| Most vertebrates including human live in social group and form various social bonds.Recently,many studies have reported the effects of social bonds interruption,such as maternal separation,paternal deprivation or social isolation on emotion,behaviors and their neuroendocrine mechanisms.But,the effects of pair-bond interruption on emotion,behaviors and its neuroendocrine mechanisms remain unknown.In adult,pair-bond is one of the most important social bonds.It facilitates the positive social interactions and in turn affects the evolution of brain and behaviors.Positive pair-bond can buffer the negative effects induced by other stress,thus decreasing the risk of suffering from mental diseases.In humans,disharmonious pair-bond or single parent family can affect the mental health and behaviors of offspring.Thus,studying the behavioral effects of pair-bond is crucial to effectively prevent and intervene in emotional disorder and behavior disorders.Besides,research on relevant neuroendocrine mechanisms can provide reference for clinic treatment.Although study has showed that in male prairie vole,pair-bond formation is one of the factors making them keep intact social recognition and pair-bond interruption can increase the anxiety or depression level,whether the pair-bond interruption could affect social recognition,sociability,and social preference or not remains unclear.Most traditional experimental animals(mice or rats)are polygamous animals without complex social behaviors.But,mandarin voles(Microtus mandarinus)can form complex social structure,have higher level of sociability and they are monogamy.It proves a good animal model for us to study the behavioral effects of pair-bond interruption and its neuroendocrine mechanisms.Our study is the first time to use the mandarin voles as animal model to study the effects of pair-bond interruption on levels of anxiety and social behaviors.First,we ensured the timewindow pair-bond formation of male mandarin voles using partner preference test.After pair-bond formation,experimental animals were randomly allocated into non-interruption group and interruption group.In order to ensure the onset time of its behavioral effects,our study designed time gradient of pair-bond interruption(respectively interruption for one week or two weeks).Behavioral methods(open field test(OFT),light-dark box(LDB),social recognition,sociability,social preference and social interaction)were conducted to study the effects of pair-bond on levels of anxiety and social behaviors.After behavioral effects were determined,the immunohistochemistry was used to study the expressions of arginine vasopressin(AVP)in the paraventricular nucleus of hypothalamus(PVN)and tyrosine hydroxylase(TH)in the ventral tegmental area(VTA).Real-time PCR was used to study the expression of vasopressin-1a receptor(V1aR)mRNA in the lateral septum(LS)and D1-like receptor(D1R)mRNA in the nucleus accumbens(NAcc).Main results for this study are as follows:1.The timewindow of pair-bond formationThe timewindow of pair-bond formation was ensured by partner preference test.The result showed that compared with paired for one day or three days,male mandarin voles exhibited higher level of side-by-side behavior toward the partners and higher level of attack behavior toward the strange females after paired for five days.The result suggested that the pair-bond could be formed after paired for five days in male mandarin voles.2.The change rate of body weightWhen the pair-bond interruption began,the body weight of experimental animals was collected for 14 straight days and the change rate of body weight was calculated.About the change rate of body weight,the change trend of both groups was similar and the significant difference between two groups was not found.The result suggested that pair-bond interruption could not affect the change rate of body weight.3.Behavior tests3.1.Anxiety-like behaviors testThe OFT and LDB were conducted to evaluate anxiety-like behavior.Result showed that after pair-bond interruption for one week,there was no significant difference between the total distance of both groups in OFT.But the percentage of time in central area of the interruption group was significantly lower than that of non-interruption group.Besides,pair-bond interruption for one week,the distance in the light box of interruption group was significantly lower than that of non-interruption group,but there was no significant difference between the percentage of time spent in the light box of both groups.After pair-bond interruption for two weeks,there was no significant difference between the total distance of both groups in OFT.But the percentage of time in central area of the interruption group was significantly lower than that of non-interruption group.Besides,after pair-bond interruption for two weeks,the distance in the light box of interruption group was significantly lower than that of non-interruption group,and there was significant difference between the percentage of time spent in the light box of both groups.The result suggested that compared with pair-bond interruption for one week,pair-bond interruption for two weeks could significantly increase the anxiety level in male mandarin voles.And the results of the both behavioral tests were consistent with each other.3.2.Social recognition testThe social recognition was tested by habituation and dishabituation paradigm.Regardless of pair-bond interruption for one week or two weeks,the investigation time of non-interruption group in dishabituation phase was significantly higher than it in habituation phase and the social recognition score was significant higher than zero.But there was no significant difference between the investigation time of interruption group in habituation phase and dishabituation phase.And there was no significant difference between the social recognition score and zero.Besides,there was a significant difference between the social recognition score of both groups.The result suggested that non-interruption group exhibited intact social recognition toward the same-sex juvenile conspecifics.But social recognition of interruption group was impaired after pair-bond interruption for one week or two weeks.Whether the pair-bond was interrupted for one week or two weeks,there was no significant difference between the fatigued score and zero in non-interruption group and interruption group.It suggested that both groups were not fatigue in this test.3.3.Sociability testThe three-chamber apparatus was used to test the sociability.Regardless of pair-bond interruption for one week or two weeks,the time spent in investigating the same-sex juvenile stimulus voles was significant higher than the time spent in investigating the empty box.The result showed that pair-bond interruption could not affect the sociability of male mandarin voles.3.4.Social preference testRegardless of pair-bond interruption for one week or two weeks,non-interruption group exhibited a significant preference to novel same-sex juvenile stimulus voles in the social preference test,but interruption group did not show this preference.The result showed that pair-bond interruption could affect social preference.3.5.Social interaction testIn the social interaction test,after pair-bond interruption for one week,the duration of attack behavior,exploration behavior and self-grooming behavior in interruption group were significantly lower than non-interruption group.But the duration of defence behavior,obedience behavior and escape behavior in interruption group were significantly higher than non-interruption group.The effects of pair-bond interruption for two weeks on the above behaviors were similar to the pair-bond interruption for one week.The result suggested that pair-bond interruption for one week or two weeks could significantly affect social interaction behaviors of male mandarin voles4.ImmunohistochemistryAfter pair-bond interruption for two weeks,the expression of AVP in the PVN and TH in the VTA were evaluated by immunohistochemistry.Compared with non-interruption group,AVP-ir neurons in the PVN of interruption group significantly increased.The TH-ir neurons in the VTA between both groups had no significant difference,but compared with interruption group,the number of TH-ir neurons in the VTA of non-interruption group exhibited increasing trend.Result showed that pair-bond interruption increased the expression of AVP in the PVN,and down-regulated the expression of TH in the VTA to a certain extent.5.Real-time PCRThe expression of V1aR mRNA in the LS and D1R mRNA in the NAcc were evaluated by Real-time PCR.The V1aR mRNA relative expression level in the LS of interruption group was significantly lower than that of non-interruption group.The D1R mRNA relative expression level in the NAcc of both groups has no significant difference.But,compared with interruption group,D1R mRNA relative expression level in the NAcc of interruption group exhibited decreased trend.The result suggested that pair-bond interruption could decrease the expression of V1aR mRNA in the LS,and down-regulate the D1R mRNA relative expression level in the NAcc to a certain extent.In conclusion,the change rate of body weight and sociability were not affected by pair-bond interruption.But pair-bond interruption could increase anxiety-like behavior,impair social recognition,decrease the social preference to strange same-sex conspecifics and decrease the aggressivity in the social interaction.The expression of AVP in the PVN increased,whereas the V1aR mRNA relative expression level decreased after pair-bond interruption.The expression of TH in the VTA and the DIR mRNA relative expression level in the NAcc were down-regulated by pair-bond interruption to a certain extent.The result suggested that the pair-bond interruption could increase the anxiety level,impair social recognition and induce various behavioral changes in the social interaction.The activity of AVP system in brain may play a certain role in the process of regulating the behavioral changes. |
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