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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.

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.

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.

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.

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.

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.

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.

Environmental Enrichment has been shown in experimental contexts to have clear and often beneficial effects on animal physiology and behavior. Housing prior to experiments can represent a large proportion of an animal’s living conditions, and improving housing conditions can be seen as “refinement” in a 3R-Context. However, large-dimensional implementation in rodent facilities often lack systematic analysis of respective refinement measures. Although enrichment is a legally binding element for housing animals according to EU law, potential effects on data variability are often neglected or not taken in consideration for experimental designs. Here, we aimed at implementing a new and innovative tool to improve wellbeing without side effects applicable for male and female mice. This poses a great challenge since the social structure in this species allows group housing in females without the risk of aggressive interaction, while in males despotic dominance hierarchy is often associated with agonistic interactions. Thus, we focused on enrichment-induced changes in behavior and stress physiology emphasizing effects on data variability in both sexes. Accordingly, recycled cage lids (resembling sex-neutral early environmental experiences in mice) were formed and three shapes of different structures were examined (‘Cage Climber’): ‘Triangle Climber’, ‘Bridge Climber’ and ‘Round Arch Climber’. The results demonstrate significant preferences of C57BL/6 N mice for any of the three structures in comparison with a neutral object when presented in a Novel Object Test. Despite intense use of enrichment, there were neither behavioral alterations detectable in a test battery assessing locomotion, anxiety and sociability nor in assessment of stress physiological parameters such as stress hormone metabolites, analyzed non-invasively from feces. Additionally, the structural supplement did not affect general variability of data in both male and female mice. To promote well-being of mice in a 3R-matched context, our study recommends the use of properly assessed structural enrichment, such as ‘Cage Climbers’ combined with nesting material to satisfy physical and thermal needs in the cage environment.

Early adverse experiences are known to influence the risk of developing psychiatric disorders later. To shed further light on the development of laboratory mice, we systematically examined the influence of a prenatal or postnatal olfactory stressor, namely unfamiliar male mouse faeces, presented to pregnant or nursing mouse dams. Maternal and offspring behaviours were then examined. Maternal behaviours relative to controls revealed changes in nest building by the pregnant dams exposed to the unfamiliar faeces. There were no differences among groups on pup retrieval or exploration by the dams. Behavioural phenotyping of male and female offspring as adults included measures of exploration, anxiety, social and depressive-like behaviours. Additionally, serum corticosterone was assessed as a marker of physiological stress response. Group differences were dependent on the sex of the adult offspring. Males raised by dams that were stressed during pregnancy presented elevated emotionality as indicated by increased numbers of faecal boluses in the open field paradigm. Consistent with the effects of prenatal stress on the males only the prenatally stressed females had higher body weights than their respective controls. Indeed, males in both experimental groups had higher circulating corticosterone levels. By contrast, female offspring of dams exposed to the olfactory stressor after parturition were more anxious in the O-maze as indicated by increased latencies in entering the exposed areas of the maze. These findings emphasize the necessity for researchers to consider the pre- and postnatal environments, even of mice with almost identical genetic backgrounds, in designing experiments and interpreting their data.

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.

Currently, the mouse represents the preferred model organism among mammals used for animal studies. Due to a great availability of mutant strains it represents a standard method to analyze in vivo the effects of targeted gene manipulations. While this – at least in theory – represents a valuable tool to elucidate the pathophysiology of certain human diseases, there are several caveats which need to be considered working with animals. In our study we aimed at elucidating, how a widely established breeding strategy, i.e. the use of “foster mothers” to save the survival of compromised mouse pups for ongoing experiments, per se, affects the emotional phenotype of the fostered offspring. Since it is a popular method to use outbred strains like NMRI to do this job, we sought to evaluate the potential effects of such an artificial postnatal condition and compare either offspring nurtured by their biological mothers or two different strains of foster mothers. Hence we analysed changes in maternal care and later on the emotional behaviour of male and female C57BL/6 mice reared by (i) their biological C57BL/6 mothers, (ii) C57BL/6 foster mothers and (iii) NMRI foster mothers in a behavioural test battery. In addition we assessed corticosterone levels as indicator for stress-physiological changes. Besides clear differences in maternal behaviour, our study indicates an altered emotional state (i.e. differences in anxiety and depressive-like features) in mice reared by different “categories” of mothers, which emphasizes the importance to embed such perinatal conditions in the evaluation of animal-deriving data.

Behavioural studies using transgenic techniques in mice usually require extensive backcrossing to a defined background strain, e.g. to C57BL/6. In this study we investigated whether backcrossing can be replaced by using the 129SvEv strain from which the embryonic stem cells are generally obtained for gene targeting strategies to analyze e.g. depression-like behaviour. For that purpose we subjected male and female 129SvEv mice to two frequently used depression tests and compared them with commonly used C57BL/6 mice. 129SvEv and C57BL/6 mice exhibited differing profiles with regard to locomotion and pain sensitivity. However, in the learned helplessness paradigm, a procedure, which represents a valid method to detect depressive-like behaviour, 129SvEv animals develop a similar level of helplessness as C57BL/6 mice. One great advantage of the 129SvEv animals though, is the fact that in this strain even females develop helplessness, which could not be produced in C57BL/6 mice. In the tail suspension test, both genders of 129SvEv exhibited more despair behaviour than C57BL/6 animals. We therefore suggest that this strain may be utilized in the establishment of new test procedures for affective diseases, since costly and time-consuming backcrossing can be prevented, depressive-like behaviour may be analyzed effectively, and gender-specific topics could be addressed in an adequate way.

The 5-HT(4) receptor is a new potential target for antidepressant treatment and may be implicated in the pathogenesis of depression. This study investigated differences in 5-HT(4) receptor and 5-HT transporter (5-HTT) binding by quantitative autoradiography of [(3)H]SB207145 and (S)-[N-methyl-(3)H]citalopram in two murine models of depression-related states, olfactory bulbectomy and glucocorticoid receptor heterozygous (GR(+/-)) mice. The olfactory bulbectomy model is characterized by 5-HT system changes, while the GR(+/-) mice have a deficit in hypothalamic-pituitary-adrenal (HPA) system control. The olfactory bulbectomized mice displayed increased activity in the open field test, a characteristic depression-like feature of this model. After bulbectomy, 5-HT(4) receptor binding was increased in the ventral hippocampus (12%) but unchanged in the dorsal hippocampus, frontal and caudal caudate putamen. Among post hoc analyzed regions, there was a 14% decrease in 5-HT(4) receptor binding in the olfactory tubercles. The 5-HTT binding was unchanged in the hippocampus and caudate putamen of bulbectomized mice but post hoc analysis showed small decreases in lateral septum and lateral globus pallidus. In comparison, GR(+/-) mice had increased 5-HT(4) receptor (11%) binding in the caudal caudate putamen and decreased 5-HTT binding in the frontal caudate putamen but no changes in dorsal and ventral hippocampus. Post hoc analysis showed increased 5-HT(4) receptor binding in the olfactory tubercles of GR(+/-) mice. In conclusion, we have found brain regional changes in 5-HT(4) receptor and 5-HTT transporter binding in two murine models of depression-related states, characterized by 5-HT and HPA system changes.

Increasing evidence suggests that depression is characterised by impaired brain plasticity that might originate from the interaction between genetic and environmental risk factors. Hence, the aim of this study was to investigate changes in neuroplasticity following exposure to stress, an environmental condition highly relevant to psychiatric disorders, in glucocorticoid receptor-deficient mice (GR +/−), a genetic model of predisposition to depression. Specifically, we have analysed the neurotrophin brain-derived neurotrophic factor (BDNF) and the immediate-early gene activity-regulated cytoskeletal-associated protein (Arc), two closely related molecules that can contribute to neuroplastic and morphological changes observed in depression. We found a region-specific influence of the GR-genotype on BDNF levels both under basal and stress conditions. Steady-state levels of BDNF mRNA were unchanged in hippocampus while up-regulated in frontal lobe of GR+/− mice. Following exposure to an acute stress, increased processing from pro- to mature BDNF was observed in hippocampal synaptosomes of wild-type mice, but not in GR mutants. Furthermore, the stress-dependent modulation of Arc was impaired in the hippocampus of GR+/− mice. These results indicate that GR+/− mice show overt differences in the stress-induced modulation of neuroplastic proteins, which may contribute to pathologic conditions that may originate following gene x environment interaction.

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.

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.

Stress-induced helplessness in rodents constitutes a well-defined model to investigate neurobiological mechanisms of depression. Neurotrophins like nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) have both been shown to be involved in neurobiological changes of physiological and pathological reactions to stress. In this study we investigated NGF and BDNF protein levels in the frontal cortex and hippocampus in mice treated with an established model of inducible helplessness via electric footshocks compared to untreated controls at various times (0 h up to 14 days after treatment). NGF levels were transiently decreased by one forth in the frontal cortex of shocked mice at 6 h after the stress treatment, whereas BDNF levels remained unchanged in the brain areas investigated throughout the time course. In addition, frontal cortex BDNF levels showed a significantly higher concentration in the right compared to the left hemisphere (up to 3-fold). This effect was detectable independently of treatment, namely in shocked and control mice at any time point measured. In conclusion, a transient decrease of frontal NGF constitutes the most striking correlate of neurobiological changes in this animal model of stress-induced change of behaviour. Interhemispherical differences of BDNF content in the frontal cortex are a new finding that might reflect intracerebral side dominance. Thus, subsequent studies of frontal cortex BDNF expression should carefully consider an interhemispherical variance to avoid misinterpretation.

Cytokine-dependent mechanisms in the CNS have been implicated in the pathogenesis of depression. Interleukin-6 is upregulated in depressed patients and dowregulated by antidepressants. It is, however, unknown whether IL-6 is involved in the pathogenesis of depression. We subjected IL-6-deficient mice (IL-6(-/-)) to depression-related tests (learned helplessness, forced swimming, tail suspension, sucrose preference). We also investigated IL-6 in the hippocampus of stressed wild-type mice. IL-6(-/-) mice showed reduced despair in the forced swim, and tail suspension test, and enhanced hedonic behavior. Moreover, IL-6(-/-) mice exhibited resistance to helplessness. This resistance may be caused by the lack of IL-6, because stress increased IL-6 expression in wild-type hippocampi. This suggests that IL-6 is a component in molecular mechanisms in the pathogenesis of depression. IL-6(-/-) mice represent tools to study IL-6-dependent signaling pathways in the pathophysiology of depression in vivo. Moreover, these mice may support the screening of compounds for depression by altering cytokine-mediated signaling.

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.

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.

Learning and memory, like most physiological processes, seem to be under the control of circadian rhythm. The recently cloned mPer1 and mPer2 genes play an important role in the regulation of the circadian rhythm. In this study, we tested mPer1 and mPer2 mutant mice in two different learning and memory paradigms, a water-maze place navigation task and contextual fear conditioning. In both learning tests, the hippocampus is critically involved. None of these learning types were affected by the mutations, suggesting that mPer1 and mPer2 do not play a major role in the regulation of hippocampus- dependent learning and memory.

The olfactory bulbectomy syndrome is thought to represent a rodent model for psychomotor agitated depression. While this model has been extensively characterized in rats, fewer studies have been conducted with mice. Therefore, the present study aimed at extending the characterization of the OBX-induced behavioral syndrome in mice, using tests like open field, novel object exploration, novel cage and T-maze learning. OBX mice exhibited hyperactivity in a brightly illuminated open field, and also in a novel home cage as well as in the T-maze. Furthermore, OBX mice demonstrated increased exploratory behavior in the novel object test and in the T-maze. The complex alterations described here with respect to locomotion and exploration are robust and can be achieved by relatively simple test procedures. The extended behavioral characterization of the murine OBX model may contribute in particular to the increasing need to test transgenic mice for the presence of depression-like behaviors.

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.

The "neurotrophin hypothesis" of depression predicts that depressive disorders in humans coincide with a decreased activity and/or expression of brain-derived neurotrophic factor (BDNF) in the brain. Therefore, we investigated whether mice with a reduced BDNF expression due to heterozygous gene disruption demonstrate depression-like neurochemical changes or behavioral symptoms. BNDF protein levels of adult BDNF(+/-) mice were reduced to about 60% in several brain areas investigated, including the hippocampus, frontal cortex, striatum, and hypothalamus. The content of monoamines (serotonin, norepinephrine, and dopamine) as well as of serotonin and dopamine degradation products was unchanged in these brain regions. By contrast, choline acetyltransferase activity was significantly reduced by 19% in the hippocampus of BDNF(+/-) mice, indicating that the cholinergic system of the basal forebrain is critically dependent on sufficient endogenous BDNF levels in adulthood. Moreover, BDNF(+/-) mice exhibited normal corticosterone and adrenocorticotropic hormone (ACTH) serum levels under baseline conditions and following immobilization stress. In a panel of behavioral tests investigating locomotor activity, exploration, anxiety, fear-associated learning, and behavioral despair, BDNF(+/-) mice were indistinguishable from wild-type littermates. Thus, a chronic reduction of BDNF protein content in adult mice is not sufficient to induce neurochemical or behavioral alterations that are reminiscent of depressive symptoms in humans.

Based on clinical and experimental observations, the neurotrophin hypothesis of depression as conceptualized originally, made the following predictions: (i) reduced activity of the CREB–BDNF–TrkB pathway is implicated in the pathogenesis of depression; (ii) activation of the CREB–BDNF–TrkB pathway is part of the molecular mechanisms of antidepressive therapy. This concept has recently been challenged, at least in part, by studies with transgenic mice. According to the neurotrophin hypothesis, mice with genetical disruptions of any part of the CREB–BDNF–TrKB pathway would be expected to display depression-like behaviors. However, none of several mouse strains investigated so far have exhibited such a behavior, some of them being even less ‘depressive’ than the controls. Despite some possible explanations for the lack of a depression-like phenotype, the present findings challenge the hypothesis that this signaling pathway plays a major role in the pathogenesis of depression. However, mice with impaired CREB signaling show reduced BDNF inducibility. Moreover, mice with impaired BDNF-TrkB signaling have a decreased behavioral response to antidepressants. Thus, despite the conflicting results on the role of the CREB–BDNF–TrkB pathway in the pathogenesis of depression, this signaling cascade seems to be directly involved in the mechanisms of antidepressive therapy. Therefore, pharmacological strategies should be developed to generate small-molecule agents that increase the expression and promote the release of BDNF more specifically and more efficiently than currently available antidepressants.

Increasing paternal age is known to be associated with a great variety of psychiatric disorders such as schizophrenia or autism. Hence the factor “age” may be taken as strategic tool to analyse specific scientific hypotheses. Additionally, this finding also needs to be addressed in rather pragmatically performed breeding protocols of model organisms, since otherwise artefacts may challenge the validity of the results. Our study was performed to investigate influences of advanced age of mouse dams (30 vs. 16 weeks) on maternal- and offspring behaviour. Adult offspring of both sexes was analysed in a test battery comprising paradigms for exploration, anxiety and depressive-like behaviours. Final blood sampling was conducted for stressphysiological analysis. Interestingly, advanced age of the mothers was associated with increased nest-building quality while maternal activity was unaffected. Moreover “maternal (mice) age” (MA) affected emotionality in the offspring, which became apparent in the dark–light box and the social recognition paradigm. These findings not only emphasize MA to model a potent risk factor with regard to emotional stability, but also underscore the vast necessity to include information about breeding protocols into the methods section of any animal study.

Working with mice represents a smart method to study pathophysiological mechanisms in vivo. However, using animals as model organisms also bears immense caveats. While many aspects in animal research are meanwhile standardized (e.g. nutrition, housing, health) the breeding environment remains unaddressed. Moreover, since the "production" of mice is mostly performed pragmatically, continuous mating (CM) represents a common method to boost the amount of offspring. This condition implies simultaneous pregnancy and lactation in presence of the male, which is associated with increased costs for the breeding dam. Facing the widely-accepted impact of perinatal conditions, our aim was to elucidate how CM affects emotional behaviour of mouse offspring. We therefore compared pregnant mice in CM with mice raising their pups without potentially disturbing influences. According to our hypothesis CM-deriving offspring should demonstrate increased anxiety and depression-like behaviour shaped by pre- and postnatal stress of the mother. Maternal care, i.e. nest building and pup retrieval, was analysed around delivery. To assess the emotional state of the offspring, males and females of either condition were exposed to a behavioural test battery for exploration, anxiety and fear, social and despair behaviour. In addition we analysed corticosterone as stressphysiological correlate. Our study demonstrates that CM affects the emotional phenotype regarding nearly all parameters addressed. These findings emphasize (i) the impact of the perinatal environment on stress-associated behaviour such as depression, and (ii) the need to imply perinatal conditions in the experimental design to decrease the risk of artefacts and increase the overall validity of animal studies.

Experimental evidence in mice indicates that environmental conditions affect females and males differently. However, in a recent study analyzing the heterozygous mutation of brain-derived neurotrophic factor (BDNF), both sexes presented a similar emotional phenotype, which became obvious only under impoverished, but not in enriched conditions suggesting an “enrichment-induced” rescue. To investigate the basis of this behavioral “rescue” effect, we analyzed neurochemical changes (BDNF expression, serotonergic changes, and corticosterone) in the hippocampus, frontal cortex and hypothalamus of animals housed under respective conditions. In male mice, enrichment induced an increase of BDNF expression in the hippocampus of both BDNF heterozygous (BDNF +/− ) and wild-types. Notably, in enriched-reared BDNF +/− mice BDNF mRNA and protein increased to levels comparable to those of wild-types in impoverished environment. In the frontal cortex of males, only wild-types presented an enrichment-induced increase of BDNF mRNA, while no effect of environment could be detected in BDNF protein levels of the male hypothalamus. A further male-specific effect of “environment” is the significant reduction of hypothalamic 5-hydroxyindoleacetic acid in enriched-housed wild-types. In female mice, environmental enrichment did not affect BDNF expression in the hippocampus and hypothalamus. However, comparable to males, an enrichment-induced increase of BDNF mRNA was detected in the frontal cortex of wild-types only. In contrast to males, no influence of environment on serotonergic parameters was observed. Male and female corticosterone levels were neither affected by “genotype” nor by “environment”. In conclusion, we propose that the rescue of the emotional phenotype by environmental enrichment in BDNF +/− mice is directed by distinct mechanisms in males and females. Only in male BDNF +/− mice the rescue is related to an increase in hippocampal BDNF expression suggesting that enrichment triggers different neuronal systems in a gender-specific manner.

According to the "neurotrophin hypothesis", brain-derived neurotrophic factor (BDNF) is an important candidate gene in depression. Moreover, environmental stress is known to represent a risk factor in the pathophysiology and treatment of this disease. To elucidate, whether changes of BDNF availability signify cause or consequence of depressive-like alterations, it is essential to look for endophenotypes under distinct genetic conditions (e.g. altered BDNF expression). Furthermore it is crucial to examine environment-driven BDNF regulation and its effect on depressive-linked features. Consequently, gene × environment studies investigating prospective genetic mouse models of depression in different environmental contexts become increasingly important. The present review summarizes recent findings in BDNF-mutant mice, which have been controversially discussed as models of depression and anxiety. It furthermore illustrates the potential of environment to serve as naturalistic stressor with the potential to modulate the phenotype in wildtype and mutant mice. Moreover, environment may exert protective effects by regulating BDNF levels as attributed to "environmental enrichment". The effect of this beneficial condition will also be discussed with regard to probable "curative/therapeutic" approaches.

Major depression is associated with both dysregulation of the hypothalamic pituitary adrenal axis and serotonergic deficiency, not the least of the 5-HT2A receptor. However, how these phenomena are linked to each other, and whether a low 5-HT2A receptor level is a state or a trait marker of depression is unknown. In mice with altered glucocorticoid receptor (GR) expression we investigated 5-HT2A receptor levels by Western blot and 3H-MDL100907 receptor binding. Serotonin fibre density was analyzed by stereological quantification of serotonin transporter immunopositive fibers. To establish an effect of GR activation on 5-HT2A levels, mature organotypic hippocampal cultures were exposed to corticosterone with or without GR antagonist mifepristone and mineralocorticoid receptor (MR) antagonist spironolactone. In GR under-expressing mice, hippocampal 5-HT2A receptor protein levels were decreased (26.3 +/- 1.6%, p < 0.05) and frontal 5-HT2A receptor binding was decreased (20 +/- 15%, p < 0.01) as compared to wild-type mice. Conversely, in over-expressing GR mice hippocampal 5-HT2A receptor protein levels were increased (60.8 +/- 4.0%, p = 0.0001) and 5-HT2A receptor binding was increased in dorsal hippocampus (77 +/- 35%, p < 0.05) as compared to wild-type mice. No difference in serotonin fibre density was observed in the GR over-expressing mice, while the GR under-expressing mice showed lower serotonergic innervation in the frontal cortex area. An effect of GR activation on 5-HT2A receptor levels was further corroborated by the culture studies as long-term exposure of 3 microM corticosterone to organotypic hippocampal cultures increased 5-HT2A receptor levels (p < 0.05). The corticosterone-induced 5-HT2A receptor up-regulation was blocked by addition of either spironolactone or mifepristone.

The “Dopamine Deficiency Hypothesis” and a considerable number of recent pharmacological studies propose to thoroughly verify and improve the standard of knowledge regarding a possible role of dopamine in the pathogenesis and treatment of depression. To elucidate the potential role of D3 receptors in the development of a depressive-like phenotype, we subjected D3 receptor knockout mice to a series of selected behavioural paradigms particularly focussing on depressive-like traits. In our experimental design we exposed animals with a deficiency of the D3 receptor (D3−/−) to a standardised behavioural test battery, in which general changes of locomotion, exploration, anxiety and depressive-like characteristics (i.e. Learned Helplessness, Sucrose Consumption and Forced Swim test) can be detected. Thorough behavioural phenotyping, however, neither revealed behavioural consequences on the basal level (locomotion, exploration) nor depressive- or anxiety-like alterations. Hence, these results do not indicate an evident involvement of the D3 receptor in the development of a depression-like syndrome in mice for now. This does, however, not exclude the D3 receptor as a potential target for pharmacotherapy.

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.

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.

Tamoxifen-induced mutagenesis via the so-called CreERT2 fusion enzyme is a key technology for the inducible gene knockout in the adult murine brain. However, it requires a subchronic transient treatment with high doses of the non-selective estrogen receptor antagonist tamoxifen. It has been shown earlier that acute tamoxifen treatment causes behavioral alterations, while the long-term behavioral effects of tamoxifen in mice are so far unknown. Therefore C57BL/6 male mice, a common strain used for targeted mutagenesis and behavioral analyses, were subjected to a tamoxifen treatment protocol as used for inducible mutagenesis in vivo, and analyzed for effects on general behavior (locomotion, exploration), emotional behavior (anxiety, depression) and on learning and memory after a drug-free interval period of 4 weeks. The results demonstrate that a test for depression-like behavior, i.e. the Forced Swim Test, is affected even more than 4 weeks after tamoxifen treatment. In contrast, in all other tests, tamoxifen treated mice showed unaltered behaviors, indicating that the currently established 5-day protocol of tamoxifen treatment (40 mg/kg bid) for inducible mutagenesis has no or little effects on the behavior of C57BL/6 male mice after a latency period of 4 weeks. These results are important for all studies using tamoxifen-induced mutagenesis since this protocol obviously does not evoke alterations in general behaviors such as locomotion, exploration or anxiety-like behaviors, which might confound more complex behavioral analyses, nor does it affect standard tests for learning and memory, such as Morris Water Maze, contextual and cued Fear Conditioning and T-Maze learning.

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.

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.

The neurotrophin and serotonin (5-HT) hypotheses of depression were studied in a mouse model of reduced glucocorticoid receptor (GR) function (GR(+/-) mice), which recently has been proven as a murine model of predisposition for depressive behaviour under stressful conditions. In this model we studied diurnal changes in neurotrophins and serotonergic function in candidate brain regions mediating depressive behaviour. Morning and evening levels of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), 5-HT and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) were analyzed in representative brain regions of GR(+/-) and wildtype mice. The diurnal variation of hippocampal BDNF in wildtypes with higher levels in the morning was absent in GR(+/-) mice. Hypothalamus and parietal cortex displayed enhanced BDNF levels in GR(+/-) mice. In the frontal cortex, striatum and hypothalamus NGF increased from morning to evening in both genotypes, with an exaggeration in GR(+/-) mice. The diurnal variation of 5-HT levels and turnover did not differ significantly between genotypes. It was only in the hypothalamus that the evening level of 5-HIAA was lower in GR(+/-) mice than in wildtype mice. In conclusion, the present data indicate a contribution of altered BDNF and NGF protein levels to the predisposition for depressive behaviour in the GR(+/-) mouse model of depression, but argue against an eminent role of the serotonergic system.

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.

The learned helplessness paradigm is a depression model in which animals are exposed to unpredictable and uncontrollable stress, e.g. electroshocks, and subsequently develop coping deficits for aversive but escapable situations (J.B. Overmier, M.E. Seligman, Effects of inescapable shock upon subsequent escape and avoidance responding, J. Comp. Physiol. Psychol. 63 (1967) 28-33 ). It represents a model with good similarity to the symptoms of depression, construct, and predictive validity in rats. Despite an increased need to investigate emotional, in particular depression-like behaviors in transgenic mice, so far only a few studies have been published using the learned helplessness paradigm. One reason may be the fact that-in contrast to rats (B. Vollmayr, F.A. Henn, Learned helplessness in the rat: improvements in validity and reliability, Brain Res. Brain Res. Protoc. 8 (2001) 1-7)--there is no generally accepted learned helplessness protocol available for mice. This prompted us to develop a reliable helplessness procedure in C57BL/6N mice, to exclude possible artifacts, and to establish a protocol, which yields a consistent fraction of helpless mice following the shock exposure. Furthermore, we validated this protocol pharmacologically using the tricyclic antidepressant imipramine. Here, we present a mouse model with good face and predictive validity that can be used for transgenic, behavioral, and pharmacological studies.

Although it has been postulated that adult neurogenesis, i.e. the generation of functional neurons from progenitor cells in the mammalian brain, is involved in both the pathogenesis of depressive disorders and the therapeutic effect of antidepressant drugs, its regulation is still poorly understood. Nitric oxide, a gaseous messenger molecule, represents a possible modulating agent as it is involved in learning and memory formation as well as synapto- and morphogenesis. Here we investigated whether adult neurogenesis is altered in mice lacking endothelial nitric oxide synthase (NOS-III). Compared to wild-type littermates, NOS-III-deficient mice showed a significant reduction in neuronal progenitor cell proliferation in the dentate gyrus, suggesting a role for NOS-III in the stimulation of neuroneogenesis. NeuN, beta-III-tubulin and GFAP double-immunolabelling demonstrated that proliferating progenitor cells differentiate preferentially into neurons but not into astrocytes. However, when the survival rate of newly formed cells was examined no difference between wild-type and NOS-III knockout mice was found, suggesting that NOS-III selectively exerts its effects on the proliferation of progenitor cells. This might be mediated by a decrease in vascular endothelial growth factor (VEGF) transcripts in the hippocampus of knockout animals. At the behavioural level, while NOS-III knockout mice displayed better and faster learning in a learned helplessness paradigm, no depression-like behaviours were observed. In conclusion, our results indicated that NOS-III is involved in the proliferation of neuronal progenitor cells, although behavioural analysis does not provide evidence for a pro-depressive effect of reduced neuroneogenesis.