Saskia S. Arndt

Individual housing of mice – Impact on behaviour and stress responses

The replicability of results derived from studies in rodents might be influenced by stress caused by inappropriate housing conditions. Here we compared the experimental behaviour and stress response (circulating corticosterone level and adrenal tyrosine hydroxylase activity) of individually-housed male and female inbred mice with that of animals housed in social groups. All mice were behaviourally tested in the modified hole board test (mHB). Male C57BL/6, BALB/c and A mice housed in groups of 3 were compared with individually-housed mice. In a subsequent experiment female C57BL/6 and BALB/c mice were housed under similar conditions. To exclude the possible effects of within-cage order of testing, only one individual per group was behaviourally tested. Neither male nor female mice housed individually showed stronger signs of stress than their socially-housed counterparts. However, we observed a within-cage order effect on the hormonal stress response (corticosterone) in socially-housed female C57BL/6 mice. No effects of individual housing on behaviour in the mHB were found.

Mild neonatal hypoxia-ischemia induces long-term motor- and cognitive impairments in mice.

To understand and potentially treat the lifelong cognitive and motor deficits in humans resulting from perinatal mild cerebral hypoxic-ischemic (HI) events, valid animal models are of high importance. Nowadays the murine model of neonatal cerebral HI-injury (unilateral carotid artery occlusion followed by hypoxia) is applied more frequently. In the present study we investigated motor, behavioral and cognitive functioning in mice with mild cerebral HI-injury (45 min of hypoxia; HI-45) in comparison to mice exposed to severe HI (HI-75) and sham-control mice. Lateralizing motor disturbances as measured using the cylinder rearing test developed in both HI-45 and HI-75 mice and was significantly more severe in HI-75 animals. To assess behavior and cognitive functions, we used the modified hole board (mHB) test in two stages. First, the ability of the animals to find the three food rewards in cued holes over time was determined. The results revealed an overall learning impairment in HI-75 mice, while HI-45 mice were not different from sham controls. In the second stage, a reversal test was performed with rewarded cylinders being non-cued and non-rewarded cylinders being cued. This reversal-task revealed impairments in cognitive flexibility in HI-45 mice as compared to sham-control animals. Our data indicate that both the cylinder rearing task and the two stages of the mHB are suitable behavioral approaches to differentiate consequences of neonatal mild and severe brain damage on executive functioning.

Identifying emotional adaptation: behavioural habituation to novelty and immediate early gene expression in two inbred mouse strains

Normal anxiety is an adaptive emotional response. However, when anxiety appears to lack adaptive value, it might be defined as pathological. Adaptation in animals can be assessed for example by changes in behavioural responses over time, i.e. habituation. We hypothesize that non‐adaptive anxiety might be reflected by impaired habituation. To test our hypothesis, we repeatedly exposed male mice from two inbred strains to a novel environment, the modified hole board. BALB/cJ mice were found to be initially highly anxious, but subsequently habituated to the test environment. In contrast, 129P3/J mice initially showed less anxiety‐related behaviour compared with the BALB/cJ mice but no habituation in anxiety‐related behaviour was observed. Notably, anxiety‐related behaviour even increased during the experimental period. Complementary, 129P3/J mice did not show habituation in other parameters such as locomotor and exploratory activity, whereas significant changes appeared in these behaviours in BALB/c mice. Finally, the expression of the immediate early gene c‐fos differed between the two strains in distinct brain areas, known to regulate the integration of emotional and cognitive processes. These results suggest that 129P3/J mice might be a promising (neuro)‐behavioural animal model for non‐adaptive, i.e. pathological anxiety.

The standardization-generalization dilemma: a way out

Recently, a debate has emerged on the use and necessity of standardization in experimental testing using animal subjects. The difficulties encountered when trying to reconcile standardization and generalization largely underlie this debate. The more specific the testing procedures are, the less one can generalize to more naturalistic situations, including to human clinical populations. If the goal of a study is to generalize to a larger population, there may be a higher risk attached to false‐positive than false‐negative results; thus the balance sways toward generalization. Heterogenization of housing conditions and of genetic makeup of experimental animals has been suggested as a possible method to increase the generalizability of results. It is important to remain cognizant, however, of situations in which false negatives can be counterproductive or even dangerous, such as when the goal is to elucidate a physiological mechanism, when expected effect sizes are small, in toxicological studies and in drug safety testing. In such cases, experiments based on standardization may provide more useful information. We pose that it is essential that the goal of the specific experiment conducted is clearly defined and that the decision to balance between standardization and generalization must be made based on the specific needs to meet the intended goal. In this light, we discuss a multi‐tiered approach to animal experimentation, in which standardization and generalizability are each given precedence during different phases of a project, depending upon the goal of the experiment.

A test to identify judgement bias in mice

Emotional states are known to affect cognitive processes. For example highly anxious individuals interpret ambiguous stimuli more negatively than low anxious people, an effect called negative judgement bias. Recently, the measurement of judgement bias has been used to try and indicate emotional states in animals. In the present experiment a potential test for judgement bias in mice was examined. Mice were trained with two distinct odour cues (vanilla or apple) predicting either a palatable or an unpalatable almond piece. Subsequently their reaction to mixtures of both odours, the ambiguous stimuli, was investigated. Mice of the BALB/cJ and 129P3/J inbred mouse strains (high initial anxiety and low initial anxiety phenotypes respectively) were tested. While BALB/cJ mice showed odour association learning and showed intermediate reactions to the ambiguous cues, 129P3/J mice did not discriminate between the cues. Additionally BALB/cJ mice that were tested under more aversive white light conditions revealed a higher latency to approach the almond piece than mice tested under less aversive red light conditions. The ambiguous stimulus however was interpreted as negative under both test conditions. Brain c-Fos expression levels (a marker for neuronal activity) differed between the BALB/c/J and 129P3/J in the lateral amygdala and the prelimbic cortex, indicating differences in ambiguous information processing between the strains. The behavioural results suggest that the present judgement bias test might be used to assess emotional states in at least BALB/c mice, however further research on both behaviour and on the involved brain mechanisms is necessary to confirm this idea.

Nociception and Conditioned Fear in Rats: Strains Matter

When using rats in pain research, strain-related differences in outcomes of tests for pain and nociception are acknowledged. However, very little is known about the specific characteristics of these strain differences. In this study four phylogenetically distant inbred rat strains, i.e. Wistar Kyoto (WKY), Fawn Hooded (FH), Brown Norway (BN) and Lewis (LE), were investigated in different tests related to pain and nociception. During Pavlovian fear conditioning, the LE and WKY showed a significantly longer duration of freezing behaviour than the FH and BN. Additionally, differences in c-Fos expression in subregions of the prefrontal cortex and amygdala between rat strains during retrieval and expression of conditioned fear were found. For example, the BN did not show recruitment of the basolateral amygdala, whereas the WKY, FH and LE did. During the hot plate test, the WKY and LE showed a lower thermal threshold compared to the BN and FH. In a follow-up experiment, the two most contrasting strains regarding behaviour during the hot plate test and Pavlovian fear conditioning (i.e. FH and WKY) were selected and the hot plate test, Von Frey test and somatosensory-evoked potential (SEP) were investigated. During the Von Frey test, the WKY showed a lower mechanical threshold compared to the FH. When measuring the SEP, the FH appeared to be less reactive to increasing stimulus intensities when considering both peak amplitudes and latencies. Altogether, the combined results indicate various differences between rat strains in Pavlovian fear conditioning, nociception related behaviours and nociceptive processing. These findings demonstrate the necessity of using multiple rat strains when using tests including noxious stimuli and suggest that the choice of rat strains should be considered. When selecting a strain for a particular study it should be considered how this strain behaves during the tests used in that study.

Susceptibility of a potential animal model for pathological anxiety to chronic mild stress

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.

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

In mice, emotional adaptation might be assessed by changes in behavioural responses towards novelty over time (i.e. habituation), with non-adaptive anxiety being expressed by a lack of habituation. Recently we found that male 129P3/J mice showed such a profound lack of habituation in comparison to male BALB/c mice. From these results we concluded that male 129P3/J mice might model non-adaptive, i.e. pathological anxiety. As a first step in the process of assessing the generalizability of our results, we investigated whether these results were robust across gender. Therefore we replicated our previous study in female individuals. Results from the present study reveal behavioural habituation towards novelty, i.e. an adaptive phenotype in female BALB/c mice. In contrast, females of the 129P3/J strain were characterised by a lack of habituation, similar as their male counterparts. Compared to female BALB/c, female 129P3/J mice showed lower neural activity in brain areas known to regulate the integration of emotional and cognitive processes. Extending the results found in males, female 129P3/J mice revealed increased post-testing plasma corticosterone levels and higher neural activity in brain areas related to emotional processing than females of the BALB/c strain. Taken together our results demonstrate that both genders of the 129P3/J mouse strain are characterised by a non-adaptive anxiety phenotype, strengthening the hypothesis that the 129P3/J strain may be a promising (neuro)-behavioural model for pathological anxiety.

The impact of transportation on physiological and behavioral parameters in Wistar rats: implications for acclimatization periods.

Transportation of laboratory rodents unavoidably causes stress. Nevertheless, very little is known about the effects of transportation and how long it takes for the animal to recuperate. In the present study, we investigated physiological and behavioral parameters before and after transportation in both transported and nontransported animals. We took blood samples to analyze plasma corticosterone and creatine kinase, and performed physiological measurements by means of telemetry, measuring heart rate, blood pressure, and activity. Behavior was measured by means of home cage observations. This study revealed that plasma corticosterone levels increased at least up to 16 days after transportation, blood pressure and heart rate showed a lasting decrease after transportation, grooming increased, and social interactions and locomotor activity decreased after transportation. With these data we demonstrate that there is a long-lasting effect of transportation on physiological and behavioral parameters. Our results show that the stressful impact of transportation embraces all parts of the procedure, including for example the packing of the animals. Researchers must be aware of this impact and provide a sufficient acclimatization period to allow for the (re-)stabilization of parameters. Insufficient acclimatization periods endanger not only the reliability of research results but also the welfare of the animal used.

Impact of anxiety profiles on cognitive performance in BALB/c and 129P2 mice

It has been suggested over the decades that dysfunctional anxiety may be caused by distinct alterations in cognitive processing. To learn more about the relation between anxiety and cognitive functioning, two mouse strains that display either adaptive (BALB/c) or nonadaptive (129P2) anxiety, as reflected by their ability to habituate when repeatedly exposed to a novel environment, were tested for their cognitive performance in the modified hole board (mHB) task. In general, both strains showed successful acquisition of the task. The initially more anxious BALB/c mice revealed rapid habituation to the test setup, followed by decreased long-term and short-term memory errors across the experimental period and fast relearning after reversal of the task. By contrast, the nonadaptive 129P2 mice made more short-term memory errors and performed worse than the BALB/c animals after reversal of the test. The results confirm the proposed interaction of anxiety and cognition: In BALB/c mice, adaptive characteristics of anxiety were paralleled by more successful cognitive performance, while in 129P2 mice nonadaptive anxiety-related behaviour was accompanied by a higher level of short-term memory errors and less cognitive flexibility. Moreover, these results support our hypothesis that the nonadaptive anxiety phenotype in 129P2 mice may be the result of impaired cognitive control of emotional processes, resulting in impaired behavioural flexibility, for example in response to novelty.