Fig. 1
Development of PA-Flp. a Schematic depicting PA-Flp reconstitution and activation upon blue light illumination, and detection of GFP signals by PA-Flp-mediated deletion of a stop cassette in a Frt-floxed construct. b PA-Flp and fDIO-YFP (Flp reporter) expression plasmids were electroporated into an embryonic mouse brain (E15), with (w/) subsequent noninvasive light stimulation (0.5 mW mm−2, blue fluorescent gun) at postnatal day 1–2 (P1–2) or maintained in dark conditions. Pup brains were harvested at P3-4. c AAV-EF1a-PA-Flp and AAV-EF1a-fDIO-YFP were co-infected into the hippocampus of 8-wk-old mice, with 30 min light (0.4 mW mm−2, 20 Hz, 20% duty cycle) or without (w/o) light stimulation 2 wks after infection and sacrificed 1 wk after light stimulation. A blue line indicates laser light path through implanted optic fiber. b, c Measurement of GFP positive cells among both mCherry and iRFP positive (GFP+/mCh+iRFP+) cells or among both DAPI and mCherry positive (GFP+/DAPI+mCh+) cells in 4–8 coronal slices at each group. Scale bar: 100 μm. Data represent means ± s.e.m. (n = 2 mice/group; ***P < 0.0001, two-tailed Student’s t-test). d, e Schematic depicting AAV infection in the mouse hippocampus and local light stimulation via implanted optic fiber (d), and AAV targeting sites (green) and optic fiber implantation sites (red) (e), in coronal sections of hippocampus. f Left: representative images showing local labeling of GFP signals from RCE:FRT mice at different depths along the rostral-caudal (RC) axis. Yellow arrowhead indicates the medial-lateral (ML) 1.35 coordinate. Right: sites (i)–(iv) at left marked in white-dashed squares are shown in higher-magnification views in the correspondingly labeled rows. Sites (i), (ii), and (iv) indicate the same ML coordinate along the RC axis; (iii) indicates a distal site with a different ML coordinate on the same coronal section of bregma (−1.88 mm). Scale bar: 100 μm. g, h Analysis of percentage (g) and intensity (h) of GFP signals along the ML axis at the same RC coordinate (bregma −1.88 mm) or RC axis on the same ML coordinate (ML 1.35). Measurement performed in each coronal slice. Data represent means ± s.e.m. (n = 1 mouse)
