Figure 3.

Dependence of intrinsic PLR on melanopsin. a, Action spectrum of mouse muscle (WT pigmented) (6 muscles), with sensitivity normalized to value at 480 nm in each experiment. Red circles are averages, and curve is an A₁-pigment spectral template¹⁷ with λ_max = 480nm. Hg light with interference filters used. b, Average flash intensity-response relation for Opn4^−/− muscle (3 muscles). WT relation from Fig. 1c also shown for comparison. Inset shows sample responses of WT and Opn4^−/− muscles to a saturating flash of 4.0×10⁹ photons μm⁻² (equivalent 480 nm). c, Melanopsin (Opn4) mRNA detected by RT-PCR in iris and retina from WT but not Opn4^−/−. PNR (photoreceptor-specific nuclear receptor) mRNA used as control to rule out contamination from retina to iris. β-actin mRNA is a positive control. Difference in melanopsin mRNA signal between iris and retina presumably reflects different fractional total-tissue mRNA coding for melanopsin. d, Immunostaining of WT and Opn4^−/− iris cross-sections for melanopsin (green), smooth-muscle α-actin (red, as muscle marker), and DAPI (blue). Anterior side (stroma) up and posterior side (posterior epithelium) down. Black arrowhead marks pupillary edge. The intensity of melanopsin immunofluorescence appeared lower in the iris than in ipRGCs (not shown). Additionally, although the Opn4^−/− mouse contains the tau-lacZ marker gene replacing Opn4¹⁸, β-galactosidase activity (by X-gal labeling) was not evident in the iris (not shown), presumably due to the low melanopsin-promoter activity. Scale bar: 20 μm. e, TdTomato signal detected in iris of Opn4:tdTomato but not WT mouse. Scale bar: 100 μm. Stimuli in b were 436-nm Hg light, and white for the two brightest intensities, although expressed in equivalent 480-nm photons. 3-mm-diameter spot covering entire muscle. All force measurements at 35–37°C. Mice in d and e were albino (C57BL/6J-Tyr^c-2J/J).