Edited by: Tomoko Soga, Monash University Sunway Campus, Malaysia
Reviewed by: Takefumi Kikusui, Azabu University, Japan; Fumihiko Maekawa, National Institute for Environmental Studies, Japan
*Correspondence: Tsuyoshi Koide, Mouse Genomics Resource Laboratory, National Institute of Genetics, 1111 Yata, Mishima, Shizuoka 411-8540, Japan e-mail:
This article was submitted to Neuroendocrine Science, a section of the journal Frontiers in Neuroscience.
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The Japanese wild-derived mouse strain MSM/Ms (MSM) retains a wide range of traits related to behavioral wildness, including high levels of emotionality and avoidance of humans. In this study, we observed that MSM showed a markedly higher level of aggression than the standard laboratory strain C57BL/6J. Whereas almost all MSM males showed high frequencies of attack bites and pursuit in the resident-intruder test, only a few C57BL/6J males showed aggressive behaviors, with these behaviors observed at only a low frequency. Sexually mature MSM males in their home cages killed their littermates, or sometimes female pair-mates. To study the genetic and neurobiological mechanisms that underlie the escalated aggression observed in MSM mice, we analyzed reciprocal F1 crosses and five consomic strains of MSM (Chr 4, 13, 15, X and Y) against the background of C57BL/6J. We identified two chromosomes, Chr 4 and Chr 15, which were involved in the heightened aggression observed in MSM. These chromosomes had different effects on aggression: whereas MSM Chr 15 increased agitation and initiation of aggressive events, MSM Chr 4 induced a maladaptive level of aggressive behavior. Expression analysis of mRNAs of serotonin receptors, serotonin transporter and
Aggression is one of the most conserved behavioral traits in the animal kingdom. It is observed in insects, fish, crustaceans, reptiles, amphibians, birds, and mammals, including humans. However, there are also large differences in the level of aggression between individuals from the same species. These differences can be caused by both environmental and genetic factors. Mouse strains can differ substantially in their levels of aggressive behavior (Ginsberg and Allee,
The neurobiological mechanisms that control aggression are widely conserved, and the involvement of the serotonin (5-HT) system in aggressive behavior has been confirmed for species from fly to human (for review, see Olivier et al.,
It has been unclear whether results from studies of laboratory mice are representative of their wild conspecifics. For example, it has been shown that the level of emotionality is attenuated and behavioral patterns are changed in laboratory strains compared with those in wild mice (Holmes et al.,
In this study, we aimed to identify (1) the genetic basis of escalated aggressive behavior and (2) the involvement of the 5-HT system in the escalated aggression of MSM. For the genetic analysis, we first characterized the aggressive behavior of MSM in comparison with that of B6 in a standard test for territorial aggression (resident-intruder test) and in the daily housing condition. Then, we analyzed a selected set of consomic strains of MSM against a background of B6 to identify the chromosomes that are involved in the escalated aggression of MSM. To examine the involvement of the 5-HT system as one of the intermediate phenotypes (endophenotypes) of the individual differences in aggression, we also examined the mRNA expression of genes for the receptors, synthesizing enzyme and transporter of 5-HT in several brain areas of the consomic strains, MSM and B6.
The MSM/Ms (MSM) strain was established and bred at the National Institute of Genetics (NIG). C57BL/6JJcl (B6) mice were purchased from CLEA Japan and bred at NIG. For F1 analysis, we made reciprocal crosses of B6 and MSM (3–4 pairs for each line) to make (B×M)F1 progeny (MSM father) and (M×B)F1 (MSM mother) progeny at NIG. A panel of B6-ChrNMSM consomic strains were established and has been maintained at NIG. The process used to establish this panel was described previously (Takada et al.,
Each resident male was housed in pairs with a female of the same strain in transparent polycarbonate cage (22 × 32 × 13.5 cm) with wood chips as bedding material. Intruder males were group-housed at 3–6 per cage in the absence of females. All animals were maintained at NIG with controlled humidity and temperature (50 ± 10%, 23 ± 2°C) under a 12/12-h light/dark cycle (lights on at 6:00 AM). Food and water were freely available. All of the behavioral testing was conducted during the dark period of the photo-cycle (from 6:00 PM to 10:00 PM). All procedures were approved (permit numbers 23-10, 24-10 and 25-10) by the Institutional Committee for Animal Care and Use of the NIG.
To follow the aggression of MSM and B6 strains in the rearing conditions, we examined their breeding records in the NIG for the previous six years. These records contain information on all the animals from after they were weaned from their parents (at about 3–4 weeks old) until they were used for other studies (at 9–10 weeks old) or used for breeding to produce the next generation. Animals that had been severely injured (lost their tails or had some signs of wounding) or died from attacks by a littermate were recorded as having been subjected to “injurious aggression.” Animals with severe injuries were euthanized once we found evidence of injurious aggression, given that such injuries often result in death within a few days. Given that these records were limited to only the animals in the maintenance colony, we could not follow the animals after they were used for other studies (after 9–10 weeks old). Therefore, there is a limitation in these breeding records insofar as there is the possibility of overlooking incidents of aggression that occur later in the life of these animals.
Resident males at the age of 7 weeks were housed in pairs with females of the same strain to enhance territorial aggression. In the case of consomic strains, B6 females were sometimes used as the pair-mate if females of the same genotype were not available. After 3 weeks of being housed with a female, the residents were studied for their territorial aggression to an intruder male by using the resident-intruder test. Animals were 10 weeks of age when their aggression was assessed (10–12 weeks in the analysis of consomic strains). Males of a different litter but the same strain were used as the intruders to estimate the aggression in B6 and MSM strains. For reciprocal F1s and consomic strains, we used B6 males as the intruders. The female and pups were removed, and an intruder male was introduced into the home cage of the resident male. Their behaviors were observed for 5 min after the first attack bite, or the intruder was removed after 5 min if no attack occurred. This encounter occurred twice, with a 48-h interval. All behaviors of the animals during the test were videotaped for subsequent behavioral analysis. During the video analysis, the frequency of attack bites and the durations of sideways threats, tail rattles, pursuit, and non-aggressive behaviors (walking, rearing, self-grooming and contact) were quantified as operationally defined and illustrated previously (Grant and Mackintosh,
Animals were euthanized by CO2 inhalation, and their brains were rapidly removed and placed on ice. Seven brain areas (olfactory bulb, prefrontal cortex, striatum, hippocampus, hypothalamus, midbrain, and cerebellum) were dissected by a surgical knife on ice. Briefly, the olfactory bulb was first dissected at the rostral tip of the prefrontal cortex, then the brain was inverted upside-down and the hypothalamus—defined as the area between optic chiasm and mammillary body—was dissected. Next, the midbrain and the cerebellum were obtained. The midbrain area was defined as a coronal section that includes both the superior and the inferior colliculus, and thus both the dorsal raphe and the median raphe nuclei were included in this area. Finally, the brain was sagittally split by the midline, and the prefrontal cortex was dissected from both hemispheres by cutting the 1 mm rostral tip of the frontal cortex at approximately a 45° angle. The whole hippocampal structure was also taken out from both hemispheres, and the striatum was dissected using scissors. These samples were homogenized on ice in Trizol (Invitrogen, USA). Total RNA was extracted and the quantity and quality were checked using a spectrophotometer (NanoDrop, USA). The RNA purity was assessed by determining the OD ratio (260/280 nm > 2) and the 28S/18S rRNA ratio by denaturing RNAs and separating them in a 1% agarose gel with ethidium bromide staining. After DNase treatment (TURBO DNA-free™ kit, Ambion, USA), cDNA was synthesized from each brain area using Primescript Reverse Transcriptase (TaKaRa Bio, Japan). All cDNA samples were stored at −20°C until analysis by real-time PCR.
The primers used in this study are listed in Table
Eight to fifteen animals in each strain at around 11–12 weeks of age were used for this analysis. Each male was housed with a female for 3 weeks and then experienced two aggressive encounters separated by a 48-h interval. Their brains were removed five days after the last aggressive encounter.
The midbrain and prefrontal cortex were sampled from males of B6 (
Samples were measured using a high performance liquid chromatography (HPLC) system equipped with an electrochemical detector (ECD-300, Eicom Co., Kyoto, Japan) and ODS column [EICOMPAC PP-ODS II (4.6 × 30 mm) at 25°C (Eicom Co.)]. To measure 5-HT, a mobile phase with 100 mM PBS (pH 5.4), 500 mg/L sodium n-Dodecyl Sulfate (SDS), 13.4 uM EDTA-2Na and 2% methanol in HPLC grade water was used. Samples were diluted by 10%, and 10 μl samples were injected into the HPLC.
Fisher's exact test was used to compare the proportion of animals that showed aggressive behaviors during the 5-min encounter in B6 with those in MSM, F1s, and consomic strains. A repeated-measures Two-Way ANOVA was performed to examine the strain difference in aggressive and non-aggressive behaviors over the two encounters. For the analysis of consomic strains, One-Way ANOVA was conducted using the average value of the first and second encounters owing to the low occurrence of aggressive behavior in the consomic strains. One-Way ANOVA was performed to examine strain differences in the expression of mRNA. When a significant
Although the records kept during the breeding of the MSM strain are incomplete (see Materials and Methods), we found an interesting trend in the differences between strains in terms of their aggression toward same-sex littermates in the home cage. As mentioned above, animals that had been severely injured or died after an attack by another littermate were recorded as having suffered from “injurious aggression.” From the records of MSM, injurious aggression was observed in 13.6% of the housing cages (24 out of 177 cages) that contained multiple male littermates (on average, three males per cage). This injurious aggression was observed after the age of 7 weeks old, when the males are sexually mature. In contrast, injurious aggression was never noted in any of the 265 cages that housed B6 animals. In addition, none of the females of either the MSM or B6 strains showed injurious aggression toward their same-sex cage mates. However, MSM males sometimes attacked their female pair-mates. Females in 9 out of 62 breeding pairs of MSM (14.5%) were injured or killed.
Mice of the MSM strain showed higher levels of inter-male aggression than their B6 counterparts in the resident-intruder test. Whereas 14 resident males out of 16 pairs (87.5%) of MSM showed attack bites at the first encounter, only 2 residents out of 19 pairs (10.5%) of B6 showed aggressive behaviors (Table
Total resident males | 19 | 19 | 16 | 16 |
Males that showed attack bites | 2 | 4 | 14 |
15 |
% aggressive males | 10.5 % | 21.1 % | 87.5 % | 93.8 % |
Attack bites (f) | 3.4 ± 2.3 | 5.7 ± 2.7 | 42.9 ± 6.0 |
33.8 ± 6.1 |
Sideways threats (d) | 2.9 ± 2.0 | 5.7 ± 4.0 | 6.2 ± 1.7 | 4.0 ± 0.9 |
Tail rattles (d) | 1.2 ± 0.8 | 2.5 ± 1.4 | 8.8 ± 1.4 |
8.1 ± 3.5 |
Pursuit (d) | 0.8 ± 0.7 | 0.7 ± 0.5 | 70.3 ± 10.2 |
50.1 ± 8.9 |
Walking (d) | 124.8 ± 8.8 | 102.5 ± 7.4 | 57.4 ± 5.8 |
62.1 ± 8.0 |
Rearing (d) | 43.1 ± 3.9 | 34.6 ± 4.6 | 18.5 ± 3.9 |
18.7 ± 4.1 |
Grooming (d) | 7.8 ± 1.8 | 8.6 ± 2.0 | 6.3 ± 3.7 | 9.3 ± 4.9 |
Contact (d) | 44.7 ± 5.5 | 39.6 ± 6.3 | 21.7 ± 12.7 | 11.9 ± 10.1 |
Attack latency | 287.9 ± 9 | 263.5 ± 18 | 172.7 ± 23 |
99.2 ± 24 |
Aggressive behaviors of the reciprocal F1 heterozygotes, (B×M)F1 and (M×B)F1, were also examined and compared with those of their parental strains, B6 and MSM (Figure
Total resident males | 10 | 10 | 10 | 10 |
Males that showed attack bites | 6 | 8 | 9 |
10 |
% aggressive males | 60 % | 80 % | 90 % | 100 % |
Attack bites (f) | 25.0 ± 8.9 | 31.6 ± 7.4 |
44.9 ± 9.7 |
42.8 ± 11.5 |
Sideways threats (d) | 8.2 ± 2.3 | 8.3 ± 1.6 | 13.5 ± 2.8 |
11.4 ± 3.3 |
Tail rattles (d) | 4.6 ± 1.6 | 12.3 ± 4.7 | 17.0 ± 6.1 |
19.8 ± 7.5 |
Pursuit (d) | 4.0 ± 1.8 |
7.6 ± 3.4 |
5.9 ± 1.9 |
4.4 ± 1.7 |
Walking (d) | 79.3 ± 5.2 |
80.5 ± 7.0 | 73.5 ± 7.2 |
55.7 ± 8.8 |
Rearing (d) | 50.3 ± 10.1 |
36.4 ± 6.8 | 32.7 ± 7.5 | 16.7 ± 6.7 |
Grooming (d) | 13.5 ± 5.5 | 9.8 ± 2.0 | 9.3 ± 4.8 | 9.5 ± 3.0 |
Contact (d) | 48.1 ± 15.4 | 26.9 ± 11.0 | 43.0 ± 13.8 | 8.4 ± 5.8 |
Attack latency | 197.0 ± 27 |
111.9 ± 33 |
89.4 ± 26 |
58.6 ± 14 |
This study examined five strains (that correspond to chromosomes Chr 4, Chr 13, Chr 15, Chr X, and Chr Y) of twenty-nine consomic strains. We chose these strains in this analysis because a previous study that used the social interaction test indicated that a subset of male pairs in the consomic strains of Chr 4, 13, 15, and 17 showed attack bites during the test, whereas the other strains did not show any aggressive behavior (Takahashi et al.,
All of the consomic strains analyzed in this study showed a low level of aggressive behavior similar to that of B6 at the first encounter (Table
Total resident males | 32 | 20 | 18 | 23 | 18 | 15 |
Males that showed attack bites | 5 (2) | 9 (5) | 4 (1) | 13 (2) |
2 (0) | 1 (0) |
% aggressive males | 15.6 % | 45.0 % | 22.2 % | 56.5 % | 11.1 % | 6.7 % |
Attack bites (f) | 2.7 ± 1.2 | 14.8 ± 5.0 |
5.8 ± 3.1 | 5.2 ± 1.5 | 1.2 ± 0.8 | 1.0 ± 1.0 |
Sideways threat (d) | 2.6 ± 1.4 | 7.8 ± 2.4 |
3.3 ± 1.9 | 4.1 ± 1.3 | 1.2 ± 0.9 | 0.6 ± 0.6 |
Tail rattles (d) | 1.1 ± 0.5 | 9.7 ± 2.8 |
5.8 ± 3.1 | 8.8 ± 2.5 |
0.5 ± 0.3 | 0.8 ± 0.8 |
Pursuit (d) | 0.4 ± 0.3 | 6.6 ± 3.4 |
0.8 ± 0.6 | 0.4 ± 0.2 | 0.3 ± 0.3 | 0.0 ± 0.0 |
Walking (d) | 108.0 ± 4.7 | 100.7 ± 6.6 | 123.1 ± 6.6 | 92.7 ± 4.1 | 140.9 ± 4.0 |
106.1 ± 7.9 |
Rearing (d) | 39.7 ± 2.4 | 37.4 ± 3.2 | 41.5 ± 3.2 | 37.6 ± 2.5 | 36.8 ± 2.2 | 37.1 ± 2.9 |
Grooming (d) | 7.6 ± 0.9 | 9.1 ± 2.4 | 5.9 ± 0.8 | 11.0 ± 1.5 | 5.5 ± 1.0 | 6.4 ± 1.1 |
Contact (d) | 44.1 ± 3.6 | 42.0 ± 5.8 | 43.0 ± 6.0 | 35.3 ± 3.6 | 32.5 ± 3.0 | 28.4 ± 4.8 |
Attack latency | 278.3 ± 11.0 | 189.6 ± 27.1 |
262.9 ± 20.3 | 219.8 ± 19.1 |
276.2 ± 16.6 | 288.3 ± 11.7 |
The escalation of aggression in the Chr 4 consomic strain was also observed in the daily housing condition according to the breeding record. During the 3 weeks of housing with a female before the test, we also checked the occurrence of injurious aggression toward a female pair-mate. The Chr 4 consomic strain showed injurious aggression toward females, and females in 8 out of 20 pairs were injured. This strain also showed injurious aggression toward male cage mates (11.9%, 15 out of 126 cages). On the other hand, we did not observe any cages with injurious aggression in the other consomic strains of Chr 13, Chr 15, Chr X, and Chr Y.
To evaluate the difference in the 5-HT system between B6 and MSM, we examined the expression level of 5-HT receptor mRNAs in seven brain areas of B6 and MSM using quantitative real-time PCR (Figure
To examine whether these strain differences observed in the expression of 5-HT-related mRNA correspond to heightened aggression in MSM, we then examined the expression of 5-HT receptors and Tph2 using five consomic strains, and its genetic correlation with aggressive behaviors (Table
Chr 4 | 86 ± 9 | 150 ± 14 |
98 ± 11 | 85 ± 9 | 97 ± 4 | 72 ± 10 | 146 ± 15 |
Chr 13 | 108 ± 12 | 146 ± 27 |
125 ± 27 | 164 ± 14 |
79 ± 6 | 55 ± 6 | 117 ± 10 |
Chr 15 | 113 ± 16 | 91 ± 13 | 84 ± 13 | 83 ± 9 | 83 ± 8 | 68 ± 13 | 124 ± 11 |
Chr X | 114 ± 12 |
92 ± 14 | 126 ± 18 | 93 ± 13 | 82 ± 10 | 39 ± 9 |
113 ± 14 |
Chr Y | 106 ± 2 | 86 ± 9 | 106 ± 26 | 81 ± 8 | 99 ± 15 | 77 ± 11 | 98 ± 10 |
% aggressive animals | −0.49 | 0.31 | −0.69 | −0.07 | −0.21 | 0.03 | 0.82 |
Attack bites | −0.46 | 0.81 |
−0.31 | 0.00 | 0.12 | 0.06 | 0.85 |
Tail rattles | −0.53 | 0.61 | −0.49 | 0.15 | −0.22 | −0.05 | 0.90 |
Pursuit | −0.32 | 0.72 | −0.23 | −0.20 | 0.31 | 0.07 | 0.74 |
To examine whether the increase of
This study revealed that a Japanese wild-derived mouse strain, MSM, has an escalated level of aggressive behavior compared with the commonly used laboratory strain B6. This aggressive behavior of MSM was characterized by frequent pursuit (chasing) behavior, in addition to attack bites. This pursuing contrasts with the behavior of not only B6 strain but other laboratory mouse lines, such as ICR and CFW (Takahashi et al.,
The analysis of reciprocal F1s showed that there is a different mode of inheritance for some indices of escalated aggression observed in MSM. Given that we did not observe any injurious aggression and also no increase of pursuit in both F1 intercrosses, these phenotypes are considered as recessive traits. On the other hand, the frequency of attack bites and tail rattles, as well as the percentage of aggressive animals, were higher in the F1 intercrosses than in B6, whereas attack latency was lower in F1 intercrosses than in B6. Thus, these behaviors have either a dominant or an additive mode of inheritance. Interestingly, we found differences between the reciprocal F1 crosses in these phenotypes: whereas (M×B)F1, which has MSM as a mother, showed a pronounced increase of aggression similar to that of MSM, (B×M)F1, which has MSM as a father, showed a level of aggression intermediate between that of B6 and MSM. The genetic differences between (M×B)F1 and (B×M)F1 are only in sex chromosomes and mitochondrial DNA; all autosomes are identically heterozygote. However, our analysis of consomic strains did not find any effect of the sex chromosomes on intermale aggression despite the sex chromosomes previously being implicated in aggressive behaviors by the analysis of both hybrid or congenic strains of Y (Selmanoff et al.,
Analysis of consomic strains identified two chromosomes, Chr 4 and Chr 15, which are involved in these different aspects of aggressive behavior. Our results indicated that Chr 15 of MSM increased the proportion of animals that initiated aggressive behavior and the frequency of tail rattles. However, the frequencies of attack bites and pursuit were similar to those in B6, and there was no injurious aggression observed in Chr 15 consomic males. These findings suggest that there is genetic locus that increases agitation and the initiation of aggressive behaviors on Chr 15. On the other hand, we found that the consomic strain of Chr 4 showed a maladaptive level of aggression. The breeding records from daily housing conditions indicated that the Chr 4 consomic males showed injurious aggression toward both their same-sex littermates and their female mates. In the resident-intruder test, Chr 4 males showed increased frequencies of attack bite and a longer duration of pursuit. On the other hand, the proportion of animals that showed aggressive behavior was not significantly different from that in B6. This indicated that Chr 4 consomic animals showed exaggerated aggressive behavior after aggression had been triggered. Thus, a genetic locus on MSM Chr 4 might be responsible for the maladaptive aspect of aggression observed in MSM. Our findings indicate that there are different genetic bases for agitation and readily provoked aggressive behavior (Chr 15) and for escalated maladaptive aggressive behavior (Chr 4). A role for Chr 4 in controlling aggression is consistent with a report that strains of A/J and B6, which carry substitutions in Chr 4, also showed severe fighting in the housing cage (Singer et al.,
Compared with MSM, all tested consomic strains showed a low level of aggression at the first encounter compared with MSM. This indicates that the genetic effect of either Chr 4 or Chr 15 is not very large and that multiple loci contribute to the escalated aggression of MSM.
Expression analysis of several 5-HT receptors,
Although Tph2 has been implicated in aggression because it directly affects the activity of 5-HT neurons, the relationship between Tph2 activity and the level of aggression seems to be complex. It has been shown that both male and female
This study identified the involvement of two chromosomes, Chr 4 and Chr 15, in different aspects of escalated aggression in MSM. Our result of a correlation between
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The authors would like to thank Kumiko Takahashi, Michiko Arii and Miyoko Umehara for the maintenance and breeding records of the consomic strains, MSM, and B6, and Akira Tanave for establishing behavioral analysis software. This research was funded by KAKENHI (22830130, 25116527, and 23683021).
The Supplementary Material for this article can be found online at: