Rabies virus-based labeling of layer 6 corticothalamic neurons for two-photon imaging in vivo

Miaoqing Gu; Xiuli Li; Shanshan Liang; Jiahui Zhu; Pei Sun; Yong He; Haipeng Yu; Ruijie Li; Zhenqiao Zhou; Jing Lyu; Sunny C Li; Eike Budinger; Yi Zhou; Hongbo Jia; Jianxiong Zhang; Xiaowei Chen

doi:10.1016/j.isci.2023.106625

Rabies virus-based labeling of layer 6 corticothalamic neurons for two-photon imaging in vivo

iScience. 2023 Apr 8;26(5):106625. doi: 10.1016/j.isci.2023.106625. eCollection 2023 May 19.

Authors

Miaoqing Gu^{1

2}, Xiuli Li³, Shanshan Liang³, Jiahui Zhu², Pei Sun³, Yong He³, Haipeng Yu⁴, Ruijie Li³, Zhenqiao Zhou⁵, Jing Lyu⁵, Sunny C Li², Eike Budinger⁶, Yi Zhou^{2

4}, Hongbo Jia^{1

5

6}, Jianxiong Zhang³, Xiaowei Chen^{3

7}

Affiliations

¹ School of Physical Science and Technology, Guangxi University, Nanning 530004, China.
² Advanced Institute for Brain and Intelligence, School of Medicine, Guangxi University, Nanning 530004, China.
³ Brain Research Center and State Key Laboratory of Trauma, Burns, and Combined Injury, Third Military Medical University, Chongqing 400038, China.
⁴ Department of Neurobiology, Chongqing Key Laboratory of Neurobiology, School of Basic Medicine, Third Military Medical University, Chongqing 400038, China.
⁵ Brain Research Instrument Innovation Center, Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou 215163, China.
⁶ Combinatorial NeuroImaging Core Facility, Leibniz Institute for Neurobiology, 39118 Magdeburg, Germany.
⁷ Guangyang Bay Laboratory, Chongqing Institute for Brain and Intelligence, Chongqing 400064, China.

Abstract

Neocortical layer 6 (L6) is less understood than other more superficial layers, largely owing to limitations of performing high-resolution investigations in vivo. Here, we show that labeling with the Challenge Virus Standard (CVS) rabies virus strain enables high-quality imaging of L6 neurons by conventional two-photon microscopes. CVS virus injection into the medial geniculate body can selectively label L6 neurons in the auditory cortex. Only three days after injection, dendrites and cell bodies of L6 neurons could be imaged across all cortical layers. Ca²⁺ imaging in awake mice showed that sound stimulation evokes neuronal responses from cell bodies with minimal contamination from neuropil signals. In addition, dendritic Ca²⁺ imaging revealed significant responses from spines and trunks across all layers. These results demonstrate a reliable method capable of rapid, high-quality labeling of L6 neurons that can be readily extended to other brain regions.

Keywords: Cellular neuroscience; Molecular neuroscience; Optical imaging.