As we hypothesized, C57BL/6J mice were more passively social than BALB/cJ mice in the home cage environment, and the largest (and only statistically significant) strain differences were at 30 days of age. The levels of C57BL/6J passive social behaviors declined to BALB/cJ levels by 69 days of age, and both the decline and the strain difference at 30 days were mostly attributable to differences in huddling among the mouse groups. Contrary to our hypothesis, 41-day-old BALB/cJ mice were apparently more actively social than 41-day-old C57BL/6J mice in the home cage environment, but this apparent difference may be a false positive, as discussed below.
C57BL/6J mice were more passively social than BALB/cJ mice in home cage environments at 30 days of age, but their passive social levels declined to BALB/cJ levels by 69 days of age. Our previous studies also indicate that at ~30 days of age, C57BL/6J mice are more sociable than BALB/cJ mice in the Social Approach Test, and this strain difference diminishes or disappears by 70 days of age [14
]. Thus, a similar strain-difference pattern manifests in what are apparently two different kinds of social behavioral repertoires, which further supports the view that BALB/cJ mice show pervasively low levels of social affiliation at around 30 days of age.
Yet this strain difference in social interaction was not entirely pervasive: it did not appear in active social behaviors in the home cage environment. Instead, the strains were actively social at comparable levels, except at 41 days and principally due to especially high levels of active sociability among BALB/cJ females. This elevated sociability may be the result of pro-affiliative factors that occur specifically around 41 days of age. But, importantly, the 41-day-old BALB/cJ mice were composed of an especially small sample size: 4 females (2 cages) and 6 males (3 cages). Furthermore, most of the elevation appears attributable specifically to a relatively high level of nose-to-nose sniffing among the 41-day-old BALB/cJ females (). Thus, the elevation may be due to a non-representative sample of a single kind of behavior with only 2 independent cases. Only another, larger sample can distinguish between these two possibilities (i.e., particularly elevated active social behaviors among 41-day-old BALB/cJ females vs. a small, non-representative sample), but a more conservative interpretation is provisionally warranted: C57BL/6J and BALB/cJ mice probably do not differ in their home cage active social behaviors at any age that was tested in this study.
Regardless of whether strain differences truly exist in home cage active social behaviors, active and passive social behaviors clearly showed different patterns of strain differences. Furthermore, the strain-difference pattern and developmental trajectory of the passive – and not the active – social behaviors resembled that of sociability in the Social Approach Test. Initially, this result seems counterintuitive. Cylinder scores of the Social Approach Test almost entirely consist of sniffing, an active social behavior. Yet the strain difference results in the Social Approach Test based on an active behavior resemble strain difference results of passive – not active – behaviors in the home cage environment.
This apparent discrepancy might be resolved by recognizing the very different social situations and environmental contexts between the two behavioral assays. Test mice in the Social Approach Test are paired with an unfamiliar stimulus mouse in a relatively unfamiliar environment. The novelty of this stimulus mouse likely promotes active social sniffing, whereas the familiar littermate in the home cage does not. Instead, any heightened tendency toward social affiliation may manifest as passive social behaviors, such as huddling together, in the home cage. It is possible that genetic variants and neural circuits contribute to a generally low social interaction phenotype in BALB/cJ mice, but that this low social interaction phenotype may manifest in different behaviors depending on the social situation and environment. However, despite the possibility of some mechanisms in common, it is also likely that mechanisms mediating active social investigation in the Social Approach Test and passive social behaviors in the home cage test are not identical. For example, it is notable that we found correlations between sociability in the Social Approach Test and other variables (i.e., brain size, litter size, litter sex composition) [14
] that we did not find between home cage social behaviors and these other variables. More importantly, social behaviors in the Social Approach Test and in the home cage did not directly correlate when examined by pairs of mice. Thus, the effects of any genetic variants and neural circuits that may influence active social investigation in the Social Approach Test and passive social behaviors in the home cage may be detectable only at a group level, where individual differences are not as important.
The strain-difference patterns of the nonsocial behaviors in the home cage did not resemble those of social behaviors in either the home cage or the Social Approach Test. In general, BALB/cJ mice reared more than C57BL/6J did, and C57BL/6J mice groomed themselves more than BALB/cJ mice did. For both of these behaviors, there were no significant strain differences at 41 days of age, but this may have been due to the small sample sizes at this age. BALB/cJ mice at 30 days appeared to dig more than C57BL/6J mice, and this difference appeared to be primarily due to the females. However, the difference, as well as the overall levels of digging and autogrooming, was not large. Digging and autogrooming never exceeded 13% of a mouse’s time, on average, whereas the mice reared substantially more. These nonsocial behaviors were of particular interest because they might be considered as repetitive behaviors [62
], which are of potential clinical relevance because ASD patients exhibit restricted and repetitive behaviors [3
]. Neither strain exceeded the other in all of the nonsocial behaviors, but BALB/cJ mice did show higher levels of rearing, which was the most abundant nonsocial behavior. Additionally, BALB/cJ mice seemed to increase their levels of rearing with age. We did not have an a priori
hypothesis about the developmental trajectory of rearing, specifically, and therefore we did not statistically test for such an effect, but this apparent trend may be worth exploring in future studies.
Despite the importance of huddling to the differences between C57BL/6J and BALB/cJ mice in passive social behaviors, we observed relatively little huddling compared to a similar study of home cage behaviors in C57BL/6J mice [63
]. Curley et al. (2010) observed that 35- to 45-day-old female and male C57BL/6J mice huddled for about 51% of scored time points. Our 30- and 41-day-old C57BL6/J mice are approximately comparable but huddled for only 9% – 19% of the time. Similarly, mice of Curley et al. (2010) rested alone for 4% – 8% of the time, while our pairs of mice almost never rested separately from each other. The differences in these two behaviors suggest that our mice exhibited an overall higher activity level than the mice reported by Curley et al. (2010). Much of the higher activity in our mice manifested as rearing, which occupied them about 21% – 28% of their time, while Curley et al.’s (2010) mice reared for less than 2% of the time. The higher activity in our mice might be attributable to our observing the mice only during the early part of the dark cycle, when mice tend to show their highest locomotor and social activity levels [64
]. By contrast, Curley et al. (2010) spread their observations through the day. Additionally, we relocated our mice from the colony room to a separate room for video recording, which might have aroused the mice to explore the partly new environment (novel room, but same cage), even though an hour elapsed between relocation and the start of the observation period.
As noted above, the home cage behaviors might vary when experimental conditions are altered. For example, Panksepp et al. [66
] showed that 30- to 35-day-old C57BL/6J and BALB/cJ mice varied in their home cage social behaviors across the circadian cycle. Similarly, different mouse strains might mature at different rates such that differences in social behaviors are observed only at particular ages. Further experiments will be useful in determining how robust our current findings are to other experimental conditions.
The classification scheme used in the present study encompasses nearly all of the social affiliative behaviors included in other studies of home cage behaviors [58
]. The most common active social behaviors, sniffing and allogrooming, are subsumed under our “active social” category, along with the less commonly observed behaviors. The primary difference between our study and others is that our behavioral scheme explicitly includes all contact between the mice, even if the contact does not conform to a specific, defined behavior. It does this by including all passive contact while a mouse is moving, rather than being limited to only passive contact while the mice are huddling, or resting together (i.e., not moving). These “passive while moving” behaviors include receiving active social behaviors from the other mouse as well as other, less-defined passive contact, such as “bumping” one another. The disadvantage of our behavioral scheme is that it is not as detailed as those in some other studies. We chose to use more general categories for two reasons. First, it reduced the labor involved in scoring the behaviors, thus making the behavioral scoring procedure potentially widely applicable to assessment of various mouse models in future studies. Second, some behaviors, such as sniffing and allogrooming, were difficult to distinguish in our video recordings. Using more general categories let us avoid especially subjective decisions in distinguishing these behaviors. This approach probably kept our inter-rater reliability relatively high and made our results more reproducible in future studies.
In summary, C57BL/6J and BALB/cJ mice show a differential developmental pattern of passive social behaviors in the home cage similar to their differential developmental pattern of social behaviors in the Social Approach Test. These results show that the relatively low sociability of 30-day-old BALB/cJ mice is pervasive across different kinds of social and environmental contexts. Importantly, the behavioral manifestation of this low sociability can vary depending on the social context: in the Social Approach test, it appears as relatively low levels of sniffing of the unfamiliar stimulus mouse – an active social behavior – whereas in the home cage with a littermate, it appears as relatively low levels of huddling – a passive social behavior. We did not find significant correlations – when viewed by pairs of mice – between social behaviors in the Social Approach Test and the home cage. Moreover, we did not find some of the correlations between home cage social behaviors and brain size, litter size, and litter sex composition that we reported earlier for Social Approach Test behaviors [14
]. Thus, although there may be some genetic and neural mechanisms that underlie low BALB/cJ sociability in both the Social Approach Test and the home cage, there are likely to be non-overlapping sets of mechanisms involved as well, in the different social contexts. Future studies are needed to identify these mechanisms. In addition to the commonly used Social Approach Test, an assessment of social interactions in the familiar home cage environment with a familiar littermate can provide a more complete profile of sociability in mouse models relevant to autism.