Arvicanthis Niloticus)

Topographic Arrangement of S-cone Photoreceptors in the Retina of the Diurnal Nile Grass Rat (Arvicanthis niloticus)

Fre´de´ric Gaillard,1 Sharee Kuny,2,3 and Yves Sauve´ 2,3

1,2 PURPOSE. The retina of Arvicanthis niloticus, a diurnal murine become available, ground squirrels, guinea pigs, or tree rodent closely related to Rattus (rats) and Mus (mice), contains shrews remain popular models for investigating structure, Ϸ30% to 35% cones and has several cone-driven functional function, and pathologic features of cone-rich mammalian ret- characteristics found in humans. In this study the organization inas. Members of the Arvicanthis genus (a genus closely re- of these cone photoreceptors was examined, with emphasis lated to Rattus and Mus, but with a diurnal lifestyle) may be a on those expressing the S-opsin photopigment (S-cones). useful alternative.3 The first immunohistochemical investiga- METHODS. Cones were labeled with antibodies against M- and tions dedicated to the visual system of two geographically S-opsins. Their topographic arrangement was examined on distant Arvicanthis species, the Sudanian (A. ansorgei) and the images of retinal flatmounts using density measures, nearest- Nile (A. niloticus) grass rats, pointed out the efficiency of neighbor distance, and Voronoi domain analysis. Partial se- preexisting antibodies in these species and established that their retinas contain Ϸ30% to 35% cones.3–5 Additional exper- quencing of the S-opsin DNA was also performed to determine 5,6 whether this visual pigment was blue/violet or UV sensitive. iments in A. niloticus showed that its retina displays post- receptoral neural features commonly observed in diurnal RESULTS. Cone photoreceptors (estimated total population Ϸ mammals and that it has several cone-driven functional char- 1.450 million) came in two distinct types that express either acteristics (as assessed with the electroretinogram) found in M/L- or S-opsin. Both types were present across the retinal Ϸ human retinas (large photopic a-wave amplitudes, photopic surface. S-cones ( 7–8% of the total cone population) hill effect, and critical flicker fusion beyond 60 Hz). In the achieved a higher density in a discrete temporodorsal sector of present study, we determined the distribution of the cone the retina. The S-cone mosaic was irregular. Finally, S-cones population (more specifically those expressing the S-opsin were likely to be UV sensitive, according to genetic analysis. photopigment) in A. niloticus with the rationale of acquiring CONCLUSIONS. The topographic arrangement of cone photore- normative data against which to compare the effects of exper- ceptors in the retina of the diurnal Nile grass rat A. niloticus imental manipulations and/or aging. represents a highly pertinent model to improve understanding of the pathologic course of and related therapy for retinal disease involving cones. (Invest Ophthalmol Vis Sci. 2009;50: MATERIAL AND METHODS 5426–5434) DOI:10.1167/iovs.09-3896 Animals one photoreceptors in human retinas achieve their highest This study was performed on young adult (2–6 months of age) Nile Cdensities in the fovea. They mediate high-spatial-resolution grass rats (A. niloticus) of both sexes derived from a breeding colony daylight vision and color discrimination. Their progressive loss established at the University of Alberta. The animals were raised on a in a variety of retinal degenerations induces a central visual 12:12 light–dark cycle (lights on at 5 AM; ambient temperature 21 Ϯ field scotoma, which almost inevitably leads to legal blindness 1°C; relative humidity Ϸ50%) and supplied ad libitum with water and (visual acuity of 20/200 or less). Ideally, such diseases would standard rodent diet (formula 5001 LabDiet; Nutrition International, be investigated in animal species with dense cone populations. Richmond, IN). Experiments were performed in accordance with the Although genetically engineered rodless mice have recently ARVO Statement for the Use of Animals in Ophthalmic and Visual Research and with the guidelines laid down by the NIH (National Institutes of Health) in the United States regarding the care and use of

1 animals for experimental procedures. The University of Alberta Animal From the Institut de Physiologie et Biologie Cellulaires, Universite´ Care and Use Committee approved the present work. de Poitiers, UMR 6187, CNRS (Centre National de la Recherche Scien- tifique), Poitiers, France; and the Departments of 2Physiology and 3Ophthalmology, University of Alberta, Edmonton, Alberta, Canada. Primary Antibodies Supported by Canadian Institutes of Health Research (CIHR) Grant Cone photoreceptors were screened using anti-M/L- and anti-S-opsin 151145; an Alberta Heritage Foundation for Medical Research (AH- FMR) equipment grant; the Canadian National Institute for the Blind; polyclonal antibodies (AB5405 and AB5407, working dilution 1:500; the Olive Young Foundation; and The Lena McLaughlin Foundation both from Chemicon, Temecula, CA;) raised in rabbit against the last (Mona & Rod McLennan). FG was supported by the International 42 and 38 amino acids respectively, at the C-terminus of recombinant Society for Clinical Electrophysiology of Vision (ISCEV; short lab visit human red/green- and blue opsins.7 These antibodies have been re- grant 2008). YS is an AHFMR Senior Scholar. ported to label the outer segments (OS) and cell membranes of specific Submitted for publication April 22, 2009; revised May 25, 2009; types of cones in human, mouse, and ground squirrel retinas.8,9 Their accepted August 5, 2009. specificity was confirmed in Western blot analyses from Nile grass rat Disclosure: F. Gaillard, None; S. Kuny, None; Y. Sauve´, None retinal tissue (described later). For double-labeling experiments, the The publication costs of this article were defrayed in part by page S-opsin AB5407 rabbit polyclonal antibody was replaced with an affin- charge payment. This article must therefore be marked “advertise- ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact. ity-purified goat polyclonal antibody raised against a 20-amino-acid Corresponding author: Yves Sauve´, Assistant Professor of Ophthal- synthetic peptide mapping within amino acids 1 to 50 of human mology and Physiology, Department of Physiology, 7-55 Medical Sci- blue-sensitive opsin (EFYLFKNISSVGPWDGPQYH; sc-14363; Santa ences Building, University of Alberta, Edmonton AB, Canada, T6G 2H7; Cruz Biotechnology Inc., Santa Cruz, CA; working dilution 1:200). This [email protected]. antibody has been used to identify S-opsin expressing cones in a range

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FIGURE 1. Cone distribution in cross sections and flatmounted retinas. (A) Western blots of A. niloticus retinal tissue. AB5405 and AB5407 antibodies detect major products at Ϸ39 and Ϸ37 kDa (lanes M and S, respectively) as expected for M/L- and S- opsins. A long exposure time (Ͼ4 minutes) may explain the faint bands (at Ϸ80 and Ϸ130 kDa) observed with the M/L-opsin antibody. (B) Near-central retinal cross section treated with M/L (AB5405; red)- and S (sc-14363; green)-opsin antibodies. These antibodies stain distinct OS. Pseudo co-localization of both opsins in the leftmost cone is due to acci- dental superimposition of two OS (the thickness of the sections is 20 ␮m). (C) M/L-cone entirely labeled with antibody AB5405. (D) S-cone as revealed with antibody sc-14363. (E) Cross section treated with anti-S-opsin antibody AB5407. (F) Same cross section treated with anti-S-opsin antibody sc-14363. (G) Merged pictures show that these two antibodies detected the same S-cone OS. (H) Ex- ample of M/L- (red) and S- (green) opsin expressing cones as viewed in a flatmounted retina (nasodorsal periphery). (I) Enlargement of the inset in (H) illustrates that expression of M/L- and S-opsins was mutually exclusive in the cones of A. niloticus. (J, K) Absence of opsin coexpression in an additional double-labeled flat- mount retina. Pictures taken in the midperiphery of the ventral quadrant where dual-pigment cones are most evident in the mouse. OS, outer segment; IS, inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; RPE, retinal pigment epithelium.

of species.10–14 Competition controls where sc-14363 was preincu- major products at Ϸ39 and Ϸ37 kDa, respectively corresponding to bated with the antigenic peptide11,13 yielded no labeling. M/L- and S-opsin (Fig. 1A).

Western Blot Analysis Immunohistochemistry Freshly dissected retinas (n ϭ 3; 2 months of age) were homogenized Cryosections were collected from 4% paraformaldehyde-fixed retinas in SDS buffer (4% [wt/vol] sodium dodecyl sulfate, 0.13 M Tris, 2% (n ϭ 2; 4 months of age) cut serially at 20 ␮m, parallel to the [vol/vol] 2-mercaptoethanol, 20% [vol/vol] glycerol; pH 6.8) with a nasotemporal axis, and mounted on glass slides (Superfrost/Plus; protease inhibitor cocktail (Complete; Roche Applied Science, Mann- Fisher Scientific, Pittsburgh, PA). After they were extensively washed ␮ heim, Germany). Samples (25 g protein) were resolved by SDS-PAGE in PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4-7H2O, 1.4 mM on 8% to 10% acrylamide gels. Proteins were transferred to polyvinyli- KH2PO4; pH 7.3), the sections were blocked for 2 hours in a medium dene fluoride (PVDF) membranes, blocked for 1 hour with 5% nonfat containing PBS ϩ0.1% Triton X-100ϩ5% nonfat milk, and reacted milk diluted in TBS-T (20 mM Tris, 137 mM NaCl, and 0.1% Tween-20; overnight with anti-M/L- and anti-S-opsin antibodies diluted appropri- pH 7.6), incubated overnight with anti-M/L-opsin (AB5405) or anti-S- ately in a 1:10 solution of the previous blocking medium. On the opsin (AB5407) primary antibodies (dilution 1:500 in the blocking following day, the sections were washed with PBS, blocked again for 1 solution), washed three times for 10 minutes each in TBS-T and reacted hour and exposed for 2 hours to goat anti-rabbit Alexa488-tagged for Ϸ1 hour with a donkey anti-rabbit IgG, HRP-conjugated enhanced secondary antibody (Molecular Probes Inc., Eugene, OR) diluted to chemiluminescence (ECL) antibody (1:5000 in the blocking solution; 1:500 in a 1:10 solution of the blocking medium. In double-staining NA934; GE Health Care, Little Chalfont, UK). After washing three times experiments, donkey anti-rabbit-Alexa594 and donkey anti-goat- for 10 minutes each in TBS-T, the protein bands were visualized using Alexa488 (1:500) were used as secondary antibodies. Sections were ECL reagent (NEL 103; Perkin Elmer, Wellesley, MA) and an imaging washed extensively in PBS, covered with an antifade solution (ProLong station (Eastman Kodak, Rochester, NY). These antibodies detected gold antifade reagent, P36939; Molecular Probes), and coverslipped.

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Control labeling without primary antibody remained negative. All re- actions were run at room temperature. Additional retinas (2.5–6 months of age) were prepared as ﬂat- mounts. After euthanatization (Euthanyl; Bimeda-MTC Animal Health Inc., Cambridge, ON, Canada), the eyes were enucleated (after a small incision was made in the dorsal margin of the ora serrata for orienta- tion). The cornea and lens were removed. The retinas were then carefully dissected from the eye cup, cut into four distinct quadrants, ﬁxed overnight at 4°C in 4% paraformaldehyde and processed with anti-M/L-opsin (n ϭ 2), anti-S-opsin (n ϭ 2), a mixture of anti-M/LϩS- opsin (n ϭ 2) antibodies, as well as (n ϭ 2) with a rabbit anti-␥- transducin antibody5 (dilution 1:1000, PAB-00801G; CytoSignal, Irvine, CA) as described earlier, with the exception that incubations with the primary and secondary antibodies were performed at 4°C and lasted up to 3 days.

Quantitative Investigations on Flatmounted Retinas Representative samples were imaged with a laser confocal microscope (LSM 510 Axiovert 100M; Carl Zeiss Meditec, Inc.) or a fluorescence microscope (DM6000 Leica; Deerfield, IL) equipped with a computer controlled motorized stage. Raw images were then processed with the use of several computer programs (ImageJ ver. 1.40g; developed by Wayne Rasband, National Institutes of Health, Bethesda, MD, and available at http://rsb.info.nih.gov/ij/, or Image-Pro ver. 4.0; Media FIGURE 2. Analysis of the cone photoreceptor populations from flat- Cybernetics, Silver Spring, MD, for quantitative measurements, and mounted retinas (same Nile grass rat specimen). (A) M/L-cones; (B) with Photoshop 6.0, Adobe, San Jose, CA, for illustrations). S-cones. Manual counts were performed at ϫ40 magnification. The The density of M/L- and S-opsin expressing cones was determined rectangular regions are of the size matching the counting frame of 2 from manual counts applied to raw images (area ϭ 0.092 mm ) taken 0.092 mm2. White rectangles: areas of higher cone density (Ͼ36,000/ with a 40ϫ oil immersion objective in the center (from 0 to 1250 ␮m mm2 for the M/L-cones and Ͼ3,600/mm2 for the S-cones, i.e., respec- from the optic nerve head [ONH]), in the mid periphery (from 1250 to tively, 28% and 68% above the average density in the rest of the retina). 2500 ␮m away from the ONH) and in the far periphery (Ͼ2500 ␮m Cone densities at specific retinal locations are indicated. (C) Isodensity from the ONH) of each retinal quadrant of the flatmounted retinas curves for the M/L-cone population (as determined from manual (Figs. 2A, 2B). Every labeled cone OS in these frames, which did not counts over a total of 76 frames). (D) Isodensity curves (modeled from 101 fields counted in ImageJ) optimally illustrate the higher S-cone touch the borders, was scored and tagged at its base for additional 15,16 density in the temporodorsal sector of an A. niloticus retina. A similar analysis (nearest-neighbor distance and Voronoi domain ) by using map (modeled from 44 fields counted in ImageJ) was obtained with the the appropriate ImageJ plug-ins. retina double-labeled for M/L- and S-cones. A complete density map of the S-cone population (Fig. 2D) was further achieved in a computer-assisted manner by using the Analyze Particles tool of ImageJ software. Threshold and object size values onds, annealing at 60°C for 30 seconds, extension at 72°C for 30 were adjusted for the S-cone population in any previous frames that seconds for 30 cycles; and final extension at 72°C for 10 minutes The could be detected accurately, and the process was applied to a mask resulting 275-bp product was visualized on a 2% TAE gel, the band was containing 101 nonoverlapping fields of interest (squares 350 ϫ excised, and the DNA was extracted with a gel extraction kit (Qiaex II, 350-␮m wide; area, 0.1225 mm2; spacing, 200 ␮m) superimposed on a cat. No. 20021; Qiagen Sciences). DNA was eluted in ultrapure water digital photographic montage of the flatmounted retina obtained by and sequenced in the forward and reverse directions using the primers the automated confocal microscope system (single focal plane; 10ϫ just shown. Sequencing was performed with genetic analyzer (3130xl; objective). Counting variability between manual detection and com- Applied Biosystems, Inc. [ABI], Foster City, CA). Amino acids at posi- puter-aided processing, as assessed a posteriori in an additional 10 tions corresponding to 46 through 114 in human S-opsin (41 through randomly selected fields of interest, was no more than 5%, which 109 in rat) were examined. confirms the reliability of the quantitative method used.17 Counts were not corrected for possible tissue shrinkage. RESULTS Estimates of cone populations were calculated for an average retinal area of 43 mm2 (mean value from flatmounts: 42.85 Ϯ 2.5 mm2; Cone Types in the Nile Grass Rat Retina Statview software; SAS Institute Inc, Cary, NC). Significance was set at In cryosections of Nile grass rat retinas, antibodies AB5405 P ϭ 0.05. All values are given as the mean Ϯ SD. (against M/L-opsin) and AB5407 (against S-opsin) stain outer segments (OS) from cone photoreceptors (negative to the Partial Sequencing of the S-opsin Gene anti-rhodopsin monoclonal antibody Rho4D2; data not Genomic DNA was prepared from ear notch biopsies of 3 Nile grass shown). The double labeling procedure shows further that: (1) rats (all at 3 weeks of age) and one mouse (for control) (DNeasy Blood OS stained with AB5405 do not label with sc-14363 (Figs. 1B, and Tissue Kit; cat. no. 69504; Qiagen Sciences, Germantown, MD). 1D); and (2) OS stained with AB5407 are also labeled with PCR was performed by using the following primers designed within sc-14363. Complete colocalization of the two labels demon- exon 1 of rat Opn1sw (GenBank accession number NM_031015; strates that both antisera recognize the same S-opsin (Figs. http://www.ncbi.nlm.nih.gov/Genbank; provided in the public do- 1E–G). Antibodies AB5405 and sc14363 also faintly stain a few main by the National Center for Biotechnology Information, Bethesda, cones (Ϸ50 per section) from OS to synaptic pedicles (Figs. MD): 5Ј-CTGGGATGGGCCTCAGTAC-3Ј (forward) and 5Ј-AGGCCTC- 1C, 1D). The reason for such whole cell labeling (which is CAGAGCACAAAC-3Ј (reverse). PCR conditions were as follows: initial absent in mouse and rat) is unclear, but likely related to the denaturation at 94°C for 5 minutes; denaturation at 94°C for 30 sec- specificity of these antibodies in A. niloticus. Of note, the

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TABLE 1. Cone Densities in the Retina of A. niloticus

Far Periphery Mid Periphery Center Estimated Total (n/mm2) (n/mm2) (n/mm2) (n)§

Single labeling M-cones* 23,580 Ϯ 2,010 (n ϭ 14) 35,130 Ϯ 1,470 (n ϭ 8) 37,700 Ϯ 1,620 (n ϭ 8) Ϸ1,350,000 S-cones* 1,660 Ϯ 240 (n ϭ 10) 2,680 Ϯ 750 (n ϭ 6) 3,200 Ϯ 560 (n ϭ 5) 100,000 S-cones† 1,720 Ϯ 540 (n ϭ 41) 2,600 Ϯ 540 (n ϭ 31) 3,100 Ϯ 590 (n ϭ 20) 100,450 Total S- and M-cones* 25,240 37,810 40,900 Ϸ1,450,000 Ratio S-cones over total cones (%) 6.6 7.1 7.6 6.8 Total Cones (␥-transducin labeling)‡ 28,350 Ϯ 1,950 (n ϭ 8) 36,400 Ϯ 2,500 (n ϭ 4) 40,300 Ϯ 2,600 (n ϭ 8) Ϸ1,495,000 Double labeling M-cones* 22,820 Ϯ 3,280 (n ϭ 4) 35,810 Ϯ 2,660 (n ϭ 4) Ϸ1,260,000 S-cones*† 1,760 Ϯ 230 (n ϭ 17) 2,350 Ϯ 240 (n ϭ 15) 3,100 Ϯ 360 (n ϭ 12) Ϸ104,670 Ratio S-cones over total cones (%) 7.1 8.0 7.7

(n) indicates the number of sampling frames examined per retinal locations. * Manual procedure. † Computer-aided procedure. ‡ Recalculated from Gaillard et al., Figure 1D.5 § For a 43-mm2 retina.

commonly used anti-S-opsin antibody JH455 (mapped at the test). Therefore, S-cones distributed preferentially along the C-terminal of the human S-opsin; Jeremy Nathans; Johns Hop- temporonasal compared with the dorsoventral axis (P ϭ 0.009, kins University School of Medicine, Baltimore, MD) also stains one-tailed t-test). Finally, S-cones were denser (3760 Ϯ 345/ S-cones entirely in several species.13,18–20 The cell bodies of mm2; n ϭ 12) in a restricted sector of the temporal retina cones are located in the two first rows (outer side) of the outer superior to the ONH (Fig. 2D), than elsewhere in the retina nuclear layer and have comparable sizes (average vertical di- (Ϸ2160 Ϯ 625/mm2; n ϭ 89; P ϭ 0.0001, one-tailed t-test). In ameter: 9.0 Ϯ 1.2 ␮m for the S-cones, n ϭ 76; 8.4 Ϯ 0.8 ␮m for this sector, S-cones represented Ϸ10% of the total cone pop- the M/L-cones, n ϭ 41). M/L- and S-opsin expressing OS have ulation. very different distributions (approximate M/L:S ratio in Fig. 1B is 13–14:1), which suggests that they reflect two distinct cone Estimated Cone Densities in the populations. Close inspection of two flatmounted retinas, pro- Double-Labeled Retina cessed with a mixture of anti-M/L- and anti-S-opsin antibodies, indeed confirmed that the retina of A. niloticus possesses no Taking advantage of the fact that the Nile rat retina has no dual-pigment cones. This is illustrated in samples taken at the dual-pigment cone, individual M/L- and S-cone distributions naso-dorsal periphery (Figs. 1H, 1I) and in the ventral quadrant were further assessed in one of the double-labeled flatmounted which contains most of the dual-pigment–expressing cones in retinas. Counts in this retina (Table 1) gave an average packing Ϯ 2 Ϸ the mouse (Figs. 1J, 1K). density of 29,320 7,400/mm (leading to a total of 1.260 million), in the M/L-cones, and an average packing density of Cone Densities in the Nile Grass Rat Retina 2,430 Ϯ 890/mm2 (leading to a total of 104,500) in the S-cones. The S-cones were also more numerous along the temporonasal M/L-cones. M/L-cones distribute across the retina of A. axis, peaking Ϸ2 mm temporally and Ϸ1 mm superior to the niloticus. Their density increased smoothly (1.6-fold) but sig- ONH (3910 Ϯ 940/mm2; n ϭ 8 compared with 2130 Ϯ 450/ Ͻ nificantly (P 0.001, Mann-Whitney U test), from the far mm2; n ϭ 36 in the rest of the retina). periphery to the ONH (Table 1). Apart from the central retina, high cone density was notable in the dorsal part of the tempo- Cone Mosaics in the Nile Grass Rat Retina ral quadrant (white rectangles, Fig. 2A; and darkest area, Fig. 2C). Variations in M/L-cone densities between quadrants and Collectively, counts of M/L- and S-cones yield Ϸ1.350 to 1.450 between the main retinal axis never reached statistical signifi- million total cones in A. niloticus, which is in accord with the cance. Given an average packing density of 31,000 Ϯ 5,700/ Ϸ1.495 million obtained previously5 after retinas were pro- mm2 (n ϭ 76), the total number of M/L-cones in A. niloticus cessed with an anti-␥-transducin antibody as a general cone retina was estimated at Ϸ1.350 million. marker.21 Of the total cone population, Ϸ92% to 93% express S-cones. As was the case for the M/L-cones, S-cones popu- M/L-opsin and Ϸ7% to 8% express S-opsin (Table 1). In addition lated the entire retina and displayed an approximately twofold to these measurements, we examined the spatial organization increase in cell density from periphery (1660 Ϯ 240/mm2)to of the respective M/L- and S-cone populations by computing center (3200 Ϯ 560 /mm2), as deduced in a manual counting for each cone the distance with its nearest neighbor (NND) and approach (Fig. 2B, Table 1). The collected values are consid- its Voronoi domain (VD; defining points in the plane that were erably lower (12–14-fold) than those obtained for the M/L- closer to that cone than to any other cone in the mosaic). VDs cones at similar retinal locations (compare Fig. 2A with 2B). intersecting the sampling frame and NNDs of cells closer to the Given an average packing density of 2320 Ϯ 820/mm2 (n ϭ frame borders than to any cells in the sample were discarded 21), the total number of S-cones in the A. niloticus retina was from the analysis. estimated at Ϸ100,000. S-cones. The regularity of S-cone distribution in the retina The computer-assisted counting procedure allowed a more of A. niloticus was examined in 15 of the sampling frames detailed analysis of the S-cone distribution in this retina. Signif- initially used for pilot density measurements (Fig. 2B), one of icant differences in S-cone numbers were observed between which was located within the temporodorsal cone-rich sector. retinal quadrants (P ϭ 0.0001, ANOVA, F ϭ 7.55, df ϭ 3). Depending on their location, these sampling frames (0.092 S-cones appeared more numerous in the temporal quadrant mm2) contain between 134 and 368 S-cones. On average, the than in any other quadrant (P Յ 0.003, Fisher PLSD post hoc NND was 11.21 ␮m Ϯ 1.75 (n ϭ 15), the regularity index (RI;

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FIGURE 3. Cone mosaics in the retina of A. niloticus.(A) Distributions of the NNDs for S-cones taken from fields T3 (periphery; temporal quadrant) and D6 (center; dorsal quadrant). Counting fields (0.092 mm2) contained respectively 179 and 329 S-cones. (B) Distributions of the NNDs for M/L-cones. Sampling areas (0.01 mm2) were taken from fields T2 (periphery; temporal quadrant) and D7 (center; dorsal quadrant) and contained, respectively, 244 and 397 M/L-cones. The Bell-shaped curve in each histogram corresponds to the Gaussian fit. (C) Two examples of Voronoi domains for S-cones from fields T3 and D6. Field positions are given in Figure 2A for the M/L-cones and Figure 2B for the S-cones.

ratio of the mean NND to the SD) was 1.94 Ϯ 0.14, and the dexes were equally high across the retina. Together these NND index (Rn; ratio of observed NND to the mean random findings indicate that the M/L-cone mosaic is more regular than distance) was 1.04 Ϯ 0.046. The NND in a given sampling the S-cone mosaic is. frame appeared to be closely related to the cone density in that Total Cone Mosaic. The total cone mosaic was ascertained frame (Figs. 3A, 3B); and the regularity indexes tended to be from two retinas processed with the anti-␥-transducin anti- lower (P ϭ 0.042, one-tailed U test) in the periphery than in body, as previously described.5 Results were essentially similar the center of the retina. Finally, distributions of the NNDs were to those obtained for the M/L-cones (Table 2). similar in each frame (Fig. 3A), and all deviated significantly from a normal distribution (P Ͻ 0.01, ␹2 test of normality). As UV Sensitivity of S-cones suggested with the NNDs, Voronoi domains were larger (P ϭ According to genetic analysis, the presence of Phe86 is exclu- 0.01, one-tailed U test) in the retinal periphery than in the sive to all UV-sensitive S-opsins, whereas this site is occupied center (Fig. 3C). Corresponding RIs (ratio of the mean Voronoi by a different amino acid in the blue/violet-sensitive S pig- area to the SD) were low, and did not vary with eccentricity ments.22–24 Sequencing the first exon of the S-opsin gene in (Table 2). Investigations in the additional, double-labeled retina three different specimens of A. niloticus returned the follow- (Figs. 1J, 1K) provided similar results: both NND and VD ing translated amino-acid sequence: 46-FVFFVGTPLNATVL regularity indexes were low (respectively, 2.20 Ϯ 0.10, n ϭ 8; VATLHYKKLRQPLNYILVNVSLGGFLFCIFSVFTVFIASCHGYFLF- and 2.02 Ϯ 0.3, n ϭ 8) and remained stable with eccentricity. GRHVCALEA-114. This sequence contains Phe46, Phe49, Together these findings indicate that the S-cone mosaic of A. Thr52, Phe86, Thr93, and Ala114, indicating that the S-opsin in niloticus is not regular. A. niloticus is likely sensitive to UV by comparison with known M/L-cones. The M/L-cone mosaic was studied on the retina mammalian sequences (Table 3). that was initially used for density measurements (Fig. 2A). On average, NND and VD values were found to be respectively 3 and 14 to 17 times smaller than those observed in the S-cone DISCUSSION population (Table 2). Regardless of the retinal position, the distributions of NND and VD values (Fig 3B) were symmetric Previous immunohistochemical investigation with an antibody around the mean (i.e., fitting a Gaussian distribution; P Ͼ 0.1 in recognizing the cone-specific G␥8 transducin subunit showed all cases, ␹2 test of normality). Corresponding regularity in- that cone photoreceptors (Ϸ30%–35% of the total photorecep-

TABLE 2. Cone Mosaics

Fields Cells in Location (n) Fields (n) NND (Mean ␮m) RI VD (Mean ␮m2) RI

S-cones Center 5 250–368 9.15 Ϯ 0.67 2.03 Ϯ 0.10 287.00 Ϯ 59.30 1.82 Ϯ 0.08 Periphery 8 134–208 12.41 Ϯ 0.9 1.84 Ϯ 0.14 528.65 Ϯ 62.20 1.82 Ϯ 0.18 M/L-cones Center 5 330–392 3.54 Ϯ 0.20 4.67 Ϯ 0.33 20.50 Ϯ 1.90 3.84 Ϯ 0.42 Periphery 7 224–280 4.15 Ϯ 0.25 4.02 Ϯ 0.18 31.00 Ϯ 4.80 3.58 Ϯ 0.28 All cones Center 5 350–395 3.55 Ϯ 0.25 4.25 Ϯ 0.18 21.00 Ϯ 2.18 3.88 Ϯ 0.20 Periphery 8 245–320 4.12 Ϯ 0.22 4.18 Ϯ 0.30 28.40 Ϯ 2.85 3.57 Ϯ 0.28

Average NND, VD, and RI found for counting ﬁelds located in the far periphery (Ͻ350 ␮m from the ora serrata) and in the center (within 1000 ␮m from the ONH) of A. niloticus retina. Field areas were 0.01 mm2 except for the S-cones (0.092 mm2).

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TABLE 3. Amino Acids at Key Positions in the S-opsin Pigment of Some Mammalian Species

Amino Acid Positions

␭ Species max 46 49 52 86 93 114 118

(1) Homo sapiens 424 Thr Leu Phe Leu Pro Gly Thr (2) Bos taurus 435 Phe Phe Thr Tyr Ile Ala Cys (3) Cavia porcellus 430 Ile Cys Thr Val Ala Gly Ser (4) Sciurus carolinensis 440 Phe Phe Thr Tyr Val Ala Ser (5) Talpa europaea UV — Phe Thr Phe Thr Ala Ser (6) Mus musculus 359 Phe Phe Thr Phe Thr Ala Ser (7) Rattus norvegicus 358 Phe Phe Thr Phe Thr Ala Ser (8) Arvicanthis niloticus — Phe Phe Thr Phe Thr Ala —

GenBank Accession numbers: (1) NM_001708; (2) NM_174567; (3) AY_552608; (4) DQ_302163; (5) EU_130563; (6) NM_007538; (7) NM_031015.

tor population) in the diurnal muridae A. niloticus are distrib- gions is not well understood.26,43 In A. niloticus, the S-cone- uted evenly across the retina and that their packing density rich region approximately matches the area where M/L-cones (Ϸ36,000/mm2 on average) increases only 1.8-fold from the far are the densest. Both cone types might thus form a broad, periphery to center.5 The present study provides new insight horizontally oriented, centrotemporally elongated elliptical into the topographic arrangement of cone photoreceptors in streak. Such retinal specialization may provide a better scan- this rodent. ning of the natural environment in the frontal visual field. Cone photoreceptors in A. niloticus segregate in two com- Further experiments are needed to examine whether there is plementary populations: a major one expressing the M/L-opsin also a corresponding higher density of rods and ganglion cells pigment and a minor one expressing the S-opsin pigment. in this retinal sector that might subserve the higher visual Their respective proportions (13–14:1) are conserved in most acuity observed in A. niloticus compared with mice and rats.5 mammalian species with rod-dominated retinas, including hu- Within each nuclear layer of the retina, neurons of a given mans (outside the fovea25,26). Of note, the M/L:S ratio in A. type are commonly spaced in an orderly manner, forming niloticus is very similar to that in the rat retina27; further proof planar arrays known as mosaics15 that are established during that this ratio is independent of the proportion of cone over development through local interactions between immediate total photoreceptor population. Dual opsin expression in a neighboring cells of the same type (for a review, see Ref. 49). single cone was not detected by immunohistochemistry. Opsin Topographic analysis of mosaic patterns may be useful to coexpression in rodents examined so far is primarily a species- pathologists: A recent study in humans50 showed for instance specific feature: it has been observed in pocket gopher, sibe- that progressive tritan color vision deficiency is accompanied rian hamster, common house mouse (Mus) and guinea pig; it is by a progressive disorganization of the S-cone mosaic, which absent in the wood mouse (Apodemus sylvaticus), squirrel, ultimately disrupts the whole cone mosaic. Knowledge of the and degu (Table 4 and related references). A recent report normal pattern of retinal cell organization in A. niloticus is indicates that opsin coexpression is also absent in agoutis.40 In therefore essential for comparative and developmental studies some instances (rat and gerbil), however, pigment coexpres- as well as for experimental manipulations.51 sion is present postnatally, but is downregulated by 1 month of Absence of cell body–specific labels for each cone subtype age.31 Further studies are needed to explore whether this is led us to rely on markers expressed in the OS to map cone also the case in the Nile grass rat. mosaics in A. niloticus. This approach revealed contrasting S-cone topography is extremely variable among mammalian spatial arrangements between cone subtypes: the S-cone mo- species, ranging from a complete absence to an even distribu- saic is distinctly irregular (with nearest-neighbor RI and Rn tion across the retina.26,43 In rodents, notable interspecies indexes just above the theoretical upper limit for random- differences have been reported in the mouse, hamster, and ness52), whereas that of the M/L-cones is more ordered. Among squirrel.25,28,31,44 S-cones in A. niloticus are distributed rodents, such dissimilar patterning has been described recently throughout the retina as in Rattus and Apodemus (Table 4). in the diurnal agouti, Dasyprocta aguti.40 This pattern seems Their density (Ϸ2320 cells/mm2 on average) increases (1.8- to be the rule in rod-dominated mammalian retinas.43 The fold) from periphery to center, and is maximum in the tem- present results further suggest that the S-cone mosaic (with porodorsal quadrant. A pioneer investigation3 in another Arvi- significantly large NND and VD values) is not integrated into canthis species, A. ansorgei, showed a similar distribution but the dominant cone mosaic in A. niloticus and does not inter- with a higher centripetal increase (threefold), a significantly fere with the regularity of the total cone mosaic (which fur- lower packing density (Ϸ1480 cells/mm2), and no topographic thermore remains stable across the retina as observed in the preference. The minor discrepancies in S-cone densities may horseshoe bat53). Such interference43 occurs probably only be related either to sampling bias or to species differences. between two largely regular and independent mosaics, as is the There is evidence, for instance, that densities of retinal cells, case in primates. S-cones in the Nile rat retina may be too including photoreceptors, differ tremendously between mice sparse to disturb the general cone pattern to any large extent. species or strains.25,31,45–47 The presence of UV photopigments in retinal cones is There is an S-cone-rich region in the temporodorsal quad- usually considered to be the ancestral template from which rant of the Nile grass rat retina. For a still unknown reason, the emerged the classic mammalian S-type pigments after multiple location of this S-cone-rich region is closer to that found in mutations.24,54 According to an extensive literature review, all ferret, lynx, and cheetah43,48 than that in most other rodents UV-sensitive S pigments in mammals have Phe86.22–24 Substi- ␭ where the S-cone rich region is localized in the ventral retina tution of Tyr86Phe indeed is sufficient to shift the max of the (Table 4). It should be noted, however, that the retinal location bovine blue/violet-sensitive opsin from 430 to 360 nm. Of the of such S-cone-rich regions is extremely variable between rodents studied so far, only diurnal squirrels, prairie dogs mammalian species and that the visual function of these re- (Sciurus carolinensis, Spermophilus beecheyi, and Cynomys

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) ludovicianus; all from the Sciuridae family), and crepuscular 2 guinea pigs (Cavia porcellus; Cavidae) have been found to possess blue/violet-sensitive S-cones.44,55,56 In general, muridae have UV-sensitive S-cones (or no S-cones at all; Tables 3, 4).

1000/mm Although diurnal, Arvicanthis retains this characteristic, like ؋ Peak Density ( its nocturnal relatives Mus, Rattus, and Apodemus, the S-opsin of A. niloticus has the exclusive Phe86 suggesting that S-cones are UV-sensitive. To determine whether these cones respond efﬁciently to UV light, the spectral transmission properties of the the lens and cornea must be assessed. Of note, however, in all the rodents studied so far having UV-sensitive S-cones, the eye optics transmit in the near-UV range.11,13,37,38,57 The function of UV vision in diurnal rodents with UV- sensitive S-cones (gerbil and degu) is a matter of debate. Con- tribution to color discrimination, improvement of visual perception at dawn and dusk (when UV light levels are 37 ata not available; N/A, not applicable. overrepresented in the solar spectrum ), as well as involve-

S-cones ment in social behavior (detection of urine and fur reﬂectance) have all been postulated.38,58,59 These postulates apply well to the Nile grass rat which has a typical dual-cone retina, is mostly Retinal diurnal with peaks of activity at dawn and dusk, and spends Distribution Peak Location long daylight periods interacting with its juvenile offspring outside densely interconnected burrows.60 Whether and how A. niloticus relies on UV-sensitive cones in its natural environment nevertheless remains an open question.

max In conclusion, the Nile grass rat A. niloticus has a dual cone retina with a majority of M/L-cones and a minority of S-cones. The S-cones are UV-sensitive, distribute throughout the retinal surface, and peak in the temporodorsal quadrant. Omitting the latter, these characteristics compare well with those found in Mongolian gerbil57 and Chilean degu,38,59 two distantly related nL diurnal rodents, but with similar lifestyles. Further investiga- Approx. tions will examine how S-cones in A. niloticus are integrated into the retinal circuitry and whether these photoreceptors may participate in some form of color perception.

Acknowledgments Ratio M/L:S The authors thank Laura Smale for generously providing breeders from her Nile grass rat colony at Michigan State University (supported by a grant from the National Institute for Mental Health, R01 MH53433) to start the colony that presently exists at the University of Alberta. The 0.5 N/A Absent N/A N/A N/A N/A %

Ͻ authors also thank Paul Freund for performing the Western Blot anal- Cones yses, Marc-Andre Filion for support in density measurements, William Ted Allison for his advice on the characterization of the S-opsin gene, Compared with Other Rodents Sue Kenney, and The Applied Genomics Centre (TAGC), Department of Medical Genetics, and The Division of Gastroenterology (CEGIIR),

SSNNNN 26.4N 10N 1–2D 1–2 1:2 (DPC)D 3C ? 1:1 (DPC)D 5–14:1 1D 650,000 12–14 N/A 2:1 (DPC) 220,000 32 25–18:1 8–17 UV, 17–19 367 ? 14:1 20:1 86 UV, Absent 95,000 360 7–8,000 (DPC)University 13:1 13:1 Even UV, UV, 365 359 130,000 20,000 N/A 14:1 Even UV, ? of 221.000 260,000Alberta, UV, ? 360 UV, 359 DVG DVG 530,000 UV, 32–37 362 N/A for Even N/Atechnical BV, 8–12 Even 430 Even BV, 436 Centroventral Even 22–24support 6.5 Even Ventronasal DVG N/A Even 0.5 in 0.85 ? Mid-ventral sequencing. 5.4–7.0 Ventral half Central, 1.5 ONH ? Central, ONH N/A 20 Centroventral ? Temporocentral Ventral Dorsonasal half rim A. niloticus References 1. Lyubarsky AL, Lem J, Chen J, Falsini B, Iannaccone A, Pugh EN Jr.

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) 2. Nikonov SS, Daniele LL, Zhu X, Craft CM, Swaroop A, Pugh EN Jr. Photoreceptors of NrlϪ/Ϫ mice coexpress functional S- and M-cone 38 10,31–34 ) )

39 opsins having distinct inactivation mechanisms. J Gen Physiol. )

40 2007;125:287–304. )

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