In vivo electroporation to physiologically identified deep brain regions in postnatal mammals.
In vivo electroporation to physiologically identified deep brain regions in postnatal mammals.
复制标题
对出生后哺乳动物的生理学鉴定的深部脑区域进行体内电穿孔。
DOI：
10.1007/s00429-014-0724-x
复制
发表时间：
2015
影响因子：
3.1
通讯作者：
Satoh T. and Hata Y.
中科院分区：
文献类型：
--
作者：
Ohmura N.;Kawasaki K.;Satoh T. and Hata Y.
遗传操作被广泛应用于中枢神经系统的研究。在少数神经元中操纵分子表达，可以详细研究特定分子对神经元功能和形态的作用。电穿孔是一种广泛应用于中枢神经系统基因转移的技术。然而，在以前的报道中，利用电穿孔技术在产后动物中靶向基因转移仅限于皮质、海马或面向脑室的区域。电穿孔瞄准脑深部结构，如丘脑，一直很困难。我们介绍了一种新的电穿孔技术，使基因转移到一个生理识别的脑深部区域使用玻璃移液管。我们使用含有编码增强型绿色荧光蛋白（EGFP）的质粒DNA的玻璃移液电极，记录了年轻成年小鼠的神经活动，以确定丘脑外侧膝状核（LGN）的位置。通过监测视觉反应来确定LGN的位置，并将质粒溶液加压注入记录部位。电压脉冲通过玻璃移液电极传递。几周后，在LGN中观察到egfp标记的体细胞和树突，在视觉皮层中发现标记的轴突。对表达egfp的结构进行了详细的观察，足以在三维上重建其形态。我们进一步证实了这项技术在猫身上的适用性。这种方法对于将各种基因转移到啮齿动物和脑回哺乳动物生理上确定的大脑区域的细胞中应该是有用的。
Genetic manipulation is widely used to research the central nervous system (CNS). The manipulation of molecular expression in a small number of neurons permits the detailed investigation of the role of specific molecules on the function and morphology of the neurons. Electroporation is a broadly used technique for gene transfer in the CNS. However, the targeting of gene transfer using electroporation in postnatal animals was restricted to the cortex, hippocampus, or the region facing the ventricle in previous reports. Electroporation targeting of deep brain structures, such as the thalamus, has been difficult. We introduce a novel electroporation technique that enables gene transfer to a physiologically identified deep brain region using a glass pipette. We recorded neural activity in young-adult mice to identify the location of the lateral geniculate nucleus (LGN) of the thalamus, using a glass pipette electrode containing the plasmid DNA encoding enhanced green fluorescent protein (EGFP). The location of the LGN was confirmed by monitoring visual responses, and the plasmid solution was pressure-injected into the recording site. Voltage pulses were delivered through the glass pipette electrode. Several EGFP-labeled somata and dendrites were observed in the LGN after a few weeks, and labeled axons were found in the visual cortex. The EGFP-expressing structures were observed in detail sufficient to reconstruct their morphology in three dimensions. We further confirmed the applicability of this technique in cats. This method should be useful for the transfer of various genes into cells in physiologically identified brain regions in rodents and gyrencephalic mammals.