三种逆行示踪工具标记小鼠前扣带回皮质传入核团的比较  

Comparative study of three retrograde tracing tools on labeling the afferent nuclei of the anterior cingulate cortex in mice

作　　者：肖昊翔 解进祎 刘明月 刘珺琛 袁滋铎 武胜昔 郭保霖 XIAO Haoxiang;XIE Jinyi;LIU Mingyue;LIU Junchen;YUAN Ziduo;WU Shengxi;GUO Baolin(Department of Neurobiology,School of Basic Medicine,Air Force Medical University,Xi’an 710032;Department of Physiology,School of Medicine,Yan’an University,Yan’an 716099;School of Basic Medicine,Shaanxi University of College Chinese Medicine,Xi’an 712046;Department of Neurobiology,School of Life Science,Yan’an University,Yan’an 716099,China)

机构地区：[1]空军军医大学基础医学院神经生物学教研室,西安710032 [2]延安大学医学院生理学教研室,延安716099 [3]陕西中医药大学基础医学院,西安712046 [4]延安大学生命科学学院神经生物学教研室,延安716099

出　　处：《神经解剖学杂志》2023年第4期393-400,共8页Chinese Journal of Neuroanatomy

基　　金：国家自然科学基金(82201699);陕西省重点研发计划(2023⁃YBSF⁃093);陕西省自然科学基础研究计划(2021JCW⁃13);陕西省高校科协青年人才托举计划(20220306)。

摘　　要：目的:通过对小鼠前扣带回皮层(ACC)立体定位注射重组腺相关病毒(rAAV2⁃retro)、荧光乳胶微球(retrobeads)及狂犬病毒(RV)的方法,比较三种逆行示踪工具对ACC上游传入核团的标记效果。方法:将rAAV2⁃retro⁃hSyn⁃EGFP⁃P2A⁃Cre⁃WPRE⁃hGH⁃PA、retrobeads(Red)等量混合后注射到小鼠ACC脑区,待病毒表达3周后灌注取脑、切片观察。另外将rAAV2/9⁃EF1α⁃DIO⁃oRVG(19G)、rAAV2/9⁃EF1α⁃DIO⁃NLS⁃mCherry⁃P2A⁃TVA⁃T2A⁃RVG、rAAV2/9⁃CaMKIIα⁃Cre混合后注射到小鼠ACC脑区,待病毒表达2周后将RV⁃EnvA⁃ΔG⁃EGFP注射到相同部位,1周后灌注取脑、切片观察,比较三种策略标记到ACC上游传入核团的神经元数量及形态。结果:retrobeads标记到的核团最多,但无法标记神经元形态及树突结构;RV标记到的核团数量次之,胞体及树突结构标记完整,而且可以观察到清晰的树突棘;rAAV2⁃retro标记到的核团最少,能标记到树突,但无法标记树突棘。三者在内侧眶皮层、腹侧眶皮层、视皮层等区域均能够高效标记。其中,RV在丘脑腹前核和腹外侧核标记效率高于其他两种工具。结论:ACC接受脑内的广泛投射,而三种逆行示踪剂在标记效率和标记神经元结构完整性上各有优劣。Objective:To compare the labeling effect and fiber projection of three retrograde tracing tools,we injec⁃ted recombinant adeno⁃associated virus(rAAV2⁃retro),retrobeads,and rabies virus(RV)into the anterior cingulate cortex(ACC)of mice.Methods:The rAAV2⁃retro⁃hSyn⁃EGFP⁃PS2A⁃Cre⁃WPre⁃hGH⁃PA and retrobeads(Red)were mixed in equal quantities and injected into the ACC region of mice.After 3 weeks of virus expression,the mice were perfused and the brains were sliced for observation.In addition,rAAV2/9⁃EF1α⁃DIO⁃oRVG(19G),rAAV2/9⁃EF1α⁃DIO⁃NLS⁃mCherry⁃P2A⁃TVA⁃T2A⁃RVG and rAAV2⁃CaMKIIα⁃Cre were mixed in quantities and injected into the ACC region of mice.After 2 weeks of virus expression,RV⁃EnvA⁃△G⁃EGFP was injected into the same site.A week later,the mice were perfused and the brains were sliced to observe the number and morphology of neurons labeled in the upstream afferent nuclei of ACC by the three strategies.Results:Retrobeads labeled the maximum number of afferent nuclei,but could not accurately label neuronal morphology and dendritic structure.The afferent nuclei labeled by RV were less than those labeled by retrobeads.However,the soma and dendrites were completely labeled by RV,and clear dendritic spines could be observed.Then rAAV2⁃retro labeled the least number of afferent nuclei and could label den⁃drites,but not dendritic spines.These three tracer strategies showed comparable upstream labeled neurons in medial orbital cortex,ventral orbital cortex,visual cortex,and other afferent nuclei.Among them,RV was more efficient than the other two tools in the ventral anterior and ventrolateral thalamic nuclei.Conclusion:ACC receives a wide range of projections in the brain,and the three retrograde tracing tools have their own advantages and disadvantages in labeling efficiency and structural integrity of labeled neurons.

关 键 词：前扣带回皮层 重组腺相关病毒 荧光乳胶微球 狂犬病毒 神经示踪 小鼠 

分 类 号：Q42[生物学—神经生物学]