Behavioral phenotyping, gene expression profiles, and cognitive aspects in a mouse model of trait anxiety

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

A hypomorphic vasopressin allele prevents anxiety-related behavior

In this study, microarray analysis, in situ hybridization, quantitative real-time PCR and immunohistochemistry revealed decreased expression of the vasopressin gene (Avp) in the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei of adult LAB mice compared to HAB, NAB (normal anxiety-related behavior) and HABxLAB F1 intercross controls, without detecting differences in receptor expression or density. By sequencing the regions 2.5 kbp up- and downstream of the Avp gene locus, we could identify several polymorphic loci, differing between the HAB and LAB lines. In the gene promoter, a deletion of twelve bp Δ(−2180–2191) is particularly likely to contribute to the reduced Avp expression detected in LAB animals under basal conditions. Indeed, allele-specific transcription analysis of F1 animals revealed a hypomorphic LAB-specific Avp allele with a reduced transcription rate by 75% compared to the HAB-specific allele, thus explaining line-specific Avp expression profiles and phenotypic features. Accordingly, intra-PVN Avp mRNA levels were found to correlate with anxiety-related and depression-like behaviors. In addition to this correlative evidence, a significant, though moderate, genotype/phenotype association was demonstrated in 258 male mice of a freely-segregating F2 panel, suggesting a causal contribution of the Avp promoter deletion to anxiety-related behavior

A Hypomorphic Vasopressin Allele Prevents Anxiety-Related Behavior

To investigate neurobiological correlates of trait anxiety, CD1 mice were selectively bred for extremes in anxiety-related behavior, with high (HAB) and low (LAB) anxiety-related behavior mice additionally differing in behavioral tests reflecting depression-like behavior. promoter deletion to anxiety-related behavior. gene promoter explains gene expression differences in association with the observed phenotype, thus further strengthening the concept of the critical involvement of centrally released AVP in trait anxiety

Stable Isotope Metabolic Labeling with a Novel 15N-Enriched Bacteria Diet for Improved Proteomic Analyses of Mouse Models for Psychopathologies

The identification of differentially regulated proteins in animal models of psychiatric diseases is essential for a comprehensive analysis of associated psychopathological processes. Mass spectrometry is the most relevant method for analyzing differences in protein expression of tissue and body fluid proteomes. However, standardization of sample handling and sample-to-sample variability are problematic. Stable isotope metabolic labeling of a proteome represents the gold standard for quantitative mass spectrometry analysis. The simultaneous processing of a mixture of labeled and unlabeled samples allows a sensitive and accurate comparative analysis between the respective proteomes. Here, we describe a cost-effective feeding protocol based on a newly developed 15N bacteria diet based on Ralstonia eutropha protein, which was applied to a mouse model for trait anxiety. Tissue from 15N-labeled vs. 14N-unlabeled mice was examined by mass spectrometry and differences in the expression of glyoxalase-1 (GLO1) and histidine triad nucleotide binding protein 2 (Hint2) proteins were correlated with the animals' psychopathological behaviors for methodological validation and proof of concept, respectively. Additionally, phenotyping unraveled an antidepressant-like effect of the incorporation of the stable isotope 15N into the proteome of highly anxious mice. This novel phenomenon is of considerable relevance to the metabolic labeling method and could provide an opportunity for the discovery of candidate proteins involved in depression-like behavior. The newly developed 15N bacteria diet provides researchers a novel tool to discover disease-relevant protein expression differences in mouse models using quantitative mass spectrometry

Enhanced Fear Expression in a Psychopathological Mouse Model of Trait Anxiety: Pharmacological Interventions

The propensity to develop an anxiety disorder is thought to be determined by genetic and environmental factors. Here we investigated the relationship between a genetic predisposition to trait anxiety and experience-based learned fear in a psychopathological mouse model. Male CD-1 mice selectively bred for either high (HAB), or normal (NAB) anxiety-related behaviour on the elevated plus maze were subjected to classical fear conditioning. During conditioning both mouse lines showed increased fear responses as assessed by freezing behaviour. However, 24 h later, HAB mice displayed more pronounced conditioned responses to both a contextual or cued stimulus when compared with NAB mice. Interestingly, 6 h and already 1 h after fear conditioning, freezing levels were high in HAB mice but not in NAB mice. These results suggest that trait anxiety determines stronger fear memory and/or a weaker ability to inhibit fear responses in the HAB line. The enhanced fear response of HAB mice was attenuated by treatment with either the α2,3,5-subunit selective benzodiazepine partial agonist L-838,417, corticosterone or the selective neurokinin-1 receptor antagonist L-822,429. Overall, the HAB mouse line may represent an interesting model (i) for identifying biological factors underlying misguided conditioned fear responses and (ii) for studying novel anxiolytic pharmacotherapies for patients with fear-associated disorders, including post-traumatic stress disorder and phobias

Profiling Trait Anxiety: Transcriptome Analysis Reveals Cathepsin B (Ctsb) as a Novel Candidate Gene for Emotionality in Mice

Behavioral endophenotypes are determined by a multitude of counteracting but precisely balanced molecular and physiological mechanisms. In this study, we aim to identify potential novel molecular targets that contribute to the multigenic trait “anxiety”. We used microarrays to investigate the gene expression profiles of different brain regions within the limbic system of mice which were selectively bred for either high (HAB) or low (LAB) anxiety-related behavior, and also show signs of comorbid depression-like behavior

Differential Stress-Induced Neuronal Activation Patterns in Mouse Lines Selectively Bred for High, Normal or Low Anxiety

There is evidence for a disturbed perception and processing of emotional information in pathological anxiety. Using a rat model of trait anxiety generated by selective breeding, we previously revealed differences in challenge-induced neuronal activation in fear/anxiety-related brain areas between high (HAB) and low (LAB) anxiety rats. To confirm whether findings generalize to other species, we used the corresponding HAB/LAB mouse model and investigated c-Fos responses to elevated open arm exposure. Moreover, for the first time we included normal anxiety mice (NAB) for comparison. The results confirm that HAB mice show hyperanxious behavior compared to their LAB counterparts, with NAB mice displaying an intermediate anxiety phenotype. Open arm challenge revealed altered c-Fos response in prefrontal-cortical, limbic and hypothalamic areas in HAB mice as compared to LAB mice, and this was similar to the differences observed previously in the HAB/LAB rat lines. In mice, however, additional differential c-Fos response was observed in subregions of the amygdala, hypothalamus, nucleus accumbens, midbrain and pons. Most of these differences were also seen between HAB and NAB mice, indicating that it is predominately the HAB line showing altered neuronal processing. Hypothalamic hypoactivation detected in LAB versus NAB mice may be associated with their low-anxiety/high-novelty-seeking phenotype. The detection of similarly disturbed activation patterns in a key set of anxiety-related brain areas in two independent models reflecting psychopathological states of trait anxiety confirms the notion that the altered brain activation in HAB animals is indeed characteristic of enhanced (pathological) anxiety, providing information for potential targets of therapeutic intervention

Behavioral parameters of HAB, NAB and LAB mice measured in the 5-min exposure to the OA.

<p>(a) Time spent in distal zone, entries into distal zone, total distance traveled. (b) Head-dip behavior. Values are expressed as mean±SEM. HAB: n = 9, NAB: n = 8, LAB: n = 8; * p<0.05, ** p<0.01.</p