Female rats that interacted with a familiar sister expressing fear to a CS displayed more social interactions during the time immediately following exposure to the CS than those who experienced the CS in the company of a less familiar, unrelated female rat. Although the sister rats showed a trend toward increased fear-conditioning by-proxy in response to the CS the following day, compared to non-sister rats, this effect was entirely driven by the post-cue social contact. We found that a number of female rats, like some male rats, acquire fear to a previously neutral cue by observing, and freely interacting with, a fear-conditioned cage mate during presentation of a CS. Like male rats, social interactions contribute to the expression of socially learned fear the following day, but the specifics of this interaction in relation to the onset and offset of the cue differ between sexes. Additionally, familiarity/kinship modulates the extent of prosocial interactions between the fear-conditioned rat and the fear-conditioned by-proxy rat during the fear-conditioning by-proxy session on day 2, and these interactions determine the degree of fear that is socially transmitted between female rats.

The ability to learn about danger from conspecifics is potentially adaptive, especially in animals living in social colonies in the wild where an individual can learn to avoid a specific situation without the threat of immediate danger. It is interesting to note that the fear-conditioning by-proxy paradigm described here consistently reveals a subset of rats that do not appear to learn fear by-proxy (as evidenced by a complete lack of freezing on long-term memory tests on day 3). The factors that determine these individual differences are the subject of further research but may be the result of differences of individual roles in the colony (or in this case, the cage) (Blanchard et al. 1988; Kavaliers et al. 2005; Shettleworth 2010), resulting in differences in either social interactions or social learning. In purely observational fear-conditioning paradigms, mice with social relations (10+ weeks as a mating pair or siblings raised together) displayed more freezing both when observing a partner fear conditioned to a context and when tested in the context the following day than mice lacking these relationships (Jeon et al. 2010). Additionally, both familiarity and relatedness were significant contributors to deer mice observing other deer mice responding defensively to a natural predator. Mice with a genetic predisposition to increased sociability (B6 mice) condition more strongly to a cue when pre-exposed to another mouse undergoing fear conditioning to the same cue (Chen et al. 2009) further supporting the idea that social factors are essential to observational fear learning. In each of these paradigms, the mice were not allowed to interact with one another, thus restricting potentially salient factors of social learning on observational learning paradigms. In the fear-conditioning by-proxy experiments performed here, we are able to measure socially transmitted fear to a Pavlovian conditioned cue while allowing the animals to freely interact socially.

By manipulating the familiarity and relatedness of the rats involved in this social learning paradigm, we found that after a CS is presented, rats interact more with a sister they had been raised with compared to a non-related, less familiar cage mate, and this interaction leads to increased fear-conditioning by-proxy. One limitation was our ability to tease apart the effects of familiarity versus kinship on freezing and social interactions. Future research will explore the unique contributions of kinship and familiarity independently in the fear-conditioning by-proxy paradigm by breeding with parent stock ordered from different sources, thereby increasing genetic diversity and allowing us to specifically investigate the role of familiarity by testing fear-conditioning by-proxy in both littermate and non-littermate triads. Additionally, raising littermates apart and looking at fear-conditioning by-proxy in sister rats that have not been housed together since weaning could test the role of genetic relatedness.

In addition to the social/genetic relationships between animals, prior fear experience has been shown to modulate the freezing response when observing another rat undergoing contextual fear conditioning (Atsak et al. 2011) as well as observing (Pereira et al. 2012) or interacting with another rat displaying a fear response to a cue (Kim et al. 2010). However, these rats were not tested the following day for retention of this socially transmitted fear making it difficult to differentiate between an emotional response to a conspecific in distress or the acquisition of a fear memory to a social stimulus. Consistent with this previous research, during day 2 of the fear-conditioning by-proxy paradigm, we noticed that previously naïve rats do not display any freezing while interacting with a fearful cage mate. By measuring freezing during long-term memory tests, we were able to examine retention of a fear memory after social acquisition and found that a subset of FCbP rats froze in response to the cue on day 3. Taking into consideration the importance of previous experience on social fear transmission, breeding rats in our own colony allowed us to better control for prior life experience.

Previous research shows a sex difference in freezing behavior after contextual fear conditioning, with males freezing more than females (Archer 1975; Gupta et al. 2001; Pryce et al. 1999; Morgan and Pfaff 2001), suggesting that estrogens modulate freezing to contextual stimuli (Gupta et al. 2001). Additionally, estradiol and progesterone both influence how female rats respond in high anxiety situations (Mora et al. 1996; Valle 1970; Nomikos and Spyraki 1988; Marcondes et al. 2001), and estradiol treatment in ovariectomized rats enhances social recognition memory (Hlinak 1993). These sexual dimorphisms in both fear and social behavior motivated us to explore the efficacy of fear-conditioning by-proxy in female rats and to assess possible influences of estrous cycle status, as ovarian hormones fluctuate substantially depending upon cycle phase (Smith et al. 1975). However, the lack of correlation of day of the cycle with the freezing response suggests that at least in the short term, cyclic fluctuations in hormones do not drive this sex difference. When the fear-conditioning by-proxy paradigm was previously performed in male rats, social contact during the cue presentation in the fear-conditioning by-proxy session was positively correlated with long-term memory freezing during day 3 by the FCbP rat (Bruchey et al. 2010). In female rats, we found that rather than socially contacting the freezing rat while the cue was playing, the FCbP females engaged in prosocial behaviors only once the cue ended. It is important to note, however, that these sex differences were detected across two different experiments and it would be beneficial to further investigate sex differences in fear-conditioning by-proxy by conducting a single experiment where both males and females are tested in parallel.

The spectrum of social behavior in the rat involves both social relationships and social interactions, and until now, these had not been systematically investigated in a Pavlovian fear-conditioning setting. Here, we demonstrate that Sprague–Dawley rats acquire more fear information about a conditioned cue from a familiar and related conspecific and that, in female rats, social interactions immediately following cue termination modulate the degree of freezing during test. The fact that there are gender-specific responses to a novel cue further underscores the importance of studying how gender and sex hormones factor into fear transmission. These results further our understanding of social transmission of Pavlovian fear in laboratory bred Sprague–Dawley rats, thereby opening the door to investigate the neural substrates involved in the fear-conditioning by-proxy paradigm as well as examining fear-conditioning by-proxy in other strains of rats or even other species of research animals.