Assessing the complex nature of behavior

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

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

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 New Way of Identifying Biomarkers in Biomedical Basic-Research Studies

A simple, nonparametric and distribution free method was developed for quick identification of the most meaningful biomarkers among a number of candidates in complex biological phenomena, especially in relatively small samples. This method is independent of rigid model forms or other link functions. It may be applied both to metric and non-metric data as well as to independent or matched parallel samples. With this method identification of the most relevant biomarkers is not based on inferential methods; therefore, its application does not require corrections of the level of significance, even in cases of thousands of variables. Hence, the introduced method is appropriate to analyze and evaluate data of complex investigations in clinical and pre-clinical basic research, such as gene or protein expressions, phenotype-genotype associations in case-control studies on the basis of thousands of genes and SNPs (single nucleotide polymorphism), search of prevalence in sleep EEG-Data, functional magnetic resonance imaging (fMRI) or others

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