Role of Environment and Experimenter in Reproducibility of Behavioral Studies With Laboratory Mice.

Behavioral phenotyping of mice has received a great deal of attention during the past three decades. However, there is still a pressing need to understand the variability caused by environmental and biological factors, human interference, and poorly standardized experimental protocols. The inconsistency of results is often attributed to the inter-individual difference between the experimenters and environmental conditions. The present work aims to dissect the combined influence of the experimenter and the environment on the detection of behavioral traits in two inbred strains most commonly used in behavioral genetics due to their contrasting phenotypes, the C57BL/6J and DBA/2J mice. To this purpose, the elevated O-maze, the open field with object, the accelerating rotarod and the Barnes maze tests were performed by two experimenters in two diverse laboratory environments. Our findings confirm the well-characterized behavioral differences between these strains in exploratory behavior, motor performance, learning and memory. Moreover, the results demonstrate how the experimenter and the environment influence the behavioral tests with a variable-dependent effect, often with mutually exclusive contributions. In this context, our study highlights how both the experimenter and the environment can have an impact on the strain effect size without altering the direction of the conclusions. Importantly, the general agreement on the results is reached by converging evidence from multiple measures addressing the same trait. In conclusion, the present work elucidates the contribution of both the experimenter and the laboratory environment in the intricate field of reproducibility in mouse behavioral phenotyping.

Retinal Temperature Determination Based on Photopic Porcine Electroretinogram.

OBJECTIVE: Subthreshold retinal laser therapy (SLT) is a treatment modality where the temperature of the retinal pigment epithelium (RPE) is briefly elevated to trigger the therapeutic benefits of sublethal heat shock. However, the temperature elevation induced by a laser exposure varies between patients due to individual differences in RPE pigmentation and choroidal perfusion. This study describes an electroretinography (ERG)-based method for controlling the temperature elevation during SLT. METHODS: The temperature dependence of the photopic ERG response kinetics were investigated both ex vivo with isolated pig retinas and in vivo with anesthetized pigs by altering the temperature of the subject and recording ERG in different temperatures. A model was created for ERG-based temperature estimation and the feasibility of the model for controlling SLT was assessed through computational simulations. RESULTS: The kinetics of the photopic in vivo flash ERG signaling accelerated between 3.6 and 4.7%/°C, depending on the strength of the stimulus. The temperature dependence was 5.0%/°C in the entire investigated range of 33 to 44°C in ex vivo ERG. The simulations showed that the method is suitable for determining the steady-state temperature elevation in SLT treatments with a sufficiently long laser exposure and large spot size, e.g., during > 30 s laser exposures with > 3 mm stimulus spot diameter. CONCLUSIONS: The described ERG-based temperature estimation model could be used to control SLT treatments such as transpupillary thermotherapy. SIGNIFICANCE: The introduced ERG-based method for controlling SLT could improve the repeatability, safety, and efficacy of the treatment of various retinal disorders.

Experiments done in Black-6 mice: what does it mean?

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.

Housing mice in the individually ventilated or open cages-Does it matter for behavioral phenotype?

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.

Behavioural characterization of C57BL/6N and BALB/c female mice in social home cage - Effect of mixed housing in complex environment.

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 (B6) 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.

Heritability of hypothyroidism in the Finnish Hovawart population.

BACKGROUND: The Hovawart is a working and companion dog breed of German origin. A few hundred Hovawart dogs are registered annually in Finland. The most common disease with a proposed genetic background in Hovawarts is hypothyroidism. The disease is usually caused by lymphocytic thyroiditis, an autoimmune disorder which destroys the thyroid gland. Hypothyroidism can be treated medically with hormone replacement. Its overall incidence could also be reduced through selection, provided that the trait shows an adequate genetic basis. The aim of this study was to estimate the heritability of hypothyroidism in the Finnish Hovawart population. RESULTS: The pedigree data for the study were provided by the Finnish Kennel Club and the hypothyroidism data by the Finnish Hovawart Club. The data included 4953 dogs born between 1990 and 2010, of which 107 had hypothyroidism and 4846 were unaffected. Prior to the estimation of heritability, we studied the effects of gender, birth year, birth month, and inbreeding on susceptibility to hypothyroidism. Heritability was estimated with the probit model both via restricted maximum likelihood (REML) and Gibbs sampling, using litter and sire of the dog as random effects. None of the studied systematic effects or level of inbreeding had a significant effect on susceptibility to hypothyroidism. The estimated heritability of hypothyroidism varied from 0.47 (SE = 0.18) using REML to 0.62 (SD = 0.21) using Gibbs sampling. CONCLUSIONS: Based on our analysis, the heritability of hypothyroidism is moderate to high, suggesting that its prevalence could be decreased through selection. Thus, breeders should notify the breed association of any affected dogs, and their use for breeding should be avoided.