Physical interaction is required in social buffering induced by a familiar conspecific

ABSTRACT In social animals, signals released from fearless conspecifics attenuate fear responses, namely social buffering. The presence of conspecific odor can suppress the expression of

freezing response of conditioned mice. The present study investigated if physical social experience is required for this social buffering effect. The mice were exposed to donors, donor

bedding (collected from cages of donors), or fresh bedding as control, respectively, for 10 days (1 hour daily) in prior to fear conditioning test. The fear expression test was examined in

presence of donor bedding. The results showed that only the donor group mice showed reduced freezing time than the other two groups in the fear memory test. This phenomenon indicated that

physical interaction might be required for the social buffering effect. SIMILAR CONTENT BEING VIEWED BY OTHERS SOCIAL BUFFERING IN RATS REDUCES FEAR BY OXYTOCIN TRIGGERING SUSTAINED CHANGES

IN CENTRAL AMYGDALA NEURONAL ACTIVITY Article Open access 07 March 2024 CONSPECIFIC INTERACTIONS PREDICT SOCIAL TRANSMISSION OF FEAR IN FEMALE RATS Article Open access 02 April 2024 SOCIAL

ENRICHMENT BY SEPARATED PAIR HOUSING OF MALE C57BL/6JRJ MICE Article Open access 07 July 2020 INTRODUCTION In social animals, presence of affiliated conspecifics influence fear responses.

For instance, signals released from fearful conspecifics aggravated fear responses1. On the other hand, fearless conspecifics could release signals that attenuate fear responses at various

levels, including behavioral, autonomic and neural levels2,3,4, namely social buffering. This buffering effect can be induced either by ‘pair-housing’ after a stressful event, or by

‘pair-exposure’ to an acute stressor with a conspecific animal5. Social buffering is recognized as a multi-sensory modality effect, consisting of direct physical contact, visual

observation6, and olfaction, for instance7,8. Notably, lesion of the main olfactory epithelium abolished this buffering effect7, indicating the importance of olfactory system underling the

social buffering phenomenon. Interestingly, the suppression of the fear responses by a familiar conspecific was greater than an unfamiliar one9. This pointed out that the both the memory for

odor identity (“what is the odor”?) and acquired value (“is the odor more rewarded”?, such as social familiarity) might be involved in this synthetic processing of the social buffering

effect10. Physical interaction is a crucial factor of individual recognition but not individual odor recognition. The prior physical interaction was required for hamster to discriminate

different individuals in across-odor habituation11,12, but not in single-odor habituation tests13. These studies indicated that there are two classes of social odor learning in relation to

distance-based response pattern14. In particular, different communication and behavior strategies are employed in the volatile distance or nose-contact distance15. In present study, we

investigated that if the prior physical contact is required for the odor based social buffering effect for fear memory expression. METHODS ANIMALS 6-week-old experimentally naïve male

wild-type C57BL/6 N mice were bred and kept in IVC. The room temperature was controlled at 24 ± 1 °C. Food and water were available ad libitum. 6 mice were assigned to singly housed donor

group that was used to odorize the wood bedding. 17 animals were divided to social group (n = 6), odor+ group (n = 6) and odor− group (n = 5). 2 animals were housed in one cage, and cage

mates were assigned to the same group. A donor would serve as a familiar donor for the subjects of social or odor+ group and as an unfamiliar donor for the subjects in odor− group. Animal

maintenance and use were performed in accordance with the National Technical Committee on Laboratory Animal Science of the Standardization Administration of China guidelines. All

experimental procedures were approved by the Institution’s Animal Care and Use Committees of Nanjing Normal University, China. ODOR STIMULI FROM DONOR CAGE 5 days prior to formal experiment,

donor rats were given 600 cc of fresh wood bedding16,17. In continuous 10 days’ odor exposure, 200 cc samples were collected and served as the odorants for the exposure. These samples

contained bedding, feces, urine, and mice food particles. Equal amount of fresh bedding was mixed into the remainder. The night before fear-expression day all bedding was collected and

stored hermetically for following test. Behavioral experiments included odor exposure phase, fear conditioning, memory retrieval, social novelty preference test and fear-expression test. All

procedures took place during 10:00 to 14:00 (see Fig. 1). ODOR EXPOSURE PHASE Subjects were exposed to odorants daily for 1 hour during 10 days as previously described18. During exposure,

subjects in social group were kept in cages which bottoms were covered by fresh wood bedding with donors together. Subjects in odor+ group and in odor− group were kept singly in cages which

bottoms were covered by samples collected from cages of donors or fresh wood bedding, respectively. FEAR CONDITIONING 24 hours after the last exposure, mice were subjected for fear

conditioning. Briefly, mice were placed in a conditioning chamber (rectangle, white) for 120 seconds before a 30 seconds’ tone stimulus (2900 Hz, 80 dB). An electrical foot shock (0.65 mA)

was presented during the last 1 second of the tone presentation and co-terminates with the tone. Five trials of conditioning were separated by inter-trial intervals randomly range from 30 

seconds to 150 seconds (on average 90 seconds). Following an additional stay for 60 second in the chamber, the animals were removed back to home cage19. Cage mates were manipulated

simultaneously at all time. MEMORY RETRIEVAL 24 hours after the fear conditioning, subjects exposed to odorants for 1 hour to retrieve memory of odor. The manipulation of memory retrieval

was the same as odor exposure. Social novelty preference test and fear-expression test following memory retrieval instantly. SOCIAL NOVELTY PREFERENCE TEST Subjects were placed in the middle

of a social chamber, with both sides connected with a small box. A piece of gauze separated the box from the chamber. In the first session, mice had a 5 minutes’ exploration in the chamber.

Then bedding collected from the familiar donor cage (FO) and from cage of another unfamiliar mouse (UO) were placed into the two boxes respectively, followed by 10 minutes’ social

exploration in the second session20. The FO was always placed in the box preferred previously. For odor− group, bedding collected from two unfamiliar mice was placed in two boxes randomly

and a similar 10 minutes’ exploration would be delivered (see Fig. 2). FEAR-EXPRESSION TEST Cued fear test was conducted in a chamber with a different context (triangle, black). The bedding

stored hermetically was laid on the salver of the fear conditioning chamber. Testing was performed with the similar procedure as in the conditioning period but engaged 90 seconds’ intervals,

without US19. DATA ANALYSES AND STATISTICAL PROCEDURES Social preference measures were taken of the amount of time spent in each side of chamber20. Preference scores were calculated by

subtracting the time spent in FO from the time spent in UO for donor and odor+ groups, or by subtracting the time spent in left from the time spent in right for odor− group. The baselines in

the first session were calculated for all subjects. Freezing behaviors defined as the complete absence of any movement except for respiration and heartbeat lasting for longer than 2 second

were timed during the testing session. The percentage of freezing time on or off tone presentation was recorded as CS+ or CS−, respectively. Repeated measurement ANOVAs were employed in

analyzing preference scores with the between-subjects factors group (social group, odor+ group, and odor− group) and the within-subjects factors time (pre, post). The same statistical test

was used for freezing percentage with the between-subjects factors (social group, odor+ group, and odor− group) and the within-subjects factors cue (CS+, CS−). LSD Post hoc test was employed

for pairwise comparisons. _p_ < 0.05 was considered statistically significant. RESULTS SOCIAL NOVELTY PREFERENCE TEST We compared preference for the side with an unfamiliar conspecific

with baseline, and found a significant main effect of time (_F__1,13_ = 15.501, _p_ < 0.01,   = 0.544). Simple main effect analysis showed that there was a significant effect of time in

social group (_F__1,13_ = 7.383, _p_ < 0.05) and odor+ group (_F__1,13_ = 8.836, _p_ < 0.05), but not in odor− group (see Fig. 3). FEAR-EXPRESSION TEST We tested whether there are some

differences in freezing ratio between groups and conditions of cue (presence or absence). There was a significant main effect of cue (_F__1,14_ = 67.830, _p_ < 0.001,   = 0.829) and a

significant interaction of cue and groups (_F__2,14_ = 7.219, _p_ < 0.01,   = 0.508), although no significant effect of groups. Simple main effect analysis showed that there was a

significant effect of cue in social group (_F__1,14_ = 6.539, _p_ < 0.05), odor+ group (_F__1,13_ = 61.552, _p_ < 0.001), and odor− group (_F__1,14_ = 15.639, _p_ < 0.01). At CS+

level, there was a significant effect of groups (_F__2,14_ = 4.839, _p_ < 0.05). The LSD post hoc tests showed significant difference between social group and odor+ group (_p_ < 0.01),

and marginal significant difference between social group and odor− group (_p_ = 0.065). But at CS− level, there was no significant effect of groups (see Fig. 4). DISCUSSION ODOR EXPOSURE

IMPROVED THE FAMILIARITY WITH THE ODOR In the first session, all groups spent equal time in each side. But social group and odor+ group spent more time in the UO later. The preferences

indicated that UO was a novel social stimulus by comparison with FO, and drew mice’s attention to explore11,12,13,20,21. In other words, the FO became a familiar odor after 10-days odor

exposure, no matter whether physical interaction took place during exposure or not. Consistent with this, subjects in odor− group did not show the preference. ODOR EXPOSURE AND PHYSICAL

INTERACTION FACILITATED SOCIAL BUFFERING All subjects showed increased freezing percentage on tone presentation, which proved that tone cue induced fear responses effectively. More

importantly, at CS+ level, social group showed reduced freezing time than other groups. Because bedding used in test were collected from the same donor, the difference between groups should

be due to the different manipulations during odor exposure. Kiyokawa and his colleague9 found that a familiar conspecific is more effective than an unfamiliar conspecific for social

buffering, as we have seen in this experiment. They assumed housing with a donor induced the plasticity in the MOB, enabling the subjects to perceive familiar odor more effectively. But some

researches indicated that only odor exposure potentiated MOB response specificity22 but depressed response intensity23,24, which did not support the hypothesis. The present result that only

odor exposure did not induce a greater social buffering in odor+ group also contradicted this assumption. Olfaction is synthetic perception processing in which odors are initially encoded

as ‘objects’25, and linked with specific biological significance26,27. Natural odors are also associated with experience-dependent significance or value28,29, which means delicious food or,

in present study, a familiar conspecific. So an alternative hypothesis emerged that specific social value of a familiar conspecific was acquired by olfactory learning30, which a phenomenon

was similar with that showed in previous researches21,31,32. Both in the previous and present study, physical interaction played a required role for acquisition of social value. It was the

prerequisite for individual recognition that physical contact was allowed in hamsters11,12,13, which could be an explanation for results that social group showed lower freezing percentage

than other groups. Social group showed considerable but not significant decline of freezing time when compared to odor− group. The reason might be a time lag detected in the odor− group, in

which the max response sometimes appeared after the tone presentation. It should be noted that different from some researches5,8,9, only freezing behavior occurred during 30 seconds’ tone

presentation was recorded as response to cue. The other explanation for our finding is that bedding collected from cages of donors did not supplied odor as effective as donor itself.

However, a significant preference to UO in odor+ group was found; and something similar to that was found in social groups. These facts indicated that olfactory learning took place in two

groups equally. Besides, odor− group without donor odor exposure showed the same lever of freezing time as odor+ group. Moreover, fear test used bedding as odorants, which had been used as

odorants for exposure in odor+ group, so social group did not have additional advantage due to consistency of learning and test. To summarize, our result suggests that physical interaction

is required in social buffering induced by a familiar conspecific. ADDITIONAL INFORMATION HOW TO CITE THIS ARTICLE: Liu, H. and Yuan, T.-F. Physical Interaction Is Required in Social

Buffering Induced by a Familiar Conspecific. _Sci. Rep._ 6, 39788; doi: 10.1038/srep39788 (2016). PUBLISHER'S NOTE: Springer Nature remains neutral with regard to jurisdictional claims

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CAS  Google Scholar  Download references ACKNOWLEDGEMENTS We thank Professor Burton Slotnick on comments to this project. This study was supported by “Hundred Talents program” of Nanjing

Normal University, “Qing Lan Project” of Jiangsu Province, Jiangsu Provincial Natural Science Foundation (BK20140917), National Natural Science Foundation of China (81501164, 81611130224).

AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * School of psychology, Nanjing Normal University, Nanjing, 210097, China Hou Liu & Ti-Fei Yuan Authors * Hou Liu View author publications You

can also search for this author inPubMed Google Scholar * Ti-Fei Yuan View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS Conception and

design: H.L. and T.Y. Analysis and interpretation of data, drafting of the article: H.L. All authors read and approved the final version the manuscript. ETHICS DECLARATIONS COMPETING

INTERESTS The authors declare no competing financial interests. RIGHTS AND PERMISSIONS This work is licensed under a Creative Commons Attribution 4.0 International License. The images or

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http://creativecommons.org/licenses/by/4.0/ Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Liu, H., Yuan, TF. Physical Interaction Is Required in Social Buffering Induced by a

Familiar Conspecific. _Sci Rep_ 6, 39788 (2016). https://doi.org/10.1038/srep39788 Download citation * Received: 06 October 2016 * Accepted: 28 November 2016 * Published: 23 December 2016 *

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