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Although enrichment is an essential part of modern zoo animal husbandry, research on effective enrichment for small mammals is scarce, albeit they are regularly kept in zoos. To develop solutions that are inexpensive, readily applicable, and adaptable for a variety of species, we investigate whether three types of simple enrichment devices (SEDs) – object, olfactory, and food-motivated – elicit positive interactions in various zoo-housed small mammal species. In this preliminary exploration study, we evaluate if amount of enrichment interactions, display of behavioral stress reactions, and display of stereotypic behaviors vary, if different types of SEDs are provided. Therefore, we observed 39 individuals of ten species, most of which are underrepresented in enrichment literature: Eastern quoll (Dasyurus viverrinus), round-eared elephant shrew (Macroscelides proboscideus), aardvark (Orycteropus afer), southern tamandua (Tamandua tetradactyla), northern Luzon giant cloud rat (Phloeomys pallidus), green acouchi (Myoprocta pratti), South African springhare (Pedetes capensis), aye-aye (Daubentonia madagascariensis), golden lion tamarin (Leontopithecus rosalia), and Javan mouse-deer (Tragulus javanicus) housed in Frankfurt Zoo, Germany. All focal individuals interacted with at least two types of SEDs and around 80% interacted with all types of SEDs, resulting in a considerable amount of time spent with the enrichment. There was a preference for food-motivated enrichment. Neither behavioral stress reactions nor stereotypic behaviors differed if different types of SEDs were present. Model selection showed that GLMs explaining enrichment interactions improved if the variable ‘individual’ instead of ‘species’ was included, suggesting that reactions towards unspecific SEDs might be more individual-driven than species-specific. Because individuals of all species chose to interact with the SEDs and we did not find behavioral indicators for stress elicited by the SEDs, we advocate for the regular provision of SEDs for small mammals in human care.

Providing structural enrichment is a widespread refinement method for laboratory rodents and other animals in captivity. So far, animal welfare research has mostly focused on the effect of increased complexity either by accumulating or combining different enrichment items. However, increasing complexity is not the only possibility to refine housing conditions. Another refinement option is to increase novelty by regularly exchanging known enrichment items with new ones. In the present study, we used pair-housed non-breeding female C57BL/6J and DBA/2N mice to investigate the effect of novelty when applying structural enrichment. We used a double cage system, in which one cage served as home cage and the other as extra cage. While the home cage was furnished in the same way for all mice, in the extra cage we either provided only space with no additional enrichment items (space), a fixed set of enrichment items (complexity), or a changing set of enrichment items (novelty). Over 5 weeks, we assessed spontaneous behaviors, body weight, and extra cage usage as indicators of welfare and preference. Our main results showed that mice with access to structurally enriched extra cages (complexity and novelty) spent more time in their extra cages and complexity mice had lower latencies to enter their extra cages than mice with access to the extra cages without any structural enrichment (space). This indicates that the mice preferred the structurally enriched extra cages over the structurally non-enriched space cages. We found only one statistically significant difference between the novelty and complexity condition: during week 3, novelty mice spent more time in their extra cages than complexity mice. Although we did not detect any other significant differences between the novelty and complexity condition in the present study, more research is required to further explore the potential benefits of novelty beyond complexity.

Touchscreen-based procedures are increasingly used in experimental animal research. They not only represent a promising approach for translational research, but have also been highlighted as a powerful tool to reduce potential experimenter effects in animal studies. However, to prepare the animals for a touchscreen-based test, an often time-consuming training phase is required that has itself been shown to cause increased adrenocortical activity and anxiety-like behavior in mice. While these findings point at a potentially negative effect of touchscreen training at first glance, results have also been discussed in light of an enriching effect of touchscreen training. The aim of the present study was therefore to shed more light on recently reported touchscreen training effects, with a particular focus on the termination of the training routine. Specifically, we investigated whether the termination of regular touchscreen training could constitute a loss of enrichment for mice. Thus, we assessed fecal corticosterone metabolites (FCMs), exploratory-, anxiety-like and home cage behavior in touchscreen-trained mice in comparison to food restricted and ad libitum fed mice, as a restricted diet is an integral part of the training process. Furthermore, we compared these parameters between mice that were continuously trained and mice whose training was terminated 2 weeks earlier. Our results confirm previous findings showing that a mild food restriction increases the animals' exploratory behavior and shifts their activity rhythm. Moreover, touchscreen training was found to increase FCM levels and anxiety-like behavior of the mice. However, no effect of the termination of touchscreen training could be detected, a finding which contradicts the enrichment loss hypothesis. Therefore, we discuss two alternative explanations for the findings. Yet, the current state of knowledge is not sufficient to draw final conclusions at this stage. In compliance with the refinement endeavors for laboratory animals, further research should assess the severity of touchscreen procedures to ensure a responsible and well-founded use of animals for experimental purposes.

Abstract Background Glucocorticoids (e.g. cortisol) are associated with variation in social behavior, and previous studies have linked baseline as well as challenge-induced glucocorticoid concentrations to dominance status. It is known that cortisol response to an acute challenge is repeatable and correlates to social behavior in males of many mammal species. However, it is unclear whether these patterns are also consistent for females. The aim of this study was to investigate whether baseline and response cortisol concentrations are repeatable in female guinea pigs (Cavia aperea f. porcellus) and whether dominance rank is stable and correlated to baseline cortisol concentration and/or cortisol responsiveness. Results Our results show that cortisol responsiveness (after 1 h: R = 0.635, 95% CI = 0.229, 0.927; after 2 h: R = 0.764, 95% CI = 0.433, 0.951) and dominance rank (R = 0.709, 95% CI = 0.316, 0.935) of females were significantly repeatable after six weeks but not correlated. Baseline cortisol was not repeatable (R = 0, 95% CI = 0, 0.690) and also did not correlate to dominance rank. Furthermore, the difference in repeatability estimates of baseline and response values was due to high within-individual variance of baseline cortisol concentration; the amount of between-individual variance was similar for baseline cortisol and the two measures of cortisol responsiveness. Conclusions Females occupying different dominance ranks did not have long-term differences in cortisol concentrations, and cortisol responsiveness does not seem to be significantly involved in the maintenance of dominance rank. Overall, this study reveals the remarkable stability of cortisol responsiveness and dominance rank in a female rodent, and it remains an open question whether the magnitude of cortisol responsiveness is adaptive in social contexts for females.

An uncontrolled reproduction of animals in human hands should be avoided. To meet this goal, animals are widely castrated, i.e., the gonads are completely removed. Since the gonads are the most important source of sex hormones, this is a serious intervention in the entire endocrine system of an organism. Sterilization is a much less invasive procedure. Thus, it could have advantages over castration. Therefore, the overall aim of this study was to analyze the effect of castration vs. sterilization on the release of glucocorticoids, i.e., an important indicator for welfare. Taking domestic guinea pigs as a model system, we studied baseline and response cortisol values (cortisol is the main glucocorticoid in guinea pigs) in castrated, sterilized, sham-operated and intact males and baseline values in their cohoused females. Whereas baseline values of males did not differ between the groups, castrated males showed significantly higher cortisol response levels than intact, sham-operated and sterilized males. Females housed with castrated, sterilized, sham-operated or intact males did not differ in their cortisol concentrations, neither shortly after being placed with the respective male or after being co-housed for several weeks. Overall, the results support the hypothesis that castrated males exhibited a higher cortisol responsiveness during acute challenge which could point to a generalized impaired welfare of castrated males in comparison to intact, sham-operated and sterilized males. Our results provide first evidence for a potential negative impact of castration on the animals' welfare, while at the same time pointing toward sterilization representing a less invasive, promising alternative. Therefore, the results may stimulate future research on this topic to further detect potential welfare-related side effects of castration.

In humans, non-right-handedness is associated with a higher incidence of psychiatric disorders. Since serotonin seems to be involved in both, the development of psychiatric disorders and lateralization, the present study focuses on the effect of the serotonin transporter (5-HTT) gene on behavioral lateralization. For this, we used the 5-HTT knockout mouse model, a well-established animal model for the study of human depression and anxiety disorders. For female mice from all three 5-HTT genotypes (wild type, heterozygous, and homozygous knockout), we repeatedly observed the direction and strength of lateralization of the following four behaviors: grid climbing (GC), food-reaching in an artificial test situation (FRT), self-grooming (SG), and barrier crossing (BC), with the FRT being the standard test for assessing behavioral lateralization in mice. We found no association between behavioral lateralization and 5-HTT genotype. However, in accordance with previous findings, the strength and temporal consistency of lateralization differed between the four behaviors observed. In conclusion, since the 5-HTT genotype did not affect behavioral lateralization in mice, more research on other factors connected with behavioral lateralization and the development of symptoms of psychiatric disorders, such as environmental influences, is needed.

[This corrects the article DOI: 10.1371/journal.pbio.3001564.].

The phenotype of an organism results from its genotype and the influence of the environment throughout development. Even when using animals of the same genotype, independent studies may test animals of different phenotypes, resulting in poor replicability due to genotype-by-environment interactions. Thus, genetically defined strains of mice may respond differently to experimental treatments depending on their rearing environment. However, the extent of such phenotypic plasticity and its implications for the replicability of research findings have remained unknown. Here, we examined the extent to which common environmental differences between animal facilities modulate the phenotype of genetically homogeneous (inbred) mice. We conducted a comprehensive multicentre study, whereby inbred C57BL/6J mice from a single breeding cohort were allocated to and reared in 5 different animal facilities throughout early life and adolescence, before being transported to a single test laboratory. We found persistent effects of the rearing facility on the composition and heterogeneity of the gut microbial community. These effects were paralleled by persistent differences in body weight and in the behavioural phenotype of the mice. Furthermore, we show that environmental variation among animal facilities is strong enough to influence epigenetic patterns in neurons at the level of chromatin organisation. We detected changes in chromatin organisation in the regulatory regions of genes involved in nucleosome assembly, neuronal differentiation, synaptic plasticity, and regulation of behaviour. Our findings demonstrate that common environmental differences between animal facilities may produce facility-specific phenotypes, from the molecular to the behavioural level. Furthermore, they highlight an important limitation of inferences from single-laboratory studies and thus argue that study designs should take environmental background into account to increase the robustness and replicability of findings.

Background: The amygdala is crucial for emotional cognitive processing. Affective or emotional states can bias cognitive processes, including attention, memory, and decision-making. This can result in optimistic or pessimistic behaviors that are partially driven by the activation of the amygdala. The resulting emotional cognitive bias is a common feature of anxiety and mood disorders, both of which are interactively influenced by genetic and environmental factors. It is also known that emotional cognitive biases can be influenced by environmental factors. However, little is known about the effects of genetics and/or gene-environment interactions on emotional cognitive biases. We investigated the effects of the genetic background and environmental enrichment on the transcriptional profiles of the mouse amygdala following a well-established cognitive bias test.Methods: Twenty-four female C57BL/6J and B6D2F1N mice were housed either in standard (control) conditions or in an enriched environment. After appropriate training, the cognitive bias test was performed on 19 mice that satisfactorily completed the training scheme to assess their responses to ambiguous cues. This allowed us to calculate an “optimism score” for each mouse. Subsequently, we dissected the anterior and posterior portions of the amygdala to perform RNA-sequencing for differential expression and other statistical analyses.Results: In general, we found only minor changes in the amygdala’s transcriptome associated with the levels of optimism in our mice. In contrast, we observed wide molecular effects of the genetic background in both housing environments. The C57BL/6J animals showed more transcriptional changes in response to enriched environments than the B6D2F1N mice. We also generally found more dysregulated genes in the posterior than in the anterior portion of the amygdala. Gene set overrepresentation analyses consistently implicated cellular metabolic responses and immune processes in the differences observed between mouse strains, while processes favoring neurogenesis and neurotransmission were implicated in the responses to environmental enrichment. In a correlation analysis, lipid metabolism in the anterior amygdala was suggested to influence the levels of optimism.Conclusions: Our observations underscore the importance of selecting appropriate animal models when performing molecular studies of affective conditions or emotional states, and suggest an important role of immune and stress responses in the genetic component of emotion regulation.

BackgroundDuring early phases of life, such as prenatal or early postnatal development and adolescence, an organism's phenotype can be shaped by the environmental conditions it experiences. According to the Match-Mismatch hypothesis (MMH), changes to this environment during later life stages can result in a mismatch between the individual's adaptations and the prevailing environmental conditions. Thus, negative consequences in welfare and health can occur. We aimed to test the MMH in the context of food availability, assuming adolescence as a sensitive period of adaptation.MethodsWe have previously reported a study of the physiological and behavioral effects of match and mismatch conditions of high (ad libitum) and low (90% of ad libitum intake) food availability from adolescence to early adulthood in female C57BL/6J mice (n = 62). Here, we performed RNA-sequencing of the livers of a subset of these animals (n = 16) to test the effects of match and mismatch feeding conditions on the liver transcriptome.ResultsIn general, we found no effect of the match-mismatch situations. Contrarily, the amount of food available during early adulthood (low vs. high) drove the differences we observed in final body weight and gene expression in the liver, regardless of the amount of food available to the animals during adolescence. Many of the differentially expressed genes and the corresponding biological processes found to be overrepresented overlapped, implicating common changes in various domains. These included metabolism, homeostasis, cellular responses to diverse stimuli, transport of bile acids and other molecules, cell differentiation, major urinary proteins, and immunity and inflammation.ConclusionsOur previous and present observations found no support for the MMH in the context of low vs high food availability from adolescence to early adulthood in female C57BL/6J mice. However, even small differences of approximately 10% in food availability during early adulthood resulted in physiological and molecular changes with potential beneficial implications for metabolic diseases.

The credibility of scientific research has been seriously questioned by the widely claimed "reproducibility crisis". In light of this crisis, there is a growing awareness that the rigorous standardisation of experimental conditions may contribute to poor reproducibility of animal studies. Instead, systematic heterogenisation has been proposed as a tool to enhance reproducibility, but a real-life test across multiple independent laboratories is still pending. The aim of this study was therefore to test whether heterogenisation of experimental conditions by using multiple experimenters improves the reproducibility of research findings compared to standardised conditions with only one experimenter. To this end, we replicated the same animal experiment in 3 independent laboratories, each employing both a heterogenised and a standardised design. Whereas in the standardised design, all animals were tested by a single experimenter; in the heterogenised design, 3 different experimenters were involved in testing the animals. In contrast to our expectation, the inclusion of multiple experimenters in the heterogenised design did not improve the reproducibility of the results across the 3 laboratories. Interestingly, however, a variance component analysis indicated that the variation introduced by the different experimenters was not as high as the variation introduced by the laboratories, probably explaining why this heterogenisation strategy did not bring the anticipated success. Even more interestingly, for the majority of outcome measures, the remaining residual variation was identified as an important source of variance accounting for 41% (CI95 [34%, 49%]) to 72% (CI95 [58%, 88%]) of the observed total variance. Despite some uncertainty surrounding the estimated numbers, these findings argue for systematically including biological variation rather than eliminating it in animal studies and call for future research on effective improvement strategies.

The accepted norm in most laboratories around the globe is feeding laboratory mice an ad libitum diet, although several health impairments are well-established. In contrast, reducing the animals' body weight by feeding them less food once per day (referred to as 24 h schedule) has been shown to enhance life span and reduce disease susceptibility. Against this background, this study aimed at systematically investigating the effects of different feeding routines. Therefore, three feeding routines were compared to the standard ad libitum feeding and effects on body weight development and welfare were investigated in male C57BL/6J mice. In particular, a 24 h schedule group, an AUTO group, characterized by an automated supply of small pieces of food all over the day, and a 4 h removal group, characterized by daily removal of food for 4 h, were studied. While the removal of food for 4 h per day did not lead to a reduction of body weight, and hence is unlikely to prevent negative effects of overfeeding, both the 24 h schedule group and the AUTO group led to the aspired body weight reduction. In the AUTO group, however, higher levels of corticosterone metabolites and stereotypies were observed, implying a rather negative impact on welfare. By contrast, no distinct negative effects of a 24 h schedule were found. Studies like this underline the general need for evidence-based severity assessments of any procedure involving living animals.

The start of actual breeding in male social mammals can occur long after individuals attain sexual maturity. Mainly prevented from reproduction by older and dominant males, young males often queue until strong enough to compete for favorable social positions and, in this way, to obtain access to females. However, to what extent maturing males also apply tactics to reproduce before this time is largely unknown. Therefore, the aim of the present study was to elucidate male socio-sexual development from onset of sexual maturity through first mating success until the achievement of a stable social position in a complex social environment. For this purpose, guinea pigs were used as a model system and reproductive success of males living in large mixed-sex colonies was assessed during their first year of life. As a reference, males in a mixed-sex pair situation were examined. Pair-housed males reproduced for the first time around the onset of sexual maturity whereas colony-housed males did so much later in life and with a considerably higher variance. In colonies, reproductive success was significantly affected by dominance status. Dominance itself was age-dependent, with older males having significantly higher dominance ranks than younger males. Surprisingly, both younger and older colony-housed males attained substantial reproductive success of comparable amounts. Thus, younger males reproduced irrespective of queuing and already before reaching a high social status. This mating success of maturing males was most likely achieved via several reproductive tactics which were flexibly applied with the onset of sexual maturity. The period of socio-sexual development before a stable social position is established may, therefore, be a time during which male mammals use flexible behavioral tactics to achieve reproductive success more frequently than commonly is presumed. In addition, the findings strongly indicate that high behavioral plasticity exists well beyond sexual maturity.

In humans, the short allele of a common polymorphism in the serotonin transporter (5-HTT) gene is associated with a higher risk to develop depression and anxiety disorders. Furthermore, individuals carrying this allele are characterized by negative judgment biases, as they tend to interpret ambiguous information in a more pessimistic way. 5-HTT knockout mice, lacking the 5-HTT gene either homo- or heterozygously, provide a widely used model organism for the study of symptoms related to human anxiety disorders. In the present study, we aimed to prove the anxiety-like phenotype of the 5-HTT mouse model, and to investigate whether 5-HTT genotype also causes differences in judgment bias. While our results confirm that homozygous 5-HTT knockout mice display highest levels of anxiety-like behavior, it was decreased in heterozygous mice. Against our expectations, we did not detect differences in the animals’ judgment bias. These results indicate that at least in mice the association between 5-HTT genotype and judgment bias is not straightforward and that other factors, including multiple genes as well as environmental influences, are implicated in the modulation of judgment biases. More research is needed to gain further insights into their function as potential endophenotypes for psychopathology.

Social experiences can have profound effects on an individual’s level of anxiety. While various studies have addressed consequences of experiences of a specific type, e.g., social defeat, a recent study in mice investigated the impact of combinations of adverse and beneficial social experiences. Quite surprisingly, mice exposed to benefits during early life phases followed by escapable adversity in adulthood displayed lowest levels of anxiety, even compared to individuals having experienced throughout beneficial conditions. The present study aimed to elucidate whether this phenomenon is restricted to these specific life phases or whether it also exists when all these experiences are made in full adulthood. For this purpose, we compared anxiety-like behavior and stress response of adult male mice exposed to escapable social defeat following beneficial social experiences to that of mice exposed to either throughout adverse or throughout beneficial conditions. More precisely, we performed three established behavioral paradigms measuring anxiety-like behavior and assessed corticosterone metabolites non-invasively via feces sampling. Interestingly, we found no effects of social experience on anxiety-like behavior. In contrast to that, the animals’ stress hormone levels were profoundly affected by current social conditions: escapable social defeat (adverse condition) led to an increase in corticosterone metabolite concentrations, whereas living with a female (beneficial condition) led to a decrease. Thus, on the one hand this study suggests the importance of the timing of social experience for affecting an individual’s level of anxiety. On the other hand, it demonstrates that anxiety and stress hormone levels can be affected separately by social experience during adulthood.

‘Animal personalities’ have been shown to exist in many species. Yet, fluctuations in the stability of these inter-individual behavioural differences are not well understood. Against this background, we wondered whether behavioural consistency over time is affected by the reproductive cycle. Female mice were tested twice at an interval of eight weeks in four paradigms assessing social interest as well as anxiety-like behaviour and exploratory locomotion. Twenty-two individuals were tested repeatedly near ovulation, whereas another twenty-two were tested repeatedly in the non-receptive phase. While we found no major behavioural effects at the group level, the reproductive state indeed had profound effects on behavioural stability over time: social interest as well as anxiety-like behaviour proved to be significantly less predictable near ovulation. It is generally believed that phenotypic plasticity is limited due to the costs it brings about. In this context, our data indicate that females accept higher costs in phases directly related to fitness maximization.

Behavioral profiles are strongly shaped by an individual’s whole life experience. The accumulation of negative experiences over lifetime is thought to promote anxiety-like behavior in adulthood (‘allostatic load hypothesis’). In contrast, the ‘mismatch hypothesis’ of psychiatric disease suggests that high levels of anxiety-like behavior are the result of a discrepancy between early and late environment. The aim of the present study was to investigate how different life histories shape the expression of anxiety-like behavior and modulate fear memory. In addition, we aimed to clarify which of the two hypotheses can better explain the modulation of anxiety and fear. For this purpose, male mice grew up under either adverse or beneficial conditions during early phase of life. In adulthood they were further subdivided in groups that either matched or mismatched the condition experienced before, resulting in four different life histories. The main results were: (i) Early life benefit followed by late life adversity caused decreased levels of anxiety-like behavior. (ii) Accumulation of adversity throughout life history led to impaired fear extinction learning. Late life adversity as compared to late life benefit mainly affected extinction training, while early life adversity as compared to early life benefit interfered with extinction recall. Concerning anxiety-like behavior, the results do neither support the allostatic load nor the mismatch hypothesis, but rather indicate an anxiolytic effect of a mismatched early beneficial and later adverse life history. In contrast, fear memory was strongly affected by the accumulation of adverse experiences over the lifetime, therefore supporting allostatic load hypothesis. In summary, this study highlights that anxiety-like behavior and fear memory are differently affected by specific combinations of adverse or beneficial events experienced throughout life.

In mammals, weaning constitutes an important phase in the progression to adulthood. It comprises the termination of suckling and is characterized by several changes in the behaviour of both mother and offspring. Furthermore, numerous studies in rodents have shown that the time point of weaning shapes the behavioural profile of the young. Most of these studies, however, have focused on 'early weaning', while relatively little work has been done to study 'late weaning' effects. The aim of the present study was therefore to explore behavioural effects of 'late weaning', and furthermore to gain insights into modulating effects of weaning age on the consistency of behavioural expressions over time. In total, 25 male and 20 female C57BL/6J mice, weaned after three (W3) or four (W4) weeks of age, were subjected to a series of behavioural paradigms widely used to assess anxiety-like behaviour, exploratory locomotion, and nest building performance. Behavioural testing took place with the mice reaching an age of 20 weeks and was repeated eight weeks later to investigate the stability of behavioural expressions over time. At the group level, W4 mice behaved less anxious and more explorative than W3 animals in the Open Field and Novel Cage, while anxiety-like behaviour on the Elevated Plus Maze was modulated by a weaning-age-by-sex interaction. Furthermore, weaning age shaped the degree of behavioural stability over time in a sex-specific way. While W3 females and W4 males displayed a remarkable degree of behavioural stability over time, no such patterns were observed in W3 males and W4 females. Adding to the existing literature, we could thus confirm that effects of weaning age do indeed exist when prolonging this phase, and were furthermore able to provide first evidence for the impact of weaning age and sex on the consistency of behavioural expressions over time.

The abilities to either flexibly adjust behavior according to changing demands (cognitive flexibility) or to maintain it in the face of potential distractors (cognitive stability) are critical for adaptive behavior in many situations. Recently, a novel human paradigm has found individual differences of cognitive flexibility and stability to be related to common prefrontal networks. The aims of the present study were, first, to translate this paradigm from humans to mice and, second, to test conceptual predictions of a computational model of prefrontal working memory mechanisms, the Dual State Theory, which assumes an antagonistic relation between cognitive flexibility and stability.Mice were trained in a touchscreen-paradigm to discriminate visual cues. The task involved ‘ongoing’ and cued ‘switch’ trials. In addition distractor cues were interspersed to test the ability to resist distraction, and an ambiguous condition assessed the spontaneous switching between two possible responses without explicit cues. While response times did not differ substantially between conditions, error rates increased from the ‘ongoing’ baseline condition to the most complex condition, where subjects were required to switch between two responses in the presence of a distracting cue. Importantly, subjects switching more often spontaneously were found to be more distractible by task irrelevant cues, but also more flexible in situations, where switching was required. These results support a dichotomy of cognitive flexibility and stability as predicted by the Dual State Theory. Furthermore, they replicate critical aspects of the human paradigm, which indicates the translational potential of the testing procedure and supports the use of touchscreen procedures in preclinical animal research.

Background: The amygdala is crucial for emotional cognitive processing. Affective or emotional states can bias cognitive processes, including attention, memory, and decision-making. This can result in optimistic or pessimistic behaviors that are partially driven by the activation of the amygdala. The resulting emotional cognitive bias is a common feature of anxiety and mood disorders, both of which are interactively influenced by genetic and environmental factors. It is also known that emotional cognitive biases can be influenced by environmental factors. However, little is known about the effects of genetics and/or gene-environment interactions on emotional cognitive biases. We investigated the effects of the genetic background and environmental enrichment on the transcriptional profiles of the mouse amygdala following a well-established cognitive bias test. Methods: Twenty-four female C57BL/6J and B6D2F1N mice were housed either in standard (control) conditions or in an enriched environment. After appropriate training, the cognitive bias test was performed on 19 mice that satisfactorily completed the training scheme to assess their responses to ambiguous cues. This allowed us to calculate an “optimism score” for each mouse. Subsequently, we dissected the anterior and posterior portions of the amygdala to perform RNA-sequencing for differential expression and other statistical analyses. Results: In general, we found only minor changes in the amygdala’s transcriptome associated with the levels of optimism in our mice. In contrast, we observed wide molecular effects of the genetic background in both housing environments. The C57BL/6J animals showed more transcriptional changes in response to enriched environments than the B6D2F1N mice.We also generally found more dysregulated genes in the posterior than in the anterior portion of the amygdala. Gene set overrepresentation analyses consistently implicated cellular metabolic responses and immune processes in the differences observed between mouse strains, while processes favoring neurogenesis and neurotransmission were implicated in the responses to environmental enrichment. In a correlation analysis, lipid metabolism in the anterior amygdala was suggested to influence the levels of optimism. Conclusions: Our observations underscore the importance of selecting appropriate animal models when performing molecular studies of affective conditions or emotional states, and suggest an important role of immune and stress responses in the genetic component of emotion regulation. [ABSTRACT FROM AUTHOR]

Disturbances in cognitive functioning are among the most debilitating problems experienced by patients with major depression. Investigations of these deficits in animals help to extend and refine our understanding of human emotional disorder, while at the same time providing valid tools to study higher executive functions in animals. We employ the "learned helplessness" genetic rat model of depression in studying working memory using an eight arm radial maze procedure with temporal delay. This so-called delayed spatial win-shift task consists of three phases, training, delay and test, requiring rats to hold information on-line across a retention interval and making choices based on this information in the test phase. According to a 2×2 factorial design, working memory performance of thirty-one congenitally helpless (cLH) and non-helpless (cNLH) rats was tested on eighteen trials, additionally imposing two different delay durations, 30 s and 15 min, respectively. While not observing a general cognitive deficit in cLH rats, the delay length greatly influenced maze performance. Notably, performance was most impaired in cLH rats tested with the shorter 30 s delay, suggesting a stress-related disruption of attentional processes in rats that are more sensitive to stress. Our study provides direct animal homologues of clinically important measures in human research, and contributes to the non-invasive assessment of cognitive deficits associated with depression.

In animal experiments, animals, husbandry and test procedures are traditionally standardized to maximize test sensitivity and minimize animal use, assuming that this will also guarantee reproducibility. However, by reducing within-experiment variation, standardization may limit inference to the specific experimental conditions. Indeed, we have recently shown in mice that standardization may generate spurious results in behavioral tests, accounting for poor reproducibility, and that this can be avoided by population heterogenization through systematic variation of experimental conditions. Here, we examined whether a simple form of heterogenization effectively improves reproducibility of test results in a multi-laboratory situation. Each of six laboratories independently ordered 64 female mice of two inbred strains (C57BL/6NCrl, DBA/2NCrl) and examined them for strain differences in five commonly used behavioral tests under two different experimental designs. In the standardized design, experimental conditions were standardized as much as possible in each laboratory, while they were systematically varied with respect to the animals' test age and cage enrichment in the heterogenized design. Although heterogenization tended to improve reproducibility by increasing within-experiment variation relative to between-experiment variation, the effect was too weak to account for the large variation between laboratories. However, our findings confirm the potential of systematic heterogenization for improving reproducibility of animal experiments and highlight the need for effective and practicable heterogenization strategies.

Depressive episodes are frequently preceded by stressful life events. Evidence from genetic association studies suggests a role for the glucocorticoid receptor (GR), an essential element in the regulation of stress responses, in the pathophysiology of the disorder. Since the stress response system is affected by pregnancy and postpartum-associated changes, it has also been implicated in the pathophysiology of postpartum depression. Using a 2 × 2 factorial design, we investigated whether a heterozygous deletion of GR would influence maternal care behavior in C57BL/6 and Balb/c mice, two inbred strains known to display qualitative differences in this behavior. Behavioral observation was carried out between postnatal days 1 and 7, followed by a pup retrieval test on postnatal days 7 or 8. While previously noted inter-strain differences were confirmed for different manifestations of caring behavior, self-maintenance and neglecting behaviors as well as the pup retrieval test, no strain-independent effect of the GR mutation was noted. However, an interaction between GR genotype and licking/grooming behavior was observed: it was down-regulated in heterozygous C57BL/6 mice to the level recorded for Balb/c mice. Home cage observation poses minimal disturbance of the dam and her litter as compared to more invasive assessments of dams' emotional behavior. This might be a reason for the absence of any overall effects of the GR mutation, particularly since GR heterozygous animals display a depressive-like phenotype under stressful conditions only. Still, the subtle effect we observed may point towards a role of GR in postpartum affective disorders.