To elucidate physiological mechanisms underlying pathological anxiety, valid animal models are essential (Cryan and Holmes 2005). Previous studies confirmed that the high anxiety-related behavior/low anxiety-related behavior (HAB/ LAB) mouse model for trait anxiety mimics many clinically relevant features typical of psychiatric patients (Krömer et al. 2005;Landgraf et al. 2007). In this model, behavioral phenotyping on the elevated-plus-maze (EPM) test was used as selection criterion for bidirectional intra-strain (CD1) breeding. Consequently, inbreeding for more than 30 generations created hyper-anxious (HAB) and hypo-anxious (LAB) mice. These animals provide robust phenotypes for extremes in anxiety-related behavior, and show high comorbidity with depression-like behavior. Inbreeding with focus on a particular trait included a strong accumulation of specific genetic material. Therefore, HAB/LAB animals serve as a potent model to analyze candidates underlying trait anxiety. One of the first protein markers shown to be differentially regulated in extremes of trait anxiety was glyoxalase1 (GLO1; lactoylglutathione lyase, EC 4.4.1.5).Mouse GLO1 is an enzyme of 21kDa, containing 184 amino acids that is found as a dimer in the cytosol of cells. Its physiological function is the catalytic conversion of Abbreviations used: AGE, advanced glycation end; ARMS, amplification refractory mutation system; CNV, copy number variant; enpp5, ectonucleotide pyrophosphatase/phosphodiesterase 5; EPM, elevatedplus-maze; GC-MS, gas chromatography-mass spectrometry; GLO1, glyoxalase1; HAB, high anxiety-related behavior; i.c.v., intracerebroventricular; LAB, low anxiety-related behavior; LTP, long-term potentiation; MG, methylglyoxal; NAB, normal anxiety-related behavior; TST, tail suspension test; WB, western blotting. AbstractMethylglyoxal (MG) is a highly reactive metabolite that forms adducts with basic amino acid side chains in proteins. MG is degraded by glyoxalase1 (GLO1), an enzyme shown to be differentially expressed in several mouse models of anxietyrelated behavior. As yet, molecular mechanisms by which altered GLO1 expression influences emotionality have not been elucidated. Here we report that both MG concentration and protein modification are altered in brain tissue of a mouse model for trait anxiety, with elevated levels in low anxiety-related behavior relative to high anxiety-related behavior animals. Accordingly, repeated intracerebroventricular injections of MG mediated anxiolysis in inbred high anxiety-related behavior and outbred CD1 mice. We found that anxiolytic-like properties of MG were independent of GLO1 expression. In contrast, antidepressant-like properties of intracerebroventricular MG were suppressed in CD1 mice carrying extra copies of the GLO1 gene. Moreover, MG treatment increased expression of GLO1 only in CD1 mice that did not have extra copies of GLO1. Taken together, these results suggest that the MG levels in brain are negatively correlated with anxiety. Thereby, we identified a novel molecular mec...

Chronic stress is a risk factor for psychiatric disorders but does not necessarily lead to uniform long-term effects on mental health, suggesting modulating factors such as genetic predispositions. Here we address the question whether natural genetic variations in the mouse CRH receptor 1 (Crhr1) locus modulate the effects of adolescent chronic social stress (ACSS) on long-term stress hormone dysregulation in outbred CD1 mice, which allows a better understanding of the currently reported genes × environment interactions of early trauma and CRHR1 in humans. We identified 2 main haplotype variants in the mouse Crhr1 locus that modulate the long-term effects of ACSS on basal hypothalamic-pituitary-adrenal axis activity. This effect is likely mediated by higher levels of CRHR1, because Crhr1 mRNA expression and CRHR1 binding were enhanced in risk haplotype carriers. Furthermore, a CRHR1 receptor antagonist normalized these long-term effects. Deep sequencing of the Crhr1 locus in CD1 mice revealed a large number of linked single-nucleotide polymorphisms with some located in important regulatory regions, similar to the location of human CRHR1 variants implicated in modulating gene × stress exposure interactions. Our data support that the described gene × stress exposure interaction in this animal model is based on naturally occurring genetic variations in the Crhr1 gene associated with enhanced CRHR1-mediated signaling. Our results suggest that patients with a specific genetic predisposition in the CRHR1 gene together with an exposure to chronic stress may benefit from a treatment selectively antagonizing CRHR1 hyperactivity.

Genomic copy number variants (CNVs) have been implicated in multiple psychiatric disorders, but not much is known about their influence on anxiety disorders specifically. Using next-generation sequencing (NGS) and two additional array-based genotyping approaches, we detected CNVs in a mouse model consisting of two inbred mouse lines showing high (HAB) and low (LAB) anxiety-related behavior, respectively. An influence of CNVs on gene expression in the central (CeA) and basolateral (BLA) amygdala, paraventricular nucleus (PVN), and cingulate cortex (Cg) was shown by a two-proportion Z-test (p = 1.6 x 10-31), with a positive correlation in the CeA (p = 0.0062), PVN (p = 0.0046) and Cg (p = 0.0114), indicating a contribution of CNVs to the genetic predisposition to trait anxiety in the specific context of HAB/LAB mice. In order to confirm anxiety-relevant CNVs and corresponding genes in a second mouse model, we further examined CD-1 outbred mice. We revealed the distribution of CNVs by genotyping 64 CD 1 individuals using a high-density genotyping array (Jackson Laboratory). 78 genes within those CNVs were identified to show nominally significant association (48 genes), or a statistical trend in their association (30 genes) with the time animals spent on the open arms of the elevated plus-maze (EPM). Fifteen of them were considered promising candidate genes of anxiety-related behavior as we could show a significant overlap (permutation test, p = 0.0051) with genes within HAB/LAB CNVs. Thus, here we provide what is to our knowledge the first extensive catalogue of CNVs in CD-1 mice and potential corresponding candidate genes linked to anxiety-related behavior in mice.

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