摘要
OBJECTIVE Social anxiety disorder,also known as social phobia,is the most common of all anxiety disorders.Despite the seriousness of this disorder,it has rarely been studied and as a consequence,little is known about its underlying neurobiology.Growing evidence suggests that inhibitory neurons dysfunction could affect the balance of neuronal activity in psychotic disorders including anxiety,SAD,etc.Ephrin-B(EB) proteins were previously demonstrated to ensure normal distribution and function of inhibitory interneurons in the brain.We thus proposed that deletion of EB2 in inhibitory neurons might destroy the homeostasis of excitatory neurons and inhibitory neurons,and induce behavioral deficits associated with psychotic diseases.METHODS(1)Mice.We generated conditional EB2 knockout mice as a model of SAD and characterized the behaviors and the biochemical changes in the brains of the knockout mice.We also generated a mouse line with a selective deletion of EB2 from vGAT neurons by breeding a v GAT-Cre line with a loxP-flanked alleles in EB2.For EB2 detection,we crossed vGAT-Cre;EphrinB2 loxp/loxp mice with Rosa26-STOP-tdTomato Cre indicator(Ai9) mice to label the vGAT neurons with tdTomato.Mice were maintained in a mixed CD1/129 genetic background.Al experiments involving mice were carried out in accordance with the Shanghai Jiaotong University.Genotypes were unlocked only after completion of analysis.(2) General procedures for behavioral testing.Open field test:Locomotor activity was measured using an open field test.The size of the open field box was 44 cm×44 cm×44 cm.Each mouse was placed in the corner of the open-field apparatus at the beginning of the test,and allowed to explore it for 15 min.The total distance traveled(in cm) and counts for stereotyped behaviors were recorded and analyzed.The total distance traveled was used as an index of locomotor activity.The percentage of center zone entries was considered as anxiety indexes.EPM test:The elevated plus maze apparatus was made of dark gray plastic and comprised two open arms(30 cm×7 cm×0.25 cm) opposed to two enclosed arms(30 cm × 7 cm × 15 cm) elevated60 cm from the floor.Animals were placed in the central area of the apparatus with their head facing an enclosed arm(test duration,5 min).Digitized video recordings with EthoVision software were used for behavioral analysis.The percentage of time spent in open arms and the percentage of open-arm entries were calculated.Social behaviors:Briefly,a top open acrylic box was divided into three compartments by two clear acrylic glass partitions with an opening in the bottom.Wire mesh cages that are either empty or have social stimulus mouse in it were put in the center of each outer compartment.Unfamiliar naive CD1/129 mice of the same age were used as stimulus mice.Prior to the test,the mouse was introduced into the center chamber for 5 min,with both gates closed.Gates were then open and the test mouse was acclimated to explore the empty three-chambered apparatus freely for another 10 min.After the initial 15 min habituation session,mice were placed in the center compartment and allowed to explore freely all the three compartments.To examine the social contact,the following activities were recorded via a video surveillance for 10 min.The number of entries to each compartment,the time spent in each compartment as well as the time and frequency each mouse spent in sniffing were analyzed with EthoVision XT 8.5(Noldus,Wageningen,Netherlands).RESULTS(1)Social preference assays were performed in WT-vGATCre,EB2-flox,and EB2-vGATCre mice respectively.To evaluate social approach behavior,we used a three-chamber social arena to evaluate animals for their social interaction.The control groups of WTvGATCre,EB2-flox mice showed a significant preference for spending time in the social chamber(P<0.05);whereas the EB2-vGATCre mice did not show this preference and spent roughly equal time in the social and nonsocial chambers.(2)To determine if the decrease in social preference could result from changes in locomotion,we compared locomotor activity in EB2-vG ATCre mice and control mice.We did not observe a significant lower locomotive activity in mice.Hence,EB2 deletion caused a change in social preference but not in locomotion.Taken together,EB2 deletion exerts abnormal psychotogenic activity of social deficits which was often considered as an important feature of SAD.(3) The time spent in the open arms is widely used to measure anxiety in mice,with greater time spent in the open arm being considered as less anxious behavior.To normalize for individual differences in overall activity level,the time spent in the open arms was divided by the total time.The ratio was higher for WT-vGATCre mice and EB2-loxp mice as compared to the EB2-vG ATCre mice(P=0.0112,P=0.0197).Thus,results showed that there was a change in anxiety levels from in EB2-vGATCre mice as compared with control mice.CONCLUSION EB2 may be a candidate risk gene for SAD and that the EB2 conditional knockout model could be a tool for studying the underlying mechanisms in SAD.
OBJECTIVE Social anxiety disorder,also known as social phobia,is the most common of all anxiety disorders.Despite the seriousness of this disorder,it has rarely been studied and as a consequence,little is known about its underlying neurobiology.Growing evidence suggests that inhibitory neurons dysfunction could affect the balance of neuronal activity in psychotic disorders including anxiety,SAD,etc.Ephrin-B(EB) proteins were previously demonstrated to ensure normal distribution and function of inhibitory interneurons in the brain.We thus proposed that deletion of EB2 in inhibitory neurons might destroy the homeostasis of excitatory neurons and inhibitory neurons,and induce behavioral deficits associated with psychotic diseases.METHODS(1)Mice.We generated conditional EB2 knockout mice as a model of SAD and characterized the behaviors and the biochemical changes in the brains of the knockout mice.We also generated a mouse line with a selective deletion of EB2 from vGAT neurons by breeding a v GAT-Cre line with a loxP-flanked alleles in EB2.For EB2 detection,we crossed vGAT-Cre;EphrinB2 loxp/loxp mice with Rosa26-STOP-tdTomato Cre indicator(Ai9) mice to label the vGAT neurons with tdTomato.Mice were maintained in a mixed CD1/129 genetic background.Al experiments involving mice were carried out in accordance with the Shanghai Jiaotong University.Genotypes were unlocked only after completion of analysis.(2) General procedures for behavioral testing.Open field test:Locomotor activity was measured using an open field test.The size of the open field box was 44 cm×44 cm×44 cm.Each mouse was placed in the corner of the open-field apparatus at the beginning of the test,and allowed to explore it for 15 min.The total distance traveled(in cm) and counts for stereotyped behaviors were recorded and analyzed.The total distance traveled was used as an index of locomotor activity.The percentage of center zone entries was considered as anxiety indexes.EPM test:The elevated plus maze apparatus was made of dark gray plastic and comprised two open arms(30 cm×7 cm×0.25 cm) opposed to two enclosed arms(30 cm × 7 cm × 15 cm) elevated60 cm from the floor.Animals were placed in the central area of the apparatus with their head facing an enclosed arm(test duration,5 min).Digitized video recordings with EthoVision software were used for behavioral analysis.The percentage of time spent in open arms and the percentage of open-arm entries were calculated.Social behaviors:Briefly,a top open acrylic box was divided into three compartments by two clear acrylic glass partitions with an opening in the bottom.Wire mesh cages that are either empty or have social stimulus mouse in it were put in the center of each outer compartment.Unfamiliar naive CD1/129 mice of the same age were used as stimulus mice.Prior to the test,the mouse was introduced into the center chamber for 5 min,with both gates closed.Gates were then open and the test mouse was acclimated to explore the empty three-chambered apparatus freely for another 10 min.After the initial 15 min habituation session,mice were placed in the center compartment and allowed to explore freely all the three compartments.To examine the social contact,the following activities were recorded via a video surveillance for 10 min.The number of entries to each compartment,the time spent in each compartment as well as the time and frequency each mouse spent in sniffing were analyzed with EthoVision XT 8.5(Noldus,Wageningen,Netherlands).RESULTS(1)Social preference assays were performed in WT-vGATCre,EB2-flox,and EB2-vGATCre mice respectively.To evaluate social approach behavior,we used a three-chamber social arena to evaluate animals for their social interaction.The control groups of WTvGATCre,EB2-flox mice showed a significant preference for spending time in the social chamber(P<0.05);whereas the EB2-vGATCre mice did not show this preference and spent roughly equal time in the social and nonsocial chambers.(2)To determine if the decrease in social preference could result from changes in locomotion,we compared locomotor activity in EB2-vG ATCre mice and control mice.We did not observe a significant lower locomotive activity in mice.Hence,EB2 deletion caused a change in social preference but not in locomotion.Taken together,EB2 deletion exerts abnormal psychotogenic activity of social deficits which was often considered as an important feature of SAD.(3) The time spent in the open arms is widely used to measure anxiety in mice,with greater time spent in the open arm being considered as less anxious behavior.To normalize for individual differences in overall activity level,the time spent in the open arms was divided by the total time.The ratio was higher for WT-vGATCre mice and EB2-loxp mice as compared to the EB2-vG ATCre mice(P=0.0112,P=0.0197).Thus,results showed that there was a change in anxiety levels from in EB2-vGATCre mice as compared with control mice.CONCLUSION EB2 may be a candidate risk gene for SAD and that the EB2 conditional knockout model could be a tool for studying the underlying mechanisms in SAD.
出处
《中国药理学与毒理学杂志》
CAS
CSCD
北大核心
2018年第9期730-732,共3页
Chinese Journal of Pharmacology and Toxicology
基金
National Natural Science Foundation of China (81501153
81571326).
