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From the Department of Integrative and Molecular Neuroscience, Division of Neuroscience and Psychological Medicine, Faculty of Medicine, Imperial College of Science, Engineering, and Medicine, London, United Kingdom.
The improver of natural science absolutely refuses to acknowledge authority, as such. For him, scepticism is the highest of duties: blind faith the one unpardonable sin. Thomas Henry Huxley, Addresses and Reviews, 1871.
The title of this article, and the topic that will dominate this review, relates to the role of the eye in regulating mammalian circadian rhythms. This review, however, will not be confined to a consideration of the circadian system alone. My objective in the following pages is to summarize the development of a line of research that has taken place over approximately 10 years and that has forced us to think very differently about the eye. What started in my group as a fairly narrow consideration of how biological clocks are regulated by light, has now expanded into a much broader interest in how the eye might collect light to generate a measure of the overall amount of light in the environment. The course of these investigations has led to the discovery that varied aspects of physiology and behavior appear to be regulated by non-rod, non-cone ocular photoreceptors. I will outline the evidence that supports this radical conclusion, review what we know about this novel photoreceptor, and draw attention to some of the broader implications of these results. Because much of this work has its origins in the study of biological clocks, this is where I start the discussion.
The primary role of the circadian system is to set the phases at which physiological and behavioral events occur with respect to the 24-hour environmental cycle or in relation to rhythmic events within the organism. Thus, the circadian system must remain synchronized, or entrained, to the solar day. When entrained, the circadian clock adopts a distinct phase relationship with the astronomical day, and each of the different expressed rhythms adopts its own phase relationships with the clock. The systematic daily change in the irradiance of light at dawn or dusk provides the most reliable indicator of the phase of the day. As a result, most organisms have evolved to use the twilight transition as the primary zeitgeber to adjust circadian phase (photoentrainment).1 But, of course, environmental irradiance does more than regulate biological time. Varied aspects of mammalian physiology, endocrinology, and behavior respond to gross changes in environmental light. For example, pineal melatonin production, pupil size, adrenal cortisol secretion, heart rate, and body temperature are all affected by irradiance.2 3 4 5 Furthermore, in our own species, increasing environmental irradiance can result in marked improvements in alertness and performance6 and changes in electroencephalic (EEG) and electro-oculogram (EOG) correlates.7
It is important to note that the circadian and physiological responses just mentioned are modified by gross changes in environmental light, rather than by specific patterns of light. In addition, the thresholds for eliciting these responses are higher, and the time over which the light stimuli are integrated is significantly longer, than for classic vision.8 For example, hamsters can recognize optical gratings at a luminance level 200 times lower than the irradiance required to induce phase shifts in locomotor rhythms.9 In addition, hamsters are relatively insensitive to an entraining light stimulus of less than 30 seconds’ duration.10 11 Such observations have led to the appreciation that the eye performs two very different light-detecting tasks that can be likened to physical detectors that measure either radiance or irradiance (Fig. 1) . For the purposes of brevity, the primary photosensory task of irradiance detection is referred to as non-image-forming (NIF) photoreception, and classic visual responses are referred to as image-forming (IF) photoreception.
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Significantly, type III ganglion cells have also been shown to innervate several other regions of the brain that respond to gross changes in environmental light. The olivary pretectal nucleus (OPN) is involved in pupilloconstriction22 23 and the consensual pupillary light reflex24 and receives substantial input from type III RGCs.21 Along with the SCN and IGL, the OPN is the only other dedicated irradiance detector described in any detail within the mammalian brain.22 23 Another target for type III RGCs is the ventrolateral preoptic nucleus (VLPO), a region of the rodent brain that appears to be involved in sleep-wake regulation.25 Environmental irradiance regulates sleep through the circadian system, but also has effects that are quite independent of the clock. For example, sustained exposure to light increases non-rapid eye movement (NREM) sleep in rats,26 27 28 and darkness delivered during the light phase of a 24-hour light-dark cycle can trigger rapid eye movement (REM) sleep.28 29 It has been argued recently that these acute effects of light on sleep may be mediated by the type III RGC projection to the VLPO.25
In contrast to the SCN, IGL, OPN, and VLPO, the IF visual system consists of subcortical pathways to the geniculate complex and superior colliculus. These projections are highly ordered and retinotopically mapped. Thus, the mammalian eye has parallel outputs, encoding predominantly NIF or IF information. Until recently, there was no reason to expect that these divergent projections from the eye would use distinct classes of photoreceptors. The very reasonable assumption was that retinal rods and cones mediate both NIF and IF light detection.
A Novel Ocular Photoreceptor in Mammals
Unlike mammals, nonmammalian vertebrates possess several photoreceptor organs in addition to their ocular photoreceptors. These extraocular photoreceptors mediate NIF responses to light and have been classified as the pineal organ or pineal body (epiphysis cerebri), which is photoreceptive in all nonmammalian vertebrates; the parapineal organ, found in many teleost fish and well-developed in the lamprey; the extracranial "third eye," called a frontal organ in frogs and the parietal eye/body in lizards (missing in birds); and deep brain photoreceptors, which are located in several sites of the brain and are found in all nonmammalian vertebrates.30 Note that these extraocular photoreceptors are more complex than one might imagine. For example, the pineal organ of fish and birds contains multiple photopigments, expressing rod- and conelike, as well as novel, photopigments within the same organ.31 32 33 34 35 Of these extraocular photoreceptors, the deep brain and pineal photoreceptors have been shown to play a critical role in the regulation of circadian clocks (for a full review see Ref. 30 ). Mammals are unique among vertebrates, in that they appear to have lost extraocular photoreceptors.36 Why this has occurred is unclear, but it may be due to the early evolutionary history of mammals and their passage through what has been called a "nocturnal bottleneck."37 Modern mammals seem to have been derived from nocturnal insectivorous or omnivorous animals approximately 100 million years ago. Extraretinal photoreceptors may not have been sufficiently sensitive to discriminate twilight changes in primitive mammals spending their days hiding in holes or crevices and then emerging at dusk.38 39
The existence of extraocular photoreceptors in nonmammals and their role in regulating circadian rhythms in these vertebrate classes reinforced the view that extraocular photoreceptors regulate NIF responses to light, whereas the eye is dedicated to the collection of IF information. By extension, it was assumed that because the mammals had lost their extraocular photoreceptors they were "forced" to use ocular rods and cones for both IF and NIF responses to light. Until recently, there was no reason to doubt this assumption; however, studies on the mammalian circadian system have provided the substrate to challenge this view.
Photoentrainment of the Mammalian Circadian System
The overwhelming mass of experimental evidence tells us that the circadian system of mammals is entrained by photoreceptors within the eye. However, some mention should be made of a report in Science that suggests that bright light of approximately 13,000 lux applied to the popliteal region (skin behind the knee) can induce a shift in circadian rhythms of body temperature and melatonin.40
There has been much reluctance to accept these results by most circadian and vision biologists, not least because eye loss in humans41
42
and other mammals (e.g., Refs. 43
44
45
) always blocks photoentrainment. Nevertheless, the implications of these findings were so important that several groups have tried, and continue to try, to provide independent support for the possibility that dermal irradiation can modify the circadian physiology of humans and other mammals. To date, however, results in all such studies have failed to support this possibility. For example, popliteal illumination produced no effect on the suppression of nocturnal levels of pineal melatonin in human subjects,46
and exposing the chest and abdomen to 13,000-lux broad-spectrum light did not shift rhythms of melatonin, cortisol, and thyrotropin.47
Although clearly ocular,48 disentangling which retinal cells mediate photoentrainment has presented a problem. The use of mice with naturally occurring genetic disorders of the eye, however, provided a way forward. The original intent was to correlate rod and cone photoreceptor loss with a loss of sensitivity in the circadian system to light. The first animals used for such studies were mice homozygous for retinal degeneration (rd/rd). Despite the massive (but not complete) loss of rods and cones in rd/rd mice49 50 these animals showed circadian responses to light that were indistinguishable to nondegenerate congenic control mice (rd+, rd+/+). The sensitivity of the circadian system to light did not parallel the loss of either rod or cone photoreceptors,51 but removal of the eyes abolished all circadian responses to light in rd/rd mice.44 Our studies on the rd/rd mice contradict an earlier report that suggested that this mutation would attenuate circadian photosensitivity.52 This earlier report showed that the threshold for entrainment in C57 wild-type mice was approximately 2 log units lower than the threshold for entrainment in C3H rd/rd mice. This difference, however, was later shown to be an artifact of comparing different strains of mice rather than an effect of the rd/rd mutation. When congenic C3H+/+ and C3H rd/rd mice are examined under the same experimental conditions, no reduction in circadian photosensitivity is observed.53 54 This example illustrates the importance of controlling for genetic background when examining the impact of a gene defect.55 56 57
Our findings in rd/rd mice and parallel findings in mice homozygous for retinal degeneration slow (rds)58 suggest that at some level the processing of light information for IF and NIF photoreception must be profoundly different. Additional support for a distinction between these photic pathways has come from studies in humans. Two reports have demonstrated that a significant subset of individuals who have eyes but have lost conscious light perception due to retinal disease have retained the ability to suppress melatonin41 and entrain circadian rhythms of behavior.42 Although the findings in retinally degenerate mice and these observations in humans were surprising, they did not demonstrate the existence of novel ocular photoreceptors. Because small numbers of rods and/or cones remain in the retina of these retinally degenerate mammals, these experiments were unable to resolve whether the circadian axis is able to retain normal photosensitivity using only very small numbers of rods and/or cones. To investigate this, we generated two lines of transgenic mice in which both rod and cone photoreceptors were absent (Fig. 2) . This was achieved by introducing a synthetic transgene (cl) consisting of an attenuated diphtheria toxin coding sequence driven by a portion of the human red cone opsin promoter59 60 into two lines of mice that had no rods. We used either rd/rd mice or mice that carried a separate diphtheria toxin-based transgene (rdta).61 Despite the absence of both rod and cone photoreceptors, rd/rd cl and rdta cl mice retain superficially normal circadian responses to light, as demonstrated both by the phase-shifting effects of a single 15-minute pulse of light on free-running rhythms of circadian locomotor activity and by the acute inhibition of pineal melatonin production.62 63 As with rd/rd mice, enucleation of the eyes in rd/rd cl or rdta cl mice abolishes these effects of light on the circadian system. These results demonstrate that the circadian system can use unidentified ocular photoreceptors. Furthermore, our most recent studies show that two other NIF light responses, pupillary constriction and acute alterations in locomotor behavior, also survive the loss of rod and cone photoreceptors.
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The Regulation of Masking Behavior
Nocturnal rodents show a marked inhibition of locomotor activity when exposed to light during the night.74
This response is thought to complement photoentrainment by ensuring that activity is restricted to the hours of darkness. As a result, it has been commonly referred to as "circadian masking" or simply "masking."75
Previous reports have indicated that masking survives significant photoreceptor loss.76
77
However, the use of rd/rd cl mice allowed us to ask whether masking can occur in animals that have no rod and cone photoreceptors.78
Both wild-type and rd/rd cl mice exhibited a marked inhibition of wheel-running activity during acute exposure to a broad-spectrum green light presented 2 hours after normal lights off (Fig. 4A)
. At high irradiances, activity was reduced to around 10% of normal wheel running in both genotypes. These effects on activity were irradiance dependent (Fig. 4B)
, and there was no significant difference in sensitivity associated with rod and cone loss.78
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A Novel Ocular Photopigment in Mammals
Beyond the fact that novel photoreceptors exist and that they may regulate diverse aspects of our "vegetative" responses to light, what do we know about these receptors? Photoreceptor characterization has traditionally been based on a number of complementary approaches (see Critical Criteria, to follow), but perhaps the most useful single technique is to conduct an action spectrum to define the nature of the photopigment. A photopigment has a discrete absorbance spectrum, which describes the probability that photons are absorbed as a function of wavelength, and the magnitude of any light-dependent response depends on the number of photons absorbed by the photopigment. Thus, a description of the spectral sensitivity profile (action spectrum) of any light-dependent response must by necessity match the absorbance spectra of the photopigment mediating the response, provided that any confounding factors, such as screening pigments or absorption of the ocular media, are taken into account.80 81
Although light can be used in a variety of ways (as an energy source, as a sensory stimulus, or as a regulatory signal), relatively few photopigments appear to have evolved.82 This may relate to the demanding task of a photopigment molecule. It must be able to absorb a photon with high probability and, again with high probability (high quantum efficiency), pass on this information to a transduction mechanism.83 In nature, a wide variety of light responses are regulated by only a few types of photopigment, and these have highly conserved absorbance spectra. For example, both the action spectra for phototaxis in the algae Chlamydomonas and visual photosensitivity in humans are described by the same standard absorbance spectrum for an opsin-vitamin A photopigment.83 In the same way, flavoprotein-based photoresponses can be described by conserved absorbance spectra.82 83 It is worth stressing that when an action spectrum is conducted correctly, it provides one of the few approaches that can be used to identify a class of protein and simultaneously link that protein to a particular cellular or behavioral function.
Because action spectra reflect photopigment absorbance spectra and because individual photopigment families have characteristic absorbance spectra, the involvement of candidate pigments can be assessed using action spectrum techniques.82 83 In an attempt to characterize the photopigment(s) mediating NIF responses to light, we have initiated a series of action spectra on rd/rd cl and wild-type mice. The first completed was an action spectrum for the PLR.71 The PLR is particularly well suited for spectral analysis, in that pupillary constriction is relatively easy to monitor and shows only a small degree of interanimal variance.71 This permitted us to generate a well-resolved action spectrum based on the irradiance required to induce a 50% pupil constriction at 10 monochromatic wavelengths between 421 and 625 nm. This action spectrum was then compared to the absorbance spectra of the known photopigments of the mouse (Fig. 5) . The results demonstrate that the PLR in rd/rd cl mice, over the range 420 to 625 nm, is driven by a single, previously uncharacterized, opsin-vitamin A-based photopigment with peak sensitivity at 479 nm (opsin photopigment [OP479]). These data represent the first functional characterization of a non-rod, non-cone photoreceptive system in the mammalian eye.71 Whether OP479 mediates all non-rod, non-cone NIF responses to light remains to be determined. A preliminary action spectrum for phase-shifting circadian rhythms of locomotor behavior in rd/rd cl mice suggests the involvement of an opsin-vitamin A-based photopigment with a wavelength of maximum sensitivity significantly different from the mouse visual opsins, but very close to that of OP479 (Thompson C, Lucas R, Foster R, unpublished observations, 2002). A more complete action spectrum for phase shifting and additional action spectra for those light responses preserved in rd/rd cl mice (e.g., melatonin suppression and masking behavior), will determine the extent to which OP479 contributes to the light responses of the mouse.
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So, which photoreceptor cells of the eye use OP479? In the absence of an outer retina in rd/rd cl mice and knowing that light information is transmitted to the SCN, IGL, and OPN through type III RGCs, it seems that these photoreceptors must reside within the inner retina. Two lines of evidence have suggested that a subclass of RGC may be directly light sensitive. Indirect and preliminary evidence comes from our studies in the aged rd/rd cl mouse.87 After 9 months of age rd/rd cl mice start to lose much of the inner retina, so that by 18 to 24 months of age, there is little left of the inner retina other than the RGC layer. Despite this loss, rd/rd cl mice show both circadian and pupillary responses to light. Furthermore, a subset of RGCs in these aged rd/rd cl mice show induction of the immediate early gene c-fos in response to light exposure. On the basis of these data, we proposed that a subset of ganglion cells house the novel ocular photoreceptors.87 More direct evidence for photosensory ganglion cells comes from an exciting study by Berson et al.88 at Brown University (Providence, RI). Rat RGCs were retrolabeled from the SCN with rhodamine beads, then recorded by whole-cell current clamp in isolated retinal flatmounts. Light was shown to tonically depolarize most SCN-projecting RGCs, and, significantly, these responses persisted when these cells were isolated from rod and cone inputs using both microsurgical isolation and a cocktail of agents that block synaptic transmission.88
Inner Retinal Photoreception in Nonmammals
Thus far, the discussion of the existence of a novel opsin-based photoreceptor within the inner retina has been limited to the mammals. However, parallel experiments on the retina of teleost fish provide independent support of the existence of a non-rod, non-cone ocular photoreceptor in vertebrates.
In an effort to characterize the extraocular photoreceptors of Atlantic salmon, we developed degenerate PCR primers to amplify opsin cDNAs from the salmon central nervous system (CNS). To optimize our PCR procedures, we first tested these degenerate primers using salmon ocular cDNA. An initial screen identified a cDNA whose conceptual translation shared only 37% to 42% identity with any of the known opsin families. This level of identity immediately isolated this opsin into its own, previously unrecognized opsin family89 (Table 1) .
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Candidate Novel Photopigment Genes in Mammals
As discussed in the foregoing section regarding a novel ocular photopigment in mammals, behavioral studies and action spectrum results in rd/rd cl mice have provided overwhelming evidence for the existence of a novel opsin-based photopigment within the eye. Results from aged rd/rd cl mice and exciting studies emerging from David Berson’s laboratory suggest that some retinal ganglion cells may be directly light sensitive. These findings in mammals, together with the VA opsin results in teleosts reviewed in the section just prior, are all consistent with the hypothesis that the rods and cones are not the only photoreceptive cells within the vertebrate retina. But further analysis of this photoreceptor pathway is going to be heavily dependent on the identification of the photopigment gene(s) and protein product. In recent years, two classes of photopigment molecule have been proposed for these novel photoreceptors, the opsins and the cryptochromes.
The Opsin Candidates
The isolation of VA opsin in fish immediately led several groups to look for orthologues of this gene in mammals. To date, this search has been unsuccessful. However, several other opsin genes have been identified and proposed as candidates.93
It is also clear from our own investigations that yet more opsin candidates will emerge from the human and mouse genome data bases. But how are we to assess the candidacy of these genes based on their sequence identity alone? What features should guide the search for a novel photopigment gene? This is more complex than might first be thought. All the known opsin-based vertebrate photopigment families share approximately 40% amino acid identity with each other, approximately 25% identity with the invertebrate photopigments, and approximately 17% with all other G-coupled receptors (Table 1) . Other than levels of overall identity, surprisingly few features of the photopigment opsins distinguish them from other members of the G-coupled receptor superfamily. Two features, however, have been considered critical: the possession of a lysine residue at position 296 for Schiff base attachment of the chromophore, and a glutamine at position 113 as a charge counterion for the chromophore.94
Unfortunately, these features cannot be considered diagnostic for an opsin photopigment. Not all photopigment opsins use a glutamine counterion. For example, some members of the short-wavelength visual pigment family use an aspartate at this site.95
Furthermore, the lysine residue at 296, which is essential for chromophore attachment, is also found in opsins that are not thought to have a photosensory function. For example, the retinal G protein-coupled receptor (RGR) differs from the photopigment opsins in that its preferred chromophore is all-trans-retinaldehyde rather than 11-cis-retinal. On exposure to light, RGR photoisomerizes the all-trans chromophore to the 11-cis configuration. This known function, coupled with the fact that RGR is expressed in the retinal pigment epithelium (RPE) and Müller cells96
and that RGR shares only 22% identity with the visual pigment opsins (Table 1)
, suggests a role as a photoisomerase rather than a sensory photopigment.97
We are left therefore with few strong guidelines to direct the search for novel opsin photopigment genes.
Of the various opsin candidates proposed recently,93 melanopsin has emerged as the most interesting.98 99 100 101 It can be argued that melanopsin is not a strong candidate based on its level of identity; it shares only 27% identity with the known vertebrate photopigments (Table 1) . Furthermore, the genomic structure of this gene bears no resemblance to the known photopigment opsins of the vertebrates.101 Both of these features contradict the argument that functionally related molecules are likely to share a close phylogenetic relationship based on both amino acid identity and genomic structure. However, melanopsin is interesting, because in many respects it resembles an invertebrate photopigment. Moreover, it is expressed in very few RGCs and in even fewer cells within the amacrine cell layer in the mouse retina. This distribution is strikingly similar to the distribution of murine RGCs that form the retinohypothalamic tract, and project to the OPN.21 In a very recent study, rat RGCs that show intrinsic photosensitivity have been shown to express melanopsin.102 Thus melanopsin, at the very least, is a good marker of a subset of inner retinal photoreceptors. The on-going analysis of this fascinating gene, including its functional expression, will determine whether melanopsin is OP479 or whether it performs some other function within the eye, perhaps acting as a photoisomerase and part of an as yet unidentified opsin photopigment system within these photosensitive RGCs.
The Cryptochromes
An alternative hypothesis to the involvement of opsin-based photopigments is that the cryptochromes act as photopigments within the inner retina. It has been argued that the mammalian eye contains a class of flavoprotein-based photopigment called cryptochrome (CRY).103
The origins of this hypothesis have, to a large extent, arisen from the presumed photoreceptive role for the CRYs in other organisms.104
CRY-like proteins are found in diverse phyla and belong to a group of proteins that bind both a pterin (methenyltetrahydrofolate [MTHF]) and a flavin (7,8-didemethyl-8-hydroxy-5-deazariboflavin [8-HDF]). On the basis of sequence similarity and defined function, these pterin-flavin-binding proteins can be subdivided into the photolyases and cryptochromes.105 Perhaps the best characterized are the photolyases, which repair UV damaged DNA and of which there are two types. The pyrimidine dimer photolyases harvest the energy of near-UV light to fix UV-induced cyclobutane pyrimidine dimerization in DNA.106 The other group is the (6-4) photolyases, which perform a similar function but act specifically on UV-induced pyrimidine (6-4) pyrimidine dimers.107 In contrast to the photolyases, the cryptochromes use near UV light for a non-DNA repair function. The cryptochromes were first named in the plants and appear to act as blue/UV photopigments and contribute to several physiological responses.108 109 110 111 Two CRY proteins have been identified in plants, CRY1 and -2.105 Disruption of cry1 attenuates a number of blue light responses, including shortening of the circadian period by constant blue light.112 Disruption of cry2 again disrupts a number of photic responses, including the regulation of flowering by day length.109 Clearly, the plant CRYs are involved in a number of blue light-regulated processes, and it has been argued that these proteins must act as photopigments.105 However, we do not currently have sufficient experimental evidence to say whether the plant CRYs are acting as photopigments or as components of a light-input pathway, perhaps coupling a photopigment to a transduction cascade (see the following section, Critical Criteria).
A single CRY-like gene has been identified in Drosophila (dcry)113 and two CRY-like genes in mammals (cry1 and cry2).114 Note that the naming of the animal cryptochrome genes is a little misleading. The animal CRYs show a much higher degree of identity with the (6-4) photolyases than to the plant cryptochromes, but despite their overall similarity to the (6-4) photolyases, they show no apparent photolyase activity. In addition, the mammalian CRYs have a COOH-terminal extension that is absent in the photolyases but is similar to the COOH extension found in the plant CRYs. These features (absence of photolyase activity and the COOH tail) were used to group the plant and animal CRYs together and then to suggest that the animal (Drosophila and mammal) CRYs are photopigments that mediate blue light’s effects on the circadian system.104 105 115 116
Genetic manipulation of dcry has been shown to modify the effects of light on the circadian system. For example, underexpression of dcry reduces the size of phase shifts,117 whereas overexpression of dcry leads to larger phase shifts.113 Additional evidence that CRY contributes to photoentrainment comes from studies of a mutation in the dcry gene that has been called crybaby (cryb).118 Although cryb mutants can entrain their locomotor rhythms almost normally and rhythmic clock gene expression can be entrained in the lateral neurons of the brain,118 mutants are unable to reset their clocks to short light pulses,118 and cryb mutants do not become arrhythmic under continuous light conditions.115 116 In Drosophila, as in plants, CRY seems to act somewhere on the light-input pathway to the clock and may even act as a photopigment, acting both to absorb light and transduce light information directly to the molecular clock. However, critical photobiological experiments have yet to be undertaken that distinguish between CRY’s acting as a photopigment or as an element of the phototransduction cascade (see Critical Criteria). The obvious approach to resolve this issue would be to match action spectra in Drosophila to CRY absorbance spectra or to the absorbance spectra of other flavoprotein photopigments.83 Unfortunately, this has not yet been accomplished with any degree of precision. It is also important to note that although CRY may act as a photopigment in the entrainment of Drosophila circadian rhythms, it is not the only photopigment. Opsin-based photopigments in the compound eyes and the Hofbauer-Buchner eyelet are strongly implicated in photoentrainment in Drosophila54 and other insects.119
In contrast to the situation in Drosophila and despite claims that the CRYs mediate all of light’s effects on the mammalian clock,103 there is no direct evidence that these proteins contribute to photoentrainment or to any other photosensitive process in mammals. The mammalian CRYs appear to play a central role in the generation of the circadian oscillation itself. The strongest evidence for this comes from the work of van der Horst et al.120 This group showed that cry1-/-cry2-/- mice are completely arrhythmic, with no indication of a functional circadian clock under conditions of continuous darkness. When exposed to a light-dark cycle, cry1-/-cry2-/- mice confined most of their activity to the dark portion of the cycle but showed no evidence of anticipating light-dark transitions (as do normal mice). Suggesting that the behavior observed was simply masking (see earlier section on regulation of masking behavior) and driven by exposure to light and dark rather than being influenced by an endogenous clock. Significantly, the disruption of the cryptochrome genes in cry1-/-cry2-/- mice does not block the light-induced expression of the two clock genes mPer1 and mPer2 in the SCN.121 Thus, on the basis of the current evidence, the mammalian CRYs appear to act as components of the molecular clock.122 We speculated that the animal CRYs may contribute to clock function by mediating REDOX reactions.123 Exciting new results from the McKnight laboratory have provided evidence that this may indeed be the case.124 125
Some researchers argue, however, that the mammalian CRYs have a dual function as both components of the oscillator and as photopigments. Indeed, a recent report appeared to support this possibility. In an attempt to define the photoreceptors that contribute to masking behavior, rd/rd mice (see foregoing section, Photoentrainment of the Mammalian Circadian System) were crossed with cry1-/-cry2-/- mice to generate mice without classic photoreceptors and CRYs (although rd/rd mice still have some cones). In contrast to young cry1-/-cry2-/- mice, which confine most (80%) of their activity to the dark portion of the light-dark cycle, the combined rd/rd cry1-/-cry2-/- mice spread their activity more evenly between the light and dark, with 37% to 40% of their activity occurring in the light phase.126 These results suggest that masking is abolished in rd/rd cry1-/-cry2-/- mice, and by extension, that classic and CRY photoreceptors contribute in a redundant manner to masking. This interpretation, however, does not take into account the following results. Approximately 30% of rd/rd cry1-/-cry2-/- mice are still able to mask. Furthermore, some mice that can mask at 5 to 6 months lose this ability by 9 to 12 months127 (Van Gelder RN, written communication, 2001). These results suggest that the loss of masking may be independent of the direct effect of losing CRY, the rod photoreceptors, and most cone photoreceptors. This conclusion is supported by two independent observations showing that masking is frequently lost in mice more than 6 months of age who lack only their CRY proteins (cry1-/-cry2-/-) (van der Horst GTJ and Mrosovsky N, written communication, 2001). Thus, the loss of masking in cry1-/-cry2-/- and rd/rd cry1-/-cry2-/- mice may be due to some secondary effect of CRY loss. One explanation could be that CRY loss precipitates an ocular disease that results in the secondary loss of the photoreceptors that mediate masking. These rather complicated arguments are summarized in Table 2 .
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We now have overwhelming evidence that a variety of NIF responses to light are regulated by non-rod, non-cone ocular photoreceptors within the mammalian eye, and evidence from pupillary action spectra show that at least one NIF response is regulated by a novel opsin-vitamin A-based photopigment. Furthermore, the recent findings showing that a subset of RGCs are intrinsically photosensitive in the rat retina supports the conclusion that the rods and cones are not the only cells within the mammalian retina capable of photoreception. However, these finding do not demonstrate that the classic rod and cone photoreceptors play no role in NIF responses to light. Indeed, there may be a close interaction between rods, cones, and novel photoreceptors in the regulation of the murine pupil.71 Furthermore, several studies have indirectly implicated classic photoreceptors in photoentrainment.133 134 135 Presumably, this multiplicity of photoreceptor inputs to the clock relates to the complex sensory task of twilight detection.1 A decade of research now allows us to move on from experiments designed to demonstrate the existence of novel ocular photoreceptors, to experiments directed toward an under standing of this unexplored photosensory system of the eye. It is clear, however, that this understanding will emerge only if the cellular and molecular analysis is undertaken in parallel with a consideration of those features of the light environment used to regulate circadian and other NIF responses to light.
Acknowledgements
I thank all my colleagues who have collaborated on these studies during the past 10 years. I want, however, to extend my particular thanks to Robert Lucas and Ignacio Provencio, without whom much of the work outlined in this review could not have been undertaken. Furthermore, I offer my thanks to Robert Lucas, James Bellingham, and Mark Hankins for their help in the preparation of the manuscript and Nicholas Mrosovsky, Bert van der Horst, and Russell Van Gelder, for communicating to me their unpublished observations in transgenic mice without cryptochrome.
Footnotes
Submitted for publication July 20, 2001; accepted August 21, 2001.
Supported by the Biotechnology and Biological Sciences Research Council, United Kingdom, and the European Union BioMed2.
Commercial relationships policy: N.
Corresponding author: Russell G. Foster, Department of Integrative & Molecular Neuroscience, Division of Neuroscience & Psychological Medicine, Faculty of Medicine, Imperial College of Science, Engineering and Medicine, Charing Cross Hospital, Fulham Palace Road, London, W6 8RF, UK; r.foster{at}ic.ac.uk.
References
max 506 nm, half bandwidth 10 nm) stimulus for wild type and rd/rd cl mice. The data are fitted with a sigmoidal function of slope 0.58 (wild type) and 1.05 (rd/rd cl).
