Research reportA novel social proximity test suggests patterns of social avoidance and gaze aversion-like behavior in BTBR T+ tf/J mice
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.
References (71)
Molecular genetics and animal models in autistic disorder
Brain Res Bull
(2002)- et al.
A new test paradigm for social recognition evidenced by urinary scent marking behavior in C57BL/6J mice
Behav Brain Res
(2008) - et al.
Colony formation of C57BL/6J mice in visible burrow system: identification of eusocial behaviors in a background strain for genetic animal models of autism
Behav Brain Res
(2007) - et al.
Neuroanatomic observations of the brain in autism: a review and future directions
Int J Dev Neurosci
(2005) - et al.
Exaggerated responses to stress in the BTBR T+ tf/J mouse: an unusual behavioral phenotype
Behav Brain Res
(2009) - et al.
The Mouse defense test battery: pharmacological and behavioral assays for anxiety and panic
Eur J Pharmacol
(2003) - et al.
Assessing autism-like behavior in mice: variations in social interactions among inbred strains
Behav Brain Res
(2007) - et al.
Social approach-avoidance behavior of inbred mouse strains towards DBA/2 mice
Brain Res
(2004) - et al.
The genetic basis of individual-recognition signals in the mouse
Curr Biol
(2007) Behavioral phenotyping strategies for mutant mice
Neuron
(2008)