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. 2007 Mar 1;53(5):677-87.
doi: 10.1016/j.neuron.2007.02.005.

Modeling the Role of Mid-Wavelength Cones in Circadian Responses to Light

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Free PMC article

Modeling the Role of Mid-Wavelength Cones in Circadian Responses to Light

Ouria Dkhissi-Benyahya et al. Neuron. .
Free PMC article

Abstract

Nonvisual responses to light, such as photic entrainment of the circadian clock, involve intrinsically light-sensitive melanopsin-expressing ganglion cells as well as rod and cone photoreceptors. However, previous studies have been unable to demonstrate a specific contribution of cones in the photic control of circadian responses to light. Using a mouse model that specifically lacks mid-wavelength (MW) cones we show that these photoreceptors play a significant role in light entrainment and in phase shifting of the circadian oscillator. The contribution of MW cones is mainly observed for light exposures of short duration and toward the longer wavelength region of the spectrum, consistent with the known properties of this opsin. Modeling the contributions of the various photoreceptors stresses the importance of considering the particular spectral, temporal, and irradiance response domains of the photopigments when assessing their role and contribution in circadian responses to light.

Figures

Figure 1
Figure 1
Cone opsins and melanopsin expression in the retina of wild-type (WT) and MW-coneless (TRβ−/−) mice, a) Confocal photomicrographs showing immunohistochemical labelling of MW (red fluorescence) and SW (green fluorescence) opsins in retinal sections. In wild-type mice, both MW and SW opsins are present with co-expression of both opsins in the outer segments of some cones (white arrow, yellow fluorescence). In TRβ−/− mice, MW-immunoreactive cones are not detected and all cones express SW opsin. Scale = 10μm b) Confocal photomicrographs of melanopsin-immunopositive ganglion cells (red fluorescence) in retinal sections of WT and MW-coneless mice showing that the relative number and distribution of melanopsin-containing ganglion cells are similar for both genotypes. Scale = 20 μm; OS: outer segment; ONL: outer nuclear layer; INL: inner nuclear layer; GCL: ganglion cell layer, c) Relative Opsins (SW, MW and rhodopsin) and melanopsin mRNA levels in the retina of WT (black bars) and MW-coneless (grey bars) mice using real time PCR. Results are expressed as mean ± SEM (n = 4 for each genotype). The TRβ−/− knockout mouse is characterized by a total absence of MW opsin and over-expression of SW opsin. The relative level of melanopsin is also up-regulated whereas rhodopsin levels are equivalent in both genotypes. Asterisks indicate a statistically significant difference (Mann-Whitney U test *:p<0.05; **: p<0.01).
Figure 2
Figure 2
Impaired photic entrainment in MW-coneless mice, a) Two representative actograms of locomotor activity for WT and MW-coneless (TRβ−/−) mice under a 12L/12D cycle, double-plotted on a 24 h timescale. The numbered lines represent successive days and the bar above the actograms indicates the original light/dark cycle. The grey rectangles indicate the dark phase of the subsequent shifted 12L/12D cycles. After 3 weeks of entrainment under a 12L/12D cycle, mice were exposed to successive 6h-delayed light/dark cycle, associated with a decrease of light intensity (from 100 to 10 lux, and from 10 to 1 lux). In the WT mice, locomotor activity had a rhythm of 24 h and was phase-locked to the onset of darkness, showing photic entrainment from 100 to 1 lux. In contrast, MW-coneless mice entrained normally at a high light level (100 lux), entrained with an abnormal phase angle at 10 lux and at 1 lux do not show stable entrainment at least after 43 days, b) Phase angles of activity onsets showing the differences in entrainment for all individual WT and MW-coneless mice (n=8 for both genotypes).
Figure 3
Figure 3
Mean (± SEM) phase angles of activity onsets in wild-type and MW-coneless mice (see figure 2b for individual data). In both genotypes (n=8), the number of days to achieve a stable phase angle of entrainment increases as the light intensity during the light phase decreases Wild-type mice achieve stable re-entrainment (phase angle = 0 hr) after the shift from 100 to 10 lux and from 10 to 1 lux (respectively 14.7 ± 2 and 23.2 ± 1.1 days). In contrast, MW-coneless (TRβ−/−) mice re-entrain with an abnormal phase angle (1.91 ± 0.04 hr, ANOVA, p<0.001) after 21.3 ± 1.1 days for the decrease from 100-10 lux (Mann-Whitney U test; p<0.05) and when the light level is decreased to 1 lux still do not display a stable phase angle of entrainment at least after 43 days.
Figure 4
Figure 4
Attenuated phase shifting response to light in MW-coneless mice, a) Representative actograms of locomotor activity of WT and MW-coneless (TRβ−/−) mice exposed to 15-min pulses of monochromatic light (370, 480 and 530 nm) at 4 different irradiances and at CT16 (white circle), b) Mean ± SEM (n=8 for each genotype) phase shifts for WT and TRβ−/− mice at the 3 wavelengths tested. A statistically significant difference was observed between the genotypes only for monochromatic stimulation at 530 nm. Control animals handled in the same way but that did not receive a light pulse show no significant difference between genotypes. Asterisks indicates a statistically significant difference between the two genotypes (ANOVA: p<0.05; post-hoc Student Newman-Keuls tests comparing genotypes at each irradiance: *:p<0.05; **:p<0.01).
Figure 5
Figure 5
Attenuated phase shifting response to short duration light pulses at 480 nm in MW-coneless mice. A significant difference between genotypes is only observed for light pulses of short duration (Means ± SEM; n=8 for each genotype). Asterisks indicate a statistically significant difference between the two genotypes (ANOVA: p<0.05; post-hoc Student Newman-Keuls tests comparing genotypes at each duration:*:p<0.05; **: p<0.01).
Figure 6
Figure 6
Model of the relative contributions of MW opsin and melanopsin responsiveness derived from the phase shifting response in wild-type and MW-coneless mice, a) The difference in the response functions are based on the mean differences in light-induced phase shifts in MW-coneless mice compared to wild-type for each irradiance at 480 and 530 nm, for values significantly different from the dark controls, respectively 6 % and 48 %. The model (a) is based on additivity of the sensitivities of the two photopigments constrained by the Lamb nomogram and multiplied by the coefficients of sensitivity in the following equations: 480nma(σMelWT)+b(σMWWT)=c(σMelMWKO)10.06530nma(σMelWT)+b(σMWWT)=c(σMelMWKO)10.48 where: a = coeff. for sensitivity of melanopsin in the wild-type mouse b = coeff. for sensitivity of MW-opsin in the wild-type mouse c = coeff. for sensitivity of melanopsin in the knockout mouse σ corresponds to the relative sensitivity of the photopigment (Lamb, 1995) The response function in the wild-type mouse (MW+MelWT, solid black line) corresponds to the combined contribution of melanopsin (MelWT, λmax = 480 nm, dashed blue line) and MW-opsin (MWWT, λmax = 508 nm, dashed green line). The response function in the MW-coneless mouse only involves melanopsin (MelMW-KO, solid blue line). The model predicts that the behavioral responsiveness in the wild-type mouse is derived from relative contributions of 1.0 melanopsin: 1.12 of MW-opsin, with a predicted maximum of sensitivity at 492 nm, which is close to the spectral sensitivity of the circadian system previously reported in the wild-type mouse (Foster et al., 1991; Yoshimura and Ebihara, 1996; Lucas et al., 2001). In a post-hoc experiment to validate the model, we measured light induced phase shifts in response to a longer wavelength (560 nm, 15 min, 2.8 × 1014 photons/cm2/s) in the two genotypes. The amplitudes of the phase shifts were entirely consistent with the predicted values for both the wild-type mouse (0.92 ± 0.16 hrs, black circle) and the MW-coneless mouse (0.21 ± 0.13 hrs, blue circle, see text for further details), (b) Using the lamb nomogram and the derived coefficients for the wild-type mouse, the model allows prediction of the relative contributions of melanopsin and MW-opsin across the spectrum. This shows that in the wild-type mouse, the relative contribution of MW cones is greater for wavelengths above 490 nm while, inversely, at wavelengths shorter than 490 nm melanopsin dominantly contributes to the response.

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