Study On Effect And Mechanism Of Liraglutide On Alcohol Addiction In Mice

Background and ObjectiveAlcohol addiction,also known as alcohol dependence,is a chronic and recurrent brain disease.Globally,alcoholism causes about 3.3 million deaths every year,and alcohol addiction accounts for up to 5% of the global burden of disease.Given the adverse effects of alcohol addiction,we need to pay more attention to it.Heavier and persistent drinking are two features of Alcohol addicts.Studies have shown that the transition from occasional moderate drinking to regular and uncontrolled drinking is mainly related to the activation of reward system.As a reward substance,alcohol can produce excitement and pleasure after ingested by an individual,which triggers the reward effect and produces craving and dependence on it.Activation of the reward system is mainly regulated by the mesolimbic dopamine system(MLDS).Midbrain ventral tegmental area(VTA)contains a large number of dopamine(DA)neurons,which can project to different downstream areas to regulate a series of behaviors(such as learning and memory,anxiety,depression-like behaviors,etc.),including nucleus accumbens(NAc),central amygdala(Ce A),medial prefrontal cortex(mPFC)and hippocampus(HP).Glucagon-like peptide-1(GLP-1)is an incretin hormone and neuropeptide.It was originally found to be synthesized in the intestine,and later it was found to be able to cross the blood-brain barrier and enter the central nervous system(CNS).Studies in recent years found that it can also be produced by neurons and microglia in the CNS.A large number of studies have shown that GLP-1 in brainstem and hypothalamus plays an important role in the regulation of food intake.However,researches also found that GLP-1 can also regulate food reward behavior through the reward loop(including VTA and NAc).In addition,it promotes neurogenesis by stimulating the production of neurotrophic factors,and reduces inflammation through regulating production of TNFα,IL-6,IL-10 and activation of microglia.Liraglutide is a selective GLP-1 receptor agonist.Because of its features of promoting insulin secretion and reducing glucagon secretion and gastric emptying,it has been approved as a drug for treating type 2diabetes in clinic.Studies have shown that Liraglutide can cross the blood-brain barrier and regulate various behaviors by targeting different brain regions,such as cognitive function,depression and anxiety.Therefore,we present a hypothesis here: Liraglutide can act on different regions of the reward circuit in mice and alleviate abnormal behavior in alcohol-addicted mice by regulating synaptic plasticity and neuroinflammation.Objective: To study the effect and mechanism of Liraglutide on alcohol addiction in mice.This experiment intends to establish a mouse model of alcohol addiction to observe the effect of Liraglutide on alcohol-related behavior in mice through intraperitoneal injection of Liraglutide,and analyze the neuronal activity of VTA and the changes in synaptic plasticity and neuroinflammation of mPFC and HP during this process.This study will provide new ideas and methods for future treatment of alcohol addiction in clinic.Methods(1)Establishment of alcohol addiction mouse modelThree alcohol addiction models were established: alcohol continuous model,alcohol intermittent model and stress intermittent model.By analyzing the alcohol consumption and preference of the three models,the best one was chosen for the next experimental study.The mice were randomly divided into four groups in subsequent experiments:control group,Liraglutide group,alcohol group and alcohol+Liraglutide group.According to methods of establishing alcohol addiction mouse model,the mice were fed with alcohol in the former four weeks.From the fifth week,each group was injected with Liraglutide or normal saline intraperitoneally,and the mice in each group were given alcohol withdrawal from the seventh week for 2 weeks.During the withdrawal period,intraperitoneal injection of Liraglutide or saline was continued.After alcohol withdrawal was completed,the mice in each group were re-drinked with alcohol for 4days.During this period,the body weight and fluid consumption were recorded every day.(2)Blood glucose testBlood glucose level: The blood glucose levels of mice before and after intraperitoneal injection of Liraglutide were detected by a blood glucose meter,and the effect of Liraglutide on the blood glucose of mice was analyzed.(3)Behavioral testsAlcohol consumption and preference: Observing the changes in alcohol consumption and preference of each group through data analysis,and analyzing the effect of Liraglutide on alcohol intake in mice;Learning,memory and anxiety-like behaviors: Water maze,open field and elevated plus maze tests were used to detect the behavioral changes of mice after alcohol withdrawal and re-drinking,and analyze the effects of Liraglutide on the learning,memory and anxiety-like behaviors of mice.(4)Detecting neural activity of VTANeural electrical activity: Recording the LFPs signal of VTA in alcohol addition mice through multi-channel electrophysiological technology in vivo,and Plex Control and Matlab softwares were used to analyze the time-frequency and power density changes of LFPs.Observing the effect of Liraglutide on the neural electrical activity of VTA in alcohol addition mice;Neural activation: Colocalization of TH and c-Fos was detected by immunofluorescence,and the effect of Liraglutide on the activity of VTA dopaminergic neurons was also analyzed.(5)Detection of synaptic plasticity in mPFC and HPChanges of dendritic spine density in mPFC and HP were analyzed by Golgi staining technique,and the effect of Liraglutide on dendritic spine density in mPFC and HP was also observed.The expression of p-Glu A1,Glu A1,VGlu T1,SYN and PSD95 protein in mPFC and HP was detected by western blot and immunofluorescence technology,and the effect of Liraglutide on the expression of synaptic transmissionrelated protein in mPFC and HP was analyzed.(6)Analysis of inflammatory cell marker and inflammation-related protein expression in mPFC and HPThe expression of GFAP,Iba1 and TNFα protein in mPFC and HP was detected by western blot and immunofluorescence techniques,and the effect of Liraglutide on the expression of inflammatory cell marker and inflammation-related proteins in mPFC and HP was observed.Result(1)Alcohol addiction mouse modelThe mortality rate of mice in the intermittent stress model was higher,so we directly eliminated them without analysis.Alcohol consumption and preference of the intermittent alcohol model were higher than those of in continuous alcohol model,so alcohol intermittent model was used to carry out follow-up experiments.(2)Blood glucose levelLiraglutide had no effect on the blood sugar in all groups.(3)Behavioral testsThe alcohol consumption and preference in the alcohol group gradually increased,and compared with the alcohol group,Liraglutide could decrease this behavior.After alcohol withdrawal,the mice in the alcohol group had impaired learning and memory ability and increased anxiety-like behaviors compared with the control group,and Liraglutide was effective in improving learning and memory ability and anxiety-like behaviors.While,the memory ability and anxiety-like behavior of mice in the alcohol group did not change after alcohol re-drinking,but the learning ability was reduced,which was also enhanced by Liraglutide.(4)VTA neural activityCompared with the control group,the power spectral density of the VTA LFPs in the other groups did not change significantly after alcohol withdrawal.Although,the power spectral density of LFPs in the VTA of alcohol group were increased after alcohol re-drinking,and decreased after Liralitide treatment.The co-localized expression of TH and c-Fos was detected in the VTA area of each group of mice.(5)Detection of synaptic plasticity in mPFC and HPCompared with the control group,the densities of dendritic spine in mPFC and HP in the alcohol group were reduced,and both areas improved after Liraglutide treatment.The total protein expression of Glu A1 in mPFC and HP in the alcohol group did not change significantly compared with the control group,while expression of pGlu A1,VGlu T1,SYN and PSD95 was decreased,and increased expression of them after Liraglutide treatment.(6)Expression of inflammatory cell marker and inflammation-related proteins in mPFC and HPCompared with the control group,the expression levels of GFAP,Iba1 and TNFαin alcohol group in mPFC and GFAP in HP were increased,and Liraglutide could reversed overexpression of these proteins.ConclusionLiraglutide alleviates the behavioral deficits of alcohol-addicted mice by regulating the electrical activity of VTA neurons and the synaptic plasticity and neuroinflammation of mPFC and HP in the reward system.