Effects of light regime and substrain on behavioral profiles of male C57BL/6 mice in three tests of unconditioned anxiety.

Substrains of the C57BL/6 inbred mouse are widely used in genetic, behavioral and physiological research, as well as models for human disease. Throughout, the choice of the respective substrain can have a large influence on experimental results. Likewise, the conditions under which experiments are performed, such as the light regime, can significantly affect the outcome of an experiment, especially when aiming at experimental behavior. Here, two commonly used mouse substrains, C57BL/6JOlaHsd and C57BL/6NCrl, were housed under either a conventional or a reverse light regime and were tested in either the light phase or the dark phase, respectively. All animals were exposed to three unconditioned anxiety-related behavior set-ups: the modified Hole Board test, the light-dark box and the elevated plus maze. Significant substrain and light regime effects were found in all three behavioral tests, with some of the latter being substrain and test specific. This signifies the importance of the choice of substrain used in for example, a mouse knockout experiment studying behavior, also in relation to light regime under which the animals are tested.

The modified hole board--measuring behavior, cognition and social interaction in mice and rats.

This protocol describes the modified hole board (mHB), which combines features from a traditional hole board and open field and is designed to measure multiple dimensions of unconditioned behavior in small laboratory mammals (e.g., mice, rats, tree shrews and small primates). This paradigm is a valuable alternative for the use of a behavioral test battery, since a broad behavioral spectrum of an animal's behavioral profile can be investigated in one single test. The apparatus consists of a box, representing the 'protected' area, separated from a group compartment. A board, on which small cylinders are staggered in three lines, is placed in the center of the box, representing the 'unprotected' area of the set-up. The cognitive abilities of the animals can be measured by baiting some cylinders on the board and measuring the working and reference memory. Other unconditioned behavior, such as activity-related-, anxiety-related- and social behavior, can be observed using this paradigm. Behavioral flexibility and the ability to habituate to a novel environment can additionally be observed by subjecting the animals to multiple trials in the mHB, revealing insight into the animals' adaptive capacities. Due to testing order effects in a behavioral test battery, naïve animals should be used for each individual experiment. By testing multiple behavioral dimensions in a single paradigm and thereby circumventing this issue, the number of experimental animals used is reduced. Furthermore, by avoiding social isolation during testing and without the need to food deprive the animals, the mHB represents a behavioral test system, inducing if any, very low amount of stress.

Chromosomal assignment of quantitative trait loci influencing baseline circulating total cholesterol level in male laboratory mice: report of a consomic strain survey and comparison with published results.

BACKGROUND: An important risk for atherosclerosis is a low level of HDL cholesterol. Baseline HDL cholesterol is under complex genetic and environmental control. Here we report on results of male mice from a consomic strain survey and the parental inbred strains for baseline circulating total cholesterol concentration, which is almost the same as HDL cholesterol in chow fed mice. The consomic strains have been derived from C57BL/6J (host strain) and A/J (donor strain) inbred lines. The work contributes to the value of the mouse as an animal model for studying the genetic background of differences in baseline circulating total and HDL cholesterol levels. RESULTS: The consomic strain survey suggested that mouse chromosomes 1, 7, 9, 14, 16, 17, 19, X, and Y contained at least one quantitative trait locus that is involved in baseline circulating total cholesterol concentration. All consomic lines, for which there is evidence that the substituted chromosome contains a quantitative trait locus, increase compared to the host strain baseline circulating total cholesterol concentration. Since there is evidence that 'body weight', 'age at blood sampling', 'time of the day blood was collected', and 'season' influence this phenotype, additional statistical analyses (with these variables as covariates) were performed. Now there is only evidence for quantitative trait loci on chromosomes 1, 8, 12, and Y. Taken the present results together with previous consomic strain surveys there is evidence that all mouse chromosomes carry quantitative trait loci that control baseline circulating total cholesterol levels. There was however little agreement between the present consomic strain results and previous sets of data. This might be explained by seasonal effects and differences in methodological variables such as age of the mice, fasting versus non-fasting, percentage of dietary fat, unanesthetized versus anesthetized mice, and the daily light-dark cycle. CONCLUSIONS: The present findings, when compared with previous consomic strain surveys, clearly illustrate the complexity of the genetic-environmental architecture for the regulation of baseline circulating total cholesterol levels in mice. Different data can be obtained from different labs and it underscores that animal geneticists should present as accurate a picture as possible of the laboratory mouse's environment.

Ethical issues associated with the use of animal experimentation in behavioral neuroscience research.

This chapter briefly explores whether there are distinct characteristics in the field of Behavioral Neuroscience that demand specific ethical reflection. We argue that although the ethical issues in animal-based Behavioral Neuroscience are not necessarily distinct from those in other research disciplines using animal experimentation, this field of endeavor makes a number of specific, ethically relevant, questions more explicit and, as a result, may expose to discussion a series of ethical issues that have relevance beyond this field of science. We suggest that innovative research, by its very definition, demands out-of-the-box thinking. At the same time, standardization of animal models and test procedures for the sake of comparability across experiments inhibits the potential and willingness to leave well-established tracks of thinking, and leaves us wondering how open minded research is and whether it is the researcher's established perspective that drives the research rather than the research that drives the researcher's perspective. The chapter finishes by introducing subsequent chapters of this book volume on Ethical Issues in Behavioral Neuroscience.

Chronic social stress does not affect behavioural habituation in male CD1 mice.

Various protocols to induce chronic stress in rodents are being used to determine the effects and underlying mechanisms of prolonged stress experience. Recently, a novel chronic social stress (CSS) protocol has been developed for mice where social instability in adolescence and early adulthood is induced. This protocol has been shown to cause an increase in HPA-axis activity and acute avoidance behaviour in the elevated plus maze. The aim of the present study was to investigate the effect of this CSS protocol on habituation to an initially novel environment in CD1 mice, since it has been shown that initially high avoidance behaviour in mice can still be followed by rapid habituation, pointing towards an adaptive response. One group of male mice, the CSS group, was exposed to the CSS protocol for 7 weeks and we compared their behavioural and physiological responses with male mice that were housed in a stable social group, the SH group. The results reveal a decrease in body weight gain and fur condition, changes in adrenal weight and decreased GR mRNA expression in the CA1 and the dentate gyrus of the hippocampus in chronically stressed CD1 animals. Irrespective of such evidence for a significantly stressful effect of the protocol, CD 1 mice, after termination of the stress procedure, revealed habituation profiles that matched those of control animals. We conclude that the physiological and central-nervous effects caused by a CSS procedure as used in this experiment fall within the coping capacities of CD1 mice at the behavioural level.

Sex Differences in Physiological Acclimatization after Transfer in Wistar Rats.

Most laboratory animals used in research are vendor-bred and transferred to research facilities. Transfer procedures might have considerable and unintended effects on research results. In the present study we compared physiological and behavioral parameters before and after external and internal transfer, as well as between transferred and non-transferred Wistar rats. The impact of both external and internal transfer on body weight, plasma corticosterone levels, heart rate, blood pressure, and locomotor activity was studied in both male and female Wistar rats, taking into account the sex differences in stress responsivity. External transfer was found to decrease body weight, increase plasma corticosterone, increase activity, increase heart rate in female rats, but decrease heart rate in male rats. Parameters showed differences between the sexes and light phases. This study shows that acclimatization after transfer is sex-specific and researchers should take the sex into consideration when determining the acclimatization period. It is recommended to allow for acclimatization of at least 8 days in males and two weeks in females after external transfer and timely (2 days before starting experiments) transfer the animals internally to the testing room.

Effects of Transfer from Breeding to Research Facility on the Welfare of Rats.

Transfer from the breeding facility to a research facility is a stressful event for laboratory animals. Heat stress has been reported to constitute one of the major concerns during transport of animals. This study measured ambient and body temperature, corticosterone and glucose levels, body weight, behavior and water and food intake before, during and after transfer in Wistar rats. Decreased body weight, water and food intake were observed on the day of transfer in rats. Environmental temperature strongly affected body temperature of rats and needs to be controlled. Male rats need to habituate for at least one week, females for two weeks after transfer.

Expression of CRFR1 and Glu5R mRNA in different brain areas following repeated testing in mice that differ in habituation behaviour.

Our recent studies revealed a profound impairment to habituate in 129P3 mice compared to BALB/c mice after repeated exposure to an initially novel environment. This was accompanied by strain-specific c-Fos expression in the prelimbic cortex, a brain area related to emotional and cognitive processing. The metabotropic glutamate receptor 5 (mGlu5R) antagonist MPEP increased c-Fos expression in brain areas related to cognition while it decreased c-Fos expression in brain areas processing emotions in 129P3 animals. We hypothesised that the non-adaptive response of 129P3 mice to a novel environment may be the result of impaired neural processing between the prelimbic cortex and emotion processing brain areas, possibly regulated by glutamatergic neurotransmission. To explore this hypothesis, we compared c-Fos activity in between naïve and repeatedly tested animals. Further, we investigated mRNA expression of CRFR1 and mGlu5R in the prelimbic cortex and amygdala, since these transmitter systems are not only involved in the regulation of anxiety, but are indicated to be co-expressed in relevant brain areas. Behavioural results confirmed strain-specific habituation profiles and strain-specific c-Fos expression in brain areas regulating cognitive and emotional processes in BALB/c and 129P3 mice. We found that repeated testing resulted in contrasting behavioural responses in both strains, and this was accompanied by strain-specific effects on c-Fos and receptor-expression. From these results it may be concluded that habituation in BALB/c mice reflects a shift from a primary emotional response to a more cognitively controlled behaviour, and that this shift over time may be impaired in 129P3 animals.

Effects of isoflurane-induced anaesthesia on cognitive performance in a mouse model of Alzheimer's disease: A randomised trial in transgenic APP23 mice.

BACKGROUND: Results from in-vitro experiments suggest that inhalational anaesthetics may have a detrimental effect on the course and incidence of Alzheimer's disease. However, case-control studies in humans show no negative impact of anaesthetics on the course of Alzheimer's disease. OBJECTIVE: To test the hypothesis that 2 h of general anaesthesia with 1 MAC isoflurane changes learning abilities of young and old transgenic Alzheimer's mice (APP23 mice). DESIGN: Randomised controlled double-blinded study in mice. SETTING: Animal laboratory and operating theatre in the Klinik für Anästhesiologie, Technische Universität München, Germany ANIMALS: Ninety-six male mice divided in four groups: young (4 months) APP23 mice and corresponding wild-type mice; old (14 to 16 months) APP23 and corresponding wild-type mice. INTERVENTION: Mice were either anaesthetised for 2 h with 1 MAC isoflurane or sham-anaesthetised ('isoflurane' or 'control'). MAIN OUTCOME MEASURES: Learning and locomotor activity during the following 8 days using the modified Hole Board Test for mice. Results are median (interquartile range) and median difference (95% confidence interval). RESULTS: Young mice, [1.0 (1.3)] as assessed by the number of omission errors, learned better than old [1.8 (1.8); age: P = 0.004, median difference 0.5 (0.2 to 1.0)]. Anaesthetised animals [0.8 (1.5)] learned better than controls [1.6 (1.7); anaesthesia: P = 0.010, median difference 0.5 (0.1 to 0.9)]. This was accompanied by higher locomotor activity in young compared to old mice as assessed by number of line crossings per minute [10 (5) min(-1) vs. 7 (3) min(-1), P < 0.001, median difference 3 (2 to 4) min(-1)]. Anaesthesia and genotype Alzheimer's disease had no impact on locomotor activity. CONCLUSION: Isoflurane may have protective, rather than detrimental, effects on cognition in Alzheimer's disease.

Not all mice are equal: welfare implications of behavioural habituation profiles in four 129 mouse substrains.

Safeguarding the welfare of animals is an important aim when defining housing and management standards in animal based, experimental research. While such standards are usually defined per animal species, it is known that considerable differences between laboratory mouse strains exist, for example with regard to their emotional traits. Following earlier experiments, in which we found that 129P3 mice show a lack of habituation of anxiety related behaviour after repeated exposure to an initially novel environment (non-adaptive profile), we here investigated four other 129 inbred mouse substrains (129S2/SvPas, 129S2/SvHsd (exp 1); 129P2 and 129X1 (exp 2)) on habituation of anxiety related behaviour. Male mice of each strain were repeatedly placed in the modified hole board test, measuring anxiety-related behaviour, exploratory and locomotor behaviour. The results reveal that all four substrains show a lack of habituation behaviour throughout the period of testing. Although not in all of the substrains a possible confounding effect of general activity can be excluded, our findings suggest that the genetic background of the 129 substrains may increase their vulnerability to cope with environmental challenges, such as exposure to novelty. This vulnerability might negatively affect the welfare of these mice under standard laboratory conditions when compared with other strains. Based on our findings we suggest to consider (sub)strain-specific guidelines and protocols, taking the (subs)train-specific adaptive capabilities into account.

Evaluation of neurobehavioral deficits following different severities of cerebral ischemia in rats: a comparison between the modified hole board test and the Morris water maze test.

OBJECTIVE: While histological injury following cerebral ischemia has been extensively characterized in rodents, evidence on the effects on executive functioning is still missing. This study was designed to evaluate neuropsychological outcome following different severities of cerebral ischemia in rats with the modified hole board test or the Morris water maze. SETTING: With institutional review board approval, anesthetized rats were exposed to bilateral carotid artery occlusion (BCAO) for escalating time intervals (0-12.5 min). Postoperatively cognitive performance was assessed using either the modified hole board test (mHB) or the Morris water maze (MWM). On postoperative day 14 rats were euthanized and intact neurons in five cerebral regions were counted. RESULTS: Rats of the 0 and 5 min groups showed normal functional outcome with mild histological damage after 5 min of BCAO. Following 7.5 min of BCAO the mHB test showed cognitive deficits reflecting histological damage of the hippocampus while the MWM revealed normal functional outcome. Rats of the 10 and 12.5 min groups showed cognitive deficits in both tests, motor dysfunction and behavioral alterations in the mHB test and profound histological damage. CONCLUSIONS: The results indicate that the mHB is not inferior to the MWM for the evaluation of cognitive impairment following incomplete forebrain ischemia in rats. As the mHB additionally investigates a variety of behavioral dimensions and motor parameters in the same test environment, it is advantageous for the evaluation of interacting and potentially confounding behavioral changes following cerebral ischemia in rats.

Impact of anxiety profiles on cognitive performance in BALB/c and 129P2 mice.

It has been suggested over the decades that dysfunctional anxiety may be caused by distinct alterations in cognitive processing. To learn more about the relation between anxiety and cognitive functioning, two mouse strains that display either adaptive (BALB/c) or nonadaptive (129P2) anxiety, as reflected by their ability to habituate when repeatedly exposed to a novel environment, were tested for their cognitive performance in the modified hole board (mHB) task. In general, both strains showed successful acquisition of the task. The initially more anxious BALB/c mice revealed rapid habituation to the test setup, followed by decreased long-term and short-term memory errors across the experimental period and fast relearning after reversal of the task. By contrast, the nonadaptive 129P2 mice made more short-term memory errors and performed worse than the BALB/c animals after reversal of the test. The results confirm the proposed interaction of anxiety and cognition: In BALB/c mice, adaptive characteristics of anxiety were paralleled by more successful cognitive performance, while in 129P2 mice nonadaptive anxiety-related behaviour was accompanied by a higher level of short-term memory errors and less cognitive flexibility. Moreover, these results support our hypothesis that the nonadaptive anxiety phenotype in 129P2 mice may be the result of impaired cognitive control of emotional processes, resulting in impaired behavioural flexibility, for example in response to novelty.

Differential effects of diazepam and MPEP on habituation and neuro-behavioural processes in inbred mice.

BACKGROUND: Previous studies have demonstrated a profound lack of habituation in 129P3 mice compared to the habituating, but initially more anxious, BALB/c mice. The present study investigated whether this non-adaptive phenotype of 129P3 mice is primarily based on anxiety-related characteristics. METHODS: To test this hypothesis and extend our knowledge on the behavioural profile of 129P3 mice, the effects of the anxiolyticdiazepam (1, 3 and 5 mg/kg) and the putative anxiolytic metabotropic glutamate receptor 5 (mGlu5R) antagonist 2-methyl-6-(phenylethynyl)pyridine (MPEP, 3, 10 and 30 mg/kg) treatment on within-trial (intrasession) habituation, object recognition (diazepam: 1 mg/kg; MPEP 10 mg/kg) and on the central-nervous expression of the immediate early gene c-Fos (diazepam: 1 mg/kg; MPEP 10 mg/kg) were investigated. RESULTS: Behavioural findings validated the initially high, but habituating phenotype of BALB/c mice, while 129P3 mice were characterized by impaired intrasession habituation. Diazepam had an anxiolytic effect in BALB/c mice, while in higher doses caused behavioural inactivity in 129P3 mice. MPEP revealed almost no anxiolytic effects on behaviour in both strains, but reduced stress-induced corticosterone responses only in 129P3 mice. These results were complemented by reduced expression of c-Fos after MPEP treatment in brain areas related to emotional processes, and increased c-Fos expression in higher integrating brain areas such as the prelimbic cortex compared to vehicle-treated 129P3 mice. CONCLUSIONS: These results suggest that the strain differences observed in (non)adaptive anxiety behaviour are at least in part mediated by differences in gamma-aminobutyric acid- A and mGluR5 mediated transmission.

A test to identify judgement bias in mice.

Emotional states are known to affect cognitive processes. For example highly anxious individuals interpret ambiguous stimuli more negatively than low anxious people, an effect called negative judgement bias. Recently, the measurement of judgement bias has been used to try and indicate emotional states in animals. In the present experiment a potential test for judgement bias in mice was examined. Mice were trained with two distinct odour cues (vanilla or apple) predicting either a palatable or an unpalatable almond piece. Subsequently their reaction to mixtures of both odours, the ambiguous stimuli, was investigated. Mice of the BALB/cJ and 129P3/J inbred mouse strains (high initial anxiety and low initial anxiety phenotypes respectively) were tested. While BALB/cJ mice showed odour association learning and showed intermediate reactions to the ambiguous cues, 129P3/J mice did not discriminate between the cues. Additionally BALB/cJ mice that were tested under more aversive white light conditions revealed a higher latency to approach the almond piece than mice tested under less aversive red light conditions. The ambiguous stimulus however was interpreted as negative under both test conditions. Brain c-Fos expression levels (a marker for neuronal activity) differed between the BALB/c/J and 129P3/J in the lateral amygdala and the prelimbic cortex, indicating differences in ambiguous information processing between the strains. The behavioural results suggest that the present judgement bias test might be used to assess emotional states in at least BALB/c mice, however further research on both behaviour and on the involved brain mechanisms is necessary to confirm this idea.

The impact of transportation on physiological and behavioral parameters in Wistar rats: implications for acclimatization periods.

Transportation of laboratory rodents unavoidably causes stress. Nevertheless, very little is known about the effects of transportation and how long it takes for the animal to recuperate. In the present study, we investigated physiological and behavioral parameters before and after transportation in both transported and nontransported animals. We took blood samples to analyze plasma corticosterone and creatine kinase, and performed physiological measurements by means of telemetry, measuring heart rate, blood pressure, and activity. Behavior was measured by means of home cage observations. This study revealed that plasma corticosterone levels increased at least up to 16 days after transportation, blood pressure and heart rate showed a lasting decrease after transportation, grooming increased, and social interactions and locomotor activity decreased after transportation. With these data we demonstrate that there is a long-lasting effect of transportation on physiological and behavioral parameters. Our results show that the stressful impact of transportation embraces all parts of the procedure, including for example the packing of the animals. Researchers must be aware of this impact and provide a sufficient acclimatization period to allow for the (re-)stabilization of parameters. Insufficient acclimatization periods endanger not only the reliability of research results but also the welfare of the animal used.

The appetitively motivated "cognitive" holeboard: a family of complex spatial discrimination tasks for assessing learning and memory.

Spatial learning and memory tasks have captured a solid position in neuroscience research. A variety of holeboard-type tasks are suitable for investigating the effects of a broad range of experimental manipulations on spatial learning and memory in a broad range of species, including fish, rodents, cats, pigs, tupaias, and humans. We summarize the concepts and procedures underlying tests of spatial discrimination learning, with special emphasis on holeboard-type tasks and task-specific characteristics. Holeboard-type tasks enable a broad range of mnemonic and cognitive variables to be measured in parallel, including cognitive processes such as habituation processes, spatial working and reference memory, and search strategies, but also non-cognitive variables, such as exploration, anxiety-related behavior, and stereotypies. These tasks are sensitive to a large number of naturally occurring differences (e.g. strain differences and age effects) and to the effects of non-genetic (e.g. specific brain lesions, stress, treatment with cognition impairers or cognition enhancers) and genetic experimental manipulations. In conclusion, holeboard-type tasks provide powerful tools to investigate multiple aspects of spatial orientation behavior in the same experimental setup. Cross-species comparison of holeboard performance shows the potential for translational studies.

Critical evaluation of the use of dogs in biomedical research and testing in Europe.

Dogs are sometimes referred to as "man's best friend" and with the increase in urbanization and lifestyle changes, dogs are seen by their owners as family members. Society expresses specific concerns about the experimental use of dogs, as they are sometimes perceived to have a special status for humans. This may appear somewhat conflicting with the idea that the intrinsic value of all animals is the same, and that also several other animal species are used in biomedical research and toxicology. This aspect and many others are discussed in an introductory chapter dealing with ethical considerations on the use of dogs as laboratory animals. The report gives an overview on the use of dogs in biomedical research, safety assessment and the drug developmental process and reflects the discussion on the use of dogs as second (non-rodent)species in toxicity testing. Approximately 20,000 dogs are used in scientific procedures in Europe every year, and their distinct genetic, physiological and behavioral characteristics may support their use as models for e.g. behavioral analysis and genetic research. Advances in the 3Rs (Replacement, Reduction and Refinement of experiments using dogs) are described, potential opportunities are discussed and recommendations for further progress in this area are made.

A framework program for the teaching of alternative methods (replacement, reduction, refinement) to animal experimentation.

Development of improved communication and education strategies is important to make alternatives to the use of animals, and the broad range of applications of the 3Rs concept better known and understood by different audiences. For this purpose, the Center for Alternatives to Animal Testing in Europe (CAAT-Europe) together with the Transatlantic Think Tank for Toxicology (t(4)) hosted a three-day workshop on "Teaching Alternative Methods to Animal Experimentation". A compilation of the recommendations by a group of international specialists in the field is summarized in this report. Initially, the workshop participants identified the different audience groups to be addressed and also the communication media that may be used. The main outcome of the workshop was a framework for a comprehensive educational program. The modular structure of the teaching program presented here allows adaptation to different audiences with their specific needs; different time schedules can be easily accommodated on this basis. The topics cover the 3Rs principle, basic research, toxicological applications, method development and validation, regulatory aspects, case studies and ethical aspects of 3Rs approaches. This expert consortium agreed to generating teaching materials covering all modules and providing them in an open access online repository.

Cognitive deficits after systemic induction of inducible nitric oxide synthase: a randomised trial in rats.

BACKGROUND: Nitric oxide acts as an important neurotransmitter as well as a sepsis mediator. During sepsis, high levels of nitric oxide, produced by the inducible form of the nitric oxide synthase (iNOS), may lead to disturbances concerning these conflicting roles and cause septic encephalopathy. To evaluate this theory, we aimed at first, to demonstrate cognitive dysfunction in a rat model based on systemic iNOS induction; second, to elucidate molecular mechanisms; and third, to prevent cognitive deficits in our sepsis model. METHODS: We used a rat systemic inflammation model that is based on the induction of iNOS by heat-killed Corynebacterium parvum in different doses (30 or 60 mg kg). NO2/NO3 plasma levels were measured to prove iNOS induction. Cognitive performance was investigated. In brain tissue, NOS protein and NOS activity were determined. To prevent cognitive deficits, two groups of rats received L-N-(1-Iminoethyl)-lysine (L-NIL), a specific iNOS inhibitor in the drinking water. RESULTS: The rats[Combining Acute Accent] cognitive performance, that is, short-term memory as well as long-term memory was impaired in C. parvum rats with a peak at the third day after injection in the 60 mg kg group. At the same day, neuronal NOS (nNOS)-protein content in the prefrontal cortex was reduced in C. parvum rats. nNOS activity was also reduced in C. parvum rats. The cognitive deficit in short-term memory could be prevented by L-NIL. CONCLUSION: We demonstrate early, reversible cognitive deficits in a rat model of systemic inflammation with increased systemic iNOS activity. As systemic inhibition of iNOS activity prevented rats from the deficit in short-term memory, an involvement of systemic iNOS induction in this deficit is likely. Whether the reduced nNOS-protein expression and nNOS activity are connected to systemic iNOS induction, however, remains unclear.