Adeno-associated Virus Is Used In The Study Of The Thalamic Parafascicular Nucleus-striatum Pathway

Objective: In this study,adeno-associated virus 2/9-green fluorescence Protein(AAV2/9-GFP)was injected into the Parafascicular nucleus(PF)or dorsolateral striatum of the rat thalamus by a self-made microinjection system.DLS was injected at different doses and different weeks of transfection to observe viral transfection and Projection in the Parafascicular nucleus-DLSiatum Pathway,to find the optimal dose of the tracer reagent and its transfection.time.At the same time,the effects of transfected virus on tissues were observed.Methods: Seventy-seven Wistar male rats weighing 280-320 g were tested for behavioral abnormalities before the experiment.The experimental rats were randomly divided into two groups according to whether or not the virus was injected,which were the experimental group(n=72)and the control group(n=5).Different doses of the tracer AAV2/9-GFP were injected into the DLS and PF by stereotaxic,and after different transfection times,they were frozen and sectioned.The nerve and brain tissues were observed under a laser confocal fluorescence microscope,and the degree points were identified.Statistical analysis was performed by Image-Pro Plus 6.0 software to optimize parameters: tracer average optical density,average cell perimeter,average cell area,optimal time of tracer injection,optimal injection dose,and tracer diffusion area.To investigate the morphological changes of DLS and PF cells in rats injected with virus.The measured data were statistically analyzed using IBM SPSS Statistics 22.0 software and considered statistically significant at P < 0.05.Results:Brain slices injected with AAV2/9-GFP were observed under laser confocal microscopy.Brain slices of the dorsolateral DLS and PF sites were analyzed by comparing Paxions & Watson rat brain maps.After 3 weeks of transfection,the adeno-associated virus traced by the tracer was clearly observed in DLS and PF.When the virus was injected into the dorsolateral part of DLS,it was observed to project to the motor cortex and the hypothalamic nucleus.Shape.When the virus is injected into the PF,it can be seen projected to the DLS,the motor cortex,and the insular leaves.Further quantitative analysis of the dose-effect and time-variation of the cumulative optical density(IOD)of DLS and PF nuclei injected with different doses and different time was carried out.The results showed that the best time for virus transfection of DLS nuclei And the dose was 0.6 ?L for 4 weeks.The optimal time and dose of virus transfection of PF nuclei was 0.4 ?L for 3 weeks.All transfected AAV2/9-GFP showed morphological features of cell membrane,cytoplasm to nucleus development.Under the light microscope,it was found that AAV2/9-GFP showed damage to the cells,which showed that the dorsolateral DLS and PF neurons of the rat virus injection were irregular in shape,and the cell body became large and free.Bubbles appear,the boundaries are not clear,and the nucleolus and Nissl are ambiguous.At the same time,the phenomenon of increased glial cells injected into the virus site was found.The results showed that compared with normal rats,the number of cells of DLS and PF cells in transfected virus rats decreased,the average cell area increased,and the average cell perimeter increased.Conclusion: The brain slices of DLS and PF sites injected with adeno-associated virus can be accurately located under laser confocal microscopy.The significant advantages of fluorescent protein are easy to identify and detect after labeling biomolecules.The optimal time and dose of virus transfection of DLS nucleus was 0.6 ?L for 4 weeks.The optimal time and dose of virus transfection of PF nuclei was 0.4 ?L for 3 weeks.Adeno-associated viruses injected into DLS and PF have a certain degree of damage to cells.