| Objective:Road traffic injury is a serious public health problem in China and even in the world.Drivers,vehicles and road conditions can affect the occurrence of road traffic accidents,and drivers are the dominant factors.In the early stage,Our team found that the 5-HTTLPR gene polymorphism may affect the psychological behavior of drivers,leading to the occurrence of road traffic accidents.To investigate the effect of 5-HTT gene polymorphism on psychological behavior and glutamate/GABA system by detecting relevant behavioral indicators and the expression of glutamate/GABA system related molecules in cerebral cortex of 5-HTT knockout mice,and to provide an theoretical basis for our team previous study on the relationship between5-HTTLPR gene polymorphism and psychological behavior of drivers.Methods:Three mouse models of 5-HTT+/+,5-HTT+/-,and 5-HTT-/-were included in this study,each with 8 mice.The Morris water maze test,open-field test and forced swimming test were used to evaluate the mouce learning and memory ability,as well as the psychological behavior of depression,tension and anxiety.Real-time PCR and western blotting were used to detect the m RNA and protein content of glutamatergic system related molecules（Glu N1,Glu R1,EAAT1,EAAT2,PSD95）and GABAergic system related molecules（Npas4,GAD67,VGAT,Gabrg2,GAT1,GAT3）in the mouse cerebral cortex.The measurement data were described by the mean±standard deviation（x±s）.The analysis of variance was used for comparison between multiple groups,the escape latency and average swimming speed in the Morris water maze test were analyzed by repeated measurement ANOVA,and the least significant difference method（LSD）was used for further pairwise comparisons.Pearson/Spearman correlation analysis method was used to analyze the correlation between related molecules in the glutamate/GABA system,and between related molecules and behavioral indicators.Results:1.The mouse behavioral test（1）In the Morris water maze test,there was no interaction between different groups and time in the escape latency and average swimming speed（P>0.05）.There was a statistically significant difference in the escape latency and average swimming speed at different time（P<0.05）.The average swimming speed,times of crossing platform,residence time in target quadrant were the highest in 5-HTT+/+group,followed by 5-HTT+/-group and the lowest in5-HTT-/-group.On the contrary,the escape latency of 5-HTT+/+group was the shortest,5-HTT+/-group’s was the second,and 5-HTT-/-group’s was the longest（P<0.05）.（2）In the forced swimming test,there was significant difference in immobility time among different groups（P<0.05）,and 5-HTT+/-group’s and5-HTT-/-group’s were longer than 5-HTT+/+group’s（P<0.05）.（3）In the open-field test,there were significant differences in the times of entering central area,the residence time in the central area among different groups（P<0.05）,and the two indexes in 5-HTT-/-group were lower than those in5-HTT+/+group and 5-HTT+/-group（P<0.05）.However,there was no significant difference in the number of modifications（P>0.05）.2.In the glutamatergic system of the mouse cerebral cortex（1）At the level of gene transcription,there were significant differences in Glu R1,EAAT1 and EAAT2 in cerebral cortex among different groups（P<0.05）,but there were no significant differences in Glu N1 and PSD95 among different groups（P<0.05）.The m RNA levels of Glu R1 and EAAT1 in 5-HTT-/-group were significantly higher than those in 5-HTT+/+group and 5-HTT+/-group（P<0.05）;EAAT2 m RNA expression in 5-HTT+/-group and 5-HTT-/-group was higher than that in 5-HTT+/+group（P<0.05）.（2）There was significant difference in EAAT1 protein level among the three groups（P<0.05）,but there was no significant difference in Glu N1,Glu R1and EAAT2 protein levels among the three groups（P>0.05）.EAAT1 protein level in 5-HTT-/-group was higher than that in 5-HTT+/+group and 5-HTT+/-group（P<0.05）.After 5-HTT knockout,the content of glun1 protein decreased,but there was no significant difference（P>0.05）.（3）At the gene transcription level,glutamate receptors（Glu N1 and Glu R1）,excitatory amino acid transporters（EAAT1 and EAAT2）,and PSD95 were positively correlated（P<0.05）.However,at the protein expression level,EAAT2showed a negative correlation with glutamate receptors（Glu N1,Glu R1）（P<0.05）.（4）Glu N1 m RNA in the mouce cerebral cortex was mainly associated with the average escape latency and average swimming speed in the Morris water maze test,and the immobility time in forced swimming test（P<0.05）;The expression level of Glu N1 protein was correlated with the average escape latency,average swimming speed,times of crossing platform in the Morris water maze test,and the times of entering central area and central area residence time in the open-field test（P<0.05）.Glu R1 m RNA was mainly associated with the average escape latency in the Morris water maze test,the residence time in target quadrant and the number of times to enter the central region in the open-field test（P<0.05）.However,it was not found that Glu R1 protein level was related to the behavior changes of mice in the Morris water maze,forced swimming and open-field test（P>0.05）.EAAT1 m RNA was negatively correlated with the number of times of entering the central region in the open-field test（P<0.05）;EAAT1 protein level was correlated with the average escape latency,average swimming speed,immobility time in the forced swimming,times of entering central area,residence time in central area,in the Morris water maze test（P<0.05）.EAAT2 m RNA level was negatively correlated with the number of crossing platforms,the number of entering the central area,the retention time in Morris water maze test（P<0.05）;EAAT2 protein level was negatively correlated with the residence time of target quadrant in the Morris water maze test（P<0.05）.There was no correlation between PSD95 m RNA level and behavioral indexes（P>0.05）.2.In the GABAergic system of the mouse cerebral cortex（1）At the gene transcription level,Npas4,VGAT,GAT1 and GAT3 were significantly different among the three groups（P<0.05）,but the m RNA levels of GAD67 and Gabrg2 were not significantly different（P>0.05）.The content of Npas4 m RNA in 5-HTT+/-group was significantly higher than that in 5-HTT+/+group（P<0.05）,but also may higher than that in 5-HTT-/-group（P=0.064）.The m RNA levels of VGAT and GAT1 in 5-HTT-/-group were higher than those in5-HTT+/+group and 5-HTT+/-group（P<0.05）.The expression of GAT3 m RNA in 5-HTT-/-group was lower than that in 5-HTT+/+group（P<0.05）.（2）There was significant difference in the protein content of VGAT between the three groups（P<0.05）,and there was no significant difference between the three groups（P>0.05）.The content of VGAT protein in 5-HTT-/-group was lower than that in 5-HTT+/+group and 5-HTT+/-group（P<0.05）,which was only 74%higher than that in 5-HTT+/+group and 5-HTT+/-group（P<0.05）.（3）At the gene transcription level,there was a significant positive correlation between Npas4 and GAD67,GAD67 and Gabrg2,GAD67 and GAT1,and VGAT and GAT1（P<0.05）.Howere,at the protein expression level,there was no correlation among GAT1,GAD67 and VGAT（P>0.05）.（4）GAD67 m RNA was negatively correlated with the number of times of entering the central region in the open-field test（P<0.05）;There was no correlation between GAD67 protein level and related behavioral indicators（P>0.05）.VGAT m RNA was negatively correlated with the residence time of target quadrant in the Morris water maze test（P<0.05）.However,the level of VGAT protein was positively correlated with the times of entering central area,residence time of central area in the open-field test（P<0.05）.GAT1 m RNA was negatively correlated with the number of times of crossing platform in the Morris water maze test（P<0.05）,but GAT1 protein level was not found to be correlated with related behavioral indicators（P>0.05）.GAT3 m RNA was positively correlated with average swimming speed（P<0.05）.In addition,there was no correlation between the transcription levels of Npas4 and Gabrg2 and the related behavioral indicators（P>0.05）.3.The relationship between glutamate and GABA systemAt the gene transcription level,Glu R1 and PSD95 in glutamate pathway are related to many signaling factors in GABA pathway,such as GAD67,VGAT,Gabrg2 and GAT1;GAT1 was positively correlated with Glu R1,EAAT1,EAAT2and PSD95;Npas4 was mainly associated with EAAT2（P<0.05）.In the level of protein expression,Glu R1 and GAD67,EAAT2 and GAT1 showed strong positive correlation（P<0.05）.Conclusions:（1）The deletion of 5-HTT gene may lead to cognitive dysfunction,depression,anxiety and other behavioral disorders in mice,and may affect the balance of glutamate/GABA system in the mouce cerebral cortex.（2）To some extent,this study may verify the previous hypothesis:the change of 5-HT signaling pathway in vivo may lead to the imbalance of glutamate/GABA system,and then lead to cognitive dysfunction and behavioral abnormalities,which may increase the risk of traffic accidents. |
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