July 2009
Volume 50, Issue 7
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Retinal Cell Biology  |   July 2009
Regulation of Retinal Photoreceptor Phagocytosis in a Diurnal Mammal by Circadian Clocks and Ambient Lighting
Author Affiliations
  • Corina Bobu
    From the Department of Neurobiology of Rhythms, UPR 3212 CNRS (Centre National de la Recherche Scientifique), Institute for Cellular and Integrative Neurosciences, Strasbourg, France.
  • David Hicks
    From the Department of Neurobiology of Rhythms, UPR 3212 CNRS (Centre National de la Recherche Scientifique), Institute for Cellular and Integrative Neurosciences, Strasbourg, France.
Investigative Ophthalmology & Visual Science July 2009, Vol.50, 3495-3502. doi:10.1167/iovs.08-3145
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      Corina Bobu, David Hicks; Regulation of Retinal Photoreceptor Phagocytosis in a Diurnal Mammal by Circadian Clocks and Ambient Lighting. Invest. Ophthalmol. Vis. Sci. 2009;50(7):3495-3502. doi: 10.1167/iovs.08-3145.

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      © 2017 Association for Research in Vision and Ophthalmology.

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Abstract

purpose. To characterize light and circadian control of photoreceptor phagocytosis in a diurnal cone-rich rodent.

methods. Diurnal Arvicanthis ansorgei were maintained under standard cyclic light (12 hours 300 lux white light [L]/12 hours dark [D]) and were divided into five groups: 1, maintained in LD; 2, transferred to constant darkness (DD); 3, transferred to constant light (LL, 300 lux); 4, subjected to 6-hour advance in light onset; and 5, subjected to 6-hour delay in light onset. Animals were killed every 3 hours during 24 hours, and their eyes were rapidly enucleated and fixed. Cryosections were stained using specific rod (rhodopsin) and cone (MW cone opsin) antibodies. Immunopositive inclusions within the retinal pigment epithelium layer were quantified for each time point.

results. In LD, both rod and cone phagocytosis showed coincident synchronized profiles with sharp peaks 1 to 2 hours after light onset. In groups 2 and 3, phagocytic activity shifted partially to the new light schedules. In DD, the temporal phagocytic profile resembled largely that of LD. On the contrary, LL animals exhibited large alterations in both rod and cone phagocytosis. There was no longer any peak, with both showing relatively uniform profiles. In addition, the number of cone phagosomes was much higher (∼250% increase) compared with LD or DD.

conclusions. These data are the first to measure photoreceptor phagocytosis in a diurnal mammal under different lighting conditions and to highlight the disruptive effects of constant light, especially on cone photoreceptor function.

Living organisms possess an intrinsic circadian time-keeping system to synchronize their physiology with the environment. The retina has a double interest in circadian biology, since on the one hand light is the most powerful zeitgeber, acting though intrinsically photosensitive retinal ganglion cells projecting to the central circadian clock in the suprachiasmatic nucleus (SCN) 1 ; and on the other, the retina exhibits numerous rhythmic physiological processes of its own, including melatonin synthesis, 2 ion channel sensitivity, 3 4 visual pigment synthesis 5 and phagocytosis of shed photoreceptor (PR) outer segments (OS). 6 PR are highly metabolically active cells, undergoing constant membrane renewal such that the OS are replaced entirely within 7 to 10 days. 7 This turnover is composed of several sequential, synchronized steps: RNA synthesis of visual pigments, protein translation and transport, and removal of aged membrane from the distal end of the OS. This latter process is achieved through phagocytosis of shed membrane by the apposing retinal pigmented epithelium (RPE). 8 Each step of this renewal process is tightly regulated, and errors in any one of them can lead to PR breakdown and death. For example, rhodopsin transcription levels are controlled precisely, with both under- 9 and overexpression 10 leading to PR degeneration. Mutations in the Mertk receptor involved in PR phagocytosis lead to retinal breakdown in animals 11 and humans. 12 Much effort has been given to defining the environmental and molecular control mechanisms of these different processes. Visual pigment synthesis and phagocytosis are both known to be controlled by light and/or circadian clocks. 5 13 14 In addition, the severity of retinal light damage is also dependent on circadian control. 15 There is evidence that some of these activities are regulated by an endogenous retinal clock, since cultured retinal explants continue to synthesize melatonin in a rhythmic manner 16 and optic nerve sectioning does not perturb phagocytosis. 17 In mammals, these phenomena have generally been studied in laboratory strains of rats and mice, which are both nocturnal species. Nocturnal mammals are rod dominant, which has led to a relative lack of information on the poorly represented cone PR. 18 19 This latter PR type is critical to human eyesight, subserving both chromatic sensitivity and high-acuity vision. To investigate photoreceptor responses to various ambient light levels, we used a diurnal murid rodent, Arvicanthis ansorgei, which we have shown to contain more than 10-fold more cones PR than mice. 20 21 In stark contrast to data on rats and mice, diurnal rodent cones showed a strong upregulation of phagocytosis and a complete loss of rhythmic activity when animals were exposed to prolonged lighting. 
Methods
Animal Care and Handling
This study was conducted with Sudanian Unstriped Grass Rats Arvicanthis ansorgei, born and reared in our Chronobiotron animal facilities from individuals captured in southern Mali in 1998. 22 Young (3–6 months) adult female Arvicanthis ansorgei were housed in individual cages under standard 12-hour light (L, 300 lux)/12 hour dark (D) cycles, lights on at 7 AM, lights off at 7 PM, with free access to food (standard rat chow) and water. Five experimental series were used. Series 1: The animals (n= 4 per time point) were taken every 3 hours through a complete 24-hour period, with light onset defined as zeitgeber time (ZT)0. At ZT1, 4, 7, 10, 13, 16, 19, and 22, the rats were anesthetized by isoflurane inhalation and decapitated, and the eyes were enucleated rapidly and fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 24 hours at 4°C. Series 2: For constant dark studies (DD), the animals were placed in total darkness for 24 hours and therefore experienced a complete cycle of subjective day and night before sample collection. Collection commenced at circadian time (CT)1 (i.e., 1 hour after subjective light onset), and continued every 3 hours through a complete 24-hour period (n = 4 per time point). Series 3: For constant light (LL), the animals were left in permanent 300-lux white light for 36 hours before collection of samples. In this way, the animals experienced a complete cycle of subjective night and day, with sampling commencing at CT12, every 3 hours through a complete 24-hour period (n = 4 per time point). Series 4: The animals (n = 4 per time point) that had been maintained in LD conditions were subjected to a 6-hour advance (lights on at ZT18) of normal light onset and were examined over the immediate following 24 hours. Eyes were taken at times corresponding to the new and expected lighting schedules (i.e., ZT19, ZT1, ZT7, ZT13, and ZT16; order of collection). Series 5: The animals (n = 4 per time point) that had been maintained in LD conditions were subjected to a 6-hour delay (lights on at ZT6) of normal light onset, and were examined over the immediate following 24 hours. The eyes were taken at times corresponding to the new and expected lighting schedules (i.e., ZT7, ZT10, ZT13, ZT19, and ZT1; order of collection). The schedules and lighting conditions used in these different series are shown in Figure 1 . All animal experimentation was performed according to institutional and national guidelines and adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Immunohistochemistry
Fixed eyes were treated for double-label immunohistochemistry, as described previously. 20 Subsequent to fixation, the eyeballs were rinsed in PBS, transferred to an ascending series of sucrose solutions (10%, 20%, and 30% each for 2 hours) and embedded (Tissue-Tek; Sakura, Torrence, CA). Cryostat sections (10 μm thick) were prepared and stored at −20°C until ready for use. The sections were permeabilized with Triton X-100 (0.1% in PBS for 5 minutes) and then saturated with PBS containing 0.1% bovine serum albumin, 0.1% Tween-20 and 0.1% sodium azide (buffer A) for 30 minutes. Sections were incubated overnight at 4°C with monoclonal anti-rhodopsin antibody Rho-4D2 23 and polyclonal anti-mouse mid-wavelength (MW)-cone opsin antibody 24 (generous gift of Cheryl Craft, Doheny Eye Institute, University of California, Los Angeles). For animals exposed to LL conditions, we performed additional immunostaining studies with rod- and cone-specific antibody markers: rod transducin α subunit for rods and cone transducin α subunit (Santa Cruz Biotechnology, Santa Cruz, CA, clones H20 and I20, respectively) and cone arrestin (generous gift of Cheryl Craft) for cones. These antibodies were used as described in our previous publications. 20 21 Secondary antibody incubation was performed at room temperature for 2 hours with Alexa (488 or 594) goat anti-rabbit or anti-mouse IgG–conjugated antibodies (Molecular Probes Inc., Eugene, OR). Cell nuclei were stained with 4,6-di-amino-phenyl-indolamine (DAPI; Molecular Probes). Slides were washed thoroughly, mounted in PBS/glycerol (1:1), and observed by fluorescence confocal microscope (LSM 510; Carl Zeiss Meditec, Dublin, CA). Images were relayed by CCD camera video capture to a dedicated computer containing image-analysis software. The number of phagosome was quantified as described previously by us. 20 Transverse sections were obtained from the central retina, covering the whole width of the retina from one periphery to the other. Four sections from one eye (at ∼0.5 and 1 mm from the optic nerve head) of each animal at each point were analyzed (i.e., total of 16 sections per time point). Taking the OS/RPE interface as a baseline, any immunopositive inclusion of ≥1 μm lying within the RPE subcellular space (visualized by faint background lighting) was scored as a phagosome. Phagosomes were counted by aligning a 150 × 150-μm grid placed within the eyepiece, parallel with the retinal pigmented epithelial (RPE) layer. Taking the optic nerve head as the reference, we displaced the grid dorsally and ventrally along the photoreceptor outer segment (OS)/RPE interface, from the posterior to the superior margin. The phagosome counts are expressed as the sum of all four sections per eye. 
Statistics
Results are presented as the mean ± SEM. Statistically significant differences among different ZT or CT groups were analyzed by one-way ANOVA (Statistica 8.0; StatSoft, Tulsa, OK) on the normalized data. When ANOVA indicated significant differences (P < 0.05) a post hoc analysis (Bonferroni or Tukey test) was performed. 
Results
We have shown previously that in a standard LD cycle, both rod and cone PRs undergo rhythmic shedding of phagosomes. 20 For the sake of completion we show similar data here: dual immunohistochemical staining revealed the presence of distinct fluorescent inclusions (phagosomes) of both rods and cones within the RPE at ZT1, but not at other time points (e.g., ZT13, Figs. 2a 2b 2c 2d 2e 2f ). These observations were very similar for animals maintained for 48 to 72 hours in DD: Both PR types showed frequent scattered immunofluorescent phagosomes at CT1 and very low or no phagosomes at other times of the day, such as at CT13 (Figs. 2g 2h 2i 2j 2k 2l) . The situation was very different for animals maintained 48 to 72 hours in LL. Scattered rod and cone phagosomes were seen in the RPE throughout the 24-hour period (e.g., CT1 and CT13, Figs. 2m 2n 2o 2p 2q 2r ). 
Quantification of phagosomes as a function of daily time in LD showed that both rods and cones had similar temporal profiles, with maximum values at 1 hour after light onset (ZT1; Fig. 3 ). Rod phagosome counts were very low for the other points, reaching a minimum in late day/early night (ZT10–13; Fig. 3a ), whereas cone activity was minimal during early night (ZT16; Fig. 3b ). Both rod and cone PR showed small nonsignificant peaks of phagosome formation in the late night, before lights on (ZT19–22). Overall, rod peak activity was approximately 25- to 30-fold higher than basal activity, and peak cone activity was 10 to 15 times the basal level. These patterns were very similar on the second day of DD conditions. A very large peak in rod and cone phagosome numbers occurred at CT1 (1 hour into subjective day), of the same or greater magnitude than in LD (∼40-fold background levels for rods, ∼20 fold for cones; Fig. 4 ). Phagosome numbers were very low for the remainder of the 24-hour period, except at CT19, when a second peak of phagosome formation was seen. The size of this peak was greater than that of LD and attained highly significant statistical difference compared with other sample times. On the other hand, after 2 days in LL, quantification of phagosomes revealed a very different pattern. There was no longer any obvious peak; instead rod and cone phagosomes were broadly distributed throughout the 24-hour period. The profile for rods was a broad double hump with maxima at ZT10 and -22, and troughs at ZT4 and -16; for cones, there was a uniform elevated level (Fig. 5)
Summation of phagosome counts for each measured time point over the entire 24-hour period demonstrated that for rods the total phagocytic burden changed by a relatively small amount in the three illumination conditions. With LD counts taken to be 100%, DD showed an increase of 46% and LL a small decrease of 14% (Table 1) . Cone phagosome counts showed a 37% increase in DD compared with LD; but they showed a much larger overall increase, 249%, in LL compared with LD. In addition, comparison of total phagosome tallies for both PR types under each lighting condition revealed that the ratio of rod-to-cone burden was roughly the same in LD and DD (∼8- to 9-fold more rod than cone phagosomes); however, in LL this ratio was more nearly equal, at twofold more rod than cone inclusions (Table 1)
To see whether changes in phagocytic activity under LL conditions could be associated with overt changes in photoreceptor structure or integrity, additional rod- and cone-specific antibody markers were used. There was no evidence of nuclear condensation or fragmentation, as seen by DAPI staining (Fig. 6a) . Immunostaining for cone arrestin revealed intense labeling of cone pedicles and moderate labeling of cell bodies and OS (Fig. 6b) . Immunoreactivity for rod transducin was intense in OS but weak throughout the rest of the cells (Fig. 6c) , whereas that of cone transducin was more uniform (Fig. 6d) . In general, there was no evidence of deleterious changes in cell structure or OS integrity compared with that in similar experiments performed in retinas from animals maintained under LD conditions. 20 21  
Animals that were subjected to a 6-hour advance in light onset and observed over the subsequent 24 hours showed a phagocytic profile that largely followed lighting conditions. Peak activity occurred 1 hour after the new lights-on time (i.e., ZT19, for both rods and cones; Fig. 7 ). However, the rod peak amplitude was notably reduced (by roughly 30%) compared with that in normal LD. There was only background activity at the anticipated time (CT1) for rods, whereas cone uptake displayed an intermediate level (∼50% maximum activity) throughout the period examined. For animals subjected to a 6-hour delay in light onset, peak activity again followed the new lighting regimen for both rods and cones (i.e., CT7). Again rod peak activity was reduced ∼50% compared with LD levels and was minimal at other times in the 24-hour period, whereas cone activity was relatively elevated, around 50% peak values (Fig. 8)
Discussion
This study presents the first quantitative data on both rod and cone PR recycling within a diurnal cone-rich mammal, as a function of daily time and ambient lighting. The results show that under LD and DD conditions, the temporal expression profiles and overall magnitude of phagosome formation were largely similar, with sharp peaks shortly after light onset (and secondary smaller peaks during subjective night). But under LL, rhythmic phagosome formation was completely lost, to be replaced by roughly uniform activity, which was downregulated in rods and greatly upregulated in cones relative to normal cyclic light. Finally, switching of animals to new lighting regimens led to immediate but only partial matching responses in phagocytic profiles. 
There are few data on retinal circadian processes in diurnal mammals, which, it can be argued, represent a closer analogy to human behavior than nocturnal species such as Mus musculus and Rattus norvegicus. Peak rod phagocytosis occurs invariably shortly after light onset in every species (both nocturnal and diurnal) that has been examined (e.g., rhesus monkey, 25 frog [Rana], 26 mouse, 27 cat, 28 rat, 14 tree squirrel, 29 ground squirrel, 30 and chicken 31 ). Data on peak cone phagocytosis are less common and show more variability. Most studies show cone peak phagocytosis as occurring within the dark period, as in diurnal chickens, 31 fence lizards, 32 tree squirrels, 29 ground squirrels, 30 and rhesus monkeys. 25 A few studies show the maximum activity being coincident with that of rods (in nocturnal species such as cat 28 and diurnal species such as Tupaia 33 or Arvicanthis 20 ). In the present study, advances or delays in the lighting regimen led to immediate adjustments in the phagocytic profiles, with peak activities after the modified light schedule, in a time course expected for light-triggered responses (although there was increased phagocytosis outside of peak times compared to LD). Previous phase-shift studies of phagocytosis in rats have shown that readjustment to new lighting schedules can take up to 2 weeks. 34 We propose that in Arvicanthis, rhythmic phagocytic behavior is governed by light under normal cyclic conditions, but an endogenous circadian mechanism rapidly assumes control of rhythmic activity in constant darkness. This switch appears to require one complete cycle of subjective day and night to become operable, since after 24 hours in constant darkness, cyclic phagocytic behavior occurs fully according to the anticipated subjective dawn. There is thus a lag phase of ∼24 hours for both rods and cones during which this process switches between the two regulatory systems. 
Several observations regarding rod and cone shedding in relation to ambient light deserve comment. The secondary smaller peak occurring during the night has been seen in several previous studies 14 25 28 35 and seems to correspond to clock activity “priming” the system to prepare for light onset and dawn. There are no specific data on the amplification effects of constant darkness on this nocturnal increase, but one paper documents a general increase in shedding when rats were housed in constant darkness. 17 Of interest, the increase calculated from their data matches closely the figure obtained in the present study (∼50% for rods). On the other hand, another study observed a reduction of phagocytic activity in DD. 34 Second, the magnitude in peak–trough differences calculated by us are in general much higher than those quoted in previous studies (in our experiments 20- to 40-fold compared with ∼5- to 10-fold in prior reports 17 28 36 ). In part, these differences may be due to our assay. The reliance on immunohistochemical detection would limit our counts to early phagosomes, before enzymatic digestion and loss of immunoreactivity. Histologic methods that are normally used would also pick up older inclusions and tend to attenuate the difference, both in absolute numbers between peak and trough, and across the 24-hour period (see Fig. 1in Ref. 31 ). In addition, phagocytic activity may be more intense in this species. Third, we consistently saw rod phagosome counts much higher (an average of eight times) than those of cone phagosomes, under LD and DD conditions. In only one previous study has an attempt been made to compare rod and cone shedding, and the conclusion was that rod and cone membrane production may be different (i.e., greater) in rods than cones. 28 Even correcting for the twofold excess of rods to cones, we observed a net fourfold excess of rods, indicating that in this species, too, rod OS membranes are more actively recycled than those of cones. Finally, previous investigators have observed a relationship between ambient lighting levels and OS length in rats. 37 Although we have not performed detailed morphometric measurements, we do not see changes in rod or cone OS length in DD (or LL). Furthermore, these earlier studies showed that such changes required at least 1 week and thus are unlikely to operate at the shorter time frame used here. 
Recent studies using mouse mutants have identified specific components of the circadian phagocytic pathway. Among the different molecules that have been identified as crucial for rod phagocytosis, the αVβ5 integrin receptor present at the RPE surface is critically involved in binding shed outer segment packets. 38 Of interest, β5-knockout mice lack the normal morning peak but instead exhibit a uniform pattern. 36 39 Knockout mice further develop age-related autofluorescent inclusions within the RPE, and exhibit age-related decline in visual responses. Subsequent studies highlighted the role of milk fat globule-EGF factor 8 as a candidate photoreceptor-bound ligand for this integrin, and revealed that mice with a gene deletion for this protein also showed uniform uptake profiles. 40 The same authors also identified a second distinct role for αVβ5 within the retina, in that it also mediates rhythmic variations in retinal adhesion. 41 These data suggest that ligand–receptor interactions may constitute one of the retinal circadian clock outputs or checkpoints that control rhythmic phagocytosis (and adhesion), although the authors did not detect any rhythmic changes in either αVβ5 or milk fat globule-EGF factor 8. There are so far no data linking specific clock genes to retinal phagocytosis or showing whether similar mechanisms underlie cone phagocytosis. 
The most dramatic findings in the present study concern the effects of constant lighting. Constant light induced complete loss in rhythmicity of rod and cone PR phagocytosis, which instead showed a broadly uniform activity. In addition, the data suggest that cone but not rod PR phagocytosis was strongly upregulated, perhaps linked to the constant functional activation of this former population by ambient light levels. Indeed, rod phagosome counts dropped to comparable levels of cone counts (when corrected for by population density differences). These data fit broadly with previous observations in frogs 42 and rats, 34 which concluded that prolonged maintenance in LL abolishes the burst of shedding. In opposition to cones, rods would largely cease to function in such prolonged bright illumination, and hence possibly do not regenerate so rapidly. On the other hand, these findings contrast sharply with previous studies on nocturnal mice, 27 which showed no changes. The apparent increase in cone phagosome counts in LL may reflect either increased shedding or uptake or decreased degradation. Immunohistochemistry of LL retinas failed to reveal obvious tissue damage or breakdown in OS structure, which may occur after protracted lighting. Staining patterns were essentially similar to those seen in animals maintained in cyclic lighting conditions (and fixed during the lighting period), with strong OS staining for rod and cone transducin, 21 and strong synaptic staining for cone arrestin. 20 One caveat for these quantitative measures concerns the 3-hour intervals between samples, such that we might under estimate the number of rod and/or cone phagosomes directly before or after the large morning peak. In a previous paper, the investigators postulated that there exists an inverse correlation between (rod) phagocytosis and ambient light levels, 43 which is in accord with our observations on rod debris. It is interesting to speculate that the cone data would fit in this scheme if it were modified to account for this population’s having a behavior opposite that of rods, related to their physiology of operating in relatively bright light. 
 
Figure 1.
 
Schematic diagram showing time schedule of lighting conditions and sampling points. Broad arrows in series 2 to 5 indicate times at which animals were switched to new lighting regimens. Thin arrows joined by a horizontal bar indicate the beginning and end of the sampling period. Series 1 is the control condition, animals maintained in LD (alternating white bars [light, 300 lux, 12 hours] and black bars [dark, 12 hours]) throughout the experiment; series 2 represents DD (alternating black dots on a white background [subjective day] and black bars); series 3 represents LL (alternating white dots on a black background [subjective night] and white bars); series 4 shows the paradigm for a 6-hour advance of light onset (shortened black bar during the second 24-hour period); and series 5 shows the paradigm for a 6-hour delay of light onset (elongated black bar at the start of the second 24-hour period). The x-axis shows time elapsed in hours since the beginning of the experimental period.
Figure 1.
 
Schematic diagram showing time schedule of lighting conditions and sampling points. Broad arrows in series 2 to 5 indicate times at which animals were switched to new lighting regimens. Thin arrows joined by a horizontal bar indicate the beginning and end of the sampling period. Series 1 is the control condition, animals maintained in LD (alternating white bars [light, 300 lux, 12 hours] and black bars [dark, 12 hours]) throughout the experiment; series 2 represents DD (alternating black dots on a white background [subjective day] and black bars); series 3 represents LL (alternating white dots on a black background [subjective night] and white bars); series 4 shows the paradigm for a 6-hour advance of light onset (shortened black bar during the second 24-hour period); and series 5 shows the paradigm for a 6-hour delay of light onset (elongated black bar at the start of the second 24-hour period). The x-axis shows time elapsed in hours since the beginning of the experimental period.
Figure 2.
 
Double immunohistochemical staining of rod and cone phagosomes in the LD, DD, and LL conditions. Arvicanthis were killed at ZT1 (a–c, g–i, m–o) and ZT13 (d–f, j–l, p–r), and cryosections subjected to double immunolabeling with anti-rhodopsin (for rods: a, d, g, j, m, p) and anti-cone MW opsin (for MW cones: b, e, h, k, n, and q) antibodies. Merged images: c, f, i, l, o, and r. Antibodies heavily labeled the rod OS and cone OS, respectively, but also inclusions of ∼1 μm present within the RPE (phagosomes, examples indicated by white arrows). In LD and DD, scattered inclusions were observed for both rods and cones at ZT1 but not at ZT13. In LL, scattered phagosomes were observed at all time points. Scale bar, r = 10 μm for all panels.
Figure 2.
 
Double immunohistochemical staining of rod and cone phagosomes in the LD, DD, and LL conditions. Arvicanthis were killed at ZT1 (a–c, g–i, m–o) and ZT13 (d–f, j–l, p–r), and cryosections subjected to double immunolabeling with anti-rhodopsin (for rods: a, d, g, j, m, p) and anti-cone MW opsin (for MW cones: b, e, h, k, n, and q) antibodies. Merged images: c, f, i, l, o, and r. Antibodies heavily labeled the rod OS and cone OS, respectively, but also inclusions of ∼1 μm present within the RPE (phagosomes, examples indicated by white arrows). In LD and DD, scattered inclusions were observed for both rods and cones at ZT1 but not at ZT13. In LL, scattered phagosomes were observed at all time points. Scale bar, r = 10 μm for all panels.
Figure 3.
 
Quantitative measure of rod and cone phagocytosis under LD conditions. For rods, quantification of rhodopsin-immunoreactive phagosomes across the 24-hour period showed a large sharp peak at ZT1, and an additional smaller but significant increase at ZT19 (a). Under the same conditions, quantification of cone MW opsin-immunoreactive phagosomes across the 24-hour period showed a very similar profile, with a sharp peak at ZT1, and a second peak at ZT19 (b). Bars above each histogram depict illumination conditions, with alternating white and black bars for day and night, respectively. Statistical significance: ***P < 0.001. Note that the scale for the y-axis in this figure and in Figures 4 7 and 8is 10-fold higher for rods than for cones.
Figure 3.
 
Quantitative measure of rod and cone phagocytosis under LD conditions. For rods, quantification of rhodopsin-immunoreactive phagosomes across the 24-hour period showed a large sharp peak at ZT1, and an additional smaller but significant increase at ZT19 (a). Under the same conditions, quantification of cone MW opsin-immunoreactive phagosomes across the 24-hour period showed a very similar profile, with a sharp peak at ZT1, and a second peak at ZT19 (b). Bars above each histogram depict illumination conditions, with alternating white and black bars for day and night, respectively. Statistical significance: ***P < 0.001. Note that the scale for the y-axis in this figure and in Figures 4 7 and 8is 10-fold higher for rods than for cones.
Figure 4.
 
Quantitative measure of rod and cone phagocytosis in DD conditions. For rods, quantification of rhodopsin-immunoreactive phagosomes across the 24-hour period showed a general profile very close to that in LD: a large sharp peak at ZT1 and an additional smaller but significant increase at ZT19 (a). Counting of cone MW opsin-immunoreactive phagosomes across the 24-hour period was also similar to that of both rods and cones in LD, with a sharp peak at ZT1, and a second peak at ZT19 (b). Bars above each histogram depict illumination conditions (alternating black and black dots on white background [subjective day]). Statistical significance: ***P < 0.001.
Figure 4.
 
Quantitative measure of rod and cone phagocytosis in DD conditions. For rods, quantification of rhodopsin-immunoreactive phagosomes across the 24-hour period showed a general profile very close to that in LD: a large sharp peak at ZT1 and an additional smaller but significant increase at ZT19 (a). Counting of cone MW opsin-immunoreactive phagosomes across the 24-hour period was also similar to that of both rods and cones in LD, with a sharp peak at ZT1, and a second peak at ZT19 (b). Bars above each histogram depict illumination conditions (alternating black and black dots on white background [subjective day]). Statistical significance: ***P < 0.001.
Figure 5.
 
In LL conditions, in stark contrast to LD and DD, rod phagosomes were present in roughly equal numbers throughout the 24-hour period, with a broad double-humped distribution (a). Note that the magnitude of the y-axis scale is 10-fold less than that for rod phagocytosis. Cone phagosome counts also displayed a uniform elevated distribution (b). Bars above each histogram depict illumination conditions (alternating white and white dots on black background [subjective night]). Statistical significance: **P < 0.01.
Figure 5.
 
In LL conditions, in stark contrast to LD and DD, rod phagosomes were present in roughly equal numbers throughout the 24-hour period, with a broad double-humped distribution (a). Note that the magnitude of the y-axis scale is 10-fold less than that for rod phagocytosis. Cone phagosome counts also displayed a uniform elevated distribution (b). Bars above each histogram depict illumination conditions (alternating white and white dots on black background [subjective night]). Statistical significance: **P < 0.01.
Table 1.
 
Total Rod and Cone Phagosome Counts in Different Lighting Environments
Table 1.
 
Total Rod and Cone Phagosome Counts in Different Lighting Environments
Lighting Condition (1) Σ Rod Phago (2) Σ Cone Phago (3) Ratio Rod Phago In LD (4) Ratio Cone Phago In LD (5) Ratio Rods/Cones
LD 4510 560 100 100 8.1
DD 6582 767 146 137 8.6
LL 3866 1953 86 349 2.0
Figure 6.
 
General retinal cell structure and immunohistochemical staining of rods and cones in LL retina. (a) General nuclear morphology, as seen after DAPI staining, was the same as in control animals, with fainter cone nuclei (cone cell bodies, CCB) and darker rod nuclei (RCB, rod cell bodies). (b) Cone arrestin antibody gave staining throughout the cone cells, at the levels of their outer (COS) and inner (CIS) segments and cell bodies (CCB), and especially strong within the synaptic pedicles (CP). (c) Rod transducin was detected within the outer (ROS) and inner (RIS) segments and cell bodies (RCB). (d) Cone transducin was also visible throughout the cells, including COS, CIS, CCB, and CP. Scale bar: 12 μm for all panels.
Figure 6.
 
General retinal cell structure and immunohistochemical staining of rods and cones in LL retina. (a) General nuclear morphology, as seen after DAPI staining, was the same as in control animals, with fainter cone nuclei (cone cell bodies, CCB) and darker rod nuclei (RCB, rod cell bodies). (b) Cone arrestin antibody gave staining throughout the cone cells, at the levels of their outer (COS) and inner (CIS) segments and cell bodies (CCB), and especially strong within the synaptic pedicles (CP). (c) Rod transducin was detected within the outer (ROS) and inner (RIS) segments and cell bodies (RCB). (d) Cone transducin was also visible throughout the cells, including COS, CIS, CCB, and CP. Scale bar: 12 μm for all panels.
Figure 7.
 
Quantitative estimation of rod and cone phagocytosis after the advance of the lighting phase. Animals were subjected to a 6-hour advance in light onset relative to normal lights on (ZT18) and killed at the times indicated. Rod (a) and cone (b) phagosome counts were estimated as described. Statistical significance: **P < 0.01.
Figure 7.
 
Quantitative estimation of rod and cone phagocytosis after the advance of the lighting phase. Animals were subjected to a 6-hour advance in light onset relative to normal lights on (ZT18) and killed at the times indicated. Rod (a) and cone (b) phagosome counts were estimated as described. Statistical significance: **P < 0.01.
Figure 8.
 
Quantitative estimation of rod and cone phagocytosis after the delay of lighting phase. Animals were subjected to a 6-hour delay in light onset relative to normal lights on (ZT6) and killed at the times indicated. Rod (a) and cone (b) phagosome counts were estimated as described. Statistical significance: *P < 0.05.
Figure 8.
 
Quantitative estimation of rod and cone phagocytosis after the delay of lighting phase. Animals were subjected to a 6-hour delay in light onset relative to normal lights on (ZT6) and killed at the times indicated. Rod (a) and cone (b) phagosome counts were estimated as described. Statistical significance: *P < 0.05.
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Figure 1.
 
Schematic diagram showing time schedule of lighting conditions and sampling points. Broad arrows in series 2 to 5 indicate times at which animals were switched to new lighting regimens. Thin arrows joined by a horizontal bar indicate the beginning and end of the sampling period. Series 1 is the control condition, animals maintained in LD (alternating white bars [light, 300 lux, 12 hours] and black bars [dark, 12 hours]) throughout the experiment; series 2 represents DD (alternating black dots on a white background [subjective day] and black bars); series 3 represents LL (alternating white dots on a black background [subjective night] and white bars); series 4 shows the paradigm for a 6-hour advance of light onset (shortened black bar during the second 24-hour period); and series 5 shows the paradigm for a 6-hour delay of light onset (elongated black bar at the start of the second 24-hour period). The x-axis shows time elapsed in hours since the beginning of the experimental period.
Figure 1.
 
Schematic diagram showing time schedule of lighting conditions and sampling points. Broad arrows in series 2 to 5 indicate times at which animals were switched to new lighting regimens. Thin arrows joined by a horizontal bar indicate the beginning and end of the sampling period. Series 1 is the control condition, animals maintained in LD (alternating white bars [light, 300 lux, 12 hours] and black bars [dark, 12 hours]) throughout the experiment; series 2 represents DD (alternating black dots on a white background [subjective day] and black bars); series 3 represents LL (alternating white dots on a black background [subjective night] and white bars); series 4 shows the paradigm for a 6-hour advance of light onset (shortened black bar during the second 24-hour period); and series 5 shows the paradigm for a 6-hour delay of light onset (elongated black bar at the start of the second 24-hour period). The x-axis shows time elapsed in hours since the beginning of the experimental period.
Figure 2.
 
Double immunohistochemical staining of rod and cone phagosomes in the LD, DD, and LL conditions. Arvicanthis were killed at ZT1 (a–c, g–i, m–o) and ZT13 (d–f, j–l, p–r), and cryosections subjected to double immunolabeling with anti-rhodopsin (for rods: a, d, g, j, m, p) and anti-cone MW opsin (for MW cones: b, e, h, k, n, and q) antibodies. Merged images: c, f, i, l, o, and r. Antibodies heavily labeled the rod OS and cone OS, respectively, but also inclusions of ∼1 μm present within the RPE (phagosomes, examples indicated by white arrows). In LD and DD, scattered inclusions were observed for both rods and cones at ZT1 but not at ZT13. In LL, scattered phagosomes were observed at all time points. Scale bar, r = 10 μm for all panels.
Figure 2.
 
Double immunohistochemical staining of rod and cone phagosomes in the LD, DD, and LL conditions. Arvicanthis were killed at ZT1 (a–c, g–i, m–o) and ZT13 (d–f, j–l, p–r), and cryosections subjected to double immunolabeling with anti-rhodopsin (for rods: a, d, g, j, m, p) and anti-cone MW opsin (for MW cones: b, e, h, k, n, and q) antibodies. Merged images: c, f, i, l, o, and r. Antibodies heavily labeled the rod OS and cone OS, respectively, but also inclusions of ∼1 μm present within the RPE (phagosomes, examples indicated by white arrows). In LD and DD, scattered inclusions were observed for both rods and cones at ZT1 but not at ZT13. In LL, scattered phagosomes were observed at all time points. Scale bar, r = 10 μm for all panels.
Figure 3.
 
Quantitative measure of rod and cone phagocytosis under LD conditions. For rods, quantification of rhodopsin-immunoreactive phagosomes across the 24-hour period showed a large sharp peak at ZT1, and an additional smaller but significant increase at ZT19 (a). Under the same conditions, quantification of cone MW opsin-immunoreactive phagosomes across the 24-hour period showed a very similar profile, with a sharp peak at ZT1, and a second peak at ZT19 (b). Bars above each histogram depict illumination conditions, with alternating white and black bars for day and night, respectively. Statistical significance: ***P < 0.001. Note that the scale for the y-axis in this figure and in Figures 4 7 and 8is 10-fold higher for rods than for cones.
Figure 3.
 
Quantitative measure of rod and cone phagocytosis under LD conditions. For rods, quantification of rhodopsin-immunoreactive phagosomes across the 24-hour period showed a large sharp peak at ZT1, and an additional smaller but significant increase at ZT19 (a). Under the same conditions, quantification of cone MW opsin-immunoreactive phagosomes across the 24-hour period showed a very similar profile, with a sharp peak at ZT1, and a second peak at ZT19 (b). Bars above each histogram depict illumination conditions, with alternating white and black bars for day and night, respectively. Statistical significance: ***P < 0.001. Note that the scale for the y-axis in this figure and in Figures 4 7 and 8is 10-fold higher for rods than for cones.
Figure 4.
 
Quantitative measure of rod and cone phagocytosis in DD conditions. For rods, quantification of rhodopsin-immunoreactive phagosomes across the 24-hour period showed a general profile very close to that in LD: a large sharp peak at ZT1 and an additional smaller but significant increase at ZT19 (a). Counting of cone MW opsin-immunoreactive phagosomes across the 24-hour period was also similar to that of both rods and cones in LD, with a sharp peak at ZT1, and a second peak at ZT19 (b). Bars above each histogram depict illumination conditions (alternating black and black dots on white background [subjective day]). Statistical significance: ***P < 0.001.
Figure 4.
 
Quantitative measure of rod and cone phagocytosis in DD conditions. For rods, quantification of rhodopsin-immunoreactive phagosomes across the 24-hour period showed a general profile very close to that in LD: a large sharp peak at ZT1 and an additional smaller but significant increase at ZT19 (a). Counting of cone MW opsin-immunoreactive phagosomes across the 24-hour period was also similar to that of both rods and cones in LD, with a sharp peak at ZT1, and a second peak at ZT19 (b). Bars above each histogram depict illumination conditions (alternating black and black dots on white background [subjective day]). Statistical significance: ***P < 0.001.
Figure 5.
 
In LL conditions, in stark contrast to LD and DD, rod phagosomes were present in roughly equal numbers throughout the 24-hour period, with a broad double-humped distribution (a). Note that the magnitude of the y-axis scale is 10-fold less than that for rod phagocytosis. Cone phagosome counts also displayed a uniform elevated distribution (b). Bars above each histogram depict illumination conditions (alternating white and white dots on black background [subjective night]). Statistical significance: **P < 0.01.
Figure 5.
 
In LL conditions, in stark contrast to LD and DD, rod phagosomes were present in roughly equal numbers throughout the 24-hour period, with a broad double-humped distribution (a). Note that the magnitude of the y-axis scale is 10-fold less than that for rod phagocytosis. Cone phagosome counts also displayed a uniform elevated distribution (b). Bars above each histogram depict illumination conditions (alternating white and white dots on black background [subjective night]). Statistical significance: **P < 0.01.
Figure 6.
 
General retinal cell structure and immunohistochemical staining of rods and cones in LL retina. (a) General nuclear morphology, as seen after DAPI staining, was the same as in control animals, with fainter cone nuclei (cone cell bodies, CCB) and darker rod nuclei (RCB, rod cell bodies). (b) Cone arrestin antibody gave staining throughout the cone cells, at the levels of their outer (COS) and inner (CIS) segments and cell bodies (CCB), and especially strong within the synaptic pedicles (CP). (c) Rod transducin was detected within the outer (ROS) and inner (RIS) segments and cell bodies (RCB). (d) Cone transducin was also visible throughout the cells, including COS, CIS, CCB, and CP. Scale bar: 12 μm for all panels.
Figure 6.
 
General retinal cell structure and immunohistochemical staining of rods and cones in LL retina. (a) General nuclear morphology, as seen after DAPI staining, was the same as in control animals, with fainter cone nuclei (cone cell bodies, CCB) and darker rod nuclei (RCB, rod cell bodies). (b) Cone arrestin antibody gave staining throughout the cone cells, at the levels of their outer (COS) and inner (CIS) segments and cell bodies (CCB), and especially strong within the synaptic pedicles (CP). (c) Rod transducin was detected within the outer (ROS) and inner (RIS) segments and cell bodies (RCB). (d) Cone transducin was also visible throughout the cells, including COS, CIS, CCB, and CP. Scale bar: 12 μm for all panels.
Figure 7.
 
Quantitative estimation of rod and cone phagocytosis after the advance of the lighting phase. Animals were subjected to a 6-hour advance in light onset relative to normal lights on (ZT18) and killed at the times indicated. Rod (a) and cone (b) phagosome counts were estimated as described. Statistical significance: **P < 0.01.
Figure 7.
 
Quantitative estimation of rod and cone phagocytosis after the advance of the lighting phase. Animals were subjected to a 6-hour advance in light onset relative to normal lights on (ZT18) and killed at the times indicated. Rod (a) and cone (b) phagosome counts were estimated as described. Statistical significance: **P < 0.01.
Figure 8.
 
Quantitative estimation of rod and cone phagocytosis after the delay of lighting phase. Animals were subjected to a 6-hour delay in light onset relative to normal lights on (ZT6) and killed at the times indicated. Rod (a) and cone (b) phagosome counts were estimated as described. Statistical significance: *P < 0.05.
Figure 8.
 
Quantitative estimation of rod and cone phagocytosis after the delay of lighting phase. Animals were subjected to a 6-hour delay in light onset relative to normal lights on (ZT6) and killed at the times indicated. Rod (a) and cone (b) phagosome counts were estimated as described. Statistical significance: *P < 0.05.
Table 1.
 
Total Rod and Cone Phagosome Counts in Different Lighting Environments
Table 1.
 
Total Rod and Cone Phagosome Counts in Different Lighting Environments
Lighting Condition (1) Σ Rod Phago (2) Σ Cone Phago (3) Ratio Rod Phago In LD (4) Ratio Cone Phago In LD (5) Ratio Rods/Cones
LD 4510 560 100 100 8.1
DD 6582 767 146 137 8.6
LL 3866 1953 86 349 2.0
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