Hanno Würbel

Dissociable effects of isolation rearing and environmental enrichment on exploration, spatial learning and HPA activity in adult rats

Male Lister hooded rats were reared from weaning either singly or in groups of three in either barren or enriched cages (n=9 each) to study effects of isolation rearing and environmental enrichment on open-field activity, object exploration, activity in the Light/Dark box (L/D box), spatial learning and memory in the Morris water maze, and hypothalamic-pituitary-adrenal (HPA) activity in response to restraint stress. Regardless of inanimate background, isolation rearing mainly enhanced activity under several conditions of environmental novelty. By contrast, environmental enrichment, regardless of social background, primarily accelerated habituation to novelty and improved spatial learning and memory. None of the treatments significantly altered basal and response levels of plasma ACTH and corticosterone. Furthermore, rats reared singly in barren cages showed persistent activity in the L/D box, indicating an interaction between isolation-induced hyperactivity and reduced habituation due to barren caging. These results show that isolation rearing and environmental enrichment affect behaviour selectively, while at the same time revealing biologically relevant interactions between social and inanimate stimulation. It is concluded that systematic variation of social and inanimate stimulation can help distinguish between effects that generalise across variation in environmental background and effects that are idiosyncratic to a specific environmental background.

Dissociation in the effects of neonatal maternal separations on maternal care and the offspring's HPA and fear responses in rats

The development of the hypothalamic–pituitary–adrenal (HPA) response to stress is influenced by the early mother–infant relationship. In rats, early handling (brief daily mother–offspring separations) attenuates the adult offsprings HPA and fear responses compared to both nonhandling (no separations) and maternal separation (prolonged daily separations). It has been proposed that variation in the amount of maternal care mediates these effects of neonatal manipulations on the adult offsprings stress and fear responses. Here we tested this hypothesis by assessing maternal care and the adult offsprings HPA and fear responses in Lister hooded rats which were subjected to either early handling (EH) or maternal separation (MS) from postnatal day 1–13, or were left completely undisturbed (nonhandled, NH) throughout this period. Both EH and MS induced a more active nursing style and elevated levels of maternal care compared to NH. Total levels of maternal care were indistinguishable between EH and MS, but diurnal distribution differed. MS dams showed elevated levels of maternal care following the 4‐h separation period, thereby fully compensating for the amount of maternal care provided by EH dams during the time MS dams were separated from their pups. However, while EH resulted in reduced HPA and fear responses in the adult offspring compared to NH, MS and NH offspring did not differ. Our findings therefore demonstrate dissociation in the effects of EH and MS on maternal care and on the stress and fear responses in the offspring. This indicates that maternal care cannot be the sole mediator of these effects.

Developmental plasticity of HPA and fear responses in rats: A critical review of the maternal mediation hypothesis

Developmental plasticity of HPA and fear responses in rats has been proposed to be mediated by environment-dependent variation in active maternal care. Here, we review this maternal mediation hypothesis based on the postnatal manipulation literature and on our own recent research in rats. We show that developmental plasticity of HPA and fear responses in rats cannot be explained by a linear single-factor model based on environment-dependent variation in active maternal care. However, by adding environmental stress as a second factor to the model, we were able to explain the variation in HPA and fear responses induced by postnatal manipulations. In this two-factor model, active maternal care and environmental stress (as induced, e.g., by long maternal separations or maternal food restriction) exert independent, yet opposing, effects on HPA reactivity and fearfulness in the offspring. This accounts well for the finding that completely safe and stable, as well as, highly stressful maternal environments result in high HPA reactivity and fearfulness compared to moderately challenging maternal environments. Furthermore, it suggests that the downregulation of the HPA system in response to stressful maternal environments could reflect adaptive developmental plasticity based on the increasing costs of high stress reactivity with increasingly stressful conditions. By contrast, high levels of environmental stress induced by environmental adversity might constrain such adaptive plasticity, resulting in non-adaptive or even pathological outcomes. Alternatively, however, developmental plasticity of HPA and fear responses in rats might be a function of maternal HPA activation (e.g., levels of circulating maternal glucocorticoid hormones). Thus, implying a U-shaped relationship between maternal HPA activation and HPA reactivity and fearfulness in the offspring, increasing maternal HPA activation with increasing environmental adversity would explain the effects of postnatal manipulations equally well. This raises the possibility that variation in active maternal care is an epiphenomenon, rather than a causal factor in developmental plasticity of HPA and fear responses in rats. Developmental plasticity of HPA and fear responses in rats and other animals has important implications for the design of animal experiments and for the well-being of experimental animals, both of which depend on the exact underlying mechanism(s). Importantly, however, more naturalistic approaches are needed to elucidate the adaptive significance of environment-dependent variation of HPA reactivity and fearfulness in view of discriminating between effects reflecting adaptive plasticity, phenotypic mismatch and pathological outcomes, respectively.

Laboratory animal welfare: Cage enrichment and mouse behaviour

Mice housed in standard cages show impaired brain development, abnormal repetitive behaviours (stereotypies) and an anxious behavioural profile, all of which can be lessened by making the cage environment more stimulating. But concerns have been raised that enriched housing might disrupt standardization and so affect the precision and reproducibility of behavioural-test results (for example, see ref. 4). Here we show that environmental enrichment increases neither individual variability in behavioural tests nor the risk of obtaining conflicting data in replicate studies. Our findings indicate that the housing conditions of laboratory mice can be markedly improved without affecting the standardization of results.

Enrichment-dependent differences in novelty exploration in rats can be explained by habituation

In rats, exploratory activity and emotional reactivity towards novel stimuli reflect independent biological functions that are modulated differently by rearing experiences. Environmental enrichment is known to improve performance in exploratory tasks, while having inconsistent effects on emotionality. This study examined the effect of environmental enrichment on the behaviour of rats in two exploratory tasks. Male rats were reared under one of four conditions, differing in social and non-social complexity. At 9 weeks of age, exploration of a novel open field, and exploration of novel objects in the same open field following 24 h habituation, was assessed. Differences in social and non-social complexity of the rearing environment had inconsistent effects on exploration in the novel open field. In contrast, when rats were faced with novel objects in an otherwise familiar environment, exploration habituated faster with increasing stimulus complexity of the non-social environment. The social environment had no effect on this latter test. These findings indicate that environmental enrichment affects exploratory activity primarily through its effect on habituation to novelty. This effect depends on relative stimulus complexity of the rearing environment, but is independent of social factors. The present results further suggest that aversive tasks can obscure the expression of enrichment-dependent differences in habituation to novelty.

Behavioral phenotyping enhanced – beyond (environmental) standardization

It is basic biology that the phenotype of an animal is the product of a complex and dynamic interplay between nature (genotype) and nurture (environment). It is far less clear, however, how this might translate into experimental design and the interpretation of animal experiments. Animal experiments are a compromise between modelling real world phenomena with maximal validity (complexity) and designing practicable research projects (abstraction). Textbooks on laboratory animal science generally favour abstraction over complexity. Depending on the area of research, however, abstraction can seriously compromise information gain, with respect to the real world phenomena an experiment is designed to model. Behavioral phenotyping of mouse mutants often deals with particularly complex manifestations of life, such as learning, memory or anxiety, that are strongly modulated by environmental factors. A growing body of evidence indicates that current approaches to behavioral phenotyping might often produce results that are idiosyncratic to the study in which they were obtained, because the interactive nature of genotype‐environment relationships underlying behavioral phenotypes was not taken into account. This paper argues that systematic variation of genetic and environmental backgrounds, instead of excessive standardization, is needed to control the robustness of the results and to detect biologically relevant interactions between the mutation and the genetic and environmental background of the animals.

Animal neuropsychology: Validation of the Intra-Dimensional Extra-Dimensional set shifting task for mice

Research in animal neuropsychology is providing an exciting new generation of behavioral tests for mice that promise to overcome many of the limitations of current high-throughput testing, and provide direct animal homologues of clinically important measures in human research. Set shifting tasks are some of the best understood and widely used human neuropsychological tasks, with clinical relevance to traumatic brain injury, schizophrenia, autism, obsessive compulsive disorder, trichotillomania, and many other disorders. Here we report the first successful modification of a human set shifting neuropsychological task, the Intra-Dimensional Extra-Dimensional (IDED) task, for use with mice. We presented mice with a series of compound discrimination and reversal tasks where one stimulus dimension consistently cued reward. Task performance improved with a new set of compound stimuli, as did reversal performance--indicating the formation of a cognitive-attentional set. We then overtrained a subset of the mice, and presented control and overtrained mice with a new compound discrimination where a novel stimulus dimension cued reward. As is the case in human control subjects, control mice persisted in responding to the now-incorrect stimulus dimension, performing poorly on this extra-dimensional shift compared with the previous intra-dimensional shift, thereby validating the task as a measure of set shifting. Furthermore, overtrained mice were impaired on this extra-dimensional shift compared with controls, further validating the task. The advantages and disadvantages of the IDED task compared to high-throughput approaches are discussed.

Double dissociation of social and environmental stimulation on spatial learning and reversal learning in rats

Environmental enrichment induces structural and biochemical changes in the brains of mammals that correlate with improved learning and memory. Research in rats suggests that social compared to inanimate stimulation might affect behavior differently, by acting upon dissociable neural substrates. Here we tested this hypothesis at the behavioral level by examining whether social and inanimate stimulation affect spatial memory formation and non-spatial discrimination reversal learning selectively. Spatial memory formation is known to depend on hippocampal-neocortical pathways, whereas reversal learning depends primarily on prefrontal cortico-striatal pathways. Male Lister hooded rats were housed singly or in groups of three in either small barren or large enriched cages, from weaning onwards. After 10 weeks of differential housing, spatial learning and memory were examined in the Morris water maze, followed by a series of tactile and odour discriminations, including discrimination reversal, in a two-choice discrimination task. Regardless of inanimate stimulation, social deprivation affected neither the acquisition of simple or complex discriminations, nor spatial memory formation, but was associated with impaired reversal learning in the two-choice discrimination task. By contrast, inanimate deprivation, regardless of social stimulation, affected neither acquisition nor reversal of two-choice discriminations, but selectively delayed the acquisition of spatial memory in the Morris water maze. This is the first demonstration of a double dissociation of early social and inanimate stimulation on two distinct behavioural functions that are mediated by dissociable underlying neural pathways. These findings strengthen the view that social and inanimate stimulation act, at least in part, upon dissociable neural substrates.

Systematic variation improves reproducibility of animal experiments

measures to compare between-experiment variation for the standardized and heterogenized design. Whereas strain differences were relatively consistent among heterogenized experiments, they varied considerably between standardized experiments (Fig. 1a–c). In 33 of 36 measures, between-experiment variation was lower in the heterogenized design, indicating better reproducibility. We also analyzed each experiment separately as if conducted independently in different laboratories and assessed the effect of ‘strain’ on each of the 36 measures using a general linear model (GLM). Based on the 2 × 2 factorial nature of the heterogenized design and cage position in the rack, we divided each replicate experiment into four ‘blocks’, each comprising one cage per strain (Supplementary Fig. 1), and included ‘block’ nested within experiment as blocking factor in the GLM (Supplementary Methods). Whereas the effect of ‘strain’ was stable in the four heterogenized experiments, outcomes of the four standardized experiments were highly variable (Supplementary Fig. 2), suggesting that withinSystematic variation improves reproducibility of animal experiments

Attentional set-shifting in mice: modification of a rat paradigm, and evidence for strain-dependent variation.

Increasingly precise molecular genetic tools are available to study in mice the cellular mechanisms underlying complex brain functions, but the behavioural paradigms to assess these functions often lack the required specificity. In this study, an attentional set-shifting paradigm to assess medial frontal cortex functions in rats was modified for use in mice and variation between two relevant mouse strains assessed. Male 129/SvEv and C57BL/6J mice and their F1 intercross (n=8 per genotype) were trained to dig in bowls for a food reward. On four consecutive days, mice performed a series of discriminations to criterion (six consecutive correct choices) between pairs of food bowls that differed along two dimensions (odour, digging medium), including a reversal, an intra-dimensional shift, and an extra-dimensional shift. Mice from the 129 strain performed significantly better than C57 mice in the initial acquisition of a simple discrimination and in the final extra-dimensional shift test, with no difference in the reversal and intra-dimensional shift. Performance of the F1 mice was intermediate or similar to that of the 129 mice. These results indicate a selective difference between these two strains in attentional selection processes that have been shown in humans, monkeys and rats to be mediated by prefrontal cortex.