Targeted genetic manipulations of neuronal subtypes using promoter-specific combinatorial AAVs in wild-type animals.
Targeted genetic manipulations of neuronal subtypes using promoter-specific combinatorial AAVs in wild-type animals.
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DOI：
10.3389/fnbeh.2015.00152
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发表时间：
2015
影响因子：
3
通讯作者：
Bass CE
中科院分区：
文献类型：
--
作者：
Gompf HS;Budygin EA;Fuller PM;Bass CE
基因控制特定神经元亚群活性的技术在阐明功能方面是有用的，但在转基因小鼠以外的翻译研究中的适用性是有限的。利用特定的启动子可以实现亚型靶向转基因的表达，但目前可用的启动子要么太大而无法包装到许多载体中，特别是腺相关病毒(AAV)，要么不能在足以改变行为的水平上驱动表达。为了允许神经元亚型特异性基因在野生型动物中表达，我们开发了一种组合AAV靶向系统，该系统结合驱动亚型特异性Cre重组酶的表达和强烈但非特异性的Cre条件转基因。利用该系统，我们证明了酪氨酸羟化酶启动子(TH-Cre-AAv)将通道视紫红质-2(Ef1α-DIO-ChR2-EYFP-AAv)的表达限制在大鼠的腹侧被盖区，或激活DREADD(hSyn-DIo-hM3Dq-mCherry-AAv)来激活大鼠蓝斑中心( ，LC)。这两个区域都获得了高水平的表达。免疫组织化学(IHC)显示VTA内大部分ChR2+神经元(>；93%)与TH共存，光刺激可引起纹状体多巴胺的释放。激活LC内的TH神经元可产生持续的脑电和行为觉醒。进一步比较了TH特异性hM3Dq在LC中的表达：(1)由强大但非特异性启动子驱动的Cre结构(非靶向)；(2)逆行运输的WGA-Cre递送机制(针对特定的投影)。IHC显示，CNO处理后LC和LC周围神经元中c-fos的激活面积似乎与由此导致的觉醒增加成正比(非靶向>；靶向>；ACC对LC的投射受到限制)。我们的双重AAV靶向系统通过从功能上分离亚型特异性和启动子强度，有效地克服了许多亚型特异性启动子普遍存在的大尺寸和弱活性障碍。
Techniques to genetically manipulate the activity of defined neuronal subpopulations have been useful in elucidating function, however applicability to translational research beyond transgenic mice is limited. Subtype targeted transgene expression can be achieved using specific promoters, but often currently available promoters are either too large to package into many vectors, in particular adeno-associated virus (AAV), or do not drive expression at levels sufficient to alter behavior. To permit neuron subtype specific gene expression in wildtype animals, we developed a combinatorial AAV targeting system that drives, in combination, subtype specific Cre-recombinase expression with a strong but non-specific Cre-conditional transgene. Using this system we demonstrate that the tyrosine hydroxylase promoter (TH-Cre-AAV) restricted expression of channelrhodopsin-2 (EF1α-DIO-ChR2-EYFP-AAV) to the rat ventral tegmental area (VTA), or an activating DREADD (hSyn-DIO-hM3Dq-mCherry-AAV) to the rat locus coeruleus (LC). High expression levels were achieved in both regions. Immunohistochemistry (IHC) showed the majority of ChR2+ neurons (>93%) colocalized with TH in the VTA, and optical stimulation evoked striatal dopamine release. Activation of TH neurons in the LC produced sustained EEG and behavioral arousal. TH-specific hM3Dq expression in the LC was further compared with: (1) a Cre construct driven by a strong but non-specific promoter (non-targeting); and (2) a retrogradely-transported WGA-Cre delivery mechanism (targeting a specific projection). IHC revealed that the area of c-fos activation after CNO treatment in the LC and peri-LC neurons appeared proportional to the resulting increase in wakefulness (non-targeted > targeted > ACC to LC projection restricted). Our dual AAV targeting system effectively overcomes the large size and weak activity barrier prevalent with many subtype specific promoters by functionally separating subtype specificity from promoter strength.
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影响因子：
5.3
作者：
Konadhode, Roda Rani;Pelluru, Dheeraj;Shiromani, Priyattam J.
通讯作者：
Shiromani, Priyattam J.
影响因子：
16.2
作者：
Lammel, Stephan;Steinberg, Elizabeth E.;Foeldy, Csaba;Wall, Nicholas R.;Beier, Kevin;Luo, Liqun;Malenka, Robert C.
通讯作者：
Malenka, Robert C.
DOI：
10.1073/pnas.0700293104
发表时间：
2007-03-20
影响因子：
11.1
作者：
Armbruster, Blaine N.;Li, Xiang;Roth, Bryan L.
通讯作者：
Roth, Bryan L.
影响因子：
48
作者：
Brown, Andrew J.;Fisher, Daniel A.;Cui, Xiaoxia
通讯作者：
Cui, Xiaoxia
影响因子：
--
作者：
Gore, Bryan B;Soden, Marta E;Zweifel, Larry S
通讯作者：
Zweifel, Larry S