氯胺酮上调谷氨酸能突触通路诱导斑马鱼成瘾  

Ketamine Upregulates the Glutamatergic Synaptic Pathway and Induces Zebrafish Addiction

作　　者：钱松 朱思琪 徐金中 方晨雨 柴尹泽 骆洋 王锴[1] 刘奕周 QIAN Song;ZHU Si-Qi;XU Jin-Zhong;FANG Cheng-Yu;CHAI Yin-Ze;LUO Yang;WANG Kai;LIU Yi-Zhou(The Criminal Science and Technology Department,Zhejiang Police College,Hangzhou 310053,Zhejiang,China;Key Laboratory of Drug Prevention and Control Technology of Zhejiang Province,Hangzhou 310053,Zhejiang,China;National Narcotic Laboratory Zhejiang Regional Center,Hangzhou 310053,Zhejiang,China;Affiliated Wenling Hospital,Wenzhou Medical University,Taizhou 317500,Zhejiang,China)

机构地区：[1]浙江警察学院刑事科学技术系,杭州310053 [2]浙江省毒品防控技术研究重点实验室,杭州310053 [3]国家毒品实验室浙江分中心,杭州310053 [4]温州医科大学附属温岭医院临床药学室,浙江台州317500

出　　处：《中国生物化学与分子生物学报》2024年第8期1153-1160,共8页Chinese Journal of Biochemistry and Molecular Biology

基　　金：浙江省基础公益研究计划(No.LTGY23H230001)和(No.LGF19H280003);浙江省毒品防控技术研究重点实验室开放课题(No.2020012)资助。

摘　　要：氯胺酮是谷氨酸NMDA受体的拮抗剂,也是当下滥用较严重的一种精神活性物质,长期滥用会引起机体多个系统的损害,探讨氯胺酮的成瘾机制并筛选相关生物标志物对毒品防控具有重要的意义。本研究先对斑马鱼胚胎/幼鱼进行氯胺酮急性暴露实验,然后取6月龄的斑马鱼,通过条件性位置偏爱实验建立氯胺酮成瘾模型,RNA-seq检测斑马鱼脑转录组,qPCR和Western印迹分别检测其中关键基因表达。结果显示:与对照组相比,氯胺酮组中斑马鱼胚胎的自主抽动、胚胎孵化率及幼鱼存活率都有不同程度的降低,移动距离更是大幅下降,由对照组的1 904.2 mm降到300μmol/L氯胺酮组的319.0 mm;条件性位置偏爱实验显示对照组斑马鱼在鱼缸的活动没有明显变化,而氯胺酮组斑马鱼在鱼缸明区的活动时间从训练前的385.2 s提高到训练后的706.4 s,时间占比提高了26.8%(^(***)P<0.001),说明斑马鱼对对氯胺酮刺激产生了偏爱;KEGG分析显示氯胺酮处理的差异基因在神经活性配体-受体相互作用通路中富集,GSEA分析进一步发现氯胺酮显著上调谷氨酸能突触通路(NES值为1.5);此外,与对照组相比,氯胺酮组的Grin2b和Gria2的mRNA水平分别升高至4.6倍、1.4倍,同时蛋白质水平分别升高至2.0倍和1.4倍。这说明氯胺酮能够诱导斑马鱼成瘾,谷氨酸能突触通路可能参与调控作用。Ketamine,an antagonist of the glutamate N-methyl-D-aspartate(NMDA)receptor,is currently one of the most widely abused psychoactive substances.Prolonged abuse can result in damages to various systems in the body,making it crucial to investigate the regulatory mechanism of ketamine addiction and screening related biomarkers.In this study,zebrafish embryos/larvae were initially exposed acutely to ketamine.Then,a ketamine addiction model was established in 6-month-old zebrafish through conditioned place preference(CPP)experiments.The zebrafish brain transcriptome was analyzed using RNA-seq,while qPCR and Western blotting were employed to detect the expression of key genes.Results revealed significant reductions in the spontaneous tail coiling,embryo hatching rate,and survival rate of zebrafish embryos in the ketamine-treated group compared to the control group.The distance moved also decreased significantly,from 1904.2 mm in the control group to 319.0 mm in the high dose of ketamine group(300μmol/L).Conditional positional preference experiments demonstrated that the control zebrafish did not exhibit significant changes in activity in the CPP tank.In contrast,the ketamine-treated group increased their activity time in the light zone of the tank from 385.2 s before training to 706.4 s after training,representing a 26.8%increase(^(***)P<0.001).This suggests a preference for ketamine stimulation in zebrafish.KEGG analysis indicated enrichment of differentially expressed genes in the neuroactive ligand-receptor interaction pathway in the ketamine-treated samples.GSEA analysis further reveals a significant upregulation of the glutamatergic synapse pathway(NES=1.5).In addition,compared with the control group,the mRNA levels of Grin2b and Gria2 in the ketamine group increased by 4.6 and 1.4 times,respectively,while the protein levels increased by 2.0 and 1.4 times,respectively.These findings suggest that ketamine can induce addiction in zebrafish,potentially through upregulation of the glutamatergic synaptic pathway.

关 键 词：氯胺酮 斑马鱼 转录组 谷氨酸能突触 成瘾 

分 类 号：Q423[生物学—神经生物学]