F. Josef van der Staay

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.

Working and reference memory of pigs in the spatial holeboard discrimination task

The present study investigated whether pigs are able to acquire a complex spatial holeboard discrimination task (4 of 16 holes baited) and whether mixing stress affects performance in this task. All pigs rapidly reduced the number of re-visits to baited holes (working memory) and to unbaited holes (reference memory). Mixing stress did not affect performance.

The appetitively motivated “cognitive” holeboard: A family of complex spatial discrimination tasks for assessing learning and memory

Spatial learning and memory tasks have captured a solid position in neuroscience research. A variety of holeboard-type tasks are suitable for investigating the effects of a broad range of experimental manipulations on spatial learning and memory in a broad range of species, including fish, rodents, cats, pigs, tupaias, and humans. We summarize the concepts and procedures underlying tests of spatial discrimination learning, with special emphasis on holeboard-type tasks and task-specific characteristics. Holeboard-type tasks enable a broad range of mnemonic and cognitive variables to be measured in parallel, including cognitive processes such as habituation processes, spatial working and reference memory, and search strategies, but also non-cognitive variables, such as exploration, anxiety-related behavior, and stereotypies. These tasks are sensitive to a large number of naturally occurring differences (e.g. strain differences and age effects) and to the effects of non-genetic (e.g. specific brain lesions, stress, treatment with cognition impairers or cognition enhancers) and genetic experimental manipulations. In conclusion, holeboard-type tasks provide powerful tools to investigate multiple aspects of spatial orientation behavior in the same experimental setup. Cross-species comparison of holeboard performance shows the potential for translational studies.

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.

The Pig as a Model Animal for Studying Cognition and Neurobehavioral Disorders

In experimental animal research, a short phylogenetic distance, i.e., high resemblance between the model species and the species to be modeled is expected to increase the relevance and generalizability of results obtained in the model species. The (mini)pig shows multiple advantageous characteristics that have led to an increase in the use of this species in studies modeling human medical issues, including neurobehavioral (dys)functions. For example, the cerebral cortex of pigs, unlike that of mice or rats, has cerebral convolutions (gyri and sulci) similar to the human neocortex. We expect that appropriately chosen pig models will yield results of high translational value. However, this claim still needs to be substantiated by research, and the area of pig research is still in its infancy. This chapter provides an overview of the pig as a model species for studying cognitive dysfunctions and neurobehavioral disorders and their treatment, along with a discussion of the pros and cons of various tests, as an aid to researchers considering the use of pigs as model animal species in biomedical research.

Effects of chronic stress: A Comparison between tethered and loose sows

The present study aimed to investigate whether long-lasting, recurrent tethering of sows leads to enduring effects on measures that may be indicative of chronic stress. Sows that had experienced tethering for about 1.5 or 4.5years and age-matched sows kept in a social housing system (loose sows) were compared. Immediately after slaughter, blood samples were taken to measure plasma cortisol levels, and the brain, spleen, and adrenals were dissected and weighed. Gene expression in the frontal cortex and hippocampus was analyzed. Plasma cortisol levels were higher in the tethered sows than in the loose sows. The older, but not the younger, tethered sows had heavier adrenal glands than their loose counterparts. The weight of the spleen was not affected by the housing conditions, but the pituitary gland was lighter in tethered sows than in loose sows. Microarray analyses revealed an increased expression of beta-globin mRNA in the hippocampus and to a lesser extent in the frontal cortex of the older tethered sows, compared with the older loose sows. Taken together, the findings indicate that chronically stressed pigs develop depression-like symptoms. However, it can be questioned whether the pig subjected to repeated, long-term stress can be regarded an animal model of major depression.

Judgement bias in pigs is independent of performance in a spatial holeboard task and conditional discrimination learning

Biases in judgement of ambiguous stimuli, as measured in a judgement bias task, have been proposed as a measure of the valence of affective states in animals. We recently suggested a list of criteria for behavioural tests of emotion, one of them stating that responses on the task used to assess emotionality should not be confounded by, among others, differences in learning capacity, i.e. must not simply reflect the cognitive capacity of an animal. We performed three independent studies in which pigs acquired a spatial holeboard task, a free choice maze which simultaneously assesses working memory and reference memory. Next, pigs learned a conditional discrimination between auditory stimuli predicting a large or small reward, a prerequisite for assessment of judgement bias. Once pigs had acquired the conditional discrimination task, optimistic responses to previously unheard ambiguous stimuli were measured in the judgement bias task as choices indicating expectation of the large reward. We found that optimism in the judgement bias task was independent of all three measures of learning and memory indicating that the performance is not dependent on the pig’s cognitive abilities. These results support the use of biases in judgement as proxy indicators of emotional valence in animals.

Overnight Social Isolation in Pigs Decreases Salivary Cortisol but Does Not Impair Spatial Learning and Memory or Performance in a Decision-Making Task

Pigs in modern farming practice may be exposed to a number of stressors, including social stressors such as mixing or isolation. This may potentially affect both cognitive abilities and stress physiology of the animals. We tested the hypothesis that overnight social isolation in pigs impairs performance in a cognitive holeboard (HB) task (Experiment 1) and the Pig Gambling Task (PGT) (Experiment 2), a decision-making task inspired by the Iowa Gambling Task. In addition, we tested the effect of overnight social isolation on salivary cortisol levels. A within-subjects approach was used in which performance in the two behavioral tasks and cortisol levels were first determined during normal social housing, followed by performance and cortisol levels after experiencing stress induced by overnight social isolation. A total of 19 female pigs with a birth weight closest to their respective litter average was selected from 10 different litters and placed in two pens after weaning. Following habituation, pigs were trained in the HB task, starting at 10 weeks of age. Then, the pigs were isolated overnight, five individuals per night, at 15, 16, and 17 weeks of age. Between these three isolations, social housing and training in the HB continued. Starting 6 weeks after the end of the HB experiment, at approximately 23 weeks of age, the pigs were trained in the PGT. The effects of overnight social isolation on performance in this task were assessed once, when the pigs were 25 weeks old. Salivary cortisol was measured from samples collected 15 min after the start of isolation and at the end of the isolation period and compared to baseline values collected before the start of social isolation. Our results did not confirm the hypothesis that isolation impaired HB performance and decision-making in the PGT. Unexpectedly, overnight social isolation decreased cortisol levels below baseline values, an effect that was not associated with changes in performance of the behavioral tasks. We hypothesized that the housing and testing conditions may have prepared the animals to cope efficiently with stress.