1. Introduction
The presence of an affiliative conspecific (or cues associated with a conspecific) reduces stress responses to a wide variety of stimuli (Davitz & Mason, 1955; Lyons et al. , 1988; da Costa et al. , 2004; Bowen et al. , 2013; Kanitz et al. , 2014). This phenomenon is known as “social buffering” (Kiyokawa, 2017; 2018; Kiyokawa & Hennessy, 2018). Numerous studies in a variety of species have demonstrated that, in addition to social buffering by the mother or mate (Hennessy et al. , 2009; Smith & Wang, 2014), social buffering can be elicited by other conspecifics (Lyons et al. , 1993; Winslow et al. , 2003; Kiyokawa et al. , 2004; Hennessy et al. , 2008). Given that these three classes of conspecifics induce social buffering via distinct neural mechanisms, specification of the type of conspecific is important when discussing the neural mechanisms underlying social buffering (Kiyokawa & Hennessy, 2018).
We demonstrated in rats that social buffering induced by a conspecific other than the mother or mate can ameliorate stress responses to an aversively conditioned stimulus (CS). When a fear-conditioned rat was exposed to the CS alone, conditioned fear responses, including increased freezing and hypothalamic-pituitary-adrenal axis activation, were observed. However, the presence of a non-conditioned unfamiliar same-sex rat (associate) completely blocked these responses (Kiyokawa et al. , 2004; Kiyokawa et al. , 2007; Kiyokawa & Takeuchi, 2017). Subsequent studies revealed a number of characteristics (Kiyokawaet al. , 2004; Ishii et al. , 2016; Mikami et al. , 2016; Kiyokawa et al. , 2018; Kiyokawa et al. , 2019; Mikamiet al. , 2020) and a possible neural pathway underlying social buffering of conditioned fear responses (Kiyokawa et al. , 2009; Kiyokawa et al. , 2012; Takahashi et al. , 2013; Kiyokawaet al. , 2014; Fuzzo et al. , 2015; Minami et al. , 2019). However, little information is available regarding the neurochemical background of social buffering.
We propose opioids, dopamine, oxytocin, and vasopressin as candidates responsible for social buffering because they play an important role in affiliative behavior toward conspecifics other than the mother or mate. For example, play behavior between same-sex adolescent rats is reduced by treatment with an opioid receptor antagonist (Beatty & Costello, 1982). Another study using social play as an unconditioned stimulus demonstrated that opioid receptor antagonists block the establishment of conditioned place preference (Trezza et al. , 2011). Dopamine is an additional candidate because treatment with a dopamine receptor antagonist was shown to reduce intrinsic play behavior between same-sex adolescent rats (Beatty et al. , 1984; Niesink & Van Ree, 1989) and optogenetically induced social investigation between female mice (Gunaydin et al. , 2014). In addition to opioids and dopamine, oxytocin and vasopressin should be included among the list of candidates based on their established roles in the formation of attachment to one’s children and mates. For example, after giving birth, ewes exhibit rejection behavior toward alien lambs. However, oxytocin infusion reverses such rejection behavior (Kendrick et al. , 1992). Similarly, in monogamous prairie voles, males and females show a preference for their mates over unfamiliar opposite-sex conspecifics. However, this partner preference is blocked by treatment with vasopressin antagonists in males (Winslow et al. , 1993) and treatment with oxytocin antagonists in females (Insel & Hulihan, 1995). Consistent with these findings, an oxytocin antagonist was found to block social buffering induced by bonded males in female prairie voles (Adam, 2014). In addition, some studies have suggested possible roles for oxytocin and vasopressin in affiliative behavior toward same-sex conspecifics. For example, treatment with an oxytocin antagonist was shown to reduce intermale social investigation both in rats and mice (Lukas et al. , 2011). A similar effect was observed in vasopressin gene–knock-down male mice (Rigney et al. , 2022). Based on these findings, we hypothesized that antagonists of one or more of these neurochemicals would block social buffering of conditioned fear responses in rats.
To elucidate the neurochemical background of social buffering, it is important to identify the sites at which responsible neurochemicals act. The presence of opioids (Stein, 2016) or dopamine (Einhorn et al. , 1991) decreases the intrinsic excitability of neurons, whereas the presence of oxytocin (Rogers-Carter et al. , 2018) or vasopressin (Liu et al. , 2003) increases the intrinsic excitability of neurons. Therefore, during antagonist-mediated blockade of social buffering, the antagonist would modulate neural activity at the site of action within the neural pathway related to social buffering and/or affiliative behavior. One methodological approach for identifying the site of modulation would be to observe the changes in Fos expression throughout the brain. We expect that Fos expression would be altered at the sites at which responsible neurochemicals act.
In this study, a series of experiments were performed to explore the neurochemical background of social buffering in rats. In Experiment 1, fear-conditioned subject rats were first injected intraperitoneally with either naloxone (a non-selective opioid receptor antagonist), haloperidol (a dopamine D2 receptor antagonist), atosiban (a oxytocin receptor antagonist), SR49059 (a vasopressin V1a receptor antagonist), or saline. The subjects were then exposed to the CS with an associate. The effect of each antagonist was evaluated by observing thebehavioral responses. Experiment 2 examined the effect of naloxone on locomotor activity during an open-field test. In Experiment 3, we analyzed Fos expression in 16 brain regions of the second cohort of subjects during naloxone-mediated blockade of social buffering.