Neural Regeneration Research ›› 2023, Vol. 18 ›› Issue (8): 1827-1833.doi: 10.4103/1673-5374.358618

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A rabies virus-based toolkit for efficient retrograde labeling and monosynaptic tracing

Kun-Zhang Lin1, 2, *, Lei Li2, Wen-Yu Ma2, 3, Xin Yang2, Zeng-Peng Han1, 2, 3, Neng-Song Luo1, 2, 4, Jie Wang2, 3, Fu-Qiang Xu1, 2, 3, 4, 5, 6, *   

  1. 1The Brain Cognition and Brain Disease Institute (BCBDI), Shenzhen Key Laboratory of Viral Vectors for Biomedicine, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences; Shenzhen-Hong Kong Institute of Brain Science-Shenzhen Fundamental Research Institutions, NMPA Key Laboratory for Research and Evaluation of Viral Vector Technology in Cell and Gene Therapy Medicinal Products, Shenzhen, Key Laboratory of Quality Control Technology for Virus-Based Therapeutics, Guangdong Provincial Medical Products Administration, Shenzhen, Guangdong Province, China; 2State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, National Center for Magnetic Resonance in Wuhan, Wuhan Institute of Physics and Mathematics, Innovation Academy for Precision Measurement Science and Technology, Chinese Academy of Sciences-Wuhan National Laboratory for Optoelectronics, Wuhan, Hubei Province, China; 3University of Chinese Academy of Sciences, Beijing, China; 4Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Wuhan, Hubei Province, China; 5Shenzhen-Hong Kong Institute of Brain Science-Shenzhen Fundamental Research Institutions, Shenzhen, Guangdong Province, China; 6Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences, Shanghai, China.
  • Online:2023-08-15 Published:2023-02-24
  • Contact: Fu-Qiang Xu, PhD, fq.xu@siat.ac.cn; Kun-Zhang Lin, PhD, kz.lin@siat.ac.cn.
  • Supported by:
    This study was supported by the National Natural Science Foundation of China, Nos. 32100899 (to KZL), 31830035 (to FQX), 31771156 (to FQX), 21921004 (to FQX); the National Science and Technology Innovation 2030, No. 2021ZD0201003 (to FQX); the Key-Area Research and Development Program of Guangdong Province, No. 2018B030331001 (to FQX); the Strategic Priority Research Program of the Chinese Academy of Sciences, No. XDB32030200 (to FQX); and the Shenzhen Key Laboratory of Viral Vectors for Biomedicine, No. ZDSYS20200811142401005 (to FQX).

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Abstract: Analyzing the structure and function of the brain’s neural network is critical for identifying the working principles of the brain and the mechanisms of brain diseases. Recombinant rabies viral vectors allow for the retrograde labeling of projection neurons and cell type-specific trans-monosynaptic tracing, making these vectors powerful candidates for the dissection of synaptic inputs. Although several attenuated rabies viral vectors have been developed, their application in studies of functional networks is hindered by the long preparation cycle and low yield of these vectors. To overcome these limitations, we developed an improved production system for the rapid rescue and preparation of a high-titer CVS-N2c-ΔG virus. Our results showed that the new CVS-N2c-ΔG-based toolkit performed remarkably: (1) N2cG-coated CVS-N2c-ΔG allowed for efficient retrograde access to projection neurons that were unaddressed by rAAV9-Retro, and the efficiency was six times higher than that of rAAV9-Retro; (2) the trans-monosynaptic efficiency of oG-mediated CVS-N2c-ΔG was 2–3 times higher than that of oG-mediated SAD-B19-ΔG; (3) CVS-N2c-ΔG could delivery modified genes for neural activity monitoring, and the time window during which this was maintained was 3 weeks; and (4) CVS-N2c-ΔG could express sufficient recombinases for efficient transgene recombination. These findings demonstrate that new CVS-N2c-ΔG-based toolkit may serve as a versatile tool for structural and functional studies of neural circuits.

Key words: functional studies, neural activity, neural circuits, projection neurons, rAAV9-Retro, rabies virus, recombination, retrograde labeling, synaptic inputs, trans-monosynaptic tracing