The anxious mouse: implications for preclinical research and animal welfare

Anxiety is an essential emotion that is highly conserved during evolution and is present in animals and humans. Although anxiety is a biological adaptive response, anxiety disorders in humans are common and affect about 10-17% of the world population. To gain more insight in the underlying neurobiological mechanisms of pathological anxiety, animal models are often used. However, commonly used animal models of anxiety are based on the single exposure to for example novelty and thus measure normal i.e. adaptive anxiety. A biological appropriate animal model for pathological anxiety would be of high use for both, translational research on human anxiety disorders and for the investigation of animal welfare aspects, since welfare might be compromised in animals suffering from non-adaptive anxiety. The main aim of the present studies was to evaluate behavioural habituation as an indicator of non-adaptive, i.e. pathological anxiety in mice. Two mouse strains BALB/c and 129P3 mice were characterized for their ability to habituate to an initially novel environment. Behavioural performance as well as stress hormones and the neural activity marker c-Fos were investigated. After behavioural testing for their ability to habituate, it was clear that initially highly anxious BALB/c mice were characterized by rapid habituation i.e. adaptive anxiety. In contrast, 129P3 mice showed a profound lack of habituation which indicated non-adaptive anxiety. This characteristic appeared to be gender-independent and sensitive to pre-exposure to mild stress. The lack of habituation in 129P3 mice was accompanied by a lower c-Fos expression in the prelimbic cortex, a brain area primarily involved in cognition. We hypothesized, that this might reflect a deficit in the integration of cognitive and emotional information, a process needed to adapt over time. Interestingly, the metabotropic glutamate 5 antagonist (mGlu5R) MPEP improved habituation and, in parallel, increased c-Fos expression in the prelimbic area. At the same time c-Fos expression decreased in brain areas known to regulate emotions, such as the amygdala, suggesting that increased glutamatergic neurotransmission might play a role in the behavioural profile of 129P3 mice. We concluded from strain-specific expression of the neuromodulator corticotropin-releasing factor (CRFR1), that CRFR1 mRNA (down) regulation in the prelimbic cortex is associated with habituation and that the non-adaptive phenotype of 129P3 mice is primarily a cognitive dysfunction, a theory which has also been suggested for human anxiety disorders. Thus 129P3 mice not only seem to be a promising translational model for human anxiety disorders. The fact that these mice show a possibly pathological phenotype suggests that their welfare may be compromised under standard housing conditions as applied in our studies. For 129P3 animals, strain-specific demands with respect to habituation processes, such as transfer to a new home cage, can be assumed. Characterizing the emotional phenotype, not only in 129P3 mice but also in other laboratory animals, would lead to a better understanding of potentially confounding factors on laboratory animal welfar

Anxiety in relation to animal environment and welfare

Negative emotions do not compromise welfare, as long as they do not exceed the individual’s adaptive  capabilities. Anxiety, though a negative emotion, is highly conserved during evolution, and essential for  enabling an individual to both escape from dangerous situations and to avoid them in the future, i.e. to adapt  to environmental challenges. However, the interactions between anxiety and environment are highly dynamic  and can result in non-adaptive anxiety responses. Non-adaptive anxiety responses not only compromise the  animal’s welfare, but may be substantially detrimental to experimental results even in non-behavioural  studies by dramatically reducing the reliability of the study results obtained. Detailed knowledge about the  emotional phenotype of experimental animals used is necessary to reach a balance between reliability of  experimental research and the welfare of laboratory animals.

Expression of CRFR1 and Glu5R mRNA in different brain areas following repeated testing in mice that differ in habituation behaviour

Item does not contain fulltextOur recent studies revealed a profound impairment to habituate in 129P3 mice compared to BALB/c mice after repeated exposure to an initially novel environment. This was accompanied by strain-specific c-Fos expression in the prelimbic cortex, a brain area related to emotional and cognitive processing. The metabotropic glutamate receptor 5 (mGlu5R) antagonist MPEP increased c-Fos expression in brain areas related to cognition while it decreased c-Fos expression in brain areas processing emotions in 129P3 animals. We hypothesised that the non-adaptive response of 129P3 mice to a novel environment may be the result of impaired neural processing between the prelimbic cortex and emotion processing brain areas, possibly regulated by glutamatergic neurotransmission. To explore this hypothesis, we compared c-Fos activity in between naive and repeatedly tested animals. Further, we investigated mRNA expression of CRFR1 and mGlu5R in the prelimbic cortex and amygdala, since these transmitter systems are not only involved in the regulation of anxiety, but are indicated to be co-expressed in relevant brain areas. Behavioural results confirmed strain-specific habituation profiles and strain-specific c-Fos expression in brain areas regulating cognitive and emotional processes in BALB/c and 129P3 mice. We found that repeated testing resulted in contrasting behavioural responses in both strains, and this was accompanied by strain-specific effects on c-Fos and receptor-expression. From these results it may be concluded that habituation in BALB/c mice reflects a shift from a primary emotional response to a more cognitively controlled behaviour, and that this shift over time may be impaired in 129P3 animals

Behavioural habituation to novelty and brain area specific immediate early gene expression in female mice of two inbred strains

When anxiety-related behaviour in animals appears to lack adaptive value, it might be defined as pathological. Adaptive behaviour can be assessed for example by changes in behavioural responses over time, i.e. habituation. Thus, non-adaptive anxiety would be reflected by a lack of habituation. Recently, we found that 129P3/J mice are characterised by non-adaptive avoidance behaviour after repeated test exposure. The present study was aimed at investigating the sensitivity of the behavioural profile of these animals to exposure to a chronic mild stress (CMS) paradigm followed by repeated exposure to the modified hole board test. If the behavioural profile of 129P3/J mice mirrors pathological anxiety, their behavioural habituation under repeated test exposure conditions should be affected by CMS treatment. The results confirm the profound lack of habituation with respect to anxiety-related behaviour in both control and CMS treated mice. Additionally, CMS treated animals revealed a lower exploratory behaviour, reduced locomotor activity and increased arousal-related behaviour over time when compared to control individuals, proving an extension of their impaired habituation behaviour. Although no effects of CMS treatment on plasma corticosterone levels were found, higher immediate early gene expression in the bed nucleus of the stria terminalis and the ventrolateral periaqueductal grey in CMS treated mice indicated that 129P3/J mice are susceptible to the negative effects of CMS treatment at both the behavioural and the functional level. These results support the hypothesis that 129P3/J mice might be an interesting model for pathological anxiety

An individual based, multidimensional approach to identify emotional reactivity profiles in inbred mice

Background Despite extensive environmental standardization and the use of genetically and microbiologically defined mice of similar age and sex, individuals of the same mouse inbred strain commonly differ in quantitative traits. This is a major issue as it affects the quality of experimental results. Standard analysis practices summarize numerical data by means and associated measures of dispersion, while individual values are ignored. Perhaps taking individual values into account in statistical analysis may improve the quality of results. New method The present study re-inspected existing data on emotional reactivity profiles in 125 BALB/cJ and 129 mice, which displayed contrasting patterns of habituation and sensitization when repeatedly exposed to a novel environment (modified Hole Board). Behaviors were re-analyzed on an individual level, using a multivariate approach, in order to explore whether this yielded new information regarding subtypes of response, and their expression between and within strains. Results Clustering individual mice across multiple behavioral dimensions identified two response profiles: a habituation and a sensitization cluster. Comparison with existing method(s) These retrospect analyses identified habituation and sensitization profiles that were similar to those observed in the original data but also yielded new information such as a more pronounced sensitization response. Also, it allowed for the identification of individuals that deviated from the predominant response profile within a strain. Conclusions The present approach allows for the behavioral characterization of experimental animals on an individual level and as such provides a valuable contribution to existing approaches that take individual variation into account in statistical analysis