Yasushi Kiyokawa

Partner's stress status influences social buffering effects in rats.

The relationship between a social partners stress status and the social buffering effect was examined in adult male Wistar rats. Fear-conditioned rats were exposed to the same context along with either a shocked or nonshocked conspecific partner. Changes in body temperature and behavioral responses were monitored in order to compare the effects of social buffering, and the Fos protein in the paraventricular nucleus was immunostained. The presence of the partner rat attenuated stress-induced hyperthermia, as well as behavioral responses and Fos expression in response to the fearful context, and nonshocked partners were more effective than shocked partners. These findings suggest that the social buffering effect depends on the stress status of the accompanying conspecific animal.

Two types of social buffering differentially mitigate conditioned fear responses

In a phenomenon known as ‘social buffering’ in various species, signals from a conspecific animal can mitigate stress responses. This buffering can be achieved either by ‘pair‐housing’ after a stressful event or by ‘pair‐exposure’ to an acute stressor with a conspecific animal. In this study, we compared the impacts of these two types of social buffering on auditory conditioned fear responses in male rats. When subjects were exposed to an auditory conditioned stimulus (CS) that had been paired with foot shocks on the previous day, they clearly exhibited behavioral (freezing), autonomic (aggravated stress‐induced hyperthermia) and neural (Fos expression) responses. Pair‐housing for 24 h with an unfamiliar rat following fear conditioning resulted in a suppressed autonomic, but not behavioral, response, with Fos expression in the lateral nucleus of the amygdala and ventrolateral periaqueductal gray. On the other hand, pair‐exposure to the CS with an unfamiliar rat eliminated the behavioral, but not the autonomic, response, with Fos expression in the basal nucleus of the amygdala and infralimbic region of the prefrontal cortex. Furthermore, subjects that had been pair‐housed and then pair‐exposed showed no behavioral, autonomic or neural responses, suggesting that the combination of the two procedures can completely block the fear conditioning sequence. These results demonstrate that two types of social buffering differentially relieve conditioned fear responses, by influencing different neural pathways in the amygdala.

Mapping the neural circuit activated by alarm pheromone perception by c-Fos immunohistochemistry.

We previously reported that the alarm pheromones released from stressed male rats exaggerated both behavioral and autonomic (stress-induced hyperthermia) responses in recipient rats that were introduced into a novel environment. Subsequent experiments provided evidence that these alarm pheromones could be divided into two functionally different categories based on the site specificity and testosterone dependency of their production. However, the neural mechanisms underlying these behavioral and physiological responses remain unknown. In the present study, we examined Fos expression in 26 brain sites of the recipient rat 60 min after the exposure to the pheromone that aggravated stress-induced hyperthermia. The alarm pheromone-exposed rats showed a concurrent increase in Fos expression, in contrast to control odor-exposed rats in the anterior division lateral and medial group of the bed nucleus of the stria terminalis, paraventricular nucleus, dorsomedial hypothalamic nucleus, anterodorsal medial, lateral and basolateral amygdaloid nucleus, ventrolateral periaqueductal gray, laterodorsal tegmental nucleus, and locus coeruleus. These results provide information about the neural mechanisms in response to a non-sexual pheromone, i.e., an alarm pheromone, and suggest that the perception of the alarm pheromone is related to stress-responsive brains structures, including the hypothalamus and brainstem, as well as to the amygdaloid nuclei.

Main olfactory system mediates social buffering of conditioned fear responses in male rats

We previously reported that the presence of a conspecific animal blocked freezing of a male rat in response to an auditory conditioned stimulus that had been paired with foot shocks, as well as associated Fos expression in the paraventricular nucleus. Here we investigated how this ‘social buffering’ is mediated by examining the contributions of both physical contact and the main olfactory system. Fear‐conditioned rats exposed to the conditioned stimulus alone responded by freezing and increased Fos expression in the paraventricular nucleus. However, the presence of another rat, but not a guinea pig, dramatically mitigated these responses, even if the dyad was separated by a wire mesh or a pair of wire meshes 5 cm apart. In contrast, social buffering was absent when a transparent acrylic board was inserted between the double wire mesh. Lesioning of the main olfactory epithelium by injection of ZnSO4 intranasally also abolished social buffering. Thus, we conclude that the main olfactory system is essential for the social buffering but does not require physical contact between the dyad.

Deprivation of mother-pup interaction by early weaning alters myelin formation in male, but not female, ICR mice.

We previously reported that early-weaned Balb/c mice develop a persistent increase in anxiety as well as aggression, and we suggested that deprivation of mother-pup interaction from postnatal days 15 to 21 might account for this phenomenon. In the present study, we investigated developmental changes in myelin formation and behavioral effects of early weaning in male and female ICR mice. Early weaning was associated with decreased numbers of open-arm entries in an elevated plus-maze for both male and female mice at 3 weeks of age (W3); this effect was persistently observed in males, but ceased after W3 in females. Compared to the brains of normally weaned mice, the brains of the early-weaned males at W8 and of the females at W5 were of lesser mass. Western blotting with whole-brain homogenates identified four isoforms of myelin basic protein (MBP; 21.5, 18.5, 17.0, and 14.0 kDa). Expression of these MBPs increased gradually in normally weaned mice. In contrast, in the early-weaned male mice, but not the early-weaned female mice, it increased robustly at W3 and then declined at W5, as compared to the normally weaned mice. These results suggest that early weaning influences not only anxiety-related behavior but also myelin formation in the brain during the developmental period, particularly between 3 and 5 weeks of age, and male mice are more vulnerable than females to early-weaning effects on behavior and myelin formation.

Modulatory role of testosterone in alarm pheromone release by male rats.

An alarm pheromone released from stressed conspecifics evokes behavioral and autonomic responses in rats. We have previously reported that male Wistar rats show behavioral changes including increased sniffing, walking and rearing, and decreased resting as well as exaggerated response of body temperature to a novel environment [known as stress-induced hyperthermia (SIH)] when they are exposed to an alarm pheromone released from other male rats receiving foot shocks. The purpose of the present study was to examine the role of testosterone in the production and release of the alarm pheromone using these behavioral and autonomic responses in recipient rats. Three groups of alarm pheromone donors were presented, namely, intact males, castrated males, and testosterone-implanted castrated males. The effects of the alarm pheromone on the autonomic responses did not differ among the three groups, regardless of the donors steroidal milieu, whereas behavioral responses were altered by castrating the donor males and the effects were restored by testosterone implantation. These results suggest that the alarm pheromone released from stressed male rats can be classified into at least two categories according to the androgen dependency of their production and/or release.

Enhancement of the acoustic startle reflex by an alarm pheromone in male rats

Recently, we reported that an alarm pheromone released from the perianal region of male rats aggravated stress-induced hyperthermia and increased defensive and risk assessment behaviors in recipient male rats. Based on these results, we hypothesized that the primary effect of the alarm pheromone is to increase anxiety; however, there is still no clear evidence for this pheromone effect. Therefore, we examined this issue by assessing the effect of the alarm pheromone on the acoustic startle reflex (ASR), which is a useful index for studying negative emotions such as anxiety in rats. The alarm pheromone enhanced the ASR for 105-dB auditory stimuli, but not for those of 90 and 120 dB, when these three intensities of sound were used randomly. The same results were obtained when one of these three intensities was used repeatedly. In addition, pretreatment with diazepam (i.p.) at doses of 0.7 and 2.0 mg/kg suppressed the ASR of the pheromone recipients, whereas the lower dose (0.2 mg/kg) slightly attenuated the pheromone effect and the control injection (vehicle) had no effect. These results indicate that the alarm pheromone enhances the ASR by increasing anxiety in recipient rats, suggesting that the primary effect of the alarm pheromone is to increase the anxiety level.

A familiar conspecific is more effective than an unfamiliar conspecific for social buffering of conditioned fear responses in male rats.

In social animals, the presence of an affiliative conspecific alleviates acute stress responses, and this is called social buffering. We previously reported in male rats that the presence of a conspecific mitigates conditioned fear responses to auditory conditioned stimulus paired with foot shocks. Subsequent studies revealed that we could observe this social buffering when rats were tested in a box odorized by a conspecific. Because we previously used an unfamiliar conspecific, the effects of familiarity with a conspecific on the intensity of social buffering remain unclear. Here, we examine this question by preparing a familiar conspecific that had been housed with a subject for 3 weeks in the same cage. We exposed fear-conditioned subjects to a conditioned stimulus in either a clean control box or a box odorized beforehand by either an unfamiliar or a familiar conspecific. When the subjects were tested in the control box, they showed freezing and Fos expression in the paraventricular nucleus. These responses were suppressed when we placed rats in the box odorized by a conspecific. However, the suppression was greater when the box was odorized by a familiar conspecific rather than by an unfamiliar conspecific. Fos expression in the lateral amygdala was also suppressed in the same manner. These results suggest that a familiar conspecific is more effective for social buffering of conditioned fear responses.

Olfactory signals mediate social buffering of conditioned fear responses in male rats.

In social animals, the presence of an affiliative conspecific alleviates acute stress responses, and this is called social buffering. We previously reported that social buffering mitigates the fear responses of male rats to auditory conditioned stimuli that had been paired with foot shocks. Subsequent studies revealed that signals that are perceived by the main olfactory epithelium are important for social buffering. Because olfactory signals are the signal perceived by the main olfactory epithelium, we hypothesized that we could induce the social buffering of conditioned fear responses by presenting olfactory signals that were derived from a conspecific. In order to test this hypothesis, we exposed fear-conditioned subjects to a conditioned stimulus either in a clean test box or in a test box that was odorized by keeping a conspecific in it as an odor donor beforehand. When the subjects were tested in the clean test box, they showed behavioral fear responses and enhanced Fos expression in the paraventricular nucleus. In contrast, the presence of conspecific olfactory signals blocked these fear responses and Fos expression. These results suggested that olfactory signals suppress conditioned fear responses. Fos expression in the posteromedial region of the olfactory peduncle and amygdala suggested that this suppression involves the same neural mechanisms as those of social buffering. Taken together, we concluded that olfactory signals mediate the social buffering of conditioned fear responses.

The neural pathway underlying social buffering of conditioned fear responses in male rats

In social animals, the presence of an affiliative conspecific alleviates acute stress responses, and this is termed social buffering. However, the neural mechanisms underlying social buffering have not been elucidated. We have reported that the main olfactory system mediates social buffering of conditioned fear responses in male rats, and this is accompanied by suppression of the lateral and central amygdala. Therefore, olfactory signals are probably transmitted from the main olfactory system to the amygdala. Because the lateral and central amygdala do not receive projections from the main olfactory bulb, the site that links the main olfactory bulb and amygdala was presumed to be located within the main olfactory system. To find the linkage site, we generated lesions within the main olfactory system, and found that a bilateral lesion in the posteromedial region of the olfactory peduncle (pmOP) blocked social buffering. Next, we determined that the pmOP receives direct projections from the main olfactory bulb. Finally, we demonstrated that the connection between the pmOP and ipsilateral amygdala is important for social buffering, and that the pmOP projects directly to the ipsilateral amygdala, including the lateral and central amygdala. On the basis of these results, we suggest that the pmOP links the main olfactory blub to the amygdala and enables social buffering of conditioned fear responses. These results provide the first comprehensive picture of the neural pathway underlying the social buffering phenomenon.