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.
Keywords: functional studies; neural activity; neural circuits; projection neurons; rAAV9-Retro; rabies virus; recombination; retrograde labeling; synaptic inputs; trans-monosynaptic tracing.