Social context affects the motivation of laboratory mice, Mus musculus, to gain access to resources

Consumer demand studies measuring the motivation that animals have for resources have almost exclusively used gregarious species, but have tested animals housed in isolation. The motivation that gregarious animals have for resources could be increased or decreased by the presence or absence of companion animals, that is, the social context. I trained a single mouse from each of six groups to perform an operant task that was quantifiable and would not be performed fortuitously or learnt by nontrained mice. The trained mice were housed with nontrained cagemates in a start cage that required them to perform the operant task to enter a resource cage providing either additional space or a running wheel. The presence of cagemates in the start cage significantly decreased the number of visits/24 h to the running wheel, the number of bouts and the duration of wheel running, but had no effect on the number of visits to additional space. The presence of cagemates increased the time spent in the resource cage whether this provided additional space or a running wheel, indicating that the mice overcompensated for the reduced frequency of visits. These results show that social context can have a significant effect on the motivation that animals have for resources, and that this effect is resource dependent. The influence of social context should therefore be considered in the design and interpretation of consumer demand studies. Copyright 2003 Published by Elsevier Ltd on behalf of The Association for the Study of Animal Behaviour.       

Do Male Mice Prefer or Avoid Each Other's Company? Influence of Hierarchy,Kinship, and Familiarity

In the laboratory, individual housing of male mice who otherwise show aggression is common practice. Because mice are a social species, the question arises whether this procedure is right from the animals' point of view. This study tested the preference of subordinate animals for their dominant cagemate, and vice versa, and the preference of subordinate animals for an unknown subordinate partner. Experiments that allowed male mice with different histories to choose either an inhabited or an empty cage have shown that the mice preferred the proximity of another male over individual housing. No differences in this respect were found between dominant and subordinate males, or between littermates and nonlittermates. The preference was most obvious when mice who were previously housed together were tested. The study concludes that separation and single housing for mice are not attractive solutions for overcoming aggression in group-housed male mice and that alternative approaches, such as improving the housing conditions, should be explored as a way of tempering intermale aggression.     

Do Male Mice Prefer or Avoid Each Other's Company? Influence of Hierarchy, Kinship, and Familiarity

In the laboratory, individual housing of male mice who otherwise show aggression is common practice. Because mice are a social species, the question arises whether this procedure is right from the animals' point of view. This study tested the preference of subordinate animals for their dominant cagemate, and vice versa, and the preference of subordinate animals for an unknown subordinate partner. Experiments that allowed male mice with different histories to choose either an inhabited or an empty cage have shown that the mice preferred the proximity of another male over individual housing. No differences in this respect were found between dominant and subordinate males, or between littermates and nonlittermates. The preference was most obvious when mice who were previously housed together were tested. The study concludes that separation and single housing for mice are not attractive solutions for overcoming aggression in group-housed male mice and that alternative approaches, such as improving the housing conditions, should be explored as a way of tempering intermale aggression.     

Preferences of group-housed female mice regarding structure of softwood bedding

Bedding influences various parameters in the housing of laboratory mice, such as health, physiology and behaviour (often considered as being integral parts of welfare). Notwithstanding existent studies about bedding preferences of individually tested mice, data about group-housed mice are still lacking. The aim of this study was to find out the structure preference for softwood bedding of group-housed mice. One hundred and eight 8-week-old female mice (C57BL6/JOlaHsd and BALB/cOlaHsd) were housed in groups of three and were given one-week free access to two different bedding structures at a time. In three test combinations, softwood shaving bedding was tested versus softwood chip bedding products of three different particle sizes (fine/medium/coarse-grained). The preference test was performed in a DoubleCage system composed of two Makrolon type IIL cages, connected by a perspex tunnel. This validated system was able to detect the crossings of each individual animal with correct crossing time and direction. On the basis of these data, dwelling times on the particular bedding structures were statistically analysed as a parameter for bedding preferences. In all three test combinations, a highly significant shaving preference was detected. On average, mice spent 70% of their dwelling time on the shavings. This preference was more explicit during the light period and in C57BL/6J mice. The relative ranking of the bedding structures was: shavings > coarse-grained chips > medium chips = fine chips. By means of these results, a shaving structure as bedding can be recommended for laboratory mice, whereas fine chip structures should be avoided.     

Time to Integrate to Nest Test Evaluation in a Mouse DSS-Colitis Model

Severity assessment in laboratory animals is an important issue regarding the implementation of the 3R concept into biomedical research and pivotal in current EU regulations. In mouse models of inflammatory bowel disease severity assessment is usually undertaken by clinical scoring, especially by monitoring reduction of body weight. This requires daily observance and handling of each mouse, which is time consuming, stressful for the animal and necessitates an experienced observer. The time to integrate to nest test (TINT) is an easily applicable test detecting disturbed welfare by measuring the time interval mice need to integrate nesting material to an existing nest. Here, TINT was utilized to assess severity in a mouse DSS-colitis model. TINT results depended on the group size of mice maintained per cage with most consistent time intervals measured when co-housing 4 to 5 mice. Colitis was induced with 1% or 1.5% DSS in group-housed WT and Cd14-deficient mice. Higher clinical scores and loss of body weight were detected in 1.5% compared to 1% DSS treated mice. TINT time intervals showed no dose dependent differences. However, increased clinical scores, body weight reductions, and increased TINT time intervals were detected in Cd14 ^-/- compared to WT mice revealing mouse strain related differences. Therefore, TINT is an easily applicable method for severity assessment in a mouse colitis model detecting CD14 related differences, but not dose dependent differences. As TINT revealed most consistent results in group-housed mice, we recommend utilization as an additional method substituting clinical monitoring of the individual mouse.     

Synergistic effect of estradiol benzoate and dihydrotestosterone on aggression in mice

Orchiectomized CD-1 mice were treated with several steroids, alone and in combination, and then tested for aggressiveness against group-housed, intact “stimulus” mice. Estradiol benzoate (EB) in combination with dihydrotestosterone (DHT) elicited aggression equivalent to that shown by intact, sham-operated animals and by testosterone-treated subjects. EB alone was less effective than EB with DHT. DHT alone elicited no more aggression than that observed in control animals. The data suggest a synergism between EB and DHT within the central nervous system in the induction of aggressive behavior.     

Time-dependent enhancement of lymphocyte activation by mitogens after exposure to isolation or water scheduling

The effects of isolation and water scheduling on mitogen induced lymphocyte proliferation were investigated. Isolated rats were animals which had been raised in group-housed conditions and then transferred to individual cages with ad lib access to water for a 1 or 2 week period. Water scheduled rats were maintained in group housing (5 rats per cage) with ad lib access to food but with access to water for a single 30 minute session each day. Responses of these groups were compared to those of animals which had been continuously group-housed with ad lib access to food and water. No differences in lymphocyte responses to phytohemagglutinin (PHA) were found 1 week after exposure to isolation. However, after 2 weeks, splenic and blood T lymphocytes from isolated animals demonstrated an increased proliferative response to suboptimum and maximum concentrations of PHA. Splenic B lymphocyte responses to lipopolysaccharide (LPS) from isolated animals were also increased by 2- to 3-fold compared to group-housed controls. Two weeks of exposure of animals to daily water scheduling similarly increased the splenic lymphocyte proliferation. This increased responsiveness to PHA was not accompanied by a significant change in the sensitivity of the lymphocytes to PHA, in the total number of white blood cells, or the proportion of splenic T or T helper lymphocytes. Our results show that the increase in lymphocyte proliferation is time-dependent, requires greater than 1 week of exposure to isolation and is due to factors other than changes in sensitivity to mitogen or T lymphocyte number.     

A Model of Alcohol Drinking under an Intermittent Access Schedule Using Group-Housed Mice

Here, we describe a new model of voluntary alcohol drinking by group-housed mice. The model employs sensor-equipped cages that track the behaviors of the individual animals via implanted radio chips. After the animals were allowed intermittent access to alcohol (three 24 h intervals every week) for 4 weeks, the proportions of licks directed toward bottles containing alcohol were 50.9% and 39.6% for the male and female mice, respectively. We used three approaches (i.e., quinine adulteration, a progressive ratio schedule and a schedule involving a risk of punishment) to test for symptoms of compulsive alcohol drinking. The addition of 0.01% quinine to the alcohol solution did not significantly affect intake, but 0.03% quinine induced a greater than 5-fold reduction in the number of licks on the alcohol bottles. When the animals were required to perform increasing numbers of instrumental responses to obtain access to the bottle with alcohol (i.e., a progressive ratio schedule), they frequently reached a maximum of 21 responses irrespective of the available reward. Although the mice rarely achieved higher response criteria, the number of attempts was ∼10 times greater in case of alcohol than water. We have developed an approach for mapping social interactions among animals that is based on analysis of the sequences of entries into the cage corners. This approach allowed us to identify the mice that followed other animals in non-random fashions. Approximately half of the mice displayed at least one interaction of this type. We have not yet found a clear correlation between imitative behavior and relative alcohol preference. In conclusion, the model we describe avoids the limitations associated with testing isolated animals and reliably leads to stable alcohol drinking. Therefore, this model may be well suited to screening for the effects of genetic mutations or pharmacological treatments on alcohol-induced behaviors.     

Involvement of diazepam binding inhibitor and its fragment octadecaneuropeptide in social isolation stress-induced decrease in pentobarbital sleep in mice.

Diazepam binding inhibitor (DBI) and its fragment, octadecaneuropeptide (ODN), are putative endogenous ligands for benzodiazepine (BZD) receptors and have been shown to act as an inverse BZD receptor agonist in the brain. A previous study suggested that the social isolation stress-induced decrease in pentobarbital sleep in mice was partly due to endogenous substances with an inverse BZD receptor agonist-like property. In this study, we examined the effects of DBI and ODN on pentobarbital sleep in group-housed and socially isolated mice to test the possible involvement of DBI and ODN in a social isolation-induced decrease in pentobarbital sleep. The socially isolated mice showed significantly shorter durations of pentobarbital (50 mg/kg, intraperitoneally, i. p.) sleep compared to the group-housed animals. When injected intracerebroventricularly (i.c.v.), DBI and ODN (3 and 10 nmol) dose-dependently shortened the pentobarbital-induced sleeping time in group-housed mice at the same dose range, but these peptides had no effect on the sleeping time in socially isolated animals. In contrast, flumazenil (16.5-33 nmol, i.c.v.), a BZD receptor antagonist, reversed the pentobarbital sleeping time in socially isolated mice to the level of group-housed animals without affecting the sleeping time in group-housed animals. The effects of DBI and ODN in group-housed mice were significantly blocked by flumazenil (33 nmol, i.c.v.). Moreover, the effect of flumazenil in socially isolated mice was significantly attenuated by DBI and ODN (10 nmol, i.c.v.). These results suggest that the changes in the activity of DBI and/or ODN are partly involved in the social isolation-induced decrease in the hypnotic action of pentobarbital in mice.     

ManR,a Transcriptional Regulator of the β-Mannan Utilization System,Controls the Cellulose Utilization System in Aspergillus oryzae

The author modified a respiratory gas analyzer to analyze the respiratory ¹³CO₂ of 12 small laboratory animals all at once. To investigate the practical use of this system, mice were orally (OR) or intravenously (IV) given glucose solutions containing three different amounts of ¹³C-labeled glucose. Expired ¹³CO₂ derived from exogenous glucose was detected within 10 minutes after administration in OR mice, but about 30 minutes in IV mice. The height of the peak of ¹³CO₂ expiration was correlated with the administered ¹³C-glucose mass.     

Testing resource value in group-housed animals: an investigation of cage height preference in laying hens

To avoid unpredictable social effects, animals' behavioural priorities are almost always tested using individuals housed singly, yet many species kept commercially are social animals housed in groups. Our aim was to develop a method of investigating environmental preference in group-housed laying hens, Gallus gallus domesticus, that maximised the external validity of our findings. In a simple test of preference, eight groups of ten hens were given free choice between furnished cages with minimum heights of 38 cm (low) and 45 cm (high). A preference for one cage height over the other would be evident as a shift from a binomial distribution of flock sizes in the two cages. No height preference was found as hens distributed evenly between the two cages more frequently than was expected. This suggests at high stocking densities maximising average inter-individual distance could be a priority over increased cage height. In a second experiment, to investigate the value that hens placed on a change in cage height; a 'cost' in the form of a narrow gap was imposed on movement from a low or high start cage to a high or low target cage, respectively. Cage height did not influence the latency of the first three hens to enter the target cage. However, latencies for subsequent hens were shorter and more hens worked to access a high target cage than a low target cage. We suggest that titrating animals' willingness to tolerate higher stocking densities against access to a resource could be an effective way to compare responses of group-housed animals to resources that are expected to satisfy the same motivational state.     

Validation of an automatic system (DoubleCage) for detecting the location of animals during preference tests

Preference tests have often been performed for collecting information about animals' acceptance of environmental refinement objects. In numerous published studies animals were individually tested during preference experiments, as it is difficult to observe group-housed animals with an automatic system. Thus, videotaping is still the most favoured method for observing preferences of socially-housed animals. To reduce the observation workload and to be able to carry out preference testing of socially-housed animals, an automatic recording system (DoubleCage) was developed for determining the location of group-housed animals in a preference test set-up. This system is able to distinguish the transition of individual animals between two cages and to record up to 16 animals at the same time (four animals per cage). The present study evaluated the reliability of the DoubleCage system. The data recorded by the DoubleCage program and the data obtained by human observation were compared. The measurements of the DoubleCage system and manual observation of the videotapes are comparable and significantly correlated (P < 0.0001) with good agreement. Using the DoubleCage system enables precise and reliable recording of the preferences of group-housed animals and a considerable reduction of animal observation time.     

The effects of long-duration, low-temperature ground transportation on physiological and biochemical indicators of stress in mice

Transportation can cause stress to laboratory animals and alter physiological characteristics that may confound experimental results. The authors investigated stress-related effects of 3-4 h of transportation by truck in two strains of mice (C57BL/6, which are known to be aggressive, and ICR, which are less aggressive). Transported mice had sufficient space and access to water, though temperature in the truck was lower than what is usually recommended. Transportation affected the following parameters in both strains of mice: (i) serum corticosterone concentrations, (ii) expression of the chaperone proteins Hsp70 and Grp78 in various tissues and (iii) concentrations of serological enzymes that are associated with liver disease. These parameters also differed substantially between the two strains of mice.     

Effects of an early handling-like procedure and individual housing on anxiety-like behavior in adult C57BL/6J and DBA/2J mice

Manipulations of rearing conditions have been used to examine the effects of early experience on adult behavior with varying results. Evidence suggests that postnatal days (PND) 15-21 are a time of particular susceptibility to environmental influences on anxiety-like behavior in mice. To examine this, we subjected C57BL/6J and DBA/2J mice to an early handling-like procedure. Pups were separated from dams from PND 12-20 for 30 minutes daily or received standard care. On PND 21, pups were weaned and either individually- or group-housed. On PND 60, anxiety-like behavior was examined on the elevated zero-maze. Although individually-housed animals took longer to enter an open quadrant of the maze, they spent more time in the open than group-housed animals. Additionally, we observed a trend of reduced anxiety-like behavior in C57BL/6J, but not DBA/2J mice that underwent the handling-like procedure.     

Quantitative Genetic Analysis of Temperature Regulation in MUS MUSCULUS. I. Partitioning of Variance

Heritabilities (from parent-offspring regression) and intraclass correlations of full sibs for a variety of traits were estimated from 225 litters of a heterogeneous stock (HS/Ibg) of laboratory mice. Initial variance partitioning suggested different adaptive functions for physiological, morphological and behavioral adjustments with respect to their thermoregulatory significance. Metabolic heat-production mechanisms appear to have reached their genetic limits, with little additive genetic variance remaining. This study provided no genetic evidence that body size has a close directional association with fitness in cold environments, since heritability estimates for weight gain and adult weight were similar and high, whether or not the animals were exposed to cold. Behavioral heat conservation mechanisms also displayed considerable amounts of genetic variability. However, due to strong evidence from numerous other studies that behavior serves an important adaptive role for temperature regulation in small mammals, we suggest that fluctuating selection pressures may have acted to maintain heritable variation in these traits.     

Therapeutic effect of CHF5074, a new γ-secretase modulator,in a mouse model of scrapie

In transmissible spongiform encephalopathies (TSEs) and Alzheimer disease (AD) both misfolding and aggregation of specific proteins represent key features. Recently, it was observed that PrP^c is a mediator of a synaptic dysfunction induced by Aβ oligomers. We tested a novel γ-secretase modulator (CHF5074) in a murine model of prion disease. Groups of female mice were intracerebrally or intraperitoneally infected with the mouse-adapted Rocky Mountain Laboratory prions. Two weeks prior infection, the animals were provided with a CHF5074-medicated diet (375 ppm) or a standard diet (vehicle) until they showed neurological signs and eventually died. In intracerebrally infected mice, oral administration of CHF5074 did not prolong survival of the animals. In intraperitoneally-infected mice, CHF5074-treated animals showed a median survival time of 21 d longer than vehicle-treated mice (p &lt; 0.001). In these animals, immunohistochemistry analyses showed that deposition of PrP^Sc in the cerebellum, hippocampus and parietal cortex in CHF5074-treated mice was significantly lower than in vehicle-treated animals. Immunostaining of glial fibrillary acidic protein (GFAP) in parietal cortex revealed a significantly higher reactive gliosis in CHF5074-treated mice compared with the control group of infected animals. Although the mechanism underlying the beneficial effects of CHF5074 in this murine model of human prion disease is unclear, it could be hypothesized that the drug counteracts PrP^Sc toxicity through astrocyte-mediated neuroprotection. CHF5074 shows a pharmacological potential in murine models of both AD and TSEs thus suggesting a link between these degenerative pathologies.     

Targeted Deletion of Kynurenine 3-Monooxygenase in Mice: A NEW TOOL FOR STUDYING KYNURENINE PATHWAY METABOLISM IN PERIPHERY AND BRAIN*

Kynurenine 3-monooxygenase (KMO), a pivotal enzyme in the kynurenine pathway (KP) of tryptophan degradation, has been suggested to play a major role in physiological and pathological events involving bioactive KP metabolites. To explore this role in greater detail, we generated mice with a targeted genetic disruption of Kmo and present here the first biochemical and neurochemical characterization of these mutant animals. Kmo^−/− mice lacked KMO activity but showed no obvious abnormalities in the activity of four additional KP enzymes tested. As expected, Kmo^−/− mice showed substantial reductions in the levels of its enzymatic product, 3-hydroxykynurenine, in liver, brain, and plasma. Compared with wild-type animals, the levels of the downstream metabolite quinolinic acid were also greatly decreased in liver and plasma of the mutant mice but surprisingly were only slightly reduced (by ∼20%) in the brain. The levels of three other KP metabolites: kynurenine, kynurenic acid, and anthranilic acid, were substantially, but differentially, elevated in the liver, brain, and plasma of Kmo^−/− mice, whereas the liver and brain content of the major end product of the enzymatic cascade, NAD⁺, did not differ between Kmo^−/− and wild-type animals. When assessed by in vivo microdialysis, extracellular kynurenic acid levels were found to be significantly elevated in the brains of Kmo^−/− mice. Taken together, these results provide further evidence that KMO plays a key regulatory role in the KP and indicate that Kmo^−/− mice will be useful for studying tissue-specific functions of individual KP metabolites in health and disease.     

Lymphocyte-induced angiogenesis factor is produced by L3T4+ murine T lymphocytes,and its production declines with age

Summary Lymphocyte-induced angiogenesis factor (LIA) is a product of T lymphocytes which has been shown to stimulate new vessel formation. Because immune senescence most profoundly affects T lymphocyte functions, we suspected that LIA production would decline with age. An assay for angiogenesis stimulated by allogeneic reaction was performed by injecting spleen cells from young or old donor mice into the skin of irradiated allogeneic recipient mice. The spleen cells from young mice induced a significantly greater number of vessels than did cells from older mice. In additional experiments, spleen cells from young and old animals were treated with a monoclonal antibody GK1.5) directed at the L3T4 antigen on murine T helper lymphocytes. Such treatment significantly reduced the new vessel formation induced by young lymphocytes but had no effect on that induced by lymphocytes from old animals. Studies employing indirect immunofluorescence demonstrated that the proportion of L3T4⁺ cells in the mononuclear fraction of splenocytes was nearly identical in both young and old mice. From these investigations we can conclude that (1) L3T4⁺ lymphocytes are responsible for LIA production, and (2) production, like that of other T lymphokines, declines with age.Supported by NIH grant AG 00332 and VA Merit Award (WBE)     

Long-term effects of husbandry procedures on stress-related parameters in male mice of two strains

In socially unstable groups of male laboratory mice, individuals may experience a chronic stress situation. Previous experiments have shown that the transfer of specific olfactory cues during cage cleaning, and the provision of nesting material decrease aggression and stress in group-housed male mice. In this study, the combined effect of these husbandry procedures were tested for their long-term effect on stress in groups of moderately aggressive (BALB/c) and severely aggressive (CD-1) male mice. The physiological and behavioural stress-related parameters used were body weight, food and water intake, spleen and thymus weight, adrenal tyrosine hydroxylase activity, urine corticosterone levels and behaviour in a cage emergence test. Long-term provision of nesting material and its transfer during cage cleaning was found to influence several stress-related physiological parameters. Mice housed in cages enriched with nesting material had lower urine corticosterone levels and heavier thymuses, and they consumed less food and water than standard-housed mice. Furthermore, marked differences were found between strains. CD-1 mice were less anxious in the cage emergence test, weighed more, ate and drank more, and had heavier thymuses but lighter spleens and lower corticosterone levels than BALB/c mice. We conclude that the long-term provision of nesting material, including the transfer of nesting material during cage cleaning, reduces stress and thereby enhances the welfare of laboratory mice.     

Identification of an Astrovirus Commonly Infecting Laboratory Mice in the US and Japan

Mice (Mus musculus) are the most commonly used laboratory animals. Viral metagenomics on tissues of immunodeficient mice revealed sequences of a novel mammalian astrovirus. Using PCR, we screened mice from 4 breeders, 4 pharmaceutical companies, 14 research institutes and 30 universities in the US and Japan. Mice from one US breeder tested positive while none from Japanese breeders were positive for MuAstV. Mice in over half of the universities (19/30), institutes (7/14) and pharmaceutical animal facilities (2/4) investigated revealed the presence of MuAstV. Nine mice strains tested positive including both immunodeficient strains (NSG, NOD-SCID, NSG-3GS, C57BL6-Timp-3 ^−/−, and uPA-NOG) and immunocompetent strains (B6J, ICR, Bash2, BALB/c). Our data indicates that MuAstV has a wide geographical, institutional and host strain distribution. Comparison of the MuAstV RdRp sequences showed numerous mutations indicating ongoing viral divergence in different facilities. This study demonstrates the need for metagenomic screening of laboratory animals to identify adventitious infections that may affect experimental outcomes.