Behavioural Brain Research 267 (2014) 189–193
Contents lists available at ScienceDirect
Behavioural Brain Research journal homepage: www.elsevier.com/locate/bbr
Research report
A familiar conspecific is more effective than an unfamiliar conspecific for social buffering of conditioned fear responses in male rats Yasushi Kiyokawa ∗ , Akira Honda, Yukari Takeuchi, Yuji Mori Laboratory of Veterinary Ethology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
h i g h l i g h t s • • • •
Social buffering by an unfamiliar conspecific suppressed conditioned fear responses. The suppression was greater in the presence of a familiar conspecific. Social buffering under both conditions shared the same neural mechanisms. Familiar conspecifics are more effective than unfamiliar ones for social buffering.
a r t i c l e
i n f o
Article history: Received 8 February 2014 Received in revised form 20 March 2014 Accepted 24 March 2014 Available online 31 March 2014 Keywords: Exposure-type social buffering Familiarity Fear conditioning Freezing HPA axis Amygdala.
a b s t r a c t 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 fearconditioned 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. © 2014 Elsevier B.V. All rights reserved.
1. Introduction In social mammals, signals from conspecific animals influence stress responses. For example, in rats, olfactory signals that are released from stressed conspecifics aggravate autonomic [1] and behavioral [2] responses to a novel environment or startle reflex to a loud noise [3]. On the other hand, signals from affiliative conspecifics could mitigate acute stress responses in a
Abbreviations: BA, basal amygdala; CeA, central amygdala; CS, conditioned stimulus; HPA, hypothalamic–pituitary–adrenal; LA, lateral amygdala; MOB, main olfactory bulb; PVN, paraventricular nucleus of the hypothalamus. ∗ Corresponding author. Tel.: +81 3 5841 7577; fax: +81 3 5841 8190. E-mail addresses: [email protected] (Y. Kiyokawa), [email protected] (A. Honda), [email protected] (Y. Takeuchi), [email protected] (Y. Mori). http://dx.doi.org/10.1016/j.bbr.2014.03.043 0166-4328/© 2014 Elsevier B.V. All rights reserved.
variety of social species [4–8]. These phenomena are called social buffering [9]. We previously reported that in response to an auditory conditioned stimulus (CS) that had been paired with foot shocks, adult male rats in the presence of an unfamiliar adult male conspecific no longer froze or had related behavioral responses, and their hypothalamic–pituitary–adrenal (HPA) axis activation as measured by Fos expression in the paraventricular nucleus of the hypothalamus (PVN) was completely blocked [10]. Separating the dyad with 2 wire-mesh screens placed 5 cm apart had no effect on this social buffering of conditioned fear responses [11,12]. In a subsequent study, by keeping a conspecific as an odor donor in the test box beforehand, we found that social buffering still occurred [13]. The neural pathway that underlies this social buffering of conditioned fear responses has also been studied; after being perceived at the main olfactory epithelium [12], olfactory signals from a conspecific
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are transmitted to the main olfactory bulb (MOB). The signals are then transmitted to the posterior complex of the anterior olfactory nucleus [13,14], and then to the lateral amygdala (LA) and/or central amygdala (CeA) [10,13]. As a result, olfactory signals suppress LA and CeA activation and block conditioned fear responses. The intensity of social buffering appears to be more conspicuous when it is induced by familiar conspecifics as compared to social buffering triggered by unfamiliar conspecifics, although clear evidence is still lacking. For example, in adult male rats, 3 familiar conspecifics tended to be more effective than 3 unfamiliar conspecifics when social buffering suppressed freezing and defecation in response to a sudden loud noise presented in a novel environment [15]. In addition, a familiar conspecific tended to be more effective than an unfamiliar conspecific in periadolescent rats when social buffering suppressed HPA axis activation in response to a novel environment [16]. Based on these previous studies, we hypothesized that a familiar conspecific is more effective than an unfamiliar conspecific for social buffering of conditioned fear responses by further suppressing LA and CeA activation. To assess this hypothesis, we prepared a familiar conspecific by housing it with a subject for 3 weeks in the same cage. In order to trap olfactory signals responsible for social buffering, either an unfamiliar or a familiar conspecific was kept in a test box as an odor donor for 2 h [13]. An identical test box in which a donor had not been kept in was used as a control box. Then, fear-conditioned subjects were exposed to the CS while they were in 1 of these 3 boxes. Observing the behavioral responses and Fos expression in the PVN allowed us to assess the intensity of social buffering. Fos expression was also observed in the amygdala of the subjects in order to assess the underlying neural mechanisms. 2. Material and methods 2.1. Animals All experiments were approved by the Animal Care and Use Committee of the Faculty of Agriculture of The University of Tokyo and were based on guidelines that were adapted from the Consensus Recommendations on Effective Institutional Animal Care and Use Committees by the Scientists Center for Animal Welfare. Experimentally naïve male Wistar rats (aged 7 weeks) were purchased from Charles River Laboratories Japan (Kanagawa, Japan). The animals were kept in an ambient temperature of 24 ± 1 ◦ C, 45 ± 5% humidity, and under a 12-h light/12-h dark cycle (lights switched on at 0800). Food and water were available ad libitum. Upon arrival, each rat was assigned to either the subject or the donor group that was used to odorize the test box (see below). A subject and a donor or 2 subjects were housed in a cage for 3 weeks until the conditioning day. Therefore, a donor could serve both as a familiar donor for the subject that was housed in the same cage and as an unfamiliar donor for the other subjects. Then, all animals were housed individually on the morning of the conditioning day. All animals were handled for 5 min per day for 3 days before the conditioning day. 2.2. Fear conditioning Fear conditioning was performed in an illuminated room between 0900 and 1700, as described in our previous studies [11,13,14]. A subject was placed in an acrylic conditioning box (28 × 20 × 27 cm) for 20 min and received 7 repetitions of a 3-s tone (8 kHz, 65 dB) that terminated concurrently with a foot shock (0.5 s, 0.65 mA). The intertrial interval randomly varied between 60 and 270 s. Each subject was returned to its home cage after the fear conditioning.
Fig. 1. Schematic diagram of the test apparatus used in this study.
2.3. Odorization and fear-expression test A fear-expression test was performed 24 h after the fear conditioning in a dark room that was illuminated with dim red light. The test box used for the fear-expression test is divided into 2 compartments (14 × 44 × 15 cm each) by a detachable acrylic board that has 175 holes (6-mm diameter) in its upper 6.5 cm and a demountable wire mesh that consists of a 1-cm2 gauge (Fig. 1). The distance between the acrylic board and wire mesh is 5 mm. Preceding the fear-expression test, the test box was odorized, as done in a previous study [13]. A donor that had been acclimatized to the soundproof chamber (36 × 54 × 35 cm; Muromachi Kikai, Tokyo, Japan) for about 20 min was placed in 1 of 2 compartments of the test box with clean bedding. Then, the test box was returned to the chamber and kept there for 2 h. We prepared the control box by keeping the test box without a donor in it. The donor was always placed in the compartment that had the wire mesh as one of the walls in order to prevent the soiled bedding from moving to the other compartment during the odorization, although the side within the test box was counterbalanced. Donor rats had been acclimatized to this odorization procedure by being kept in the test box for 1 h on a day before the conditioning day and on the conditioning day, and for 30 min just before the odorization. All donors were used twice, once as a familiar donor and once as an unfamiliar donor. After the odorization, the donor rat was removed and the test box was placed on an experimental table. The subjects were then placed in a different compartment from the one the donor was kept in and underwent the fear-expression test. After a 2-min acclimation period, a CS was presented 5 times for 3 s each at 1-min intervals during the first half of the 10-min experimental period. The subjects were divided into the following 3 groups based on the type of the box: the subjects that underwent the fear-expression test in the odorized box by an unfamiliar donor (unfamiliar group, n = 10), in the odorized box by a familiar donor (familiar group, n = 9), and in the control box (control group, n = 11). We recorded the behavior of the subjects during the acclimation and experimental periods with a video camera (DCR-TRV18; Sony, Tokyo, Japan) and an HD-DVD recorder (DVR-77H; Pioneer, Kanagawa, Japan). After the fear-expression test, the subjects were returned to their home cages and kept in a colony room.
Y. Kiyokawa et al. / Behavioural Brain Research 267 (2014) 189–193
2.4. Immunohistochemistry for c-Fos Each subject was deeply anesthetized with sodium pentobarbital (Somnopentyl, Schering-Plough Animal Health, Harefield, UK) and perfused intracardially with 0.9% saline, which was followed by 4% paraformaldehyde in 0.1 M phosphate buffer 48 min after the fear-expression test, that is, 60 min after the beginning of the acclimation period. The brain was removed, immersed overnight in the same fixative, and then placed in 30% sucrose/phosphate buffer for cryoprotection. The avidin–biotin–peroxidase method was used for detection of the immunohistochemistry, as previously described [13,17]. Briefly, 6 successive 30-m sections that contained the PVN (Bregma –1.80 mm) and LA, CeA and BA (Bregma – 2.76 mm) were collected, and the second and fifth sections were stained with cresyl violet in order to confirm the location of the nucleus, while the remaining sections were used for freefloating immunohistochemistry. The sections were incubated with primary antibody to Fos protein (PC38, Merck Millipore, Billerica, MA) for 65 h and biotinylated anti-rabbit secondary antibody (BA-1000, Vector Laboratories, Burlingame, CA) for 2 h, and the sections were then processed with the ABC kit (Vector Laboratories) and developed using a diaminobenzidine solution with nickel intensification. 2.5. Data analyses and statistical procedures The data are expressed as means ± standard error of means, and significance was set at P < 0.05 for all statistical tests. A researcher, blinded to the experimental conditions, recorded the duration of the behaviors of freezing (immobile posture, with cessation of skeletal and vibrissae movement except in respiration) and sniffing (regular movement of vibrissae with exploring), and the frequency of walking (number of steps taken with the hind paws) by the subjects by using Microsoft Excel-based Visual Basic software that records the duration and number of pressing keys, as was done in our previous studies [11,13,17,18]. The behavioral data during the initial acclimation and experimental period of the subjects were analyzed by MANOVA followed by Fisher’s PLSD post hoc test. For immunohistochemical analyses, sections of each region were captured using a microscope equipped with a digital camera (DP30BW, Olympus, Tokyo, Japan). We confirmed that our analyses were in our regions of interest according to a brain atlas [19]. To analyze Fos expression in the PVN, the number of Fosimmunoreactive cells was counted bilaterally in 4 sections with ImageJ 1.45s software by an experimenter blinded to the experimental groups. The area of the nucleus was also measured using the same software. Then, the mean value of density (number of cells/mm2 ) for each subject was calculated and was analyzed by ANOVA followed by Fisher’s PLSD post hoc test. To analyze Fos expression in the LA, CeA, and basal amygdala (BA), we randomly chose 2 sections for analyzing left amygdala and 2 sections for right amygdala. Then, Fos-immunoreactive cells were counted within a 0.5-mm square of each region. When the designated region was smaller than the boundaries of a 0.5-mm square, only the cells in the region of interest were counted. The mean value of the number for each subject was calculated and was analyzed by ANOVA followed by Fisher’s PLSD post hoc test. 3. Results 3.1. Conditioned fear responses to the CS The behavioral responses during the acclimation period were not different between the groups [F(6,50) = 1.26; P = 0.294] (Table 1). However, the behavioral responses during the experimental period
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Table 1 Behavioral responses during the acclimation period of subjects. Groups Freezing Sniffing Walking
Control (11)
Unfamiliar (10)
7.5 ± 3.7 83.8 ± 8.6 25.5 ± 3.3
4.3 ± 1.9 86.3 ± 10.6 31.4 ± 3.4
Familiar (9) 0.2 ± 0.2 107 ± 4 33.9 ± 2.2
Data are expressed as mean ± SEM. The numbers of rats were shown in parentheses.
were significantly different between the groups [F(6,50) = 5.12; P < 0.01]. A post hoc test revealed that the unfamiliar (P < 0.05) and familiar groups (P < 0.01) showed decreased freezing as compared to the control group. In addition, the familiar group showed decreased freezing as compared to the unfamiliar group (P < 0.01) (Fig. 2A top). The unfamiliar and familiar groups also showed increased sniffing (unfamiliar, P < 0.05; familiar, P < 0.01) and walking (unfamiliar, P < 0.05; familiar, P < 0.01) as compared to the control group. However, these behaviors were not different between the familiar and unfamiliar groups, although the familiar group tended to show increased sniffing as compared to the unfamiliar group (P = 0.0634) (Fig. 2A middle and bottom). Fos expression in the PVN was different between the groups [F(2,27) = 21.2; P < 0.01]. A post hoc test revealed that the unfamiliar (P < 0.01) and familiar groups (P < 0.01) showed decreased Fos expression as compared to the control group. In addition, the familiar group showed decreased Fos expression as compared to the unfamiliar group (P < 0.05) (Fig. 2B). 3.2. Fos expression in the amygdala In the amygdala (Fig. 3A), Fos expression in the LA [F(2,27) = 11.3; P < 0.01], CeA [F(2,27) = 3.40; P < 0.05], and BA [F(2,27) = 3.99; P < 0.05] was different between the groups. A post hoc test revealed that the unfamiliar (P < 0.05) and familiar groups (P < 0.01) showed decreased Fos expression in the LA as compared to the control group. Moreover, the familiar group showed decreased Fos expression as compared to the unfamiliar group (P < 0.05) (Fig. 3B top). In the CeA, the familiar group, but not the unfamiliar group, showed decreased Fos expression as compared to the control group (P < 0.05). However, Fos expression was not different between the familiar and unfamiliar groups (Fig. 3B middle). In the BA, Fos expression was not different between the unfamiliar and control groups, and between the familiar and control groups. However, the familiar group showed decreased Fos expression as compared to the unfamiliar group (P < 0.01) (Fig. 3B bottom). 4. Discussion When fear-conditioned subjects were tested in the clean test box, they showed freezing and Fos expression in the PVN. These responses were suppressed when fear-conditioned subjects were tested in the odorized box by a conspecific. In addition, the suppression was more pronounced when the box was odorized by a familiar conspecific rather than by an unfamiliar conspecific. These results suggest that social buffering was more conspicuous when we tested in the box odorized by a familiar conspecific. To assess potential underlying mechanisms of this phenomenon, we examined Fos expression in the amygdala. We found that Fos expression in the LA was suppressed when we tested in the conspecific-odorized box and that the suppression was greater when the box was odorized by a familiar conspecific. These results suggest that social buffering induced by unfamiliar and familiar conspecifics share the same neural mechanisms. Taken together, we conclude that a familiar conspecific is more effective for social buffering of conditioned fear responses.
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Fig. 2. Conditioned fear responses to the auditory conditioned stimulus (CS) of the subjects. (A) Duration of freezing and sniffing and frequency of walking (mean + SEM) and (B) the density of Fos-immunoreactive cells (mean + SEM) in the paraventricular nucleus (PVN) of the fear-conditioned subjects that underwent the fear-expression test in a test box odorized by unfamiliar (unfamiliar group) or familiar (familiar group) donor rats, or in a clean test box (control group). The letters indicate the significant differences (P < 0.05) according to MANOVA followed by Fisher’s PLSD post hoc test for behavioral results, and according to ANOVA followed by Fisher’s PLSD post hoc test for Fos expression in the PVN.
Because the same donor served both as an unfamiliar conspecific and as a familiar conspecific depending on the subjects, the present results might be ascribed to the familiarity of a subject with the olfactory signals from the donor. One possible explanation for the present results might be that olfactory signals from a familiar donor more effectively activated the neural pathway underlying social buffering [14]. For example, plasticity in the MOB is reported to play an important role in recognizing odors as familiar [20,21]. In addition, plasticity in the MOB increased the ability to detect odors similar to those that induced the plasticity [22]. Therefore, it is possible that housing with a donor induced the plasticity in the MOB, enabling the subjects to perceive olfactory signals from the familiar donor more effectively. However, it is also possible that olfactory
Fig. 3. The expression of Fos in the amygdala. (A) Schematic diagram of the location of the brain regions (open square) in which Fos-immunoreactive cells were counted. For simplification, the location was shown only in one side. Abbreviations: BA: basal amygdala; CeA: central amygdala; LA: lateral amygdala. The number of Fos-immunoreactive cells (mean + SEM) in the lateral, central, and basal amygdala of the fear-conditioned subjects that underwent the fear-expression test in an test box odorized by an unfamiliar (unfamiliar group) or familiar (familiar group) donor rats, or in a clean test box (control group). The letters indicate the significant differences (P < 0.05) according to ANOVA followed by Fisher’s PLSD post hoc test.
signals from a familiar conspecific additionally suppressed amygdalar activation through a different, but parallel, neural pathway. Further research would clarify how a familiar conspecific enhances the intensity of social buffering. In the present study, Fos expression in the BA was greater in subjects placed in the box odorized by an unfamiliar donor than in subjects placed in the box odorized by a familiar donor. However, it still remains unclear how the BA is involved in our experimental model. Although we have repeatedly observed increased Fos expression in the BA during social buffering by an unfamiliar conspecific in one condition [10,13,17], Fos expression in the BA was not increased in another condition even if an unfamiliar conspecific induced social buffering in the same way [10], suggesting that the increase of Fos expression in the BA may be unrelated to the familiarity of the conspecific or the induction of social
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buffering. In addition, although the BA is a brain area important for processing contextual information [23] or suppressing fear responses by extinction training [24], these roles do not account for the incremental change seen in our experimental model. To the best of our knowledge, this is the first clear evidence that a familiar conspecific is more effective for social buffering. In previous studies, the difference between the intensity of social buffering by an unfamiliar and familiar conspecific did not reach statistical significance [15,16]. One possible reason for this might be that the presence of an unfamiliar conspecific suppresses most of the stress responses. Therefore, the floor effects prevented us from observing any additional suppression by a familiar conspecific. However, it remains unclear why Armario et al. repeatedly observed in Sprague-Dawley adult male rats that the presence of a familiar conspecific aggravated, rather than suppressed, corticosterone response to a novel environment, as well as why the presence of an unfamiliar conspecific did not induce social buffering [25,26]. In contrast, other researchers using the same experimental model with the same strain of adult male [27] and periadolescent rats [16] or with other rat strains [11,28–30] showed that social buffering is induced by an unfamiliar conspecific. Another important finding in our present study is that olfactory signals responsible for social buffering are volatile. In our previous study, we used a test box without a partition so that a donor and a subject shared the same space [13]. Therefore, it remained unclear whether volatile signals in the air or nonvolatile signals in the soiled bedding are important for social buffering. To clarify this point, we divided the test box into 2 compartments by a partition that allowed only volatile signals to penetrate, and placed the subject and donor in a different compartment. As seen in the previous study, we found that we could induce social buffering in this divided box. Therefore, these results suggest that olfactory signals responsible for social buffering are volatile. 5. Conclusions In conclusion, we showed that compared to an unfamiliar conspecific, a familiar conspecific is more effective for social buffering of conditioned fear responses in male rats. Because we believe that affiliation to a familiar conspecific produced the differences observed in this study, to understand the neurobiology of affiliation in animals, further analyses using the present experimental model would be most helpful. Acknowledgements This study was supported by JSPS KAKENHI Grant Numbers 21228006 and 23688035. References [1] Kiyokawa Y, Kikusui T, Takeuchi Y, Mori Y. Modulatory role of testosterone in alarm pheromone release by male rats. Horm Behav 2004;45:122–7. [2] Kiyokawa Y, Shimozuru M, Kikusui T, Takeuchi Y, Mori Y. Alarm pheromone increases defensive and risk assessment behaviors in male rats. Physiol Behav 2006;87:383–7.
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[3] Inagaki H, Kiyokawa Y, Kikusui T, Takeuchi Y, Mori Y. Enhancement of the acoustic startle reflex by an alarm pheromone in male rats. Physiol Behav 2008;93:606–11. [4] da Costa AP, Leigh AE, Man MS, Kendrick KM. Face pictures reduce behavioural, autonomic, endocrine and neural indices of stress and fear in sheep. Proc Biol Sci 2004;271:2077–84. [5] Davitz JR, Mason DJ. Social facilitated reduction of a fear response in rats. J Comp Physiol Psychol 1955;48:149–56. [6] Grignard L, Boissy A, Boivin X, Garel JP, Le Neindre P. The social environment influences the behavioural responses of beef cattle to handling. Appl Anim Behav Sci 2000;68:1–11. [7] Guzman YF, Tronson NC, Guedea A, Huh KH, Gao C, Radulovic J. Social modeling of conditioned fear in mice by non-fearful conspecifics. Behav Brain Res 2009;201:173–8. [8] Kirschbaum C, Klauer T, Filipp SH, Hellhammer DH. Sex-specific effects of social support on cortisol and subjective responses to acute psychological stress. Psychosom Med 1995;57:23–31. [9] Hennessy MB, Kaiser S, Sachser N. Social buffering of the stress response: diversity, mechanisms, and functions. Front Neuroendocrinol 2009;30:470–82. [10] Kiyokawa Y, Takeuchi Y, Mori Y. Two types of social buffering differentially mitigate conditioned fear responses. Eur J Neurosci 2007;26:3606–13. [11] Kiyokawa Y, Hiroshima S, Takeuchi Y, Mori Y. Social buffering reduces male rats’ behavioral and corticosterone responses to a conditioned stimulus. Horm Behav 2014;65:114–8. [12] Kiyokawa Y, Takeuchi Y, Nishihara M, Mori Y. Main olfactory system mediates social buffering of conditioned fear responses in male rats. Eur J Neurosci 2009;29:777–85. [13] Takahashi Y, Kiyokawa Y, Kodama Y, Arata S, Takeuchi Y, Mori Y. Olfactory signals mediate social buffering of conditioned fear responses in male rats. Behav Brain Res 2013;240:46–51. [14] Kiyokawa Y, Wakabayashi Y, Takeuchi Y, Mori Y. The neural pathway underlying social buffering of conditioned fear responses in male rats. Eur J Neurosci 2012;36:3429–37. [15] Taylor GT. Fear and affiliation in domesticated male rats. J Comp Physiol Psychol 1981;95:685–93. [16] Terranova ML, Cirulli F, Laviola G. Behavioral and hormonal effects of partner familiarity in periadolescent rat pairs upon novelty exposure. Psychoneuroendocrinology 1999;24:639–56. [17] Kiyokawa Y, Kodama Y, Takeuchi Y, Mori Y. Physical interaction is not necessary for the induction of housing-type social buffering of conditioned hyperthermia in male rats. Behav Brain Res 2013;256:414–9. [18] Kodama Y, Kiyokawa Y, Takeuchi Y, Mori Y. Twelve hours is sufficient for social buffering of conditioned hyperthermia. Physiol Behav 2011;102:188–92. [19] Paxinos G, Watson C. The Rat Brain in Stereotaxic Coordinates. Sixth ed Tokyo, Japan: Elsevier; 2007. [20] Mak GK, Weiss S. Paternal recognition of adult offspring mediated by newly generated CNS neurons. Nat Neurosci 2010;13:753–8. [21] Shea SD, Katz LC, Mooney R. Noradrenergic induction of odor-specific neural habituation and olfactory memories. J Neurosci 2008;28:10711–9. [22] Mandairon N, Stack C, Kiselycznyk C, Linster C. Broad activation of the olfactory bulb produces long-lasting changes in odor perception. Proc Natl Acad Sci USA 2006;103:13543–8. [23] Onishi BK, Xavier GF. Contextual, but not auditory, fear conditioning is disrupted by neurotoxic selective lesion of the basal nucleus of amygdala in rats. Neurobiol Learn Mem 2010;93:165–74. [24] Herry C, Ciocchi S, Senn V, Demmou L, Muller C, Luthi A. Switching on and off fear by distinct neuronal circuits. Nature 2008;454:600–6. [25] Armario A, Luna G, Balasch J. The effect of conspecifics on corticoadrenal response of rats to a novel environment. Behav Neural Biol 1983;37: 332–7. [26] Armario A, Ortiz R, Balasch J. Corticoadrenal and behavioral response to open field in pairs of male rats either familiar or non-familiar to each other. Experientia 1983;39:1316–7. [27] Latane B. Stimulus determinants of social attraction in rats. J Comp Physiol Psychol 1972;79:13–21. [28] File SE, Peet LA. The sensitivity of the rat corticosterone response to environmental manipulations and to chronic chlordiazepoxide treatment. Physiol Behav 1980;25:753–8. [29] Wilson JH. A conspecific attenuates prolactin responses to open-field exposure in rats. Horm Behav 2000;38:39–43. [30] Wilson JH. Prolactin in rats is attenuated by conspecific touch in a novel environment. Cogn Affect Behav Neurosci 2001;1:199–205.
Contents lists available at ScienceDirect
Behavioural Brain Research journal homepage: www.elsevier.com/locate/bbr
Research report
A familiar conspecific is more effective than an unfamiliar conspecific for social buffering of conditioned fear responses in male rats Yasushi Kiyokawa ∗ , Akira Honda, Yukari Takeuchi, Yuji Mori Laboratory of Veterinary Ethology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
h i g h l i g h t s • • • •
Social buffering by an unfamiliar conspecific suppressed conditioned fear responses. The suppression was greater in the presence of a familiar conspecific. Social buffering under both conditions shared the same neural mechanisms. Familiar conspecifics are more effective than unfamiliar ones for social buffering.
a r t i c l e
i n f o
Article history: Received 8 February 2014 Received in revised form 20 March 2014 Accepted 24 March 2014 Available online 31 March 2014 Keywords: Exposure-type social buffering Familiarity Fear conditioning Freezing HPA axis Amygdala.
a b s t r a c t 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 fearconditioned 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. © 2014 Elsevier B.V. All rights reserved.
1. Introduction In social mammals, signals from conspecific animals influence stress responses. For example, in rats, olfactory signals that are released from stressed conspecifics aggravate autonomic [1] and behavioral [2] responses to a novel environment or startle reflex to a loud noise [3]. On the other hand, signals from affiliative conspecifics could mitigate acute stress responses in a
Abbreviations: BA, basal amygdala; CeA, central amygdala; CS, conditioned stimulus; HPA, hypothalamic–pituitary–adrenal; LA, lateral amygdala; MOB, main olfactory bulb; PVN, paraventricular nucleus of the hypothalamus. ∗ Corresponding author. Tel.: +81 3 5841 7577; fax: +81 3 5841 8190. E-mail addresses: [email protected] (Y. Kiyokawa), [email protected] (A. Honda), [email protected] (Y. Takeuchi), [email protected] (Y. Mori). http://dx.doi.org/10.1016/j.bbr.2014.03.043 0166-4328/© 2014 Elsevier B.V. All rights reserved.
variety of social species [4–8]. These phenomena are called social buffering [9]. We previously reported that in response to an auditory conditioned stimulus (CS) that had been paired with foot shocks, adult male rats in the presence of an unfamiliar adult male conspecific no longer froze or had related behavioral responses, and their hypothalamic–pituitary–adrenal (HPA) axis activation as measured by Fos expression in the paraventricular nucleus of the hypothalamus (PVN) was completely blocked [10]. Separating the dyad with 2 wire-mesh screens placed 5 cm apart had no effect on this social buffering of conditioned fear responses [11,12]. In a subsequent study, by keeping a conspecific as an odor donor in the test box beforehand, we found that social buffering still occurred [13]. The neural pathway that underlies this social buffering of conditioned fear responses has also been studied; after being perceived at the main olfactory epithelium [12], olfactory signals from a conspecific
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are transmitted to the main olfactory bulb (MOB). The signals are then transmitted to the posterior complex of the anterior olfactory nucleus [13,14], and then to the lateral amygdala (LA) and/or central amygdala (CeA) [10,13]. As a result, olfactory signals suppress LA and CeA activation and block conditioned fear responses. The intensity of social buffering appears to be more conspicuous when it is induced by familiar conspecifics as compared to social buffering triggered by unfamiliar conspecifics, although clear evidence is still lacking. For example, in adult male rats, 3 familiar conspecifics tended to be more effective than 3 unfamiliar conspecifics when social buffering suppressed freezing and defecation in response to a sudden loud noise presented in a novel environment [15]. In addition, a familiar conspecific tended to be more effective than an unfamiliar conspecific in periadolescent rats when social buffering suppressed HPA axis activation in response to a novel environment [16]. Based on these previous studies, we hypothesized that a familiar conspecific is more effective than an unfamiliar conspecific for social buffering of conditioned fear responses by further suppressing LA and CeA activation. To assess this hypothesis, we prepared a familiar conspecific by housing it with a subject for 3 weeks in the same cage. In order to trap olfactory signals responsible for social buffering, either an unfamiliar or a familiar conspecific was kept in a test box as an odor donor for 2 h [13]. An identical test box in which a donor had not been kept in was used as a control box. Then, fear-conditioned subjects were exposed to the CS while they were in 1 of these 3 boxes. Observing the behavioral responses and Fos expression in the PVN allowed us to assess the intensity of social buffering. Fos expression was also observed in the amygdala of the subjects in order to assess the underlying neural mechanisms. 2. Material and methods 2.1. Animals All experiments were approved by the Animal Care and Use Committee of the Faculty of Agriculture of The University of Tokyo and were based on guidelines that were adapted from the Consensus Recommendations on Effective Institutional Animal Care and Use Committees by the Scientists Center for Animal Welfare. Experimentally naïve male Wistar rats (aged 7 weeks) were purchased from Charles River Laboratories Japan (Kanagawa, Japan). The animals were kept in an ambient temperature of 24 ± 1 ◦ C, 45 ± 5% humidity, and under a 12-h light/12-h dark cycle (lights switched on at 0800). Food and water were available ad libitum. Upon arrival, each rat was assigned to either the subject or the donor group that was used to odorize the test box (see below). A subject and a donor or 2 subjects were housed in a cage for 3 weeks until the conditioning day. Therefore, a donor could serve both as a familiar donor for the subject that was housed in the same cage and as an unfamiliar donor for the other subjects. Then, all animals were housed individually on the morning of the conditioning day. All animals were handled for 5 min per day for 3 days before the conditioning day. 2.2. Fear conditioning Fear conditioning was performed in an illuminated room between 0900 and 1700, as described in our previous studies [11,13,14]. A subject was placed in an acrylic conditioning box (28 × 20 × 27 cm) for 20 min and received 7 repetitions of a 3-s tone (8 kHz, 65 dB) that terminated concurrently with a foot shock (0.5 s, 0.65 mA). The intertrial interval randomly varied between 60 and 270 s. Each subject was returned to its home cage after the fear conditioning.
Fig. 1. Schematic diagram of the test apparatus used in this study.
2.3. Odorization and fear-expression test A fear-expression test was performed 24 h after the fear conditioning in a dark room that was illuminated with dim red light. The test box used for the fear-expression test is divided into 2 compartments (14 × 44 × 15 cm each) by a detachable acrylic board that has 175 holes (6-mm diameter) in its upper 6.5 cm and a demountable wire mesh that consists of a 1-cm2 gauge (Fig. 1). The distance between the acrylic board and wire mesh is 5 mm. Preceding the fear-expression test, the test box was odorized, as done in a previous study [13]. A donor that had been acclimatized to the soundproof chamber (36 × 54 × 35 cm; Muromachi Kikai, Tokyo, Japan) for about 20 min was placed in 1 of 2 compartments of the test box with clean bedding. Then, the test box was returned to the chamber and kept there for 2 h. We prepared the control box by keeping the test box without a donor in it. The donor was always placed in the compartment that had the wire mesh as one of the walls in order to prevent the soiled bedding from moving to the other compartment during the odorization, although the side within the test box was counterbalanced. Donor rats had been acclimatized to this odorization procedure by being kept in the test box for 1 h on a day before the conditioning day and on the conditioning day, and for 30 min just before the odorization. All donors were used twice, once as a familiar donor and once as an unfamiliar donor. After the odorization, the donor rat was removed and the test box was placed on an experimental table. The subjects were then placed in a different compartment from the one the donor was kept in and underwent the fear-expression test. After a 2-min acclimation period, a CS was presented 5 times for 3 s each at 1-min intervals during the first half of the 10-min experimental period. The subjects were divided into the following 3 groups based on the type of the box: the subjects that underwent the fear-expression test in the odorized box by an unfamiliar donor (unfamiliar group, n = 10), in the odorized box by a familiar donor (familiar group, n = 9), and in the control box (control group, n = 11). We recorded the behavior of the subjects during the acclimation and experimental periods with a video camera (DCR-TRV18; Sony, Tokyo, Japan) and an HD-DVD recorder (DVR-77H; Pioneer, Kanagawa, Japan). After the fear-expression test, the subjects were returned to their home cages and kept in a colony room.
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2.4. Immunohistochemistry for c-Fos Each subject was deeply anesthetized with sodium pentobarbital (Somnopentyl, Schering-Plough Animal Health, Harefield, UK) and perfused intracardially with 0.9% saline, which was followed by 4% paraformaldehyde in 0.1 M phosphate buffer 48 min after the fear-expression test, that is, 60 min after the beginning of the acclimation period. The brain was removed, immersed overnight in the same fixative, and then placed in 30% sucrose/phosphate buffer for cryoprotection. The avidin–biotin–peroxidase method was used for detection of the immunohistochemistry, as previously described [13,17]. Briefly, 6 successive 30-m sections that contained the PVN (Bregma –1.80 mm) and LA, CeA and BA (Bregma – 2.76 mm) were collected, and the second and fifth sections were stained with cresyl violet in order to confirm the location of the nucleus, while the remaining sections were used for freefloating immunohistochemistry. The sections were incubated with primary antibody to Fos protein (PC38, Merck Millipore, Billerica, MA) for 65 h and biotinylated anti-rabbit secondary antibody (BA-1000, Vector Laboratories, Burlingame, CA) for 2 h, and the sections were then processed with the ABC kit (Vector Laboratories) and developed using a diaminobenzidine solution with nickel intensification. 2.5. Data analyses and statistical procedures The data are expressed as means ± standard error of means, and significance was set at P < 0.05 for all statistical tests. A researcher, blinded to the experimental conditions, recorded the duration of the behaviors of freezing (immobile posture, with cessation of skeletal and vibrissae movement except in respiration) and sniffing (regular movement of vibrissae with exploring), and the frequency of walking (number of steps taken with the hind paws) by the subjects by using Microsoft Excel-based Visual Basic software that records the duration and number of pressing keys, as was done in our previous studies [11,13,17,18]. The behavioral data during the initial acclimation and experimental period of the subjects were analyzed by MANOVA followed by Fisher’s PLSD post hoc test. For immunohistochemical analyses, sections of each region were captured using a microscope equipped with a digital camera (DP30BW, Olympus, Tokyo, Japan). We confirmed that our analyses were in our regions of interest according to a brain atlas [19]. To analyze Fos expression in the PVN, the number of Fosimmunoreactive cells was counted bilaterally in 4 sections with ImageJ 1.45s software by an experimenter blinded to the experimental groups. The area of the nucleus was also measured using the same software. Then, the mean value of density (number of cells/mm2 ) for each subject was calculated and was analyzed by ANOVA followed by Fisher’s PLSD post hoc test. To analyze Fos expression in the LA, CeA, and basal amygdala (BA), we randomly chose 2 sections for analyzing left amygdala and 2 sections for right amygdala. Then, Fos-immunoreactive cells were counted within a 0.5-mm square of each region. When the designated region was smaller than the boundaries of a 0.5-mm square, only the cells in the region of interest were counted. The mean value of the number for each subject was calculated and was analyzed by ANOVA followed by Fisher’s PLSD post hoc test. 3. Results 3.1. Conditioned fear responses to the CS The behavioral responses during the acclimation period were not different between the groups [F(6,50) = 1.26; P = 0.294] (Table 1). However, the behavioral responses during the experimental period
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Table 1 Behavioral responses during the acclimation period of subjects. Groups Freezing Sniffing Walking
Control (11)
Unfamiliar (10)
7.5 ± 3.7 83.8 ± 8.6 25.5 ± 3.3
4.3 ± 1.9 86.3 ± 10.6 31.4 ± 3.4
Familiar (9) 0.2 ± 0.2 107 ± 4 33.9 ± 2.2
Data are expressed as mean ± SEM. The numbers of rats were shown in parentheses.
were significantly different between the groups [F(6,50) = 5.12; P < 0.01]. A post hoc test revealed that the unfamiliar (P < 0.05) and familiar groups (P < 0.01) showed decreased freezing as compared to the control group. In addition, the familiar group showed decreased freezing as compared to the unfamiliar group (P < 0.01) (Fig. 2A top). The unfamiliar and familiar groups also showed increased sniffing (unfamiliar, P < 0.05; familiar, P < 0.01) and walking (unfamiliar, P < 0.05; familiar, P < 0.01) as compared to the control group. However, these behaviors were not different between the familiar and unfamiliar groups, although the familiar group tended to show increased sniffing as compared to the unfamiliar group (P = 0.0634) (Fig. 2A middle and bottom). Fos expression in the PVN was different between the groups [F(2,27) = 21.2; P < 0.01]. A post hoc test revealed that the unfamiliar (P < 0.01) and familiar groups (P < 0.01) showed decreased Fos expression as compared to the control group. In addition, the familiar group showed decreased Fos expression as compared to the unfamiliar group (P < 0.05) (Fig. 2B). 3.2. Fos expression in the amygdala In the amygdala (Fig. 3A), Fos expression in the LA [F(2,27) = 11.3; P < 0.01], CeA [F(2,27) = 3.40; P < 0.05], and BA [F(2,27) = 3.99; P < 0.05] was different between the groups. A post hoc test revealed that the unfamiliar (P < 0.05) and familiar groups (P < 0.01) showed decreased Fos expression in the LA as compared to the control group. Moreover, the familiar group showed decreased Fos expression as compared to the unfamiliar group (P < 0.05) (Fig. 3B top). In the CeA, the familiar group, but not the unfamiliar group, showed decreased Fos expression as compared to the control group (P < 0.05). However, Fos expression was not different between the familiar and unfamiliar groups (Fig. 3B middle). In the BA, Fos expression was not different between the unfamiliar and control groups, and between the familiar and control groups. However, the familiar group showed decreased Fos expression as compared to the unfamiliar group (P < 0.01) (Fig. 3B bottom). 4. Discussion When fear-conditioned subjects were tested in the clean test box, they showed freezing and Fos expression in the PVN. These responses were suppressed when fear-conditioned subjects were tested in the odorized box by a conspecific. In addition, the suppression was more pronounced when the box was odorized by a familiar conspecific rather than by an unfamiliar conspecific. These results suggest that social buffering was more conspicuous when we tested in the box odorized by a familiar conspecific. To assess potential underlying mechanisms of this phenomenon, we examined Fos expression in the amygdala. We found that Fos expression in the LA was suppressed when we tested in the conspecific-odorized box and that the suppression was greater when the box was odorized by a familiar conspecific. These results suggest that social buffering induced by unfamiliar and familiar conspecifics share the same neural mechanisms. Taken together, we conclude that a familiar conspecific is more effective for social buffering of conditioned fear responses.
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Fig. 2. Conditioned fear responses to the auditory conditioned stimulus (CS) of the subjects. (A) Duration of freezing and sniffing and frequency of walking (mean + SEM) and (B) the density of Fos-immunoreactive cells (mean + SEM) in the paraventricular nucleus (PVN) of the fear-conditioned subjects that underwent the fear-expression test in a test box odorized by unfamiliar (unfamiliar group) or familiar (familiar group) donor rats, or in a clean test box (control group). The letters indicate the significant differences (P < 0.05) according to MANOVA followed by Fisher’s PLSD post hoc test for behavioral results, and according to ANOVA followed by Fisher’s PLSD post hoc test for Fos expression in the PVN.
Because the same donor served both as an unfamiliar conspecific and as a familiar conspecific depending on the subjects, the present results might be ascribed to the familiarity of a subject with the olfactory signals from the donor. One possible explanation for the present results might be that olfactory signals from a familiar donor more effectively activated the neural pathway underlying social buffering [14]. For example, plasticity in the MOB is reported to play an important role in recognizing odors as familiar [20,21]. In addition, plasticity in the MOB increased the ability to detect odors similar to those that induced the plasticity [22]. Therefore, it is possible that housing with a donor induced the plasticity in the MOB, enabling the subjects to perceive olfactory signals from the familiar donor more effectively. However, it is also possible that olfactory
Fig. 3. The expression of Fos in the amygdala. (A) Schematic diagram of the location of the brain regions (open square) in which Fos-immunoreactive cells were counted. For simplification, the location was shown only in one side. Abbreviations: BA: basal amygdala; CeA: central amygdala; LA: lateral amygdala. The number of Fos-immunoreactive cells (mean + SEM) in the lateral, central, and basal amygdala of the fear-conditioned subjects that underwent the fear-expression test in an test box odorized by an unfamiliar (unfamiliar group) or familiar (familiar group) donor rats, or in a clean test box (control group). The letters indicate the significant differences (P < 0.05) according to ANOVA followed by Fisher’s PLSD post hoc test.
signals from a familiar conspecific additionally suppressed amygdalar activation through a different, but parallel, neural pathway. Further research would clarify how a familiar conspecific enhances the intensity of social buffering. In the present study, Fos expression in the BA was greater in subjects placed in the box odorized by an unfamiliar donor than in subjects placed in the box odorized by a familiar donor. However, it still remains unclear how the BA is involved in our experimental model. Although we have repeatedly observed increased Fos expression in the BA during social buffering by an unfamiliar conspecific in one condition [10,13,17], Fos expression in the BA was not increased in another condition even if an unfamiliar conspecific induced social buffering in the same way [10], suggesting that the increase of Fos expression in the BA may be unrelated to the familiarity of the conspecific or the induction of social
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buffering. In addition, although the BA is a brain area important for processing contextual information [23] or suppressing fear responses by extinction training [24], these roles do not account for the incremental change seen in our experimental model. To the best of our knowledge, this is the first clear evidence that a familiar conspecific is more effective for social buffering. In previous studies, the difference between the intensity of social buffering by an unfamiliar and familiar conspecific did not reach statistical significance [15,16]. One possible reason for this might be that the presence of an unfamiliar conspecific suppresses most of the stress responses. Therefore, the floor effects prevented us from observing any additional suppression by a familiar conspecific. However, it remains unclear why Armario et al. repeatedly observed in Sprague-Dawley adult male rats that the presence of a familiar conspecific aggravated, rather than suppressed, corticosterone response to a novel environment, as well as why the presence of an unfamiliar conspecific did not induce social buffering [25,26]. In contrast, other researchers using the same experimental model with the same strain of adult male [27] and periadolescent rats [16] or with other rat strains [11,28–30] showed that social buffering is induced by an unfamiliar conspecific. Another important finding in our present study is that olfactory signals responsible for social buffering are volatile. In our previous study, we used a test box without a partition so that a donor and a subject shared the same space [13]. Therefore, it remained unclear whether volatile signals in the air or nonvolatile signals in the soiled bedding are important for social buffering. To clarify this point, we divided the test box into 2 compartments by a partition that allowed only volatile signals to penetrate, and placed the subject and donor in a different compartment. As seen in the previous study, we found that we could induce social buffering in this divided box. Therefore, these results suggest that olfactory signals responsible for social buffering are volatile. 5. Conclusions In conclusion, we showed that compared to an unfamiliar conspecific, a familiar conspecific is more effective for social buffering of conditioned fear responses in male rats. Because we believe that affiliation to a familiar conspecific produced the differences observed in this study, to understand the neurobiology of affiliation in animals, further analyses using the present experimental model would be most helpful. Acknowledgements This study was supported by JSPS KAKENHI Grant Numbers 21228006 and 23688035. References [1] Kiyokawa Y, Kikusui T, Takeuchi Y, Mori Y. Modulatory role of testosterone in alarm pheromone release by male rats. Horm Behav 2004;45:122–7. [2] Kiyokawa Y, Shimozuru M, Kikusui T, Takeuchi Y, Mori Y. Alarm pheromone increases defensive and risk assessment behaviors in male rats. Physiol Behav 2006;87:383–7.
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