Background

Like humans, rats are considered a social species; they tend to thrive with conspecifics rather than in isolation. Generally, rats can relay information through ultrasonic vocalizations and pheromones. Given their highly developed olfactory systems, a wide array of information may be conveyed through the sense of smell. Releasing and receiving pheromonal signals play critical roles in both neurobiological and ethological aspects of rodent behaviour. These include but are not limited to the selection of mating partners (Kumar et al., 2014), spreading alarm signals (Kiyokawa et al., 2013), novel food acquisition (Posadas-Andrews and Roper, 1983), territory marking (Brain et al., 2009), demonstrating social status (Krames et al., 1969), and maternal interaction (Bind et al., 2013). Pheromonal signaling can actually interfere with several biological functions in a laboratory setting; the presence of unfamiliar males can prevent pregnancy (Bruce, 1959), female estrous cycles may become prolonged when exposed to urine from a lactating female (Hoover and Drickamer, 1979), and male urine can cause acceleration of puberty in females (Vandenbergh, 1975). It has been known for some time that certain pheromones can carry information regarding sexual behaviours (Hayashi and Kimura, 1974; Gawienowski et al., 1975), but more recently it has been conclusively shown that mammals can also communicate information about stress via alarm pheromones (Inagaki et al., 2014; Kiyokawa, 2017).

Social fear learning is a growing field in neuroscience and many models have been used to attempt to study how it works. The subtle differences in approach give clues as to how rodents are actually able to communicate with one another. Knapska et al. (2006) exposed rats to shock in a novel context then allowed their cagemate, the “observer”, to interact with this “demonstrator” rat. They found that observer rats housed with a demonstrator that had been fear conditioned spent more time interacting with the demonstrator and an enhanced acoustic startle response following this interaction, suggesting a transfer of emotional states between the two animals. Bruchey et al. (2010) developed a fear conditioning by proxy paradigm whereby a naïve rat watches its cagemate respond to a previously fear conditioned auditory stimulus. When the trained cagemate freezes in response to the tones, this causes the naïve rat to develop a fear reaction to subsequent presentations of the conditioned stimulus. Although the naïve rat had never received a shock, witnessing fearful behavior of its cagemate in response to the tone was enough to cause a conditioned response (albeit weaker than the classically conditioned fear) in some of the rats. The degree of success in fear conditioning by proxy was correlated with the duration of social interaction during the procedure. Again, it is clear that interaction with a stressed conspecific is sufficient to induce some behavioural change in a rat’s emotional response.

Since rats rely so heavily on their sense of smell, it is likely that this communication occurs through an olfactory mode, likely via pheromones, as opposed to ultrasonic vocalizations. Debiec and Sullivan (2014) found that a stressed mother’s scent delivered through an olfactometer paired with a novel odor caused avoidance to that odor in pups. A series of experiments from the Mori group has identified a rat alarm pheromone that causes stress-induced hyperthermia in recipient rats, as well as increased defensive and risk assessment behaviours combined with a decrease in exploratory and grooming behavior (Kikusui et al., 2001; Kiyokawa et al., 2006). This rat alarm pheromone has been isolated and two of its components, 4-methylpentanal and hexanal, produce the same behavioural effects as the pheromone itself (Inagaki et al., 2014). Two things are clear from the previous research: 1) information can be conveyed regarding emotional responses between conspecific rats and 2) interaction between a naïve rat and either a conditioned rat or a pheromonal component of its odor is important for this communication.

The current protocol describes the use of same sex 8-10-week old Sprague Dawley rat pairs as odor + shock (O⁺/S⁺) and companion (O⁺/Comp) conditions in a paradigm by which a conditioned fear of a specific odor is communicated from the O⁺/S⁺ rat to its companion. This paradigm produces a socially conditioned fear in a rat whose cagemate has been classically conditioned to associate an odor with a shock. Unshocked O⁺/SB rats are also able to acquire this fear in the absence of a cage mate by being exposed to the soiled bedding of an O⁺/S⁺ rat along with the conditioned odor. In fact, pairing previously identified alarm pheromone components with the conditioned odor also produces this phenomenon in unshocked O⁺/Ph rats. Together, these experiments provide strong support to the notion that rats are able to communicate a specific fear through pheromones. As opposed to other methodologies, the current protocol may reflect actual communication through pheromones rather than just a transfer of emotional states, as the socially acquired fear is specific to the trained odor and is not generalized to a different stimulus if presented after the interaction. The protocol described below does not require expensive equipment or materials, and can be completed within one week.