In Vivo Two-Photon Imaging of Dendritic Spines in Marmoset Neocortex.
In Vivo Two-Photon Imaging of Dendritic Spines in Marmoset Neocortex.
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DOI：
10.1523/eneuro.0019-15.2015
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发表时间：
2015-07
期刊：
影响因子：
3.4
通讯作者：
Yamamori T
中科院分区：
文献类型：
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作者：
Sadakane O;Watakabe A;Ohtsuka M;Takaji M;Sasaki T;Kasai M;Isa T;Kato G;Nabekura J;Mizukami H;Ozawa K;Kawasaki H;Yamamori T
双光子显微镜与人工表达荧光蛋白的技术相结合，使人们能够直接观察活体大脑中的树突棘。然而，这种方法在灵长类动物大脑中的应用一直受到阻碍，缺乏适当的标记技术可视化树突棘。在这里，我们开发了一种基于腺相关病毒载体的荧光蛋白表达系统，用于在体内观察绒猴新皮层中的树突棘。为了使每个棘的清晰可视化，报告荧光蛋白的表达应该既稀疏又强烈。为了满足这些要求，我们使用四环素反式激活因子（tTA）-四环素响应元件系统，并通过滴定Thy 1 S启动子驱动的tTA稀疏表达的量来放大荧光信号。通过这种方法，我们能够在体内用双光子显微镜观察绒猴皮层中的树突棘，并分析前额叶皮层中棘的周转。我们的研究结果表明，在绒猴皮层的短刺往往比长刺更频繁地变化。体内样品与固定样品的比较表明，我们没有检测到所有现有的棘通过我们的方法。虽然我们发现神经胶质细胞增殖，但从有或没有窗口结构的样品之间的脊柱长度的比较来看，窗口结构引起的组织损伤相对较小。我们的新标记技术的双光子成像可视化在体内的绒猴新皮层的树突棘可以适用于检查电路重组和突触可塑性的灵长类动物。
Two-photon microscopy in combination with a technique involving the artificial expression of fluorescent protein has enabled the direct observation of dendritic spines in living brains. However, the application of this method to primate brains has been hindered by the lack of appropriate labeling techniques for visualizing dendritic spines. Here, we developed an adeno-associated virus vector-based fluorescent protein expression system for visualizing dendritic spines in vivo in the marmoset neocortex. For the clear visualization of each spine, the expression of reporter fluorescent protein should be both sparse and strong. To fulfill these requirements, we amplified fluorescent signals using the tetracycline transactivator (tTA)–tetracycline-responsive element system and by titrating down the amount of Thy1S promoter-driven tTA for sparse expression. By this method, we were able to visualize dendritic spines in the marmoset cortex by two-photon microscopy in vivo and analyze the turnover of spines in the prefrontal cortex. Our results demonstrated that short spines in the marmoset cortex tend to change more frequently than long spines. The comparison of in vivo samples with fixed samples showed that we did not detect all existing spines by our method. Although we found glial cell proliferation, the damage of tissues caused by window construction was relatively small, judging from the comparison of spine length between samples with or without window construction. Our new labeling technique for two-photon imaging to visualize in vivo dendritic spines of the marmoset neocortex can be applicable to examining circuit reorganization and synaptic plasticity in primates.