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Free keywords:
anxiety, stress, epigenetics, Crhr1, YY1, methylation
Abstract:
The use of selectively bred mouse models of enhanced fear and/or anxiety-related behavior provides a unique opportunity to identify genetic targets that contribute to pathological anxiety. However, dealing with animal models needs accurate information about their phenotypes. Accordingly, high (HAB), normal (NAB) and low (LAB) anxiety-related behavior mice ? a validated model of anxiety disorders - were repeatedly tested in a variety of behavioral paradigms. Whereas most tests to assess anxiety traits are based on fear of novel and open spaces, we took advantage of the inborn fear and associated avoidance of the predator odor (trimethylthiazoline (TMT)) as a measure of anxiety-related behavior. We were able to show that avoidance of TMT reflects the high anxiety phenotype of HAB mice, indicated by the decreased time animals spent in the chamber with TMT compared to NAB and LAB mice. Importantly, this result is not confounded by any deficit of the olfactory system, since mice responded to both the pleasant odor of female urine and the repugnant odor of butyric acid.
To take the influence of environmental stimuli on inborn anxiety further, we next studied the impact of environmental manipulations on the genetically driven phenotype of LAB mice. Therefore, animals were exposed to a series of chronic unpredictable mild stressors (CMS). CMS-treated mice displayed increased anxiety in the TMT-avoidance test, elevated plus-maze (EPM) and light-dark box (LDB). Moreover, these animals were characterized by increased depression-like behavior and a blunted neuroendocrine regulation. Furthermore, TMT-exposure promoted a higher activation of immediate early gene expression, e.g. c-fos, in the amygdala, especially in the basolateral nuclei (BLA). c-Fos expression pattern correlated with anxiety-related behavior after CMS. Importantly, our electrophysiological studies also indicated a higher activation of amygdala in LAB mice after CMS treatment.
Since corticotropin releasing hormone (CRH) is one of the most important mediators of amygdala activity and is largely involved in the regulation of the anxiety-related behavior, we hypothesized that environmental influences are translated via an altered CRH system. Previous experiments had shown that enriched environment (EE) induced a down-regulation of Crhr1. Here, we report that CMS induced higher expression of Crhr1 in the BLA of LAB mice, in contrast to EE. Thus, these data indicate, that Crhr1 expression might be plastic in response to both, beneficial and detrimental, environmental factors.
Thereafter, we studied the role of DNA methylation as a probable mechanism behind the different gene expression. Using pyrosequencing of the bisulfite-converted DNA, one specific CpG site (CpG1) of Crhr1 was found to be higher methylated after both treatments. In order to evaluate functional importance of this modification, we tested the impact of CpG1 methylation on promoter activity using the luciferase assay and observed that the presence of methylation reduced promoter activity. Moreover, elevated methylation decreased the binding efficiency of the transcription factor Yin Yang 1 (YY1) as indicated by electrophoretic mobility shift assay (EMSA). Furthermore, we analyzed whether a higher expression of YY1 in the BLA of LAB mice, observed after CMS, contributed to the elevation of Crhr1. Indeed, overexpression of YY1 in the neuronal cell culture enhanced both Crhr1 expression and Crhr1 promoter activity. Finally, we estimated the effects of combininig CpG1 site-specific methylation with YY1 overexpression on Crhr1 promoter activity and tested whether in vitro overexpression of YY1 induced methylation of CpG1.
Altogether, our data suggest that even a rigid genetic predisposition to low anxiety-related behavior could be rescued by environmental modification and provide evidence that the epigenetic regulation of Crhr1 expression in the BLA is a possible underlying mechanism behind.
