A novel social proximity test suggests patterns of social avoidance and gaze aversion-like behavior in BTBR T+ tf/J mice

Abstract

The BTBR T+ tf/J (BTBR) inbred mouse strain displays a low sociability phenotype relevant to the first diagnostic symptom of autism, deficits in reciprocal social interactions. Previous studies have shown that BTBR mice exhibit reduced social approach, juvenile play, and interactive behaviors. The present study evaluated the behavior of the BTBR and C57BL/6J (B6) strains in social proximity. Subjects were closely confined and tested in four experimental conditions: same strain male pairs (Experiment 1); different strain male pairs (Experiment 2); same strain male pairs and female pairs (Experiment 3); same strain male pairs treated with an anxiolytic (Experiment 4). Results showed that BTBR mice displayed decreased nose tip-to-nose tip, nose-to-head and upright behaviors and increased nose-to-anogenital, crawl under and crawl over behaviors. These results demonstrated avoidance of reciprocal frontal orientations in the BTBR, providing a parallel to gaze aversion, a fundamental predictor of autism. For comparative purposes, Experiment 3 assessed male and female mice in a three-chamber social approach test and in the social proximity test. Results from the three-chamber test showed that male B6 and female BTBR displayed a preference for the sex and strain matched conspecific stimulus, while female B6 and male BTBR did not. Although there was no significant interaction between sex and strain in the social proximity test, a significant main effect of sex indicated that female mice displayed higher levels of nose tip-to-nose tip contacts and lower levels of anogenital investigation (nose-to-anogenital) in comparison to male mice, all together suggesting different motivations for sociability in males and females. Systemic administration of the anxiolytic, diazepam, decreased the frequency of two behaviors associated with anxiety and defensiveness, upright and jump escape, as well as crawl under behavior. This result suggests that crawl under behavior, observed at high levels in BTBR mice, is elicited by the aversiveness of social proximity, and possibly serves to avoid reciprocal frontal orientations with other mice.

Figure 1

BTBR mice confined in the social proximity test. The side walls of the clear colorless testing chamber are tinted a false gray to emphasize the area of confinement.

Figure 2

Male B6 and BTBR mice were confined with a same strain partner in the social proximity test. Behavioral frequencies are expressed as mean ± S.E.M. Behaviors measured were nose tip-to-nose tip contact (NN), nose-to-anogenital contact (NA), crawl over (CO), crawl under (CU), nose-to-head contact (NH), upright (U) and jump escape (JE). Significant differences are indicated by (*), p < 0.01 and (**), p < 0.001.

Figure 3

Male B6 and BTBR mice were paired together in the social proximity test. Behavioral frequencies are expressed as mean ± S.E.M. Behaviors measured were nose tip-to-nose tip contact (NN), nose-to-anogenital contact (NA), crawl over (CO), crawl under (CU), nose-to-head contact (NH), upright (U) and jump escape (JE). Significant differences are indicated by (*), p < 0.001.

Figure 4

Male and female B6 and BTBR mice were assessed in the social approach test. Duration of time spent in each chamber is expressed as mean ± S.E.M. Significant differences in duration of time spent in the chamber containing the conspecific stimulus are indicated by (*), p < 0.01 and (**), p < 0.001.

Figure 5

Male and Female B6 and BTBR mice were confined with a same sex and same strain partner in the social proximity test. Behavioral frequencies are expressed as mean (± S.E.M. Behaviors measured were nose tip-to-nose tip contact (NN), nose-to-anogenital contact (NA), crawl over (CO), crawl under (CU), nose-to-head contact (NH), upright (U) and jump escape (JE). (*) indicates a significant difference between BTBR and B6 mice, p < 0.001. (^#) indicates a significant difference between males and females, p < 0.05. (^$) indicates a significant difference from all other groups, p < 0.05.

Figure 6

Male B6 and BTBR mice were administered vehicle or diazepam and confined with a same strain partner in social proximity test. Behavioral frequencies are expressed as mean ± S.E.M. Behaviors measured were nose tip-to-nose tip contact (NN), nose-to-anogenital contact (NA), crawl over (CO), crawl under (CU), nose-to-head contact (NH), upright (U) and jump escape (JE). (*) indicates a significant difference between BTBR and B6 mice, p < 0.05. (^#) indicates a significant difference between vehicle and diazepam treatment, p < 0.05. (^$) indicates a significant difference from all other groups, p < 0.05.