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Fast and effective anesthesia is the key for refining many invasive procedures in fish and gaining reliable data. For fish as for all vertebrates, it is also required by European law to reduce pain, suffering, and distress to the unavoidable minimum in husbandry and experiments. The most often used substance to induce anesthesia in zebrafish is tricaine (MS-222). When properly prepared and dosed, tricaine causes rapid loss of mobility, balance and reaction to touch. These signs are interpreted as a stage of deep anesthesia although its effects on the central nervous system have not convincingly been shown. Therefore, it might be possible that tricaine first acts only on the periphery, resulting in a paralyzed instead of an anesthetized fish. This has severe implications for animals undergoing procedures. To investigate the effects of tricaine on the central nervous system, we used zebrafish larvae [Tg(elavl3:H2B-GCaMP6s)] at 4 days post fertilization (dpf), expressing a calcium indicator (GCaMP6s) in all neurons, that allows monitoring and quantifying the neuronal activity. After treating larvae with 168 mg/L tricaine, a rapid loss of neuronal activity in the forebrain was observed in confocal microscopy. In contrast, only mild effects were seen in the midbrain and hindbrain. In conclusion, the different larval brain areas showed differences in the sensitivity to tricaine treatment. The effects on the central nervous system are indicative of tricaine’s anesthetic function and are consistent with behavioral observations of inactivity and unresponsiveness to touch.

Background Due to the lack of complexity and variety of stimuli, conventional housing conditions of laboratory mice do not allow these animals to fully express their behavioral repertoire, including manipulative and cognitive activities. Therefore, we designed mechanical puzzles, so-called lockboxes, for mice that can be provided in their home cages. We investigated the impact of the lockbox enrichment on their phenotype and affective state when compared to conventional housing (CH) and super-environmental enrichment (SEE). Methods Young adult female C57BL/6JCrl mice were examined before and after 2-month exposure to the different types of enrichment in a phenotyping test battery, including tests for trait and state anxiety-related behavior, calorimetric measurements, body weight measurements, the analysis of stress hormone metabolite concentrations, and sequential problem-solving abilities with a novel lockbox. At the end of the study, adrenal gland weights were determined and pathohistological evaluation was performed. For all continuous variables, the relative variability was calculated. Results While the different types of enrichment affected trait anxiety-related behavior, neither state anxiety-related behavior nor physiological variables (i.e., bodyweight, resting metabolic rate, stress hormone metabolite concentrations, adrenal gland weights) were influenced. LE improved sequential problem-solving (i.e., solving novel lockboxes) when compared to SEE. Regardless of the housing condition, the relative variability increased in most variables over time, although the coefficient of variation decreased for some variables, especially in animals with access to LE. There was no evidence of toxicopathological effects associated with the material from which the lockboxes were made. Conclusions All lockboxes are available as open-source tool. LE revealed beneficial effects on the affective state of laboratory mice and their performance in solving novel lockboxes. Neither relevant phenotype of the mice nor reproducibility of the data were compromised by LE, similar to SEE. The lockboxes may also be used as novel approach for assessing cognition in mice.

Abstract 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.

Abstract 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.

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.

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.

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.

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.

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.

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.

Laboratory mice are predominantly used for one experiment only, i.e., new mice are ordered or bred for every new experiment. Moreover, most experiments use relatively young mice in the range of late adolescence to early adulthood. As a consequence, little is known about the day-to-day life of adult and aged laboratory mice. Here we present a long-term data set with three consecutive phases conducted with the same male mice over their lifetime in order to shed light on possible long-term effects of repeated cognitive stimulation. One third of the animals was trained by a variety of learning tasks conducted up to an age of 606 days. The mice were housed in four cages with 12 animals per cage; only four mice per cage had to repeatedly solve cognitive tasks for getting access to water using the IntelliCage system. In addition, these learner mice were tested in standard cognitive tests outside their home-cage. The other eight mice served as two control groups living in the same environment but without having to solve tasks for getting access to water. One control group was additionally placed on the test set-ups without having to learn the tasks. Next to the cognitive tasks, we took physiological measures (body mass, resting metabolic rate) and tested for dominance behavior, and attractivity in a female choice experiment. Overall, the mice were under surveillance until they died a natural death, providing a unique data set over the course of virtually their entire lives. Our data showed treatment differences during the first phase of our lifetime data set. Young learner mice showed a higher activity, less growth and resting metabolic rate, and were less attractive for female mice. These effects, however, were not preserved over the long-term. We also did not find differences in dominance or effects on longevity. However, we generated a unique and valuable set of long-term behavioral and physiological data from a single group of male mice and note that our long-term data contribute to a better understanding of the behavioral and physiological processes in male C57Bl/6J mice.

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.

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.

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.

Reliability of data has become a major concern in the course of the reproducibility crisis. Especially when studying animal behavior, confounding factors such as novelty of the test apparatus can lead to a wide variability of data which may mask treatment effects and consequently lead to misinterpretation. Habituation to the test situation is a common practice to circumvent novelty induced increases in variance and to improve the reliability of the respective measurements. However, there is a lack of published empirical knowledge regarding reasonable habituation procedures and a method validation seems to be overdue. This study aimed at setting up a simple strategy to increase reliability of behavioral data measured in a familiar test apparatus. Therefore, exemplary data from mice tested in an Open Field (OF) arena were used to elucidate the potential of habituation and how reliability of measures can be confirmed by means of a repeatability analysis using the software R. On seven consecutive days, male C57BL/6J, BALB/cJ and 129S1/SvImJ mice were tested in an OF arena once daily and individual mouse behavior was recorded. A repeatability analysis was conducted with regard to repeated trials of habituation. Our data analysis revealed that monitoring animal behavior during habituation is important to determine when individual differences of the measurements are stable. Repeatability values from distance travelled and average activity increased over the habituation period, revealing that around 60% of the variance of the data can be explained by individual differences between mice. The first day of habituation was significantly different from the following 6 days. A three-day habituation period appeared to be sufficient in this study. Overall, these results emphasize the importance of habituation and in depth analysis of habituation data to define the correct starting point of the experiment for improving the reliability and reproducibility of experimental data.

Assessing the well-being of an animal is hindered by the limitations of efficient communication between humans and animals. Instead of direct communication, a variety of parameters are employed to evaluate the well-being of an animal. Especially in the field of biomedical research, scientifically sound tools to assess pain, suffering, and distress for experimental animals are highly demanded due to ethical and legal reasons. For mice, the most commonly used laboratory animals, a valuable tool is the Mouse Grimace Scale (MGS), a coding system for facial expressions of pain in mice. We aim to develop a fully automated system for the surveillance of post-surgical and post-anesthetic effects in mice. Our work introduces a semi-automated pipeline as a first step towards this goal. A new data set of images of black-furred laboratory mice that were moving freely is used and provided. Images were obtained after anesthesia (with isoflurane or ketamine/xylazine combination) and surgery (castration). We deploy two pre-trained state of the art deep convolutional neural network (CNN) architectures (ResNet50 and InceptionV3) and compare to a third CNN architecture without pre-training. Depending on the particular treatment, we achieve an accuracy of up to 99% for the recognition of the absence or presence of post-surgical and/or post-anesthetic effects on the facial expression.

It has become mandatory for the application for allowance of animal experimentation to rate the severity of the experimental procedures. In order to minimize suffering related to animal experimentation it is therefore crucial to develop appropriate methods for the assessment of animal suffering. Physiological parameters such as hormones or body weight are used to assess stress in laboratory animals. However, such physiological parameters alone are often difficult to interpret and leave a wide scope for interpretation. More recently, behavior, feelings and emotions have come increasingly into the focus of welfare research. Tests like preference tests or cognitive bias tests give insight on how animals evaluate certain situations or objects, how they feel and what their emotional state is. These methods should be combined in order to obtain a comprehensive understanding of the well-being of laboratory animals.

Disruption of the Heparan sulfate (HS)-biosynthetic gene N-acetylglucosamine N-Deacetylase/N-sulfotransferase 1 (Ndst1) during nervous system development causes malformations that are composites of those caused by mutations of multiple HS binding growth factors and morphogens. However, the role of Ndst function in adult brain physiology is less explored. Therefore, we generated mice bearing a Purkinje-cell-specific deletion in Ndst1 gene function by using Cre/loxP technology under the control of the Purkinje cell protein 2 (Pcp2/L7) promotor, which results in HS undersulfation. We observed that mutant mice did not show overt changes in the density or organization of Purkinje cells in the adult cerebellum, and behavioral tests also demonstrated normal cerebellar function. This suggested that postnatal Purkinje cell development and homeostasis are independent of Ndst1 function, or that impaired HS sulfation upon deletion of Ndst1 function may be compensated for by other Purkinje cell-expressed Ndst isoforms. To test the latter possibility, we additionally deleted the second Purkinje-cell expressed Ndst family member, Ndst2. This selectively abolished reproductive capacity of compound mutant female, but not male, mice, suggesting that ovulation, gestation, or female reproductive behavior specifically depends on Ndst-dependent HS sulfation in cells types that express Cre under Pcp2/L7 promotor control.

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 Aβ 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 Aβ42/40 ratio towards Aβ40 and up-regulation of Aβ 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 Aβ, while therapeutic enrichment mitigates growth and fusion of plaque seeds to large plaques by inhibiting further Aβ aggregation. This study provides an experimental basis for application of physical/cognitive training in both prophylaxis and therapy of Alzheimer's disease.

Cognitive bias, the altered information processing resulting from the background emotional state of an individual, has been suggested as a promising new indicator of animal emotion. Comparable to anxious or depressed humans, animals in a putatively negative emotional state are more likely to judge an ambiguous stimulus as if it predicts a negative event, than those in positive states. The present study aimed to establish a cognitive bias test for mice based on a spatial judgment task and to apply it in a pilot study to serotonin transporter (5-HTT) knockout mice, a well-established mouse model for the study of anxiety- and depression-related behavior. In a first step, we validated that our setup can assess different expectations about the outcome of an ambiguous stimulus: mice having learned to expect something positive within a maze differed significantly in their behavior towards an unfamiliar location than animals having learned to expect something negative. In a second step, the use of spatial location as a discriminatory stimulus was confirmed by showing that mice interpret an ambiguous stimulus depending on its spatial location, with a position exactly midway between a positive and a negative reference point provoking the highest level of ambiguity. Finally, the anxiety- and depression-like phenotype of the 5-HTT knockout mouse model manifested--comparable to human conditions--in a trend for a negatively distorted interpretation of ambiguous information, albeit this effect was not statistically significant. The results suggest that the present cognitive bias test provides a useful basis to study the emotional state in mice, which may not only increase the translational value of animal models in the study of human affective disorders, but which is also a central objective of animal welfare research.

The most prominent brain region evaluating the significance of external stimuli immediately after their onset is the amygdala. Stimuli evaluated as being stressful actuate a number of physiological processes as an immediate stress response. Variation in the serotonin transporter gene has been associated with increased anxiety- and depression-like behavior, altered stress reactivity and adaptation, and pathophysiology of stress-related disorders. In this study the instant reactions to an acute stressor were measured in a serotonin transporter knockout mouse model. Mice lacking the serotonin transporter were verified to be more anxious than their wild-type conspecifics. Genome-wide gene expression changes in the amygdala were measured after the mice were subjected to control condition or to an acute stressor of one minute exposure to water. The dissection of amygdalae and stabilization of RNA was conducted within nine minutes after the onset of the stressor. This extremely short protocol allowed for analysis of first wave primary response genes, typically induced within five to ten minutes of stimulation, and was performed using Affymetrix GeneChip Mouse Gene 1.0 ST Arrays. RNA profiling revealed a largely new set of differentially expressed primary response genes between the conditions acute stress and control that differed distinctly between wild-type and knockout mice. Consequently, functional categorization and pathway analysis indicated genes related to neuroplasticity and adaptation in wild-types whereas knockouts were characterized by impaired plasticity and genes more related to chronic stress and pathophysiology. Our study therefore disclosed different coping styles dependent on serotonin transporter genotype even directly after the onset of stress and accentuates the role of the serotonergic system in processing stressors and threat in the amygdala. Moreover, several of the first wave primary response genes that we found might provide promising targets for future therapeutic interventions of stress-related disorders also in humans.

The purpose of this study was to evaluate whether spatial hippocampus-dependent learning is affected by the serotonergic system and stress. Therefore, 5-HTT knockout (-/-), heterozygous (+/-) and wildtype (+/+) mice were subjected to the Barnes maze (BM) and the Morris water maze (WM), the latter being discussed as more aversive. Additionally, immediate early gene (IEG) expression, hippocampal adult neurogenesis (aN), and blood plasma corticosterone were analyzed. While the performance of 5-HTT-/- mice in the BM was undistinguishable from both other genotypes, they performed worse in the WM. However, in the course of the repeated WM trials 5-HTT-/- mice advanced to wildtype level. The experience of a single trial of either the WM or the BM resulted in increased plasma corticosterone levels in all genotypes. After several trials 5-HTT-/- mice exhibited higher corticosterone concentrations compared with both other genotypes in both tests. Corticosterone levels were highest in 5-HTT-/- mice tested in the WM indicating greater aversiveness of the WM and a greater stress sensitivity of 5-HTT deficient mice. Quantitative immunohistochemistry in the hippocampus revealed increased cell counts positive for the IEG products cFos and Arc as well as for proliferation marker Ki67 and immature neuron marker NeuroD in 5-HTT-/- mice compared to 5-HTT+/+ mice, irrespective of the test. Most differences were found in the suprapyramidal blade of the dentate gyrus of the septal hippocampus. Ki67-immunohistochemistry revealed a genotype x environment interaction with 5-HTT genotype differences in naïve controls and WM experience exclusively yielding more Ki67-positive cells in 5-HTT+/+ mice. Moreover, in 5-HTT-/- mice we demonstrate that learning performance correlates with the extent of aN. Overall, higher baseline IEG expression and increased an in the hippocampus of 5-HTT-/- mice together with increased stress sensitivity may constitute the neurobiological correlate of raised alertness, possibly impeding optimal learning performance in the more stressful WM.

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.

Anxiety and anxiety disorders are influenced by both, environmental and genetic factors. One genetic factor under scrutiny for anxiety disorders is the genetically encoded variation of the serotonin transporter (5-HTT). The aim of this study was to elucidate the effects of a threatening environment during early phases of life on anxiety-like (ANX) and exploratory behavior (EXP) in adult mice, varying in serotonin transporter (5-HTT) genotype. For this purpose, pregnant and lactating 5-HTT +/- dams were repeatedly exposed to olfactory cues of unfamiliar adult males by introducing small amounts of soiled bedding to their home cage. These stimuli signal the danger of infanticide and simulate a threatening environment. Control females were treated with neutral bedding. The offspring (5-HTT +/+, +/-, -/-) were examined for their ANX and EXP. The main results were: (1) a main effect of genotype existed, with 5-HTT -/- showing higher levels of ANX and lower levels of EXP than 5-HTT +/- and wildtypes. (2) When mothers had lived in a threatening environment, their offspring showed increased ANX and reduced EXP compared to controls. (3) These effects were most pronounced in 5-HTT -/- mice. By applying a new ecologically relevant paradigm we conclude: If 5-HTT +/- mothers live in a threatening environment during pregnancy and lactation, their offspring behavioral profile will, in principle, be shaped in an adaptive way preparing the young for an adverse environment. This process is, however, modulated by 5-HTT genotype, bearing the risk that individuals with impaired serotonergic neurotransmission (5-HTT -/-) will develop an exaggerated, potentially pathological level of anxiety from gene x environment interactions.