Establishment of an in vivo electroporation method into postnatal newborn neurons in the dentate gyrus.

Electroporation-mediated gene transfer has been developed for the analysis of mammalian brain development in vivo. Indeed, in utero electroporation method is widely used for the investigation of the mouse embryonic cortical development while in vivo electroporation using neonatal mouse brain is employed for the analysis of the rostral migratory stream (RMS) and postnatal olfactory neurogenesis. In the present study, we established a stable gene-transfer method to dentate gyrus (DG) neurons by carefully determining the in vivo electroporation conditions, such as position and direction of electrode, voltage for electric pulses, and interval between electroporation and sample preparation. Consequently, GFP-positive cells in DG were observed to extend branched dendrites and long axons into the molecular layer and the hilus, respectively, 21 days after electrporation. They were morphologically identified as dentate granule neurons with many protrusions on dendrites, and some of them had wide head and thin neck that resembled matured mushroom spines. Expression of GFP in dentate neurons sustained for at least 9 months after electroporation under our experimental conditions. Taken together, the method developed here could be a powerful new tool for the analysis of the postnatal DG development.