Differences In Neurotropism And Neurotoxicity Among Neurontropic Viral Tracers

Neurotropic viruses are a class of viruses that are highly sensitive to the nervous system.Many neurotropic viruses have strict neurotropism and can spread across synapses in a specific direction after infection.With the rapid development of genetic engineering and reverse genetics technology,neurotropic viruses have been transformed into various forms of neuron circuit tracing tools to map the neural network connection of the central nervous system.The neurotropic virus has a certain preference for the direction of transport in neurons.The herpes simplex virus(HSV)HSV129 strain and the vesicular stomatitis virus(VSV)are mainly transported in the antegrade direction,and have been used as an anterograde neuronal circuit tracer.Rabies virus(RABV),canine adenovirus type 2(CAV),pseudorabies virus(PRV)Bartha strain and retrograde adeno-associated virus(rAAV2-retro)are retrogradely transported along axons,and engineered for retrogradely labeling.According to the replication ability of the virus,it can be divided into two types: multi-trans-synaptic and mono-synaptic viral tracers.The mono-synaptic virus is a class of replication-defective virus,such as the G-deficient RABV,the E1 protein-deleted canine adenovirus and rAAV2-retro.The mono-synaptic viruses are usually been used to identify the direct monosynaptic inputs and output of a nucleus.The multi-trans-synaptic virus is a class of replication-intensive virus,including the Bartha strain of PRV,the fixed strain of RABV,the H29 strain of HSV.The multi-trans-synaptic viruses are usually been used to trace the multi-trans-synaptic circuits in the central nervous system.Neurotropic virus-based tracers are most widely utilized for delineating the fine architecture and function of neural circuits.How to select an effective virus tools in different studies depends on our understanding of different virus characteristics.Different types of viruses have different characteristics,including viral structure,genome,receptor protein,replication and packaging,etc.The viruses only invade neurons expressing the corresponding receptor,and then interact with the host protein to complete its replication and packaging.Neural networks are made up of hundreds of millions of different types of neurons with different gene expression.These factors may cause the virus to have different neurotropism for different groups of neurons in the nervous system.However,there have no systematic studies on the neurotropism of different viral tracer.This study systematically analyzes the neurotropism and neurotoxicity of viruses commonly used in neuron circuit mapping,and provide important reference for appropriate application of viral tracers to delineate the landscape and dissect the function of neural network.1.Comparison of neurotropism of multi-trans-synaptic viral tracerswe compared the neurotropic actions of a fixed RABV derivative B2C-EGFP with those of the PRV-Bartha derivative PRV-152.These GFP expressing viruses were injected into mouse popliteal lymph nodes to trace the upstream multi-trans-synaptic circuits in the central nervous system,respectively.Virus invades the nerves that innervate the lymph nodes,and then trans-synaptically hop to the high order neuron.Through the whole brain slice imaging,neuron circuit tracing by the two viruses were analyzed.Our results showed that PRV and RABV exhibit differential neurotropism when they were used for central neural circuit tracing from popliteal lymph nodes.2.Discrepancies in Circuit Labeling with Different Mono-synaptic Retrograde Tracing VirusesWe injected Several replication-defective viruses SAD-?G,rAAV2-retro,CAV2-Cre and a chemical tracer into the same nucleus of the mouse brain,following comparing the direct upstream circuits mapping by these tracers.It was found that the neural circuits mapping with different virus were not completely consistent.By using the Cre/Lox recombinase system and co-injection of different viral tracer we still observed the selecting labelling of neuron circuits.This suggests that SAD-?G,rAAV2-retro,CAV2-Cre indeed displayed discrepant neurotropic property.Statistical analysis of the numbers of neurons in different nucleus suggests rAAV2-retro demonstrated preference in cerebral cortex,whereas SAD-?G prefer to label basal ganglia and hypothalamus.3.Exploration of the mechanism of virus neurotropismDifferent types of viruses recognize and bind with their corresponding receptor to infect neuron.There are many kinds of neurons with different gene expression pattern in the brain.Therefore,we speculate that the heterogeneous expression of viral receptors at the synaptic terminals of the neurons is an important factor for the different neurotropism.Complementation of TVA receptor gene in SAD-resistant neurons enabled EnvA-pseudotyped RABV infection,supporting receptors attribution to viral neurotropism.To investigate the heterogeneous gene expression profiles of the neurons labeled by different retrograde viral tracers,which may have contributed to the observed differences in viral neurotropism,we performed single-cell transcriptome sequencing of the infected neurons.We analyzed 21 groups of rAAV2-retro-labeled neurons and 22 groups of SAD-?G-labeled neurons with high quality to Screening of Candidate Genes Contributing to Neurotropism.4.Differences in the Neurotoxicity of rAAV2-retro and SAD-?G TracersAs neurotropic viruses have also been extensively applied in the recording of circuit functions,and viral infection and gene expression can induce certain toxicity,we aimed to systematically analyze the neurotoxicity of these viruses at the injection site and in retrogradely labeled nuclei.First,the injection site,and retrogradely-labeled tissue were dissected for RNA-seq.Bioinformatic analysis of the gene profiles from different groups revealed that infection with retrograde viral tracers mainly influenced genes associated with the immune response,and that the impact of SAD-?G infection on gene expression is more pronounced.Then we performed Iba1 immunohistochemistry to evaluate microglial activation induced by SAD-?G and rAAV2-retro infection at the injection site and in retrogradely labeled nuclei.The result showed microglial infiltration was markedly increased at the injection site(LHA)and in the retrogradely traced region following SAD-?G injection.Microglial activation due to rAAV2-retro infection was significantly milder,indicating that SAD-?G is much more toxic to both the injection site and retrogradely labeled regions.At the same time,we also found that the SAD-?G infection can caused high expression of the oxytocin gene.5.Strategy to dissect the input circuits of by combination of different viral tracersOur data demonstrated that application of rAAV2-retro or SAD-?G for retrograde tracing only reveals a portion of the direct upstream regions.Thus,to achieve a more comprehensive map of higher-order circuits involving a specific target nucleus,we proposed a strategy by combing the different viral tracers.