Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

摘要

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the first born neurons remain closer to the ventricle while the last born neurons migrate past the first born neurons towards the surface of the brain¹. In addition to neuronal migration², a key process for normal cortical function is the regulation of neuronal morphogenesis³. While neuronal morphogenesis can be studied in vitro in primary cultures, there is much to be learned from how these processes are regulated in tissue environments.We describe techniques to analyze neuronal migration and/or morphogenesis in organotypic slices of the cerebral cortex^4,6. A pSilencer modified vector is used which contains both a U6 promoter that drives the double stranded hairpin RNA and a separate expression cassette that encodes GFP protein driven by a CMV promoter^7-9. Our approach allows for the rapid assessment of defects in neurite outgrowth upon specific knockdown of candidate genes and has been successfully used in a screen for regulators of neurite outgrowth⁸. Because only a subset of cells will express the RNAi constructs, the organotypic slices allow for a mosaic analysis of the potential phenotypes. Moreover, because this analysis is done in a near approximation of the in vivo environment, it provides a low cost and rapid alternative to the generation of transgenic or knockout animals for genes of unknown cortical function. Finally, in comparison with in vivo electroporation technology, the success of ex vivo electroporation experiments is not dependant upon proficient surgery skill development and can be performed with a shorter training time and skill.