Targeted patch-clamp recordings and single-cell electroporation of unlabeled neurons in vivo
Targeted patch-clamp recordings and single-cell electroporation of unlabeled neurons in vivo
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DOI：
10.1038/nmeth1150
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发表时间：
2008-01-01
期刊：
影响因子：
48
通讯作者：
Hausser, Michael
中科院分区：
文献类型：
--
作者：
Kitamura, Kazuo;Judkewitz, Benjamin;Hausser, Michael
在这里，我们描述了一种方法，使有针对性的膜片钳记录单神经元在体内，可视化的双光子显微镜。贴片电极用于用荧光染料灌注感兴趣的神经元周围的细胞外空间，从而使神经元能够被可视化为负像（“阴影”），并基于其体树突结构来识别。然后，在视觉控制下，将相同的电极放置在神经元上，以形成千兆密封（“阴影修补”）。我们证明了shadowpatching的可靠性和多功能性，从视觉识别的神经元在大鼠和小鼠的新皮层和小脑进行全细胞记录。我们还表明，该方法可用于有针对性的在体内单细胞电穿孔质粒DNA到确定的细胞类型，导致稳定的转基因表达。这种方法有利于记录，标记和遗传操作的单个神经元在完整的天然哺乳动物的大脑，而不需要预先标记的神经元群体。
Here we describe an approach for making targeted patch-clamp recordings from single neurons in vivo, visualized by two-photon microscopy. A patch electrode is used to perfuse the extracellular space surrounding the neuron of interest with a fluorescent dye, thus enabling the neuron to be visualized as a negative image ('shadow') and identified on the basis of its somatodendritic structure. The same electrode is then placed on the neuron under visual control to allow formation of a gigaseal ('shadowpatching'). We demonstrate the reliability and versatility of shadowpatching by performing whole-cell recordings from visually identified neurons in the neocortex and cerebellum of rat and mouse. We also show that the method can be used for targeted in vivo single-cell electroporation of plasmid DNA into identified cell types, leading to stable transgene expression. This approach facilitates the recording, labeling and genetic manipulation of single neurons in the intact native mammalian brain without the need to pre-label neuronal populations.