Behavioural differences between C57BL/6 and 129S6/SvEv strains are reinforced by environmental enrichment

Housing in enriched environment has been advocated as a means for controlled variation of environmental conditions in transgenic studies to explore interactions between genes and surroundings. In the present study, behavioural phenotypes of C57Bl/6 (B6) and 129S6/SvEv (129) mice, housed in either standard laboratory conditions or environmentally enriched conditions, were explored. Housing in enriched conditions increased exploratory activity in the plus-maze and reduced habituation in the locomotor activity test in B6 mice, whereas in 129 mice increased hot plate latencies and reduced aggression were observed. Compared to B6, 129 strain displayed lower exploratory activity in the plus-maze and locomotor activity test, longer hot plate latencies, spent more time immobile in the forced swim test and engaged more in social interaction. These behavioural differences between the two strains were reproducible independent of pre-experimental housing conditions. Moreover, environmental enrichment accentuated dissimilarities between the strains in the plus-maze, locomotor activity, hot plate and forced swim test. By contrast, strain differences in anxiety-like behaviours in the plus-maze test and in aggressive encounters in the resident-intruder test were not reproducible in mice housed in alternative environmental conditions, suggesting a strong contribution of environmental factors to the development of these phenotypes. It is concluded that the application of environmental enrichment in addition to standard housing conditions is a meaningful approach for testing reproducibility of behavioural findings within one laboratory.     

Strain and gender differences in the behavior of mouse lines commonly used in transgenic studies

The present study was aimed at establishing behavioral differences between three inbred mouse strains (129S2/SvHsd, C57BL/6JOlaHsd, FVB/NHsd) and two F1 hybrid lines derived from them (129 x C57BL/6 and 129 x FVB). The choice of the given strains was based on the frequent use of these mice in transgenic research. For the behavioral phenotyping, we employed a test battery consisting of the following models: elevated plus-maze (EPM), open field (OF), light-dark exploration, spontaneous locomotor activity, rota-rod (RR), Porsolt's forced-swimming test (FST), and Morris water task. Significant variations between the strains were established in all tests. Anxiety-like behavior was more pronounced in the 129S2/Sv and 129 x C57BL/6 mice, the FVB/N mice were spontaneously hyperactive, the best coordination ability was demonstrated by the C57BL/6 and 129 x C57BL/6 groups. A good performance in the learning test was established in both hybrid lines and the 129S2/Sv mice, whereas the well-known visual impairment of the FVB strain was confirmed by low performance in spatial and non-spatial tasks. Differences related to the gender were revealed occasionally; most importantly, 129 x C57BL/6 males had a higher anxiety level than their female counterparts in the EPM. Several other gender dissociations suggest the strain and task specificity. In conclusion, we would like to highlight the importance of the genetic background and gender of mice for the molecular biological and pharmacological studies and also the need for well-established testing protocols to obtain wide information at the first stage of behavioral screening of genetically modified mice.     

Improved generation of germline-competent embryonic stem cell lines from inbred mouse strains

Genetically altered mice may exhibit highly variable phenotypes due to the variation in genetic background, which can only be circumvented by generation of inbred, isogenic gene-targeted and control mice. Here we report that an embryonic stem (ES) cell culture medium conditioned by a rabbit fibroblast cell line transduced with genomic rabbit leukemia inhibitory factor allows efficient derivation and maintenance of ES cell lines from all of 10 inbred mouse strains tested, including some that were presumed to be nonpermissive for ES cell derivation (129/SvEv, 129/SvJ, C57BL/6N, C57BL/6JOla, CBA/CaOla, DBA/2N, DBA/1Ola, C3H/HeN, BALB/c, and FVB/N). Germline transmission was established by blastocyst injection of established ES cell lines after 10 or more passages from all of seven strains tested (129/SvJ, C57BL/6N, C57BL/6JOla, DBA/2N, DBA/1Ola, BALB/c, and FVB/N), by diploid aggregation of ES cell lines from all of four strains tested (129/SvEv, C57BL/6N, CBA/ CaOla, and FVB/N), or by tetraploid aggregation of ES cell lines from all of three strains tested (129/SvEv, C57BL/6N, and CBA/CaOla). Thus, these inbred ES cell lines may constitute useful tools to derive gene-targeted mice and isogenic controls in selected genetic backgrounds.     

Deficiency of Vlgr1 resulted in deafness and susceptibility to audiogenic seizures while the degree of hearing impairment was not correlated with seizure severity in C57BL/6- and 129-backcrossed lines of Vlgr1 knockout mice

Vlgr1 (very large G-protein coupled receptor 1) knockout mice against hybrid backgrounds of the 129/Ola and C57BL/6 mouse strains show hearing deficit and high susceptibility to audiogenic seizures. The present study examined how hearing impairment and susceptibility to audiogenic seizures in Vlgr1-deficient mice change according to the genetic background of 129 and C57BL/6 mouse strains, which are popular strains for genetic studies. C57BL/6 mice have normal hearing ability during adolescence and are resistant to audiogenic seizures, and the 129S1/SvImJ substrain does not have a severe hearing deficit or convulsions as a result of audiogenic seizures; therefore, these strains were chosen for the present backcross study. C57BL/6-backcrossed Vlgr1 knockout mice and 129 (129S1/SvImJ)-backcrossed Vlgr1 knockout mice were established and their phenotypes investigated. Vlgr1 knockout mice showed hearing loss and high susceptibility to audiogenic seizures regardless of their genetic backgrounds. 129-backcrossed Vlgr1 knockout mice exhibited 10–20 dB more severe hearing loss than C57BL/6-backcrossed Vlgr1 knockout mice. In general, 129-backcrossed Vlgr1 knockout mice showed a higher incidence of wild running than C57BL/6-backcrossed Vlgr1 knockout mice, and this incidence became smaller as they matured. However, C57BL/6-backcrossed Vlgr1 knockout mice showed a significantly higher mortality rate as a result of auditory stimulation 3 weeks postnatally than 129-backcrossed mice.     

The mTPH2 C1473G single nucleotide polymorphism is not responsible for behavioural differences between mouse strains

Tryptophan hydroxylase 2 (TPH2) is the rate limiting enzyme of serotonin synthesis in the brain. A recently described functional (C1473G) single nucleotide polymorphism in mouse TPH2 resulting in vitro in a strongly decreased enzymatic activity was suspected to be responsible for the observed differences in 5-HT levels and behaviour between mice strains. We bred two substrains of C57BL/6 mice carrying the two isoforms and could show that both exhibit equal TPH activity, brain 5-HT content and behaviour. These data indicate that the distinct behavioural characteristics of mouse strains are not due to differences in TPH2 activity, but to other variations in the genetic background.     

Home cage activity and activity-based measures of anxiety in 129P3/J, 129X1/SvJ and C57BL/6J mice

We investigated the home cage activity and emotional behavior in mouse strains used as background for many studies of altered genes [C57BL/6J (B6, n=20), 129X1/SvJ (X1, n=20) and 129P3/J (P3, n=19)]. In their home cages, X1 and P3 mice exhibited less locomotion than did B6 mice, and the X1 mice showed significantly greater rearing than B6 and P3 mice did. A battery of three tests conducted in an open field (open field, emergence and novel object) revealed strain rankings of B6>X1>P3 or B6>X1=P3 in most activity variables. Significant correlations were found between home cage activity and activity in each of three tests, but not in all observation periods. Strain rankings on the elevated zero maze test were B6=X1>P3 in the number of stretched-attend body postures (SAPS) during the initial 6-min exposure for naive mice. Naive and nonnaive mice showed significantly different behaviors on the elevated zero maze. The results suggest that rankings on anxiety are P3>X1>B6 and that B6 mice have greater exploration in a novel environment compared with X1 and P3 mice. However, anxiety-like behaviors differed among strains in open-field-based tests and in the zero maze, and testing experience impacted performance on the zero maze. The findings illustrate that test variations and experience can influence performance and suggest the need for the consideration of how these factors interact with background strains in assessing gene-altered mice.     

Behavioral responses of 129/Sv, C57BL/6J and DBA/2J mice to a non-predator aversive olfactory stimulus

We examined the behavior of three inbred mouse strains (129/SvPasIco, C57BL/6J, and DBA/2J) exposed to an object soaked with the chemical component of the aversive scent (toluquinone odor) emitted by a myriapod species (Ommatoiulus sabulosus) in the presence of a predator. Subjects were exposed to the odor for three consecutive days. Behavioral responses to the toluquinone odor were characterized both by an approach phase of risk assessment and by a repeated series of approach-avoid episodes. Results indicate that toluquinone exposure reduced completely, and in a strain independent fashion, selected behaviors such as crouching, catching and eating object. Other responses were strain-dependent: the DBA (DBA/2J) strain displayed defensive burying at high levels, C57 (C57BL/6J) mice performed high levels of withdrawal while the 129/Sv (129/SvPasIco) strain showed also high levels of stretch attend posture. Compared to other tasks, this test is ethological, simple, cheap and is not affected by strain differences in appetitive-sensory responses, as shown by some strain-independent responses. These features make this task as a good complement to any exploration-anxiety test battery.     

Genetic background modifies nuclear mutant huntingtin accumulation and HD CAG repeat instability in Huntington's disease knock-in mice

Genetically precise models of Huntington's disease (HD), Hdh CAG knock-in mice, are powerful systems in which phenotypes associated with expanded HD CAG repeats are studied. To dissect the genetic pathways that underlie such phenotypes, we have generated Hdh(Q111) knock-in mouse lines that are congenic for C57BL/6, FVB/N and 129Sv inbred genetic backgrounds and investigated four Hdh(Q111) phenotypes in these three genetic backgrounds: the intergenerational instability of the HD CAG repeat and the striatal-specific somatic HD CAG repeat expansion, nuclear mutant huntingtin accumulation and intranuclear inclusion formation. Our results reveal increased intergenerational and somatic instability of the HD CAG repeat in C57BL/6 and FVB/N backgrounds compared with the 129Sv background. The accumulation of nuclear mutant huntingtin and the formation of intranuclear inclusions were fastest in the C57BL/6 background, slowest in the 129Sv background and intermediate in the FVB/N background. Inbred strain-specific differences were independent of constitutive HD CAG repeat size and did not correlate with Hdh mRNA levels. These data provide evidence for genetic modifiers of both intergenerational HD CAG repeat instability and striatal-specific phenotypes. Different relative contributions of C57BL/6 and 129Sv genetic backgrounds to the onset of nuclear mutant huntingtin and somatic HD CAG repeat expansion predict that the initiation of each of these two phenotypes is modified by different genes. Our findings set the stage for defining disease-related genetic pathways that will ultimately provide insight into disease mechanism.     

Behavioral differences among 129 substrains: implications for knockout and transgenic mice

Most knockout (KO) mice are produced with embryonic stem cells derived from a 129 strain. Because most KO strains are backcrossed to B6 yet retain a portion of their genome from 129, especially around the ablated target locus, phenotypes previously attributed to the ablated locus may be due to passenger 129 genes. Thus, the authors decided to test several 129 substrains for their behavioral characteristics. Seven 129 substrains were put through a battery of tasks to determine their behavioral profiles. Differences were found in anxiety-related behaviors in the zero-maze, habituation to the open field, and cued fear conditioning. All strains successfully performed the rotorod task. The behavioral differences observed may have important implications for the interpretation of data and show divergence of behavioral performance in these 129 substrains.     

Heroin-induced locomotor activity and conditioned place preference in C57BL/6J and 129P3/J mice

Differences in the locomotor stimulating and rewarding properties of drugs of abuse have been described in several inbred strains of mice, and comparisons of inbred strains with differing responses to drugs of abuse may provide crucial insight into the question of individual vulnerability to the effects of drugs of abuse. The present study was designed to examine the rewarding and locomotor-stimulating effects of heroin in C57BL/6J and 129P3/J mice. Heroin produced a robust dose-dependent locomotor stimulation in both strains. Both strains also developed conditioned place preference to heroin, again in a dose-dependent manner. However C57BL/6J mice developed conditioned place preference to only the two lowest doses of heroin tested, while the 129P3/J counterparts showed conditioned place preference to only the three highest doses tested. These studies indicate that 129P3/J mice are less sensitive to the rewarding effects of heroin than are age-matched C57BL/6J mice.     

The rat exposure test: a model of mouse defensive behaviors

In order to facilitate behavioral, and potentially pharmacological, analyses of risk assessment behaviors in mice, a rat exposure test (RET) was devised and evaluated. This test provides a home chamber connected via a tunnel to a rat (predator) exposure area. Familiar substrate is provided to permit burying, and mouse subjects are habituated to the apparatus prior to exposure to an amphetamine-activated rat. In comparison to toy-rat-exposed controls, rat-exposed BALB/c mice showed significantly more risk assessment [stretch attend posture (SAP) and stretch approach], freezing, and avoidance (time in the home chamber), and less time in contact with the wire mesh screen between itself and the threat stimulus. When BALB/c, C57BL/6, CD-1, and Swiss-Webster mice were compared in this test, the two inbred strains (BALB/c and C57BL/6) tended to show more extreme values of particular defensive behaviors, compared to the two outbred strains (Swiss-Webster and CD-1). C57BL/6 mice showed more avoidance and higher levels of SAP, freezing, and burying than BALB/c and more than one or both outbred strains as well. BALB/c mice showed little defensive burying, both in comparison to toy-exposed controls (Experiment 1), and in comparison to the three other strains in Experiment 2. These findings are somewhat at variance with characterizations of anxiety in C57BL/6 and BALB/c mice, based on tests utilizing novel areas and noxious stimuli, suggesting strain differences in defensiveness to such stimuli, compared to antipredator defense levels. Nonetheless, with the exception of burying in BALB/c mice, all strains showed all defensive behaviors measured to the rat stimulus. In particular, SAP levels were substantial in all strains tested, suggesting the usefulness of this test in assessment of the role of risk assessment in defense.     

Progesterone and 3alpha,5alpha-THP enhance sexual receptivity in mice

Progesterone (P) and its metabolites' effects on sexual receptivity (lordosis) of mice was examined. P dosages that produced normal circulating concentrations of P and 5alpha-pregnan-3alpha-ol-20-one (3alpha,5alpha-THP) enhanced lordosis of ovariectomized, sexually experienced C57BL/6J (C57), +/+ C57BL/6Jx129SvEv (C57x129), and -/- C57BL/6Jx129SvEv (PRKO) mice. Only C57 and C57x129 mice had increases in progestin receptor (PR)-immunoreactivity (PR-IR) in the hypothalamus. RU38486, a PR antagonist, attenuated lordosis of C57 and C57x129, but not PRKO, mice; epostane, a progestin biosynthesis inhibitor, reduced plasma progestins; and finasteride, a P metabolism inhibitor, reduced plasma 3alpha,5alpha-THP and attenuated lordosis of all mice. For sexually naive mice, greater lordosis on initial sexual experience corresponded to greater concentrations of plasma and central progestins and increased central binding of a GABAA agonist, muscimol, compared with that seen in mice with lower lordosis on initial mating. Thus, P-facilitated receptivity in mice involves P and 3alpha,5alpha-THP and their actions at PRs and GABAA receptors.     

Nutrient preference and diet-induced adiposity in C57BL/6ByJ and 129P3/J mice

Purified carbohydrates and fats are usually palatable to humans and other animals, and their consumption often induces weight gain and accumulation of fat. In this study, we examined consumption of complex carbohydrates (cornstarch and Polycose) and fats (soybean oil and margarine) in mice from two inbred strains, C57BL/6ByJ and 129P3/J. At lower concentrations of liquid nutrients tested using two-bottle tests, when the amounts consumed had negligible energy content, the C57BL/6ByJ mice had higher acceptance of Polycose and soybean oil. This was probably due to strain differences in chemosensory perception of Polycose and oil. At higher concentrations, the mice consumed a substantial part of their daily energy from the macronutrient sources, however, there were no or only small strain differences in nutrient consumption. These small differences were probably due to strain variation in body size. The two strains also did not differ in chow intake. Despite similar energy intakes, access to the nutrients resulted in greater body weight (BW) gain in the C57BL/6ByJ mice than in the 129P3/J mice. The diet-induced weight gain was examined in detail in groups of 2-month-old C57BL/6ByJ and 129P3/J mice given ether chow, or chow and margarine to eat. Access to margarine did not increase total energy consumption of either strain. It increased BW and adiposity of the C57BL/6ByJ mice, but only after they reached the age of approximately 3 months. There were no differences in BW and adiposity between control and margarine-exposed 129P3/J mice. The results suggest that diet-induced adiposity in the B6 mice depends on age and does not depend on hyperphagia.     

Hyalinosis and Ym1/Ym2 gene expression in the stomach and respiratory tract of 129S4/SvJae and wild-type and CYP1A2-null B6, 129 mice

The C57BL/6, 129, and B6,129 mouse strains or stocks have been commonly used to generate targeted mutant mice. The pathology of these mice is not well characterized. In studies of these aging mice, we found high incidences of hyalinosis (eosinophilic cytoplasmic change) in the glandular stomach, respiratory tract, bile duct, and gall bladder of B6,129 CYP1A2-null and wild-type mice as well as in both sexes of the background 129S4/SvJae strain. The gastric lesions of the glandular stomach were found in 95.7% of female CYP1A2-null mice as well as in 45.7% of female 129S4/SvJae animals. The eosinophilic protein isolated from characteristic hyaline gastric lesions was identified as Ym2, a member of the chitinase family. Immunohistochemistry, using rabbit polyclonal antibodies to oligopeptides derived from the Ym1 sequence, detected focal to diffuse reactivity within both normal and abnormal nasal olfactory and respiratory epithelium, pulmonary alveolar macrophages, bone marrow myeloid cells, and the squamous epithelium of the forestomach and epithelium of the glandular stomach. Alveolar macrophages in acidophilic pneumonia, a major cause of death of aging 129 mice, and in mice with the me mutation also were highly immunoreactive. The possible cause of this protein excess in gastric and other lesions and its possible functions are discussed.     

Phenotypic characterization of C57BL/6J mice carrying the Disc1 gene from the 129S6/SvEv strain

Disruption of disrupted-in-schizophrenia 1 (DISC1), a candidate susceptibility gene for schizophrenia, was first identified in a large Scottish family in which many members suffered from various psychiatric disorders, including schizophrenia. To model the Scottish DISC1 truncation, we established a Disc1 mutant mouse line in which the 129S6/SvEv 25-bp deletion variant was transferred into the C57BL/6J strain by backcrossing. A battery of behavioral tasks was conducted to evaluate the basic behaviors and cognitive function of these mice. In heterozygote and homozygote Disc1 mutant (Het and Homo) mice, behavioral impairments were noted in working memory test which is thought to be mediated by the function of the medial prefrontal cortex (mPFC). The properties of mPFC neurons were characterized in both morphological and physiological aspects. The dendritic diameters were decreased in layer II/III mPFC pyramidal neurons of Het and Homo mice, whereas a significant reduction in spine density was observed in Homo mice. Neuronal excitability was declined in layer II/III mPFC pyramidal neurons of Het and Homo mice, yet increased transmitter release was identified in Homo mice. Thus, the structural and functional alterations of the mPFC in Het and Homo mice might account for their cognitive impairment. Since most of the gene knockout mice are generated from 129 substrain-derived embryonic stem cells, potential Disc1 deficiency should be considered.     

Loss of Aggression, After Transfer onto a C57BL/6J Background, in Mice Carrying a Targeted Disruption of the Neuronal Nitric Oxide Synthase Gene

Phenotypic differences among mice with disrupted genes and those with wild-type alleles have not provided the necessary evidence for desired gene/phenotype correlations. These differences could be due to "passenger genes" from the donor 129 strains that are used to produce stem cells. Three variations of attack behavior were measured, using mice carrying a disruption of the neural nitric oxide synthase gene. In the first population, the disrupted gene had been maintained on a mixed background including C57BL/6J and 129 alleles. We have developed a second population in which the disrupted gene was transferred onto a C57BL/6J background during five backcross generations. On the mixed C57BL/6J-129 background, mice homozygous for disrupted Nos1 alleles attacked more frequently, had shorter attack latencies, and presented a greater number of attacks than mice carrying nondisrupted alleles. On the C57BL/6J background, no significant difference persisted between the carriers of the disrupted gene and their noncarrier siblings. The noncarriers on the mixed C57BL/6J-129 background, and the carriers or noncarriers on the C57BL/6J background, did not differ from C57BL/6J. The frequency of attacking males was identical in the homozygous carriers of the disrupted gene, in the mixed C57BL/6J-129 background, and in the 129/SvPas, which approximates the 129/SvJae strain from which the stem cells were derived to produce the disrupted Nos1 gene. These results suggest that Nos1 disruption was not implicated in attack behavior. A possible passenger-gene effect from the 129 donor strain is discussed.     

zeta-/- Thalassemic mice are affected by two modifying loci and display unanticipated somatic recombination leading to inherited variation

Thalassemia is a disease caused by a variety of mutations affecting both the adult and embryonic alpha- and beta-globin loci. A mouse strain carrying an embryonic zeta-globin gene disrupted by the insertion of a PGK-Neo cassette displays an alpha-thalassemia-like syndrome. Embryonic survival of this zeta-null mouse is variable and strongly influenced by genetic background, the 129/SvEv mouse strain displaying a more severe phenotype than C57BL/6. We have identified two modifying loci on C57BL/6 chromosomes 2 and 5, which affect the penetrance of embryonic lethality in the 129/SvEv mouse. Through this work, we were able to observe an interesting effect on somatic recombination events in thalassemic embryos. We show that these events can occur on multiple chromosomes in very early embryonic cells, prior to their allocation to the germline. Our results demonstrate that somatic recombination events can be transmitted to subsequent generations.     

Antibody class switching differs among SJL, C57BL/6 and 129 mice

Inbred mouse strains used for gene manipulation studies vary in many respects, including immune system function. These differences can interfere with data interpretation unless the mice are well backcrossed. Here, we show that antibody class switching to IgG3 in cultured splenic B cells from Swiss James Lambert (SJL) and 129/Sv mice is 2- to 6-fold less efficient compared with C57BL/6 (B6). Under optimal stimulation conditions, IgA switching is also 2- to 19-fold lower in SJL and 129/Sv B cells. Splenic B cells from SJL mice express higher levels of CD19 and CD21 compared with B6, and their CD21(high)CD23(low) B cells have little CD9 expression, suggesting atypical marginal zone (MZ) B cells. However, sort purification of splenic B cell subsets did not equalize in vitro class switching to IgG3 or IgA between SJL and B6. 129/Sv spleens have a 3-fold greater number of MZ B cells compared with B6, with similar CD9 expression. Poor IgG3 switching by 129/Sv B cells is specific to CD23(high) follicular B cells, whereas similar changes in IgA switching are seen in both CD21(high) and CD23(high) B cell subsets from 129/Sv. Therefore, the functions and phenotypes of mature B cells differ among three common strains of mice.     

Behavioral Differences among C57BL/6 Substrains: Implications for Transgenic and Knockout Studies

Separate breeding colonies of C57BL/6 (“B6”) mice maintained at the Jackson Laboratories (“J”) and NIH (“N”) have led to the emergence of two distinct substrains of C57BL/6 mice: C57BL/6J and C57BL/6N. Molecular genetic studies indicate simple sequence-length polymorphisms, single-nucleotide polymorphisms, and copy-number variants among B6 substrains that may contribute to phenotypic differences. We examined differences in motor coordination, pain sensitivity, and conditional fear in the C57BL/6J strain and three N strains: C57BL/6NCrl (Charles River), C57BL/6NTac (Taconic), and C57BL/6NHsd (Harlan Sprague Dawley). Male C57BL/6J mice demonstrated enhanced motor coordination, as measured by the rotarod assay, markedly enhanced pain sensitivity in two assays of acute thermal nociception (e.g., tail withdrawal and hot plate), and a reduced level of conditional fear. The tail withdrawal result was confirmed in a separate laboratory. We also provide a table reviewing previously reported behavioral differences among various B6 substrains and discuss the significance of environmental differences due to obtaining mice form different vendors. These data may be seen as a potential problem and as a potential opportunity. Great care must be taken when working with mice engineered by using B6 embryonic stem cell lines because control groups, backcrosses, and intercrosses could inadvertently introduce behaviorally significant polymorphic alleles or environmental confounds. On the other hand, deliberate crosses between B6 substrains may provide an opportunity to map polymorphic loci that contribute to variability in a trait on largely homogenous backgrounds, which has the potential to improve mapping resolution and aid in the selection of candidate genes.