The arousal-promoting and sleep-promoting effects of nocturnal light exposure in mice depend upon different neural pathways.

<p>In response to blue light, M1 pRGC projections to the SCN result in activation of the adrenal gland via the ANS. This alertness promoting pathway is associated with corticosterone release and increased waking. This pathway is strongly activated by blue light due to the spectral sensitivity of melanopsin which peaks ~480 nm. Loss of melanopsin results in reduced activation of this pathway, resulting in enhanced sleep induction. Additional arousal-promoting pathways undoubtedly contribute to this response. By contrast, green light results in activation of the sleep-promoting VLPO pathway, most likely via non-M1 melanopsin pRGCs that are more dependent upon rod and cone input. As melanopsin plays a critical role in rod and cone adaptation, under bright light conditions, loss of melanopsin results in attenuated sleep induction via this pathway. Moreover, the resultant saturation of rod and cone pathways also results in a loss of chromatic responses.</p

Molecular responses to light in SCN and VLPO are wavelength-dependent.

<p>A <i>Fos</i> induction in response to a 30 min light pulse was studied in wildtype mice exposed to blue or green light in both the SCN and VLPO (two-way ANOVA for wavelength x brain region interaction F_(2,31) = 10.968 <i>p</i> ≤ 0.001). Mice exposed to blue light show greater <i>Fos</i> induction in the SCN compared with green light (posthoc Tukey dark versus blue <i>p</i> = 0.001, dark versus green <i>p</i> = 0.035, blue versus green <i>p</i> = 0.008). By contrast, in the VLPO, a greater response to green light than blue light was observed (dark versus blue <i>p</i> = 0.880, dark versus green <i>p</i> = 0.015). As a control, <i>Gal</i> induction was also studied, showing no induction in the SCN but significant induction in the VLPO (two-way ANOVA for wavelength and x brain region interaction F_(2,30) = 3.774 <i>p</i> = 0.035). This response was only evident in response to green light (posthoc Tukey dark versus blue <i>p</i> = 0.742, dark versus green <i>p</i> = 0.011, blue versus green <i>p</i> = 0.003). Histograms show mean ± SEM, <i>n</i> = 5–8/group. Significant differences indicated by *** <i>p</i> ≤ 0.001, ** <i>p</i> ≤0.01, * <i>p</i> ≤0.05, NS = not significant. The data used to make this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002482#pbio.1002482.s006" target="_blank">S6 Data</a>.</p

Wavelength-dependent effects of light on sleep are abolished in melanopsin-deficient (<i>Opn4</i>^<i>-/-</i>) mice.

<p>(<b>A–C</b>) Sleep induction in response to violet (<b>A</b>), blue (<b>B</b>) and green (<b>C</b>) light. Sleep was studied in <i>Opn4</i>^<i>-/-</i> mice (open symbols, <i>n</i> = 6–10/group) and compared with wildtype mice (WT, solid symbols). (<b>D</b>) In comparison with wildtype mice, <i>Opn4</i>^<i>-/-</i> mice show delayed sleep onset in response to green and violet light but advanced sleep onset in response to blue light. Two-way ANOVA for wavelength and genotype, wavelength x genotype interaction F_(2,43) = 12.143, <i>p</i> ≤ 0.001. Posthoc Tukey wildtype versus <i>Opn4</i>^<i>-/-</i> violet <i>p</i> = 0.011, blue <i>p</i> = 0.013, green <i>p</i> ≤ 0.001. Comparison of <i>Opn4</i>^<i>-/-</i> responses in sleep induction as well as duration show no statistical differences due to wavelength. (<b>E</b>) However, in comparison to wildtype mice <i>Opn4</i>^<i>-/-</i> mice exposed to green and violet light show reduced sleep duration. Two-way ANOVA for wavelength and genotype, wavelength x genotype interaction F_(2.44) = 5.142, <i>p</i> = 0.010, posthoc Tukey wildtype versus <i>Opn4</i>^<i>-/-</i> violet <i>p</i> = 0.017, blue <i>p</i> = 0.259, green <i>p</i> = 0.003. Despite the difference in sleep onset under blue light in <i>Opn4</i>^<i>-/-</i> and wildtype mice, there was no difference in sleep duration. Solid horizontal bars illustrate light pulse duration from ZT14 until ZT15. Data plotted as mean ± SEM. Significant differences indicated by *** <i>p</i> ≤ 0.001, ** <i>p</i> ≤ 0.01, * <i>p</i> ≤ 0.05, NS = not significant. The data used to make this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002482#pbio.1002482.s002" target="_blank">S2 Data</a>.</p

Wavelength-dependent effects on behavioural light aversion.

<p>(<b>A</b>) Mice entrained to an LD12:12 cycle were exposed to 405 nm (violet), 470 nm (blue), and 530 nm (green) light at ZT14 in a light/dark box for 10 min. (<b>B</b>) In the first 1 min of the test, mice spent significantly more time in the hidden zone under blue light compared to violet, green, or control (dark) conditions. One-way repeated measures ANOVA for light condition, F_(3.25) = 20.696, <i>p</i> ≤ 0.001. Post hoc Tukey dark versus violet <i>p</i> = 0.006, dark versus blue <i>p</i> = 0.021, dark versus green <i>p</i> ≤ 0.001, blue versus violet <i>p</i> ≤ 0.001, blue versus green <i>p</i> ≤ 0.001. (<b>C</b>) In the following 9 min, mice exposed to blue light continued to spend more time in the hidden zone compared to violet or green light. One-way repeated measures ANOVA light condition over time 2–-10 mins, F_(3,25) = 11.721, <i>p</i> ≤ 0.001. Post hoc Tukey dark versus violet <i>p</i> = 0.635, dark versus blue <i>p</i> = 0.021, dark versus green <i>p</i> = 0.635, violet versus blue <i>p</i> ≤ 0.001, blue versus green <i>p</i> ≤ 0.001. (<b>D</b>) <i>Opn4</i>^<i>-/-</i> mice (open bars) showed different responses to both blue and green light. In the first minute of the test, in comparison with wildtype mice, <i>Opn4</i>^<i>-/-</i> mice spent less time in the hidden zone in response to blue light. However, <i>Opn4</i>^<i>-/-</i> animals spent more time in the hidden zone compared with wildtype under green light. Two-way repeated measures ANOVA for light condition and genotype, light condition x genotype interaction F_(1,26) = 20.585, <i>p</i> ≤ 0.001. Post hoc Tukey wildtype versus <i>Opn4</i>^<i>-/-</i> blue <i>p</i> = 0.010, green <i>p</i> = 0.001. (<b>E</b>) Whilst the attenuated response to blue light in <i>Opn4</i>^<i>-/-</i> mice persisted over the remaining time course of the test (one-way repeated measures ANOVA for effect of genotype over time, F_(1.13) = 14.376, <i>p</i> = 0.002), no difference was observed between wildtype and <i>Opn4</i>^<i>-/-</i> mice under green light (one-way repeated measures ANOVA for effect of genotype over time, F_(1.13) = 0.26, <i>p</i> = 0.617). <i>Opn4</i>^<i>-/-</i> mice showed no statistical difference in responses to wavelength in the first minute or whole time of the test (one-way repeated measures ANOVA for effect time over wavelength F_(9,81) = 0.976 <i>p</i> = 0.466). Histograms reflect mean percentage ± SEM of time spent in the dark box of the light dark box during the 10 min trial (<i>n</i> = 6–-10/group wildtype, <i>n</i> = 4–-10 <i>Opn4</i>^<i>-/-</i>). Significant differences indicated by *** <i>p</i> ≤ 0.001, ** <i>p</i> ≤ 0.01, * <i>p</i> ≤ 0.05, NS = not significant. The data used to make this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002482#pbio.1002482.s003" target="_blank">S3 Data</a>.</p

Wavelength-dependent effects on light on sleep induction and sleep duration.

<p>(<b>A</b>) Mice exposed to different wavelengths (405 nm violet, 470 nm blue, or 530 nm green) for 1 hr at ZT14 exhibited differences in sleep onset and sleep duration. (<b>B</b>) Sleep induction is delayed in response to blue light exposure. One-way ANOVA for wavelength, F_(2.23) = 18.791, <i>p</i> ≤ 0.001. Posthoc Tukey violet versus blue <i>p</i> = 0.003, violet versus green <i>p</i> = 0.041, blue versus green <i>p</i> ≤ 0.001. (<b>C</b>) Total sleep duration during the 1 h light pulse is reduced in response to blue light. Data plotted as mean percentage ± SEM (<i>n</i> = 8–10/group). Horizontal black-white-black bar illustrates the light pulse condition from ZT14 until ZT15. One-way ANOVA for wavelength, F_(2.23) = 4.391, <i>p</i> = 0.024. Posthoc Tukey violet versus blue <i>p</i> = 0.046, violet versus green <i>p</i> = 0.517, blue versus green <i>p</i> = 0.008. Statistical differences indicated by *** <i>p</i> ≤ 0.001, ** <i>p</i> ≤ 0.01, * <i>p</i> ≤ 0.05, NS = not significant. The data used to make this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002482#pbio.1002482.s001" target="_blank">S1 Data</a>.</p

Wavelength-dependent effects on plasma corticosterone.

<p>(<b>A</b>) Plasma corticosterone levels were assessed from terminal blood following a 30 min light pulse at ZT14. All three wavelengths produced significant rise in plasma corticosterone levels. However, blue light evoked significantly higher corticosterone secretion compared to violet, green, and dark conditions (one-way ANOVA for wavelength, F_(3.30) = 39.80, <i>p</i> ≤ 0.001. Posthoc Tukey dark versus violet <i>p</i> ≤ 0.001, dark versus blue <i>p</i> ≤ 0.001, dark versus green <i>p</i> = 0.002, violet versus blue <i>p</i> ≤ 0.001, and blue versus green <i>p</i> ≤ 0.001). Responses to green light were significantly lower compared to either violet or blue light. (<b>B</b>) <i>Opn4</i>^<i>-/-</i> mice (open bars) show not only an overall elevation of plasma corticosterone levels in response to light (two-way ANOVA for wavelength and genotype, wavelength effect F_(2.36) = 42.135 <i>p</i> ≤ 0.001, posthoc Tukey <i>Opn4</i>^<i>-/-</i> dark versus blue <i>p</i> = 0.001, <i>Opn4</i>^<i>-/-</i> dark versus green <i>p</i> = 0.013) but also showed different responses to wildtype mice (open bars, wavelength x genotype interaction F_(2.36) = 13.809 <i>p</i> ≤ 0.001). Responses to blue light were significantly attenuated (wildtype versus <i>Opn4</i>^<i>-/-</i> blue <i>p</i> = 0.004) whereas responses to green light were elevated (<i>p</i> = 0.029). As such, there was no significant difference in corticosterone response between blue and green light in <i>Opn4</i>^<i>-/-</i> mice. Baseline levels of corticosterone at ZT14 were significantly elevated in <i>Opn4</i>^<i>-/-</i> mice compared to wildtype controls (<i>p</i> = 0.017). Histograms reflect mean ± SEM, <i>n</i> = 5–10/group. Significant differences indicated by *** <i>p</i> ≤ 0.001, ** <i>p</i> ≤ 0.01, * <i>p</i> ≤ 0.05, NS = not significant. The data used to make this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002482#pbio.1002482.s005" target="_blank">S5 Data</a>.</p