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Melanopsin Regulates Both Sleep-Promoting and Arousal-Promoting Responses to Light

Fig 4

Wavelength-dependent effects on immediate early gene expression in the SCN and adrenal gland.

(A) Relative mRNA expression of Fos, Per1, and Per2 after 1 hr monochromatic light pulse in the SCN and adrenal gland. One-way ANOVA for wavelength, Fos (F(3.18) = 23.615 p ≤ 0.001), posthoc Tukey dark versus violet p ≤ 0.001, dark versus blue p ≤ 0.001, dark versus green p ≤ 0.001; Per1 (F(3.23) = 10.366, p ≤ 0.001) dark versus violet p = 0.036, dark versus blue p ≤ 0.001, dark versus green p = 0.007); Per2 (F(3.19) = 5.715 p = 0.006) dark versus violet p = 0.196, dark versus blue p ≤ 0.001, dark versus green p = 0.265). A significant effect of wavelength was observed in the SCN for all three genes (one-way ANOVA for wavelength, Fos F(2,14) = 8.603, p = 0.004; Per1 F(2,19) = 4.740, p = 0.021; Per2 F(2,14) = 8.565, p = 0.004), with blue light showing significantly greater Fos, Per1, and Per2 expression in the SCN compared to either violet (posthoc Tukey Fos p = 0.006, Per1 p = 0.014, Per2 p = 0.008) or green light (posthoc Tukey Fos p = 0.012, Per1 p = 0.028, Per2 p = 0.008). In the adrenal gland, responses to blue light were significantly greater than either green or violet light. Both blue and green light induced Per1 expression (one-way ANOVA for wavelength, Per1 F(3,36) = 13.070, p < 0.001; posthoc Tukey dark versus violet p = 0.098, dark versus blue p < 0.001, dark versus green p = 0.023). Blue light produced significantly greater Per1 induction compared to either green or violet light (green versus blue p = 0.001, violet versus blue p < 0.001). Fos and Per2 expression were also induced by blue light but not violet or green light (one way ANOVA for wavelength Fos F(3.31) = 2.516 p = 0.076, dark versus blue p = 0.045; Per2 F(3.31) = 7.033 p = 0.001, dark versus blue p = 0.004). Neither violet nor green light produced a significant induction of Fos or Per2 expression (Fos dark versus violet p = 0.063, dark versus green p = 0.979, Per2 dark versus violet p = 0.961, dark versus green p = 0.998). (B) Responses to blue light were subsequently studied in Opn4-/- mice (open bars) compared with wildtype mice (solid bars). Opn4-/- mice showed light induced gene expression for all three genes in the SCN, but responses were significantly attenuated in comparison to wildtype animals (unpaired t test wildtype versus Opn4-/- Fos p = 0.029, Per1 p = 0.007, Per2 p = 0.029). In the adrenal gland Per1 and Per2 (but not Fos) were induced in response to light. All three genes showed attenuated responses in comparison to wildtype mice (unpaired t test wildtype versus Opn4-/- Fos p < 0.001, Per1 p < 0.001, Per2 p = 0.002). The geometric mean of three housekeeping genes Actb, Gapdh, and Arbp were used for normalisation. Statistically significant changes in gene expression versus control (dark) conditions are shown by symbols directly above histogram bars, whereas comparisons between wavelengths are indicated by horizontal lines. Data plotted as mean ± SEM. N = 5–12/group. Significant differences indicated by *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05, NS = not significant. The data used to make this figure can be found in S4 Data.

Fig 4