Podcasts about hab lab

With the advances in genome wide screening arrays and sequencing techniques scientists were enabled to examine genetic variations and their effects on behavioral phenotypes. While single nucleotide polymorphisms (SNPs) are the most widely studied form of genomic variations to date, another type of variants has become increasingly important in recent research, the copy number variants (CNVs). These large segments of DNA that can comprise up to several megabasepairs and differ in copy number with respect to a reference genome have been associated with several disorders and behavioral phenotypes before. This study investigated the influence of CNVs on anxiety related behavior. The detection of these variants turned out to be a major challenge since all methods available are biased by limitations of the design of the approach and the subsequent computational analyses. Therefore, three different techniques (next generation sequencing and two distinct whole genome genotyping arrays) were employed to identify CNVs in a CD 1 derived mouse model consisting of two mouse strains showing high (HAB) and low (LAB) anxiety related behavior, respectively. By comparing CNVs in HAB vs. LAB mice with expression data of four distinct brain regions of high relevance to the limbic system (central and basolateral amygdala, cingulate cortex and the hypothalamic paraventricular nucleus), it was shown that CNVs can influence the expression of protein coding genes by the alteration of the genes’ copy number per se. Therefore, the genes mapping into regions where CNVs were detected in HAB vs. LAB mice (by all three detection methods) were suggested to be possible effectors of anxiety related behavior. Amongst these candidate genes those were considered to be the most interesting ones that were additionally found to map into regions of CNVs associated with anxiety related behavior in CD 1 mice. CNVs in these mice were detected by means of a whole genome genotyping array and subsequent processing of the raw data with a novel computational approach that was adapted from existing analysis methods. Furthermore, to test the effect of a specific CNV on anxiety related behavior in vivo, a breeding approach was used to generate animals with a full genetic background of HAB mice except for one LAB derived locus harboring a CNV that included the Glo1 gene. No direct effect on the phenotype could be observed, however, the respective CNV might be involved in the manipulation of anxiety related behavior taken into account the interaction with other factors. Taken together, this study provides not only a comprehensive catalogue of CNVs in HAB/LAB mice but also the evidence that these variants can influence anxiety related behavior. Furthermore, it gives a first insight into the functionality of CNVs with respect to anxiety related behavior. Therefore, this thesis provides a profound basis for multiple advanced studies.

Anxiety disorders are among the most common psychiatric diseases and contribute to the development of other psychiatric conditions, such as major depression, leading to a high impairment of daily life quality. Although it is obvious that the physiological architecture of neuronal networks and its modifications are essential for the ability of the brain to process incoming information and to control highly organized behaviour, the mechanisms underlying anxiety disorders still remain poorly understood. We focused our attention on two brain structures, which are strongly involved in emotional responses of mammals, namely the hippocampus and the amygdala. Both structures belong to the limbic system and play fundamental roles in information processing. Recent findings indicate that alterations in neuronal network properties of these two brain areas critically contribute to the development of such disorders. To potentially uncover changes in neuronal network features associated with abnormal anxiety, we performed experiments in a well-established animal model of extremes in trait anxiety, the high vs. low anxiety-related behaviour (HAB/LAB) mice. HAB mice exposed to an enriched environment (HAB E.E.) and stressed LAB mice (LAB Str.) were also used in the present study. HAB E.E. animals showed decreased anxiety compared to standard HABs, whereas LAB Str. animals displayed an increase in anxiety levels compared to standard LABs. Anxiety levels were measured by means of the elevated plus maze. For our investigations, we employed classical electrophysiological techniques and high-speed voltage-sensitive dye imaging (VSDI) in acute hippocampal (dorsal & ventral) and amygdalar brain slices. Field potential recordings revealed that HAB animals exhibit weaker long-term potentiation at CA3-CA1 synapses (CA1 LTP) in the dorsal hippocampus and an increased LTP in the ventral hippocampus compared to LAB and control CD1 mice. These observations could support the idea of an exacerbated activation of the “emotional” (ventral) hippocampus concomitantly with a decreased activity in the “cognitive” (dorsal) hippocampus, findings that have also been made in patients suffering from anxiety disorders. To examine whether neuronal activity propagation through the amygdala differs between HAB, HAB E.E., LAB and LAB Str. mice, we used a quantitative VSDI approach. Our results demonstrate that HAB animals exhibit stronger neuronal activity propagation through the amygdala compared to LAB mice. This indicates that differences in anxiety levels may correlate with the effectiveness of neuronal activity flow through the amygdalar network. Our study also provides strong evidence that environmentally induced shifts in trait anxiety are associated with changes in intrinsic amygdalar network properties. To summarize, HAB animals showed increased “excitability” in the ventral hippocampus and in the amygdalar network, both structures known to be involved in the control of emotional states and in the stress response in mammals. In addition, the differences in amygdalar network activity were rescued by environmental conditions (enriched environment). Dysregulation of these structures could lead to the “pathologic anxiety-like” behaviour, which can be observed in HAB animals.

To unravel the molecular pylons of innate anxiety, a well established animal model has been characterized using transcriptome- and sequence-based analyses. The animal model – hyper (HAB) and hypo (LAB) anxious mice – has been created by selective inbreeding based on outbred CD1 mice using the extreme values the mice spent on the open arm of the elevated plus-maze, a test also used to screen drugs for anxiolytic or anxiogenic effects. These mice proved a robust phenotypic divergence, also for depression-like behavior and stress-axis reactivity. In a first assay, brain regions unambiguously involved in regulating anxiety-related behavior were screened for gene expression differences between HAB and LAB animals in a microarray experiment covering the whole genome. This led to the identification of thousands of differentially expressed transcripts. The highest significant results were further validated by quantitative PCR or other techniques focusing either on protein quantification or enzyme activity. Applying this strategy, differential regulation of 15 out of 28 transcripts could be validated: vasopressin, tachykinin 1, transmembrane protein 132D, RIKEN cDNA 2900019G14 gene, ectonucleotide pyrophosphatase/phosphodiesterase 5, cathepsin B, coronin 7, glyoxalase 1, pyruvate dehydrogenase beta, metallothionein 1, matrix metallopeptidase 15, zinc finger protein 672, syntaxin 3, solute carrier family 25 member 17 and ATP-binding cassette, sub-family A member 2. Additionally, analysis of cytochrome c oxidase activity resulted in the identification of differences in long-term activity between HAB and LAB mice in the amygdala and the hypothalamic paraventricular nucleus pointing to an important role of these brain regions in shaping the anxiety-related extremes in these mice. In a second genome-wide screening approach, 267 single nucleotide polymorphisms were identified to constantly differ between HAB and LAB animals (i.e. to carry the opposite homozygous genotype at these loci) and subsequently genotyped in 520 F2 mice, the offspring of reciprocally mated HABxLAB animals. These F2 mice have been previously phenotyped in a broad variety of behavioral tests and show – as descendants of intermediate heterozygotes for all polymorphic genomic loci between HAB and LAB mice – a free segregation of all alleles, thus allowing genotype-phenotype associations based on whole-genome analysis. Only focusing on the most significant findings, associations have been observed between anxiety-related behavior and loci on mouse chromosomes 5 and 11, between depression-like behavior and chromosome 2 and between stress-axis reactivity and chromosome 3. The locus on chromosome 11 is marked by a polymorphism located in the 3’ untranslated region of zinc finger protein 672, a gene also markedly overexpressed in LAB mice and expressed at lower levels in HAB mice leading to a probable causal involvement in shaping the phenotype. Further associations on chromosome 5 include two functional polymorphisms in enolase phosphatase 1 that result in a different mobility of the enzyme in proteomic assays and with a polymorphism located in the transmembrane protein 132D gene. Furthermore, independently, an association of a polymorphism in this particular gene, together with the resulting gene expression differences has been observed in a group of panic disorder patients, highlighting this gene as a causal factor underlying anxiety-related behavior and disorders in both the HAB/LAB mouse model and human patients. The combination of expression profiling and confirmation by quantitative PCR, single nucleotide polymorphism analysis and F2 association studies, i.e. unbiased and hypothesis driven approaches were key to the identification and functional characterization of loci, genes and polymorphisms causally involved in shaping anxiety-related behavior. Thus, it provides an overview of some new promising targets for future pharmaceutic treatment and will contribute to a better understanding of the molecular processes that shape anxiety and thereby also animal and human behavior.

Anxiety reflects a fundamental emotion, essential for survival. However, if it occurs unpredictably and exaggerated for a long period of time, it becomes pathological, confining a normal course of life. Anxiety disorders are among the most disabling psychiatric diseases, with increasing incidence. They are complex and occur as a combination of both, inherited and stress-related phenomena, whose origin and underlying mechanisms are still poorly understood. Besides clinical studies, extensive preclinical research is strongly focusing on the genetic, environmental, and developmental underpinnings of both, “physiological” and “pathological” anxiety. Thus, in the year 2000, two mouse lines were generated by bi-directional selective inbreeding, reflecting extremes in trait anxiety. These phenotypic extremes, independent of gender, display either high (HAB) or low (LAB) anxiety-related behavior as measured in the elevated plus-maze test and a variety of other paradigms. Since anxiety is not considered as a single entity, but covers multiple facets, the studies presented in this thesis address behavioral, neuroendocrine, genetic, developmental as well as cognitive aspects in this mouse model of trait anxiety. Comprehensive phenotyping confirmed the phenotypic divergence of the mouse lines. Although selection pressure was only exerted on anxiety-related behavior, the mouse lines exhibited comorbid depression-like and altered explorative behavior. Moreover, expression profiling of genes well described in the regulation of emotionality at the level of the hypothalamo-pituitary-adrenocortical axis and synaptic neurotransmission, as well as pharmacological intervention, highlighted arginine-vasopressin (AVP), corticotropin-releasing hormone (CRH), and synaptotagmin 4 (Syt4) as potential mediators contributing to the observed behavioral differences. AVP has been identified to be under-expressed in several brain regions of LAB mice associated with their non-anxious and non-depression-like behaviors. In addition, several genetic polymorphisms have been identified that are likely to play a critical role in the AVP under-expression of these animals. In contrast, the highly anxious HAB animals revealed a CRH over-expression in various brain areas. The significance of CRH over-expression in mediating the HAB-specific phenotype was pharmacologically validated via CRH receptor 1 antagonist administration. Synaptic release, indicated by Syt4 expression, was found to be altered in both inbred mouse lines in opposite directions, indicating a dysregulation in both extremes of trait anxiety. Furthermore, glyoxalase1 (Glx1), a cellular detoxification enzyme, has been identified to be differently expressed already at early postnatal developmental stages in association with the phenotypic divergence. Thus, Glx1 might act as a biomarker suitable for the early prediction of pathological anxiety. As anxiety disorders have often been described to be accompanied by alterations in cognitive abilities, this coherency was also addressed in the HAB/LAB model. Indeed, HAB mice showed a superior ability in a social learning paradigm and displayed delayed extinction in a classical fear-conditioning study, the latter being similarly observed in patients suffering from posttraumatic stress disorder. Taken together, the HAB/LAB mouse model covers many clinical core symptoms of anxiety disorders at different levels, including behavioral emotionality, gene expression, and cognitive alterations. Therefore, it provides a valuable and promising tool to elucidate the neurobiological basis of the continuum from vital to pathological anxiety.

Das Neuropeptid Vasopressin (AVP) ist zentral an der Ausprägung von Emotionalität, Kognition und der HPA-Achsen-Aktivität im Kontinuum von physiologischer Funktion bis hin zu pathophysiologischer Dysfunktion beteiligt. Vorliegende Studien untersuchen die Rolle des vasopressinergen Systems in der Vulnerabilität für und in der Genese von Psychopathologien an einem etablierten Rattenmodell für Angsterkrankungen sowie komorbid depressionsbezogene und dysfunktionale neuroendokrine Parameter. Die verhaltensbiologische Charakterisierung zeigte dabei weitere Facetten des Tiermodells in der Reflexion von psychopathologischen Erscheinungsbildern wie Anhedonie sowie erhöhte Stressvulnerabilität und fehlregulierte Kognition im sozialen Kontext. Neben der Etablierung eines neuartigen Phänomens der HPA-Achsen-Regulierung im Zusammenhang mit der Bewältigung von sozialem Stress konnte die pathologisch überdurchschnittliche soziale Kognitionsleistung grundlegend auf das septale AVP-System zurückgeführt werden. Die Untersuchungen zur Ausprägung des im Rattenmodell vorliegenden psychopathologischen angst- und depressionsbezogenen Verhaltens bestätigten die zentrale Rolle anxiogenen AVPs. Während neuropeptiderge Kandidaten für die extrazelluläre Regulation des hypothalamischen AVP-Systems identifiziert werden konnten, war kein quantitativer Einfluss eines mutierten AVP-Gens per se in transgen gezüchteten Tieren wie auch in einer frei segregierenden F2-Generation nachzuweisen.