Peer Rotation Affects The Anxiety, Social Behavior And Neuroendocrine Mechanism Of Male C57BL/6 Mice

In the whole life course of humans and animals,there are all kinds of complex social-bonds,mainly including parental-infant bond,pair bond and companion bond.Stable social-bonds play a very important role for human and animal health,and sociality development in the future.Thus,the study of social-bonds is of vital importance.However,there is often the phenomenon of disturbance of social-bonds in humans or social animals,and its effect on emotion,social behavior and mechanism is not very clear.Because of the characteristics of many uncontrollable and complex factors,the long growth period and ethical constraint in humans,problems concerning disturbance of social-bonds appear in society which are usually analyzed and studied by direct observation and induction.As a result,there are obvious advantages in experimental conditions such as controllability,operability which used the experimental animal as model to study social-bonds.Until now,a lot of researches have focused on paternal or maternal deprivation,spouse deprivation,social isolation,the formation of the parental-infant bond and pair bond,and so on.But the study of companion bond is short relatively.And the study of companion bond concentrates on the negative effects and neural mechanisms of partner deprivation,social isolation,the increase of comemsal animals,social crowded,etc.In the subadult period,the rodent brain is in the developmental stage and is particularly sensitive to stress.Therefore,the brain has highly plasticity in the aspects such as the level of gene and behavior.But for now,there is very little research on companion bond in the subadult period.Social cohabitation of animals can reverse the negative effect of stress on behavior,physiology,etc,and can exert a positive effect.If the number of companions living with the animal remains the same while the objects of social interaction have taken place change whether the expression of the animal’s emotion,cognition and social behavior changes accordingly?What is the neural mechanism involved in causing this change?These studies are rarely reported.Oxytocin(OT)is a neurotransmitter closely related to social-bonds.OT not only can increase animal’s perception of social cognition,social memory,pro-social behavior,but also can engage in emotional processing and have function of anti-anxiety.More oxytocin receptors(OTR)were expressed in the medial prefrontal cortex(mPFC),nucleus accumbens(NAcc),medial amygdala(MeA)brain area of the limbic system.These brain regions activate the brain reward mechanism by participating in social interactions,so as to promote further expression of positive social behavior.Close social interactions can activate the brain’s reward system,in turn,the reward system also can promote the expression of behavior.Dopamine(DA)is the core of the reward system,and participates in the regulation of emotion and social memory.Activation of D1-like receptor(D1R)is directly associated with motivation mediated by the reward system and plays a critical role in it.To sum up,OT and DA are closely related to social interaction.However,the companion rotation is an important model of unstable social-bond disturbance on the basis of social interaction,and it causes changes in animal behaviors that cannot leave the OT and DA system.But at present,there is little research done in this aspect.In the current study,conducted in male C57BL/6 mice,we replaced the objects of the animal’s social interaction twice a week and continued this for 7 weeks to build the model of companion rotation.Then we explored that the model’s impact on anxiety-like behavior,social behaviors.In addition,we detected the changes of density and the classification of dendritic spines in mPFC,NAcc,MeA and the changes of OT and DA.Our experimental results are as follows:1.Behavior testing(1)The level of anxiety in the male C57BL/6 mice was evaluated by using open-field test,light-dark box test and elevated and plus maze test.The results showed that the percentage of time in central area of mice after the companions rotated and the total moving distance in light box were increased significantly.The percentage of time and frequency in the open arm were obviously increased compared with the control group.The results suggested that companion rotation can reduce the anxiety level of mice.(2)The sociality in the male C57BL/6 mice was tested with three-chamber apparatus.The results indicated that the percentage of time spent exploring strange mice in both groups increased significantly compared to the empty boxes.The social index of mice in the rotation group showed an increasing trend.The results indicated that companion rotation improved the sociality of male mice.(3)The social preference in the male C57BL/6 mice was detected with three-chamber apparatus.Compared with familiar stimulus mice,the percentage of time of investigating unfamiliar stimulus mice markedly increased in both control and rotation groups.The social preference index of the rotation group had an increasing trend.This showed that in a certain extent companion rotation may enhance the social preference ability of male mice.(4)The social recognition in the male C57BL/6 mice was studied using habituation and dishabituation paradigm.We found that the investigation time of both groups had significant differences between familiar and unfamiliar stimulus mice.There was no significant difference in cognitive index between two groups.But overall,there was a trendency which the investigation time of the rotation group was higher than the control group.These implied that companion rotation may enhance the social recognition ability of male mice.(5)In the social interaction test,as an obvious result,the investigation and physical contact behaviors of the rotation group were significantly higher than the control group.In the aggressive and defensive behaviors including chase,attack,stare,escape etc,there was no significant difference between the two groups.The results showed that companion rotation can significantly increase the animal’s prosocial behavior,and had no obvious effect on the anti-social behavior.2.The studies of density and classification of dendritic spinesGolgi staining method was used to study the effects of companion rotation on the dendritic spines of neurons.Results showed that the total dendritic spines of mice after the companions rotated in mPFC,NAcc,MeA were noticeablely higher than the control group.In addition,the dendritic spine density of mushroom type of the rotation group was significantly increased in mPFC,NAcc,and that was extremely significantly increased in MeA.This indicated companion rotation can facilitate the fonnation of mature dendritic spines of the related brain regions in male C57BL/6 mice.3.The studies of OT and DA(1)The immunohistochemistry results found that the expression of OT in the rotation group at PVN and SON was significantly higher than the control group,and the expression of TH in the rotation group at VTA was significantly higher than that in the control group,suggesting companion rotation can significantly increase the expression of OT at PVN,SON and the expression of TH at VTA.(2)The real-time PCR results showed that compared with control group,the relative expression of OTR mRNA of the rotation group mice at mPFC could increase dramatically,the expression of OTR showed a clear upward trend at NAcc,and the expression of OTR of the rotation group at MeA was an increased.The relative expression of DIR mRNA of the rotation group mice at mPFC was increased at certain degree compared with the control group.At Nacc,the mRNA relative expression of D1R of the rotation group mice was significantly increased.These suggested companion rotation could improve the mRNA relative expression of OTR and DIR to varying degrees at mPFC,NAcc,MeA.(3)The Western Blot results found that,the protein expression of OTR in the rotation group mice was remarkablely higher than the control group at mPFC,Nacc.There was no significant difference about the protein expression of OTR in MeA.The protein expression of D1R of the rotation group mice at mPFC,Nacc had no significant difference.It is showed that companion rotation would significantly improve the protein expression of OTR but had no significant difference for the protein expression of D1R at mPFC,Nacc.And companion rotation had no significant effect on the protein expression of OTR at MeA.In conclusion,companion rotation of the subadult period could lower anxiety levels of adult C57BL/6 mice,in certain degree increase sociability and improve social preferences for strange individual as well as social recognition ability,and enhance the animal’s prosocial behavior in social interaction.And these behavioral changes most probably are strongly associated with increased density of dendritic spines of the relevant mouse brain,increased expression of OT and TH,improvement of expression level of corresponding mRNA and protein during the modeling.Therefore,companion rotation is likely to be as a enriched social environment which provides more opportunities of social interaction and emotional experience so that has a positive impact on their emotion and social behavior.At the same time,this effect may be mediated by increased density of dendritic spines of the relevant mouse brain and activity in the OT and TH system.