@phdthesis{Kriegebaum2009,
  author    = {Kriegebaum, Claudia},
  title     = {Spatio-temporal Expression Patterns of the Serotonin Synthesis Enzymes TPH1 and TPH2 and Effects of Acute Stress},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-40839},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2009},
  abstract  = {Several lines of evidence implicate a dysregulation of tryptophan hydroxylase (TPH)-dependent serotonin (5-HT) synthesis in emotions and stress and point to their potential relevance to the etiology and pathogenesis of various neuropsychiatric disorders. However, the differential expression pattern of the two isoforms TPH1 and TPH2 which encode two forms of the rate-limiting enzyme of 5-HT synthesis is controversial. Here, a comprehensive spatio-temporal analysis clarifies TPH1 and TPH2 expression during pre- and postnatal development of the mouse brain and in adult human brain as well as in peripheral organs including the pineal gland. Four different methods (real time PCR, in situ hybridization, immunohistochemistry and Western blot analysis) were performed to systematically control for tissue-, species- and isoform-specific expression on both the pre- and posttranslational level. TPH2 expression was consistently detected in the raphe nuclei, as well as in fibres in the deep pineal gland and in the gastrointestinal tract. Although TPH1 expression was found in these peripheral tissues, no significant TPH1 expression was detected in the brain, neither during murine development, nor in mouse and human adult brain. Also under conditions like stress and clearing the tissue from blood cells, no changes in expression levels were detectable. Furthermore, the reuptake of 5-HT into the presynaptic neuron by the serotonin transporter (SERT) is the major mechanism terminating the neurotransmitter signal. Thus, mice with a deletion in the Sert gene (Sert KO mice) provide an adequate model for human affective disorders to study lifelong modified 5-HT homeostasis in interaction with stressful life events. To further explore the role of TPH isoforms, Tph1 and Tph2 expression was studied in the raphe nuclei of Sert deficient mice under normal conditions as well as following exposure to acute immobilization stress. Interestingly, no statistically significant changes in expression were detected. Moreover, in comparison to Tph2, no relevant Tph1 expression was detected in the brain independent from genotype, gender and treatment confirming expression in data from native animals. Raphe neurons of a brain-specific Tph2 conditional knockout (cKO) model were completely devoid of Tph2-positive neurons and consequently 5-HT in the brain, with no compensatory activation of Tph1 expression. In addition, a time-specific Tph2 inducible (i) KO mouse provides a brain-specific knockdown model during adult life, resulting in a highly reduced number of Tph2-positive cells and 5-HT in the brain. Intriguingly, expression studies detected no obvious alteration in expression of 5-HT system-associated genes in these brain-specific Tph2 knockout and knockdown models. The findings on the one hand confirm the specificity of Tph2 in brain 5-HT synthesis across the lifespan and on the other hand indicate that neither developmental nor adult Tph2-dependent 5-HT synthesis is required for normal formation of the serotonergic system, although Tph1 does not compensate for the lack of 5-HT in the brain of Tph2 KO models. A further aim of this thesis was to investigate the expression of the neuropeptide oxytocin, which is primarily produced in the hypothalamus and released for instance in response to stimulation of 5-HT and selective serotonin reuptake inhibitors (SSRIs). Oxytocin acts as a neuromodulator within the central nervous system (CNS) and is critically involved in mediating pain modulation, anxiolytic-like effects and decrease of stress response, thereby reducing the risk for emotional disorders. In this study, the expression levels of oxytocin in different brain regions of interest (cortex, hippocampus, amygdala, hypothalamus and raphe nuclei) from female and male wildtype (WT) and Sert KO mice with or without exposure to acute immobilization stress were investigated. Results showed significantly higher expression levels of oxytocin in brain regions which are involved in the regulation of emotional stimuli (amygdala and hippocampus) of stressed male WT mice, whereas male Sert KO as well as female WT and Sert KO mice lack these stress-induced changes. These findings are in accordance with the hypothesis of oxytocin being necessary for protection against stress, depressive mood and anxiety but suggest gender-dependent differences. The lack of altered oxytocin expression in Sert KO mice also indicates a modulation of the oxytocin response by the serotonergic system and provides novel research perspectives with respect to altered response of Sert KO mice to stress and anxiety inducing stimuli.},
  subject      = {Serotonin},
  language  = {en}
}
@phdthesis{Kiser2019,
  author    = {Kiser, Dominik Pascal},
  title     = {Gene x Environment Interactions in Cdh13-deficient Mice: CDH13 as a Factor for Adaptation to the Environment},
  doi       = {10.25972/OPUS-17959},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-179591},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Neurodevelopmental disorders, including attention-deficit/hyperactivity disorder (ADHD) and autism spectrum disorder (ASD) are disorders of mostly unknown etiopathogenesis, for which both genetic and environmental influences are expected to contribute to the phenotype observed in patients. Changes at all levels of brain function, from network connectivity between brain areas, over neuronal survival, synaptic connectivity and axonal growth, down to molecular changes and epigenetic modifications are suspected to play a key roles in these diseases, resulting in life-long behavioural changes. Genome-wide association as well as copy-number variation studies have linked cadherin-13 (CDH13) as a novel genetic risk factor to neuropsychiatric and neurodevelopmental disorders. CDH13 is highly expressed during embryonic brain development, as well as in the adult brain, where it is present in regions including the hippocampus, striatum and thalamus (among others) and is upregulated in response to chronic stress exposure. It is however unclear how CDH13 interacts with environmentally relevant cues, including stressful triggers, in the formation of long-lasting behavioural and molecular changes. It is currently unknown how the environment influences CDH13 and which long term changes in behaviour and gene expression are caused by their interaction. This work therefore investigates the interaction between CDH13 deficiency and neonatal maternal separation (MS) in mice with the aim to elucidate the function of CDH13 and its role in the response to early-life stress (ELS). For this purpose, mixed litters of wild-type (Cdh13+/+), heterozygous (Cdh13+/-) and homozygous knockout (Cdh13-/-) mice were maternally separated from postnatal day 1 (PN1) to postnatal day 14 (PN14) for 3 hours each day (180MS; PN1-PN14). In a first series of experiments, these mice were subjected to a battery of behavioural tests starting at 8 weeks of age in order to assess motor activity, memory functions as well as measures of anxiety. Subsequently, expression of RNA in various brain regions was measured using quantitativ real-time polymerase chain reaction (qRT-PCR). A second cohort of mice was exposed to the same MS procedure, but was not behaviourally tested, to assess molecular changes in hippocampus using RNA sequencing. Behavioural analysis revealed that MS had an overall anxiolytic-like effect, with mice after MS spending more time in the open arms of the elevated-plus-maze (EPM) and the light compartment in the light-dark box (LDB). As a notable exception, Cdh13-/- mice did not show an increase of time spent in the light compartment after MS compared to Cdh13+/+ and Cdh13+/- MS mice. During the Barnes-maze learning task, mice of most groups showed a similar ability in learning the location of the escape hole, both in terms of primary latency and primary errors. Cdh13-/- control (CTRL) mice however committed more primary errors than Cdh13-/- MS mice. In the contextual fear conditioning (cFC) test, Cdh13-/- mice showed more freezing responses during the extinction recall, indicating a reduced extinction of fear memory. In the step-down test, an impulsivity task, Cdh13-/- mice had a tendency to wait longer before stepping down from the platform, indicative of more hesitant behaviour. In the same animals, qRT-PCR of several brain areas revealed changes in the GABAergic and glutamatergic systems, while also highlighting changes in the gatekeeper enzyme Glykogensynthase-Kinase 3 (Gsk3a), both in relation to Cdh13 deficiency and MS. Results from the RNA sequencing study and subsequent gene-set enrichment analysis revealed changes in adhesion and developmental genes due to Cdh13 deficiency, while also highlighting a strong link between CDH13 and endoplasmatic reticulum function. In addition, some results suggest that MS increased pro-survival pathways, while a gene x environment analysis showed alterations in apoptotic pathways and migration, as well as immune factors and membrane metabolism. An analysis of the overlap between gene and environment, as well as their interaction, highlighted an effect on cell adhesion factors, underscoring their importance for adaptation to the environment. Overall, the stress model resulted in increased stress resilience in Cdh13+/+ and Cdh13+/- mice, a change absent in Cdh13-/- mice, suggesting a role of CDH13 during programming and adaptation to early-life experiences, that can results in long-lasting consequences on brain functions and associated behaviours. These changes were also visible in the RNA sequencing, where key pathways for cell-cell adhesion, neuronal survival and cell-stress adaptation were altered. In conclusion, these findings further highlight the role of CDH13 during brain development, while also shedding light on its function in the adaptation and response during (early life) environmental challenges.},
  subject      = {Cadherine},
  language  = {en}
}
@phdthesis{Nietzer2010,
  author    = {Nietzer, Sarah},
  title     = {Gene and environment interactions in serotonin transporter knockout mice - how stress influences gene expression and neuronal morphology},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-54391},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2010},
  abstract  = {Serotonin (5-HT) is an important modulator of many physiological, behavioural and developmental processes and it plays an important role in stress coping reactions. Anxiety disorders and depression are stress-related disorders and they are associated with a malfunction of the 5-HT system, in which the 5-HT transporter (5-HTT) plays an important role. 5-Htt knockout (KO) mice represent an artificially hyperserotonergic environment, show an increased anxiety-like behaviour and seem to be a good model to investigate the role of the 5-HT system concerning stress reactions and anxiety disorders. As synaptic proteins (SPs) seem to be involved in stress reactions, the effect of acute immobilization stress on the expression of the three SPs Synaptotagmin (Syt) I, Syt IV and Syntaxin (Stx) 1A was studied in the 5-Htt KO mouse model as well as the expression of the two immediate early genes (IEGs) FBJ osteosarcoma oncogene (c-Fos) and fos-like antigen 2 (Fra-2). Additionally, the expression of the corticotrophin releasing hormone (CRH) and its two receptors CRHR1 and CRHR2 was investigated as part of the hypothalamic-pituitary-adrenal (HPA) stress system. Based on gender- and genotype-dependent differences in corticosterone levels, expression differences in the brain were investigated by performing a quantitative real time-PCR study using primer pairs specific for these SPs and for the IEGs c-Fos and Fra-2 in five different brain regions in 5-Htt KO and 5-Htt wild-type (WT) mice. Mainly gender-dependent differences could be found and weaker stress effects on the expression of SPs could be demonstrated. Regarding the expression of IEGs, stress-, gender- and genotype-dependent differences were found mainly in the hypothalamus. Also in the hypothalamus, gender effects were found concerning the expression of CRH and its both receptors. Additionally, in a second study, male 5-Htt WT and male 5-Htt deficient mice were subjected to a resident-intruder-paradigm which stresses the animals through a loser experience. The morphological changes of neurons were subsequently analyzed in Golgi-Cox-stained sections of limbic brain areas in stressed and unstressed animals of both genotypes using the computer-based microscopy system Neurolucida (Microbrightfield, Inc.). While no differences concerning dendritic length, branching patterns and spine density were found in the hippocampus and no differences concerning dendritic length and branching patterns could be shown in the cingulate cortex (CG), pyramidal neurons in the infralimbic cortex (IL) of stressed 5-Htt WT mice displayed longer dendrites compared to unstressed 5-Htt WT mice. The results indicate that, although in this model drastic alterations of neuronal morphology are absent, subtle changes can be found in specific brain areas involved in stress- and anxiety-related behaviour which may represent neural substrates underlying behavioural phenomena.},
  subject      = {Serotonin},
  language  = {en}
}