Application Of CUBIC Tissue Transparency Technique In Neuronal Imaging

Objective:In recent years,brain structure dysplasia becoming a hotspot in the research of the development of the brain functional disease,we’re playing more and more attention to the temporal and spatial characteristics of functional neuron development and migration.However,the existing optical sectioning can’t represent the neural circuits and their characteristics effectively,Therefore,this study intends to realize three-dimensional imaging of whole brain structure through the exploration of CUBIC(clear,unobstructed brain/body imaging cocktails and computational analysis)tissue transparency technology,and also provide technical support for revealing the temporal and spatial distribution characteristics of functional neurons in the brain.Methods:1.Male C57BL/6J mice aged 10 weeks were used to perfuse and fix under anesthesia.The brain tissue,liver tissue and heart tissue of the mice were extracted respectively,and the organs were post-fixed.The tissues of the each organ were cleared with CUBIC-L and CUBIC-R tissue clearing solution,and then observed the relative transparency of each tissue.2.The whole brain of mice were transparently treated.Furthermore,small fluorescent dye Propidium iodide(PI)was used to stain the nuclei of mice brain neurons.Subsequently,BX-63 automatic scanning microscope and light sheet fluorescence microscopy(LSFM)were used to obtain two-dimensional and three-dimensional scanning images of the nuclear volume of the left and right cerebral hemispheres and cerebellar brainstem of mice.3.In order to explore the conditions of immunofluorescence labeling of dopamine neurons,the lipids in the substantia nigra region of mouse brain were cleared by the method of transparency.The labeled neurons were imaged by LSM880 laser confocal microscope for three-dimensional imaging.4.To tag neurons at specific anatomical sites,microinjection of adeno-associated virus(AAV)-U6-enhanced green fluorescent protein(EGFP)was performed in the prefrontal lobe,hippocampus and substantia nigra of mice,respectively.The labeled mouse brain was delipidated and matched with refractive index(RI).The image was obtained using LSM880 laser confocal microscope and LSFM.Results:1.After transparent treatment,the brain tissue showed a transparent gelatinous state under naked eye observation.There was no obvious morphological deformation on the whole organ;the removal efficiency of CUBIC tissue clearing in brain tissue was higher than that in liver and heart tissue.2.Using LSFM,the PI labeled nuclei can be directly imaged as a whole volume three-dimensional image,after the lamellar segmentation,the cell structure in the brain could be presented layer by layer.And the nucleolus structure could be observed at high magnification.3.The tyrosine hydroxylase(TH)positive dopamine neurons were successfully labeled by tissue clearing and immunofluorescence labeling,and mesoscopic nerve connections such as axons and dendrites of neurons can be observed.4.AAV can clearly label neurons in different locations.After transparent processing,the spatial distribution of these neurons can be detected,and the physical projection of neurons in different locations can be tested.Conclusion:CUBIC tissue transparency technology can be used for three-dimensional imaging of neurons in the brain,at the same time the transparent method can also be compatible with immunofluorescence labeling and AAV markers such as technology,which made it possible to characterize the structure and spatial distribution of neurons.This method has important scientific value for explaining the integrity of brain function and structure.