CIRCADIAN RHYTHMS:
The Clock Plot Thickens

Marcia Barinaga

Volume 284, Number 5413 Issue of 16 Apr 1999, pp. 421 - 422

Researchers prove that a nonvisual light sensor sets our daily clock; a likely candidate for that role appears to fulfill other clock functions, too

One of our most indispensable biological machines is our circadian clock, which acts like a multifunction timer to regulate sleep and activity, hormone levels, appetite, and other bodily functions with 24-hour cycles. The clock generally runs a bit fast or slow and must be reset daily by sunlight. Although many components of the clockwork are known, the crucial photoreceptor that passes light's signal to the clock is still at large.

Two suspects, the light-sensitive pigments in the rod and cone cells of the mammalian eye, are eliminated by two papers in this issue. "The really important conclusion from these experiments is that there is another photoreceptor" affecting the clock, says circadian biologist Michael Menaker of the University of Virginia, Charlottesville.

One candidate for that photoreceptor is a protein called cryptochrome. But a report in yesterday's issue of Nature puts an intriguing wrinkle in that story, fingering cryptochrome as a likely part of the clock itself. In mice that lack cryptochrome, the group found, the clock doesn't run at all. "We have never seen [in mice] a mutant like this, where there is instant arrhythmicity," says clock researcher Steve Kay of The Scripps Research Institute in La Jolla, California. That means cryptochrome is essential for clock function, but leaves open the question of whether it is the long-sought circadian photoreceptor in mammals.

Biologists have known since the 1960s that the clock-setting light signal in mammals normally comes via the eyes, because eyeless rodents and humans with few exceptions are unable to reset their clocks to light. One obvious possibility is that the molecules that capture light for vision--the opsins in the rod and cone cells of the retina--also send light signals to the clock.

Evidence against that has mounted as researchers have found that mice lacking either rods or cones have clocks that respond to light. But the chance remained that rods and cones both can do the job, and either can do it alone. The reports on pages 502 and 505 by Russell Foster of the Imperial College of Science, Technology and Medicine in London and his colleagues rule that out.

The researchers introduced genes that destroy retinal rod and cone cells into mice. They found that in those mice, just as in normal mice, light resets the clock and suppresses production of the clock-controlled nocturnal hormone, melatonin. "That says that you don't need rods and cones" for the light response, says Menaker, and means another photoreceptor must do the job.

Cryptochrome, which is found in the eye, became a hot candidate for the photoreceptor last fall, when three teams reported that it seems to help light reset the clock in plants, fruit flies, and mice (Science, 27 November 1998, p. 1628). A group led by Aziz Sancar at the University of North Carolina, Chapel Hill, and Joseph Takahashi at Northwestern University in Chicago mutated cry2, one of two mammalian cryptochrome genes, in mice. The animals' clocks lost some light responsiveness, suggesting that Cry2 is a light sensor, but not the only one. Researchers wondered if Cry1 might be the other, and waited to see if mice missing both cryptochromes could adapt to light.

In Nature this week, Jan Hoeijmakers at Erasmus University in Rotterdam, Netherlands, Akira Yasui of Tohoku University in Sendai, Japan, and their co-workers report the first tests on such mice. But instead of providing an answer about light response, the results delivered a surprise: The mice have no clock. Under conditions of 12 hours of light followed by 12 hours of dark, they act like normal mice, running in their exercise wheels in the dark and sleeping when it is light. But in constant darkness, when the clock would normally maintain the alternating cycles, their behavior loses that pattern; they run on and off around the clock.

Those results suggest the animals' clocks fail in constant darkness. But further tests show they actually have no clock at all. When normal animals are subjected to a new light-dark pattern, they begin to adapt their clocks, a slow process as any jet-lagged traveler knows. But the mutant mice instantly adjust to any light pattern; they run when the lights go out and stop when they come on. That, says clock researcher Jeff Hall of Brandeis University in Waltham, Massachusetts, is the kind of behavior observed in clockless animals. Without a clock to control their behavior, it is driven directly by the light. Sancar and Takahashi, working with Takeshi Todo of Kyoto University in Japan, have also made double cryptochrome knockout mice and have preliminary results similar to those of the Dutch-Japanese team.

Slight abnormalities in cry2 mutant mice that Sancar and Takahashi reported last fall suggested that cry2 might play a central role in the clock, but most researchers were surprised to learn it is essential for the clock to work. That creates a new mystery: What is the cryptochrome doing in the clock? But it does little to solve the old puzzle of whether cryptochrome transmits light signals.

"Perhaps both functions, the clock and the light input, are being taken out" in the double mutants, says Takahashi. Ironically, the lack of a clock in the mutant mice makes it hard to test that hypothesis; one can't measure the effect of light on a nonexistent clock. But Kay notes that even if the clock is disabled, some of its molecular parts remain and should be able to respond to light. He suggests the authors check the behavior of those proteins to see if a light signal is getting through, something both groups plan to do. Wherever the search for the circadian photopigment leads as it moves beyond the rods and cones, one thing is for sure: Cryptochrome has guaranteed itself a place in the story of the circadian clock.

Regulation of Mammalian Circadian Behavior by Non-rod, Non-cone, Ocular Photoreceptors

Melanie S. Freedman, 1 Robert J. Lucas, 1 Bobby Soni, 1 Malcolm von Schantz, 1* Marta Muñoz, 2 Zoë David-Gray, 1 Russell Foster 1dagger

Volume 284, Number 5413 Issue of 16 Apr 1999, pp. 502 - 504

Circadian rhythms of mammals are entrained by light to follow the daily solar cycle (photoentrainment). To determine whether retinal rods and cones are required for this response, the effects of light on the regulation of circadian wheel-running behavior were examined in mice lacking these photoreceptors. Mice without cones (cl) or without both rods and cones (rdta/cl) showed unattenuated phase-shifting responses to light. Removal of the eyes abolishes this behavior. Thus, neither rods nor cones are required for photoentrainment, and the murine eye contains additional photoreceptors that regulate the circadian clock.

1 Department of Biology, Alexander Fleming Building, Imperial College of Science, Technology and Medicine, London, SW7 2AZ, UK.
2 Departmento de Morfologia y Biologia Cellular, Universidad de Oviedo, Oviedo, Spain 33072.
*   Present address: School of Biological Sciences, University of Surrey, Guildford, Surrey GU2 5XH, UK.

dagger    To whom correspondence should be addressed. E-mail: r.foster@ic.ac.uk

Mammals use light both to generate a visual image of their environment and to provide time-of-day information. Internal circadian time is synchronized (entrained) with the solar day by light-induced resetting (photoentrainment) mechanisms, which correct for deviations in the period and phase of the endogenous clock (1). Eye loss in both human and nonhuman mammals abolishes photoentrainment, demonstrating that the eyes provide the primary source of light information to the clock (2). However, the retinal projections that convey light information to the visual and circadian centers of the brain are quite distinct (3), and visual blindness due to partial loss of rod and cone photoreceptors is not necessarily associated with an attenuation of circadian responses to light (2). Collectively, these findings have led to speculation that (i) the mammalian circadian system can maintain normal photosensitivity with only small numbers of rods or cones; and (ii) the eye contains unrecognized photoreceptors that mediate, or help mediate, the effects of light on the circadian system (4). In the absence of an experimental model completely lacking rods and cones, distinguishing between these alternatives has been problematic.

Two mouse models have been used previously to examine the impact of rod photoreceptor loss on circadian physiology: (i) mice homozygous for retinal degeneration (rd/rd) gradually lose all rod photoreceptors but retain normal circadian responses to light (5); and (ii) transgenic mice (rdta) undergo specific ablation of rod photoreceptors during early development (6) and are also circadian photosensitive. The responses of rdta mice were about twice as great as those of wild-type and rd/rd mice of the same genetic background (7). Loss of the eyes in both rd/rd and rdta mice abolished the effects of light on the circadian system. Collectively, these results showed that rods are not required for circadian photoentrainment and that the photoreceptors mediating these responses are ocular.

Both rd/rd and rdta mice sustain a secondary degeneration of cone photoreceptors. However, limited numbers of cones remain into old age (6, 8), making them strong candidates for the regulation of temporal physiology. The murine retina contains two populations of cones, sensitive in the green [maximum wavelength (lambda max) = 508 nm] (9) and ultraviolet (UV) (lambda max = 359 nm) (10). Both cone classes have been implicated in photoentrainment by action spectrum studies (11). Moreover, the identification of a fully functional "green" photopigment (lambda max = 534 nm) within the eyes of the blind mole rat (Spalax ehrenbergi) provides indirect evidence for the involvement of cones in circadian regulation (12).

To determine the impact of cone photoreceptor loss on photoentrainment, we used mice in which cone photoreceptors were ablated by the introduction of a synthetic transgene (cl) (13). This construct consists of a portion of the human red cone opsin promoter, attached to an attenuated diphtheria toxin gene (14). The retinas of these mice have normal numbers of rods and a substantially reduced number of UV cones (>95% lost) and appear to lack green cones (<1% remain in some retinas) (13). Our molecular (Fig. 1) (15) and immunocytochemical analysis (16) of the cl retina confirms these findings. Despite this massive insult to cone photoreceptors, cl mice showed unattenuated circadian responses to monochromatic 509-nm light (Fig. 2A). Bilateral enucleation abolished the ability of cl mice to entrain to a 12 hour light:12 hour dark cycle and to phase shift their circadian locomotor rhythm in response to a light pulse (17). In view of the loss of green cones, these data suggest that green cone photoreceptors are not required for photoentrainment. Moreover, the insensitivity of UV cones to 509-nm light (10) suggests that a non-cone photoreceptor is involved in this process. As rods remain unaffected in cl mice, under these circumstances, rods might mediate photoentrainment. Although previous studies with both rd/rd and the rdta mouse models indicate that rod photoreceptors are not required for circadian photoentrainment (5, 7), our results might reflect redundancy of photoreceptor inputs to the clock, with both rod and cone photoreceptors providing photic input to the circadian system. Hence, the absence of either cell type might be compensated for by the presence of the other. To resolve this issue, we generated mice that carry lesions to both rod and cone photoreceptors by introduction of the cl transgene into mice heterozygous for the rodless (rdta) transgene. Immunocytochemical and mRNA analyses of rdta/cl mouse retinas (15) indicate that both rod and green cone photoreceptors and their associated photopigments are eliminated from the retinas of these mice (Figs. 1 and 3). Despite the absence of rods and green-sensitive cones, rdta/cl mice show unattenuated circadian phase shifts in response to a 15-min monochromatic light (509 nm) pulse of varying irradiance (Fig. 2B).

Fig. 1. Effect of transgenic ablation on the expression of photoreceptor genes in cl and rdta/cl mice. Northern blot (A through C) and RT-PCR (D through F) detection of mRNA-encoding green cone opsin (A and D), UV cone opsin (B and E), and rod opsin (C and F) in wild-type and transgenic retinas (15). Introduction of the cl transgene rendered green cone opsin mRNA undetectable by Northern blot (A) in either cl or rdta/cl genotypes. RT-PCR techniques also failed to amplify a band visible on an ethidium bromide-stained agarose gel in either genotype (D). The effect of the cl transgene on UV cones was less marked, with UV cone opsin mRNA detectable in both cl and rdta/cl mice by Northern blotting (B) and RT-PCR techniques (E). Rod photoreceptors were unaffected by the cl transgene. By contrast, the rdta/cl retina contained no rod opsin transcript (C and F); bp, base pairs.

  

 
Fig. 2. Irradiance-dependent phase shifts of circadian locomotor activity (17). (A) Phase shifts of locomotor activity in cl mice. Phase shifts (mean ± SEM) of wild-type and cl mice, after exposure to a defined irradiance, 15-min monochromatic light (509 nm) pulse delivered at CT16 (n = 6 to 15 animals per genotype at each irradiance). There were no significant differences between cl or wild-type mice at irradiances that produce either saturating or subsaturating phase shifts [two-way analysis of variance (ANOVA): P > 0.05]. (B) Phase shifts of locomotor activity in rdta/cl transgenic mice. Phase shifts (mean ± SEM) of wild-type and rdta/cl mice, after exposure to a 15-min monochromatic light (509 nm) pulse delivered at CT16 (n = 5 to 7 animals per genotype at each irradiance). Both genotypes showed an irradiance-dependent increase in the amplitude of phase shifts. However, at an irradiance of 5.7 µW/cm2, phase shifts were significantly enhanced in rdta/cl mice, compared with wild-type mice (two-way ANOVA: P < 0.001; post hoc Student-Newman-Keuls tests comparing genotypes at each irradiance: *, P < 0.05). For further discussion, see (23).

Fig. 3. Histological analysis of serial sections from wild-type (A through C) and rdta/cl (D through F) retinas (15). Immunocytochemical staining failed to identify rod or cone photoreceptors in the retinas of rdta/cl mice. Tissue was fixed with Bouins (75% picric acid, 25% formalin, and 5% acetic acid) for 24 hours and paraffin-embedded, and 8-µm sections were treated with antibodies recognizing rod (A and D), rod and green cone (B and E), and UV cone (C and F) photoreceptors. Visualization was accomplished with ABC methods (Vectastain Elite, Vector Labs). GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; ONL, outer nuclear layer; IS, inner segments; OS, outer segments; and RPE, retinal pigment epithelium. Scale bar, 40 µm.

These results demonstrate that the mammalian eye contains non-rod, non-cone photoreceptors capable of regulating circadian behavioral responses to light. Published data suggest strongly that these receptors use a vitamin A-based photopigment (11, 18). Nonetheless, their molecular basis has been the subject of considerable recent speculation (19-22). The rdta/cl model provides an opportunity to address this issue by determining the spectral sensitivity of these uncharacterized photoreceptors.

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21 December 1998; accepted 15 March 1999