As hypothesized, for two of the three strain comparisons and in the pooled analysis, we observed that B6 mice showed a significant strain by time period statistical interaction in the time they spent in the same chamber with ‘typical’ versus ‘atypical’ mice. For the pooled analysis, Bonferroni post-test suggested that the statistical interaction was driven by a preference for spending time in the chamber with the ‘typical’ mouse in the last 10-minute period of the PTSIT. No significant statistical interactions were seen for time spent within 1 cM of the pencil cup in any of the comparisons.
The findings for 129S6 judge mice were not as striking as for B6; however one of the strain comparisons and the pooled analysis did reveal a significant strain by time period statistical interaction in the time that they spent in the same chamber with the ‘typical’ versus ‘atypical’ mice. Higher variability is also seen for the 129S6 judges, reflecting a tendency for some of these animals to remain stationary for long periods of time after the initial period of exploration (Veenstra-VanderWeele, et al., 2012
). Again, no significant interactions were seen for time spent within 1 cM of the pencil cup in any of the comparisons. This suggests that 129S6 mice do not show as strong a preference for chambers containing ‘typical’ mice.
The pattern of preference for typical social interaction in both the B6 and the 129S6 mice suggests that there may be different motivations for different time periods within the PTSIT. We would hypothesize that the initial 10-minute time period represents social exploration, as reflected in more sniffing behavior during this time period. This time period may not be as dependent upon the stimulus mouse’s social behavior as it is dependent upon the presence of a social odor to investigate. Previous findings are consistent with the interpretation that the first 5–10 minutes of the three-chamber task are dominated by olfactory social exploration. Over a 10-minute three-chamber test, the Crawley lab found that B6 mice show no preference for a mouse in an inverted pencil cup over a nestlet containing social odors (Ryan, et al., 2008
). Further, they found that B6 mice actually prefer a nestlet containing social odors to a mouse confined in a plexiglass cylinder that is impermeant to odor (Ryan, et al., 2008
). The Brodkin lab has found that time sniffing an odor-permeant cylinder containing a mouse is a more sensitive and reliable measure than time spent in the chamber with the mouse stimulus across a 10-minute three-chamber test (Fairless, et al., 2011
). The significant statistical interaction findings on the PTSIT may indicate that, once the initial social exploration has been completed, mice prefer to spend time in a chamber with a ‘typical’ over an ‘atypical’ mouse.
The lack of difference observed in time within 1 cM of the two stimulus animals further suggests that the effects seen for chamber time are not primarily driven by close social exploration or contact. Alternatively, hand scoring of close social contacts between the “judge” and “stimulus” animals could reveal subtle differences in social contacts that cannot be observed by AnyMaze coding of time within 1 cM of the pencil cup. It is possible that the effects seen for chamber time do not reflect differences in time spent in social interaction at all; although further data on vocalizations or olfactory communication would be necessary to assess this more fully. Various interpretations of the chamber time differences are possible. Late in the test, when considerable time has already been available for exploration of the social stimuli, mice may prefer the “typical” chamber because their contacts with the typical mouse have been rewarding in some way, or because they feel safer in the chamber containing a typical mouse, or because the atypical mouse chamber is aversive in some way. Ultimately, these data don’t allow a clear distinction between these possibilities, but they do suggest that C57 animals show a different pattern of behavior that may distinguish between “typical” and “atypical” mice.
The Crawley lab has found that B6 mice show preference for multiple inbred mouse strains (B6, BTBR, A/J, 129Sv/ImJ) over a novel object on the 10-minute three-chamber sociability test (Nadler, et al., 2004
; Yang, Abrams, et al., 2012
). Of further relevance to our findings, they found that B6 mice modulate their contacts with freely moving stimulus animals depending upon the stimulus strain, showing less nose-to-nose sniffing and frontal approach with BTBR in comparison to B6 stimulus animals (Yang, Abrams, et al., 2012
These initial findings suggest that mice may prefer some social interactions to others, with their preferences following a pattern that other tests differentiate as ‘typical’ versus ‘atypical’ for mouse social behavior. There are also some important limitations to these findings. First, we used limited sample sizes that only allow us to detect very large effect sizes. We reasoned that only substantial effect sizes would drive utility of this paradigm for future studies. The effect sizes we actually observed were smaller than those seen for the three-chamber test when comparing a social stimulus to a non-social stimulus. This may reflect the complexities of differentiating social aptitude from social approach behavior. Second, we only evaluated three lines of ‘atypical’ mice. Further testing could evaluate whether these findings would extend to the broader array of mouse lines with atypical social behavior. Third, we used the same ‘judge’ animals in multiple experiments, reasoning that this would allow us to evaluate the robustness of the effect across repeated testing. Fourth, we re-used stimulus animals in the assay, which could lead to changes in their behavior; although each “judge” mouse only encountered a stimulus animal a single time. Finally, we did not use littermate comparisons for these experiments, which is not possible for inbred strains of mice. It is therefore possible that the ‘atypical’ social behavior present in these mouse lines is determined by early social experience, rather than genetic background itself (Yang, Perry, et al., 2011
These findings also raise interesting questions for future study. We approached the initial experiments as an opportunity to evaluate whether mice show preference for ‘typical’ versus ‘atypical’ social interactions. Other approaches to this hypothesis would include free interactions with different strains of mice followed by a conditioned place preference approach (Panksepp & Lahvis, 2007
). Turning the paradigm on its head, it may also be possible to evaluate whether particular lines of mice show preference for typical versus atypical social interactions as judges in this paradigm, which could also indicate social aptitude. Our initial findings suggest that B6 animals show more robust preference for ‘typical’ social interactions than 129S6; although we did not explicitly evaluate this possibility. Overall, our observations suggest that the PTSIT may be a useful addition to a battery of social tests, including the three-chamber sociability test, as well as tests of activity, anxiety-like, and other autism-relevant behavior. Further work will be necessary to evaluate the degree to which it correlates with patterns of mouse free interaction or other potential measures of social aptitude.