Revisiting sociability: Factors facilitating approach and avoidance during the three-chamber test

•Factors influencing sociability scores were examined in the three-chamber test.•Sexual cue induced investigatory approach in both B6 and BTBR strains.•Object novelty induced avoidance in BTBR mice but approach in B6 mice.•Single-housing augments social cue sensitivity in BTBR mice.•Sociability scores indicate social distance but not implying social deficit. The three-chamber test, the so-called sociability test, has been widely used to assess social deficits based on impaired socially oriented investigations in rodent models. An innate motivation for investigating conspecifics is theoretically a prerequisite for gaining sociability scores in this paradigm. However, several relevant factors mediating investigatory motives, such as familiarity, attractiveness, and aggression, may affect sociability scores, which must be verified to obtain an adequate evaluation of the psychiatric phenotypes exhibited by disease-relevant rodent models. We assessed the social and non-social factors that mediate proximity preference by the three-chamber test with standard C57BL/6 J (B6) mice and low sociability BTBR+ltpr3tf/J (BTBR) mice. Strains of the opponents had no effect. Sexual cues (i.e., opposite sex) increased proximity preference in both strains of mice; in contrast, novel objects induced an approach in B6 mice but avoidance in BTBR mice. Single-housing before testing, stimulated social motive, affected BTBR mice but not B6 mice. BTBR females showed increased proximity preference across the sessions, and BTBR males showed increased preference toward a male B6 stimulus, but not a male BTBR stimulus. The male preference was restored when the male BTBR stimulus was anesthetized. In addition, self-grooming was facilitated by social and non-social novelty cues in both strains. B6 mice predominantly exhibited an investigatory approach toward social or non-social stimuli, whereas BTBR mice recognized social cues but tended to show avoidance. The three-chamber test could evaluate approach-avoidance strategies in target mouse strains that comprise innate social distance between mice.