Department of Molecular and Systems Neurobiology, Graduate School of Medicine, The University of Tokyo, Japan.
Mol Cell Neurosci. 2011 Nov;48(3):246-57. doi: 10.1016/j.mcn.2011.08.005. Epub 2011 Aug 22.
To understand the fine-scale structures and functional properties of individual neurons in vivo, we developed and validated a rapid genetic technique that enables simultaneous investigation of multiple neuronal properties with single-cell resolution in the living rodent brain. Our technique PASME (promoter-assisted sparse-neuron multiple-gene labeling using in uteroelectroporation) targets specific small subsets of sparse pyramidal neurons in layer 2/3, layer 5 of the cerebral cortex and in the hippocampus with multiple fluorescent reporter proteins such as postsynaptic PSD-95-GFP and GFP-gephyrin. The technique is also applicable for targeting independently individual neurons and their presynaptic inputs derived from surrounding neurons. Targeting sparse layer 2/3 neurons, we uncovered a novel subpopulation of layer 2/3 neurons in the mouse cerebral cortex. This technique, broadly applicable for probing and manipulating neurons with single-cell resolution in vivo, should provide a robust means to uncover the basic mechanisms employed by the brain, especially when combined with in vivo two-photon laser-scanning microscopy and/or optogenetic technologies.
为了理解单个神经元的精细结构和功能特性，我们开发并验证了一种快速的遗传技术，该技术能够在活体啮齿动物大脑中以单细胞分辨率同时研究多个神经元的特性。我们的技术 PASME（使用子宫内电穿孔的启动子辅助稀疏神经元多基因标记）以大脑皮层 2/3 层、5 层和海马体中的特定小部分稀疏锥体神经元为目标，使用多个荧光报告蛋白，如突触后 PSD-95-GFP 和 GFP-gephyrin。该技术也适用于靶向独立的单个神经元及其来自周围神经元的突触前输入。通过靶向稀疏的 2/3 层神经元，我们在小鼠大脑皮层中发现了一个新的 2/3 层神经元亚群。这项技术广泛适用于在活体中以单细胞分辨率探测和操纵神经元，应该为揭示大脑所采用的基本机制提供一种强大的手段，特别是当与活体双光子激光扫描显微镜和/或光遗传学技术结合使用时。
