Fig. 1
From: Restoration of high-sensitivity and adapting vision with a cone opsin
Expression and function of MW-opsin in HEK293 cells and RGCs of rd1 mouse retina. a, b Representative traces of activation of homotetramer GIRK(F137S) channels by photo-stimulation of rhodopsin (a) or MW-opsin (b) measured in whole cell patch in 50 mM [K+]ext at VH = −80 mV in response to low intensity (1 mW cm−2) pulses of light at 535 nm (for MW-opsin) or 500 nm (for rhodopsin). c Decay of photo-response (Tau OFF) for rhodopsin (blue) and MW-opsin (green). Values are mean + SEM; n = 6 (rhodopsin), 8 (MW-opsin) cells. d Viral DNA expression cassette. MW-opsin with YFP (green) under control of hSyn-1 promoter. e Schematic of a degenerated rd1 mouse retina with targeted RGCs highlighted (green). ONL outer nuclear layer, IPL inner plexiform layer. Photoreceptor degeneration denoted in light gray and red cross. f, g En face view of flat mount (f) and transverse slice (g) confocal images of MW-opsin expression of rd1 mouse retina 4 weeks after intravitreal injection of AAV2/2-hSyn-MW-opsin-YFP. Images of YFP fused to C-terminal end of MW-opsin (green) show pan-retinal distribution (f) in RGC layer in relation to DAPI staining of nuclei (d, blue). Scales 60 μm (f) and 20 μm (g). h, i MEA recordings from representative uninjected control (e) and MW-opsin expressing (f) rd1 mouse retina. (Top) Raster plot with spikes for each RGC (e: n = 92 cells; g: n = 68 cells). (Bottom) Peristimulus time histogram (PSTH). Light stimulation protocol: 4 pulses of light of 100 ms duration (λ = 535 nm, enlarged green bars) separated by 60 s dark intervals. j Normalized Light response Index (LRI) for rd1 retina without (gray) and with MW-opsin expression (green) (gray: N = 6 retinas, n = 295 cells; green: N = 8 retinas, n = 323 cells). LRI for 1st and 5th light flash without (N = 3 retinas, n = 106 cells) and with (N = 6 retinas, n = 257 cells) 9-cis retinal. Light intensity 2 mW cm−2. Wavelength: λ = 535 nm (MW-opsin), Values are mean + SEM. Cells are sorted units. Statistical significance assessed using Mann–Whitney U test (*p ≤ 0.01)
