Anxiety-like behavior and protein nitrosylation in a heterozygous glutathione reductase mouse model

Anxiety disorders are a group of psychiatric disorders characterized by excessive fear and anxiety that disrupts normal life. They are very common, having an estimated life-time prevalence of 16.6 %, and they start often at an early age. Anxiety disorders have a clear genetic component, but the onset is also largely affected by the environment. Although several brain regions and neurotransmitter systems have been shown to take part in the regulation of anxiety, the exact pathophysiological mechanisms behind anxiety disorders are largely unknown. Mouse models are a good tool when studying the genetic component behind anxiety disorders. Different behavioral tests measuring the anxiety-like behavior of mice exist, and these tests can be used to model certain aspects of pathological anxiety. Individual gene effects on anxiety can be studied by using transgenic mouse models, which was done in this study, where a knockout mouse model lacking the functional version of glutathione reductase (Gsr) was used to study the effect of Gsr in anxiety. Because of the reduced viability of the homozygotic knockout mice, our study was done using heterozygotic knockout mice, which have 64 % of normal glutathione reductase activity. Glutathione is one of the most important endogenous antioxidants, which protects cells from oxidative stress by getting oxidized by reactive oxygen species. Glutathione reductase takes part in this process by transforming glutathione back to its functional reduced form. Gsr expression in amygdala and cingulate cortex has been previously shown to correlate with anxiety-like behavior in mice such that mice with upregulated Gsr expression were more anxious. This and other results obtained from mice and human studies suggest that oxidative stress may be involved in the pathogenesis of anxiety. In this study we wanted to investigate, whether the heterozygous Gsr knockout mice have lower anxiety-like behavior than the wild-type mice and whether there is more oxidative damage present in the brain of heterozygous Gsr knockout mice compared to the wild-type mice. To assess the first question, we set up and validated two behavioral tests (novelty-induced hypophagia and marble burying) and tested the Gsr mice using these tests. To assess the second question, we used nitrotyrosine as a marker for oxidative damage and measured the amount of this marker from brain tissue samples of Gsr mice by a nitrotyrosine immunoblotting assay. We discovered that both our novelty-induced hypophagia and marble burying test settings were able to detect some behavioral differences between inbred mouse strains, but were not optimal in distinguishing highly anxious strains from less anxious strains. When testing the Gsr mice, no behavioral difference between the heterozygous knockout and wild-type mice was observed, but a significant difference in the behavior between sexes was detected. We obtained preliminary results showing that the nitrotyrosine levels are slightly increased in the cortex of male heterozygous Gsr knockout mice. Whether this is the case also in other brain regions and in female mice still needs to be tested. These results and previous results obtained by our group suggest that heterozygous Gsr knockout mice do not have a striking anxiety phenotype under baseline conditions, but might have slightly reduced anxiety-like behavior and slightly increased brain oxidative stress status when compared to the wild-type mice