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Background: The hypothalamic-pituitary-adrenal (HPA) axis is essential to control physiological stress responses in mammals. Its dysfunction is related to several mental disorders, including anxiety and depression. The aim of this study was to identify genetic loci underlying the endocrine regulation of the HPA axis., Method: High (HAB) and low (LAB) anxiety-related behaviour mice were established by selective inbreeding of outbred CD-1 mice to model extremes in trait anxiety. Additionally, HAB vs. LAB mice exhibit comorbid characteristics including a differential corticosterone response upon stress exposure. We crossbred HAB and LAB lines to create F1 and F2 offspring. To identify the contribution of the endocrine phenotypes to the total phenotypic variance, we examined multiple behavioural paradigms together with corticosterone secretion-based phenotypes in F2 mice by principal component analysis. Further, to pinpoint the genomic loci of the quantitative trait of the HPA axis stress response, we conducted genome-wide multipoint oligogenic linkage analyses based on Bayesian Markov chain Monte Carlo approach as well as parametric linkage in three-generation pedigrees, followed by a two-dimensional scan for epistasis and association analysis in freely segregating F2 mice using 267 single-nucleotide polymorphisms (SNPs), which were identified to consistently differ between HAB and LAB mice as genetic markers., Results: HPA axis reactivity measurements and behavioural phenotypes were represented by independent principal components and demonstrated no correlation. Based on this finding, we identified one single quantitative trait locus (QTL) on chromosome 3 showing a very strong evidence for linkage (2ln (L-score) > 10, LOD > 23) and significant association (lowest Bonferroni adjusted p < 10-28) to the neuroendocrine stress response. The location of the linkage peak was estimated at 42.3 cM (95% confidence interval: 41.3 - 43.3 cM) and was shown to be in epistasis (p-adjusted < 0.004) with the locus at 35.3 cM on the same chromosome. The QTL harbours genes involved in steroid synthesis and cardiovascular effects., Conclusion: The very prominent effect on stress-induced corticosterone secretion of the genomic locus on chromosome 3 and its involvement in epistasis highlights the critical role of this specific locus in the regulation of the HPA axis.

MAP kinase signaling has been implicated in brain development, long-term memory, and the response to antidepressants. Inducible Braf knockout mice, which exhibit protein depletion in principle forebrain neurons, enabled us to unravel a new role of neuronal MAPK signaling for emotional behavior. Braf mice that were induced during adulthood showed normal anxiety but increased depression-like behavior, in accordance with pharmacological findings. In contrast, the inducible or constitutive inactivation of Braf in the juvenile brain leads to normal depression-like behavior but decreased anxiety in adults. In juvenile, constitutive mutants we found no alteration of GABAergic neurotransmission but reduced neuronal arborization in the dentate gyrus. Analysis of gene expression in the hippocampus revealed nine downregulated MAPK target genes that represent candidates to cause the mutant phenotype.Our results reveal the differential function of MAPK signaling in juvenile and adult life phases and emphasize the early postnatal period as critical for the determination of anxiety in adults. Moreover, these results validate inducible gene inactivation as a new valuable approach, allowing it to discriminate between gene function in the adult and the developing postnatal brain.

The lifetime prevalence of panic disorder (PD) is up to 4% worldwide and there is substantial evidence that genetic factors contribute to the development of PD. Single-nucleotide polymorphisms (SNPs) in TMEM132D, identified in a whole-genome association study (GWAS), were found to be associated with PD in three independent samples, with a two-SNP haplotype associated in each of three samples in the same direction, and with a P-value of 1.2e-7 in the combined sample (909 cases and 915 controls). Independent SNPs in this gene were also associated with the severity of anxiety symptoms in patients affected by PD or panic attacks as well as in patients suffering from unipolar depression. Risk genotypes for PD were associated with higher TMEM132D mRNA expression levels in the frontal cortex. In parallel, using a mouse model of extremes in trait anxiety, we could further show that anxiety-related behavior was positively correlated with Tmem132d mRNA expression in the anterior cingulate cortex, central to the processing of anxiety/fear-related stimuli, and that in this animal model a Tmem132d SNP is associated with anxiety-related behavior in an F2 panel. TMEM132D may thus be an important new candidate gene for PD as well as more generally for anxiety-related behavior.

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. We identified and confirmed sex-independent differences in the basal expression of 13 candidate genes, using tissue from the entire brain, including coronin 7 (Coro7), cathepsin B (Ctsb), muscleblind-like 1 (Mbnl1), metallothionein 1 (Mt1), solute carrier family 25 member 17 (Slc25a17), tribbles homolog 2 (Trib2), zinc finger protein 672 (Zfp672), syntaxin 3 (Stx3), ATP-binding cassette, sub-family A member 2 (Abca2), ectonucleotide pyrophosphatase/phosphodiesterase 5 (Enpp5), high mobility group nucleosomal binding domain 3 (Hmgn3) and pyruvate dehydrogenase beta (Pdhb). Additionally, we confirmed brain region-specific differences in the expression of synaptotagmin 4 (Syt4).Our identification of about 90 polymorphisms in Ctsb suggested that this gene might play a critical role in shaping our mouse model's behavioral endophenotypes. Indeed, the assessment of anxiety-related and depression-like behaviors of Ctsb knock-out mice revealed an increase in depression-like behavior in females. Altogether, our results suggest that Ctsb has significant effects on emotionality, irrespective of the tested mouse strain, making it a promising target for future pharmacotherapy.

Background: 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., Methodology/ Principal Findings: 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 Delta(-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., Discussion: Thus, the identification of polymorphisms in the Avp 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.