Study On The Mechanism Of Maternal Separation With Early Weaning To Increase Alcohol Dependence In Rats

Background and objectiveIn the early stage of mammal life,the mother gives the cubs nutrition,heat,sensory stimulation and other aspects of care,so as to maintain a close relationship between mother and infant.If the mother is separated from the cub at this time,it will bring a series of physiological and psychological changes to the cub,and this change can last until adulthood.At present,there are many evidences that mother separation can lead to increased alcohol consumption in adult rats,but the mechanism is still unclear.Recently,the analysis of RNA SEQ data in the prefrontal cortex of rhesus monkeys showed that the genes in the alcohol addiction related pathway and the genes in the myelination related pathway were highly overlapped.It has been reported that the myelin sheath of prefrontal cortex of weaned rats at the early stage of maternal infant separation has pathological changes and the expression of myelin basic protein is decreased.The damage of myelin sheath in white matter is similar to that in many addicts.The relationship between the decrease of white matter and the increase of addiction vulnerability suggests that myelin damage may promote alcohol addiction.Therefore,we suggest that the neural mechanism of maternal separation enhancing alcohol dependence in SD rats may be due to the injury of myelin sheath in prefrontal cortex.In order to prove this hypothesis,this study took SD rats as the research object,and intended to detect the alcohol dependence of SD rats weaned at the early stage of maternal separation by two experimental methods of alcohol conditioned place preference and voluntary consumption of alcohol.The expression of myelin basic protein in prefrontal cortex was detected by immunofluorescence method.Then,the rats were treated with valproic acid and lysophosphatidylcholine to simulate the damage of prefrontal cortex caused by early weaning after mother infant separation,and to explore the relationship between myelin sheath and alcohol dependence.Methods:1.Experimental animals: 45 pregnant SD rats of 2 weeks gestation and 30 male SD rats of 6 weeks gestation were purchased from Hunan Shrek Jingda experimental company.2.Experimental groups:(1)normal feeding group(HC group);(2)In the early weaning group(MSEW group),6 newborn rats,6 female rats and 6 male rats were selected respectively to ensure that each female rat took care of 12 newborn rats.Mother infant separation was started from the third day of birth(PD2)and continued until the 18 th day(pd17).During the isolation period,each suckling rat was separately placed in a box paved with paper towel to ensure complete isolation between each suckling rat.Pd2-pd5,the temperature of incubator was controlled at 34 ℃,and the isolation time of suckling mice was 4 hours;Pd6-pd9,the temperature of incubator was controlled at 32 ℃,and the isolation time of suckling mice was 6 hours;PD10days to pd13 days,the temperature is controlled at 30 ℃,and the isolation time is 6hours;Pd14-pd17 days,the temperature was controlled at 28 ℃ and the isolation time was 6 hours;Pd17 started weaning.);(3)VPA group(VPA group,300 mg / kg of valproic acid,an inhibitor of histone acetylase,was injected intraperitoneally from the third day to the 21 st day of birth);(4)In SN group,normal saline was injected intraperitoneally every day from the 3rd day to the 21 st day;(5)In LPC group,7-week-old male SD rats were injected with 1% lysophosphatidylcholine in the medial prefrontal cortex;(6)Artificial cerebrospinal fluid group(ACSF group,7-week-old male SD rats were injected with artificial cerebrospinal fluid in medial prefrontal cortex).3.Behavioral experiments: open field experiment,elevated cross maze,alcohol conditioned place preference,voluntary consumption of alcohol.4.Expression of myelin basic protein in medial prefrontal cortex: before behavioral test,2 rats in each group were randomly selected for immunofluorescence test.5.Data processing and analysis: t-test or two-way ANOVA was used to compare the two groups of experimental data.Results1.The anxiety like behavior of rats weaned at the early stage of mother infant separation decreased both in adolescence and adulthood.2.Rats weaned at the early stage of mother infant separation were more dependent on alcohol in adolescence and adulthood.3.After systemic injection of valproate,an inhibitor of histone acetylase,the expression of myelin basic protein in medial prefrontal cortex decreased after blocking Wnt signaling pathway.4.The alcohol dependence of rats was enhanced after treatment with valproic acid,a histone acetylase inhibitor.5.The damage of myelin sheath in medial prefrontal cortex can lead to the decrease of anxiety like behavior and the increase of alcohol consumption in weaned rats at the early stage of maternal infant separation.6.Rats with medial prefrontal cortex myelin sheath injury showed stronger "preload" effect in intermittent alcohol consumption.ConclusionWe found that the alcohol dependence of SD rats weaned at the early stage of mother infant separation was stronger than that of the control group,and it is the first time to prove that the injury of myelin sheath in medial prefrontal cortex may be one of the neural mechanisms of the alcohol dependence enhancement of SD rats weaned at the early stage of mother infant separation,Moreover,the damage of myelin sheath and the enhancement of alcohol dependence may be related to the notch and Wnt signaling pathway mediated by histone acetylase.This new discovery can provide a new perspective to explore the treatment of alcohol addiction in the future.