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 th...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.展开更多
Behavioral studies play a crucial role in unraveling the mechanisms underlying brain function.Recent advances in optogenetics,neuronal typing and labeling,and circuit tracing have facilitated the dissection of the neu...Behavioral studies play a crucial role in unraveling the mechanisms underlying brain function.Recent advances in optogenetics,neuronal typing and labeling,and circuit tracing have facilitated the dissection of the neural circuitry involved in various important behaviors.The identification of monosynaptic connections,both upstream and downstream of specific neurons,serves as the foundation for understanding complex neural circuits and studying behavioral mechanisms.However,the practical implementation and mechanistic understanding of monosynaptic connection tracing techniques and functional identification remain challenging,particularly for inexperienced researchers.Improper application of these methods and misinterpretation of results can impede experimental progress and lead to erroneous conclusions.In this paper,we present a comprehensive description of the principles,specific operational details,and key steps involved in tracing anterograde and retrograde monosynaptic connections.We outline the process of functionally identifying monosynaptic connections through the integration of optogenetics and electrophysiological techniques,providing practical guidance for researchers.展开更多
基金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)+2 种基金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)the Shenzhen Key Laboratory of Viral Vectors for Biomedicine,No.ZDSYS20200811142401005(to FQX)。
文摘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.
基金National Key Research and Development Program of China(2022YFC2402703)Scientific and Technological Innovation 2030 Program of China,major projects(2021ZD0200408)+3 种基金National Natural Science Foundation of China(32171007 and 31800880)Non-profit Central Research Institute Fund of the Chinese Academy of Medical Sciences(2018PT31041)Science and Technology Planning Project of Guangdong Province(2018B030331001)Fundamental Research Funds for the Central Universities(K20210089).
文摘Behavioral studies play a crucial role in unraveling the mechanisms underlying brain function.Recent advances in optogenetics,neuronal typing and labeling,and circuit tracing have facilitated the dissection of the neural circuitry involved in various important behaviors.The identification of monosynaptic connections,both upstream and downstream of specific neurons,serves as the foundation for understanding complex neural circuits and studying behavioral mechanisms.However,the practical implementation and mechanistic understanding of monosynaptic connection tracing techniques and functional identification remain challenging,particularly for inexperienced researchers.Improper application of these methods and misinterpretation of results can impede experimental progress and lead to erroneous conclusions.In this paper,we present a comprehensive description of the principles,specific operational details,and key steps involved in tracing anterograde and retrograde monosynaptic connections.We outline the process of functionally identifying monosynaptic connections through the integration of optogenetics and electrophysiological techniques,providing practical guidance for researchers.
