There is a strong need to gain systematic knowledge about the potential impact of routine procedures on laboratory animals to ensure animal welfare and reliability of experimental results. Blood sampling is a frequently used procedure within laboratory animal research. Two of the most common anesthesia-free techniques to obtain blood in mice are the saphenous venipuncture and submandibular bleeding. It is common practice that these methods are applied repeatedly to 1 individual. The impact of both techniques, either performed singly or repeatedly, on the animal’s welfare is not fully known yet. We directly compared the effects of both sampling methods on physiological stress responses, behavior, tissue damage, and procedure duration in 2 frequently used mouse strains: C57BL/6 and BALB/c mice (n = 45/strain). The mice were randomly assigned to 3 treatment groups (saphenous venipuncture, submandibular bleeding, control) of 15 mice/strain and were sampled weekly (80 μl/ blood sample) during 4 wk for the experimental groups and 5 wk for the control group. To evaluate the acute physiological stress response, plasma corticosterone levels were determined. As an indicator of chronic stress, body weights were recorded once a week and the thymus was weighed after necropsy. Behavior was recorded using video cameras in front of the cages and afterwards analyzed by a well-trained observer using event logging software. Tissue damage was evaluated by scoring pre-and postmortem hemorrhages and hematomas at the injection site and by measuring alanine transaminase, aspartate aminotransferase, and creatine kinase plasma levels. Submandibular bleeding appeared to be less time consuming (P<0.05) while providing higher sample volumes. However, this method led to increased acute stress responses in C57BL/6 mice (P = 0.03). All animals of both strains undergoing this procedure expressed more pain-related behavior (P < 0.05) and the extent of tissue damage at the injection site was significantly higher when compared to the saphenous venipuncture (P < 0.05). Thus, in order to avoid the undesirable effects of stress on experimental outcomes and to reduce the extent of tissue damage, saphenous venipuncture should be preferred above submandibular bleeding whenever possible.

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.

BACKGROUND:Selecting chromosome substitution strains (CSSs, also called consomic strains/lines) used in the search for quantitative trait loci (QTLs) consistently requires the identification of the respective phenotypic trait of interest and is simply based on a significant difference between a consomic and host strain. However, statistical significance as represented by P values does not necessarily predicate practical importance. We therefore propose a method that pays attention to both the statistical significance and the actual size of the observed effect. The present paper extends on this approach and describes in more detail the use of effect size measures (Cohen's d, partial eta squared - η p (2) ) together with the P value as statistical selection parameters for the chromosomal assignment of QTLs influencing anxiety-related behavior and locomotion in laboratory mice.RESULTS:The effect size measures were based on integrated behavioral z-scoring and were calculated in three experiments: (A) a complete consomic male mouse panel with A/J as the donor strain and C57BL/6J as the host strain. This panel, including host and donor strains, was analyzed in the modified Hole Board (mHB). The consomic line with chromosome 19 from A/J (CSS-19A) was selected since it showed increased anxiety-related behavior, but similar locomotion compared to its host. (B) Following experiment A, female CSS-19A mice were compared with their C57BL/6J counterparts; however no significant differences and effect sizes close to zero were found. (C) A different consomic mouse strain (CSS-19PWD), with chromosome 19 from PWD/PhJ transferred on the genetic background of C57BL/6J, was compared with its host strain. Here, in contrast with CSS-19A, there was a decreased overall anxiety in CSS-19PWD compared to C57BL/6J males, but not locomotion.CONCLUSIONS:This new method shows an improved way to identify CSSs for QTL analysis for anxiety-related behavior using a combination of statistical significance testing and effect sizes. In addition, an intercross between CSS-19A and CSS-19PWD may be of interest for future studies on the genetic background of anxiety-related behavior.

Magnesium (Mg) has been described to possess an anxiolytic function, but a number of studies present inconsistent results on this matter. In this study the effect of Mg deficiency on anxiety-related behavior, brain and blood plasma Mg in young adult male C57BL/6JOlaHsd and C57BL/6NCrl mice was studied. The animals were put on a control or Mg deficient diet from day 0 and significant hypomagnesaemia was evident from day 12 onwards in the test animals. Housing and test conditions were under either conventional light regime (white light behavioral test conditions) or reverse light regime (red light behavioral test conditions). The animals were tested in three tests for unconditioned anxiety: the modified Hole Board (day 14), the light-dark test (day 21) and the elevated plus maze (day 28). Overall integrated behavioral z-scores were calculated over these three behavioral tests. Mg showed a structure dependent distribution at the level of the brain, that differed between C57BL/6 substrain and light regime (conventional versus reverse), respectively. Likewise, total brain Mg did differ between substrain and light regime, but was not affected by the diet. Animals on the Mg deficient diet housed under conventional light regime had a higher final (day 28) blood plasma corticosterone level as compared to controls. Animals housed under reverse light regime exhibited no diet effect of plasma corticosterone levels. The significant hypomagnesaemia at blood plasma level resulted in an effect of Mg deficiency on avoidance, but not overall anxiety-related behavior. Significant differences regarding avoidance behavior were found between the two substrains and light regimes, respectively.

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.

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.

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.

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.

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.

Currently assessment and management of animal welfare are based on the supposition that welfare status is something experienced identically by each individual animal when exposed to the same conditions. However, many authors argue that individual welfare cannot be seen as an 'objective' state, but is based on the animal's own self-perception; such perception might vary significantly between individuals which appear to be exposed to exactly the same challenges. We argue that this has two implications: (1) actual perceived welfare status of individuals in a population may vary over a wide range even under identical environmental conditions; (2) animals that appear to an external observer to be in better or poorer welfare condition may all in fact perceive their own individual status as the same. This would imply that optimum welfare of a social group might be achieved in situations where individual group members differ markedly in apparent welfare status and perceive their own welfare as being optimal under differing circumstances. Welfare phenotypes may also vary along a continuum between self-regarding and other-regarding behaviour; a variety of situations exist where (social) individuals appear to invest in the welfare of other individuals instead of maximising their own welfare; in such a case it is necessary to re-evaluate individual welfare within the context of a social group and recognise that there may be consequences for the welfare of individuals, of decisions made at the group level or by other group members.

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.

Much consideration of animal welfare issues, as well as the bulk of welfare legislation, is closely related to context, with clear distinction between defined responsibilities towards farm-, laboratory-, companion- and wild animals. It has however been recognised that such distinction leads to inconsistencies, in that it might lead to different welfare management practices for the same rat (for example) as a pet, laboratory animal or pest. We suggest that if legal provisions recognize animals as having moral status, then that implies certain responsibilities towards the animal that cannot be context-dependent. Although we argue the moral responsibility to consider an animal’s welfare is context- independent, we suggest that the requirement to take action (and the form of that action) may alter with context. From a biological perspective, we argue that there exists an obligation to take action to address potential welfare problems only when a challenge exceeds the animal’s adaptive capacity, such that it cannot adequately respond by changes of its behaviour. The obligation to intervene further depends on whether or not there are practical (and economically feasible) options available for mitigation. Beyond this, additional constraints may be posed by anthropocentric aims or objectives, where any suffering may be adjudged avoidable (in theory), but necessary (because of specific human interests). Finally, whatever may be determined necessary or unavoidable by the actual manager or agent himself/herself, such judgement is inevitably subjective and possibly subject to self-interest. Thus any decision must be further evaluated within the context of what is considered by a wider society to be (morally) acceptable or unacceptable.

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.

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.

The general concept of animal welfare embraces a continuum between negative/bad welfare and positive/good welfare. Early approaches to defining animal welfare were mainly based on the exclusion of negative states, neglecting the fact that during evolution animals optimised their ability to interact with and adapt to their environment(s). An animal's welfare status might best be represented by the adaptive value of the individual's interaction with a given environmental setting but this dynamic welfare concept has significant implications for practical welfare assessments. Animal welfare issues cannot simply be addressed by means of objective biological measurements of an animal's welfare status under certain circumstances. In practice, interpretation of welfare status and its translation into the active management of perceived welfare issues are both strongly influenced by context and, especially, by cultural and societal values. In assessing whether or not a given welfare status is morally acceptable, animal welfare scientists must be aware that scientifically based, operational definitions of animal welfare will necessarily be influenced strongly by a given society's moral understanding.

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.

Psychiatrische Störungen sind komplexe pathologische Phänomene, die zu einer drastischen Beeinträchtigung des alltäglichen Lebens der betroffenen Patienten führen. Aufgrund ihrer Komplexität ist nach wie vor wenig über ihre Entstehung und die zugrunde liegenden Mechanismen bekannt. Daher sind Tiermodelle für psychiatrische Störungen auch heute noch unverzichtbar, und jedes Tiermodell, das unser Verständnis dieser Erkrankungen auch nur im Bezug auf Teilaspekte dieser Störungen erweitern kann, ist äußerst bedeutsam.

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.