Optical Approaches to Brain Function Laboratory, Department of Neuroscience and Brain Technologies, Istituto Italiano di Tecnologia, Via Morego 30, 16163 Genova, Italy.
School of Physics and Astronomy, Italy-Israel Joint Neuroscience Laboratory, Tel Aviv University, 69978 Tel Aviv, Israel.
Sci Rep. 2017 Jan 5;7:40041. doi: 10.1038/srep40041.
Genetically encoded calcium indicators and optogenetic actuators can report and manipulate the activity of specific neuronal populations. However, applying imaging and optogenetics simultaneously has been difficult to establish in the mammalian brain, even though combining the techniques would provide a powerful approach to reveal the functional organization of neural circuits. Here, we developed a technique based on patterned two-photon illumination to allow fast scanless imaging of GCaMP6 signals in the intact mouse brain at the same time as single-photon optogenetic inhibition with Archaerhodopsin. Using combined imaging and electrophysiological recording, we demonstrate that single and short bursts of action potentials in pyramidal neurons can be detected in the scanless modality at acquisition frequencies up to 1 kHz. Moreover, we demonstrate that our system strongly reduces the artifacts in the fluorescence detection that are induced by single-photon optogenetic illumination. Finally, we validated our technique investigating the role of parvalbumin-positive (PV) interneurons in the control of spontaneous cortical dynamics. Monitoring the activity of cellular populations on a precise spatiotemporal scale while manipulating neuronal activity with optogenetics provides a powerful tool to causally elucidate the cellular mechanisms underlying circuit function in the intact mammalian brain.
基因编码的钙指示剂和光遗传学驱动器可以报告和操纵特定神经元群体的活动。然而，即使将这些技术结合起来可以提供一种强大的方法来揭示神经回路的功能组织，在哺乳动物大脑中同时应用成像和光遗传学仍然很难实现。在这里，我们开发了一种基于图案化双光子照明的技术，允许在完整的小鼠大脑中同时进行 GCaMP6 信号的快速无扫描成像，同时使用 Archaerhodopsin 进行单光子光遗传学抑制。通过结合成像和电生理记录，我们证明在高达 1 kHz 的采集频率下，可以在无扫描模式下检测到单个和短爆发的动作电位。此外，我们证明我们的系统可以大大减少单光子光遗传学照明引起的荧光检测中的伪影。最后，我们通过验证我们的技术来研究 PV 阳性（PV）中间神经元在皮层自发动力学控制中的作用。在利用光遗传学操纵神经元活动的同时，在精确的时空尺度上监测细胞群体的活动，为阐明完整哺乳动物大脑中回路功能的细胞机制提供了有力工具。
