| Objective:Alcohol addiction is a common mental disease that causes serious harm and consequences to individuals and society.During the occurrence and maintenance of addiction,the brain’s response to addictive drugs includes adaptive changes in neurons and changes in synaptic plasticity.In this study,we established alcohol two-bottle choice model and other alcohol dependence-related behavioral models in mice to observe the effect of Synaptotagmin 1 knockout on alcohol dependence-related behavior in mice,and analyze the neuroadaptive changes and their possible mechanisms in this process,so as to provide a new idea for exploring the new molecular regulation mechanism of alcohol dependence and abuse.Methods:1.The Syt1 knockout mice(Syt1+/-)were bred and the total DNA was extracted from the tail.The genotypes of the mice were identified and selected by PCR method.The knockout of Syt1 was detected by q PCR and Western blot experiments.2.Two-bottle choice test of mice:8-week-old WT and Syt1+/-mice were divided into WT group and Syt1+/-group with 9 mice in each group.（1）Alcohol two-bottle choice experiment:Each mouse was fed in a separate rat cage,and two identical bottles were placed in each cage,one water bottle and one alcohol bottle.Mice drank alcohol at concentrations of 2.5%,5%,10%,10%and 10%in turn.The alcohol consumption and alcohol preference of each mouse were detected during the process of increasing the alcohol concentration in a gradient manner,and the total liquid consumption was observed.（2）The effect of Syt1 on taste preference（saccharin and quinine）of mice was detected,and the total amount of drinking liquid and preference of mice in WT group and Syt1+/-group were observed.3.10 WT and 10 Syt1+/-mice were selected for 8 weeks to detect the effect of Syt1 knockout on blood alcohol concentration in mice.4.The effects of Syt1 deletion on conditioned place preference and righting reflex behavior in mice were detected.5.Exploration of related mechanism:WT mice and Syt1+/-mice were divided into four groups:WT+Water group,Syt1+/-+Water group,WT+Alcohol group and Syt1+/-+Alcohol group,with 6 mice in each group.Among them,3 were used in LC-MS experiment,and the other 3 in each group were used in western blot experiment.（1）The contents of glutamate andγ-aminobutyric acid in the amygdala of each group were detected by LC-MS.（2）The relative expression of Syt1 in the amygdala of each group was detected by Western blot.（3）The effect of Syt1 on synaptic related proteins（NR1 and NR2B）in the amygdala was analyzed by Western blot method.Results:1.Syt1+/-mice were propagated and identified.By fluorescence quantitative PCR and Western blot,we found that Syt1 was expressed in many parts of the brain of wild-type mice,especially in the amygdala,prelimbic cortex,hippocampus and accumbens nucleus.Compared with WT mice,the expression of m RNA and protein in Syt1+/-mice was down-regulated by about 55%and 41%respectively,and the difference was statistically significant.2.Alcohol two-bottle choice behavior test（1）Alcohol two-bottle choice experiment:When the alcohol concentration of mice was 2.5%,there was no significant difference in alcohol consumption and alcohol preference between WT group and Syt1+/-group.When the alcohol concentration gradually increased to 10%,the alcohol consumption of the two groups of mice gradually increased.After maintaining 10%alcohol concentration,it was found that the alcohol consumption and alcohol preference of mice in the Syt1+/-group were significantly lower than those in the WT group（P<0.05）.In the course of the experiment,there was no significant difference in total liquid consumption between the two groups.（2）Two-bottle choice experiment of saccharin and quinine:In the saccharin drinking experiment,there was no difference in saccharin consumption between the two groups for different concentrations of saccharin（0.04%and 0.08%）.At the same time,with the increase of saccharin concentration,the saccharin consumption of the two groups increased significantly（P<0.01）.However,there was no significant difference in saccharin preference and total liquid consumption between the two groups.In the quinine drinking experiment,there was no difference in quinine consumption of different concentrations（30μM and 60μM）between the two groups,but the quinine consumption in WT group increased with the increase of quinine concentration（P<0.05）,and there was no significant difference in quinine drinking preference and total liquid consumption between the two groups.3.In the blood alcohol concentration test,there was no significant difference in blood alcohol concentration between WT group and Syt1+/-group,but in both WT group and Syt1+/-group,the blood alcohol concentration at 2 h after injection was significantly lower than that at 1 h（P<0.05 and P<0.01）.4.In the conditioned place preference behavior experiment:After the mice with excessive preference for one side were eliminated in the pre-adaptation period,there was no significant difference in the preference of the mice to the companion medicine box between the two groups.After the training period,alcohol induced a significant preference for the compartment paired with the drug（P<0.001）.During the test period,it was found that the stay time of Syt1+/-mice in the companion medicine box was significantly increased（P<0.01）.The results of righting reflex test showed that there was no difference in the sedation latency between Syt1+/-mice and WT mice after alcohol injection,but the duration of alcohol sedation was significantly increased（P<0.001）.5.Exploration of related mechanisms:（1）Neurotransmitter detection experiment:The results of glutamate content test showed that the glutamate content in the amygdala of Syt1+/-mice was significantly higher than that of WT mice（P<0.01）,while that of Syt1+/-mice was significantly lower than that of WT mice after drinking alcohol（P<0.05）.The glutamate content of WT mice increased after drinking alcohol,while that of Syt1+/-mice decreased significantly after drinking alcohol（P<0.01）.The results ofγ-aminobutyric acid test showed that the content ofγ-aminobutyric acid in the amygdala increased in both WT and Syt1+/-mice after drinking alcohol（P<0.05）.（2）Expression of Syt1 in mice in each group:The content of Syt1 in the amygdala of each group showed that the expression of Syt1 in WT mice increased significantly after drinking alcohol（P<0.05）.In both drinking water mice and drinking alcohol mice,the expression of Syt1 in Syt1+/-mice was significantly lower than that in WT mice（P<0.05 and P<0.01）.（3）Detection of synaptic-related proteins:There were significant statistical differences in the expression of NR2B in the amygdala among the groups.In water-drinking and alcohol-drinking mice,the expression of NR2B in Syt1+/-mice was lower than that in WT mice（P<0.05 and P<0.01）,while the expression of NR2B in WT mice was significantly increased after drinking alcohol compared with that before drinking alcohol（P<0.05）.The expression of NR1 in the amygdala had the same trend as NR2B.Conclusion:1.Syt1 was expressed to a certain extent in mouse brain,especially in amygdala,prelimbic cortex,hippocampus and accumbens nucleus.However,the expression of Syt1 in Syt1+/-mice decreased in different degrees in each brain region.2.Syt1 deficiency can reduce alcohol consumption and alcohol preference in mice,and this preference has nothing to do with taste preference;in addition,Syt1deficiency does not affect the rate of alcohol metabolism in mice.But Syt1 deletion interferes with the establishment of alcohol rewarding effects and context-specific associations and increases mice’s tolerance to the sedative effects of alcohol.3.The behavioral changes of mice caused by Syt1 deletion can cause changes in neuronal adaptability by affecting the release of glutamate andγ-aminobutyric acid,and can regulate the content of NR1 and NR2B subunits of NMDAR,resulting in changes in synaptic plasticity. |
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