We expressed ChR2-venus in layer 2/3 pyramidal neurons over visual cortex via in utero electroporation onto C57Bl6 × CD1 mice at embryonic day 15.5. We used the offspring of a cross between CD1 females and C57BL/6 males (Charles River, UK), taking advantage of the fertility and fostering capability of CD1 females. Crossed mice had brown or black coats as described previously11 (link) and showed normal features in the pigmented epithelium of eye, confirmed with fundus images and sectioned images (data not shown). E15.5 timed-pregnant CD-1 mice were anesthetized with 2% isoflurane in oxygen. Up to 1 μl of DNA solution with Fast Green (Sigma, UK) was pressure-injected into left lateral ventricle of embryos. The solution2 (link),11 (link),21 (link) contained pCAGGS-ChR2-Venus (Addgene 15753, 1.5 μg/μl) and pCAG-mCherry (0.5 μg/μl). Electroporation was achieved with 5 square pulses (50 V, 50 ms, 1 Hz, CUY21, NepaGene, Japan). mCherry fluorescence was used to screen for positive animals at P0 under a fluorescent stereoscopic microscope (MVX10, Olympus). Images showing ChR2-venus expression in a whole brain in vivo and in sectioned slices are available in our previous study (Fig. 1d,e in Ref. 2 (link)).
Animals were maintained with a light-dark cycle of 12:12 h, and up to four mice were kept in one cage after weaning.
Animals were maintained with a light-dark cycle of 12:12 h, and up to four mice were kept in one cage after weaning.