Mechanisms Of The Central Oxytocin System Regulating The Behavioral Effects Induced By Mate Bond Interruption In Male Brown Vole

The stable and positive social bonds,such as parent-infant bonds,sibling bonds and pair bonds are vitally important to the growth and development of human,and have buffering effects on anxiety,depression,stress and drug abuse.In addition,it can also reduce the prevalence of cardiovascular disease,asthma and infectious diseases,but,the failure to form and maintain these positive social bonds can produce these various mental disorders.Studies on social isolation had shown that unstable social bonds had long-lasting effects on both individual development and behavior.Pair bond is one of the most important social bonds after sexual maturity of humans and monogamous animals,which can influence the psychology,physiology and behavior of humans and animals.Because only about 5 percent of mammals are monogamous,the further studies on pair bond formation,maintenance,and effects of pair bond interruption are difficult because of limited animal models.At present,the neural mechanisms underlying effects of pair bond interruption on behaviors are poorly understood.A few studies had focused on the effects of pair bond interferences(such as pair bond interruption)on emotions,but the effects of pair bond interruption on stereotyped behaviors,social recognition,sociality,social preference,and social interaction are not well understood,besides,the neuroendocrine mechanisms that regulate these behavior changes remains elusive.Oxytocin(OT)is a major regulator of anxiety and depression.Studies had shown that OT reduced anxiety-like behavior in female prairie voles,mice,and rats.Many brain regions are involved in regulating the antianxiety effects of OT,such as injecting OT into the medial prefrontal cortex(mPFC),which reduced anxiety levels in rats.OT can also reduce anxiety-like behavior in animals by reducing activity in the medial amygdala(MeA).In addition,OT also plays an important role in regulating social behavior.For example,injecting OT into the intracerebroventricular reversed the social avoidance behavior in socially defeated male mice.The injection of OT into the nucleus accumbens(NAcc)of prairie vole promoted social preferences and social investigation behavior.To sum up,the OT system plays an important role in regulating emotions and social behaviors.Whether the OT system is involved in behavioral effects of pair bond interruption remains unclear.Traditional laboratory animals,such as rats and mice,are polygamous without complex social behaviors.The study used monogamous mandarin voles(Microtus mandarinus)work as a subject,besides monogamy,the high sociality also made it to be an ideal animal model in this research.In the present study,after five days cohabitation,socially monogamous male mandarin voles were randomly divided into three groups:control,sibling and interruption.Two weeks after modeling,following behavioral tests were conducted in turn:open flied test(OFT),light-dark box test(LDB),marble burying test(MBT),social recognition test,sociability test,social preference test,social interaction test,tail suspension test(TST),forced swimming test(FST).After the behavioral tests,golgi staining was used to detect the density changes of dendritic spine in the mPFC,NAcc Shell,MeA,CA1,CA3 and dentate gyrus(DG).The immunofluorescence was used to explore the expression of OT and of c-FOS immunopositive neurons in paraventricular nucleus(PVN)and supraoptic nucleus(SON),as well as OT fiber projection density in mPFC,NAcc Shell and MeA.The mRNA relative expression of oxytocin receptor(OTR)in mPFC,NAcc Shell and MeA were examined by real-time PCR.Western Blot was used to measure the OTR protein level in mPFC,NAcc Shell and MeA.Finally,the important role of the central OT system in regulating the behavioral effects induced by pair bond interruption was verified by the pharmacological method via injection of exogenous OT or OT receptor antagonist(OTR-A)into NAcc Shell.The experimental results are shown as follows:1.Effects of pair bond interruption on behaviors in male mandarin voles1.1 Effects on emotionUsing OFT and LDB to detect anxiety-like behaviors in animals.The results showed that the percentage of time in central area and light box of the males in sibling and interruption groups was significantly decreased compared with control group.These results revealed that pair bond interruption can significantly increased the anxiety level of male mandarin voles.Using TST and FST to evaluate depression-like behaviors in animals.The results displayed that the duration of immobility in interruption and sibling groups was significantly higher than control group in TST.In FST,the duration of immobility in interruption group was significantly increased compared to control and sibling groups.These results suggested that pair bond interruption can increase the depression levels in male mandarin voles.1.2 Effects on stereotypical behaviorUsing MBT to explore stereotypical behavior in animals.The results indicated that the number of buried marbles in sibling and interruption groups was significantly increased.These results manifested that pair bond interruption can enhance the stereotyped behavior of male mandarin voles.1.3 Effects on social behaviorsUsing habituation/dishabituation paradigm to study the social recognition ability in animals.The results demonstrated that the investigation time in the fourth stage of(P4)control group was significantly lower than that in the first stage(P1),and the investigation time in the fifth stage(P5)was significantly higher than that in the fourth stage(P4).In addition,the recognition index in control group was significantly higher than sibling and interruption groups,and has significant difference when compared with zero.These results implied that pair bond interruption can impair social recognition in male mandarin voles.Using three-chamber device to research sociality and social preference in animals.The results showed that in the first stage of three-chamber,the time spent in exploring strange vole in three groups was obviously more than empty cages.In addition,the social index of the interruption group was significantly lower than that of control group,and the social index of sibling group also showed a downward trend.These results revealed that pair bond interruption impaired the sociality of male mandarin voles to some extent.In the second stage of three-chamber,the males in control group spent more time exploring the strange vole than the familiar vole,in addition,the social preference index of control group was significantly higher than that of sibling and interruption groups.These results suggested that pair bond interruption can inhibit the social preference of male mandarin voles in response to strange stimuli vole.Using social interaction test to assess various social behaviors in animals.The results indicated that compared with control group,the duration of attack behaviors in sibling and interruption groups were decreased,while the duration of defense and escape behaviors were significantly increased.In addition,the investigation behavior,self-grooming behavior and exploration behavior of interruption group were significantly lower than control group,but showed high obedience behavior.These results manifested that the pair bond interruption can suppress the expression of prosocial behaviors and promote the expression of antisocial behaviors in male mandarin voles to a certain extent.In behavior tests results,we concluded that pair bond interruption could elevate the levels of anxiety,depression,increase the stereotyped behavior,impair the social recognitive ability,meanwhile,reduce the sociability and social preference,and change the variety of behaviors in social interaction,at the same time,these effects were mainly caused by pair bond interruption,rather than isolation.Therefore,in subsequent mechanism studies,we grouped the animals to control and interruption groups,but excluded sibling group.2.Effeects of pair bond interruption on the density of dendritic spinesUsing golgi staining test to detect the density of dendritic spines in related brain regions.Compared with control group,there was no significant difference in the total dendritic spine density in interruption group at mPFC,NAcc Shell,MeA,CA1,CA3,and DG,but the density of mushroom dendritic spine decreased significantly.The results demonstrated that pair bond interruption could inhibit the formation of mature dendritic spines in related brain regions of male mandarin voles.3.Effects of pair bond interruption on OT-ir neurons and OT-ir nerve fibersUsing immunofluorescence test to examine the expression of OT neurons and OT fibers in related brain regions.The results showed that there was no significant difference in the expression of OT neurons in PVN between two groups,but the expression of c-FOS and the OT neurons expressing c-FOS in interruption group were significantly higher than those in control group.In addition,compared with control group,the OT fiber projection to NAcc Shell was significantly lower in interruption group.The results implied that pair bond interruption significantly increased the expression of active OT neurons at PVN and down-regulated the projection of OT fibers at NAcc Shell in male mandarin voles.4.Effects of pair bond interruption on the expression of OTRUsing real-time PCR test to research the expression of OTR mRNA in related brain regions.The results suggested that,compared with control group,the OTR mRNA in mPFC of interruption group decreased significantly,while the OTR mRNA in NAcc Shell and MeA showed no significant difference between two groups.These results revealed that the pair bond interruption could significantly decreased the expression of OTR mRNA in mPFC of male mandarin voles.Using Western blot test to explore the expression of OTR protein in related brain regions.The results displayed that the OTR protein expression in NAcc Shell of interruption group was significantly.lower than that in control group,while the OTR protein content in mPFC and MeA showed no significant difference from that in control group.These results demonstrated that pair bond interruption can significantly reduce the OTR protein in NAcc Shell of male mandarin voles.5.Effects of exogenous OT/OTR-A on animal behaviorsDifferent doses of OTR-A were given to control group and different doses of OT were given to interruption group.The results displayed that the behavior of control group was consistent with that of interruption group after injection to two doses of OTR-A.The behavior of interruption group was consistent with that of control group after injection to high dose of OT.Subsequently,the interruption group was injected with low dose OTR-A and high dose OT,its behavioral expression was consistent with interruption group.The above results indicated that NAcc shell injection of 10ng OTR-A or 100ng OTR-A can simulate the effects of pair bond interruption,while injection of 10ng OT can reverse the behavioral effects caused by pair bond interruption.Subsequently,the interruption group was injected with lOng OTR-A and 10ng OT,the behavioral expression did not change,suggesting that in the NAcc shell,OT combined with OTR to regulate the behavioral effects caused by pair bond interruption.In conclusion,the pair bond interruption in male mandarin voles could significantly improve the anxiety and depression levels,increase the expression of stereotypical behaviors,damage the social recognitive ability,decrease the sociability,inhibit the social preference for strangers,and change the expression of multiple behaviors in social interaction.These behavioral changes were mainly caused by the absence of a mate,not by the effect of isolation.These behavioral changes were associated with decreased density of mature dendritic spines in related brain regions,increased number of activated OT neurons,decreased OT fiber projection,OTR mRNA expression,and OTR protein.In addition,this study also found that give central exogenous OTR-A can induce behavioral effects which were similar with pair bond interruption,with same way given agonist can reverse the behavioral effects induced by pair bond interruption,OT is mainly combined with OTR to exert its regulating function of behavioral changes induced by pair bond interruption.