Long-lasting single-neuron labeling by in vivo electrop oration without microscopic guidance
Long-lasting single-neuron labeling by in vivo electrop oration without microscopic guidance
复制标题
通过体内电穿孔实现持久的单神经元标记，无需显微镜引导
DOI：
10.1016/j.jneumeth.2013.06.004
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发表时间：
2013
影响因子：
3
通讯作者：
Ken-Ichiro Tsutsui
中科院分区：
文献类型：
--
作者：
Kei Oyama;Shinya Ohara;Sho Satoa;Fuyuki Karube;Fumino Fujiyama;Yoshikazu Isomura;Hajime Mushiake;Toshio Iijima;Ken-Ichiro Tsutsui
为了使神经系统的形态学和功能研究之间的直接联系，我们建立了一个实验方案，通过注射编码荧光蛋白的质粒电穿孔后，记录其细胞外的活动持续标记个别神经元没有显微镜的指导。使用装有含有编码绿色荧光蛋白（GFP）的质粒的电解质溶液的玻璃移液管，对麻醉大鼠进行单神经元记录和电穿孔。当在记录完成时进行电穿孔时，通过监测记录的动作电位的轨迹的变化和电极电阻的增加来调节目标神经元和电极尖端之间的接触程度。GFP的表达及其用多克隆抗体的免疫染色使我们能够清楚地看到基本的结构成分，如细胞体，轴突，树突，甚至更小的成分，如刺。每个标记的神经元的形态亚型的识别是可能的。标记的最佳条件是电极电阻增加30%，标记后3天评价的标记成功率为40%。贴标签后一个月评估的比率仅略低（33%）。我们还通过实验证实，这种记录和标记程序在头部固定的行为大鼠中也同样成功。这项新的实验方案将是系统神经科学的一个突破，因为它将单个神经元的形态和行为相关活动直接联系起来。
In order to make a direct link between the morphological and functional study of the nervous system, we established an experimental protocol for labeling individual neurons persistently without microscopic guidance by injecting a plasmid encoding fluorescent protein electroporatively after recording their activity extracellularly. Using a glass pipette filled with electrolyte solution containing a plasmid encoding green fluorescent protein (GFP), single-neuron recording and electroporation were performed on anesthetized rats. When performing the electroporation at the completion of recording, the degree of contact between the target neuron and the electrode tip was adjusted by monitoring the change of the trace of recorded action potentials and the increase of electrode resistance. The expression of GFP and its immunostaining with a polyclonal antibody enabled us to clearly see the basic structural components such as cell bodies, axons, dendrites, and even smaller components such as spines. Identification of the morphological subtypes of neurons was possible with every labeled neuron. The optimum condition for labeling was a 30% increase of the electrode resistance, and the labeling success rate evaluated 3 days after labeling was 40%. The rate evaluated one month after labeling was only slightly lower (33%). We also confirmed experimentally that this recording and labeling procedure can be similarly successful in head-fixed behaving rats. This new experimental protocol will be a breakthrough in systems neuroscience because it makes a direct link between the morphology and behavior-related activity of single neurons.