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

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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.

Research highlights

▶ BTBR mice display gaze aversion-like behavior. ▶ A novel social proximity test shows clear behavioral discrimination between BTBR T+ tf/J and C57BL/6J mice. ▶ Diazepam alters mouse social behavior in social proximity.

Introduction

Autism is a lifelong neurodevelopmental disorder that is diagnosed by observable behaviors rather than specific biomarkers [2], [33]. The diagnostic symptoms of autism form a triad of behavioral deficits consisting of abnormal social interactions, impaired communication and repetitive and stereotyped patterns of behavior [2]. A strong genetic component for the disorder [1], [5], [29] is evident in the high (70–90%) concordance rates for autism in monozygotic twins [24], [37], [64] and the markedly high heritability of the individual diagnostic symptoms [58], [59]. Neuropathologies such as increased gross brain volume [7], [17], [30], [51], [55] and reduction in size of the corpus callosum [13], [22], [25], [39], [52] are also frequently associated with the disorder.

Because the etiology of autism may involve complex and heterogenous genetic and experiential interactions, mouse models are critical to isolating its underlying mechanisms [1], [21], [32], [43], [44]. Current animal models of autism include both inbred mouse strains that exhibit relevant behavioral characteristics [45], [48] and mouse lines engineered with targeted mutations of candidate genes [43], [46]. Inbred mouse strains are particularly useful in relating existing phenotypes to potential genetic or physiological abnormalities. However, the complexity of mouse social behavior presents a challenge to designing tasks to assess social interactions relevant to autism. A combination of social interaction tests have been used to identify low social responsiveness in inbred candidate strains [4], [11], [12], [18], [19], [20], [41], [45], [46], [48], [54], [70]. Such tests quantify social tendencies in mice by measuring the duration of proximity with another animal and the frequency of behaviors which characterize the nature of interactions. Results from previous social interaction tests have indicated that the inbred BTBR T+ tf/J (BTBR) mouse strain displays several social deficits congruent with the diagnostic criteria of autism including reduced social approach [41], [69], huddling, social investigation [11], [54] and juvenile play [41], [70]. In comparison to the C57BL/6J (B6) mouse strain, BTBR mice additionally displayed restricted interest in objects [50], repetitive grooming [50], [63], abnormal patterns of scent marking and unusual vocalizations [60], [62], [67], offering face validity to the core symptoms of autism.

Previous studies have reported inconsistent results for anxiety in BTBR mice. Baseline anxiety measured in the elevated plus maze (EPM) yielded conflicting results across studies: the duration of open arm time has been reported as reduced [53] and not different [8], [48], [68] from B6 mice; the number of open arm entries has been reported as increased [68], decreased [53] and not different [48] from those of B6 mice. Results from testing in the elevated zero maze indicated that BTBR mice consistently spent more time in the open arms [41], [53]. In addition, BTBR mice displayed heightened stress reactivity on the elevated plus maze following tail suspension [8]. Thus, the possible influence of anxiety on BTBR social behavior remains uncertain. The lack of preference for a social stimulus over a non-social stimulus [41], [48], [53], [69] and the low levels of social approach displayed by BTBR mice [11], [54] suggest social anxiety [38], [40] in this strain. Moreover, administration of an anxiolytic, diazepam, rescued BTBR preference for a social stimulus [53], suggesting that social approach is inhibited, at least in part, by anxiety.

The aim of the present study was to characterize BTBR behavior in a social proximity test and investigate the role of anxiety in that context. BTBR and B6 mice were tested in various pair configurations in a novel social proximity test that placed mice together in a small enclosure that required some physical contact of the animals. Specific avoidance behaviors, such as facial avoidance, are difficult to detect in contexts that permit substantial social distance, as mice are able to avoid contact with specific body parts by maintaining a comfortable distance from others. Assessment in social proximity conditions enables clearer analysis of specific components of avoidance behaviors. Potential investigatory, orientation, escape and avoidance behaviors are exposed by restricting social distancing of subjects.

Section snippets

Animals

Subjects were 12–14-week-old C57 BL/6J (B6) and BTBR T+ tf/J (BTBR) mice (n = 14/group/experiment, Experiments 1–3; n = 8 or 10/group, Experiment 4). All subjects were male except for Experiment 3, where both males and females were used. Naïve animals were used in each experiment, and except for Experiment 3 where males and females were initially run in the social approach test and later paired in the social proximity test, only a single test was run per animal. Animals were bred in-house from

Experiment 1—social proximity tests in BTBR and B6 male pairs

Confining subjects within the social proximity apparatus (Fig. 1) elicited markedly different behaviors in the BTBR and B6 strains (Fig. 2). BTBR males showed significantly less nose tip-to-nose tip contact [t(26) = 5.688, p < 0.0001] while displaying reliably higher nose-to-anogenital contact [t(26) = 3.011, p < 0.01] than B6 males. Upright behaviors, which often accompany contact with the mystacial vibrissae of the other animal, were displayed by only 3 of 14 BTBR mice, and were significantly

Discussion

The overarching goal of the present study was to determine how BTBR and B6 mice differ in response to enforced social proximity. Deficiencies in social behavior have been consistently reported for BTBR mice. In a three-chamber social approach test, BTBR mice failed to spend more time in the chamber containing a mouse within a cup compared to the chamber containing an empty cup [41], [48], [53], [63], [68], [69], [70]. BTBR mice also spent less time engaged in social interactions compared to B6

Acknowledgement

The present study was supported by the National Institute of Mental Health (NIMH) grant MH081845-01A2 to RJB.

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