Application Of Virus Tracing Tools In The Visual Circuit

Part 1 The number and morphology difference of cells labelled by FG or AAVObject Compare the cells traced by traditional tracers(like FG)or virus tracers(like AAV).Methods C57BL/J male mice were divided into two groups,Group FG and GroupAAV,4 mice in each group.In the superior colliculus(SC)of each mouse,fluorogold(FG group)or adeno-associated virus tracer(AAV2/2-EF1α-EYFP,AAV group)was injected.After a certain period for tracer to label cells(3 days for Group FG and 3weeks for GroupAAV),these mice were sacrificed and their retina were isolated,expanded into flat-mounted on slides,immunofluorescent stained and observed under fluorescence microscope.The number and morphology difference of cells between these two group were compared.Results 1)The cells labelled by FG is more than that by AAV at Posterior pole and midperipheral retina with statistical significance(Group FG: 3254.30±260.77/mm~2,3352.56±215.76/mm~2.GroupAAV: 466.79±95.1/mm~2,960.71±134.04/mm~2),while the cell numbers were similar at peripheral retina(Group FG: 2023.37±285.96/mm~2;GroupAAV: 1604.51±302.42 /mm~2).2)The fluorescence signal of cells labelled by AAV is stronger than that by FG.The cell morphology,such as dendrite and axons,of GroupAAV was much clear than that of Group FG.Conclusions 1)FG labelled more cells than AAV.2)AAV tools could be used to study the detailed morphological features of cells.Part 2 The morphology and number of GABAergic RGCs labelled by recombined AAV tracersObject Compare the morphology and number of RGCs labelled by two different AAV tracers.Methods GAD2-Cre male mice were divided into two groups,Group DIO and Group fDIO,4 mice in each group.AAV2-EF1α-DIO-EYFP-EYFP was injected into the SC of the mice in Group DIO.AAV2-EF1a-DIO-FLP-WPRE-pA was injected into the SC and AAV2-EF1a-FDIO-EYFP-WPRE-pA was injected into vitreous chamber of the mice in fDIO.Four weeks later,these mice were sacrificed,and their retina were isolated,expanded into flat-mounted on slides,immunofluorescent stained and observed under fluorescence microscope.The number and morphology difference of cells between these two group were compared.Results 1)The labelled GABAergic RGCs of both Group DIO and Group fDIO displayed detailed morphology results of cell bodies,dendrite and axons.2)The labelled GABAergic RGCs of Group DIO is more than that of Group fDIO with statistical significance(DIO: 115.02±7.29/mm~2;fDIO: 91.84±4.84/mm~2).Conclusions 1)In Group DIO,one AAV tracer could labelled GABAergic RGCs independently,while two AAV tracers need cooperate in Group fDIO for labeling GABAergic RGCs more specifically.2)The labelled cells of Group DIO were more than that of Group fDIO.Part 3 Label visual circuits transsynapticallyObject Label visual circuits by virus tracers that can spread transsynaptically.Methods C57BL/J male mice were divided into two groups,Group PRV and Group H129,4 mice in each group.PRV-UBC-GFP was injected into the SC of the mice in Group PRV and H129-G4 was injected into the vitreous chamber of the mice in Group H129.At later 2-6 days,these mice were sacrificed,and their retina(Group PRV)and brain(Group H129)were isolated,frozen sectioned,immunofluorescent stained and observed under fluorescence microscope.Results 1)Four days after PRV injection,many cells in ONL,INL and GCL were labelled.They may be bipolar cells,amacrine cells,Müller cells and photoreceptors according to their location and morphology.2)72 hours after H129 injection,some cells in SC but little in visual cortex(V1)were labelled.96 hours after H129 injection,much more cells in SC and V1 were labelled.Conclusions 1)Both PRV and H129 could be used to labelled visual circuits,retrograde(PRV)or anterograde(H129).2)The lethality of both PRV and H129 would kill mice within a week,limiting the application in long period experiments.Part 4 Label interneurons that transmit signals to SC via RGCsObject Applying RV tracers to label the visual circuit,interneurons-RGCs-SC.Methods AAV helpers(AAV2-DIO-EGFP-TVA and AAV2-DIO-RVG)were injected into the SC of Thy1-Cre male mice.Four weeks later,RV-Env A-ΔG-Ds Red was injected into the same site.Another 10-12 days later,these mice were sacrificed,and their retina and brain were isolated,frozen sectioned,immunofluorescent stained and observed under fluorescence microscope.Results 1)At the superficial layer of SC(the virus injection site),many neurons were labelled by AAV-EGFP and RV-Ds Red.They were the starter cells for RV to spread retrogradely.At the other layers of SC,many neurons were labelled only by RVDs Red,which came from the co-labelled cells in superficial layer of SC.2)A few cells in GCL were labelled by AAV-EGFP and RV-Ds Red(starter cells).And some cells at the INL and GCL were only labelled by RV-Ds Red,which came from the starter cells in retina or SC.Conclusions RV could be used to labelled visual circuits,but the lethality killed the mice within 2 weeks,leading to not enough cells get labelled.