Circadian photoreception in the retinally degenerate mouse (rd/rd ...

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Circadian photoreception in the retinally degenerate mouse (rd/rd) R.G. Foster a ,, I. Provencio 1, D. Hudson 1, S. Fiske 1, W. De Grip z, and M. Menaker 1 1 Department of Biology, Gilmer Hall, University of Virginia, Charlottesville, VA 22901, USA z Department of Biochemistry, Center for Eye Research, University of Nijmegen, P.O. Box 9101, NL-6500 HB Nijmegen, The Netherlands Accepted April 25, 1991

Summary. We have examined the effects of light on circadian locomotor rhythms in retinally degenerate mice (C57BL/6J mice homozygous for the rd allele: rd/rd). The sensitivity of circadian photoreception in these mice was determined by varying the irradiance of a 15 min light pulse (515 nm) given at circadian time 16 and measuring the magnitude of the phase shift of the locomotor rhythm. Experiments were performed on animals 80 days of age. Despite the loss of visual photoreceptors in the rd/rd retina, animals showed circadian responses to light that were indistinguishable from mice with normal retinas (rd/+ and + / +). While no photoreceptor outersegments were identified in the retina of rd/rd animals (80-100 days of age), we did identify a small number of perikarya that were immunoreactive for cone opsins, and even fewer cells that contained rod opsin. Using HPLC, we demonstrated the presence and photoisomerization of the rhodopsin chromophore 11-cis retinaldehyde. The rd/rd retinas contained about 2% of 11-cis retinaldehyde found in + / + retinas. We have yet to determine whether the opsin immunoreactive perikarya or some other unidentified cell type mediate circadian light detection in the rd/rd retina.

Key words: Photoreception - Retinally degenerate Mouse - Circadian - Rods - Cones - 11-cis retinaldehyde - Immunocytochemistry - HPLC

Introduction All non-mammalian vertebrates possess two functionally distinct classes of photoreceptors: visual photoreceptors, which capture light to build-up a spatial pattern of the environment, and non-visual photoreceptors, which deAbbreviations: HPLC high-performance liquid chromatographyy

* To whom offprint requests should be sent

tect light: dark cycles and synchronize (entrain) circadian and reproductive events with environmental time (see Menaker and Underwood 1976; review volume: Experientia, 1982, vol 38: 989-1128). At least some of these non-visual photoreceptors are located within the pineal complex and yet others are in unknown areas of the brain. In contrast to the rest of the vertebrates, mammals use their eyes for all forms of light detection (review: Foster et al. 1989b), but it remains unclear whether the rods and cones of the retina mediate both visual and non-visual photoreception. The circadian system of the golden hamster shows peak spectral sensitivity around 500 nm and the shape of the action spectrum very closely resembles that of a vertebrate rod rhodopsin (Takahashi et al. 1984). However, this does not demonstrate that circadian photoreception is mediated by retinal rods. Several lines of evidence suggest that non-mammalian vertebrates have non-visual photoreceptors also based upon rod-like rhodopsin photopigments that are clearly not retinal rods (e.g. Deguchi 1981 ; Foster and Follett 1985; Foster et al. 1989a). There have been relatively few studies of mammalian circadian photoreception, but all have shown important differences between visual and circadian light detection. For example, the route by which light information reaches the circadian system (the retinohypothalamic tract) is anatomically, developmentally and physiologically distinct from the visual pathway (Frost et al. 1979; Moore and Klein 1974; Pickard et al. 1982). In addition, two physiological features of circadian photoreception in the hamster differ from rod mediated visual responses: 1) the threshold for phase shifting the locomotor rhythm with light pulses is high; and 2) the intensity-duration reciprocity relationship holds for stimuli of very long duration [up to 45 min (Takahashi et al. 1984)]. Taken together the available data suggest that although mammalian circadian photoreceptors are located within the eye they may be different from the image forming rods and cones of the retina. To test the hypothesis that there are specialized nonvisual photoreceptors within the mammalian retina we

40 studied circadian photoreception in mice with genetically abnormal retinas. Mice, homozygous for the autosomal recessive allele rd, develop a severe form of retinal photoreceptor degeneration (Noell 1965; Sidman and Green 1965). This degeneration of the visual ceils has been associated with an accumulation of cGMP in photoreceptors of rd animals due to a deficiency in phosphodiesterase activity (Farber and Lolley 1974; Lolley et al. 1977). Recent studies by Bowes et al. (1990) have shown that the/3 subunit of rod cGMP-phosphodiesterase is defective in the rd mouse. Retarded development of the photoreceptor elements in the rd retina are noted as early as the 4th postnatal day (Sanyal and Bal 1973). By the 8th postnatal day the outer segments start to degenerate and are quickly followed by the inner segments and photoreceptor cell bodies of the outer nuclear layer. By day 21 all that remains of the photoreceptor layer is a single scattered row of perikarya, which slowly and continuously decrease in number over the following months (Blanks et al. 1974; Caley et al. 1972; Caravaggio and Bonting 1963; Carter-Dawson et al. 1978; Cohen 1960; La Vail and Sidman 1974; Noell 1958; Sanyal and Bal 1973; Sorsby et al. 1954). While the rd mutation causes a rapid decline in rod perikarya, cone perikarya survive much longer; by 5 weeks all rod perikarya have disappeared from the central retina and after 9 weeks the peripheral retina has lost all rod perikarya. In contrast, by 4 months of age the central retina contains 1.3% of the original number of cone perikarya and the peripheral retina 14%; by one year of age only 0.3-0.4% of cone perikarya remain in the entire retina (Carter-Dawson et al. 1978). It should be stressed that none of the surviving cones have traces of the outer segment (Carter-Dawson et al. 1978). Carter-Dawson etal. (1986) have shown that amounts of interstitial retinol binding protein (IRBP), rhodopsin and stored retinyl ester decline in rd mice as the photoreceptors degenerate. In a recent development study of the rd eye, Bowes et al. (1988) reported that the remaining perikarya of the outer nuclear layer showed immunostaining with rod opsin and transducin antibodies up to 21 days after birth, but that between 21 days and 2 months all immunostaining disappeared. In addition, immunostaining with antibodies against the 48-kDa retinal protein (common to rod and cone photoreceptors) identified perikary from birth to 2 months of age but by 6 months no perikarya were immunostained within the retina. Bowes et al. (1988) suggest that these 'longer lasting' perikarya are probably derived from cones. In the same study the authors reported that, mRNAs coding for opsin and the e, /3 and 7 subunits of transducin each have the same onset of expression in normal and rd retinas, but in rd retinas 11-12 days after birth these mRNAs begin to decline. Retinal structures outside the photoreceptive layers and the central visual centers are hardly affected by the rd mutation. Only after several months do the inner retinal layers show any signs of degeneration (Sorsby et al. 1954), and these are thought to be secondary to the photoreceptor degeneration, associated perhaps with the

R.G. Foster et al. : Circadian photoreception in mice progressive breakdown of the retinal circulation. Blanks et al. (1974) have provided evidence that bipolar cell development is modified by the rd mutation, but again this seems to be secondary to photoreceptor loss. Injections of radioactive tracers into the eye, followed by autoradiography, demonstrated that central visual structures appeared normal (Dr/iger and Hubel 1978). Analysis of the functional capabilities of the rd eye have used both behavioral and electrophysiological methods. Despite considerable photoreceptor degeneration, behavioral studies have shown that rd mice are capable of limited visual responses. Nagy and Misanin (1970) used the 'visual cliff task' to assess visual capabilities in rd mice at different stages of development. Animals seemed capable of pattern recognition in bright light up to 40 days of age, but shortly there after this ability disappears. In further experiments, 100 day old animals were still able to discriminate between bright (100 ftc) and dim (1 ftc) light. Schnitzer and Beane (1976) studied the ability of rd mice in bright light (5000 lux) to discriminate between horizontal or vertical rectangles. Animals between 65-93 days of age, could discriminate between these visual patterns, however the authors point out that rd mice may not be capable of form vision per se but rather may be utilizing the brightness characteristic of the visual stimuli. The consensus from the behavioral analysis of the visual capabilities of the rd mouse appears to be that at an early age vision is good but after some 40 days pattern recognition is lost and only responses to large changes in brightness remain. Detailed studies on aged animals (beyond 6 months) are lacking. Several studies have examined electroretinographic responses (ERG) in rd mice (Karli 1954; Keeler et al. 1928; Noell 1958; Yamasaki and Suga 1969). The ERG in rd mice (as in normal mice), can first be detected during the second week after birth, but after approximately day 15 the ERG amplitude declines in the mutant animals and disappears around the 4th week. In view of the behavioral findings, these results were perhaps surprising. However, the presence or absence of the ERG is a poor indicator of retinal function. Karpe (1949) showed that in humans with retinitis pigmentosa the ERG is gone while patients still report satisfactory vision. In rats, Arden and Ikeda (1966) found that genetic or light-induced photoreceptor damage resulted in disappearance of the ERG but cortical evoked potentials could be recorded after light flashes. In a detailed study by Dr/iger and Hubel (1978), single unit recordings from the visual cortex and tectum were made in rd mice to assess visual abilities. Recording from the tectum before 21 days of age showed that retinotopic topography and receptive fields were normal, but by 24 days no receptive fields could be recorded from parts of the tectum representing the central 90-100 ~ of the visual field, whereas in the peripheral retina responses remained fairly normal. Over the following 4 months receptive field responses slowly faded to nothing. Sensitivity was assessed by making recordings from the tectal surface while exposing the dark adapted rd eye to different light intensities. Dark adapted thresholds in rd mice

R.G. Foster et al. : Circadian photoreception in mice rose with age; at 90 days threshold levels were some 2-3 log units above the threshold levels reached in normal mice. By 5 months of age, the majority o f visual cortical and tectal cells showed only sustained activity, although in a few cases tectal cells did show suppression of sustained activity when the whole eye was exposed to very intense illumination (Drfiger and Hubel 1978). Ebihara and Tsuji (1980) studied the lowest light intensities required for two strains of mice to entrain to a light:dark cycle (LD cycle). They report that C3H mice homozygous for rd (rd/rd) with degenerate retinas could be entrained by an LD cycle in which the light in the L phase was approximately 1 lux; light intensities below this level failed to entrain. By contrast, wild-type C57BL mice were much more sensitive to light and could be entrained by an L D cycle in which the L phase was only 0.01 lux. In these experiments C57BL mice with normal retinas showed a 100 fold greater sensitivity to light than did rodless C3H mice with degenerate retinas. Ebihara and Tsuji (1980) concluded that the difference in sensitivity was probably due to the absence of rods in the C3H mice. On the other hand the difference in light sensitivity could reflect differences between the two strains and not the contribution of the rod and cone photoreceptors, an explanation which, based on the resuits reported below we are inclined to favor. In the study reported here we have examined behavioral circadian responses to light as well as several photoreceptor characteristics of the retina in one strain of mouse (C57BL/6J). In this strain we have studied the effects of the rd mutation by examining 3 groups of mice: mice homozygous for the rd allele (rd/rd); mice heterozygous for the rd allele (rd/+); mice homozygous for the wild type allele at this locus ( + / + ) . We have restricted our detailed investigation to mice 80-100 days of age because in rd/rd animals o f this age visual responses are absent and all that remains of the retinal photoreceptors are scattered cone perikarya with no outer segments. Our investigations address 3 basic questions: 1) Does the circadian system of C75BL/6J mice homozygous for rd (rd/rd) respond to light and if so does the circadian photosensitivity shown by these animals differ from the sensitivity of visually normal C57BL/6J mice heterozygous for rd (rd/+) or wild-type ( + / + ) ? Our assay for circadian light detection was the shifting effect of a single pulse of light on the phase of locomotor rhythmicity in animals 80 days o f age; 2) In view of the massive photoreceptor degeneration, what elements of the rd/rd retina remain that could account for circadian light detection? We used immunocytochemical techniques, employing antibodies directed against rod and cone opsins and cellular retinal-binding protein ( C R A L B P ) to describe the condition o f the known photoreceptor elements in the retina of animals between 90-100 days of age; 3) Is there biochemical evidence for 'light detection' within the rd/rd eye? H P L C techniques were employed to investigate whether the rhodopsin chromophore (ll-cis retinaldehyde) is present and whether photoisomerization of the chromophore occurs (11-cis to all-trans) in the retina o f mutant animals 90-100 days of age.

41

Materials and methods

Experimental animals. We maintained a breeding colony of mice (Mus domesticus/C57BL/6J) in 3 groups; homozygous for the rd allele (rd/rd), homozygous for wild type allele at this locus ( + / + ) and heterozygous (rd/+). The rd mice carry the linked recessive gene le for light ears, which we were able to use as a marker for rd/rd. The homozygous dominant ( + / + ) stock was maintained by sibling matings. To obtain heterozygous (rd/+) and homozygous animals (rd/rd) for experiments we mated heterozygous animals (rd/+) to homozygous (rd/rd) to yield approximately equal numbers of both rd/+ (dark ears) and rd/rd (light ears; le/le). Periodically eyes were removed and examined histologically to check for separation of the two traits. Prior to experimentation animals were housed in a colony room maintained at 22 ~ 50% humidity, on a 12 h:12 h light:dark cycle with food and water provided ad libitum. Animals were bred by placing a male in a female's cage for 5 days. Litters were weaned at 21 days and at about 28 days they were separated according to ear color (light or dark) and sex. Animals from the same litter and with the same trait (rd/rd; rd/+ ; + / + ) and sex were group housed (4 animals/cage) until they were used for breeding or in an experiment.

Analysis of circadian locomotor behavior. As an assay of circadian photoreception we measured the magnitude of the phase shift of the freerunning locomotor rhythm produced by a single light pulse delivered when the animals were 80 +2 days of age. At 58 days of age experimental animals were moved from the colony room into individual running wheel cages housed in lighttight boxes, and the light cycle was continued for 5-7 days. The light cycles were then discontinued; between the 14th and 16th day in darkness, each animal was removed from its cage using an infrared viewer (head mounted FIND-R-SCOPE), placed into the light pulse apparatus (Fig. 1) and exposed to a 15 min pulse of monochromatic light using an interference filter with a wavelength of maximum transmission at 515 nm; half-peak bandwidth 10 nm (Oriel Corporation, Stratford, CT). The animal was returned to constant darkness for an additional 8-10 days. The pulses were delivered to each animal 4 h after the time of locomotor activity onset (circadian time 16), the phase of the freerunning rhythm at which light pulses elicit maximum phase delays (Daan and Pittendrigh 1976). As the period of the freerunning locomotor rhythm (tau) varied slightly among individuals, different animals were at different circadian times at the same local time on the same day.

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42

R.G. Foster et al. : Circadian photoreception in mice

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Fig. 2A, B. Locomotor activity records from: A A C57BL/6J mouse wild type at the rd locus ( + / + ) with a normal retina, and B A C57BL/6J mouse homozygous for the rd gene (rd/rd) with a degenerate retina. Wheel running activity is shown for 29 days (1 line= 1 day); animals were housed within a light-tight box. For the first 5 days, each mouse was exposed to a light-dark cycle, dawn at 06:00, dusk at 18:00 (EST). The mouse was then transferred to constant darkness. On the 16th day in constant darkness, when the animal was 80 days old, the mouse was removed from its light-tight box, placed in the pulse apparatus and exposed to a 15-min light pulse at CT 16 (o on the records). After a further 8 days the phase shift was determined. In both A) and B) the light pulse caused a delaying phase shift of about 90 rain (zx on the record). In the records shown the period (tau) of freerunning activity is longer in the + / + mouse than in the rd/rd mouse. We do not find that this is a consistent difference between + / + and rd/rd animals

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This temporal spread enabled us to administer light pulses to 10-20 animals on the same day within a 20 min window in circadian time. The magnitude of the phase shift produced by the light pulse was measured as the difference between the steady state phase of the freerunning rhythm before and after the pulse (Fig. 2). Data analysis software for the analysis of locomotor behavior was developed by M.A. Vogelbaum, Department of Biology, University of Virginia. To characterize the sensitivity of the photoreceptors mediating phase shifts, we measured the effects of varying the irradiance of the monochromatic light pulse on the magnitude of the phase shift. Irradiance was varied using neutral density filters (Oriel Cor-

24 poration) and measured at the surface of the glass opal (see Fig. 1) in p~W/cm2 (United Detector Technology, CA; model 4~ $350).

Immunocytochemistry. Retinas (rd/rd; +/+ ) were prepared for immunocytochemistry when the mice were between 80 and 100 days of age. Animals were over-anaesthetized with Halothane and perfused with 10 ml of 0.9% physiological saline containing 15000 IU heparin/1000 ml, followed by 200 ml of Zamboni's fixative [3.5% paraformaldehyde, 13% saturated picric acid in 0.01 M phosphate buffered saline (PBS), sodium dihydrogen orthophosphate; di-sodium hydrogen orthophosphate] at pH 7.2-7.4. The eyes were removed, a small incision made in the cornea, and post-

R.G. Foster et al. : Circadian photoreception in mice fixed for 6 h. Eyes were then processed for cryostat sectioning and immunocytochemistry. The eyes were placed in 20% sucrose in 0.1 M phosphate buffer (pH 7.2) for 24 h, frozen in liquid nitrogen and serial sections were cut at 15-20 lam on a cryostat. All sections were collected on gelatin-coated slides, and air-dried (30 min) before washing in PBS containing 0.2% Triton X-100 (PBS-T) for 30 min. Endogenous peroxidase was blocked by immersing the sections in 0.3% hydrogen peroxide in PBS (30 rain) followed by 3 washes in PBS-T. Possible background staining was blocked by incubating the sections for 20-30 min in normal goat serum (1/30 in PBS-T). Sections were then transferred into one of 3 primary antisera: a) CERN-JS839 raised in rabbits and directed against purified lipid-free bovine rhodopsin (for details Margry et al. 1983; Schalken and De Grip 1986). This antiserum is monospecific for rod opsin (Jansen et al. 1987) and is used at a dilution of 1:1600 in PBS-T. Sections were incubated for 72 h at 4 ~ b) A newly produced antibody, CERN-874 raised in rabbits and directed against purified cone opsin. Chicken cone pigments were isolated according to Matsumoto and Yoshizawa (1982); Fager and Fager (1982); Yen and Fager (1984) with several modifications. The photoreceptor outer segments were isolated on a linear sucrose gradient (34-40%, w/w) and extracted in buffer (20 m M Pipes, 2 m M CaC12, 100 m M NaC1, 0.1 m M EDTA, 1 m M DTE, pH 6.5) containing 20 m M fl-l-dodecylmaltoside as detergent (De Grip and Bovee-Geurts 1979). The pigments were then purified over ConA-Sepharose (Pharmacia). Cone pigments were eluted with 2 m M a-methylmannoside. The cone pigment fraction had a 280/560 ratio of 3.5 + 0.3. According to spectroscopic, SDS-Page and immunoblot analysis this fraction contained 60-70% iodopsin, approximately 30% blue and green cone pigments and approximately 5% rod pigment. Antisera against the cone pigment fractions, described above, were raised in female New Zealand rabbits according to procedures described previously (Margry et al. 1983; Schalken and De Grip 1986). Animals were injected under dim red light with approximately 2 nmol of pigment mixed with 1 volume of Complete Freund Adjuvant (Difco Labs, Detroit, USA) and boosted after 5, 7, and 9 weeks with the same amount of pigment in the Incomplete Freund Adjuvant (Difco). The serum titers were determined by means of an ELISA-based titration assay (Schalken and De Grip 1986). The specificity of the raised antiserum (anti-cone: CERN 874) was evaluated using immunoblot analysis of cone (chicken, quail) and rod (chicken, bovine, mouse) pigments and immunocytochemical analysis of the retina of various species (chicken, rat, mouse, monkey). The antiserum showed some reaction with rod pigment upon immunoblot analysis, but only at low dilution (1/< 100). At higher dilution (1/> 500) this antiserum was specific for cone pigments and produced strong staining of outer segments of cone photoreceptors only (De Grip et al., unpublished observation). In this study CERN-874 was used at a dilution of 1 : 8000 in PBS-T. Sections were incubated for 72 h at 4 ~ c) Rabbit anti-bovine CRALBP (cellular retinal-binding protein; supplied by J.C. Saari, for details see Bunt-Milam and Saari 1983) was used at a dilution of 1:100 in PBS-T. Sections were incubated for 72 h at 4 ~ Immunocytochemical controls involved omitting the primary antisera and substituting non-immune serum. Following treatment with the primary antiserum, sections were either processed using peroxidase-antiperoxidase (PAP) or goat anti-rabbit IgG-fluorescein isothiocyanate (FITC). For PAP, sections were washed in PBS-T (30 min) before incubation (1 h at room temperature) with goat anti-rabbit IgG (ICN Biomedicals, Inc.) diluted 1 : 100 in PBS-T, followed by washes in PBS-T (30 min) before incubation (1 h at room temperature) with a PAP-complex (ICN Biomedicals, Inc.) diluted 1 : 100 in PBS-T. The sections were then washed in 0.05 M Tris buffer (Trizma base; no saline), pH 7.4 (15 min) and incubated in a solution of 0.025% 3,3' diaminobenzidine containing 0.03% hydrogen peroxide in Tris buffer. Following dehydration sections were cover-slipped with 'Permount', For FITC, sections were washed in PBS-T (30 min) before incubation (1 h at room temperature) with goat anti-rabbit IgG-fluorescein

43 isothiocyanate ICN Biomedicals, Inc.) diluted 1 : 100 in PBS-T, followed by washes in PBS-T (30 min) and then PBS (30 rain). Sections were cover-slipped with 80% glycerol in PBS. Immunostained sections were viewed and photographed using a Zeiss Axiophot photomicrographic microscope system.

Extraction and HPLC analysis of retinaldehydes. Extraction and analysis of the retinaldehydes was carried out using a modification of the approaches developed by Groenendijk et al. (1980). This approach allows the quantitative extraction of retinaldehyde in its original isomeric configuration from tissue samples and depends upon the fact that in the presence of excess hydroxylamine, retinaldehyde is converted into the corresponding retinaloxime with complete retention of geometric configuration. The retinaloximes, which are far less sensitive to chemical isomerization, can then be extracted with dichloromethane and analyzed by high-performance liquid chromatography (HPLC). Each isomer of retinaldehyde gives rise to two stereoisomeric oxime derivatives, the synand anti-isomer. Under dim red (R620 filter from Schott, Mainz, FRG, cut-off wavelength 610 nm; irradiance approximately 6 x 10 -1 ~tW/em 2) or under normal laboratory levels of illumination (irradianee approximately 8 x 101 BW/em2), mice (rd/rd; + / + ) between 90 and 100 days old were over-anaesthetized with Halothane and the eyes collected. Extraction was performed under the corresponding lighting conditions. Whole eyes were homogenized in PBS (1.5 ml added to 2 wild-type eyes; 1.5 ml added to 10 rd/rd eyes) and to the resulting suspension an excess of hydroxylamine (as a 1 M solution buffered to pH 6.5 with bicarbonate) was added (100 ~tl/1.5 ml eye suspension). The suspension was vortexed for 2 min and left for 10 min at room temperature. Under these conditions free retinaldehydes were very rapidly converted into retinaloxime. Methanol (2 ml/1.5 ml eye extract) was then added to the extracts to free the visual pigment bound retinaldehydes which are then converted to the oximes. After vortexing for 2 rain, 2 ml of dichloromethane were added. The resulting suspension was vortexed for 2 min and centrifuged at 4000 rpm for 10 min at 4 ~ to separate the two phases. The lower organic layer (dichloromethane), which contained the retinaloximes, was collected using a syringe. The upper layer was once more extracted with 2 ml of dichloromethane. The organic layers were combined and evaporated with oxygen-free nitrogen and the residue dissolved in 200 ~tl of hexane/dioxane (95:5, v/v). Analysis of the retinaloximes was performed by straight-phase HPLC (using a Beckman System Gold HPLC system or more recently a Waters 510 pump with 484 absorbance detector and 991 photodiode array detector) in the isocratic mode equipped with a Lichrosorb Si-60 column (100 x 3 mm, Chrompack cartridge) with detection for the single wavelength detectors set at 360 nm. Hexane: dioxane (95: 5 v/v) was used as eluent. Usually 70 Bl of the 200 p.1 extract were injected per analysis. Identification of the eluted retinaloximes (isometric configuration and syn- or anti-form) was achieved by comparison with reference compounds prepared by reaction of defined retinals with hydroxylamine in 70% methanol and purified by HPLC. Quantitative determination of all-trans and ll-cis retinaloximes was obtained by converting peak areas into absolute amounts using known amounts of reference compounds (Groenendijk et al. 1979).

Results

Analysis of circadian locomotor behavior T h e i r r a d i a n c e - r e s p o n s e c h a r a c t e r i s t i c s for all 3 g r o u p s o f m i c e (rd/rd; rd/+ ; + / + ) are s h o w n i n Fig. 3. F o r e a c h p o i n t a m i n i m u m o f 7 a n i m a l s w a s used. A l t h o u g h the i r r a d i a n c e - r e s p o n s e c h a r a c t e r i s t i c s o f the d i f f e r e n t g r o u p s a r e n o t i d e n t i c a l , t h e y are n e a r l y so: the irrad i a n c e r e q u i r e d to p r o d u c e b o t h s a t u r a t i n g a n d a half-

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Fig. 3A-D. Comparison of the effectsof a 15 rain monochromatic (515 nm) light pulse of increasing irradiance in phase shiftingthe circadian rhythms of mice with: A normal retinas (+/+); B normal retinas (rd/+); C degenerate retinas (rd/rd). For comparison the 3 irradiance response curves (A, B, C) have been superimposed

in D. Despite the substantial damage to the visual photoreceptors in rd/rd mice, these animals show circadian responses to light that are indistinguishable from those o f mice with normal retinas ( + / + and rd/+). Error bars + S E M

saturating response are essentially the same for all groups (Fig. 3 D). That the site of circadian photoreception is located within the eye was demonstrated by bilaterally enucleating rd/rd animals just prior to placing them in running-wheel cages at 58 days of age. These animals failed to entrain to light cycles or respond to light pulses.

we did observe immunostaining with the cone (CERN874) and rod (CERN JL839) antibodies. In rd/rd animals 80-100 days old, cells scattered in both the peripheral and central region of the retina and located at the junction between the inner nuclear layer and pigment epithelium, showed cone opsin immunostaining (Fig. 5A, B). Cone opsin labelling appeared most intense in the plasma membrane of the cells. In several cases we observed immunostaining in short, thin projections emerging from the perikarya, which might be a remnant of the photoreceptor cell inner segment. We found no evidence of cone outer segments. However, we did occasionally see immunoreactive structures that were always smaller than perikarya and were spheroid in shape (Fig. 5A, B). Similar 'opsin-positive vesicular structures' have been identified within the degenerating retinas of rds/rds mice (Jansen et al. 1987) and probably represent 'photoreceptor debris '. Retinal sections from rd/rd mice that were incubated with non-immune serum showed a very weak fluorescence in the inner nuclear layer (Fig. 5 C). To our surprise we also found evidence of rod opsin immunostaining within the rd/rd retinas in a very few immunoreactive perikarya (Fig. 5D). In the 4 whole retinas examined we identified no more than 8 rod immunoreactive cells

Immunocytochemistry Using the rod specific antibody (CERN-JS839) on the retinas of + / + animals 80-100 days of age, we found strong immunostaining in the rod outer-segments of the photoreceptor layer (Fig. 4A). Immunostaining with the cone specific antibody (CERN-874) on the retinas of + / + animals 80 100 days of age showed typically short, conically shaped outer-segments, intermixed with the rod inner segments and proximal portion of the rod outer-segments (Fig. 4 B). The cytology and immunostaining of the retinas from rd/rd animals were dramatically different (cf. Fig. 4 C, D). In rd/rd eyes the outer segment layer of the retina had disappeared and the outer nuclear layer had been reduced to a single layer of scattered cells resting upon the inner nuclear layer (Fig. 4 C). Despite the profound degeneration of the rd/rd retina,


45

Fig. 4A-D. Histology and immunocytochemistry of normal (+ / +) and mutant (rd/rd) retinas: A Rod opsin (CERN-JS839) immunostaining in the wild type mouse retina (+/+). B Cone opsin (CERN-874) immunostaining in the wild type mouse retina (+/+). C The retina of an rd/rd mouse 80 days of age, stained with 0.2% thionin. Note the loss of outer segments and outer nuclear layer. The pigment epithelium is distorted and not clearly visible in this

section. The inner nuclear layer and ganglion cell layer seem unaffected by the rd/rd mutation. D The retina of a + / + mouse retina 80 days of age immunostained with rod antibody (CERN-JS839) and counter stained with 0.2% Thionin. Choroid (Ch); inner segments (IS); outer nuclear layer (ON); outer segments (OS); pigment epithelium (PE). Scale bar = 20 ~tm

within a single retina, and these were located within the peripheral retina. These cells lacked outer segments but seemed to have some remnant o f an inner segment. Immunostaining with antibodies against C R A L B P (cellular retinal-binding protein) in the normal retina ( + / + ) was found in two cell types, the pigment epithelium and the Miiller glial cells. Our results with this antibody were identical to those of Bunt-Milam and Saari (1983). In the degenerate retina we also found strong immunostaining with these antibodies within the distorted pigment epithelium and Mfiller cells (Fig. 5 E).

(total of 50 eyes), 7 extractions of dark adapted eyes (total of 66 eyes). The resultant H P L C chromatograms demonstrated the presence of the rhodopsin chromophore in the retinas of both + / + and rd/rd animals. For eyes of both genotypes the ratio o f the ll-cis and all-trans isomers depended on whether the tissue had been light adapted and isolated in the light or dark adapted and isolated under dim red illumination (Fig. 6). For each separate H P L C assay, the area under the 11-cis peak (syn) was expressed as a percentage of the total retinaldehyde content of the tissue (11-cis syn and alltrans syn). Mean values _+SEM of 11-cis retinaldehyde were calculated for the different groups of eyes (Fig. 7). The dark adapted + / + eye contained 84.42+4.90% retinaldehyde in the ll-cis state, while the light adapted + / + eye contained only 11.45_+0.54% in the ll-cis state. The rd/rd eye also showed a light dependent shift in ll-cis retinaldehyde, but the ratio was different; in the dark adapted state ll-cis retinaldehyde accounted

Extraction and HPLC analysis of retinaldehydes A series of extractions was performed on both + / + and rd/rdeyes between 90 and 100 days old; for + / + ; 5 extractions of light adapted eyes (total of 10 eyes), 4 extractions of dark adapted eyes (total o f 8 eyes); and for rd/rd eyes: 5 extractions of light adapted eyes

46

Fig. 5A-E. Immunofluorescence in the mutant mouse retina: A, B Different regions of the retina in an 83 day old rd/rd mouse that have been immunostained with cone opsin antibodies (CERN874). Cone immunoreactive cells were identified in both the peripheral and central regions of the retina, and were located at the junction between the inner nuclear layer and pigment epithelium. By adjusting the plain of focus, immunostaining appeared most intense in the cell membrane. In several cases a projection could be seen emerging from cells that resembled a photoreceptor inner segment (arrow). We identified immunoreactive structures that were variably sized and spheroid, and assume that these represent 'photoreceptor debris' (see text for details). No immunoreactive outer segments were ever identified. C Immunocytochemical control; the retina from an rd/rd mouse incubated with non-immune serum. Very weak fluorescence was observed in the inner nuclear

for 74.52_+ 2 . 0 0 % o f the r e t i n a l d e h y d e present, while in the light a d a p t e d state 34.41 +_ 3.00% o f t o t a l r e t i n a l d e h y d e was in the 11-cis form. In these e x t r a c t i o n s , light a d a p t e d eyes were e x p o s e d to r o o m light at a n i r r a d i a n c e o f a p p r o x i m a t e l y 8 • 101 ktW/cm 2. To see if we c o u l d isomerize m o r e 11-cis r e t i n a l d e h y d e , a n a d d i t i o n a l exp e r i m e n t was p e r f o r m e d . Eyes f r o m + / + (4 eyes) a n d rd/rd (8 eyes) a n i m a l s were e x p o s e d to light f r o m a q u a r t z - h a l o g e n source (passed t h r o u g h a h e a t filter) at an i r r a d i a n c e o f 4 • 104 ~tW/cm 2 for 15 m i n p r i o r to ext r a c t i o n . In this e x p e r i m e n t , the p e r c e n t a g e o f 1 l - c i s ret i n a l d e h y d e in rd/rd eyes was a p p r o x i m a t e l y 1 3 % ; for the + / + eyes the p e r c e n t a g e o f 11-cis r e t i n a l d e h y d e was also a p p r o x i m a t e l y 13% (as this e x p e r i m e n t r e p r e s e n t s one e x t r a c t i o n no s t a n d a r d e r r o r s are given). Q u a n t i t a t i v e d e t e r m i n a t i o n o f a l l - t r a n s a n d 11-cis ret i n a l o x i m e / r e t i n a l d e h y d e was o b t a i n e d b y c o n v e r t i n g p e a k a r e a s (syn a n d anti) into a b s o l u t e a m o u n t s using k n o w n a m o u n t s o f reference c o m p o u n d s a n d t a k i n g di-


layer of the retina. D Rod opsin (CERN-JS839) immunofluorescence in the rd/rd mouse retina. This animal was 87 days of age. Rod opsin immunoreactive cells were rarely identified and were only found in the peripheral part of the retina; no more than 8 of these cells were ever identified within an eye. These cells lack an outer segment but seem to have remnants of an inner segment (arrow). E Cellular retinal-binding protein (CRALBP) immunofluorescence in the retina of an 82 day old rd/rd mouse. Immunostaining was identified in two cell types, the pigment epithelium and the Mfiller cells, which span the retina from the pigment epithelium to the ganglion cells. Choroid (Ch); ganglion cells (G); immunoreactive perikarya (P); inner plexiform layer (IP); Miiller cells (34); Pigment epithelium (PE). In A the star (,) indicates two immunoreactive perikarya close together. Scale bar = 20 ~tm

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Fig. 6A-D. HPLC elution profile of dark and light adapted eyes from mice 100 days old. A Dark adapted + / + mouse retinas (n = 4 eyes in this extraction); B Light adapted + / + mice retinas (n=4 eyes); C Dark adapted rd/rd mouse retinas (n=10 eyes); D Light adapted rd/rd mouse retinas (n= 10 eyes). In each case 20 ~tl of the extract was injected onto the HPLC column. Peaks were identified by comparison with reference compounds on the diode array detector. The molar extinction coefficient of 11-cis retinaloxime at 360 nm (Ea6o) is 35000. For all-trans retinaloxime E36o = 54900 (Groenendijk et al. 1980). As a result, molar equivalents of 1l-cis retinaloxime and all-trans retinaloxime will not give identical peak areas. In order to quantify 11-cis retinaloxime using all-trans retinaloxime standards, one must multiply the peak area of the ll-cis peak by the molar extinction coefficient ratio which is derived as follows: = 54900/35000 = 1.56857. Peak classification: 1) This peak includes retinylesters; 2) 1l-cis retinaldehyde (syn); 3) all-trans retinaldehyde (syn); 4) Unidentified peak, probably carotenoid; 5) ll-cis retinaldehyde (anti); 6) all-trans retinaldehyde (anti) Ea60(all-trans)/E360(11-cis}

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