A systematic review of the development and application of home cage monitoring in laboratory mice and rats.

BACKGROUND: Traditionally, in biomedical animal research, laboratory rodents are individually examined in test apparatuses outside of their home cages at selected time points. However, the outcome of such tests can be influenced by various factors and valuable information may be missed when the animals are only monitored for short periods. These issues can be overcome by longitudinally monitoring mice and rats in their home cages. To shed light on the development of home cage monitoring (HCM) and the current state-of-the-art, a systematic review was carried out on 521 publications retrieved through PubMed and Web of Science. RESULTS: Both the absolute (~ × 26) and relative (~ × 7) number of HCM-related publications increased from 1974 to 2020. There was a clear bias towards males and individually housed animals, but during the past decade (2011-2020), an increasing number of studies used both sexes and group housing. In most studies, animals were kept for short (up to 4 weeks) time periods in the HCM systems; intermediate time periods (4-12 weeks) increased in frequency in the years between 2011 and 2020. Before the 2000s, HCM techniques were predominantly applied for less than 12 h, while 24-h measurements have been more frequent since the 2000s. The systematic review demonstrated that manual monitoring is decreasing in relation to automatic techniques but still relevant. Until (and including) the 1990s, most techniques were applied manually but have been progressively replaced by automation since the 2000s. Independent of the year of publication, the main behavioral parameters measured were locomotor activity, feeding, and social behaviors; the main physiological parameters were heart rate and electrocardiography. External appearance-related parameters were rarely examined in the home cages. Due to technological progress and application of artificial intelligence, more refined and detailed behavioral parameters have been investigated in the home cage more recently. CONCLUSIONS: Over the period covered in this study, techniques for HCM of mice and rats have improved considerably. This development is ongoing and further progress as well as validation of HCM systems will extend the applications to allow for continuous, longitudinal, non-invasive monitoring of an increasing range of parameters in group-housed small rodents in their home cages.

A systematic mapping review of the evolution of the rat Forced Swim Test: Protocols and outcome parameters.

As depression is projected to become the leading mental disease burden globally by 2030, understanding the underlying pathology, as well as screening potential anti-depressants with a higher efficacy, faster onset of action, and/or fewer side-effects is essential. A commonly used test for screening novel antidepressants and studying depression-linked aspects in rodents is the Porsolt Forced Swim Test. The present systematic mappping review gives a comprehensive overview of the evolution and of the most prevalently used set-ups of this test in rats, including the choice of animals (strain, sex, and age), technical aspects of protocol and environment, as well as reported outcome measures. Additionally, we provide an accessible list of all existing publications, to support informed decision-making for procedural and technical aspects of the test, to thereby enhance reproducibility and comparability. This should further contribute to reducing the number of unnecessarily replicated experiments, and consequently, reduce the number of animals used in future.

Determining the value of preferred goods based on consumer demand in a home-cage based test for mice.

From the preference of one good over another, the strength of the preference cannot automatically be inferred. While money is the common denominator to assess the value of goods in humans, it appears difficult at first glance to put a price tag on the decisions of laboratory animals. Here we used consumer demand tests to measure how much work female mice expend to obtain access to different liquids. The mice could each choose between two liquids, one of which was free. The amount of work required to access the other liquid, by contrast, increased daily. In this way, the value of the liquid can be determined from a mouse's microeconomic perspective. The unique feature is that our test was carried out in a home-cage based setup. The mice lived in a group but could individually access the test-cage, which was connected to the home-cage via a gate. Thereby the mice were able to perform their task undisturbed by group members and on a self-chosen schedule with minimal influence by the experimenter. Our results show that the maximum number of nosepokes depends on the liquids presented. Mice worked incredibly hard for access to water while a bitter-tasting solution was offered for free whereas they made less nosepokes for sweetened liquids while water was offered for free. The results demonstrate that it is possible to perform automated and home-cage based consumer demand tests in order to ask the mice not only what they like best but also how strong their preference is.

O mouse, where art thou? The Mouse Position Surveillance System (MoPSS)-an RFID-based tracking system.

Existing methods for analysis of home cage-based preference tests are either time-consuming, not suitable for group management, expensive, and/or based on proprietary equipment that is not freely available. To correct this, we developed an automated system for group-housed mice based on radio frequency identification: the Mouse Position Surveillance System (MoPSS). The system uses an Arduino microcontroller with compatible components; it is affordable and easy to rebuild for every laboratory because it uses free and open-source software and open-source hardware with the RFID readers as the only proprietary component. The MoPSS was validated using female C57BL/6J mice and manual video comparison. It proved to be accurate even for fast-moving mice (up to 100% accuracy after logical reconstruction), and is already implemented in several studies in our laboratory. Here, we provide the complete construction description as well as the validation data and the results of an example experiment. This tracking system will allow group-based preference testing with individually identified mice to be carried out in a convenient manner. This facilitation of preference tests creates the foundation for better housing conditions from the animals' perspective.

Start early! Does social instability during the pre- and early postnatal development prepare male wild cavies for social challenge later in life?

BACKGROUND: The social environment the mother experiences during pregnancy and lactation can powerfully influence the offspring's behavioural profile. Our previous studies in wild cavies show that two different social environments during pregnancy and lactation bring about different behavioural strategies of male offspring later in life: An unstable social environment leads to a behavioural camouflage strategy, hypothesised to be beneficial at times of socially challenging situations. A stable social environment during early phases of life, however, leads to an early reproduction strategy, expected to be more successful at times of social stability. In the present study, we observed the behavioural strategies of the two types of males in direct comparison in a socially challenging situation: Two adolescent males were placed simultaneously in an unknown social group consisting of one adult male and two females in a semi-naturalistic environment. Cortisol as well as testosterone concentrations and activity levels were compared. Furthermore, paternities were analysed after the males reached sexual maturity. We hypothesised that sons showing a behavioural camouflage strategy are better adapted to cope with this socially challenging situation compared to those displaying an early reproduction strategy. RESULTS: At the beginning of the experiment, no differences in plasma cortisol concentrations between the males were found, both showed a highly significant increase due to the challenging situation. From day 5 until the end of the experiment (duration = 40 days) sons showing an early reproduction strategy had significantly higher plasma cortisol concentrations compared with those showing a behavioural camouflage strategy. Plasma testosterone concentrations did not differ significantly. Activity levels decreased significantly over time independently of the male's behavioural strategy. Both types of males did not sire offspring during the observation period. CONCLUSION: Higher cortisol values from day 5 until the end of the experiment in sons showing an early reproduction strategy indicate higher levels of stress in these males compared to those displaying a camouflage strategy. We conclude that the modulation of the males behavioural strategy due to an unstable social environment during early development facilitates the endocrine adaptation to a comparable social situation later in life.

The glass is not yet half empty: agitation but not Varroa treatment causes cognitive bias in honey bees.

Honey bees (Apis mellifera) are prone to judge an ambiguous stimulus negatively if they had been agitated through shaking which simulates a predator attack. Such a cognitive bias has been suggested to reflect an internal emotional state analogous to humans who judge more pessimistically when they do not feel well. In order to test cognitive bias experimentally, an animal is conditioned to respond to two different stimuli, where one is punished while the other is rewarded. Subsequently a third, ambiguous stimulus is presented and it is measured whether the subject responds as if it expects a reward or a punishment. Generally, it is assumed that negative experiences lower future expectations, rendering the animals more pessimistic. Here we tested whether a most likely negatively experienced formic acid treatment against the parasitic mite Varroa destructor also affects future expectations of honey bees. We applied an olfactory learning paradigm (i.e., conditioned proboscis extension response) using two odorants and blends of these odorants as the ambiguous stimuli. Unlike agitating honey bees, exposure to formic acid did not significantly change the response to the ambiguous stimuli in comparison with untreated bees. Overall evidence suggests that the commonest treatment against one of the most harmful bee pests has no detrimental effects on cognitive bias in honey bees.

Lifetime development of behavioural phenotype in the house mouse (Mus musculus).

With each trajectory taken during the ontogeny of an individual, the number of optional behavioural phenotypes that can be expressed across its life span is reduced. The initial range of phenotypic plasticity is largely determined by the genetic material/composition of the gametes whereas interacting with the given environment shapes individuals to adapt to/cope with specific demands. In mammalian species, the phenotype is shaped as the foetus grows, depending on the environment in the uterus, which in turn depends on the outer environment the mother experiences during pregnancy. After birth, a complex interaction between innate constitution and environmental conditions shapes individual lifetime trajectories, bringing about a wide range of diversity among individual subjects. In laboratory mice inbreeding has been systematically induced in order to reduce the genetic variability between experimental subjects. In addition, within most laboratories conducting behavioural phenotyping with mice, breeding and housing conditions are highly standardised. Despite such standardisation efforts a considerable amount of variability persists in the behaviour of mice. There is good evidence that phenotypic variation is not merely random but might involve individual specific behavioural patterns consistent over time. In order to understand the mechanisms and the possible adaptive value of the maintenance of individuality we review the emergence of behavioural phenotypes over the course of the life of (laboratory) mice. We present a literature review summarizing developmental stages of behavioural development of mice along with three illustrative case studies. We conclude that the accumulation of environmental differences and experiences lead to a "mouse individuality" that becomes increasingly stable over the lifetime.

Rating enrichment items by female group-housed laboratory mice in multiple binary choice tests using an RFID-based tracking system.

Laboratory mice spend most of their lives in cages, not experiments, so improving housing conditions is a first-choice approach to improving their welfare. Despite the increasing popularity of enrichment, little is known about the benefits from an animal perspective. For a detailed analysis, we categorized enrichment items according to their prospective use into the categories 'structural', 'housing', and 'foraging'. In homecage-based multiple binary choice tests 12 female C57BL/6J mice chose between enrichment items within the respective categories over a 46-hour period. A new analyzing method combined the binary decisions and ranked the enrichment items within each category by calculating worth values and consensus errors. Although there was no unequivocal ranking that was true in its entire rank order for all individual mice, certain elements (e.g. lattice ball, second plane) were always among the top positions. Overall, a high consensus error in ranking positions reflects strong individual differences in preferences which could not be resolved due to the relatively small sample size. However, individual differences in the preference for enrichment items highlights the importance of a varied enrichment approach, as there does not seem to be one item that satisfies the wants and needs of all individuals to the same degree. An enrichment concept, in which the needs of the animals are central, contributes to a more specific refinement of housing conditions.

Effects of more natural housing conditions on the muscular and skeletal characteristics of female C57BL/6J mice.

BACKGROUND: Enrichment of home cages in laboratory experiments offers clear advantages, but has been criticized in some respects. First, there is a lack of definition, which makes methodological uniformity difficult. Second, there is concern that the enrichment of home cages may increase the variance of results in experiments. Here, the influence of more natural housing conditions on physiological parameters of female C57BL/6J mice was investigated from an animal welfare point of view. For this purpose, the animals were kept in three different housing conditions: conventional cage housing, enriched housing and the semi naturalistic environment. The focus was on musculoskeletal changes after long-term environmental enrichment. RESULTS: The housing conditions had a long-term effect on the body weight of the test animals. The more complex and natural the home cage, the heavier the animals. This was associated with increased adipose deposits in the animals. There were no significant changes in muscle and bone characteristics except for single clues (femur diameter, bone resorption marker CTX-1). Additionally, the animals in the semi naturalistic environment (SNE) were found to have the fewest bone anomalies. Housing in the SNE appears to have the least effect on stress hormone concentrations. The lowest oxygen uptake was observed in enriched cage housing. CONCLUSIONS: Despite increasing values, observed body weights were in the normal and strain-typical range. Overall, musculoskeletal parameters were slightly improved and age-related effects appear to have been attenuated. The variances in the results were not increased by more natural housing. This confirms the suitability of the applied housing conditions to ensure and increase animal welfare in laboratory experiments.

Challenges and advanced concepts for the assessment of learning and memory function in mice.

The mechanisms underlying the formation and retrieval of memories are still an active area of research and discussion. Manifold models have been proposed and refined over the years, with most assuming a dichotomy between memory processes involving non-conscious and conscious mechanisms. Despite our incomplete understanding of the underlying mechanisms, tests of memory and learning count among the most performed behavioral experiments. Here, we will discuss available protocols for testing learning and memory using the example of the most prevalent animal species in research, the laboratory mouse. A wide range of protocols has been developed in mice to test, e.g., object recognition, spatial learning, procedural memory, sequential problem solving, operant- and fear conditioning, and social recognition. Those assays are carried out with individual subjects in apparatuses such as arenas and mazes, which allow for a high degree of standardization across laboratories and straightforward data interpretation but are not without caveats and limitations. In animal research, there is growing concern about the translatability of study results and animal welfare, leading to novel approaches beyond established protocols. Here, we present some of the more recent developments and more advanced concepts in learning and memory testing, such as multi-step sequential lockboxes, assays involving groups of animals, as well as home cage-based assays supported by automated tracking solutions; and weight their potential and limitations against those of established paradigms. Shifting the focus of learning tests from the classical experimental chamber to settings which are more natural for rodents comes with a new set of challenges for behavioral researchers, but also offers the opportunity to understand memory formation and retrieval in a more conclusive way than has been attainable with conventional test protocols. We predict and embrace an increase in studies relying on methods involving a higher degree of automatization, more naturalistic- and home cage-based experimental setting as well as more integrated learning tasks in the future. We are confident these trends are suited to alleviate the burden on animal subjects and improve study designs in memory research.

Development of an IntelliCage-based cognitive bias test for mice.

The cognitive bias test is used to measure the emotional state of animals with regard to future expectations. Thus, the test offers a unique possibility to assess animal welfare with regard to housing and testing conditions of laboratory animals. So far, however, performing such a test is time-consuming and requires the presence of an experimenter. Therefore, we developed an automated and home-cage based cognitive bias test based on the IntelliCage system. We present several developmental steps to improve the experimental design leading to a successful measurement of cognitive bias in group-housed female C57BL/6J mice. The automated and home-cage based test design allows to obtain individual data from group-housed mice, to test the mice in their familiar environment, and during their active phase. By connecting the test-cage to the home-cage via a gating system, the mice participated in the test on a self-chosen schedule, indicating high motivation to actively participate in the experiment. We propose that this should have a positive effect on the animals themselves as well as on the data. Unexpectedly, the mice showed an optimistic cognitive bias after enrichment was removed and additional restraining. An optimistic expectation of the future as a consequence of worsening environmental conditions, however, can also be interpreted as an active coping strategy in which a potential profit is sought to be maximized through a higher willingness to take risks.

The "WWHow" Concept for Prospective Categorization of Post-operative Severity Assessment in Mice and Rats.

The prospective severity assessment in animal experiments in the categories' non-recovery, mild, moderate, and severe is part of each approval process and serves to estimate the harm/benefit. Harms are essential for evaluating ethical justifiability, and on the other hand, they may represent confounders and effect modifiers within an experiment. Catalogs and guidelines provide a way to assess the experimental severity prospectively but are limited in adaptation due to their nature of representing particular examples without clear explanations of the assessment strategies. To provide more flexibility for current and future practices, we developed the modular Where-What-How (WWHow) concept, which applies findings from pre-clinical studies using surgical-induced pain models in mice and rats to provide a prospective severity assessment. The WWHow concept integrates intra-operative characteristics for predicting the maximum expected severity of surgical procedures. The assessed severity categorization is mainly congruent with examples in established catalogs; however, because the WWHow concept is based on anatomical location, detailed analysis of the tissue trauma and other intra-operative characteristics, it enables refinement actions, provides the basis for a fact-based dialogue with authority officials and other stakeholders, and helps to identify confounder factors of study findings.

Bored at home?-A systematic review on the effect of environmental enrichment on the welfare of laboratory rats and mice.

Boredom is an emotional state that occurs when an individual has nothing to do, is not interested in the surrounding, and feels dreary and in a monotony. While this condition is usually defined for humans, it may very well describe the lives of many laboratory animals housed in small, barren cages. To make the cages less monotonous, environmental enrichment is often proposed. Although housing in a stimulating environment is still used predominantly as a luxury good and for treatment in preclinical research, enrichment is increasingly recognized to improve animal welfare. To gain insight into how stimulating environments influence the welfare of laboratory rodents, we conducted a systematic review of studies that analyzed the effect of enriched environment on behavioral parameters of animal well-being. Remarkably, a considerable number of these parameters can be associated with symptoms of boredom. Our findings show that a stimulating living environment is essential for the development of natural behavior and animal welfare of laboratory rats and mice alike, regardless of age and sex. Conversely, confinement and under-stimulation has potentially detrimental effects on the mental and physical health of laboratory rodents. We show that boredom in experimental animals is measurable and does not have to be accepted as inevitable.

Preventive and therapeutic types of environmental enrichment counteract beta amyloid pathology by different molecular mechanisms.

Combined preventive and therapeutic physical/cognitive stimulation starting before disease onset and continuing over its progression reduce Alzheimer-related pathology in transgenic mice. We now report that exposure of TgCRND8 mice to an enriched environment as either a preventive or therapeutic approach is also capable to reduce Aß burden, though with different plaque and cerebral amyloid angiopathy (CAA) morphology. Preventive treatment resulted in fewer and smaller plaques without affecting CAA, whereas in therapeutically treated mice beside reduction of CAA extent, numerous plaques of strongly diminished size were found, so that total plaque loads declined as well. These effects seemed to be mediated by distinct molecular pathways. In preventive but not therapeutic group a shift of Aß(42/40) ratio towards Aß(40) and up-regulation of Aß clearing and degrading molecules were found. Contrariwise anti-oxidative defense mechanisms were induced only in therapy but not preventive group. We hypothesize that preventive enrichment lowers the amounts of plaque seeds and decelerates plaque growth by degradation and clearance of Aß, while therapeutic enrichment mitigates growth and fusion of plaque seeds to large plaques by inhibiting further Aß aggregation. This study provides an experimental basis for application of physical/cognitive training in both prophylaxis and therapy of Alzheimer's disease.

Living in a dangerous world decreases maternal care: a study in serotonin transporter knockout mice.

Adverse early experiences can profoundly influence the adult behavioral profile. When pregnant and lactating mice are confronted with soiled bedding of unfamiliar males (UMB), these stimuli signal the danger of infanticide and thus simulate a "dangerous world". In a previous study, offspring of UMB treated mothers were shown to display increased anxiety-like behavior and reduced exploratory locomotion as adults, compared to mice treated with neutral bedding (NB, "safe environment"). The aim of this study was to elucidate the mechanisms conveying these effects of living in a "dangerous world" to offspring behavior. We hypothesized the mother to be the major link and focused on the influence of UMB on maternal stress hormones and behavior. Thus, we investigated fecal corticosterone metabolites (CM) and maternal care of pregnant and lactating mice either treated with NB or UMB. The offspring were subsequently tested for their anxiety-like and exploratory behavior. Mothers treated with UMB showed a significantly higher increase of fecal CM following the initial treatment, than NB treated mothers, indicating that the odor cues of potentially infanticidal males represented an ethologically relevant stimulus. Whereas the hormonal stress response habituated, living in a "dangerous world" led to a distinct and consistent reduction of maternal care behavior, particularly concerning the duration of licking and grooming the pups. Surprisingly, we could not confirm our former findings of altered phenotypes in the offspring of UMB treated mothers. In summary, we hypothesize that the frequently described effects of early life adversity on the offspring's behavioral profile are mediated primarily by maternal care in altricial rodents.

"Personality" in laboratory mice used for biomedical research: a way of understanding variability?

The mouse, including countless lines of transgenic and knockout mice, has become the most prominent model organism in biomedical research. Behavioral characterization is often conducted in batteries of short tests on locomotion, anxiety, learning and memory, etc. In such tests, any individual differences within groups are usually considered to be disturbing variance. In order to reduce variance in experimental animal research enormous efforts of standardization have been made. While a substantial reduction of variability has been reached compared to the earlier years of experimental animal studies a considerable amount of inter-individual differences still seems to escape standardization. This effect is demonstrated and evaluated by re-analyzing data from two experiments conducted in our laboratory with inbred mice. Interestingly, behavioral patterns of individual animals seem to be correlated across context and time. In evolutionary biology, "animal personalities" have been discussed recently to comprise such stable patterns. We argue here, that nonrandom behavioral correlations across contexts and time might underlie the variability commonly found in biomedical mouse studies.

Roaming in a Land of Milk and Honey: Life Trajectories and Metabolic Rate of Female Inbred Mice Living in a Semi Naturalistic Environment.

Despite tremendous efforts at standardization, the results of scientific studies can vary greatly, especially when considering animal research. It is important to emphasize that consistent different personality-like traits emerge and accumulate over time in laboratory mice despite genetic and environmental standardization. To understand to what extent variability can unfold over time, we conducted a long-term study using inbred mice living in an exceptionally complex environment comprising an area of 4.6 m2 spread over five levels. In this semi-naturalistic environment (SNE) the activity and spatial distribution of 20 female C57Bl/6J was recorded by radio-frequency identification (RFID). All individuals were monitored from an age of 11 months to 22 months and their individual pattern of spatial movement in time is described as roaming entropy. Overall, we detected an increase of diversification in roaming behavior over time with stabilizing activity patterns at the individual level. However, spontaneous behavior of the animals as well as physiological parameters did not correlate with cumulative roaming entropy. Moreover, the amount of variability did not exceed the literature data derived from mice living in restricted conventional laboratory conditions. We conclude that even taking quantum leaps towards improving animal welfare does not inevitably mean a setback in terms of data quality.

Evaluation of different types of enrichment - their usage and effect on home cage behavior in female mice.

Numerous studies ascertained positive effects of enriched environments on the well-being of laboratory animals including behavioral, physiological and neurochemical parameters. Conversely, such conclusions imply impaired animal welfare and health in barren husbandry conditions. Moreover, inappropriate housing of laboratory animals may deteriorate the quality of scientific data. Recommendations for housing laboratory animals stipulate that cages should be enriched to mitigate adverse effects of barren housing. In this context, it is not only unclear what exactly is meant by enrichment, but also how the animals themselves interact with the various items on offer. Focal animal observation of female C57BL/6J mice either housed in conventional (CON) or enriched (ENR) conditions served to analyze the impact of enriching housing on welfare related behavior patterns including stereotypical, maintenance, active social, and inactive behaviors. CON conditions resembled current usual housing of laboratory mice, whereas ENR mice received varying enrichment items including foraging, housing and structural elements, and a running disc. Active and inactive use of these elements was quantitatively assessed. CON mice showed significantly more inactive and stereotypical behavior than ENR mice. ENR mice frequently engaged with all enrichment elements, whereby riddles to obtain food reward and the running disc preferably served for active interactions. Offering a second level resulted in high active and inactive interactions. Structural elements fixed at the cagetop were least attractive for the mice. Overall, the presented data underline the positive welfare benefits of enrichment and that mice clearly differentiate between distinct enrichment types, demonstrating that the perspective of the animals themselves should also be taken into account when specifying laboratory housing conditions. This is particularly important, as the ensuring of animal welfare is an essential prerequisite for reliable, reproducible, and scientifically meaningful results.

Current Methods to Investigate Nociception and Pain in Zebrafish.

Pain is an unpleasant, negative emotion and its debilitating effects are complex to manage. Mammalian models have long dominated research on nociception and pain, but there is increasing evidence for comparable processes in fish. The need to improve existing pain models for drug research and the obligation for 3R refinement of fish procedures facilitated the development of numerous new assays of nociception and pain in fish. The zebrafish is already a well-established animal model in many other research areas like toxicity testing, as model for diseases or regeneration and has great potential in pain research, too. Methods of electrophysiology, molecular biology, analysis of reflexive or non-reflexive behavior and fluorescent imaging are routinely applied but it is the combination of these tools what makes the zebrafish model so powerful. Simultaneously, observing complex behavior in free-swimming larvae, as well as their neuronal activity at the cellular level, opens new avenues for pain research. This review aims to supply a toolbox for researchers by summarizing current methods to study nociception and pain in zebrafish. We identify treatments with the best algogenic potential, be it chemical, thermal or electric stimuli and discuss options of analgesia to counter effects of nociception and pain by opioids, non-steroidal anti-inflammatory drugs (NSAIDs) or local anesthetics. In addition, we critically evaluate these practices, identify gaps of knowledge and outline potential future developments.