Abstract
Understanding the connecting structure of brain network is the basis to reveal the principle of the brain function and elucidate the mechanism of brain diseases. Trans-synaptic tracing with neurotropic viruses has become one of the most effective technologies to dissect the neural circuits. Although the retrograde trans-synaptic tracing for analyzing the input neural networks with recombinant rabies and pseudorabies virus has been broadly applied in neuroscience, viral tools for analyzing the output neural networks are still lacking. The recombinant vesicular stomatitis virus (VSV) has been used for the mapping of synaptic outputs. However, several drawbacks, including high neurotoxicity and rapid lethality in experimental animals, hinder its application in long-term studies of the structure and function of neural networks. To overcome these limitations, we generated a recombinant VSV with replication-related N gene mutation, VSV-NR7A, and examined its cytotoxicity and efficiency of trans-synaptic spreading. We found that by comparison with the wild-type tracer of VSV, the NR7A mutation endowed the virus lower rate of propagation and cytotoxicity in vitro, as well as significantly reduced neural inflammatory responses in vivo and much longer animal survival when it was injected into the nucleus of the mice brain. Besides, the spreading of the attenuated VSV was delayed when injected into the VTA. Importantly, with the reduced toxicity and extended animal survival, the number of brain regions that was trans-synaptically labeled by the mutant VSV was more than that of the wild-type VSV. These results indicated that the VSV-NR7A, could be a promising anterograde tracer that enables researchers to explore more downstream connections of a given brain region, and observe the anatomical structure and the function of the downstream circuits over a longer time window. Our work could provide an improved tool for structural and functional studies of neurocircuit.
Highlights
Analyzing the connection of brain neural networks, including input and output neural pathways, is the basis of Mapping brain connectome requires both retrograde and anterograde trans-synaptic neuronal circuit tracers [3, 12, 13]
After mutating the seventh amino acid of the N protein (VSV-NR7A), the replication speed of the virus decreased by 2–3 orders of magnitude within 24 h [24, 25], which might be used as a better tracer, but whether it still has the ability of anterograde trans-synaptic labeling is still unknown
Mutant-Vesicular stomatitis virus (VSV) can effectively infect neurons in vivo Based on the characteristics of rapid amplification, high infection efficiency and expression level of exogenous genes, VSV has been developed into an important gene transfer vector [7, 19]
Summary
Analyzing the connection of brain neural networks, including input and output neural pathways, is the basis of Mapping brain connectome requires both retrograde and anterograde trans-synaptic neuronal circuit tracers [3, 12, 13]. Envelope glycoprotein of VSV can be replaced by glycoproteins of other viruses to achieve different labeling characteristics and direction controllable trans-synaptic transfer [21, 22]. These VSVs have high cytotoxicity that can lead to rapid death of mice, which limits long-term structural observation and gene manipulation or function research, restricting their applications in neuroscience [23]. After mutating the seventh amino acid of the N protein (VSV-NR7A), the replication speed of the virus decreased by 2–3 orders of magnitude within 24 h [24, 25], which might be used as a better tracer, but whether it still has the ability of anterograde trans-synaptic labeling is still unknown
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