Involvement Of The Central Dopamine System In Regulating The Effects Of Long-term Social Isolation During Adolescence On Mood And Social Behavior In C57 Mice

Adverse events that humans and animals experience early in life(such as maternal and infant separation or social isolation)can generate long-lasting deleterious effects on brain development.The establishment and maintenance of social bonds is essential for the survival and reproduction of social species.Isolation rearing induces social isolation,is defined as the deprivation of social contact and interaction.Compared to other social stress paradigm,social isolation stress model has less physical injury and less intervention to human,which can better model the course of developmental mental illness under natural conditions.Adolescence is a period of development when plastic capacity is maximal,and is critical time for the maturation of the neural circuits that control stress responses of an organism.A large number of studies have shown that experiencing acute or chronic social isolation can lead to a range of psychiatric and neurological disorders characterized by anxiety,depression,schizophrenia,epilepsy,memory loss,and increase susceptibility to psychoactive drugs and so on.Teenagers with support and close peer relationships are more likely to get a higher level of self recognition ability,social ability,self-worth,school performance and more active and positive,but lack of normal interpersonal communication can make teenagers suffer all kinds of bad consequences,including decline of academic performance,anxiety,depression and loneliness for a long time,so.as to increase the risk of depression or schizophrenia and other mental illnesses.Social behavior contains multiple components,including social affiliation,interaction,and recognition/discrimination.However,few studies have focused on effect of chronic social isolation on social behaviors,and the underlying neurobiological mechanisms are still unclear.C57 mice is a classic model animals,no study has comprehensively evaluated the effects of chronic isolation during adolescence on its emotional and social behaviors.Researches show that the mesolimbic dopamine system is involved in stress,but its regulation in emotional and social behaviors after chronic social isolation is poorly understood.In this research,we investigated the effects of four weeks of chronic social isolation on emotional,sociality and social recognition in adolescent male C57 mice and focused on the important regulatory role of the mesencephalic limbic dopamine system in this process.The experiment included four parts,as follows:1.The effect of adolscent chronic social isolation stress on behaviorsThe effect of chionic social isolation stress(CSIS)on anxiety-like behavior and motility in mice were examined in the open field test and elevated plus maze test.Tail suspension test was used to detect the effect of CSIS on depression-like behavior in mice.The first phase of the three-chamber test and social interaction test were used to examine the effect of CSIS on sociality in mice.The second phase of the three-chamber test and 4+1 social recognition test were used to examin the effect of CSIS on social cognition in mice.The main results are as follows:1.1 Open field testThere was no significant difference in the percentage of central area time between the control mice and isolated mice,and the total movement distance and average speed of the isolated mice were significantly higher than that of the control mice.The results showed that CSIS had no effect on anxiety-like behavior but increased spontaneous movement in mice.1.2 Elevated plus maze testThe time spent in the open arms and time spent in the closed arms between control mice and isolated mice had on significant difference.The results showed that CSIS had no effect on anxiety-like behavior in mice.1.3 Tail suspension testComperated to control mice,isolated mice had longer immobility time.Thus,CSIS increased depression-like behaviors in mice.1.4 Three-chamber testIn the first phase of three-chamber test(sociality):in the control mice,the time spent exploring stranger 1 is extremely significant bigger than exploring empty cage.The isolated mice spent significant longer time exploring stranger 1 than exploring empty cage.In the second phase of three-chamber test(social recognition):control mice spent more time on exploring stranger 2 than stranger 1.However,there was no significant difference between the time exploring stranger 2 and stranger 1 in isolated mice.The results of the three-chamber test indicated that CSIS did not induce the sociality deficit,but impaired social recognition in mice.1.5 4+1 social recognition test The control mice showed normal social memory in the five trilas of exploration,while the isolated mice showed no significant difference in the length of exploration the stimulated mice in five trials of exploration.The results showed that CSIS impaired social cognition in mice.1.6 Social interaction testThe control mice spent significantly more time exploring the environment than the isolated mice,and about 40%of the isolated mice showed aggressive-like behaviors.The behaviors time of chasing and retreating stimulate mouse in isolated mice were significantly longer than that of the control mice.No aggressive-like behaviors appeared in the control mice.The results showed that CSIS increased aggression in mice.2.The effct of adolscent chronic social isolation stress on neuroendocrine parametersImmunofluorescence experiment was used to detecte the number of TH immunoreactive neurons(TH-ir)in the VTA and the DRN areas in mice after exposed to CSIS.Western Blot experiment was used to detect the levels of D1 receptor and D2 receptor in the VTA,mPFC,DRN,NAcc core and NAcc shell brain areas and the TH levels in VTA、DRN、NAcc core and NAcc shell brain areas in mice.Changes in serum corticosterone content after CSIS were detected by ELISA experiment.Golgi staining was used to detect changes in neuron morphology in mPFC and NAcc areas in mice after exposed to CSIS.The main results were as follows:2.1 ImmunofluorescenceThere were no significant differences on the number of TH-ir neurons in VTA and DRN areas between control and isolated mice.Indicated that CSIS had no effects on the number of TH-ir neurons in the VTA and DRN areas.2.2 Western BlotThere was no significant difference in D1R levels in VTA,mPFC,NAcc core,NAcc shell and DRN areas between control mice and isolated mice.Compared with control mice,D2R levels in mPFC area and D2R and TH levels in NAcc shell area decreased significantly in isolated mice.The results showed that CSIS induced D2R levels down-regulation in mPFC and D2R and TH levels down-regulation in NAcc shell in mice.2.3 Golgi stainingCompared with control mice,the number of secondary dendritic spines extremely significantly decreased in mPFC and NAcc brain region areas in isolated mice.The results showed that CSIS down-regulated the number of secondary dendritic spines in mPFC and NAcc brain regions in mice.2.4 ELISACompared with control mice,serum corticosterone content of isolated mice increased significantly.Indicated that chronic social isolation stress is a kind of strong stressor that could activate HPA axis in mice.3.Effects of D2R agonists and antagonists injections on behaviorsPharmacological experiment was used to examine the effects of infusion quinpirole(an agonist of D2 receptor with dose of 0,30,100,300 ng/200 nl)and raclopride(an antagonist of D2 receptor with dose of 0,100 ng/200 n1)into the shell of NAcc on emotional and social behaviors in mice.Three-chamber test:In isolated mice with 100 ng quinpirole infusion into the NAcc shell,the time spend in exploring stranger 1 was significantly longer than that of exploring empty cage,and the time spend in exploring strange 2 was significantly longer than exploring stranger 1.In control mice with 100 ng raclopride infusion into the NAcc shell,the time of exploring the stranger 1 was significantly longer than exploring empty cage,and the time spend in exploring stranger 1 was significantly longer than exploring stranger 2.The results showed that D2R agonist quinpirole microinjection significantly improved the social recognition of isolated mice,while D2R antagonist raclopride microinjection induced the social recognition deficit in control mice.Tail suspension test:In isolated mice with 300 ng quinpirole infusion into the NAcc shell,the immobile duration of tail suspension experiment decreased significantly.Injection of 100 ng quinpirole extremely significantly decreased the immobile duration of tail suspension experiment.In addition,after injecting 100 ng raclopride into NAcc shell of control mice,the immobile duration of tail suspension experiment was increased significantly.In conclusion,D2R agonist quinpirole microinjection decreased depression-like behavior in isolated mice.Besides,D2R antagonist raclopride microinjection induced depression-like behavior in control mice.Social interaction test:100 ng quinpirole infusion into NAcc shell in isolated mice stoped aggressive-like behaviors appear,significantly decreased the number of individuals with aggressive behaviors and increased the time of exploring environment.100 ng raclopride infusion into NAcc shell in control mice induced aggressive-like behaviors,significantly decreased the time of exploring environment.The results showed that D2R agonist quinpirole and D2R antagonist raclopride microinjection changed the social interaction behaviors in mice.4.Calcium imaging experimentCalcium imaging experiment was used to detect the changes of neuronal activity in NAcc shell in mice when exploring different stimulis.The main results are as follows:Both control and isolated mice showed extremely significant increase in NAcc shell neuronal activity when exploring the stranger 1 than exploring the empty cage.However,the NAcc shell neuronal activity was significant increased when exploring stranger 2 only in the control mice.The NAcc shell neuronal activity had no significant changes when exploring stranger 2 and strange 1 in isolated mice.In addition,when exploring the stranger 2,NAcc shell neuronal activity in control mice was significantly increased than isolated mice.The results showed that CSIS decreased the neuronal activity in NAcc shell area in mice when exploring unfamiliar social stimuli.In conclusion,four weeks of chronic social isolation during adolescence increased depression-like behavior,spontaneous movement and aggression,also impaired social recognition in mice.These behavior abnormalities are related to the down-regulation of D2R level in mPFC and down-regulation of D2R and TH levels in NAcc shell,the down-regulation of the number of secondary dendritic spines of neurons in mPFC and NAcc areas,and the increased serum corticosterone content.100 ng quinpirole infusion into the NAcc shell improved the social recognition deficits,decreased depression-like behavior extremely,significantly reduced the number of individuals with aggressive behaviors in isolated mice.Besides,100 ng raclopride infusion into the NAcc shell induced behavior abnormalities in control mice.Indicated that the NAcc shell DA system plays an important role in regulating effects of adolescent chronic social isolation stress on emotion and social behaviors in mice.In addition,our study also demonstrated that adolescent chronic social isolation stress did not change the levels of DIR,D2R and TH in DRN area,and reduced the neuronal activity in the NAcc shell when exploring unfamiliar social stimuli.