Irradiance–response relations (a) and action spectrum (b) for circadian phase shifting in rd/rd cl mice by monochromatic light between 420 nm and 580 nm, assayed by wheel-running (n = 4–7 animals per irradiance at each wavelength). The derived action spectrum in b for circadian photo-entrainment is best fitted (R² = 0.976) by the absorption spectrum of a vitamin A₁-based photopigment with λ_max = 481 nm (continuous curve).

Normal retinal morphology and presence and central connectivity of melanopsin-expressing RGCs in triple-knockout (Opn4^−/− Gnat1^−/− Cnga3^−/−) mice. a, Retinal cross-sections from Opn4^+/− and triple-knockout (KO) mice. Giemsa stain shows the various layers. Both are similar in morphology and thickness to wild type. GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; IS, inner segment layer; ONL, outer nuclear layer; OPL, outer plexiform layer; OS, outer segment layer. b, Flat-mount view of a triple-knockout retina stained with X-gal (blue). c–e, Coronal sections of triple-knockout mouse brain showing the normal innervations of the SCN, IGL and OPN by X-gal-labelled axons. Dorsal side is up in each case.

Disabling of rods, cones and melanopsin-positive RGCs essentially eliminates the pupillary light reflex. a, Pupil area (mean ± s.e.m.) as a percentage of dark-adapted aperture area (recorded just before light exposure, time 0 on graph) for Opn4^−/− Gnat1^−/− Cnga3^−/− (n = 6), Opn4^+/− Gnat1^+/− Cnga3^+/− (n = 4), Opn4^−/− (n = 8; data reproduced from ref. 8) and Gnat1^−/− Cnga3^−/− (n = 1) mice over the course of 1 min when exposed to 480-nm light at 86–140 µW cm⁻². The wild-type data are from ref. 8. For comparison, the pupil size of triple-knockout mice exposed to an infrared light pulse (n = 6) is also shown. On close scrutiny, a small, transient pupil constriction was detected in two out of six triple-knockout mice exposed to the intense 480-nm stimulus. The averaged 480-nm data shown for the triple knockouts are from mice dark-adapted for 3 days before light exposure, but are representative of similar recordings from the same group after 1-h dark adaptation. Neither the amplitude nor the frequency of occurrence of the residual pupillary response in the triple-knockout animals were increased with bright white-light stimulus (30 mW cm⁻²), or with monochromatic stimuli ranging from 360 to 660 nm (70 µW cm⁻² at 360 nm; 19 µW cm⁻² at 420 nm; 86 µW cm⁻² at 480 nm; 100 µW cm⁻² at 550 nm; 68 µW cm⁻² at 660 nm). Nor did these parameters show any significant change when tested repeatedly over a 24-h period. b, Application of 100-mM carbachol resulted in a complete pupil constriction in triple-knockout mice.

Actograms of wheel running for mice under a 16/8-h light/dark cycle, double-plotted on a 24-h timescale. The illumination was approximately 800 lx white light. The numbered lines represent successive days. Activity levels (in total number of revolutions in 10-min bins) are given in 15 quantiles, with the first being 1–55 revolutions, the second 56–110, and so on. The bar below the actograms indicates light (white) and dark (black) periods. a, Triple heterozygote (Opn4^+/− Gnat1^+/− Cnga3^+/−). The locomotor activity had a cycle very close to 24 h and was phase-locked to darkness, showing photo-entrainment. b, Triple-knockout (Opn4^−/− Gnat1^−/− Cnga3^−/−). The animal free-ran with a period of less than 24 h, showing lack of photo-entrainment.

Actograms of wheel running plotted on a 7-h timescale. The mice were subjected to an ultradian 3.5/3.5-h light/dark cycle (see text). Illumination was approximately 800 lx white light. The numbered lines represent successive 7-h cycles. The activity quantiles and time bins are the same as in Fig. 4 a, b, Two triple-heterozygous mice (Opn4^+/− Gnat1^+/− Cnga3^+/−) showing a negative and positive masking effect, respectively. The percentage of total revolutions in darkness was 99.5% and 32.7%, respectively. b, Two triple-knockout mice (Opn4^−/− Gnat1^−/− Cnga3^−/−). The percentage of total revolutions in darkness was 40.8% and 44.7%, respectively, not far from the 50% expected from randomness; that is, no masking effect of light.