Search

Traditionally, in biomedical animal research, laboratory rodents are individually examined in test apparatuses outside their home cages at selected time points. However, the outcome of such tests can be influenced by the novel environment, the time of day, separation from the social group, or the presence of an experimenter. Moreover, valuable information may be missed when the animals are only monitored in short periods. These issues can be overcome by longitudinal 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. Both the absolute (∼ ×26) and relative (∼ ×7) number of HCM-related publications increased from 1974 to 2020. In both mice and rats, 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. More than 70 % of the studies did not involve a disease model, but the percentage of studies using disease models increased since the 2000s. In most studies, animals were kept for short (up to 4 weeks) length periods in the HCM systems; intermediate length 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 hours, while 24-hour measurements have been more frequently since the 2000s. The systematic review demonstrated that manual monitoring is decreasing 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 publication year, 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 could be investigated in the home cage in recent times.Over the period covered in this study, techniques for HCM of mice and rats has improved considerably. This development is ongoing and further progress and 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.

Across sciences the lack of reproducibility has raised concerns that shake disciplinary foundations. In this article, the need for institutional solutions as one possible antidote to reproducibility issues is suggested, and Reproducibility Networks (RNs), as a case in point, are explained. In particular, we reflect on the establishment of the Finnish RN as part of a growing international network of RNs, and outline a bottom-up approach, which aims at helping to overcome the reproducibility crisis by distributing awareness of ethical, practical, and other domains of relevant knowledge in places where the future of science is being made: the pedagogical structures of research institutions and societies.

The key goal in biomedical research is a better understanding of disease aetiologies, which ideally results in strategies and recommendations for the prevention of diseases before they arise, and in the development of effective therapies. However, many concerns have been expressed about the reproducibility and the translational validity of preclinical research in animal models to inform clinical trials in humans. It has been proposed that improving internal, external and construct validity of animal studies will lead to improved translatability. Automated behaviour monitoring in the animal’s home cage, which allows for longitudinal assessment of individual trajectories over sufficiently long intervals for (chronic) drug treatment or phenotype progression, is a promising solution to these problems.

The mice and rats account as majority of laboratory animals used in biomedical research. However, their conventional housing conditions are often considered as impoverished, potentially leading to suffering, abnormal behavior and compromised welfare, which in turn may undermine the validity and generalizability of research data. The animals must have a possibility to express species-specific behavior but it is questionable if the current "standards" allow for that. With this study, we want to examine how different inbred strains react to the enriched environment (larger cages containing various items increasing cage complexity and promoting species-specific needs) and whether intermittent access to such cages is beneficial as compared to living in standard cage., The animals will arrive from the commercial vendor (Janvier) at the age of 4 weeks and upon arrival will be assigned to the groups in the Individually Ventilated Cage System (Tecniplast): standard cage (Tecniplast Greenline GM500); enriched cage (Tecniplast Greenline GR900); standard cage with intermittent transfer (2x / week) to enriched cage.For 8 weeks, the animals will be monitored in their cages with weekly body weight measurements and assessment of general appearance (body condition, fur, aggression, grooming / barbering).When the animals are 12 weeks old, behavioural testing will be caried out - light-dark test, open field with novel object, sociability, marble burrowing, time to integrate to nest, classical fear conditioning.Thereafter, the animals will be euthanized., A landmark study in 2001 (Würbel, H. (2001). "Ideal homes? Housing effects on rodent brain and behaviour." Trends Neurosci 24(4): 207-211.) challenged the standards of animal housing in research laboratories. Since then, a number of improvements have been invented and applied. In European Union, the minimum requirements for housing laboratory animals are defined by Directive 2010/63/EU. Accordingly, all animals shall be provided with space of sufficient complexity to allow expression of a wide range of normal behaviour. However, in practice this is often limited only to nesting material. Also, better (non-aversive) handling techniques for mice were recommended more than 10 years ago (Hurst, J. L. and R. S. West (2010). "Taming anxiety in laboratory mice." Nat Methods 7: 825-826.) but still many facilities and researchers are not used to this practice. Obviously, even if there are changes, they are slow. Recent systematic reviews have again called attention to the inadequate housing conditions of mice (Cait, J., A. Cait, R. W. Scott, C. B. Winder and G. J. Mason (2022). "Conventional laboratory housing increases morbidity and mortality in research rodents: results of a meta-analysis." BMC Biology 20(1): 15.). At the same time, it is interesting to note that vast majority of researchers seem to be satisfied with the current standards for housing and caretaking of laboratory animals (Walker, R. L., K. W. Saylor, M. Waltz and J. A. Fisher (2022). "Translational science: a survey of US biomedical researchers’ perspectives and practices." Lab Animal 51(1): 22-35.). For evaluation of enrichment protocols, it would be extremely important to develop animal-centric view (Hobbiesiefken, U., P. Mieske, L. Lewejohann and K. Diederich (2021). "Evaluation of different types of enrichment - their usage and effect on home cage behavior in female mice." PLOS ONE 16(12): e0261876.; Hobbiesiefken, U., B. Urmersbach, A. Jaap, K. Diederich and L. Lewejohann (2021). "Rating enrichment items by group-housed laboratory mice in multiple binary choice tests using an RFID-based tracking system." bioRxiv: 2021.2010.2020.465117.). However, it may be difficult if impossible to find the enrichment which suites equally well the number of inbred strains with distinct phenotype. Nevertheless, improvement and refinement is needed because inadequate housing will in the end negatively affect the reliability and validity of basic research with animal models. Rat playpens are gaining popularity and have been shown to have positive influence on the welfare (Hinchcliffe, J. K., M. G. Jackson and E. S. J. Robinson (2022). "The use of ball pits and playpens in laboratory Lister Hooded male rats induces ultrasonic vocalisations indicating a more positive affective state and can reduce the welfare impacts of aversive procedures." Laboratory Animals: 00236772211065920.) and recent evidence suggests that also conventionally-housed mice find voluntary playpen access rewarding.The aim of the proposed study is to evaluate the impact of three different housing conditions in male and female mice of four inbred strains. Behavioural testing, focusing on approach/avoidance and species-specific behaviors will be carried out after 8 weeks of housing mice in either standard cages, enriched cages (larger, containing various items like swings, running wheel, shelter) or in standard cage with intermittent placement in enriched cages. The results may be useful in further refinement of housing for laboratory mice.

Glial cell line-derived neurotrophic factor (GDNF) supports function and survival of dopamine neurons that degenerate in Parkinson’s disease (PD). Ectopic delivery of GDNF in clinical trials to treat PD is safe but lacks significant therapeutic effect. In pre-clinical models, ectopic GDNF is effective but causes adverse effects, including downregulation of tyrosine hydroxylase, only a transient boost in dopamine metabolism, aberrant neuronal sprouting, and hyperactivity. Hindering development of GDNF mimetic increased signaling via GDNF receptor RET by activating mutations results in cancer. Safe and effective mode of action must be defined first in animal models to develop successful GDNF-based therapies. Previously we showed that about a 2-fold increase in endogenous GDNF expression is safe and results in increased motor and dopaminergic function and protection in a PD model in young animals. Recently, similar results were reported using a novel Gdnf mRNA-targeting strategy. Next, it is important to establish the safety of a long-term increase in endogenous GDNF expression. We report behavioral, dopamine system, and cancer analysis of five cohorts of aged mice with a 2-fold increase in endogenous GDNF. We found a sustained increase in dopamine levels, improvement in motor learning, and no side effects or cancer. These results support the rationale for further development of endogenous GDNF-based treatments and GDNF mimetic., Graphical Abstract, Ectopic GDNF delivery raises concerns about efficacy in clinical trials of Parkinson’s disease and safety issues in preclinical studies. Andressoo and colleagues showed that a long-term, 2-fold increase in endogenous GDNF levels in mice is safe and improves motor and dopaminergic system function, supporting further development of endogenous GDNF-based therapies.

International audience; Academic Core Facilities are optimally situated to improve the quality of preclinical research by implementing quality control measures and offering these to their users.

Early life stress (ELS) is a well-characterized risk factor for mood and anxiety disorders. GABAergic microcircuits in the amygdala are critically implicated in anxiety; however, whether their function is altered after ELS is not known. Here we identify a novel mechanism by which kainate receptors (KARs) modulate feedforward inhibition in the lateral amygdala (LA) and show that this mechanism is downregulated after ELS induced by maternal separation (MS). Specifically, we show that in control rats but not after MS, endogenous activity of GluK1 subunit containing KARs disinhibit LA principal neurons during activation of cortical afferents. GluK1 antagonism attenuated excitability of parvalbumin (PV)-expressing interneurons, resulting in loss of PV-dependent inhibitory control and an increase in firing of somatostatin-expressing interneurons. Inactivation of Grik1 expression locally in the adult amygdala reduced ongoing GABAergic transmission and was sufficient to produce a mild anxiety-like behavioral phenotype. Interestingly, MS and GluK1-dependent phenotypes showed similar gender specificity, being detectable in male but not female rodents. Our data identify a novel KAR-dependent mechanism for cell-type and projection-specific functional modulation of the LA GABAergic microcircuit and suggest that the loss of GluK1 KAR function contributes to anxiogenesis after ELS.

While high risk of failure is an inherent part of developing innovative therapies, it can be reduced by adherence to evidence-based rigorous research practices. Numerous analyses conducted to date have clearly identified measures that need to be taken to improve research rigor. Supported through the European Union's Innovative Medicines Initiative, the EQIPD consortium has developed a novel preclinical research quality system that can be applied in both public and private sectors and is free for anyone to use. The EQIPD Quality System was designed to be suited to boost innovation by ensuring the generation of robust and reliable preclinical data while being lean, effective and not becoming a burden that could negatively impact the freedom to explore scientific questions. EQIPD defines research quality as the extent to which research data are fit for their intended use. Fitness, in this context, is defined by the stakeholders, who are the scientists directly involved in the research, but also their funders, sponsors, publishers, research tool manufacturers and collaboration partners such as peers in a multi-site research project. The essence of the EQIPD Quality System is the set of 18 core requirements that can be addressed flexibly, according to user-specific needs and following a user-defined trajectory. The EQIPD Quality System proposes guidance on expectations for quality-related measures, defines criteria for adequate processes (i.e., performance standards) and provides examples of how such measures can be developed and implemented. However, it does not prescribe any pre-determined solutions. EQIPD has also developed tools (for optional use) to support users in implementing the system and assessment services for those research units that successfully implement the quality system and seek formal accreditation. Building upon the feedback from users and continuous improvement, a sustainable EQIPD Quality System will ultimately serve the entire community of scientists conducting non-regulated preclinical research, by helping them generate reliable data that are fit for their intended use.

Low replicability of animal experiments is perceived as a major hurdle in the field of biomedicine. Attempts to enhance the replicability and to reduce the variability in basic research has led to the recommendation to use isogenic mice. The C57BL/6 strain has evolved as a gold standard strain for this purpose. However, C57BL/6 mice are maintained as substrains by multiple vendors. Evidence exists that the subtle differences between these mouse lines have not been systematically investigated and are often ignored. In the present study, we characterized the female mice of two closely related substrains (C57BL/6J and C57BL/6N) from three vendors in Europe (Charles River Laboratories, Envigo, Janvier Labs) in a battery of behavioral tests. Our data show and confirm substantial behavioral differences between the C57BL/6J and C57BL/6N mice. Importantly, the substrain differences were largely affected by the origin of the animals, as a significant effect of vendor or interaction between the substrain and vendor occurred in all tests. This work highlights the importance of adhering to precise international nomenclature in all publications reporting animal experiments. Moreover, the generalization of research findings from a single mouse substrain can be seriously limited due to genetic drift and environmental variables occurring at different vendors. However, heterogenization of samples, by including animals of different substrains, can enhance generalizability. These issues need to be seriously addressed to improve reproducibility, replicability, and the translational potential of the mouse models.

NETO1 and NETO2 are auxiliary subunits of kainate receptors (KARs). They interact with native KAR subunits to modulate multiple aspects of receptor function. Variation in KAR genes has been associated with psychiatric disorders in humans, and in mice, knockouts of the Grik1 gene have increased, while Grik2 and Grik4 knockouts have reduced anxiety-like behavior. To determine whether the NETO proteins regulate anxiety and fear through modulation of KARs, we undertook a comprehensive behavioral analysis of adult Neto1(-/-) and Neto2(-/-) mice. We observed no differences in anxiety-like behavior. However, in cued fear conditioning, Neto2(-/-), but not Neto1(-/-) mice, showed higher fear expression and delayed extinction compared to wild type mice. We established, by in situ hybridization, that Neto2 was expressed in both excitatory and inhibitory neurons throughout the fear circuit including the medial prefrontal cortex, amygdala, and hippocampus. Finally, we demonstrated that the relative amount of synaptosomal KAR GLUK2/3 subunit was 20.8% lower in the ventral hippocampus and 36.5% lower in the medial prefrontal cortex in Neto2(-/-) compared to the Neto2(+/+) mice. The GLUK5 subunit abundance was reduced 23.8% in the ventral hippocampus and 16.9% in the amygdala. We conclude that Neto2 regulates fear expression and extinction in mice, and that its absence increases conditionability, a phenotype related to post-traumatic stress disorder and propose that this phenotype is mediated by reduced KAR subunit abundance at synapses of fear-associated brain regions.

Parvalbumin-positive interneurons (PV+) are a key component of inhibitory networks in the brain and are known to modulate memory and learning by shaping network activity. The mechanisms of PV+ neuron generation and maintenance are not fully understood, yet current evidence suggests that signalling via the glial cell line-derived neurotrophic factor (GDNF) receptor GFR alpha 1 positively modulates the migration and differentiation of PV+ interneurons in the cortex. Whether GDNF also regulates PV+ cells in the hippocampus is currently unknown. In this study, we utilized a Gdnf "hypermorph" mouse model where GDNF is overexpressed from the native gene locus, providing greatly increased spatial and temporal specificity of protein expression over established models of ectopic expression. Gdnf(wt/hyper) mice demonstrated impairments in long-term memory performance in the Morris water maze test and an increase in inhibitory tone in the hippocampus measured electrophysiologically in acute brain slice preparations. Increased PV+ cell number was confirmed immunohistochemically in the hippocampus and in discrete cortical areas and an increase in epileptic seizure threshold was observed in vivo. The data consolidate prior evidence for the actions of GDNF as a regulator of PV+ cell development in the cortex and demonstrate functional effects upon network excitability via modulation of functional GABAergic signalling and under epileptic challenge.

Aims Recently we developed a model to study alcohol-seeking behaviour after withdrawal in a social context in female mice. The model raised several questions that we were eager to address to improve methodology. Methods In our model, female mice were group-housed in automated cages with three conditioned (CS+) corners and water in both sides of one separate non-conditioned corner. Water was available with opened doors at all the time of training. We established conditioning by pairing alcohol drinking with light cues. Here, we introduced prolonged access to increasing concentrations of alcohol instead of intermittent access. To study motivation to drink alcohol, we carried out the extinction tests on withdrawal days 1 (WD1) and 10 (WD10). During tests, the light cues were present in conditioned corners, but there was no liquid in the bottles. Results We found that the number of visits and nosepokes in the CS+ corner in the alcohol group was much higher than in the water group. Also, during training, the consumption of alcohol was increasing. In the extinction tests, we found that the number of nosepokes in the CS+ corner increased in the alcohol group on both WD1 and WD10. Conclusions Our study supports that alcohol-seeking behaviour after withdrawal can be modelled and studied in group-housed animals and environments without social isolation.

Animal models of neurodegenerative and neuropsychiatric disorders require extensive behavioral phenotyping. Currently, this presents several caveats and the most important are: (i) rodents are nocturnal animals, but mostly tested during the light period; (ii) the conventional behavioral experiments take into consideration only a snapshot of a rich behavioral repertoire; and (iii) environmental factors, as well as experimenter influence, are often underestimated. Consequently, serious concerns have been expressed regarding the reproducibility of research findings on the one hand, and appropriate welfare of the animals (based on the principle of 3Rs-reduce, refine and replace) on the other hand. To address these problems and improve behavioral phenotyping in general, several solutions have been proposed and developed. Undisturbed, 24/7 home-cage monitoring (HCM) is gaining increased attention and popularity as demonstrating the potential to substitute or complement the conventional phenotyping methods by providing valuable data for identifying the behavioral patterns that may have been missed otherwise. In this review, we will briefly describe the different technologies used for HCM systems. Thereafter, based on our experience, we will focus on two systems, IntelliCage (NewBehavior AG and TSE-systems) and Digital Ventilated Cage (DVC®, Tecniplast)-how they have been developed and applied during recent years. Additionally, we will touch upon the importance of the environmental/experimenter artifacts and propose alternative suggestions for performing phenotyping experiments based on the published evidence. We will discuss how the integration of telemetry systems for deriving certain physiological parameters can help to complement the description of the animal model to offer better translation to human studies. Ultimately, we will discuss how such HCM data can be statistically interpreted and analyzed.

The neural circuits regulating motivation and movement include midbrain dopaminergic neurons and associated inhibitory GABAergic and excitatory glutamatergic neurons in the anterior brainstem. Differentiation of specific subtypes of GABAergic and glutamatergic neurons in the mouse embryonic brainstem is controlled by a transcription factor Tal1. This study characterizes the behavioral and neurochemical changes caused by the absence of Tal1 function. The Tal1cko mutant mice are hyperactive, impulsive, hypersensitive to reward, have learning deficits and a habituation defect in a novel environment. Only minor changes in their dopaminergic system were detected. Amphetamine induced striatal dopamine release and amphetamine induced place preference were normal in Tal1cko mice. Increased dopamine signaling failed to stimulate the locomotor activity of the Tal1cko mice, but instead alleviated their hyperactivity. Altogether, the Tal1cko mice recapitulate many features of the attention and hyperactivity disorders, suggesting a role for Tal1 regulated developmental pathways and neural structures in the control of motivation and movement.

Peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) is a master regulator of mitochondria biogenesis and cell stress playing a role in metabolic and degenerative diseases. In the brain PGC-1α expression has been localized mainly to GABAergic interneurons but its overall role is not fully understood. We observed here that the protein levels of γ-aminobutyric acid (GABA) type A receptor-α2 subunit (GABARα2) were increased in hippocampus and brain cortex in transgenic (Tg) mice overexpressing PGC-1α in neurons. Along with this, GABARα2 expression was enhanced in the hippocampus of the PGC-1α Tg mice, as shown by quantitative PCR. Double immunostaining revealed that GABARα2 co-localized with the synaptic protein gephyrin in higher amounts in the striatum radiatum layer of the hippocampal CA1 region in the Tg compared with Wt mice. Electrophysiology revealed that the frequency of spontaneous and miniature inhibitory postsynaptic currents (mIPSCs) was increased in the CA1 region in the Tg mice, indicative of an augmented GABAergic transmission. Behavioral tests revealed an increase for anxiety-like behavior in the PGC-1α Tg mice compared with controls. To study whether drugs acting on PPARγ can affect GABARα2, we employed pioglitazone that elevated GABARα2 expression in primary cultured neurons. Similar results were obtained using the specific PPARγ agonist, N-(2-benzoylphenyl)-O-[2-(methyl-2-pyridinylamino) ethyl]-L-tyrosine hydrate (GW1929). These results demonstrate that PGC-1α regulates GABARα2 subunits and GABAergic neurotransmission in the hippocampus with behavioral consequences. This indicates further that drugs like pioglitazone, widely used in the treatment of type 2 diabetes, can influence GABARα2 expression via the PPARγ/PGC-1α system.

Cerebral dopamine neurotrophic factor (CDNF) is neuroprotective for nigrostriatal dopamine neurons and restores dopaminergic function in animal models of Parkinson's disease (PD). To understand the role of CDNF in mammals, we generated CDNF knockout mice (Cdnf(-/-)), which are viable, fertile, and have a normal life-span. Surprisingly, an age-dependent loss of enteric neurons occurs selectively in the submucosal but not in the myenteric plexus. This neuronal loss is a consequence not of increased apoptosis but of neurodegeneration and autophagy. Quantitatively, the neurodegeneration and autophagy found in the submucosal plexus in duodenum, ileum and colon of the Cdnf(-/-) mouse are much greater than in those of Cdnf(+/+) mice. The selective vulnerability of submucosal neurons to the absence of CDNF is reminiscent of the tendency of pathological abnormalities to occur in the submucosal plexus in biopsies of patients with PD. In contrast, the number of substantia nigra dopamine neurons and dopamine and its metabolite concentrations in the striatum are unaltered in Cdnf(-/-) mice; however, there is an age-dependent deficit in the function of the dopamine system in Cdnf(-/-) male mice analyzed. This is observed as D-amphetamine-induced hyperactivity, aberrant dopamine transporter function, and as increased D-amphetamine-induced dopamine release demonstrating that dopaminergic axon terminal function in the striatum of the Cdnf(-/-) mouse brain is altered. The deficiencies of Cdnf(-/-) mice, therefore, are reminiscent of those seen in early stages of Parkinson's disease.

Visual Abstract, Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an endoplasmic reticulum (ER) localized protein that regulates ER homeostasis and unfolded protein response (UPR). The biology of endogenous MANF in the mammalian brain is unknown and therefore we studied the brain phenotype of MANF-deficient female and male mice at different ages focusing on the midbrain dopamine system and cortical neurons. We show that a lack of MANF from the brain led to the chronic activation of UPR by upregulation of the endoribonuclease activity of the inositol-requiring enzyme 1α (IRE1α) pathway. Furthermore, in the aged MANF-deficient mouse brain in addition the protein kinase-like ER kinase (PERK) and activating transcription factor 6 (ATF6) branches of the UPR pathways were activated. Neuronal loss in neurodegenerative diseases has been associated with chronic ER stress. In our mouse model, increased UPR activation did not lead to neuronal cell loss in the substantia nigra (SN), decrease of striatal dopamine or behavioral changes of MANF-deficient mice. However, cortical neurons lacking MANF were more vulnerable to chemical induction of additional ER stress in vitro. We conclude that embryonic neuronal deletion of MANF does not cause the loss of midbrain dopamine neurons in mice. However, endogenous MANF is needed for maintenance of neuronal ER homeostasis both in vivo and in vitro.

Glial cell line-derived neurotrophic factor (GDNF) promotes the survival of dopaminergic neurons in vitro and in vivo. For this reason, GDNF is currently in clinical trials for the treatment of Parkinson’s disease (PD). However, how endogenous GDNF influences dopamine system function and animal behavior is not fully understood. We recently generated GDNF hypermorphic mice that express increased levels of endogenous GDNF from the native locus, resulting in augmented function of the nigrostriatal dopamine system. Specifically, Gdnf wt/hyper mice have a mild increase in striatal and midbrain dopamine levels, increased dopamine transporter activity, and 15% increased numbers of midbrain dopamine neurons and striatal dopaminergic varicosities. Since changes in the dopamine system are implicated in several neuropsychiatric diseases, including schizophrenia, attention deficit hyperactivity disorder (ADHD) and depression, and ectopic GDNF delivery associates with side-effects in PD models and clinical trials, we further investigated Gdnf wt/hyper mice using 20 behavioral tests. Despite increased dopamine levels, dopamine release and dopamine transporter activity, there were no differences in psychiatric disease related phenotypes. However, compared to controls, male Gdnf wt/hyper mice performed better in tests measuring motor function. Therefore, a modest elevation of endogenous GDNF levels improves motor function but does not induce adverse behavioral outcomes.

Drug addiction is a chronic brain disease and drugs of abuse cause long lasting neuroadaptations. Addiction is characterized by the loss of control over drug use despite harmful consequences, and high rates of relapse even after long periods of abstinence. Neurotrophic factors (NTFs) are well known for their actions on neuronal survival in the peripheral nervous system. Moreover, NTFs have been shown to be involved in synaptic plasticity in the brain. Brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) are two of the most studied NTFs and both of them have been reported to increase craving when administered into the mesocorticolimbic dopaminergic system after drug self-administration. Here we review recent data on BDNF and GDNF functions in addiction-related behavior and discuss them in relation to previous findings. Finally, we give an insight into how new technologies could aid in further elucidating the role of these factors in drug addiction.

Individually ventilated caging (IVC) systems for rodents are increasingly common in laboratory animal facilities. However, the impact of such substantial change in housing conditions on animal physiology and behavior is still debated. Most importantly, there arise the questions regarding reproducibility and comparison of previous or new phenotypes between the IVC and open cages. The present study was set up for detailed and systematic comparison of behavioral phenotypes in male and female mice of three widely used inbred strains (C57BL/6JRccHsd, DBA/2JRccHsd, 129S2/SvHSd) after being kept in two housing environments (IVC and open cages) for 6 weeks (since 4 weeks of age) before behavioral testing. The tests addressed exploratory, anxiety‐like and stress‐related behavior (light‐dark box, open field, forced swim test, stress‐induced hyperthermia), social approach and species‐specific behavior (nest building, marble burying). In all tests, large and expected strain differences were found. Somewhat surprisingly, the most striking effect of environment was found for basal body temperature and weight loss after one night of single housing in respective cages. In addition, the performance in light‐dark box and open field was affected by environment. Several parameters in different tests showed significant interaction between housing and genetic background. In summary, the IVC housing did not invalidate the well‐known differences between the mouse strains which have been established by previous studies. However, within the strains the results can be influenced by sex and housing system depending on the behavioral tasks applied. The bottom‐line is that the environmental conditions should be described explicitly in all publications., The individually ventilated caging housing did not invalidate the well‐known differences between the mouse strains established by previous studies. However, within the strains the results can be influenced by sex and housing system depending on the behavioral tasks applied.

Prolyl oligopeptidase (PREP) has been implicated in neurodegeneration and neuroinflammation and has been considered a drug target to enhance memory in dementia. However, the true physiological role of PREP is not yet understood. In this paper, we report the phenotyping of a mouse line where the PREP gene has been knocked out. This work indicates that the lack of PREP in mice causes reduced anxiety but also hyperactivity. The cortical volumes of PREP knockout mice were smaller than those of wild type littermates. Additionally, we found increased expression of diazepam binding inhibitor protein in the cortex and of the somatostatin receptor-2 in the hippocampus of PREP knockout mice. Furthermore, immunohistochemistry and tail suspension test revealed lack of response of PREP knockout mice to lipopolysaccharide insult. Further analysis revealed significantly increased levels of polysialylated-neural cell adhesion molecule in PREP deficient mice. These findings might be explained as possible alteration in brain plasticity caused by PREP deficiency, which in turn affect behaviour and brain development.

Mulibrey nanism (MUL) is a rare autosomal recessive multi-organ disorder characterized by severe prenatal-onset growth failure, infertility, cardiopathy, risk for tumors, fatty liver, and type 2 diabetes. MUL is caused by loss-of-function mutations in TRIM37, which encodes an E3 ubiquitin ligase belonging to the tripartite motif (TRIM) protein family and having both peroxisomal and nuclear localization. We describe a congenic Trim37 knock-out mouse (Trim37−/−) model for MUL. Trim37−/− mice were viable and had normal weight development until approximately 12 months of age, after which they started to manifest increasing problems in wellbeing and weight loss. Assessment of skeletal parameters with computer tomography revealed significantly smaller skull size, but no difference in the lengths of long bones in Trim37−/− mice as compared with wild-type. Both male and female Trim37−/− mice were infertile, the gonads showing germ cell aplasia, hilus and Leydig cell hyperplasia and accumulation of lipids in and around Leydig cells. Male Trim37−/− mice had elevated levels of follicle-stimulating and luteinizing hormones, but maintained normal levels of testosterone. Six-month-old Trim37−/− mice had elevated fasting blood glucose and low fasting serum insulin levels. At 1.5 years Trim37−/− mice showed non-compaction cardiomyopathy, hepatomegaly, fatty liver and various tumors. The amount and morphology of liver peroxisomes seemed normal in Trim37−/− mice. The most consistently seen phenotypes in Trim37−/− mice were infertility and the associated hormonal findings, whereas there was more variability in the other phenotypes observed. Trim37−/− mice recapitulate several features of the human MUL disease and thus provide a good model to study disease pathogenesis related to TRIM37 deficiency, including infertility, non-alcoholic fatty liver disease, cardiomyopathy and tumorigenesis., Summary: A congenic Trim37-deficient mouse model recapitulates several features of the human disorder Mulibrey nanism, and thus provides a good model to study disease pathogenesis related to TRIM37 deficiency.

Visual Abstract, NETO2 is an auxiliary subunit for kainate-type glutamate receptors that mediate normal cued fear expression and extinction. Since the amygdala is critical for these functions, we asked whether Neto2−/− mice have compromised amygdala function. We measured the abundance of molecular markers of neuronal maturation and plasticity, parvalbumin-positive (PV+), perineuronal net-positive (PNN+), and double positive (PV+PNN+) cells in the Neto2−/− amygdala. We found that Neto2−/− adult, but not postnatal day (P)23, mice had 7.5% reduction in the fraction of PV+PNN+ cells within the total PNN+ population, and 23.1% reduction in PV staining intensity compared with Neto2+/+ mice, suggesting that PV interneurons in the adult Neto2−/− amygdala remain in an immature state. An immature PV inhibitory network would be predicted to lead to stronger amygdalar excitation. In the amygdala of adult Neto2−/− mice, we identified increased glutamatergic and reduced GABAergic transmission using whole-cell patch-clamp recordings. This was accompanied by increased spine density of thin dendrites in the basal amygdala (BA) compared with Neto2+/+ mice, indicating stronger glutamatergic synapses. Moreover, after fear acquisition Neto2−/− mice had a higher number of c-Fos-positive cells than Neto2+/+ mice in the lateral amygdala (LA), BA, and central amygdala (CE). Altogether, our findings indicate that Neto2 is involved in the maturation of the amygdala PV interneuron network. Our data suggest that this defect, together with other processes influencing amygdala principal neurons, contribute to increased amygdalar excitability, higher fear expression, and delayed extinction in cued fear conditioning, phenotypes that are common in fear-related disorders, including the posttraumatic stress disorder (PTSD).

Developing reliable mouse models for social behaviour is challenging. Different tests have been proposed, but most of them consist of rather artificial confrontations of unfamiliar mice in novel arenas or are relying on social stress induced by aggressive conspecifics. Natural social interaction in home cage in laboratory has not been investigated well. IntelliCage is a fully automated home-cage system, where activity of the group-housed mice can be monitored along with various cognitive tasks. Here we report the behavioural profile of C57BL/6N (86) and BALB/c (BALB) female mice in IntelliCage when separated by strain, followed by monitoring of activity and formation of 'home-base' after mixing two strains. For that purpose, 3 cages were connected. Significant differences between the strains were established in baseline behaviour in conventional tests and in IntelliCage. The B6 mice showed reduced anxiety-like behaviour in open field and light-dark box, slightly enhanced exploratory activity in IntelliCage during initial adaptation and clearly distinct circadian activity. Mixing of two strains resulted in reduction of body weight and anhedonia in B6 mice. In addition, the B6 mice showed clear preference to previous home-cage, and formed a new home-base faster than BALB mice. In contrast, BALB mice showed enhanced activity and moving between the cages without showing any preference to previous home-cage. It could be argued that social challenge caused changes in both strains and different coping styles are responsible for behavioural manifestations. Altogether, this approach could be useful in modelling and validating mouse models for disorders with disturbed social behaviour.

To evaluate permanent effects of hippocampal and prefrontal cortex lesion on spatial tasks, lesioned and sham-operated female C57BL/6 mice were exposed to a series of conditioning schemes in IntelliCages housing 8–10 transponder-tagged mice from each treatment group. Sequential testing started at 51–172 days after bilateral lesions and lasted for 154 and 218 days in two batches of mice, respectively. Spontaneous undisturbed behavioral patterns clearly separated the three groups, hippocampals being characterized by more erratic hyperactivity, and strongly impaired circadian synchronization ability. Hippocampal lesions led to deficits in spatial passive avoidance, as well as in spatial reference and working memory tasks. Impairment was minimal in rewarded preference/reversal schemes, but prominent if behavioral responses required precise circadian timing or included punishment of wrong spatial choices. No differences between sham-operated and prefrontally lesioned subjects in conditioning success were discernible. These results corroborate the view that hippocampal dysfunction spares simple spatial learning tasks but impairs the ability to cope with conflicting task-inherent spatial, temporal or emotional cues. Methodologically, the results show that automated testing and data analysis of socially kept mice is a powerful, efficient and animal-friendly tool for dissecting complex features and behavioral profiles of hippocampal dysfunction characterizing many transgenic or pharmacological mouse models., Behavioural Brain Research, 352, ISSN:0166-4328, ISSN:1872-7549

Mounting evidence suggests the association of immune genes with complex neuropsychiatric diseases, such as schizophrenia. However, immune gene expression in the brain and their involvement in schizophrenia-related behavior in animal models have not been well studied so far. We analyzed the social (resident-intruder) and sensorimotor gating (pre-pulse inhibition (PPI) of acoustic startle) behaviors, and expression profiles of several brain immune genes in adult C57BL/6N and DBA/2J female mice. Compared to C57BL/6N mice, DBA/2J mice exhibited less social interaction in the resident-intruder test and reduced pre-pulse inhibition. The mRNA levels of Il1b and Il6 genes were significantly higher in the cortex and hypothalamus, while the mRNA level of C1qb was lower in the cortex, hippocampus and hypothalamus of DBA/2J mice compared to C57BL/6N mice. Furthermore, Tnfsf13b was up-regulated in the cortex and hippocampus, and so did Cd47 in the hippocampus, while Cx3cl1 was down-regulated in the cortex of DBA/2J mice. Our study demonstrates the differential expression of several immune genes in C57BL/6N and DBA/2J strains and more importantly provides clues on their potential importance in regulating schizophrenia-related endophenotypes in animal models.

It is about half a century ago when the so-called "Wise model" to study alcohol drinking behavior in rats was established. The model was based on voluntary intermittent access to increasing concentrations of alcohol. We aimed to establish a model of alcohol craving and used an extinction test on withdrawal days 1 and 10 to study motivation for alcohol. For this purpose, the alcohol drinking training was paired with light cues to establish conditioning. The extinction test was carried out without alcohol but in the presence of light cues and empty bottles. The outcome measures were number of visits, nosepokes, and licks in the conditioned corner where the number of nosepokes represents how much mice "want" alcohol and number of licks shows how much mice "like" alcohol. The number of nosepokes during withdrawal is a measure of craving. Late withdrawal craving was found when intermittent alcohol access was carried out in the automated cages. In this case, we observed a significant increase in the number of nosepokes on both withdrawal days 1 and 10 as compared to water control. The number of nosepokes in the withdrawal days did not correlate with alcohol dose, but number of nosepokes on withdrawal day 1 correlated with the number of nosepokes on the last training day. Although we did not observe incubation of alcohol craving after withdrawal, the craving was increased at the late time point. We conclude that we have established a new tool to study alcohol drinking behavior and craving in female mice.

Brain development is a complex process regulated by genetic programs and activity-dependent neuronal connectivity. Anesthetics profoundly alter neuronal excitability, and anesthesia during early brain development has been consistently associated with neuroapoptosis, altered synaptogenesis, and persistent behavioral abnormalities in experimental animals. However, the depth, and even more the duration and developmental time point(s) of exposure to anesthesia determine the neuropathological and long-term behavioral consequences of anesthetics. Here, we have investigated adulthood phenotypic changes induced by repeated but brief (30 min) isoflurane anesthesia delivered during two distinct developmental periods in male mice. A set of animals were subjected to anesthesia treatments at postnatal days 7, 8 and 9 (P7-9) when the animals are susceptible to anesthesia-induced neuroapoptosis and reduced synaptogenesis. To control the potential influence of (handling) stress, a separate group of animals underwent repeated maternal separations of similar durations. Another set of animals were exposed to the same treatments at postnatal days 15, 16 and 17 (P15-17), a developmental time period when anesthetics have been shown to increase synaptogenesis. Starting from postnatal week 9 the mouse phenotype was evaluated using a battery of behavioral tests that assess general locomotor activity (home cage activity, open field), learning and memory (water maze) and depression- (saccharin preference, forced swim test), anxiety- (light-dark box, stress-induced hyperthermia) and schizophrenia- (nesting, prepulse inhibition) related endophenotypes. Apart from mild impairment in spatial navigation memory, exposure to anesthesia treatments during P7-9 did not bring obvious behavioral alterations in adult animals. Importantly, maternal separation during the same developmental period produced a very similar phenotype during the water maze. Mice exposed to anesthesia during P15-17 showed mild hyperactivity and risk-taking behavior in adulthood, but were otherwise normal. We conclude that significantly longer administration periods are needed in order for early-life repeated exposures to anesthetics to produce behavioral alterations in adult mice.

Light-dark box and open field are conventional tests for assessment of anxiety-like behavior in the laboratory mice, based on approach-avoidance conflict. However, except the basic principles, variations in the equipment and procedures are very common. Therefore, contribution of certain methodological issues in different settings was investigated. Three inbred strains (C57BL/6, 129/Sv, DBA/2) and one outbred stock (ICR) of mice were used in the experiments. An effect of initial placement of mice either in the light or dark compartment was studied in the light-dark test. Moreover, two tracking systems were applied - position of the animals was detected either by infrared sensors in square box (1/2 dark) or by videotracking in rectangular box (1/3 dark). Both approaches revealed robust and consistent strain differences in the exploratory behavior. In general, C57BL/6 and ICR mice showed reduced anxiety-like behavior as compared to 129/Sv and DBA/2 strains. However, the latter two strains differed markedly in their behavior. DBA/2 mice displayed high avoidance of the light compartment accompanied by thigmotaxis, whereas the hypoactive 129 mice spent a significant proportion of time in risk-assessment behavior at the opening between two compartments. Starting from the light side increased the time spent in the light compartment and reduced the latency to the first transition. In the open field arena, black floor promoted exploratory behavior - increased time and distance in the center and increased rearing compared to white floor. In conclusion, modifications of the apparatus and procedure had significant effects on approach-avoidance behavior in general whereas the strain rankings remained unaffected.

Immune activation in the brain has been shown to contribute to neurodevelopmental and pathological progression of mental disorders, and microglia play a central role in these processes. But how genetic predisposition and environmental risk factors may act in combination to affect microglial activation and the underlying molecular mechanisms are largely unclear. In this work, we studied the inflammatory profile of microglia across four inbred strains of mice with different anxiety traits: C57BL/6J, FVB/N, DBA/2J, and 129S2/Sv. Importantly, we found that a high-anxiety strain, naïve DBA/2J mice, had significantly more M1 (MHCII(+)CD206(-))-polarized microglia, whereas another high-anxiety strain, naïve 129S2/Sv mice, expressed significantly more activated (MHCII(+)) perivascular macrophages than the other strains. After a systemic LPS challenge, polarization to M1 microglia in DBA/2J and 129S2/Sv mice was even more prominent than in C57BL/6J and FVB/N mice, and was correlated with their anxiety-like behaviors. Macrophage M1/M2 polarization in the spleen showed a similar pattern in DBA/2J and 129S2/Sv mice in response to LPS stimulation. Furthermore, DBA/2J mice expressed higher mRNA levels of Il1b, Il6, and Tnf, and higher Nos2/Arg1 ratio but lower Chi3l3 level in the hypothalamus before and after LPS stimulation, respectively. In comparison, 129S1/Sv, a sibling line of 129S2/Sv, expressed significantly higher levels of other immune-related genes in the brain. We further discovered a group of myeloid transcription factors that may underpin the strain-specific differences in microglial activation. We conclude that proinflammatory microglial activation reflects anxiety traits in mice, especially after a peripheral innate immune challenge. Our work sheds new light in understanding the potential molecular mechanisms of stress-induced microglial activation and polarization.

Summary: All forms of diabetes mellitus (DM) are characterized by the loss of functional pancreatic β cell mass, leading to insufficient insulin secretion. Thus, identification of novel approaches to protect and restore β cells is essential for the development of DM therapies. Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an endoplasmic reticulum (ER)-stress-inducible protein, but its physiological role in mammals has remained obscure. We generated MANF-deficient mice that strikingly develop severe diabetes due to progressive postnatal reduction of β cell mass, caused by decreased proliferation and increased apoptosis. Additionally, we show that lack of MANF in vivo in mouse leads to chronic unfolded protein response (UPR) activation in pancreatic islets. Importantly, MANF protein enhanced β cell proliferation in vitro and overexpression of MANF in the pancreas of diabetic mice enhanced β cell regeneration. We demonstrate that MANF specifically promotes β cell proliferation and survival, thereby constituting a therapeutic candidate for β cell protection and regeneration. : Potent strategies for diabetes mellitus (DM) prevention and treatment are urgently needed. MANF is an endoplasmic reticulum stress-inducible protein, but its physiological role in mammals is currently unknown. Lindahl et al. now find that MANF-deficient mice develop diabetes. Importantly, MANF protein enhances β cell proliferation in vitro and pancreatic MANF overexpression in diabetic mice enhances β cell regeneration. Because MANF is expressed in human β cells, it might have therapeutic potential for diabetes treatment.

Stressful life events are major environmental risk factors for anxiety disorders, although not all individuals exposed to stress develop clinical anxiety. The molecular mechanisms underlying the influence of environmental effects on anxiety are largely unknown. To identify biological pathways mediating stress-related anxiety and resilience to it, we used the chronic social defeat stress (CSDS) paradigm in male mice of two inbred strains, C57BL/6NCrl (B6) and DBA/2NCrl (D2), that differ in their susceptibility to stress. Using a multi-omics approach, we identified differential mRNA, miRNA and protein expression changes in the bed nucleus of the stria terminalis (BNST) and blood cells after chronic stress. Integrative gene set enrichment analysis revealed enrichment of mitochondrial-related genes in the BNST and blood of stressed mice. To translate these results to human anxiety, we investigated blood gene expression changes associated with exposure-induced panic attacks. Remarkably, we found reduced expression of mitochondrial-related genes in D2 stress-susceptible mice and in exposure-induced panic attacks in humans, but increased expression of these genes in B6 stress-susceptible mice. Moreover, stress-susceptible vs. stress-resilient B6 mice displayed more mitochondrial cross-sections in the post-synaptic compartment after CSDS. Our findings demonstrate mitochondrial-related alterations in gene expression as an evolutionarily conserved response in stress-related behaviors and validate the use of cross-species approaches in investigating the biological mechanisms underlying anxiety disorders., Author summary Genetic and environmental factors contribute to the etiology of psychiatric diseases but the underlying mechanisms are poorly understood. Chronic psychosocial stress is a well-known risk factor for anxiety disorders. To identify biological pathways involved in psychosocial stress-induced anxiety and resilience to it, we used a well-characterized mouse model of chronic social defeat stress (CSDS) in two inbred mouse strains, C57BL/6NCrl (B6) and DBA/2NCrl (D2), which differ in their susceptibility to stress. We focused on the bed nucleus of the stria terminalis, a key brain region behind stress-response and anxiety, and carried out genome-wide analysis of mRNA, and miRNA expression, and protein abundance. Bioinformatic integration of these data supported differences in mitochondrial pathways as a major stress response. To translate these findings to human anxiety, we investigated blood cell gene expression in mice and in panic disorder patients exposed to fearful situations and experiencing panic attacks. Concurring with our brain findings, expression of mitochondrial pathways was also affected in mouse and human blood cells, suggesting that the observed stress response mechanisms are evolutionarily conserved. Therefore, chronic stress may critically affect cellular energy metabolism, a finding that may offer new targets for therapeutic interventions of stress-related diseases.

There has been a long-standing need to develop efficient and standardized behavioral test methods for evaluating higher-order brain functions in mice. Here, we developed and validated a behavioral flexibility test in mice using IntelliCage, a fully automated behavioral analysis system for mice in a group-housed environment. We first developed a "behavioral sequencing task" in the IntelliCage that enables us to assess the learning ability of place discrimination and behavioral sequence for reward acquisition. In the serial reversal learning using the task, the discriminated spatial patterns of the rewarded and never-rewarded places were serially reversed, and thus, mice were accordingly expected to realign the previously acquired behavioral sequence. In general, the tested mice showed rapid acquisition of the behavioral sequencing task and behavioral flexibility in the subsequent serial reversal stages both in intra- and inter-session analyses. It was found that essentially the same results were obtained among three different laboratories, which confirm the high stability of the present test protocol in different strains of mice (C57BL/6, DBA/2, and ICR). In particular, the most trained cohort of C57BL/6 mice achieved a markedly rapid adaptation to the reversal task in the final phase of the long-term serial reversal test, which possibly indicated that the mice adapted to the "reversal rule" itself. In conclusion, the newly developed behavioral test was shown to be a valid assay of behavioral flexibility in mice, and is expected to be utilized in tests of mouse models of cognitive deficits.

Despite its key role in Alzheimer pathogenesis, the physiological function(s) of the amyloid precursor protein (APP) and its proteolytic fragments are still poorly understood. Previously, we generated APPsα knock-in (KI) mice expressing solely the secreted ectodomain APPsα. Here, we generated double mutants (APPsα-DM) by crossing APPsα-KI mice onto an APLP2-deficient background and show that APPsα rescues the postnatal lethality of the majority of APP/APLP2 double knockout mice. Surviving APPsα-DM mice exhibited impaired neuromuscular transmission, with reductions in quantal content, readily releasable pool, and ability to sustain vesicle release that resulted in muscular weakness. We show that these defects may be due to loss of an APP/Mint2/Munc18 complex. Moreover, APPsα-DM muscle showed fragmented post-synaptic specializations, suggesting impaired postnatal synaptic maturation and/or maintenance. Despite normal CNS morphology and unaltered basal synaptic transmission, young APPsα-DM mice already showed pronounced hippocampal dysfunction, impaired spatial learning and a deficit in LTP that could be rescued by GABAA receptor inhibition. Collectively, our data show that APLP2 and APP are synergistically required to mediate neuromuscular transmission, spatial learning and synaptic plasticity.