| Most mammals live in social organizations,forming complex social structures and social-bonds including sibling-bond,parent-bond and pair-bond.Long-term and stable social relationships are very important for growth and development.Interruption of social-bonds could affect the development of individual and the performance over the long term.At present,research on social-bonds has mainly focused on the formation of parental relationship,maternal separation,paternal deprivation,early social deprivation,and the pair-bond formation.Survey shows that spousal death is an important factor in the development of mental disorders such as anxiety and depression,pair-bond interruption threatens physiological and health to the body.However,a stable pair-bond is crucial to the individual's survival and social stability.At present,research on the unstable pair-bond is mainly focused on the impact of pair-bond interruption on emotions.So whether the pair-bond interruption could affect social recognition,sociality,social preference and its related neural mechanisms is poorly understood.In adult female prairie voles,the pharmacological activity of oxytocin receptor(OTR)in nucleus accumbens(NAcc)promotes the formation of partner preference,whereas occlusion of OTR impairs partner preference behavior resulting from mating or OT injection.The female prairie voles oxytocin(OT)in medial prefrontal cortex(mPFC)is also involved in the regulation of pair-bond.In addition,OT has an anxiolytic effect,plays an important role in regulating prosocial behavior and OT at mouse medial amygdala(MeA)is necessary for social recognition.The(Dopamine,DA)at NAcc regulates the pair-bond in a receptor-specific manner,D1-like receptor(D1R)participates in the maintenance of the pair-bond,and D2-like receptor(D2R)participates in the formation of the pair-bond.In addition,the DA system is also involved in the regulation of anxiety/depression and social memory.The D1R receptor at NAcc can enhance the memory of rewards,aversion,and warning events.The D1R receptor antagonist at NAcc impairs conditional taste aversion memory.In summary,the OT and DA neurotransmitters associated with the pair-bond are also involved in the regulation of animal emotions and social behavior.So,the OT and DA systems may be used as a neuroendocrine mechanism to explain how the pair-bond interruption affects emotions and social behavior.Therefore,we chose OT,DA and their receptors for experimental studies.The mandarin vole is a highly social,monogamy rodent.In the adult mandarin voles,pair-bond is one of the most important relationship.Therefore,we choose female mandarin voles as the animal model to study the effect of pair-bond interruption on emotion,behavior and its neural mechanism.Firstly,investigate the estrous cycle of female mandarin voles.The female were paired in the estrus.Then ensuring the time-window of pair-bond formation.After pair-bond was formed,they were randomly divided into control,sibling,and interruption group.In order to determine the onset time of behavioral effects after the pair-bond interruption,test voles were given either one week or two weeks pair-bond interruption.The open field test,light and dark box test,social recognition test,sociality,social preferences,social interaction,tail suspension test and forced swimming test were used to detect the effect of pair-bond interruption on emotion and social behaviors in female mandarin voles one by one.After the behavioral tests,the Golgi staining was used to investigate the changes of dendritic spines density in mPFC,NAcc and MeA.The expression of OT in paraventricular nucleus(PVN)and supraoptic nucleus(SON),tyrosine hydroxylase(TH)in ventral tegmental area(VTA)were investigated by using Immunohistochemistry.Real-time PCR was used to detect the mRNA expression of OTR in the mPFC,NAcc,MeA and the expression of DIR mRNA in NAcc.The results are as follows:1.Estrus cycle explorationVaginal smear method was used to explore the estrus cycle of female mandarin voles.The results show that the female mandarin voles could enter estrous period after exposing them at least two days to soiled bedding and nesting material from an unfamiliar male.So before pairing,giving the females soiled bedding and nesting material of male,paired in estrous period.2.The time-window of pair-bond formationUsing the partner preferences test to ensure the time-window of pair-bond formation.The results show that after female mandarin voles and males paired for 5 days,the females significantly attacked the strangers,the time of side-by-side with the familiar males were significantly higher than that with the strangers,but paired 3 days without this phenomenon.So female mandarin voles paired with males for 5 days could form a pair-bond.3.The change rate of body weightDuring the pair-bond interruption period,the voles body weight was measured once a day.The results showed that the rate of body weight change in the sibling group was significantly increased after one week pair-bond interruption.Although there was no significant difference in weight change rate between the interruption group and the control group,but there is an upward trend.There was no significant difference in body weight changes between the three groups after two weeks of pair-bond interruption.The results suggested that the pair-bond interruption of one-week may lead to a certain increase in the rate of body weight for female mandarin voles,this effect disappeared in two weeks.4.Behavior testing4.1.Anxiety-like behavior testAn open field and light and dark box test were used to test the level of anxiety.The results of the open field showed that after the pair-bond was interrupted for one week,the total moving distance of the sibling group was significantly lower than the control group,indicated that pair-bond interruption could reduce the motility of the female mandarin voles in the sibling group.In addition,the frequency and percentage of time did not differ significantly,indicated that pair-bond interruption did not induce anxiety in female mandarin voles.There was no significant difference in the frequency and total distance traveled between the three groups in the central region after two weeks of interruption,but compared with the control group,the percentage of time in the central region of the disruption group was significantly higher than that of the control group.The results of the light and dark box experiments showed that there was no significant difference in the frequency of target voles entering the light box or the total moving distance and the percentage of time in the bright box.Therefore,female mandarin voles of the sibling group could reduce the motility after one week pair-bond interruption and reduced anxiety-like behavioral expression of the interrupted group after two weeks pair-bond interruption.4.2.Depression-like behavior testWe used tail suspension and forced swimming test to detect depression levels,pair-bond interrupted one week or two weeks after swimming and forced swimming time percentage were not significantly different between the three groups,indicating that interruption of the pair-bond could not induce female mandarin voles depression-like behavior.4.3.Social recognition testUsing habituation and dishabituation paradigm studied the social recognition.The results showed that social recognition index in the control group was significantly greater than 0 after the pair-bond interruption for one week and two weeks,while there was no significant difference between the social recognition index in the sibling group and the interruption group compared with 0.Fatigue index results showed that the control,sibling,interruption group fatigue index has no significant difference compared with 0.The results suggest that the control group had complete social recognition ability,while the sibling group and the interruption group did not show complete social recognition ability.4.4.Sociability testStudying on the sociality of female mandarin voles with three-chamber apparatus.After one week and two weeks of pair-bong interruption,the control,sibling,and interruption groups all significantly tended to investigate the same-sex juvenile stimulus voles as compared to the empty boxes.The results suggested that pair-bond interruption does not affect the sociality of female mandarin voles.4.5.Social preference testAfter one week or two weeks of the pair-bong interruption,the control,sibling and interruption groups tended to investigating unfamiliar juvenile stimulus voles.The results suggested that female mandarin voles in three groups had normal social preference ability.4.6.Social interaction testIn the social interaction test,after one week pair-bond interruption,aggression and retreat behavior in interruption group was significantly increased,the sibling group exploration time was significantly increased.Two weeks later,compared with the control group,investigating behavior in sibling group was significantly reduced,staring behavior increased significantly.Compared with the control group,the retreat behavior of the interruption group increased.These results suggested that after pair-bond interruption of one week or two weeks could increase antisocial behavior of femalemandarin voles.Besides,two weeks of pair-bond interruption could reduce the prosocial behavior of the sibling group.5.Study of the neural structural plasticity(Golgi-Cox stained)There was no significant difference in the densities of all dendritic spines between mPFC after one week pair-bond interruption,but the densities of stabby and mushroom dendritic spines of the control group were significantly higher than other two groups.The densities of all dendritic spines in MeA were not significantly different among the three groups,but the densities of mushroom dendritic spines in the control group were significantly higher than other two groups.The density of filopodia in control group was significantly higher than that in the sibling group.The densities of all dendritic spines at NAcc did not differ significantly among the three groups,but the densities of the mushroom dendritic spines in the control group were significantly higher than other two groups.Therefore,the interruption of pair-bond could reduce the mature dendritic spine density of female mandarin voles.6.Study of theneurological function plasticity6.1.immunohistochemistryAfter one week of pair-bong interruption,the expression of OT in the interruption group at PVN was significantly higher than that in the control group and the sibling group.The expression of OT in the interrupted group was significantly higher than that of the sibling group at SON.There was no significant difference between the three groups in the expression of TH at VTA.The results suggested that the pair-bond interruption could significantly increase the expression of OT at PVN,and had no significant effect on the expression of TH at VTA.6.2.Real-time PCROne week after the pair-bond interruption,the mRNA relative expression of OTR at mPFC group was significantly higher than that in the control group and interruption group.The expression of the sibling group and the interruption group at MeA were significantly higher than those in control group.The expression of the sibling group and the interruption group at NAcc significantly lower than the control group.The mRNA expression of DIR at NAcc was significantly lower in the sibling group and the interruption group than in the control group.The results suggested that the pair-bond interruption could significantly increase the relative mRNA expression of OTR at MeA and decrease the mRNA expression of OTR and D1R at NAcc.To sum up,pair-bond interruption had little effect on female mandarin voles,mainly affecting social behavior,especially social recognition and social interaction.Pair-bond interruption could damage female mandarin voles social recognition,increase antisocial behavior and reduce the prosocial behavior.The above behavioral changes may be closely related to the decrease in the density of mature dendritic spines and the down-regulation of mRNA expression in animal-related brain regions during model establishment. |
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