A new test paradigm for social recognition evidenced by urinary scent marking behavior in C57BL/6J mice

Abstract

Olfaction is a major sensory element in intraspecies recognition and communication in mice. The present study investigated scent marking behaviors of males of the highly inbred C57BL/6J (C57) strain in order to evaluate the ability of these behaviors to provide clear and consistent measures of social familiarity and response to social signals. C57 males engage in scent marking when placed in a chamber with a wire mesh partition separating them from a conspecific. Male mice (C57 or outbred CD-1 mice) showed rapid habituation of scent marking (decreased marking over trials) with repeated exposure at 24-h intervals, to a stimulus animal of the C57 or CD-1 strains, or to an empty chamber. Subsequent exposure to a genetically different novel mouse (CD-1 after CD-1 exposure, or CD-1 after C57 exposure) or to a novel context (different shaped chamber) produced recovery of marking, while responses to a novel but genetically identical mouse (C57 after C57 exposure) or to the empty chamber did not. This finding demonstrated that male mice differentiate familiar and novel conspecifics as expressed by habituation and recovery of scent marking, but neither C57 or CD-1 mice can differentiate new vs. familiar C57 males; likely due to similarities in their odor patterns. The data also indicate that scent marking can differentiate novel from familiar contexts.

Introduction

The ability to differentiate and respond to individuals has advantages in many social contexts, enabling animals to adjust their behavior in accord with this information [8], [31], [33], [42]. Among mammals, including mice, urinary scent plays a significant role in olfactory social signaling [2], [12], [34]. Urinary odors are used both to discriminate between individual conspecifics [7], [24], [32], and to communicate information such as dominance and health [27], [29], [35], [61]. Scent marking is also used by males to advertise their male quality to potential mates [26], [46], and to advertise territory ownership [18], [25], [30]. When territory owners encounter scent from other males within their territory, they increase their own rate of scent marking to countermark the alien scent [27], [45], but show no such response to their own scent marks or to these from males genetically identical to themselves [28], [41], [42].

The tendency for rodents to investigate conspecifics has long been used as a paradigm to evaluate social attraction and social memory [17], [56], [57]. This social investigation paradigm has been used for a variety of mouse models associated with neurocognitive disorders including autism [44], [58], Fragile X syndrome [38], [55] and Rett syndrome [50], as well as studies of the effects of neuropeptides including oxytocin [21], [22] and vasopressin [49]. A habituation–dishabituation paradigm for social investigation to conspecifics can provide a measure of social memory [56], [57]. In this task, an adult male is allowed to investigate a juvenile or ovariectomized female that is introduced to its cage. When the stimulus animal is removed and later re-introduced, subjects display reduced investigation compared to the first encounter [6]. A three-chamber test modeled on place preference paradigms has been shown to provide quantitative, replicable, data on investigation of social and nonsocial stimuli, demonstrating preference for social stimuli, and habituation to individual social stimuli over repeated exposures [9], [10], [39], [40].

The present experiments aim to assess a complementary approach to analysis of social behaviors and memory, through scent marking. Conspecific communication in mice involves both the emission of olfactory social signals (scent marking) and response to such social signals through additional scent marking, often overmarking the initial scent [2], [12], [25], [34]. The present experiments of scent marking utilized C57BL/6J (C57) mice, an inbred strain often used as a background for transgenic mice [15], [16], [51], in order to facilitate the potential use of this technique for genetic studies of animal models of neurocognitive disorders. Experiments 1, 2 and 3 investigated scent marking response of adult male mice to inbred (C57) or outbred (CD-1) mice, and the habituation of this response over repeated exposures. Experiment 4 examined a potential confound in these studies; scent marking and habituation of marking to an initially novel context, and recovery of marking to a novel context. Experiment 5 attempted to separate habituation to a context from habituation to a stimulus mouse, utilizing a paradigm that evaluated these changes independently.