To ascertain how reliably this blue–yellow colour signal alone could be used to estimate phase of twilight, relative to simple measures of irradiance, we next compared the day-to-day variability of colour and irradiance measurements across our dataset ( Fig 1D ). Surprisingly, we found that colour was in fact more predictive of sun position across twilight (-7 to 0° below horizon) than was irradiance (78.5 ± 0.1% versus 75.8 ± 0.1% of variance explained by solar angle; mean ± SD). Accordingly, for any fixed solar angle, the range of observed colour values was considerably more tightly clustered than those for irradiance. These observations most likely reflect the fact that cloud cover can change overall brightness quite dramatically, but exerts only relatively minor effects on spectral composition.

We next calculated the extent to which this change in spectral composition was detectable to the mammalian visual system. Taking the mouse as a representative species, we employed previously validated approaches to quantify the relative excitation of its ultraviolet and medium wavelength sensitive (UVS/MWS) cone opsins [ 12 , 19 , 20 ]. This analysis revealed a robust change in the ratio of excitation between the two pigments ( Fig 1C ) that would constitute substantial changes in apparent colour along the blue–yellow axis. These changes were restricted to the twilight transition, with the UVS:MWS ratio fairly invariant throughout the day, indicating that measuring the change in colour could provide a useful method of tracking the progression of dawn and dusk.

( 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 .

As expected, these measurements revealed highly predictable changes in both irradiance and spectral composition as a function of solar angle (Fig 1A and 1B ). In particular, we observed a progressive enrichment of short-wavelength light across negative solar angles: a result of the increasing amount of ozone absorption and consequent Chappuis band filtering of green–yellow light when the sun was below the horizon [ 14 ].

We first set out to determine whether changes in spectral composition associated with the earth’s rotation could provide reliable information about solar angle that the circadian clock could use to estimate time of day. To this end, we obtained high resolution measurements of natural variations in spectral irradiance across multiple days (Manchester, August–October 2005, n = 36 d).

Colour Coding in the SCN

Importantly, then, measuring colour could provide a more reliable estimate of the approach of night or day than measuring irradiance. However, while the mammalian circadian clock is certainly known to respond to diurnal variations in irradiance [8–13], there has been no investigation of whether the SCN also receives colour signals. Accordingly, we next asked whether the central clock showed electrophysiological responses to changes in colour by recording extracellular activity in the mouse SCN.

In order to identify colour-sensitive cells, we set out to generate test stimuli which differentially modulated the UVS and MWS mouse cone opsins. While, in principle, producing such stimuli is straightforward, the close spectral sensitivity of mouse opsins makes it difficult to achieve this aim without concomitant changes in the activation of rods and/or melanopsin. To circumvent this problem, we employed a well-validated transgenic model in which the native mouse MWS opsin is replaced by the human long-wavelength sensitive (LWS) opsin (Opn1mwR; [8,12,19]). Cones in these animals develop and function normally, with LWS opsin expression entirely recapitulating that of the native MWS opsin [21]. Importantly, however, the resultant shift in cone spectral sensitivity in Opn1mwR mice facilitates the generation of stimuli that provide selective modulation of individual opsin classes [22].

Using this Opn1mwR model, we first established a background lighting condition (using a three-primary LED system), whose spectral composition recreated a wild-type mouse’s experience of natural daylight (S1A Fig). We next designed a set of manipulations of this background spectrum that allowed us to modulate excitation of one or both cone opsins without any concomitant change in rod or melanopsin activation. Under these conditions, we were then able to unambiguously distinguish colour-sensitive neurons based on the following criteria: (1) the presence of larger responses to chromatic versus achromatic changes in cone excitation and (2) responses of opposite sign to selective activation of UVS and LWS opsin in isolation (i.e., excitatory/ON versus inhibitory/OFF).

To achieve the largest possible change in colour, we started by selectively modulating UVS and LWS cone opsin excitation in antiphase (“colour”; S1B Fig). We then compared responses to this stimulus with those elicited by one in which the change in UVS and LWS opsin activation occurred in unison (“brightness”; S1B Fig). Any spectrally opponent cells should be more responsive to the “colour” as opposed to “brightness” condition. We found that 17/43 SCN units (from 15 mice) that responded to these stimuli showed a significant preference for the pair in which UVS and LWS activation was modulated in antiphase (Fig 2A and 2B; paired t test, p<0.05, n = 17). As there were an additional 26 visually responsive units that did not respond to either of these analytical stimuli (paired t tests, p>0.05), these data indicate that at least one quarter of light-responsive SCN neurones show chromatic opponency.

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larger image TIFF original image Download: 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. https://doi.org/10.1371/journal.pbio.1002127.g002

Interestingly, we found that cone inputs exerted a much more powerful influence over the firing activity of cells exhibiting a preference for chromatic stimuli relative to than achromatic cells (Fig 2B; absolute change for responses of chromatic cells = 8.1 ± 2.3 spikes/s versus 1.9 ± 0.5 spikes/s for achromatic cells, n = 17 and 26 respectively; t test: p<0.01). Moreover, we found that the spiking activity for the majority (13/17) of colour-sensitive SCN neurons was highest during the stimulus phase biased towards UVS opsin activation (Fig 2A), and that these cells exhibited especially robust and sustained changes in firing (Fig 2A and 2B).

Our data above therefore indicate that cone inputs constitute a dominant influence on the firing activity of colour-sensitive SCN neurons and that most of these cells exhibit blue-ON/yellow-OFF colour opponency. We confirmed this by selectively modulating brightness for each of these cone opsins independently (stimulus shown in S2A Fig); as expected, these cells reliably increased firing in response to selective increases in UVS opsin activation and decreased firing following increases in LWS opsin activation (Fig 2C). Conversely, the remaining colour-sensitive cells exhibited the opposite preference (yellow-ON/blue-OFF; Fig 2C).

An aspect of mouse retinal organisation that poses a challenge to colour vision is that most cones in this species co-express UVS and MWS opsin [23]. The exceptions are rare “primordial S-cones” that only express UVS opsin [24] and peripheral cones that may express either pigment alone [25]. One might expect that chromatic opponency would rely on comparisons between these rare single pigment cones. If this were the case, then responses to LWS- and UVS-specific stimuli of defined contrast should be insensitive to changes in the spectral composition of the background light. In fact, we found that this was not the case (S2A–S2C Fig), indicating involvement of the more common opsin co-expressing cones in the chromatic responses of SCN neurons.

By contrast with chromatic SCN neurons, none of the cells identified as achromatic exhibited any overt OFF response to selective activation of either UVS or LWS cone opsin. Instead, these achromatic cells exhibited pure ON responses to stimuli targeting one or both opsin classes, such that on average the population showed little bias towards UVS/LWS opsin-driven responses under background spectra resembling natural daylight (Fig 2C). Adjusting the background spectra to equalise basal activation of the two cone opsins skewed responses in favour of UVS opsin, however (S2D Fig), consistent with previous suggestions that ipRGCs are relatively enriched in the UVS opsin-biased dorsal retina [26].

We next asked whether colour opponent cells also received irradiance information from the melanopsin-expressing ipRGCs that dominate retinal input to the SCN [4,6,7]. To this end, we used changes in spectral composition to selectively modulate melanopsin excitation (see Methods; 14/15 mice above tested with these stimuli). When presented with large steps in melanopsin excitation (92% Michelson contrast) generated in this way, “blue”-ON cells showed slow and sustained increases in firing (Fig 3A; peak response = 3.2 ± 0.8 spikes/s above baseline; paired t test, p<0.01, n = 13), as previously described for melanopsin-driven responses [8,27,28]. The behaviour of the rare “yellow” ON cells to this stimulus was variable (Fig 3B; n = 4), while colour-insensitive cells showed the expected excitatory response (Fig 3A; peak response = 1.8 ± 0.2 spikes/s above baseline; paired t test, p<0.01, n = 23). These data therefore reveal that both chromatic and achromatic cells have access to melanopsin-dependent information about irradiance.

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larger image TIFF original image Download: 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. https://doi.org/10.1371/journal.pbio.1002127.g003

Of note, for the smaller changes in opsin excitation applied above (70% Michelson; ~0.75 log units), we found that the inclusion of melanopsin contrast had little impact on the integrated cellular response. Thus for both chromatic and achromatic populations, responses evoked by spectrally neutral increases in irradiance (“energy”) were very similar in magnitude to those observed where changes in irradiance were restricted to just cone opsins (Fig 3A; subtraction: energy − “brightness”). This was true even for steady-state components of the SCN response (last 1 s of step)—we found no significant difference in responses to two conditions (paired t test; p>0.05 for both blue-ON and achromatic populations). Thus SCN responses to relatively modest changes in light intensity and/or spectral composition are, in fact, dominated by those originating with cones.

How then do chromatic and irradiance responses interact to encode time of day under more natural conditions? To address this question, we produced stimuli that recreated, for Opn1mwR mice, the change in irradiance and colour experienced by wild-type (green cone) mice across the twilight to daylight transition (Fig 4A). We presented these as discrete light steps from darkness, to simulate the challenge in telling time of day faced by a rodent emerging from a subterranean burrow to sample the light environment. Due to their scarcity, we were unable to determine the behaviour of yellow-ON cells under these conditions. However, blue ON cells reliably exhibited a near linear increase in firing rate as a function of simulated solar angle (Fig 4B and 4C; n = 9 from 7 mice), indicating that their sensitivity is well suited to track changes in colour/irradiance occurring across the twilight to daylight transition. Interestingly, the range of solar angles to which these neurons responded was substantially greater than that for achromatic cells recorded in the same set of mice (Fig 4D and 4E; see also S3 Fig; n = 8) indicating that they may be an especially important source of temporal information for the clock around twilight.

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larger image TIFF original image Download: 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. https://doi.org/10.1371/journal.pbio.1002127.g004

To determine the extent to which this ability of blue-ON cells to encode solar angle relied upon their chromatic opponency, we next presented stimuli that recreated the natural change in irradiance over twilight but in which colour was invariant. Two versions of these stimuli were produced, in which colour was fixed either to that at the lowest solar angle for which data was available (“night”) or to that recorded in daylight (“day”; Fig 4A). Whereas achromatic cells were unable to distinguish between these two stimulus sets (Fig 4D and 4E; F-test, p = 0.72), the relationship between solar angle and blue-ON cell firing rate was consistently disrupted under these conditions (Fig 4B and 4C; F-test, p = 0.009; see also S3A Fig). Thus, firing was reliably higher for “night” and lower for “day” conditions than appropriate for that time of day. These effects are consistent with the blue-ON nature of the chromatic units and confirm that these cells employ a combination of colour and irradiance signals in order to encode time of day.