Study Of Dendritic Spines And Axonal Projection In The Mouse Cerebral Cortex Using In Utero Electroporation

Objective: In recent years, in utero electroporation(IUE)technique is a powerful tool for analyzing brain development. This method allows us to perform gene expression or knockdown in neuronal precursor cells of embryonic cerebral cortices and to observe proliferation, migration, and morphology of transfected neurons in subsequent developmental stages. In this article we investigate the development of neurons in cerebral cortex, including dendritic spines and axonal projections in vivo with IUE.Method: IUE was performed to transfect the green fluorescent protein(GFP) with the cytomegalovirus immediate early enhancer and chicken β-actin promoter fusion(CAG) promoter into the newborn neurons of embryonic mouse brains at embryonic day( E) 15.5. After perfused intracardially with ice-cold 4% paraformaldehyde(PFA) at postnatal day(P) 0 and P28, brains were sectioned on a vibratome. Sections were processed by immunocytochemical and fluorescence staining and the results were observed with confocal laser scanning microscope. At last, the results were performed by Image J for analysis.Results:1. The method of the IUE was constructed successfully. The number of samples is 10 for IUE, after the day of birth, the mice survival rate was 90%(9/10), embryo survival rate was 67.5%( 27/40), survival embryonic GFP-positive expression rate was 74.1%(20/27).2. Long-term expression of the GFP construct with pCAG promoter in el-ectroporated brains.A large number of neurons were labeled with GFP proteins, and clearly seen at P0 and P28 after electroporation at E15.5. It showed the long-term expression of the GFP construct with pCAG promoter in electr-oporated brains.3. GFP-positive cells were later-born neurons. The sections were used double immu-nohistochemical staining for GFP and Neuron-specific Nuclear Protein(NeuN).The majority of GFP-positive cells were NeuN-positive cells that indicated they were neurons indeed. These GFP-positive cells had morphous of typical neurons, that were located in cerebral cortex with their somas in layer Ⅲ/Ⅳ. It showed the GFP-positive cells were later-born neurons, and that was consistent with the transf-ection time.4. GFP-labeled neuron with a lot of dendritic spines.The GFP-labeled cells with IUE presented a typical neuron morphological characteristics, abundance of dendrites with dendritic spines of different sizes and shapes on the surface grow out of the cell body of the neuron.5. GFP-labeled axons were traced in the contralateral cortex and hippocampus. A significant number of GFP-expressing fibers were located in the hippocampal and corpus callosum. The positive axons were slender and their collaterals were traced in the hippoca-mpus and the contralateral cerebral cortex crossing the corpus callosum.Conclusion: The neurons can be transfected successfully with IUE. Long-term expression of the GFP cDNA plasmid with pCAG promoter visualized cardinal features defining the phenotype of cortical neurons, including axonal projection and the morphology of dendritc, axon and dendritic spine. This in utero electroporation method can be used for functional assays of genes in the development of dendritic spines and axonal projection.