 / Chapter 3.5.10.3 --- Haematoxylin and eosin staining --- p.104 / Chapter 3.6 --- Data analysis --- p.104 / Chapter Chapter4 --- Results --- p.113 / Chapter 4.1 --- Expression profile of 5-HT receptors and metablism of endogenous 5-HT --- p.113 / Chapter 4.1.1 --- Expression profiles of 5-HT receptors in stem cells --- p.113 / Chapter 4.1.2 --- Biosynthesis of endogenous 5-HT --- p.115 / Chapter 4.2 --- Effects of 5-HT on proliferation of mouse ES cells and NS cells --- p.115 / Chapter 4.2.1 --- Effects of 5-HT on proliferation ofES cells --- p.115 / Chapter 4.2.2 --- Effects of 5-HT on proliferation ofNP and NS cells --- p.117 / Chapter 4.3 --- Effects of 5-HT on differentiation of mouse ES cells and NS cells --- p.119 / Chapter 4.3.1 --- Neural differentiation ofES cells --- p.119 / Chapter 4.3.2 --- Effects of 5-HT on differentiation ofES cells --- p.119 / Chapter 4.3.3 --- Neural differentiation ofNP and NS cells --- p.120 / Chapter 4.3.4 --- Effects of 5-HT on differentiation ofNP and NS cells --- p.121 / Chapter 4.4 --- 5-HT metabolism in mouse ES cells and NS cells --- p.122 / Chapter 4.4.1 --- Expression of key 5-HT metablic genes in stem cells --- p.122 / Chapter 4.4.2 --- Detection ofROS generation in mouse NS cells --- p.123 / Chapter 4.4.3 --- Effects of 5-HT on expression level of MAO-A, MAO-B and SERT --- p.123 / Chapter 4.5 --- Anti-apoptotic effect of 5-HT on NP and NS cells in neural induction --- p.127 / Chapter 4.6 --- Potential signaling pathways mediated by 5-HT --- p.130 / Chapter 4.7 --- Therapeutic effects of 5-HT treated mouse ES cell-derived cells in a stoke model --- p.130 / Chapter 4.7.1 --- Induction of global ischemia by transient BCCAO --- p.130 / Chapter 4.7.1.1 --- HE staining of post ischemic brain --- p.131 / Chapter 4.7.1.2 --- TUNEL analysis of cell apoptosis at post ischemia day 3 --- p.132 / Chapter 4.7.2 --- Cell labelling --- p.132 / Chapter 4.7.3 --- Cognition monitoring post transplantation --- p.133 / Chapter 4.7.4 --- Survival, migration and differentiation of transplanted neural cells --- p.135 / Chapter Chapter5 --- Discussion --- p.180 / Chapter Chapter6 --- Conclusions --- p.192 / References --- p.195

Die Aktivierung der Speichelsekretion erfolgt in der innervierten Speicheldrüse der Schabe <i>Periplaneta americana</i> durch die biogenen Amine Dopamin (DA) und Serotonin (5-HT). Die Acini der Speicheldrüse sezernieren einen Primärspeichel, der in den Ausführgängen modifiziert wird. Die durch DA und 5-HT aktivierten Signalwege sowie die an der Elektrolyt- und Flüssigkeitssekretion bzw. Speichel-modifikation beteiligten Transportmechanismen sind weitgehend unbekannt.<br> Mikrofluorometrische Ca²⁺-, Na⁺- und pH-Messungen in Kombination mit pharmakologischen Experimenten, biochemische Messungen der Aktivitäten von Ionentransport-ATPasen sowie videomikroskopische Analysen zu transepithelialen Wasserbewegungen wurden in dieser Arbeit durchgeführt. Sie sollten Informationen über die an der Speichelbildung und -modifikation beteiligten Transportmechanismen und die Signalwege liefern, welche durch DA und/oder 5-HT aktiviert werden. <br><br> Wesentliche Ergebnisse dieser Arbeit waren:<br><br> <ul> <li>Messungen des intrazellulären pH (pH_i) in Gangzellen zeigten, dass isolierte Ausführgänge mit Acini bei Stimulierung mit DA und 5-HT stark ansäuerten. In isolierten Ausführgängen ohne Acini verursachte nur DA eine schwache Ansäuerung. Da nur die Ausführgänge dopaminerg innerviert sind, die Acini jedoch dopaminerg und serotonerg, zeigt dieses Ergebnis, dass die DA- und/oder 5-HT-induzierte Primärspeichelbildung die Ursache für die pHi-Änderungen in den Gangzellen ist. pH_i-Messungen in den Gangzellen geben also auch Hinweise auf Transportvorgänge in den Acini.</li> <li> Der Na⁺-K⁺-2Cl^--Symporter und der Cl^--HCO₃^--Antiporter, gekoppelt mit dem Na⁺ H⁺-Antiporter (NHE) waren an der NaCl-Aufnahme in die peripheren Zellen der Acini zur Bildung des NaCl-reichen Primärspeichels beteiligt. Die Aktivität dieser Transporter hing von der CO₂/HCO₃^--Verfügbarkeit ab und war Ca²⁺-abhängig.</li> <li>Die starke Ansäuerung in den Gangzellen hing nicht von der Aktivität der apikalen vakuolären Protonen-ATPase (V-H⁺-ATPase), aber von der Aktivität der basolateralen Na⁺-K⁺-ATPase ab, die anscheinend in den Ausführgängen die Speichelmodifikation energetisiert.</li> <li>In isolierten Ausführgängen mit Acini waren die V-H⁺-ATPase und Na⁺-abhängige Transporter (u. a. NHE) an der Erholung von einer DA-induzierten oder einer NH₄Cl-Vorpuls-induzierten Ansäuerung in den Gangzellen beteiligt. Bei der Regulation des pH_i in unstimulierten Gangzellen spielten diese Transporter keine Rolle.</li> <li>In isolierten Ausführgängen mit Acini induzierte DA in den Gangzellen einen Anstieg der [Na⁺]_i und, zeitlich verzögert, auch der [Ca²⁺]_i. Der [Na⁺]_i-Anstieg war von der Aktivität der Acini abhängig und erfolgte möglicherweise über apikale Na+-Kanäle. Der [Ca²⁺]_i-Anstieg war graduiert und tonisch. Der DA-induzierte [Na⁺]_i-Anstieg in den Gangzellen und deren Depolarisation führten dazu, dass der basolaterale Na⁺-Ca²⁺-Antiporter in den Ca²⁺-Influx-Modus umkehrte. Die daraus resultierende tonische [Ca²⁺]_i-Erhöhung könnte an der Regulation der Na⁺-Rückresorption beteiligt sein.</li> <li>Zum Nachweis transepithelialer Flüssigkeitsbewegungen in isolierten Ausführgängen wurde eine videomikroskopische Methode entwickelt. Isolierte Ausführgänge ohne Acini resorbierten im unstimulierten Zustand Flüssigkeit aus dem Ausführganglumen. Möglicherweise sezernieren die Acini auch im unstimulierten Zustand mit geringerer Rate einen Primärspeichel, der in den Ausführgängen resorbiert wird. Die Resorption war ATP-abhängig. Der ATP-verbrauchende Transportmechanismus konnte nicht identifiziert werden. Weder die Na⁺-K⁺-ATPase noch die V-H⁺-ATPase waren an der Resorption beteiligt.</li> </ul> <br> Diese Arbeit trug zur Kenntnis der komplexen Funktionsweise von Speicheldrüsen in Insekten bei und erweiterte das lückenhafte Wissen über die zellulären Wirkungen biogener Amine in Insekten. Zudem wurden in dieser Arbeit viele Parallelen zu Funktionsweisen der Speicheldrüsen in Vertebraten deutlich. / The acinar salivary glands in the cockroach <i>Periplaneta americana</i> are innervated by dopaminergic and serotonergic fibers and secrete a NaCl-rich primary saliva upon stimulation with the biogenic amines dopamine (DA) or serotonin (5-HT). The ducts downstream of the acini are thought to modify the primary saliva by Na⁺reabsorption and K⁺ secretion. The electrolyte and fluid transport processes activated by DA and 5-HT as well as the second messenger pathways mediating between the biogenic amine receptors and the effector transport mechanisms are poorly understood.In this sudy, microfluorometrical Ca²⁺, Na⁺ and pH measurements were performed in combination with pharmacological experiments. Furthermore, ATPase activity assays and microscopical analyses of transepithelial fluid transport were done. The aim of this work has been the characterisation of the DA-induced transport mechanisms in the cockroach salivary glands in order to improve our understanding of the cellular actions of biogenic amines in insects. <br><br> Intracellular pH measurements in duct cells of isolated small lobes of salivary glands consiting of several acini and ducts showed a strong intracellular acidification upon DA or 5-HT stimulation. On the other hand, only a small intracellular acidification could be recognised in isolated ducts without acini. The acini are innervated by dopaminergic and serotonergic fibers, whereas the ducts are innervated only by dopaminergic fibers. Thus, this result demonstrates, that the DA- or 5-HT-induced production of primary saliva in the acini causes the intracellular pH changes in the ducts. Consequently, intracellular pH measurements in ducts are also useful to characterise transport processes in the acini.<br><br> The Na⁺-K⁺-2Cl^- cotransport and/or the Cl^--HCO₃^- exchange combined with the Na⁺ H⁺ exchange (NHE) were responsible for the NaCl uptake at the basolateral membrane in the peripheral cells of the acini during production of primary saliva. The activity of these transporters was regulated by the CO₂/HCO₃^--availability and was Ca²⁺-dependent. The activity of the basolateral Na⁺-K⁺-ATPase, but not of the apical vacuolar-type proton pump (V-H⁺-ATPase) in the duct cells was necessary for the strong intracellular acidification in the ducts with acini. Thus, the Na⁺-K⁺-ATPase seems to energise the saliva modification in the ducts. In ducts with acini, the V-H⁺-ATPase and Na⁺-dependent transporters (e.g. NHE) were responsible for the pH-recovery after a DA- or NH₄Cl-induced intracellular acidification in the duct cells. In the regulation of the intracellular resting pH these transporters played a minor role. In addition, DA induced an increase in the intracellular Na⁺ concentration, followed by an increase in the intracellular Ca²⁺ concentration in duct cells with acini, but never in duct cells without acini. The Na⁺ elevation was probably the result of the activity of apical Na⁺ channels. The DA-induced Na⁺ elevation and a depolarisation of the basolateral membrane of the duct cells reversed a Na⁺-Ca²⁺ exchange activity into the reverse mode causing a graded Ca²⁺ elevation in duct cells. The Ca²⁺ elevation is probably involved in the regulation of the Na⁺ reabsorption during saliva modification. Transepithelial fluid transport in isolated ducts was detected with a fluorescent microscopical method. Already unstimulated isolated ducts reabsorbed fluid from the duct lumen to the bath side. Perhaps unstimulated acini possess a basic secretion rate and this primary saliva is than reabsorbed in the ducts. The fluid reabsorption was ATP-dependent, but the ATP-consuming transport mechanism could not be identified. Neither the basolateral Na⁺-K⁺-ATPase, nor the apical V-H⁺-ATPase were involved in fluid reabsorption. This work extends our knowledge about the complex function of insect salivary glands and about the cellular action of biogenic amines in insects. Additionally, it indicates lots of similarities between the functions of salivary glands in vertebrates and invertebrates.

Within the field of depression the “monoamine hypothesis” has been the leading theory to explain the biological basis of depression. This theory proposes that the biological basis of depression is due to a deficiency in one or more of three key neurotransmitter systems, namely noradrenaline, dopamine and serotonin which are thought to mediate the therapeutic actions of virtually every known antidepressant agent. Citalopram is a selective serotonin-reuptake inhibitor (SSRI) used for the treatment of depression and anxiety disorders. Citalopram is a racemic compound, in other words composed of a 50:50 mixture of two enantiomers (S-(+)-citalopram and R-(-)-citalopram) and with one of the enantiomers (S-(+)-citalopram) accounting for the inhibitory effect. At the time of introduction of citalopram the physician needed a therapeutic drug monitoring service to identify patients with interactions, compliance problems and for handling questions concerning polymorphic enzymes and drug metabolism. An achiral analytical separation method based on solid-phase extraction followed by high-performance liquid chromatography (HPLC) was developed for routine therapeutic drug monitoring (TDM) of citalopram and its two main demethylated metabolites. As the data available on citalopram were from achiral concentration determinations and to be able to further investigate citalopram enantiomers effects and distribution, a chiral method for separation of the enantiomers of citalopram and its demethylated metabolites was established. The advances within chiral separation techniques have made measurement of the concentrations of the individual enantiomers in biological fluids possible. The process behind enantioselective separation is however not fully understood and the mechanism behind the separation can be further scrutinized by the use of multivariate methods. A study of the optimization and characterization of the separation of the enantiomers of citalopram, desmethylcitalopram and didesmethylcitalopram on an acetylated ß-cyclodextrin column, by use of two different chemometric programs - response surface modelling and sequential optimization was performed. Sequential optimization can be a quicker mean of optimizing a chromatographic separation; response surface modelling, in addition to enabling optimization of the chromatographic process, also serves as a tool for learning more about the separation mechanism. Studies of the antidepressant effect and pharmacokinetics of citalopram have been performed in adults, but the effects on children and adolescents have only been studied to a minor extent, despite the increasing use of citalopram in these age groups. A study was initiated to investigate adolescents treated for depression, with respect to the steady-state plasma concentrations of the enantiomers of citalopram and its demethylated metabolites. The ratios between the S- and R-enantiomers of citalopram and didesmethylcitalopram were in agreement with studies involving older patients. The concentrations of the S-(+)- and R-(-) enantiomers of citalopram and desmethylcitalopram were also in agreement with values from earlier studies. The results indicate that the use of oral contraceptives may have some influence on the metabolism of citalopram. This might be because of an interaction of the contraceptive hormones with the polymorphic CYP2C19 enzyme. Even though the SSRIs are considered less toxic compared with older monoamine-active drugs like the tricyclic/tetracyclic antidepressants, the risk of developing serious side effects such as ECG abnormalities and convulsions has been seen for citalopram, when larger doses have been ingested. Furthermore, fatal overdoses have been reported where citalopram alone was the cause of death. Data on the toxicity of each of the enantiomers in humans have not been reported and no data on blood levels of the enantiomers in cases of intoxication have been presented. An investigation was initiated on forensic autopsy cases where citalopram had been found at the routine screening and these cases were further analysed with enantioselective analysis to determine the blood concentrations of the enantiomers of citalopram and metabolites. Furthermore the genotyping regarding the polymorphic enzymes CYP2D6 and CYP2C19 were performed. In 53 autopsy cases, we found increasing S/R ratios with increasing concentrations of citalopram. We found also that high citalopram S/R ratio were associated with high parent drug to metabolite ratio and may be an indicator of recent intake. Only 3.8 % were found to be poor metabolizers regarding CYP2D6 and for CYP2C19 no poor metabolizer was found. Enantioselective analysis of citalopram and its metabolites can provide valuable information about the time that has elapsed between intake and death. Genotyping can be of help in specific cases but the possibility of pharmacokinetic interactions is apparently a far greater problem than genetic enzyme deficiency. / On the day of the public defence the status of article IV was: Submitted.