The Rise of Three Rs Centres and Platforms in Europe.

Public awareness and discussion about animal experiments and replacement methods has greatly increased in recent years. The term 'the Three Rs', which stands for the Replacement, Reduction and Refinement of animal experiments, is inseparably linked in this context. A common goal within the Three Rs scientific community is to develop predictive non-animal models and to better integrate all available data from in vitro, in silico and omics technologies into regulatory decision-making processes regarding, for example, the toxicity of chemicals, drugs or food ingredients. In addition, it is a general concern to implement (human) non-animal methods in basic research. Toward these efforts, there has been an ever-increasing number of Three Rs centres and platforms established over recent years - not only to develop novel methods, but also to disseminate knowledge and help to implement the Three Rs principles in policies and education. The adoption of Directive 2010/63/EU on the protection of animals used for scientific purposes gave a strong impetus to the creation of Three Rs initiatives, in the form of centres and platforms. As the first of a series of papers, this article gives an overview of the European Three Rs centres and platforms, and their historical development. The subsequent articles, to be published over the course of ATLA's 50th Anniversary year, will summarise the current focus and tasks as well as the future and the plans of the Three Rs centres and platforms. The Three Rs centres and platforms are very important points of contact and play an immense role in their respective countries as 'on the ground' facilitators of Directive 2010/63/EU. They are also invaluable for the widespread dissemination of information and for promoting implementation of the Three Rs in general.

The Current Status and Work of Three Rs Centres and Platforms in Europe.

The adoption of Directive 2010/63/EU on the protection of animals used for scientific purposes has given a major push to the formation of Three Rs initiatives in the form of centres and platforms. These centres and platforms are dedicated to the so-called Three Rs, which are the Replacement, Reduction and Refinement of animal use in experiments. ATLA's 50th Anniversary year has seen the publication of two articles on European Three Rs centres and platforms. The first of these was about the progressive rise in their numbers and about their founding history; this second part focuses on their current status and activities. This article takes a closer look at their financial and organisational structures, describes their Three Rs focus and core activities (dissemination, education, implementation, scientific quality/translatability, ethics), and presents their areas of responsibility and projects in detail. This overview of the work and diverse structures of the Three Rs centres and platforms is not only intended to bring them closer to the reader, but also to provide role models and show examples of how such Three Rs centres and platforms could be made sustainable. The Three Rs centres and platforms are very important focal points and play an immense role as facilitators of Directive 2010/63/EU 'on the ground' in their respective countries. They are also invaluable for the wide dissemination of information and for promoting the implementation of the Three Rs in general.

Evaluation of Potential Sustainable Bedding Substrates Focusing on Preference, Behavior, and Stress Physiology in Rats-A Pilot Study.

Ensuring optimal housing conditions for laboratory animals is a crucial prerequisite for high-quality and ethically justifiable in vivo science. In addition to guaranteeing animal welfare and promoting scientific validity, environmental sustainability is also increasingly gaining attention in laboratory animal facilities. Consequently, comprehensive management of such aspects is one of the core tasks of any research vivarium. Hygienic monitoring and adhering to standardized experimental protocols have been highlighted in the past; nevertheless, various environmental aspects of housing animals still need to be evaluated in greater depth. In this pilot study, we aimed at assessing the suitability of spelt and corncob as economical and ecologically friendly bedding substrates as compared with commonly used aspen wood chips. Therefore, following a descriptive study design, we examined the preferences of male and female Wistar rats for corncob and spelt under specific conditions. In addition, we evaluated potential effects on behavior, metabolism, and stress physiology. The type of bedding did not seem to influence behavior in the observed parameters but did have time- and sex-dependent effects on blood glucose. Furthermore, housing animals on spelt led to a significant reduction in food consumption, probably compensated for by the intake of spelt, and although it did not influence glucose levels, it may have certainly impacted the nutrient supply. Our descriptive pilot study, therefore, highlights the importance of a thorough condition-associated evaluation of even seemingly marginal environmental factors, when balancing potential cost-benefit advances in sustainability and questions of standardization and reproducibility of experimental protocols.

Cre-Activation in ErbB4-Positive Neurons of Floxed Grin1/NMDA Receptor Mice Is Not Associated With Major Behavioral Impairment.

Extensive evidence suggests a dysfunction of the glutamate NMDA receptor (NMDAR) in schizophrenia, a severe psychiatric disorder with putative early neurodevelopmental origins, but clinical onset mainly during late adolescence. On the other hand, pharmacological models using NMDAR antagonists and the clinical manifestation of anti-NMDAR encephalitis indicate that NMDAR blockade/hypofunction can trigger psychosis also at adult stages, without any early developmental dysfunction. Previous genetic models of NMDAR hypofunction restricted to parvalbumin-positive interneurons indicate the necessity of an early postnatal impairment to trigger schizophrenia-like abnormalities, whereas the cellular substrates of NMDAR-mediated psychosis at adolescent/adult stages are unknown. Neuregulin 1 (NRG1) and its receptor ErbB4 represent schizophrenia-associated susceptibility factors that closely interact with NMDAR. To determine the neuronal populations implicated in "late" NMDAR-driven psychosis, we analyzed the effect of the inducible ablation of NMDARs in ErbB4-expressing cells in mice during late adolescence using a pharmacogenetic approach. Interestingly, the tamoxifen-inducible NMDAR deletion during this late developmental stage did not induce behavioral alterations resembling depression, schizophrenia or anxiety. Our data indicate that post-adolescent NMDAR deletion, even in a wider cell population than parvalbumin-positive interneurons, is also not sufficient to generate behavioral abnormalities resembling psychiatric disorders. Other neuronal substrates that have to be revealed by future studies, may underlie post-adolescent NMDAR-driven psychosis.

Systematic analysis of severity in a widely used cognitive depression model for mice.

Animal models in psychiatric research are indispensable for insights into mechanisms of behaviour and mental disorders. Distress is an important aetiological factor in psychiatric diseases, especially depression, and is often used to mimic the human condition. Modern bioethics requires balancing scientific progress with animal welfare concerns. Therefore, scientifically based severity assessment of procedures is a prerequisite for choosing the least compromising paradigm according to the 3Rs principle. Evidence-based severity assessment in psychiatric animal models is scarce, particularly in depression research. Here, we assessed severity in a cognitive depression model by analysing indicators of stress and well-being, including physiological (body weight and corticosterone metabolite concentrations) and behavioural (nesting and burrowing behaviour) parameters. Additionally, a novel approach for objective individualised severity grading was employed using clustering of voluntary wheel running (VWR) behaviour. Exposure to the paradigm evoked a transient elevation of corticosterone, but neither affected body weight, nesting or burrowing behaviour. However, the performance in VWR was impaired after recurrent stress exposure, and the individual severity level increased, indicating that this method is more sensitive in detecting compromised welfare. Interestingly, the direct comparison to a somatic, chemically induced colitis model indicates less distress in the depression model. Further objective severity assessment studies are needed to classify the severity of psychiatric animal models in order to balance validity and welfare, reduce the stress load and thus promote refinement.

Reduced hippocampal neurogenesis in the GR(+/-) genetic mouse model of depression.

Glucocorticoid receptor (GR) heterozygous mice (GR(+/- )) represent a valuable animal model for major depression. GR(+/- ) mice show a depression-related phenotype characterized by increased learned helplessness on the behavioral level and neuroendocrine alterations with hypothalamo-pituitary-adrenal (HPA) axis overdrive characteristic of depression. Hippocampal brain-derived neurotrophic factor (BDNF) levels have also been shown to be reduced in GR(+/- ) animals. Because adult hippocampal neurogenesis has been implicated in the pathophysiology of affective disorders, we studied here the effects of the GR(+/- ) genotype on neurogenesis in vivo. In a 2 x 2 design, GR(+/- ) mice and GR(+/+) littermate controls were either subjected to 1 h of restraint stress or left undisturbed in their home cages after intraperitoneal injection of BrdU. Stress exposure and BrdU injections were performed once daily for 7 days and neurogenesis analyzed 4 weeks later. BrdU cell counts were significantly reduced as an effect of GR(+/- ) genotype and as an effect of stress. Majority of the BrdU+ cells showed co-labeling with mature neuronal marker NeuN or astrocytic marker S100beta with no further significant effect of either experimental condition or of genotype. In sum, this results in reduced neurogenesis in GR(+/- ) mice which is further repressed by restraint stress. Our results, thus, reinforce the link between reduced neurogenesis, stress, neurotrophins, and behavioral symptoms of and susceptibility to depression.

Glucocorticoid receptor transgenic mice as models for depression.

In the era of mutant mice generated as molecular in vivo models for complex pathogenetic and therapeutic aspects of particular human diseases, glucocorticoid receptor transgenic mice represent an interesting and promising tool. Animals carrying mutations of this receptor show alterations in the hypothalamic-pituitary-adrenal (HPA)-system, which are comparable to those observed in depressed patients. Furthermore, similarities that may model the human disease have been described on the behavioral and pharmacological level, which increase the impact of such mutants. In this review we summarize different approaches used to alter or eliminate glucocorticoid receptor expression and function, and discuss their relevance as models for depression.

Effect of three different forms of handling on the variation of aggression-associated parameters in individually and group-housed male C57BL/6NCrl mice.

Mice are social animals hence group-housing of mice is preferred over individual housing. However, aggression in group-housed male mice under laboratory housing conditions is a well-known problem leading to serious health issues, including injury or death. Therefore, group-housed mice are frequently separated for welfare reasons. In this study, we investigated the effect of 3 different handling methods (tail, forceps, tube) in 2 different housing conditions (single vs. group) on the variance of aggression-associated parameters in male C57BL/6NCrl mice over 8 weeks. Blood glucose concentration, body weight, body temperature, plus number and severity of bite wounds and barbering intensity in group-housed mice were recorded. An assessment of nest complexity was also performed weekly. Feces were collected in week 3 and 7 for analysis of corticosterone metabolites. We also monitored the level of aggression by recording the behavior of group-housed animals after weekly cage cleaning. An open field test followed by a social novel object test, a light/dark box test, a hotplate and a resident-intruder test were performed at the end of the 8-week handling period. Post-mortem, we assessed organ weights. We found that forceps-handled mice, independent of the housing condition, had significantly higher levels of stress-induced-hyperthermia and enhanced aggression after cage cleaning, and they performed worse in the nest complexity test. In addition, handling male mice by the tail seems to be most effective to reduce aggressiveness after transferring animals into new cages, thereby representing an appropriate refinement.

Effects of Soy in Laboratory Rodent Diets on the Basal, Affective, and Cognitive Behavior of C57BL/6 Mice.

Soy is one of the most common sources of protein in many commercial formulas for laboratory rodent diets. Soy contains isoflavones, which are estrogenic. Therefore, soy-containing animal diets might influence estrogen-regulated systems, including basal behavioral domains, as well as affective behavior and cognition. Furthermore, the isoflavone content of soy varies, potentially unpredictably confounding behavioral results. Therefore researchers are increasingly considering completely avoiding dietary soy to circumvent this problem. Several animal studies have investigated the effects of soy free diets but produced inconsistent results. In addition, most of these previous studies were performed in outbred rat or mouse strains. In the current study, we assessed whether a soy-free diet altered locomotion, exploration, nesting, anxiety-related behaviors, learning, and memory in C57BL/6 mice, the most common inbred strain used in biomedical research. The parameters evaluated address measures of basic health, natural behavior, and affective state that also are landmarks for animal welfare. We found minor differences between feeding groups but no indications of altered welfare. We therefore suggest that a soy-free diet can be used as a standard diet to prevent undesirable side effects of isoflavones and to further optimize diet standardization, quality assurance, and ultimately increase the reproducibility of experiments.

Mice that under- or overexpress glucocorticoid receptors as models for depression or posttraumatic stress disorder.

Modern molecular and pathophysiological concepts suggest that glucocorticoid receptors (GRs) play a crucial role for the pathogenesis, course and therapy of affective or emotional disorders. Specifically, an impairment of GR signaling has been associated with major depression, whereas overactivity or hyperresponsiveness of GRs have been conceptualized for posttraumatic stress disorder (PTSD). Recently, several research groups have generated transgenic mouse strains that under- or overexpress GRs, respectively. These animals seem to represent valuable tools for studying the foregoing hypotheses. Indeed, first results indicate that mice with a deficit in GR expression show a depression-like behavioral phenotype as well as characteristic neuroendocrinological changes observed in depressive patients. Particularly, GR heterozygous mice with a 50% reduction of GR expression represent a model for combined effects of both genetic and environmental manipulations, since their depression-like behavior becomes only manifest after stress-exposure. Thus, the phenotype of this strain mimics the human situation in depressive disorders, in which individuals at risk are predisposed to develop depressive episodes after stress. It is currently less clear whether, and in which way, mice that overexpress GRs can serve as models for PTSD, or mimic at least specific aspects of the clinical syndrome. The latter strains have still to be subjected to specific tests analyzing conditioning and sensitization processes in fearful situations. So far, mice with compromised GR expression seem to be a good tool to further study molecular, pathophysiological and cellular/structural alterations that underlie specific behavioral features such as despair or helplessness. A major challenge is to decipher which signs and symptoms in patients correspond to these animal behavioral constructs, and to elucidate whether it is possible to gain insights from the animals' response to specific treatments for human therapy.

Evaluation of effects of previous exposure to an acute stressor before testing for depression-like behaviours in mice.

Test batteries are an essential and broadly used tool for behavioural phenotyping, especially with regard to mouse models of particular diseases, such as depression. Facing the problem of an often limited number of mutant animals, it therefore seems crucial to develop and optimise such test batteries in terms of an ideal throughput of subjects. This study aimed to characterize several common stressors, which are used for the investigation of depressive-like features with regard to their capability of each of them to affect performance in a subsequent behavioural test. Here we investigated swim-, restraint- and footshock-stress in male C57/BL6 mice, focusing on post-stress corticosterone elevations as well as potential effects on the behavioural level. The stressors increased circulating corticosterone levels when assessed 1 h after exposure. On the behavioural level, no test interactions could be detected, which suggests, that in general, combining these test conditions in experiments with a restricted availability of animals seems to be rather unproblematic.

Nature vs. nurture: can enrichment rescue the behavioural phenotype of BDNF heterozygous mice?

In earlier experiments we have demonstrated that group-housing in a rather impoverished "standard" environment can be a crucial stress factor in male C57Bl/6 mice. The present study aimed at investigating the effect of combining a probable genetic vulnerability--postulated by the "Neurotrophin Hypothesis of Depression"--with the potentially modulating influence of a stressful environment such as "impoverished" standard housing conditions. For that purpose mice with a partial deletion of brain-derived neurotrophic factor (BDNF) were group-housed under standard and enriched housing conditions and analysed in a well-established test battery for emotional behaviours. Standard group-housing affected emotional behaviour in male and female BDNF heterozygous mice, causing an increase in anxiety, changes in exploration as well as nociception. Providing the animals' cages with supplementary enrichment, however, led to a rescue of emotional alterations, which emphasises the significance of external factors and their relevance for a valid investigation of genetic aspects in these mutants as well as others, which may be examined in terms of stress-responsiveness or emotionality.

CREB-regulated diurnal activity patterns are not indicative for depression-like symptoms in mice and men.

Activation of the transcription factor CREB by Ser142 phosphorylation is implicated in synchronizing circadian rhythmicity, which is disturbed in many depressive patients. Hence, one could assume that emotional behaviour and neuroendocrinological markers would be altered in CREB(S142A) mice, in which serine 142 is replaced by alanine, preventing phosphorylation at this residue. Moreover, associations of CREB Ser142 and seasonal affective disorder (SAD) might be detectable by the analysis of single-nucleotide polymorphisms (SNPs) in the CREB gene close to the Ser142 residue in SAD patients. However, neither CREB(S142A) mice demonstrate features of depression, nor there is evidence for an association of SAD with the CREB genotypes. Nevertheless, in humans there is an association of a global seasonality score and circadian rhythmicity with the CREB genotypes in healthy control probands, but not SAD patients. This parallels the phenotype of CREB(S142A) mice, presenting alterations of circadian rhythm and light-induced entrainment. Thus it is reasonable to assume that CREB Ser142 represents a molecular switch in mice and men, which is responsible for the (dys)regulation of circadian rhythms.

Marble burying as compulsive behaviors in male and female mice.

Marble burying is considered an, albeit controversial, animal model of the compulsive like behaviors of obsessive-compulsive disorder (OCD). Hallmark features of OCD patients are similarities and, more prominent, differences from anxiety disorders, e.g., the absence of sex differences and resistance to spontaneous remission. We report an experiment on marble burying by male and female C57/BL6/N mice. Animals were administered either the classic anxiolytic drug, diazepam, that targets the GABA receptor or a "pure" inhibitor of the serotonin transporter, escitalopram, that has been reported to be particularly effective in OCD. A burying paradigm that more precisely mimics the human condition was used, e.g., testing in the home environment, chronic drug exposure and acknowledging individual differences by pre-selecting for high marble burying. Results were that there were no sex differences in groups treated with drugs or in control mice. Both diazepam and escitalopram decreased numbers of marbles buried compared to vehicle-only controls in the absence of correlated changes in anxiety. Diazepam, however, was more effective than escitalopram in suppressing MB. The conclusion is that along with serotonin, GABA is involved in regulating compulsive behaviors. The marble burying paradigm may prove more useful for pharmacological drugs tests of impulsivity or attention deficit because of the involvement of serotonin and GABA in both disorders.

May the use of different background strains 'strain' the stress-related phenotype of GR+/- mice?

Genetically altered mice are available on different background strains. While respective backcrosses are often performed for pragmatic reasons, e.g. references, comparability, or existing protocols, the interaction between the mutations per se and the background strain often remains a neglected factor. The heterozygous mutation of the glucocorticoid receptor gene (GR) represents a well-examined model for depressive-like behavior in mice. To address the question in how far a robust depressive-like phenotype on a distinct background strain may allow a generalized conclusion, we analyzed respective phenotypes in two commonly used inbred strains: i.) C57BL/6N and ii.) BALB/c. Beside the use of different genetic models, we also extended our approach by applying two alternative paradigms to induce a depressive-like phenotype. Our study therefore comprised the model of 'unpredictable chronic mild stress' (UCMS) for four weeks and 'learned helplessness' (LH), which were used to study the role of GR, a key player in the development of depression. In the course of the experiment two cohorts of male GR+/- mice on either C57BL/6N or BALB/c background strain underwent a behavioral test battery to assess basal and depressive-like features. While both stress paradigms were functional in inducing depressive-like changes, the results were strictly strain-dependent. The genetic consequences became even more obvious under non-stress conditions with significant effects detected in BALB/c mice, which indicates a different basal stress predisposition due to differences in the genetic background.

Mice with genetically altered glucocorticoid receptor expression show altered sensitivity for stress-induced depressive reactions.

Altered glucocorticoid receptor (GR) signaling is a postulated mechanism for the pathogenesis of major depression. To mimic the human situation of altered GR function claimed for depression, we generated mouse strains that underexpress or overexpress GR, but maintain the regulatory genetic context controlling the GR gene. To achieve this goal, we used the following: (1) GR-heterozygous mutant mice (GR+/-) with a 50% GR gene dose reduction, and (2) mice overexpressing GR by a yeast artificial chromosome resulting in a twofold gene dose elevation. GR+/- mice exhibit normal baseline behaviors but demonstrate increased helplessness after stress exposure, a behavioral correlate of depression in mice. Similar to depressed patients, GR+/- mice have a disinhibited hypothalamic-pituitary-adrenal (HPA) system and a pathological dexamethasone/corticotropin-releasing hormone test. Thus, they represent a murine depression model with good face and construct validity. Overexpression of GR in mice evokes reduced helplessness after stress exposure, and an enhanced HPA system feedback regulation. Therefore, they may represent a model for a stress-resistant strain. These mouse models can now be used to study biological changes underlying the pathogenesis of depressive disorders. As a first potential molecular correlate for such changes, we identified a downregulation of BDNF protein content in the hippocampus of GR+/- mice, which is in agreement with the so-called neurotrophin hypothesis of depression.

Stress-resistant mice overexpressing glucocorticoid receptors display enhanced BDNF in the amygdala and hippocampus with unchanged NGF and serotonergic function.

Dysfunctional glucocorticoid receptor (GR) signaling has been shown to be involved in the pathogenesis of depressive behavior in mice and humans. In accordance with this hypothesis GR overexpressing mice are less susceptible to develop depressive-like behavior when subjected to stressful events. Here, we analyzed GR overexpressing mice for morning and evening content of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and the tissue levels of serotonin and its metabolite 5-hydroxyindoleacetic acid) in brain areas suspected to be involved in stress adaptation. BDNF concentrations in the hippocampus and amygdala/piriform cortex were significantly enhanced in GR overexpressing mice (by maximally +103%) compared to wildtype animals. Diurnal variations, as detected for NGF in the hypothalamus, for BDNF in the frontal cortex and striatum and for serotonergic function in the frontal cortex and hypothalamus, were not affected by the genotype. In conclusion, GR overexpression-dependent increases of hippocampal and amygdala BDNF content presumably represent a dynamic correlate of enhanced stress resistance.

Effects of Embryo Transfer on Emotional Behaviors in C57BL/6 Mice.

Microbiologic standardization plays a key role in the management of animal facilities because contamination of stock could affect the health status and wellbeing of animals and thereby induce artifacts in biomedical research. One common method to avoid the dissemination of pathogens is embryo transfer (ET). Although disturbances in the perinatal environment may cause long-lasting effects on the behavior and physiology of mouse offspring, the influences of ET during this sensitive phase have not yet been addressed. Our study investigated the effects of various components of ET (anesthesia, surgery, recipient strain) on the behavior of dams (exploration, nest-building) and offspring (nest-building, exploration, anxiety, and social and depressive-like behaviors). For ET, the donor strain C57BL/6N and a standard protocol were used. Whereas treatment with anesthesia-analgesia did not affect maternal behavior, female offspring demonstrated overall effects on weight gain and corticosterone levels. Compared with naturally delivered female offspring, dams obtained through ET demonstrated decreased exploration and nest-building. In addition, female ET-derived offspring had enhanced levels of anxiety and increased social interest. Furthermore, ET-derived dams obtained by using NMRI as the recipient strain showed increased exploratory behavior compared with that of dams obtained by using C57 mice as recipients. Compared with using C57 as recipients, both sexes of offspring transferred into NMRI recipients weighed more, and female mice showed a depressive-like phenotype. Our findings suggest that ET, now considered to be a routine procedure in animal husbandry, bears the risk of introducing artifacts.

Mutant mouse models of depression: candidate genes and current mouse lines.

Depression is a multifactorial and multigenetic disease. At present, three main theories try to conceptualize its molecular and biochemical mechanisms, namely the monoamine-, the hypothalamus-pituitary-adrenal- (HPA-) system- and the neurotrophin-hypotheses. One way to explore, validate or falsify these hypotheses is to alter the expression of genes that are involved in these systems and study their respective role in animal behavior and neuroendocrinological parameters. Following an introduction in which we briefly describe each hypothesis, we review here the different mouse lines generated to study the respective molecular pathways. Among the many mutant lines generated, only a few can be regarded as genetic depression models or as models of predisposition for a depressive syndrome after stress exposure. However, this is likely to reflect the human situation where depressive syndromes are complex, can vary to a great extent with respect to their symptomatology, and may be influenced by a variety of environmental factors. Mice with mutations of candidate genes showing depression-like features on behavioral or neurochemical levels may help to define a complex molecular framework underlying depressive syndromes. Because it is conceivable that manipulation of one single genetic function may be necessary but not sufficient to cause complex behavioral alterations, strategies for improving genetic modeling of depression-like syndromes in animals possibly require a simultaneous targeted dysregulation of several genes involved in the pathogenesis of depression. This approach would correspond to the new concept of 'endophenotypes' in human depression research trying to identify behavioral traits which are thought to be encoded by a limited set of genes.

Social and structural housing conditions influence the development of a depressive-like phenotype in the learned helplessness paradigm in male mice.

Structural and social factors are known to play a crucial role in the pathogenesis of depression. Since animal models of depression are a major tool to gain insights into the mechanisms involved in the pathophysiology of this disease it is important not only to exploit but also to be aware of factors that may affect these models. As housing represents a fundamental external factor, which is controversially debated to affect the animals' emotionality, this study aimed to investigate the impact of different social and structural housing conditions on the development of a depressive-like syndrome in the learned helplessness paradigm. Group housing in an impoverished environment led to an increased vulnerability in the learned helplessness paradigm. Groups that were housed enriched, however, were less helpless. Furthermore impoverished conditions did not increase the vulnerability in single housed animals. Regarding emotionality in the animals, basal anxiety was reduced and the exploration was enhanced by group housing and enriched environment. These results suggest that housing conditions significantly influence the outcome of learned helplessness studies.