| Background and objectiveBinge drinking in adolescence predicts increased risk of lifetime alcohol use disorder(AUD)and a vulnerability to cognition disorders in adulthood,while the neural substrates underlying this relationship remain poorly understood.In China,the rate of adolescent alcoholism is 20-40%,and it is increasing year by year.In clinical practice,the treatment of neuropsychiatric disorders caused by alcoholism and related psychological education are often ignored,and there is a lack of scientific research and perfect laws and regulations.The adolescent brain is extremely plastic,especially the delayed prefrontal cortex(PFC),which is related to decision-making,control,impulsivity,learning and memory.Alcohol abuse during adolescence causes atrophy of gray matter in the mPFC and alters synaptic remodeling,leading to long-term cognitive impairment in adulthood.Synaptic plasticity is the basis of learning and memory,and Brain-derived neurotrophic factor(BDNF)is highly expressed in mPFC,which is of great significance in the survival and development of neurons,neurite outgrowth,and synaptic plasticity.The expression of BDNF is partly regulated by transcription factor cyclic adenosine monophosphate(c AMP)response element binding protein(CREB),and extracellular signal-regulated kinase(ERK)is a key regulator of effects of alcohol on CREB.As a phosphatase that can dephosphorylate tyrosine and serine/threonine residues,dual-specific phosphatase 6(DUSP6)can specifically bind to and inactivate classic ERK1/2.Does DUSP6 regulate the ERK-CREB-BDNF pathway,thereby affecting adolescent alcohol exposure-induced behavioral impairment in adulthood? This study intends to establish an adolescent intermittent alcohol(AIA)exposure mice model,and to detect the changes of emotion and cognitive function in adult mice after alcohol withdrawal.Then,the effects of AIA on the expression of DUSP6,p-ERK/ERK,p-CREB/CREB and BDNF in mPFC in adult mice was detected.Furthermore,r AAV vector was used to interfere with the expression of DUSP6 in mPFC to explore the molecular mechanism and structural basis of long-term cognitive impairment induced by adolescent alcohol exposure,and to provide experimental and theoretical basis for the treatment of alcohol-related diseases.Methods1.Male C57BL/6J mice from 28 to 55 days old were divided into AIA group and drinking water control group to establish the model of chronic intermittent alcohol exposure during puberty.The body weight,water consumption,alcohol intake and blood alcohol concentrations of mice were detected.2.Alcohol was removed when mice were adults.From 84 days after birth,anxiety-like behavior,short-term recognition memory and spatial working memory were detected by using elevated plus maze(EPM),novel object recognition(NOR)test and three chamber sociability test(3CST),respectively.The above experiments were designed to explore effects of AIA on emotional and cognitive abilities in adult mice.3.Immunofluorescence(IF)was used to detect the microtubule association protein-2(MAP2)and the co-expression of DUSP6 and neuron marker Neu N.Western blot was conducted to detect the protein expression of p-ERK/ERK,p-CREB/CREB,BDNF,NR2 A,PSD95 and DUSP6.The above experiments were designed to explore effects of AIA on DUSP6 and ERK-CREB-BDNF pathway in the mPFC of adult mice.4.Adolescent mice were randomly divided into four groups: Water + AAV-control group,Water + DUSP6-sh RNA group,Alcohol + AAV-control group,Alcohol + DUSP6-sh RNA group,and they were subjected to AIA exposure.Alcohol was removed during adulthood(postnatal 56 days),then control AAV and DUSP6 knock-down AAV were micro-injected into mPFC mice respectively.After AAV was fully expressed,the cognitive abilities of adult mice were evaluated by NOR tests and 3CST.The morphological changes of dendrites in mPFC were detected by IF.Protein expression of p-ERK/ERK,p-CREB/CREB,BDNF,NR2 A,PSD95 and DUSP6 were detected by western blot,and the ultrastructure of synapses in mPFC was detected by transmission electron microscope(TEM).The above experiments were designed to evaluate effects and molecular mechanism of inhibiting DUSP6 in mPFC on adolescent alcohol exposure induced-cognitive impairment in adulthood.Results1.During adolescent intermittent alcohol exposure period,AIA had no significant effect on the weight of mice,and the body weight of two groups increased synchronously.In AIA group,alcohol consumption remained stable,and the blood alcohol concentration remained within a certain range.2.Withdrawal of AIA did not induce anxiety-like behavior in adulthood,but significantly damaged recognitive memory.3.Compared with the drinking water group,the dendritic density and branching of neurons in mPFC decreased and the DUSP6 positive neurons increased significantly after alcohol exposure in adolescents.And the AIA inhibited the activities of ERK,CREB,BDNF,NR2 A and PSD95 but significantly increased the expression of DUSP6.4.Inhibition of DUSP6 in mPFC during adult alcohol withdrawal significantly improved the impairment of adult recognition induced by alcohol exposure during adolescence,restored the normal activity of ERK,CREB,BDNF,NR2 A and PSD95,reversed the thinning of postsynaptic density in adult mPFC,and alleviated the dendrite injury in mPFC.ConclusionAdolescent mice were exposed to 10% v/v alcohol intermittently for 28 days,which did not affect the basic metabolism and spontaneous activity of mice,and did not induce tolerance to alcohol.After intermittent alcohol exposure during adolescence,even when the mice were no longer exposed to alcohol in adults,they did not exhibit anxiety-like behavior,but AIA significantly impaired their short-term recognition memory and social recognition memory.The long-term higher expression of DUSP6 in adult mPFC induced by alcohol exposure during adolescence inhibited the activity of ERK-CREB-BDNF,which may be one of the important molecular mechanisms leading to the ultrastructural changes of synapses and impairment of recognitive memory in adult mPFC. |
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