Physiology and Pharmacology Effect of Circadian Clock Gene Mutations on Nonvisual Photoreception in the Mouse Leah Owens,1 Ethan Buhr,2 Daniel C. Tu,1 Tamara L. Lamprecht,1,2,3 Janet Lee,1 and Russell N. Van Gelder1,2,3 PURPOSE. Mice lacking rods and cones retain pupillary light cifically. The circadian clock modulates the sensitivity of non- reflexes that are mediated by intrinsically photosensitive reti- visual photoreception. (Invest Ophthalmol Vis Sci. 2012;53: nal ganglion cells (ipRGCs). Melanopsin is necessary and suffi- 454–460) DOI:10.1167/iovs.11-8717 cient for this nonvisual photoreception. The mammalian inner retina also expresses the potential blue light photopigments ice lacking all classical visual photoreceptors (rods and cryptochromes 1 and 2. Previous studies have shown that Mcones) continue to evince a number of light-mediated outer retinal degenerate mice lacking cryptochromes have behaviors and physiology, including entrainment of circadian lower nonvisual photic sensitivity than retinal degenerate rhythms,1–3 pupillary light responses,4,5 and photic suppres- mice, suggesting a role for cryptochrome in inner retinal pho- sion of pineal melatonin.6 These effects are mediated by a toreception. population of intrinsically photosensitive retinal ganglion cells 7 METHODS. Nonvisual photoreception (pupillary light responses, (ipRGCs) that project specifically to nonvisual centers such as circadian entrainment, and in vitro sensitivity of intrinsically the suprachiasmatic nuclei of the hypothalamus and the olivary 8 photosensitive retinal ganglion cells) were studied in wild- pretectum. 9,10 type, rd/rd, and circadian clock-mutant mice with and without ipRGCs express the opsin family member melanopsin, rd/rd mutation. an invertebrate-like opsin that forms a functional photopig- ment when expressed in heterologous cell culture11–14 or in RESULTS. Loss of cryptochrome in retinal degenerate mice re- non-ipRGC ganglion cells.15 Retinal degenerate mice lacking duces the sensitivity of the pupillary light response at all wave- melanopsin lose all nonvisual photoreception,16,17 and ipRGCs lengths but does not alter the form of the action spectrum, lacking melanopsin do not show intrinsic light responses.17–19 suggesting that cryptochrome does not function as a photopig- Thus melanopsin appears both necessary and sufficient for ment in the inner retina. The authors compounded the rd/rd ipRGC photosensitivity. retinal degeneration mutation with mutations in other essential Ϫ/Ϫ The murine inner retina also expresses cryptochrome fam- circadian clock genes, mPeriod and Bmal1. Both mPeriod1 ; 20 Ϫ/Ϫ Ϫ/Ϫ ily members mCry1 and mCry2. Cryptochromes are a con- mPeriod2 ;rd/rd and Bmal1 ;rd/rd mice showed signif- served family of photopigments that mediate blue-light growth icantly lower pupillary light sensitivity than rd/rd mice alone. in plants21,22 and circadian rhythm entrainment in insects23,24 A moderate amplitude (0.5 log) circadian rhythm of pupil- (see Ref. 25 for review). We have previously shown that, lary light responsiveness was observed in rd/rd mice. Mul- compared with retinal degenerate mice, retinal degenerate tielectrode array recordings of ipRGC responses of mCryp- Ϫ/Ϫ Ϫ/Ϫ Ϫ/Ϫ mice lacking cryptochromes have reduced behavioral synchro- tochrome1 ;mCryptochrome2 and mPeriod1 ; nization to light-dark cycles, reduced light-mediated c-fos in- mPeriod2Ϫ/Ϫ mice showed minimal sensitivity decrement Ϫ/Ϫ duction in the suprachiasmatic nuclei, and reduced pupillary compared with wild-type animals. mCryptochrome1 ;mCrypto- light responses,26–28 suggesting a role for cryptochromes in chrome2Ϫ/Ϫ;rd/rd, mPeriod1Ϫ/Ϫ;mPeriod2Ϫ/Ϫ;rd/rd and Ϫ/Ϫ inner retinal photoreception. Bmal1 ;rd/rd mice all showed comparable weak behavioral syn- In mammals, cryptochromes are essential components of chronization to a 12-hour light/12-hour dark cycle. the time-delayed transcription-translation feedback loop that CONCLUSIONS. The effect of cryptochrome loss on nonvisual underlies circadian pacemaking; mice lacking both mCry1 and photoreception is due to loss of the circadian clock nonspe- mCry2 lose all free-running circadian rhythms.29–31 This raises the question whether the observed additivity of loss of cryp- tochrome and outer retinal degeneration on nonvisual re- sponses reflects a role for cryptochrome as an auxiliary pho- From the Departments of 1Ophthalmology and Visual Sciences 3 toreceptive protein in the inner retina or whether such and Molecular Biology and Pharmacology, Washington University additivity is a nonspecific result of loss of circadian rhythmicity Medical School, St. Louis, Missouri; and the 2Departments of Ophthal- mology and Biological Structure, University of Washington School of in the whole animal. To distinguish these possibilities, we have Medicine, Seattle, Washington. further analyzed the physiology of cryptochrome mutant mice Supported by National Institutes of Health Grants R01EY014988 and mice lacking circadian rhythms from mutations in the (RNVG) and P30EY01730, a Burroughs-Wellcome Translational Scien- Period and Bmal1 families of circadian clock genes. tist Award (RNVG), the Medical Scientist Training Program (DCT), and an unrestricted grant from Research to Prevent Blindness. Submitted for publication October 1, 2011; accepted November MATERIALS AND METHODS 21, 2011. Disclosure: L. Owens, None; E. Buhr, None; D.C. Tu, None; T.L. Mice Lamprecht, None; J. Lee, None; R.N. Van Gelder, None Corresponding author: Russell N. Van Gelder, Department of C3H/HeJ mice (rd/rd; Jackson Laboratories, Bar Harbor, ME) were Ϫ/Ϫ Ϫ/Ϫ Ϫ/Ϫ Ophthalmology, Campus Box 359608, University of Washington Med- crossed with circadian clock mutants mCry1 ;mCry2 , mPer1 ; Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ ical School, 325 Ninth Avenue, Seattle, WA 98104; mPer2 / , and Bmal1 / and were backcrossed to create mCry1 / ; [email protected]. mCry2Ϫ/Ϫ;rd/rd, mPer1Ϫ/Ϫ;mPer2Ϫ/Ϫ;rd/rd, and Bmal1Ϫ/Ϫ;rd/rd Investigative Ophthalmology & Visual Science, January 2012, Vol. 53, No. 1 454 Copyright 2012 The Association for Research in Vision and Ophthalmology, Inc. Downloaded from jov.arvojournals.org on 09/27/2021 IOVS, January 2012, Vol. 53, No. 1 Nonvisual Photoreception in Clock-Mutant Mice 455 mice. Genotypes were verified by PCR analysis of distal tail snips, as these animals17 is well fit with an opsin template with peak previously described (mCry1Ϫ/Ϫ30; mCry2Ϫ/Ϫ31; mPer1Ϫ/Ϫ and sensitivity of approximately 480 nm. Identical action spectra mPer2Ϫ/Ϫ32; BmalϪ/Ϫ33). C57Bl/6 mice were used as wild-type controls. have been reported for ipRGC responses in vitro.7,18 To deter- Mice were housed individually in running wheel cages enclosed in light- mine whether cryptochrome contributes to the shape of this tight cabinets. Wheel running activity was monitored in 6- to 8-month-old action spectrum, irradiance response relationships were mea- mice and analyzed using a personal computer platform (Actimetrics, sured for seven wavelengths of light in rd/rd and rd/rd; Evanston, IL). All experiments were conducted under an approved animal mCry1Ϫ/Ϫ;mCry2Ϫ/Ϫ mice (Supplementary Fig. S1, http:// studies protocol and the ARVO Statement for the Use of Animals in www.iovs.org/lookup/suppl/doi:10.1167/iovs.11-8717/-/DC Ophthalmic and Vision Research. Supplemental). Retinal degenerate mice lacking cryptochrome showed approximately 10-fold reduced sensitivity at all wave- Histology lengths compared with rd/rd mice alone. However, the shape Eyes enucleated from euthanized 6- to 8-month-old mice were imme- of the resultant action spectrum was identical for retinal de- diately fixed in 10% formalin at room temperature overnight. Fixed generate mice with and without cryptochromes (Fig. 1). This globes were embedded in glycol methacrylate, cut into 3-␮m sections, suggests either that cryptochrome does not substantially par- and stained with toluidine blue. Inner and outer retinal cell counts ticipate in the photoreceptive event in inner retinal photore- were determined by counting nuclei at a location one 40 ϫ field away ception (at least that mediating the pupillary light response) or from the optic nerve. ANOVA was performed on counts averaged from that the action spectrum of cryptochrome is indistinguishable at least six different sections. from that of melanopsin. The latter possibility appears unlikely given the flavin-based spectrum associated with cryptochrome, Melanopsin Staining which is not fit by an opsin template.36 Enucleated globes of adult mice, age 1 to 8 months, were fixed in 4% paraformaldehyde and flat-mounted. Retinas were incubated overnight Pupillary Light Responses of Circadian Clock in 1:5000 of N15 anti-melanopsin antibody,10 followed by rhodamine- Gene Mutant Mice with and without Outer Retinal conjugated secondary antibody. Retinas were scored by a masked Degeneration observer for a total number of brightly stained cells per retina using an To determine whether the reduced photic sensitivity of retinal epifluorescence microscope (Olympus, Tokyo, Japan). degenerate mice lacking cryptochrome is specific to crypto- chrome or generic to genes causing loss of free running circadian Pupillary Light Recordings Ϫ Ϫ rhythmicity, we intercrossed rd/rd mice with mPeriod1 / ; Six- to 8-month-old mice were dark-adapted overnight and then ex- mPeriod2Ϫ/Ϫ (mPer1Ϫ/Ϫ;mPer2Ϫ/Ϫ) and Bmal1Ϫ/Ϫ mice and posed to various fluences of narrow bandpass-filtered 470-nm light for then backcrossed these mice to generate Bmal1Ϫ/Ϫ;rd/rd and 60 seconds. Nonanesthetized mice were video recorded in a com- mPer1Ϫ/Ϫ;mPer2Ϫ/Ϫ;rd/rd