Female C57BL/6 and BALB/c mice differently use the acoustic features of male ultrasonic vocalizations for social preferences.

Male mice emit ultrasonic vocalizations (USVs) in response to the presence of female mice and their urine. Male USVs attract females, enhancing female reproductive functions, and are thus considered as the courtship song. Previous studies have shown that female mice exhibit disassortative social preferences for male USVs. However, it remains unclear what acoustic features female mice use for the development of these preferences. To address this, we examined social preferences of female C57BL/6 and BALB/c mice using the three-chamber preference test using recorded male USVs. To dissociate the peak frequencies of these USVs from their syllable structure, we digitally manipulated the peak frequencies accordingly. We found that female mice preferred USVs that were dissimilar to those of their own strain. We also observed that, while female C57BL/6 mice were sensitive to changes in the syllable structure and the peak frequency, female BALB/c mice were sensitive to differences in the syllable structure. Our results demonstrate that female C57BL/6 and BALB/c mice differently use the acoustic features such as the peak frequency and the syllable structure for exhibiting disassortative social preferences.

Inhibitory control in BALB/c mice sub-strains during extinction learning.

Dysregulation of executive function (EF) involves alterations in cognitive flexibility / control and is underscored by learning impairments in neurodevelopmental disorders. Here, we examine cognitive inflexibility in BALB/cJ mice (a mouse model showing diminished sociability, increased anxiety and inattentive behaviour) and closely related "reference" BALB/cByJ mice. We used an appetitive extinction paradigm to investigate if cognitive flexibility measures are different between learning acquisition and extinction. The two BALB/c sub-strains learned to respond to a stimulus in a touchscreen operant chamber, after which the reward was removed and responses should be inhibited. Both mice sub-strains showed a different rate of learning while acquiring the task, in which the BALB/cJ mice were faster learners compared to the BALB/cByJ mice. This was not observed during the extinction phase, in which the BALB/cJ mice were able to extinguish responding to unrewarded stimuli equally. Within the BALB/cJ sub-strain, variation in the ability to inhibit a learnt response was observed when comparing them to similar grouped BALB/cByJ mice: BALB/cJ animals that reached the criterion were more reward driven, while BALB/cJ mice failing to reach the set criterion during extinction processing make more mistakes. Additionally, the changes observed during acquisition, were driven by animals not reaching the extinction criterion. Our results suggest that the BALB/c mice sub-strains may use different strategies to learn during appetitive extinction. This may be useful in the phenotypic dissection of cognitive flexibility in BALB/c sub-strains and their mapping on genetic variance revealed by next-generation sequencing in future studies.

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.

Wheat grain consumption and selection by inbred and outbred strains of mice.

Food selection and avoidance are driven primarily by orosensory cues. Previous studies with C57BL/6J mice indicated marked differences in selection and consumption of individual grains of different wheat varieties when presented in binary mixtures. The present study examined the patterns of mouse grain selection across four strains of laboratory mice: two inbred, BALB/c and C57BL/6J, and two outbred, Swiss-Webster and CD1. Four pairs of wheat varieties that were known to vary a priori for consumption preference or seed coat ('bran') color were tested. Two variety pairs were near-isogenic (>98% similar) with contrasting red and white seed coat coloration/pigmentation. All four mice strains exhibited similar preferences between wheat variety pairs, whereas consumption was not highly related to mouse body weight. This result indicates a more generalized phenomenon regarding how mice select and then consume individual wheat grains. The study supported the continued use of C57BL/6J as an effective strain model system to study food perception.

Behavioral characterization of a model of differential susceptibility to obesity induced by standard and personalized cafeteria diet feeding.

Despite the increase in obesity prevalence over the last decades, humans show large inter-individual variability for susceptibility to diet-induced obesity. Understanding the biological basis of this susceptibility could identify new therapeutic alternatives against obesity. We characterized behavioral changes associated with propensity to obesity induced by cafeteria (CAF) diet consumption in mice. We show that Balb/c mice fed a CAF diet display a large inter-individual variability in susceptibility to diet-induced obesity, such that based on changes in adiposity we can classify mice as obesity prone (OP) or obesity resistant (OR). Both OP and OR were hyperphagic relative to control-fed mice but caloric intake was similar between OP and OR mice. In contrast, OR had a larger increase in locomotor activity following CAF diet compared to OP mice. Obesity resistant and prone mice showed similar intake of sweet snacks, but OR ate more savory snacks than OP mice. Two bottle sucrose preference tests showed that OP decreased their sucrose preference compared to OR mice after CAF diet feeding. Finally, to test the robustness of the OR phenotype in response to further increases in caloric intake, we fed OR mice with a personalized CAF (CAF-P) diet based on individual snack preferences. When fed a CAF-P diet, OR increased their calorie intake compared to OP mice fed the standard CAF diet, but did not reach adiposity levels observed in OP mice. Together, our data show the contribution of hedonic intake, individual snack preference and physical activity to individual susceptibility to obesity in Balb/c mice fed a standard and personalized cafeteria-style diet.

Response, use and habituation to a mouse house in C57BL/6J and BALB/c mice.

Animal welfare depends on the possibility to express species-specific behaviours and can be strongly compromised in socially and environmentally deprived conditions. Nesting materials and refuges are very important resources to express these behaviours and should be considered as housing supplementation items. We evaluated the effects of one item of housing supplementation in standard settings in laboratory mice. C57BL/6JOlaHsd (B6) and BALB/cOlaHsd (BALB) young male and female mice, upon arrival, were housed in groups of four in standard laboratory cages and after 10 days of acclimatization, a red transparent plastic triangular-shaped Mouse House™ was introduced into half of the home cages. Animals with or without a mouse house were observed in various contexts for more than one month. Body weight gain and food intake, home cage behaviours, emotionality and response to standard cage changing procedures were evaluated. The presence of a mouse house in the home cage did not interfere with main developmental and behavioural parameters or emotionality of BALB and B6 male and female mice compared with controls. Both strains habituated to the mouse house in about a week, but made use of it differently, with BALB mice using the house more than the B6 strain. Our results suggest that mice habituated to the mouse house rather quickly without disrupting their home cage activities. Scientists can thus be encouraged to use mouse houses, also in view of the implementation of the EU Directive (2010/63/EU).

Delay-specific stimuli and genotype interact to determine temporal discounting in a rapid-acquisition procedure.

The importance of delay discounting to many socially important behavior problems has stimulated investigations of biological and environmental mechanisms responsible for variations in the form of the discount function. The extant experimental research, however, has yielded disparate results, raising important questions regarding Gene X Environment interactions. The present study determined the influence of stimuli that uniquely signal delays to reinforcement on delay discounting in two inbred mouse strains using a rapid-acquisition procedure. BALB/c and C57BL/6 mice responded under a six-component, concurrent-chained schedule in which the terminal-link delays preceding the larger-reinforcer were presented randomly across components of an individual session. Across conditions, components were presented either with or without delay-specific auditory stimuli, i.e., as multiple or mixed schedules. A generalized matching-based model was used to incorporate the impact of current and previous component reinforcer-delay ratios on current component response allocation. Sensitivity to reinforcer magnitude and delay were higher for BALB/c mice, but within-component preference reached final levels faster for C57Bl/6 mice. For BALB/c mice, acquisition of preference across blocks of a component was faster under the multiple than the mixed schedule, but final levels of sensitivity to reinforcement were unaffected by schedule. The speed of acquisition of preference was not different across schedules for C57Bl/6 mice, but sensitivity to reinforcement was higher under the multiple than the mixed schedule. Overall, differences in the acquisition and final form of the discount function were determined by a Gene X Environment interaction, but the presence of delay-specific stimuli attenuated genotype-dependent differences in magnitude and delay sensitivity.

A 1-night operant learning task without food-restriction differentiates among mouse strains in an automated home-cage environment.

Individuals are able to change their behavior based on its consequences, a process involving instrumental learning. Studying instrumental learning in mice can provide new insights in this elementary aspect of cognition. Conventional appetitive operant learning tasks that facilitate the study of this form of learning in mice, as well as more complex operant paradigms, require labor-intensive handling and food deprivation to motivate the animals. Here, we describe a 1-night operant learning protocol that exploits the advantages of automated home-cage testing and circumvents the interfering effects of food restriction. The task builds on behavior that is part of the spontaneous exploratory repertoire during the days before the task. We compared the behavior of C57BL/6J, BALB/cJ and DBA/2J mice and found various differences in behavior during this task, but no differences in learning curves. BALB/cJ mice showed the largest instrumental learning response, providing a superior dynamic range and statistical power to study instrumental learning by using this protocol. Insights gained with this home-cage-based learning protocol without food restriction will be valuable for the development of other, more complex, cognitive tasks in automated home-cages.

Comparative analysis of the behavioral and biomolecular parameters of four mouse strains.

The use of mice as experimental models in pharmacological and biochemical research began over 100 years ago, during which time different mice strains with specific features were developed. Numerous studies demonstrate that the pharmacological efficacy of various compounds significantly varies among different animal strains, a factor which must be considered when analyzing experimental data. The Sabra strain, developed more than 35 years ago, is widely used for research in Israel but has an unclear origin and is not characterized as well as other strains. Comparative analyses of the molecular characteristics of Sabra and other strains should help to understand their characteristics and to enhance the validity of their experimental use. Thus, four mouse strains-outbred ICR and Sabra as well as inbred C57Bl/6J and Balb/c were compared. Animals' weight, blood corticosterone and hippocampal BDNF mRNA levels were measured, and animals' behavior was compared using the EPM, open field, FST, and hot plate tests. We found that although Sabra mice are bigger and heavier than other tested lines, this is not reflected in behavior or in biomolecular features, wherein Sabra mice lay within the diapason of other tested animals. Thus, behavioral tests of anxiety-like behavior and locomotor activity revealed that Sabra mice scored close to the mean of all tested lines. Analysis of blood corticosterone levels did not show significant differences among tested strains. We also found a correlation between general and locomotor activity of the tested strains and their hippocampal BDNF mRNA expression. In summary, we may conclude that Sabra mice have traits similar to the better known lines, and therefore they are good subjects for neuroscience research.

Sociability and brain development in BALB/cJ and C57BL/6J mice.

Sociability--the tendency to seek social interaction--propels the development of social cognition and social skills, but is disrupted in autism spectrum disorders (ASD). BALB/cJ and C57BL/6J inbred mouse strains are useful models of low and high levels of juvenile sociability, respectively, but the neurobiological and developmental factors that account for the strains' contrasting sociability levels are largely unknown. We hypothesized that BALB/cJ mice would show increasing sociability with age but that C57BL/6J mice would show high sociability throughout development. We also hypothesized that littermates would resemble one another in sociability more than non-littermates. Finally, we hypothesized that low sociability would be associated with low corpus callosum size and increased brain size in BALB/cJ mice. Separate cohorts of C57BL/6J and BALB/cJ mice were tested for sociability at 19-, 23-, 31-, 42-, or 70-days-of-age, and brain weights and mid-sagittal corpus callosum area were measured. BALB/cJ sociability increased with age, and a strain by age interaction in sociability between 31 and 42 days of age suggested strong effects of puberty on sociability development. Sociability scores clustered according to litter membership in both strains, and perinatal litter size and sex ratio were identified as factors that contributed to this clustering in C57BL/6J, but not BALB/cJ, litters. There was no association between corpus callosum size and sociability, but smaller brains were associated with lower sociability in BALB/cJ mice. The associations reported here will provide directions for future mechanistic studies of sociability development.

Omission of the habituation procedure in the acquisition of a working memory task - evidence from Balb/c, C57/BL6J, and CD-1 mice.

Training animals in spatial mazes have always been preceded by prior habituation to the test apparatus and testing conditions with the main goal to reduce fear and anxiety from exposure to the unfamiliar maze environment. This approach makes assumptions about the baseline level of emotionality in animals without actual objective measurements. It also ignores that genetic factors and experimental manipulations can reduce or prolong fear and anxiety from novelty, hence affecting the acquisition of a memory task. In the present study, C57, CD-1 and Balb/c mice were introduced to a working memory task in a radial-arm maze without habituation. Fear-induced anxiety from exposure to the novelty in this maze is demonstrated by a very low number of arm entries. Animals have to climb onto a bridge in order to reach an arm of the maze. In the first session block, Bab/c mice made very few arm entries and made more arm repeats than CD-1 and C57 mice, and CD-1 made few arm entries and made more arm repeats than C57/BL6J mice. In the second session block, all three strains of mice did make 8 arm entries. Balb/c mice seem to perform better than C57 and CD-1 mice as shown by a low number of arm repeats in the second session block, a high number of correct choices before first errors in the third session block, and low number of errors and sessions to criterion. In the present case, a high baseline level of emotionality did not prevent Balb/c mice to perform better than C57 and CD-1 mice.

Paternal transmission of complex phenotypes in inbred mice.

BACKGROUND: Inbred mice are genetically identical but nonetheless demonstrate substantial variability in complex behaviors such as activity levels in a novel environment. This variability has been associated with levels of parental care experienced early in development. Although maternal effects have been reported in biparental and uniparental strains, there have been no investigations of paternal effects in non-biparental strains in which offspring are reared exclusively by mothers. METHODS: In the uniparental inbred Balb/cJ mouse strain, we examined the relationship of paternal open-field activity to the activity of both male and female offspring in the open-field. Potential mediators of paternal transmission of behavior were examined, including maternal care, growth parameters, litter characteristics, and time the father was present with the pregnant mother prenatally. RESULTS: An association of paternal open-field activity with the open-field activity of female but not male offspring was found. Variation in maternal postnatal care was associated with female but not male offspring activity in the open-field but did not mediate paternal effects on offspring behavior. Paternal effects on offspring growth parameters were present, but these effects also did not mediate paternal effects on behavior. CONCLUSIONS: Paternal transmission of complex traits in genetically identical mice reared only by mothers suggests a nongenetic mechanism of inheritance potentially mediated by epigenetic factors. The exclusion of multiple mediators of paternal effects on offspring suggests the possibility of germline paternal inheritance via sperm of complex phenotypes in inbred mice. Future studies are required to examine these interesting possibilities.

Early life stress alters adult serotonin 2C receptor pre-mRNA editing and expression of the alpha subunit of the heterotrimeric G-protein G q.

Infant maternal separation, a paradigm of early life stress in rodents, elicits long-lasting changes in gene expression that persist into adulthood. In BALB/c mice, an inbred strain with spontaneously elevated anxiety and stress reactivity, infant maternal separation led to increased depression-like behavioral responses to adult stress and robustly increased editing of serotonin 2C receptor pre-mRNA. Chronic fluoxetine treatment of adult BALB/c mice exposed to early life stress affected neither their behavioral responses to stress nor their basal 5-HT2C pre-mRNA editing phenotype. However, when fluoxetine was administered during adolescence, depression-like behavioral responses to stress were significantly diminished in these mice, and their basal and stress-induced 5-HT2C pre-mRNA editing phenotypes were significantly lower. Moreover, when BALB/c mice exposed to early life stress were raised in an enriched postweaning environment, their depression-like behavioral responses to adult stress were also significantly diminished. However, their 5-HT2C pre-mRNA editing phenotype remained unaltered. Hence, the similar behavioral effects of enrichment and fluoxetine treatment during adolescence were not accompanied by similar changes in 5-HT2C pre-mRNA editing. Enriched and nonenriched BALB/c mice exposed to early life stress also exhibited significantly increased expression of mRNA and protein encoding the G alpha q subunit of G-protein that couples to 5-HT2A/2C receptors. In contrast, G alpha q expression levels were significantly lower in fluoxetine-treated mice. These findings suggest that compensatory changes in G alpha q expression occur in mice with persistently altered 5-HT2C pre-mRNA editing and provide an explanation for the dissociation between 5-HT2C receptor editing phenotypes and behavioral stress responses.

Maternal influences on adult stress and anxiety-like behavior in C57BL/6J and BALB/cJ mice: a cross-fostering study.

The quality of maternal care during early life has a dramatic impact on later stress reactivity and anxiety. Two inbred mouse strains, C57BL/6J and BALB/cJ, differ in levels of maternal care, stress reactivity, and anxiety-like behavior in adulthood. However, the relative contribution of early environmental factors and genetic predisposition to differences in these strains is not known. Maternal care, plasma corticosterone levels, emotionality, and hippocampal and paraventricular nucleus (PVN) glucocorticoid receptor mRNA levels were measured in adult C57BL/6J and BALB/cJ mice. Litters were then cross-fostered and anxiety-like behavior and stress reactivity was assessed in adulthood. Significantly less maternal care and elevated stress-induced corticosterone and emotionality was observed in BALB/cJ compared to C57BL/6J mice. Yet, no strain differences were found in hippocampal or paraventricular nucleus glucocorticoid receptor mRNA levels. Cross-fostering did alter anxiety-like behavior and basal corticosterone levels, which suggests that while genetic differences account for some of the variations between these two strains early rearing conditions also contribute.

Short-term effects of a disturbed light-dark cycle and environmental enrichment on aggression and stress-related parameters in male mice.

In the laboratory setting, environmental factors have a major influence on the well-being of laboratory animals. The present study shows the importance of a semi-natural light-dark cycle. In this experiment one cohort of mice was kept with a continuous lighting for one week. After the first week the artificial light-dark cycle was 12:12 with lights on at 07:00 h. The second cohort of mice was kept with this 12:12 h light-dark cycle from the start. Half of each cohort received environmental enrichment. In order to analyse corticosterone levels, urine samples were collected. To measure agonistic behaviour, the behaviour of the mice was recorded on videotape immediately after cage cleaning. A significant difference in corticosterone levels between cohorts was found during disturbed lighting, but not after lighting conditions were reset to 12:12 h. In the first test week, mice subjected to disturbed lighting also showed a significantly shorter agonistic latency than control mice. This difference had disappeared when in the second test week all mice experienced 12:12 h lighting. No effects of enriched housing were found. This experiment has shown that disturbed lighting for socially-housed male mice caused physiological and behavioural changes indicative of stress, not only leading to much higher levels of corticosterone but also to shorter agonistic latency within the groups.

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.

Preference for social contact versus environmental enrichment in male laboratory mice.

Due to their aggressive nature, male mice are less frequently used than female mice in biomedical research. When aggressive males are being used, individual housing is common practice. The question arises whether this is an acceptable housing for a social species. The present study was designed to gain more insight into the nature of inter-male social contact and into the potential of a form of environmental enrichment (nesting material) to compensate for the lack of social contact. In a series of tests, we analysed whether male mice of different ages preferred to spend time (1) near a familiar cage mate versus an empty cage, or (2) near to a familiar cage mate versus direct contact with nesting material (tissues). Dwelling time in each of the test cages and sleeping sites was recorded, as was the behaviour of the test mice. Results indicated that when other conditions were similar, male mice preferred to sleep in close proximity to their familiar cage mate. Furthermore, the need to engage in active social behaviour increased with age. Tissues were used to a large extent for sleeping and sleep-related behaviour. It is concluded that single housing in order to avoid aggression between male mice is a solution with evident negative consequences for the animals. When individual housing is inevitable due to excessive aggressive behaviour, the presence of nesting material could partly compensate for the deprivation of social contact.

Floor space needs for laboratory mice: BALB/cJ males or females in solid-bottom cages with bedding.

Studies were initiated to determine the effects of restricted (32.2 cm2 per mouse), normal (96.8 cm2), or excess floor space (129.0 cm2) allowances by using a model of three mice per cage. BALB/cJ mice were bred on-site and weaned at 3 weeks of age into specially designed polycarbonate shoebox cages modified to each space allowance. Cages contained aspen shavings for bedding, and mice were fed and watered ad libitum. Body weight gains, feed and water use, and immunologic measures largely were not effected by floor space allowances. Female BALB/cJ mice were heavier and had increased lymphocyte blastogenesis to phytohemagglutinin (20 microg/mL) when given 32.2 cm2/mouse than when given 129 cm2/mouse. Female mice showed an increase in grooming and sitting behaviors when given 32.2 cm2/mouse, but male mice with restricted floor space spent more time lying down but showed no change in grooming or sitting behaviors compared to mice given more space. Among male mice, limited floor space did not significantly influence growth rates, but male mice given 32.2 cm2/mouse had less mortality than did mice given more space. We conclude that floor spaces as limited as 32.2 cm2/mouse did not cause behavior, health, immune or performance problems for BALB/cJ mice.

Behavioral, clinical, and physiologic analysis of mice used for ascites monoclonal antibody production.

BACKGROUND AND PURPOSE: The effects of pristane inoculation, ascites accumulation, peritoneocentesis, and analgesics on the well-being of mice used in monoclonal antibody (MAb) production protocols were investigated. METHODS: Four experiments, each containing 17 to 21, 6- to 8-week-old male Balb/c mice, were conducted. Each experiment involved a period in which baseline data were collected, followed by intraperitoneal injections of pristane or phosphate-buffered saline (PBS) inoculations into each mouse. One week later mice received intraperitoneal inoculations of either hybridoma cells or PBS. Parameters used to assess well-being throughout each of these periods included: wheel-running activity, food and water consumption, open-field box activity, clinical observation, and plasma corticosterone concentration. RESULTS: Compared to controls, pristane inoculation had slight to no affect on mice. There was no evidence of distress in cell-inoculated mice prior to their gaining 25% of their baseline body weight. The number of times (up to three) that peritoneocentesis was performed did not have a significant impact on mice's well-being, but ascites yields were greater when multiple harvests were performed. Cell-inoculated mice that gained weight slowly or developed high-particulate ascites were at higher risk of being distressed. CONCLUSION: Ascites yields can be maximized by performing multiple harvests; however, the well-being of mice used in such protocols should be closely monitored, as suggested here.