Gene-environment interplay of extreme anxiety-related behavior

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.