Virus-mediated swapping of zolpidem-insensitive with zolpidem-sensitive GABA(A) receptors in cortical pyramidal cells.
Virus-mediated swapping of zolpidem-insensitive with zolpidem-sensitive GABA(A) receptors in cortical pyramidal cells.
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病毒介导的Zolpidem对皮质锥体细胞中对唑吡恩敏感的GABA（A）受体的交换。
DOI：
10.1113/jphysiol.2012.227538
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发表时间：
2012-04-01
期刊：
影响因子：
--
通讯作者：
Shigemoto R
中科院分区：
文献类型：
--
作者：
Sumegi M;Fukazawa Y;Matsui K;Lorincz A;Eyre MD;Nusser Z;Shigemoto R
近年来发展起来的药物遗传学和光遗传学方法各有优缺点，已成为现代神经科学研究中不可或缺的工具。在此，我们采用了先前描述的敲入小鼠系（GABAARγ 277 Ilox），其中GABAA受体（GABAAR）的γ2亚基突变为唑吡坦不敏感（γ 277 I），并使用病毒载体将γ 277 I与野生型唑吡坦敏感的γ2亚基（γ 277 F）交换。验证病毒引入的γ2亚基的密度和亚细胞分布不变需要它们的选择性标记。为此，我们产生了6个N-和6个C-末端标记的γ2亚基，用慢病毒转导GABAARγ2−/−小鼠的皮质培养物。我们发现，N-末端AU 1标签导致出色的免疫检测和未受损的突触定位。用全细胞膜片钳记录来自培养细胞的自发IPSC，证明了AU 1标记的γ2（AU 1 γ 277 F）通道的动力学特性未发生改变。接下来，我们将含有Cre重组酶和AU 1 γ 277 F亚基的慢病毒和腺相关病毒（Cre-2A-AU 1 γ 277 F）立体定位注射到GABAARγ 277 Ilox小鼠的新皮质中。光学显微镜免疫荧光和电子显微镜冷冻断裂复制免疫金标记证明了AU 1标签的有效免疫检测和体周GABA能突触中AU 1 γ 277 F亚基的正常富集。与此一致，从转导细胞记录的微型和动作电位诱发的IPSC全细胞具有不变的振幅、动力学和恢复的唑吡坦敏感性。我们的结果得到了广泛的结构和功能验证方法揭示了γ 277 I和AU 1 γ 277 F GABAARs在皮质锥体细胞中的亚细胞分布和功能特性没有改变。这种转基因-病毒药物遗传学方法的优点在于，它不需要任何可能赋予遗传修饰细胞一些不可预见的改变的外源蛋白。此外，这种基于病毒的方法开辟了在不同脑区修饰多种细胞类型和进行替代性基于重组的交叉遗传操作的可能性。
Recently developed pharmacogenetic and optogenetic approaches, with their own advantages and disadvantages, have become indispensable tools in modern neuroscience. Here, we employed a previously described knock-in mouse line (GABAARγ277Ilox) in which the γ2 subunit of the GABAA receptor (GABAAR) was mutated to become zolpidem insensitive (γ277I) and used viral vectors to swap γ277I with wild-type, zolpidem-sensitive γ2 subunits (γ277F). The verification of unaltered density and subcellular distribution of the virally introduced γ2 subunits requires their selective labelling. For this we generated six N- and six C-terminal-tagged γ2 subunits, with which cortical cultures of GABAARγ2−/− mice were transduced using lentiviruses. We found that the N-terminal AU1 tag resulted in excellent immunodetection and unimpaired synaptic localization. Unaltered kinetic properties of the AU1-tagged γ2 (AU1γ277F) channels were demonstrated with whole-cell patch-clamp recordings of spontaneous IPSCs from cultured cells. Next, we carried out stereotaxic injections of lenti- and adeno-associated viruses containing Cre-recombinase and the AU1γ277F subunit (Cre-2A-AU1γ277F) into the neocortex of GABAARγ277Ilox mice. Light microscopic immunofluorescence and electron microscopic freeze-fracture replica immunogold labelling demonstrated the efficient immunodetection of the AU1 tag and the normal enrichment of the AU1γ277F subunits in perisomatic GABAergic synapses. In line with this, miniature and action potential-evoked IPSCs whole-cell recorded from transduced cells had unaltered amplitudes, kinetics and restored zolpidem sensitivity. Our results obtained with a wide range of structural and functional verification methods reveal unaltered subcellular distributions and functional properties of γ277I and AU1γ277F GABAARs in cortical pyramidal cells. This transgenic–viral pharmacogenetic approach has the advantage that it does not require any extrinsic protein that might endow some unforeseen alterations of the genetically modified cells. In addition, this virus-based approach opens up the possibility of modifying multiple cell types in distinct brain regions and performing alternative recombination-based intersectional genetic manipulations.
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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.
DOI：
10.1126/science.1187958
发表时间：
2010-05-14
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Lorincz A;Nusser Z
通讯作者：
Nusser Z
影响因子：
2.5
作者：
Hagiwara, A;Fukazawa, Y;Shigemoto, R
通讯作者：
Shigemoto, R
影响因子：
25
作者：
Essrich, C;Lorez, M;Lüscher, B
通讯作者：
Lüscher, B
DOI：
10.1073/pnas.0706773104
发表时间：
2007-09-25
影响因子：
11.1
作者：
Limon, Agenor;Reyes-Ruiz, Jorge Mauricio;Miledi, Ricardo
通讯作者：
Miledi, Ricardo