腺相关病毒与生物素葡聚糖胺顺行标记小鼠皮质脊髓束的比较
|
摘要
目的比较腺相关病毒(AAV)与生物素葡聚糖胺(BDA)用于顺行标记小鼠皮质脊髓束(CST)的差异,探索AAV病毒应用于小鼠皮质脊髓束的顺行神经示踪的优势。方法通过立体定位注射将AAV8-CAG-GFP-2A病毒与BDA分别注射到正常小鼠以及脊髓损伤小鼠大脑体感运动皮质,通过免疫组织化学法检测AAV与BDA标记到的CST轴突,并对轴突束(bundle)及轴突分支(branch)的荧光强度进行定量分析。结果在正常小鼠的脊髓颈段、胸段及脊髓损伤小鼠胸段,AAV与BDA均可标记到经典的CST轴突束,及投射到脊髓灰质中的轴突分支。相较于BDA,AAV在小鼠脊髓颈段和胸段均能标记到更广泛的CST轴突分支。结论 AAV对于CST轴突的标记效果优于传统的神经示踪剂BDA,可作为一种更好的神经示踪方法应用于小鼠皮质脊髓束及相关神经环路的形态学研究。
Objective To compare the differences between adeno associated virus( AAV) and biotinylated dextran amine( BDA) in anterograde tracing of mouse corticospinal tract( CST),and to explore the advantages of the application of AAV in anterograde tracing of mouse CST axons as a neural tracer. Methods AAV8-CAG-GFP-2 A and BDA were stereotactically injected into the mouse sensorimotor cortex of normal and spinal cord injury mice respectively,and immunohistochemistry was employed to observe the distribution of the labeled CST axons. The relative fluorescence of axon bundles and branches was analyzed. Results Both AAV and BDA labeled the bundles of corticospinal tract and surrounding axon branches projecting into the gray matter of cervical and thoracic cords in both coronal and saggital sections of normal and spinal cord injury mice. Meanwhile,more axon branches were labeled in the AAV injected mice as compared with those in the BDA injected ones. Conclusions AAV exhibits more superior performance in mouse CST axon tracing in comparison with BDA,and can be applied as a better neural tracer for the morphological study of mouse CST and related neural circuits.
Objective To compare the differences between adeno associated virus( AAV) and biotinylated dextran amine( BDA) in anterograde tracing of mouse corticospinal tract( CST),and to explore the advantages of the application of AAV in anterograde tracing of mouse CST axons as a neural tracer. Methods AAV8-CAG-GFP-2 A and BDA were stereotactically injected into the mouse sensorimotor cortex of normal and spinal cord injury mice respectively,and immunohistochemistry was employed to observe the distribution of the labeled CST axons. The relative fluorescence of axon bundles and branches was analyzed. Results Both AAV and BDA labeled the bundles of corticospinal tract and surrounding axon branches projecting into the gray matter of cervical and thoracic cords in both coronal and saggital sections of normal and spinal cord injury mice. Meanwhile,more axon branches were labeled in the AAV injected mice as compared with those in the BDA injected ones. Conclusions AAV exhibits more superior performance in mouse CST axon tracing in comparison with BDA,and can be applied as a better neural tracer for the morphological study of mouse CST and related neural circuits.
引文
[1]Kadoya K,Lu P,Nguyen K,et al.Spinal cord reconstitution with homologous neural grafts enables robust corticospinal regeneration[J].Nat Med,2016,22:479-487.
[2]Hantman AW,Jessell TM.Clarke's column neurons as the focus of a corticospinal corollary circuit[J].Nat Neurosci,2010,13:1233-1239.
[3]Boubker Z,Edgley SA,Soteropoulos DS,et al.Changes in descending motor pathway connectivity after corticospinal tract lesion in macaque monkey[J].Brain,2012,135:2277-2289.
[4]Jiang YQ,Zaaimi B,Martin JH.Competition with primary sensory afferents drives remodeling of corticospinal axons in mature spinal motor circuits[J].J Neurosci,2016,36:193-203.
[5]Glover JC,Petursdottir G,Jansen JKS.Fluorescent dextran-amines used as axonal tracers in the nervous system of the chicken embryo[J].J Neurosci Methods,1986,18:243-254.
[6]Mccarty DM,Monahan PE,Samulski RJ.Self-complementary recombinant adeno-associated virus(sc AAV)vectors promote efficient transduction independently of DNA synthesis[J].Gene Ther,2001,8:1248-1254.
[7]Zhang J,Xi M,Fung SJ,et al.Projections from the central nucleus of the amygdala to the nucleus pontis oralis in the rat:An anterograde labeling study[J].Neurosci Lett,2012,525:157-162.
[8]Lazarov NE.Neuroanatomical tract-tracing using biotinylated dextran amine.[J].Methods Mol Biol,2013,1018:323-334.
[9]Reiner A,Veenman CL,Medina L,et al.Pathway tracing using biotinylated dextran amines.[J].J Neurosci Methods,2000,103:23-37.
[10]Cabantous S,Terwilliger TC,Waldo GS.Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein[J].Nat Biotechnol,2005,23:102-107.
[11]Li K,Zhou T,Liao L,et al.βCa MKII in lateral habenula mediates core symptoms of depression[J].Science,2013,341:1016-1020.
[12]Liu K,Lu Y,Lee JK,et al.PTEN deletion enhances the regenerative ability of adult corticospinal neurons[J].Nat Neurosci,2010,13:1075-1081.
[13]Tye KM,Deisseroth K.Optogenetic investigation of neural circuits underlying brain disease in animal models[J].Nat Rev Neurosci,2012,13:251-266.
[14]Zhang F,Wang LP,Brauner M,et al.Multimodal fast optical interrogation of neural circuitry[J].Nature,2007,446:633-639.
[15]Zingg B,Chou X,Zhang Z,et al.AAV-mediated anterograde transsynaptic tagging:mapping input-defined functional neural pathways for defense behavior[J].Neuron,2017,93:33-47.
[2]Hantman AW,Jessell TM.Clarke's column neurons as the focus of a corticospinal corollary circuit[J].Nat Neurosci,2010,13:1233-1239.
[3]Boubker Z,Edgley SA,Soteropoulos DS,et al.Changes in descending motor pathway connectivity after corticospinal tract lesion in macaque monkey[J].Brain,2012,135:2277-2289.
[4]Jiang YQ,Zaaimi B,Martin JH.Competition with primary sensory afferents drives remodeling of corticospinal axons in mature spinal motor circuits[J].J Neurosci,2016,36:193-203.
[5]Glover JC,Petursdottir G,Jansen JKS.Fluorescent dextran-amines used as axonal tracers in the nervous system of the chicken embryo[J].J Neurosci Methods,1986,18:243-254.
[6]Mccarty DM,Monahan PE,Samulski RJ.Self-complementary recombinant adeno-associated virus(sc AAV)vectors promote efficient transduction independently of DNA synthesis[J].Gene Ther,2001,8:1248-1254.
[7]Zhang J,Xi M,Fung SJ,et al.Projections from the central nucleus of the amygdala to the nucleus pontis oralis in the rat:An anterograde labeling study[J].Neurosci Lett,2012,525:157-162.
[8]Lazarov NE.Neuroanatomical tract-tracing using biotinylated dextran amine.[J].Methods Mol Biol,2013,1018:323-334.
[9]Reiner A,Veenman CL,Medina L,et al.Pathway tracing using biotinylated dextran amines.[J].J Neurosci Methods,2000,103:23-37.
[10]Cabantous S,Terwilliger TC,Waldo GS.Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein[J].Nat Biotechnol,2005,23:102-107.
[11]Li K,Zhou T,Liao L,et al.βCa MKII in lateral habenula mediates core symptoms of depression[J].Science,2013,341:1016-1020.
[12]Liu K,Lu Y,Lee JK,et al.PTEN deletion enhances the regenerative ability of adult corticospinal neurons[J].Nat Neurosci,2010,13:1075-1081.
[13]Tye KM,Deisseroth K.Optogenetic investigation of neural circuits underlying brain disease in animal models[J].Nat Rev Neurosci,2012,13:251-266.
[14]Zhang F,Wang LP,Brauner M,et al.Multimodal fast optical interrogation of neural circuitry[J].Nature,2007,446:633-639.
[15]Zingg B,Chou X,Zhang Z,et al.AAV-mediated anterograde transsynaptic tagging:mapping input-defined functional neural pathways for defense behavior[J].Neuron,2017,93:33-47.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |