Laboratory for Molecular Analysis of Higher Brain Function, Brain Science Institute, RIKEN, Wako, Saitama 351-0198, Japan ; Division of Brain Biology, National Institute for Basic Biology , Aichi 444-8585, Japan.
Department of Ultrastructural Research, National Center of Neurology and Psychiatry, National Institute of Neuroscience , Tokyo 187-8502, Japan.
eNeuro. 2015 Sep 17;2(4). doi: 10.1523/ENEURO.0019-15.2015. eCollection 2015 Jul-Aug.
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.
双光子显微镜结合人工表达荧光蛋白的技术，使我们能够直接观察活体大脑中的树突棘。然而，由于缺乏用于可视化树突棘的适当标记技术，该方法在灵长类动物大脑中的应用受到了限制。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，用于在食蟹猴新皮层中可视化体内的树突棘。为了清楚地观察到每个棘突，报告荧光蛋白的表达应该稀疏且强烈。为了满足这些要求，我们使用四环素转录激活剂（tTA）-四环素反应元件系统放大荧光信号，并通过滴定 Thy1S 启动子驱动的 tTA 的量来实现稀疏表达。通过这种方法，我们能够通过活体双光子显微镜观察食蟹猴皮层中的树突棘，并分析前额叶皮层中棘突的周转。我们的结果表明，食蟹猴皮层中的短棘突比长棘突更容易频繁变化。体内样本与固定样本的比较表明，我们的方法并没有检测到所有现有的棘突。虽然我们发现了神经胶质细胞的增殖，但从有窗和无窗样本之间棘突长度的比较来看，窗口构建对组织的损伤相对较小。我们用于可视化食蟹猴新皮层体内树突棘的双光子成像的新标记技术可适用于检查灵长类动物的回路重组和突触可塑性。
