Colour as a signal for entraining the mammalian circadian clock

Abstract

Twilight is characterised by changes in both quantity ("irradiance") and quality ("colour") of light. Animals use the variation in irradiance to adjust their internal circadian clocks, aligning their behaviour and physiology with the solar cycle. However, it is currently unknown whether changes in colour also contribute to this entrainment process. Using environmental measurements, we show here that mammalian blue-yellow colour discrimination provides a more reliable method of tracking twilight progression than simply measuring irradiance. We next use electrophysiological recordings to demonstrate that neurons in the mouse suprachiasmatic circadian clock display the cone-dependent spectral opponency required to make use of this information. Thus, our data show that some clock neurons are highly sensitive to changes in spectral composition occurring over twilight and that this input dictates their response to changes in irradiance. Finally, using mice housed under photoperiods with simulated dawn/dusk transitions, we confirm that spectral changes occurring during twilight are required for appropriate circadian alignment under natural conditions. Together, these data reveal a new sensory mechanism for telling time of day that would be available to any mammalian species capable of chromatic vision.

Fig 1. Spectral composition of ambient illumination is predictive of solar angle.

(A) Mean (±SD) total optical power of ambient illumination around dawn/dusk as a function of solar angle relative to horizon (n = 36 d, Aug–Sep 2005; Lat.: 53.47, Long.: -2.23, Elevation 76 m). (B, top) Normalised mean spectral power distribution observed at solar angles ±6° relative to horizon. Note relative enrichment of short-wavelength light at negative solar angles. Bottom panel shows mouse ultraviolet and medium wavelength sensitive (UVS and MWS) cone opsin sensitivity profiles after correction for prereceptoral filtering. (C) Mean (±SD) “yellow–blue” colour index (effective activation of MWS/UVS opsin) as a function of solar angle around dawn/dusk. (D) Relationship between colour and irradiance (see Methods for definition), corrected according to mean for each solar angle (n = 994; -7 to 0° in 0.5° bins × 71 dawn/dusk observations; for clarity, six observations with especially high relative brightness (2.6–4.5) but normal colour (1.3–1.5) are not shown in the scatter plot). Note tighter distribution for relative “colour” versus irradiance. The data used to make this figure can be found in S1 Data.

Fig 2. Colour opponent responses in suprachiasmatic neurons.

(A: left) Example responses of 5 SCN neurons to stimuli modulating UVS and LWS opsin excitation in antiphase (“colour”) or in unison (“brightness”; both 70% Michelson contrast). Individual cells were preferentially excited by blue–yellow transitions (“blue ON”; pink traces), yellow-blue transitions (“yellow ON”; green traces) or dim-bright transitions (“achromatic”; black traces). Shaded areas represent blue/yellow or dim/bright stimulus phase; y-axis scale bars reflects peak firing rate in spikes/s; x-axis scale bars indicate temporal profile of UVS/LWS opsin excitation. (A: right) Normalised mean (±SEM) change in firing for cells classified as “blue-ON”, “yellow-ON” or achromatic (n = 13, 4 and 26 respectively). Conventions as above. (B) Mean (±SEM) firing rates of SCN cell populations tested with colour and brightness stimuli immediately following transitions (0–500 ms) from “blue”–”yellow”/”dim”–”bright” or vice versa. Data were analysed by paired t test; *** p<0.001, ** p<0.01. (C: left) Responses of cells from A to selective modulation of LWS or UVS opsin excitation, indicating “blue”-ON/”yellow-OFF”, “yellow-ON”/”blue-OFF” or non-opponent responses (conventions as in A). (C: right) Normalised mean (±SEM) change in firing for SCN cell populations evoked by LWS and UVS opsin isolating stimuli. Note, normalisation and scaling for data in A and C is identical. The data used to make this figure can be found in S2 Data.

Fig 3. Melanopsin signals influence both colour- and brightness-sensitive cells.

(A) Normalised mean (±SEM) response of blue-ON colour-sensitive (n = 13) and achromatic cells (n = 23 tested) to stimuli targeting melanopsin and/or cones. Melanopsin-isolating stimuli presented a 92% Michelson contrast change (~1.4 log units), all other stimuli were 70% Michelson contrast (see S1A Fig for details of “colour” and “brightness” stimuli). The energy condition reflects a spectrally neutral modulation in light intensity, providing 70% Michelson contrast for all retinal opsins. Far right panels reflect the predicted melanopsin contribution to the 70% energy condition (obtained by subtracting the responses to UVS + LWS only −”brightness”). Responses were normalised on a within-cell basis across all three stimulus conditions and are plotted on the same scale to highlight relative response amplitude. X-axis scale bars indicate temporal profile of UVS/LWS opsin and melanopsin excitation. (B) Example responses of yellow-ON colour-sensitive cells (bottom panels) to stimuli targeting melanopsin and/or cones or melanopsin. Melanopsin-isolating contrast had more heterogeneous effects in yellow-ON cells, with 1/4 cells exhibiting a reduction in firing and 2 cells displaying no obvious response (not shown). Conventions as above except that data are presented as raw firing rates. Y-axis scale bars represent peak firing in spikes/s. The data used to make this figure can be found in S3 Data.

Fig 4. Colour-signals control irradiance coding in suprachiasmatic neurons.

(A) Stimuli used to examine twilight coding: top panel indicates natural change in effective photon flux for each mouse opsin as a function of solar angle (0° represents sunrise/sunset), indicated points were recreated using a three-primary LED system. Note: values for LWS opsin stimulation were chosen to replicate those calculated for the wild-type MWS opsin under natural conditions. Bottom panels indicate control stimuli, which replicated the “natural” change in irradiance but lacked changes in colour (UVS opsin excitation held at a constant ratio relative to LWS, to mimic “day” or “night” spectra). (B) Mean (±SEM) normalised responses of blue-ON cells (n = 9) to 30-s light steps recreating the indicated stages of twilight. Responses were normalised on a within-cell basis according to the largest response observed across all three stimulus sets. (C) Initial (0–10 s) responses of cells from B as a function of simulated solar angle, fit with four-parameter sigmoid curves. Note influence of twilight spectral composition on the solar angle response curve (F-test for difference in curve parameters; p = 0.009; direct comparisons between each pair of curves also revealed significant differences p<0.05). (D and E) Responses of achromatic cells (n = 8), conventions as in B and C. Achromatic cell responses to the three stimulus sets were statistically indistinguishable (F-test; p = 0.72). The data used to make this figure can be found in S4 Data.

Fig 5. Colour changes associated with natural twilight influence circadian entrainment.

(A) Top: Example body temperature traces from two Opn1mw ^R mice. Mice were exposed to sequential 14 d epochs of (i) simulated “natural” twilight (replicating natural changes in irradiance and colour during a northern-latitude summer), (ii) 18:6 square wave LD cycle, and (iii) a twilight photoperiod which lacked changes in colour (irradiance profile identical to “natural” but relative cone opsin excitation fixed to mimic night spectra). Dotted red lines indicate timing of peak body temperature from last 9 d in each photoperiod. Bottom plot indicates timing of peak body temperature for each individual (n = 10); bars represent median. Temperature cycles were significantly phase-advanced under the irradiance-only versus natural twilight (paired t test; p = 0.003). (B) Mice lacking functional cone phototransduction (Cnga3 ^-/-) exhibit identical phase of entrainment under both photoperiods (conventions as in A; paired t test; p = 0.51, n = 9) with peak body temperature occurring significantly earlier versus wild-type mice under natural but not irradiance-only twilight (unpaired t tests, p = 0.005 and 0.91 respectively). The data used to make this figure can be found in S5 Data.

Fig 6. Twilight spectral composition regulates photoperiodic encoding in the suprachiasmatic nuclei.

(A–B) Phasing of SCN firing rhythms from ex vivo multielectrode array recordings of Opn1mw ^R mice housed under natural (A) or irradiance-only (B) twilight photoperiods. Left panels show Rayleigh vector plots for peak firing activity (n = 124 and 170 SCN electrodes from seven and six slices in A and B respectively). Grey-shaded areas correspond to timing of night/twilight transitions, red dotted lines indicate central 50% of the data distribution, arrows indicate mean vector direction. Right panels show representative multiunit traces. Consistent with body temperature data (Fig 5), SCN activity peaks later in mice housed under “natural” relative to irradiance-only twilight (p<0.001 based on bootstrap percentiles). The data used to make this figure can be found in S6 Data.