Development And Application Of Retrograde Neurotropic Viral Tracers

Neutropic virus has been widely used in the study of neuroscience.Taking advantage of different neurotropic viruses to unravel the structural and functional mystery of neural circuits is of great importance for investigators.Rabies virus(RV)is a common retrograde neurotropic tool used to mark the input network of neurons in specific brain regions.Herpes simplex virus type 1 H129 strain(HSV1 H129)has been widely used as a useful anterograde neuronal circuit tracing tool which can label output neural network.However,it has also been reported that H129 also infects neuron through axon terminal take-up which indicates H129 also possess retrograde-labeling phenotype.The study focused on the retrograde labeling property of RV and HSV,and mainly carried out two aspects of work.Firstly,we designed and rescued a replication-defective RV carrying soma-targeted channelrhodopsin(stChR2).Secondly,we found that there existed anterograde transsynaptic spreading of HSV1 H129 tracer after axon terminal infection.It indicates that current HSV anterograde transsynaptic tracer may have the problem of non-specific labeling.In addition,we discussed how to use this property to carry out labeling experiments.(1)Optogenetics provides a powerful approach to manipulate neural circuit.RV vector expressing the light-sensitive protein channelrhodopsin-2(ChR2)is widely used for optogenetics manipulation.However,since ChR2 is diffusely expressed in all the structures of neurons,potential confounding source of light-induced electrical responses may generate by unintended activation of pass-by fibers.To solve the problem,we utilized a targeting signal peptide originated from the Kv2.1 potassium channel,which can uniquely localize protein on neuronal soma and proximal dendrites.A recombinant RV vector expressing soma-targeted channelrhodopsin(stChR2)was generated by inserting the signal peptide sequence of Kv2.1 and ChR2 gene into the specific site of the recombinant RV infection plasmid vector.The spatial confinement of ChR2 expression contributes to reduce the potential confounding source of light-induced electrical responses.(2)It has been reported that H129 could infect upstream input neurons through axon terminal uptake,except local neuronal cell bodies infection.In the present study,we evaluated the retrograde labeling of H129 and defined whether the retrogradely infected H129 could replicate and following undergo trans-multisynaptic spread in the anterograde direction,using the Cre recombinase-dependent,conditionally replicated H129 tracer(H129ATK-TT)and rAAV helper virus expressing Cre for tracing experiment n mice.The results demonstrated that H129?TK-TT could invade axon terminals and retrogradely transport to the upstream input neuronal cell bodies.Moreover,these retrogradely transported viruses could efficiently replicate and subsequently transsynaptically spread in the anterograde direction.Taken together,the data implied that replication-competent,trans-multisynaptic HSV1 H129 tracing results might be mixed neural networks from two types of starter cells,interfering with the accurate translation of tracer results.Therefore,more rigorous H129 tracer system needs to be developed.Finally,we discuss how to use this property to label output networks with specific projective neurons.