Characterization And Systematic Improvement Of Neurotropic Virus HSV1 H129 As Anterograde Trans-synaptic Tracers

To understand how the brain works,it is essential to map the brain connectome.Therefore,tools that could spread in the specific direction between neurons are urgently needed.Neurotropic viral transsynaptic tracing is an increasingly powerful technique for dissecting the structure and function of neurocircuits.HSV1 strain H129,proved to be an anterograde tracer,has been widely used to trace neuronal networks in neuroscience research.However,H129 tracers still have several shortcomings,including nonspecific infection and low sensitivity for visualization.Here,this thesis aimed to resolve these two main drawbacks of H129 tracers.First,whether H129 is a rigorous anterograde tracer and undergoes anterograde-only spreading are questions of significant interest.In the present study,the retrograde labeling efficiency of H129 was evaluated using a TK and ICP34.5 dual deleted H129 recombinant(named as H306)which was replication-deficient in non-dividing postmitotic neurons.The novel tracer was tested in vitro and in vivo for evaluating its invasion properties and tracing capacities.The results demonstrated that H306 could efficiently label the neurons in the injection site.However,H306 could also efficiently infect upstream innervating neurons through axon terminal uptake and displayed obvious retrograde labeling phenotype,regardless of 3-days or 10-days tracing.The data implied that replication-competent,transmultisynaptic H129 tracing results might be a mixed neural networks from two types of starter cells,because the retrogradely infected neurons would also replicate H129 and spread virus anterogradely through their axon collaterals(ectopic starter sites),as the locally infected neurons in the injection site(true starter site).Therefore,the interpretation of the anterogradely tracing neural networks by current H129 tools at longer post-inoculation intervals need to be cautious,and effective modification strategies are needed to avoid or block the axon terminal invasion process of H129,which is important for rigorous anterograde H129 tracer.Next,to develop rigorous H129 anterograde tracers,this thesis was inspired by the strategy used in retargeting of oncolytic HSV(o HSV)to eliminate the capacity of H129 in axon terminal uptaking.Here,a recombinant H129,termed as Hs06,that carrying the antiHER2 sc Fv in glycoprotein D(g D)gene was generated to target genetically defined neurons.With the usage of helper virus complementarily expressing HER2 and wild-type g D,the labeling and visualization of direct projection regions of either a given brain nucleus or a specific neuron type could be realized.The anterograde tracing ability of Hs06 was verified by tracing the output network of primary visual cotex.Further,the labeling results of LH and VTA GABAergic neurons were also consistent with previous reports.Thus,Hs06 was capable of mapping the direct projectome of specific neuron type in the given brain regions,combining with transgenic(e.g.,cre-line)mice.Finally,a highly bright H129 anterograde tracer was constructed by increasing the expression of exogenous genes carried by H129 viruses.Using a Trojan horse-like strategy,an H129/AAV(adeno-associated virus)chimaera termed H8 was generated to enhance the expression of a fluorescent marker.Both in vitro and in vivo assays showed that the exogenous gene was efficiently replicated and amplified by the synergism of the HSV vector and introduced AAV replication system.H8 reporting fluorescence was significantly brighter than that of currently available H129 tracers,and thus could be used for fast and effective anterograde tracing without additional immunostaining.These results demonstrated that foreign gene expression in HSV tracers could be enhanced by integrating HSV with AAV replication system.This approach may be useful as a general strategy for HSV-based tracing tools or gene delivery vectors to enhance the expression of exogenous genes.In summary,the defects of H129 tracer were systematically studied and improved.The novel anterograde tracer constructed in this paper can realize the rigorous analysis of neuron output network and is expected to play an important role in the study of neural circuits.In this paper,a fluorescence enhancement strategy suitable for the existing H129 tracer has been established,which will play an important role in shortening the experimental time.