Exploration Of Rabies Therapy And Neurological Application Of Related Virus

Rabies is a zoonotic disease with a lethality of up to 100%,which is caused by rabies virus(RABV)infection of the animal nervous system to result in severe neurological dysfunction.Rabies is widely distributed around the world,and the current prevention and control of rabies is still not complete.Every year,there is still thousands of rabies patients died.At present,the research on rabies prevention and treatment mainly focused on RABV vaccine development,field epidemic prevention,pathogenesis and drug control.However,it is still lacking of effective drugs and methods for the treatment of rabies.Due to the dysfunction of nervous system caused by rabies virus infection,in a short period of time,it is a serious threat to the lives of patients.In order to alleviate the rabies virus-induced neurological dysfunction and win the valuable time for immune system to clean up rabies virus,we use memantine,a neural protective drug,to study whether the drug can relieve rabies symptoms.The study will provide new ideas and methods for the clinical treatment of rabies.RABV is one of the most widely used retrograde tracing viruses due to its high neurotropism.Developing more efficient circuit tracing tool based on RABV will contribute to the study on the structure and function of neural circuit.Therefore,we modified the viral genome of RABV by reverse genetics,and rescued the glycoprotein-deleted recombinant rabies virus(rRABV?G)for tracing and functional study of neural circuit.By these tracing viruses,we resolved the fine structure and function of visual circuit.The major works are as follows: 1.Memantine relieves rabies symptomsMemantine,a drug capable of passing through the blood-brain barrier,non-competitively and reversibly binds N-methyl-D-aspartic acid receptors(NMDA receptors).The drug is used for the treatment of Alzheimer's disease due to the protective effect on neurons.To explore whether the drug can alleviate the neurological disturbances caused by rabies virus,we have carried out the following experiments:(1)For primary neurons cultured in vitro,the addition of memantine showed protective effect on neurons with RABV(CVAB-24 strain)infection.(2)In the RABV challenge experiments,memantine had limited therapeutic effect which mildly extend the survival time of mice.In contrast,memantine significantly prolonged the survival time of mice and reduced intravascular cuff and inflammatory cell infiltration in mice with Japanese encephalitis virus(JEV)infection.Meanwhile,memantine treatment also decreased the JEV virus amount in mice brain.2.Resolving the fine structure of circuit from primary visual cortex to thalamus by rabies tracer virus(rRABV?G)Through reverse genetics,we modified rRABV-SAD strain by replacing glycoprotein gene with a variety of fluorescent protein genes to get multi-fluorescence rRABV tracing tool.We injected the multi-fluorescence rRABV to mouse thalamic nuclei including lateraldorsal of thalamus nucleus(LD),lateralposterior of thalamus nucleus(LP)and lateral geniculate nucleus(dLGN),which are projection targets of primary visual cortex(V1),to retrogradely label V1 neurons.Based on different fluorescence of V1 neurons targeting on specific thalamic nucleus,the V1 neurons are sorted and mapped.Meanwhile,we observed some neurons can target on multiple nuclei of thalamus by showing co-localization of different fluorescence,which we called multi-target neuron in V1.Further,we tracked the fine structure and whole brain projection of single V1 multi-target neurons by fluorescence Micro-Optical Sectioning Tomography(fMOST)imaging.In order to analyze the upstream neurons of V1-LD,V1-LP and V1-d LGN circuits,we supplied the G protein through the AAV virus at V1.Then the rRABV?G in V1 neuron successfully trans-monosynapse spread to label the upstream neurons of V1 to thalamus circuits.We analyzed the distribution and number of labeled upstream neurons,and finally delineated fine structure of V1-thalamus circuit.3.Function exploration of V1-thalamus circuits by rRABV?GBy combining rRABV?G virus tracing with in vivo calcium two-photon microscopy,we detected the functional activity of V1-thalamus circuit.We mainly detected the spontaneous activity in dendritic spine and dendrites of V1 neuron targeting on specific thalamic nucleus,to analyze the difference of upstream input on the different V1-thalamus circuit.4.The plasticity of V1-thalamus circuit under monocular deprivationUnder monocular deprivation(MD),we investigated the plastic change of V1-thalamus circuits by rRABV?G virus labeling,optogenetics and in vivo fiber photometry technique.We found the labeled V1 neuron in MD mice was significantly reduced as compared with that in normal mice by rRABV?G-eGFP virus tracing from contralateral thalamic nuclei,which indicated the monocular deprivation in the critical period caused the decrease of V1-thalamus projection.Furthermore,we functionally verified the plasticity changes of these circuits by combining the optogenetical stimulation to V1 neuron and fiber photometry detection of thalamic neuron calcium activity.Our study on the memantine alleviation of rabies will contribute to the exploration of rabies treatment methods.At the same time,we research on the fine structure and plasticity of primary visual cortex to thalamus circuit,will help for the visual functional study,and help to analyze the transmission and integration of visual information between visual cortex and thalamus,and also enrich our understanding on the visual circuit plasticity.