4. Discussion
In the present study, a series of experiments were performed to explore the neurochemical background of social buffering of conditioned fear responses in rats. In Experiment 1, pretreatment with naloxone, but not haloperidol, SR49059, or atosiban, increased freezing and decreased walking and investigation behaviors. These results suggest that naloxone blocks social buffering, whereas the other three antagonists do not. In Experiment 2, naloxone did not affect walking steps during the test. Therefore, the results of Experiment 1 are less likely due to decreased activity of naloxone. In Experiment 3, Fos expression was increased in the PVN, supporting the blockade of social buffering by naloxone. In addition, Fos expression was decreased in the NAc shell, ACC, and IC. Furthermore, Fos expression in the NAc core tended to be decreased. Naloxone thus appears to affect these four regions and/or upstream regions during the blockade of social buffering. From these collective results, we conclude that naloxone blocks social buffering of conditioned fear responses in rats.
Based on the present findings, we suggest that certain strains of rats can induce social buffering because they stimulate opioid receptors. Animals show distinct social behaviors when they evaluate an individual as to whether it is a member of the same group and recognize social similarity to that individual. In rats, the strain can serve as the group with regard to recognizing social similarity (Ben-Ami Bartalet al. , 2014; Han et al. , 2019; Kogo et al. , 2021; Kiyokawa et al. , 2022). We previously found that social buffering is affected by the strain of associates (Nakamura et al. , 2016). Specifically, social buffering in Wistar rats can be induced by unfamiliar Wistar, Sprague-Dawley, Long-Evans, or Lewis rats, but not by unfamiliar Fischer344 or Brown-Norway rats. In addition, autoradiography data suggested that opioid receptors are stimulated when rats interact with other rats of the same strain (Panksepp & Bishop, 1981). Furthermore, the stimulation of opioid receptors is known to ameliorate stress responses. For example, although rats show an augmented anxiety response after undergoing acute restraint, this augmentation is blocked by morphine, an opioid receptor agonist (Joshi et al. , 2014). These findings suggest that the recognition of social similarity stimulates opioid receptors, which in turn induces social buffering as a part of ingroup favoritism. This conclusion is further supported by the finding that naltrexone, an opioid receptor antagonist, reduces feelings of social connection in humans (Inagaki et al. , 2016).
We also found in the present study that reduced Fos expression in the Nac, ACC, and IC accompanied the blockade of social buffering by naloxone. Based on these results, we hypothesize that opioid receptors in the NAc play an important role in social buffering. It is known that the benefit of social interaction is produced by the stimulation of opioid receptors in the NAc. For example, rats prefer to stay in a compartment where they had previously interacted with conspecifics (Tzschentke, 1998; Thiel et al. , 2008). This socially conditioned place preference was blocked by the administration of the mu-opioid receptor antagonist CTAP into the NAc immediately before social interaction (Trezza et al. , 2011). Given that social buffering is one of the benefits of social interaction, it is possible that social buffering is also induced by the stimulation of opioid receptors in the NAc. However, the observed decrease in Fos expression was unexpected. Stimulation of opioid receptors is known to decrease neuronal excitability (Stein, 2016). Therefore, Fos expression would be expected to increase when an antagonist binds to opioid receptors. An alternative possibility is that opioid receptors responsible for social buffering are located in brain regions upstream of the NAc. For example, the ventral tegmental area (VTA) is a region that projects into the NAc and expresses opioid receptors (Le Merrer et al. , 2009). In addition, microinjection of the mu-opioid receptor agonist DAMGO into this region was shown to increase Fos expression in the NAc (Bontempi & Sharp, 1997). Therefore, it is possible that opioid receptors in the VTA, rather than the NAc, are responsible for social buffering. Furthermore, it is also possible opioid receptors in the ACC and IC or their upstream regions contribute to social buffering because the ACC and IC are known to contribute to empathy (Apps et al. , 2016) and stress identification (Rogers-Carter et al. , 2018; Djerdjaj et al. , 2022), respectively, in rats. Further research is needed to determine the location of the opioid receptors responsible for social buffering.