It has been appreciated for several decades that the circadian oscillator in the SCN can be entrained to the day–night cycle by virtue of light–evoked signaling originating in the eyes33
, even in the absence of rod and cone photoreceptors. These observations led to the discovery of a specialized class of retinal ganglion cells, ipRGCs, that themselves are light sensitive and send projections to the SCN3, 5
. Subsequent work has been focused on accounting for how the ipRGCs, and the rod and cone photoreceptors, contribute to various circadian functions34
. While ipRGCs have been recognized as the only input to the SCN for relaying light–evoked signals for circadian photoentrainment1, 2, 4
, melanopsin phototransduction is relatively insensitive. Instead the rods and cones must provide input to the ipRGCs to account for the robust operating range of non–image forming functions7, 8, 35
. However, for circadian photoentrainment, the relative contribution of the three retinal photoreceptors has proven quite controversial12, 14–16, 20, 21, 36
. Here we distinguished the role of rods and cones (and ipRGCs) to circadian functions and found: 1) Gnat1–/–
mice lacking functional rods, but retaining both cone and melanopsin phototransduction pathways, do not photoentrain at scotopic light levels demonstrating that rods are necessary for circadian photoentrainment at low light intensities, 2) the ability of rod–only type 2 animals to photoentrain at all tested light levels indicating that light detection by rods is sufficient for photoentrainment across a surprisingly wide range of light intensities (ranging from approximately 102
), 3) rod–only type 3 animals entrain at low but not high light intensities suggesting that rod photoreceptors use two distinct pathways for photoentrainment; the primary rod bipolar pathway at low light intensities and electrical coupling to cones in the rod–cone pathway at high light intensities, 4) the relatively high threshold for phase shifting response is mediated by rods through the rod–cone pathway and the intrinsic photosensitivity of ipRGCs, whereas the more sensitive rod bipolar pathway can support photoentrainment with prolonged scotopic illumination.
Despite previous evidence that responses from both rods and cones are necessary for circadian light responses, we show that rods are the major contributor to photoentrainment, reconciling several published observations. First the peak of the action spectra for circadian responses in both humans37–39
is near 500 nm, closely resembling the spectral sensitivity of rods but not cones. Second, RPE65–/–
animals, which lack a key component of the visual cycle leading to complete loss of cone function and attenuated rod function, still show photoentrainment in the background of the melanopsin knockout41
. In fact, even with the highly attenuated rod function, these animals show better photoentrainment than animals with fully functional cones36
, although their photoentrainment is impaired compared to WT animals41
. Third, our results show that rods can continue to entrain the circadian oscillator into photopic light intensities even under conditions when the persistent activity of the rods renders them incapable of supporting spatial vision.
Of interest is the fact that rod–only type 1 animals failed to photoentrain at low light intensities, despite demonstrating normal vision at scotopic light intensities. This apparent contradiction may result from the ‘continuous light’ condition in rod–only type 1 mice resulting from the deletion of CNG channels in the cone outer segments, which we propose adapts the retinal circuit that signals to ipRGCs. Thus, the adapted state in rod–only type 1 animals may not be sufficient to influence image formation, which will largely depend on encoding contrast, but it might influence circadian photoentrainment whereby rod signals feed through cone circuits to signal absolute irradiance levels in the environment.
In rod–only type 3 animals, we surprisingly observed responses in OFF cone bipolar cells despite the absence of cone photoreceptors; signals that we suspect originate from the rod spherules themselves. In the rodent retina a third rod pathway has been identified whereby OFF cone bipolar cells synapse onto the rod spherule28, 42
, and the sensitivity of this pathway is comparable to the rod–cone pathway43
. Under conditions where the cones are absent we postulate that the cone bipolar cells are reflecting signals through this third rod pathway. Interestingly, this pathway on its own appears insufficient for circadian photoentrainment at high light intensity.
Several studies have inferred a contribution of cone phototransduction to circadian light responses based on the action spectrum for photoentrainment16, 44
. One study, using mice that lack M–cones (515nm), found attenuated phase shifts in response 530nm but not 480nm13
. This data lead to a model whereby cones and ipRGCs account fully for light effects on the circadian oscillator13
. However, since photoreceptors have a broad absorption spectrum, the light intensities used in this study would have activated rods. Furthermore the developmental loss of M–cones might have hindered rod signals from using the rod–cone pathway thereby attenuating phase shift responses at 530nm.
Two recent studies attempted to exploit the differences in the spectral sensitivity of rod and cone photopigments to drive one photoreceptor type in preference of the other to determine their relative roles in non–image forming functions. One of these studies was carried out in humans17
, whereas the other utilized a mouse line that substituted transgenically the human L cone opsin in mouse M cones allowing them to increase spectral separation between the rod and cone light responses18
. Despite using similar strategies to drive selectively rod and cone phototransdution, both groups reached opposing conclusions about the role of rods and cones in circadian functions. It should be appreciated that despite greater efficacy in activating one photoreceptor type versus another, these approaches also don’t fully separate the light–evoked activity of rods and cones17
. Our strategy of selectively eliminating rod or cone phototransduction, or cone photoreceptors altogether, provides a stringent separation of these photoreceptors’ contributions to circadian functions.
Since rods use the cone circuits to drive photoentrainment, it seems paradoxical that that cone phototransduction alone fails to photoentrain animals. The tremendous capacity of cone phototransduction to adapt to increases in light intensity may ultimately be responsible for this phenomenon, which will prevent their photocurrent from saturating even under bright, bleaching light conditions. The recovery of dark current in cones allows both the resting membrane potential and thus glutamate release to recover toward basal levels in darkness. As such we propose that cone phototransduction would be much less able to signal steady light intensity. The persistent hyperpolarization of rods during bright, bleaching light exposures45, 46
may thus be better suited to signaling irradiance through the cone pedicle to ipRGCs which influence circadian photoentrainment. Consistent with this notion, cone adaptation impairs their ability to signal light for non–image functions, especially under prolonged light treatments17, 18
These results provide a simple model (Fig. S3
) for how the outer retinal photoreceptors and ipRGCs account for photoentrainment. At low light intensity, ipRGCs lack sensitivity while rods are known to respond to increasing light levels and thus reliably relay this information to higher centers. Rods will continue to signal persistent light exposure through the rod–cone pathway even under conditions where their photocurrent is saturated. Finally, at high light intensities and for prolonged light exposures, melanopsin phototransduction in ipRGCs will extend the range of light intensities that allow circadian photoentrainment. Ultimately the properties of rod and melanopsin phototransduction, as well as the rod pathways that impinge on ipRGCs, can account fully for the ability of mammals to photoentrain throughout physiologically relevant light conditions5, 10