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Anxiety and depressive disorders result from a complex interplay of genetic and environmental factors and are common mutual comorbidities. On the level of cellular signaling, regulator of G protein signaling 2 (Rgs2) has been implicated in human and rodent anxiety as well as rodent depression. Rgs2 negatively regulates G protein-coupled receptor (GPCR) signaling by acting as a GTPase accelerating protein towards the Gα subunit.
The present study investigates, whether mice with a homozygous Rgs2 deletion (Rgs2-/-) show behavioral alterations as well as an increased susceptibility to stressful life events related to human anxiety and depressive disorders and tries to elucidate molecular underlying’s of these changes.
To this end, Rgs2-/- mice were characterized in an aversive-associative learning paradigm to evaluate learned fear as a model for the etiology of human anxiety disorders. Spatial learning and reward motivated spatial learning were evaluated to control for learning in non-aversive paradigms. Rgs2 deletion enhanced learning in all three paradigms, rendering increased learning upon deletion of Rgs2 not specific for aversive learning. These data support reports indicating increased long-term potentiation in Rgs2-/- mice and may predict treatment response to conditioning based behavior therapy in patients with polymorphisms associated with reduced RGS2 expression. Previous reports of increased innate anxiety were corroborated in three tests based on the approach-avoidance conflict. Interestingly, Rgs2-/- mice showed novelty-induced hypo-locomotion suggesting neophobia, which may translate to the clinical picture of agoraphobia in humans and reduced RGS2 expression in humans was associated with a higher incidence of panic disorder with agoraphobia. Depression-like behavior was more distinctive in female Rgs2-/- mice. Stress resilience, tested in an acute and a chronic stress paradigm, was also more distinctive in female Rgs2-/- mice, suggesting Rgs2 to contribute to sex specific effects of anxiety disorders and depression.
Rgs2 deletion was associated with GPCR expression changes of the adrenergic, serotonergic, dopaminergic and neuropeptide Y systems in the brain and heart as well as reduced monoaminergic neurotransmitter levels. Furthermore, the expression of two stress-related microRNAs was increased upon Rgs2 deletion. The aversive-associative learning paradigm induced a dynamic Rgs2 expression change. The observed molecular changes may contribute to the anxious and depressed phenotype as well as promote altered stress reactivity, while reflecting an alter basal stress level and a disrupted sympathetic tone. Dynamic Rgs2 expression may mediate changes in GPCR signaling duration during memory formation.
Taken together, Rgs2 deletion promotes increased anxiety-like and depression-like behavior, altered stress reactivity as well as increased cognitive function.
Die Amygdala ist ein Kernkomplex, der dicht von serotonergen Afferenzen innerviert wird. Sowohl bei Tieren als auch beim Menschen spielen Interaktionen zwischen dem serotonergen System und der Amygdala bei der Verarbeitung von Reizen, die mit Angst oder Stress assoziiert sind, eine zentrale Rolle. Genetische Variationen im serotonergen System und/oder dauerhafter Stress können dazu führen, dass diese Verarbeitungsprozesse fehlerhaft ablaufen, wodurch Verhaltensanormalitäten bzw. die Entstehung psychiatrischer Erkrankungen begünstigt werden. Die Zielneurone der serotonergen Transmission in der Amygdala, die molekularen Mechanismen möglicher Interaktionen und strukturelle Konsequenzen der Störungen dieser Interaktionen sind jedoch bis zum heutigen Zeitpunkt noch nicht vollständig bekannt. Daher bestand ein Ziel der vorliegenden Arbeit darin, den Einfluss eines Ungleichgewichts im serotonergen System (5-Htt KO) sowie von wiederholtem, sozialem Stress auf die neuronale Morphologie der Amygdala zu analysieren und Zielneurone serotonerger Afferenzen zu identifizieren und zu charakterisieren, um die neuronalen Netzwerke der Emotionsverarbeitung besser verstehen zu können. Um vom 5-Htt–Genotyp abhängige und stressbedingte neuromorphologische Veränderungen zu untersuchen, wurden dreidimensionale Rekonstruktionen von Neuronen der laterobasalen Amygdala von männlichen, adulten Wildtyp (WT)- und 5-Htt KO-Mäusen angefertigt und bezüglich verschiedener morphologischer Parameter ausgewertet. An den Pyramidenzellen wurden nur geringfügige Veränderungen der dendritischen Komplexität, jedoch, im Vergleich zu WT-Mäusen, eine wesentliche Erhöhung der Dornendichte an spezifischen dendritischen Kompartimenten bei gestressten WT-Mäusen, sowie nicht gestressten und gestressten 5-Htt KO-Mäusen nachgewiesen. Im Vergleich zu nicht gestressten WT–Mäusen war die dendritische Dornendichte aller anderen Gruppen gleichermaßen erhöht. Die Sternzelle, zeigten bezüglich der untersuchten Parameter keine morphologischen Veränderungen auf. Eine besondere Subpopulation der Interneurone stellen die NeuropeptidY (NPY)–Neurone der laterobasalen Amygdala dar, da sie in diesen Nuclei anxiolytisch wirken. Es gibt nur wenige Anhaltspunkte darüber, durch welche Systeme NPY–Neurone moduliert werden. Da sowohl NPY–Neurone in der laterobasalen Amygdala als auch das serotonerge System an angstregulierenden Prozessen beteiligt sind, sollte im zweiten Teil der vorliegenden Arbeit untersucht werden, ob es sich bei diesen Neuronen um Zielstrukturen des serotonergen Systems handelt. Mittels licht- und elektronenmikroskopischer Analysen wurden synaptische Kontakte zwischen serotonergen Afferenzen und NPY-immunreaktiven Neuronen in der laterobasalen Amygdala von Ratten verifiziert. Da der funktionelle Einfluss der serotonergen Innervation auf diese Zielneurone von deren Serotoninrezeptor (5-HTR)-Ausstattung abhängt, wurden Koexpressionsanalysen von NPY mRNA mit den mRNAs verschiedener 5-HTR durchgeführt. Die Analysen ergaben, dass NPY mRNA–reaktive Neurone in der laterobasalen Amygdala 5-HT1A und 5-HT2C, jedoch nicht 5-HT3 mRNA koexprimieren. Die in der vorliegenden Arbeit erzielten Resultate liefern neue Erkenntnisse über den Einfluss des serotonergen Systems auf die laterobasale Amygdala von Mäusen und Ratten. Bei den Veränderungen der dendritischen Dornendichte nach sozialen Stresserfahrungen könnte es sich um neuroadaptive bzw. kompensatorische Mechanismen der Pyramidenzellen handeln, die WT-Mäusen eine Anpassung an sich ändernde, negative Umweltbedingungen ermöglicht. Die erhöhte Dornendichte könnte dabei die Ausbildung eines „emotionalen Gedächtnisses“ repräsentieren, das eine flexible Verhaltensantwort auf ein erneutes Auftauchen von Gefahr erlaubt. Eine solche Modulation der Erregbarkeit der laterobasalen Amygdala könnte beispielsweise über eine situationsentsprechende Hemmung des Outputs der Pyramidenzellen durch differentiell aktive inhibitorische Netzwerke erfolgen. Eine differentielle Aktivierung kann z. B. über unterschiedliche Rezeptorausstattungen, wie es in der Subpopulation der NPY–Neurone in der vorliegenden Arbeit nachgewiesen wurde, erfolgen. Das erhöhte angstähnliche Verhalten der 5-Htt KO-Mäuse nach wiederholtem Stress könnte mit der Unfähigkeit zusammenhängen, in entsprechenden Situationen durch Neubildung von Dornen zu reagieren, da die Dornendichte bei diesen Tieren schon unter stressarmen Umweltbedingungen ihr Maximum erreicht hat. Sowohl Fehlfunktionen der neuronalen Plastizität als auch mögliche Fehlfunktionen der differentiellen Inhibierung der Pyramidenzellen durch Interneurone, die durch genetische Variationen und/oder Stress bedingt sein können, könnten eine „offene Tür“ repräsentieren, die zu manifesten Auffälligkeiten im Verhalten bei Tieren führt bzw. auch zur Entstehung bestimmter psychiatrischer Erkrankungen beim Menschen beiträgt.
Serotonin (5-HT) has been implicated in the regulation of emotions as well as in its pathological states, such as anxiety disorders and depression. Mice with targeted deletion of genes encoding various mediators of central serotonergic neurotransmission therefore provides a powerful tool in understanding contributions of such mediators to homeostatic mechanisms as well as to the development of human emotional disorders. Within this thesis a battery of electrophysiological recordings were conducted in the dorsal raphe nucleus (DRN) and the hippocampus of two murine knockout lines with deficient serotonergic systems. Serotonin transporter knockout mice (5-Htt KO), which lack protein responsible for reuptake of 5-HT from the extracellular space and tryptophan hydroxylase 2 knockout (Tph2 KO) mice, which lack the gene encoding the neuronal 5-HT-synthesising enzyme. First, 5-HT1A receptor-mediated autoinhibition of serotonergic neuron firing in the DRN was assessed using the loose-seal cell-attached configuration. Stimulation of 5-HT1A receptors by a selective agonist, R-8-hydroxy-2-(di-n-propylamino)tetralin (R-8-OH-DPAT), showed a mild sensitisation and a marked desensitisation of these receptors in Tph2 KO and 5-Htt KO mice, respectively. While application of tryptophan, a precursor of 5-HT and a substrate of Tph2, did not cause autoinhibition in Tph2 KO mice due to the lack of endogenously produced 5-HT, data from 5-Htt KO mice as well as heterozygous mice of both KO mice lines demonstrated the presence of autoinhibitory mechanisms as normal as seen in wildtype (WT) controls. When the Tph2-dependent step in the 5-HT synthesis pathway was bypassed by application of 5-hydroxytryptophan (5-HTP), serotonergic neurons of both Tph2 KO and 5-Htt KO mice showed decrease in firing rates at lower concentrations of 5-HTP than in WT controls. Elevated responsiveness of serotonergic neurons from Tph2 KO mice correspond to mild sensitisation of 5-HT1A receptors, while responses from 5-Htt KO mice suggest that excess levels of extracellular 5-HT, created by the lack of 5-Htt, stimulates 5-HT1A receptors strong enough to overcome desensitisation of these receptors. Second, the whole-cell patch clamp recording data from serotonergic neurons in the DRN showed no differences in basic electrophysiological properties between Tph2 KO and WT mice, except lower membrane resistances of neurons from KO mice. Moreover, the whole-cell patch clamp recording from CA1 pyramidal neurons in the hippocampus of 5-Htt KO mice showed increased conductance both at a steady state and at action potential generation. Lastly, magnitude of long-term potentiation (LTP) induced by the Schaffer collateral/commissural pathway stimulation in the ventral hippocampus showed no differences among Tph2 KO, 5-Htt KO, and WT counterparts. Taken together, lack and excess of extracellular 5-HT caused sensitisation and desensitisation of autoinhibitory 5-HT1A receptors, respectively. However, this may not directly translate to the level of autoinhibitory regulation of serotonergic neuron firing when these receptors are stimulated by endogenously synthesised 5-HT. In general, KO mice studied here showed an astonishing level of resilience to genetic manipulations of the central serotonergic system, maintaining overall electrophysiological properties and normal LTP inducibility. This may further suggest existence of as-yet-unknown compensatory mechanisms buffering potential alterations induced by genetic manipulations.
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.