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Retinal Cell Biology  |   July 2013
Impairment of Intrinsically Photosensitive Retinal Ganglion Cells Associated With Late Stages of Retinal Degeneration
Author Affiliations & Notes
  • Gema Esquiva
    Department of Physiology, Genetics and Microbiology, University of Alicante, Alicante, Spain
  • Pedro Lax
    Department of Physiology, Genetics and Microbiology, University of Alicante, Alicante, Spain
  • Nicolás Cuenca
    Department of Physiology, Genetics and Microbiology, University of Alicante, Alicante, Spain
    Institute Ramón Margalef, University of Alicante, Alicante, Spain
  • Correspondence: Nicolás Cuenca, Department of Physiology, Genetics and Microbiology, University of Alicante, San Vicente University Campus, E-03080 Alicante, Spain; cuenca@ua.es
Investigative Ophthalmology & Visual Science July 2013, Vol.54, 4605-4618. doi:10.1167/iovs.13-12120
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      Gema Esquiva, Pedro Lax, Nicolás Cuenca; Impairment of Intrinsically Photosensitive Retinal Ganglion Cells Associated With Late Stages of Retinal Degeneration. Invest. Ophthalmol. Vis. Sci. 2013;54(7):4605-4618. doi: 10.1167/iovs.13-12120.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: To evaluate quantitative and qualitative age-related changes in intrinsically photosensitive melanopsin-containing retinal ganglion cells (ipRGCs) in transgenic P23H rats, an animal model of autosomal dominant retinitis pigmentosa (RP) was examined.

Methods.: ipRGC density, morphology, and integrity were characterized by immunohistochemistry in retinas extracted from P23H and Sprague–Dawley (SD) rats aged 4, 12, and 18 months. Differences between SD and P23H rats throughout the experimental stages, as well as the interactions among them, were morphologically evaluated.

Results.: In rat retinas, we have identified ipRGCs with dendrites stratifying in either the outer margin (M1) or inner side (M2) of the inner plexiform layer, and in both the outer and inner plexuses (M3). A small group of M1 cells had their somas located in the inner nuclear layer (M1d). In SD rats, ipRGCs showed no significant changes associated with age, in terms of either mean cell density or the morphologic parameters analyzed. However, the mean density of ipRGCs in P23H rats fell by approximately 67% between 4 and 18 months of age. Moreover, ipRGCs in these animals showed a progressive age-dependent decrease in the dendritic area, the number of branch points and terminal neurite tips per cell, and the Sholl area.

Conclusions.: In the P23H rat model of retinitis pigmentosa, density, wholeness, and dendritic arborization of melanopsin-containing ganglion cells decrease in advanced stages of the degenerative disease.

Introduction
In the mammalian retina, a population of ganglion cells has been found to express the photopigment melanopsin. 1,2 These melanopsin-containing ganglion cells are widely distributed throughout the ganglion cell layer of the retina, with a few melanopsin-positive cells located in the inner nuclear layer. 1,3,4 The cells form an extensive network of opsin-containing dendrites, 2,5 with five morphologic cell subtypes described in mice (M1 to M5) based on their dendritic stratifications in the inner plexiform layer (IPL). 610 Melanopsin-expressing cells are intrinsically photoreceptive (i.e., they respond to light independently from rod and cone photoreceptors) 5 and this intrinsic photosensitivity is lost in melanopsin-knockout animals. 11 Despite the fact that intrinsically photosensitive retinal ganglion cells (ipRGCs) do not require synaptic inputs to generate light-induced signals, they receive these inputs from bipolar and amacrine cells, 5,12 suggesting that rod and/or cone signals may be capable of modifying the animal's intrinsic response to light. 
Photosensitive retinal ganglion cells are involved in circadian photoentrainment and the pupillary light reflex 5,1317 through projections to the suprachiasmatic nucleus (SCN) and other diencephalic and midbrain regions. 3,4,1820 Mice with degenerated rods and cones are able to shift their circadian rhythms according to the external light–dark cycle, 21,22 although melanopsin is not essential for the circadian clock to receive photic inputs. Melanopsin-null mice (Opn4 −/−) still retain nonvisual photoreception capabilities, although they display severely attenuated phase resetting in response to brief pulses of monochromatic light. 15,16 Mice with both outer retinal degeneration and deficiency in melanopsin exhibit a complete loss of both circadian oscillator photoentrainment and pupillary light responses. 17  
This may be relevant for the study of retinitis pigmentosa (RP), a heterogeneous group of inherited neurodegenerative retinal disorders characterized by a progressive peripheral vision loss and night vision difficulties, subsequently leading to central vision impairment. More than 100 different mutations in the rhodopsin-encoding gene (RHO) are associated with RP, together accounting for 30% to 40% of autosomal dominant cases. The P23H mutation in this gene is the most prevalent cause of RP, 23 which alone accounts for approximately 12% of autosomal dominant cases of the disease in the United States. 24 P23H transgenic albino rats suffer from progressive rod degeneration, initially associated with normal cone function, which is consistent with the clinical findings in P23H patients. 2527 The loss of photoreceptors is accompanied by degenerative changes in the inner retina, 2630 and a substantial degeneration of retinal ganglion cells (RGCs). 3133 Thus, it has been demonstrated that homozygous P23H rats have a lower number of RGCs than their homologous SD rats, and retinal degeneration causes RGC axonal damage and RGC death. 33 In a previous study, we demonstrated that retinal degeneration in P23H rats positively correlates with the occurrence of circadian dysfunctions in these animals, 34 and it has been shown that pupil responses to red and blue light stimuli are significantly reduced in patients with RP. 35  
The aim of this study was to investigate quantitative and morphologic changes in melanopsin-containing ganglion cells throughout progressive retinal degeneration in the P23H rat autosomal dominant model of RP. 
Materials and Methods
Animals
Homozygous P23H line 3 rats aged 4, 12, and 18 months, obtained from Matthew LaVail (UCSF School of Medicine; http://www.ucsfeye.net/mlavailRDratmodels.shtml), were used as a model of RP. Age-matched Sprague–Dawley (SD) rats were used as wild-type control animals. Seven retinas were used for each experimental group. All animals were bred in a colony at the University of Alicante and maintained under controlled humidity (55%–60%), temperature (23 ± 1°C), and photoperiod (LD 12:12) conditions. Light was provided by two fluorescents lamps, with an intensity of 350 to 400 lux at cage level. Dry food and water were made available without restriction. All animals were handled in accordance with current regulations for the use of laboratory animals (National Institutes of Health, ARVO, and European Directive 2010/63/UE), to minimize animal suffering and limit the numbers used for the experiments. 
Antibodies
A primary polyclonal antibody was raised in rabbits against the first 19 amino acids of the NH2-terminal sequence of the rat melanopsin (PA1‐780; Affinity BioReagents, Inc., Golden, CO). The specificity of this antibody has been previously confirmed by Western blot experiments with both melanopsin expressed in transfected cells 3,36 and melanopsin extracted from rat retinas. 37,38 Secondary antibodies were Alexa Fluor 488 (green)–conjugated donkey anti-rabbit IgG (A21206; Molecular Probes, Eugene, OR), for immunofluorescence, or biotinylated goat anti-rabbit IgG (111‐065‐144; Jackson ImmunoResearch Laboratories, West Grove, PA), for immunoperoxidase labeling. 
Retinal Histology
Retinal Immunofluorescence.
Animals were euthanized in the morning, between 10:00 AM and 12:00 noon, by being administered a lethal dose of pentobarbital. After marking the dorsal margin of the limbus with a stitch, eyes were enucleated and fixed in 4% (w/v) paraformaldehyde for 1 hour at room temperature (RT). After being washed in PBS, eyes were cryoprotected in 15%, 20%, and 30% sucrose. The cornea, lens, and vitreous body were removed, and the eyecups were processed for vertical sections or whole mounts. For cryostat sections, eyecups were frozen in optimal cutting temperature compound with liquid N2. Sections (14 μm thick) were then obtained using a commercial cryostat (Leica CM 1900; Leica Microsystems, Wetzlar, Germany), mounted on slides (Superfrost Plus; Gerhard Menzel Glasbearbeitungswerk GmbH & Co. KG, Braunschweig, Germany), and air-dried. Sections were incubated in normal donkey serum diluted 1:100 in PBS containing 0.5% Triton X-100 for 1 hour, and then subjected to single immunostaining overnight at RT, using rabbit antimelanopsin antibody 1:2000 in PBS containing 0.5% Triton X-100. After several washes in PBS, Alexa Fluor 488 (green)–conjugated donkey anti-rabbit IgG secondary antibody was applied at a 1:100 dilution in PBS containing 0.5% Triton X-100 for 1 hour. The sections were finally washed in PBS, mounted in antifadent mounting medium (Citifluor; Citifluor Ltd., London, UK), and coverslipped for laser-scanning confocal microscopy viewing (Leica TCS SP2 system; Leica Microsystems). Immunohistochemical controls were performed by omission of either the primary or secondary antibodies. Final images from control and experimental subjects were processed in parallel, using photo-editing software (Adobe Photoshop 10.0; Adobe Systems, Inc., San Jose, CA). 
Immunoperoxidase Labeling.
After fixation, whole-mount retinas were dissected out from the choroid and put through a freeze–thaw procedure by dipping them in liquid nitrogen–cooled isopentane for a few seconds. After thawing in 30% sucrose, retinas were washed in PBS. Endogenous peroxidase activity was suppressed by immersion in 1% hydrogen peroxide (H1009; Sigma, St. Louis, MO) in PBS (10 minutes, RT). To break aldehyde bonds and enhance the permeability of the tissue, the retinas were incubated first in 2.28% sodium metaperiodate (S1878; Sigma) in PBS (5 minutes, RT) and then in 0.02% sodium borohydride (163314; Panreac, Barcelona, Spain) in PBS (5 minutes, RT). After a blocking step (10% normal goat serum in PBS plus 0.5% Triton X-100 for 1 hour), flat-mount retinas were incubated for 2 days at 4°C in a 1:2000 dilution of the rabbit antimelanopsin primary antibody (PA1‐780), washed four times in PBS (5 minutes, RT), and then incubated for 1 day at 4°C in biotinylated goat anti-rabbit secondary IgG antibody at 1:100 dilution in PBS plus 0.5% Triton X-100. The retinas were washed before being transferred to an ABC kit solution (PK-6100, Vectastain Elite ABC Kit; Vector Laboratories Ltd., Cambridgeshire, UK) in PBS containing 0.5% Triton X-100 for 1 day. Finally, the retinas were washed in PBS and preincubated under agitation in the dark with 3,3′-diaminobenzidine tetrahydrochloride (DAB, D5637; 0.5 mg/mL in PBS; Sigma) for 15 minutes and further incubated with fresh DAB solution with 0.01% H2O2 and 0.025% ammonium nickel (II) sulfate hexahydrate (A1827; Sigma). The DAB reaction was stopped by being washed with distilled water. 39,40 Whole retinas were flat mounted in mounting medium (Citifluor; Citifluor Ltd.) with the ganglion layer side up, and coverslipped for optical microscopy viewing on a high-resolution digital microscope (Leica DMR; Leica Microsystems). To determine the spatial distribution and total number of ipRGCs stained in each retina examined, every one of the cell bodies labeled with immunoperoxidase in each entire retina was manually traced using a camera lucida attached to the Leica DMR microscope (Leica Microsystems). The images created were subsequently digitalized using image-editing software (Photoshop 10.0; Adobe Systems, Inc.). 
Morphologic Analysis.
To reconstruct the soma and dendritic profiles of individual ipRGCs, we manually traced the outlines of the cell body and the minimal convex polygon enclosing the dendritic field of each labeled cell. The areas of each somatic and dendritic profiles were measured with the aid of ImageJ software (available at http://rsbweb.nih.gov/ij/index.html; developed by Wayne Rasband, National Institutes of Health, Bethesda, MD). Soma sizes were expressed as equivalent diameters (the diameter of a circle of equal area), calculated from measurements of the cross-sectional surface area of the cell body. 
To analyze ipRGC neurite morphology, we used the Bonfire program, developed in the Firestein laboratory at Rutgers University. 41 Using digitized neuritic arbors, this software allowed us to estimate the following parameters: number of branch points per cell, number of terminal neurite tips per cell, and Sholl analysis. 42 The Sholl analysis is performed conceptually by drawing concentric circles around the cell body at incrementally increasing radii and counting the number of times each circle crosses a neuritic segment. The Sholl profile is obtained by plotting the number of intersections versus the radial distance from the cell soma. The Sholl area (arbitrary units) represents the area under the Sholl profile. 
Statistical Analysis
Statistical analyses were performed using commercial software (SPSS 18.0 software; IBM, Armonk, NY). A MANOVA was used to evaluate differences between SD and P23H rats throughout the experimental stages (4, 12, and 18 months of age), as well as the interactions among them. When a 0.05 level of significance was found, post hoc pairwise comparisons using Bonferroni's test were performed. Normal distributions and homogeneity of variance were found for all analyzed categories. Values of P < 0.05 were considered statistically significant. Data were plotted as the average ± SEM. 
Results
Melanopsin-Containing Ganglion Cells in SD Rat Retinas
To examine melanopsin expression on the rat retina, vertical retinal sections were immunostained with primary polyclonal antibody against melanopsin. Figures 1A, 1B show the immunofluorescence of the melanopsin-positive retinal ganglion cells found in vertical sections of the rat retina. Melanopsin immunoreactivity was present at comparable intensity on the soma, axon, and dendrites of a subpopulation of RGCs. Immunofluorescent cell bodies appeared in the ganglion cell layer (GCL) and, to a lesser extent, within the innermost cell row of the inner nuclear layer (INL). Two plexuses of immunofluorescent dendrites were evident within the IPL: one occupying the outer margin (outer part of the OFF-sublamina, stratum S1) and the other occupying the inner side (inner part of the ON-sublamina, stratum S5). 
Figure 1
 
Melanopsin-positive ganglion cells on the rat retina. (A, B) Representative images of rat retinas vertical sections labeled with anti-melanopsin antibody. Note that melanopsin is present on the soma and neurites of the RGC subpopulation. Inmunolabeled dendrites stratified in strata S1 and S5 of the IPL. Scale bar: 20 μm. (CF) Examples of the ipRGC types found in whole-mount rat retinas from SD rats. (C) M1 cell, with their soma placed in the GCL and dentrites in the stratum S1 of the IPL. (D) M1 displaced cell (M1d), with their cell body placed in the INL and dendrites in the stratum S1 of the IPL. (E) M2 cell, with their cell body located in the GCL and dendrites in S5 of the IPL. (F) M3 cell, with their cell body placed in the GCL and dendrites in both S1 and S5 of the IPL. Each image is the projection of five to ten focal planes between the GCL and the stratum S1 of the IPL. Scale bar: 100 μm.
Figure 1
 
Melanopsin-positive ganglion cells on the rat retina. (A, B) Representative images of rat retinas vertical sections labeled with anti-melanopsin antibody. Note that melanopsin is present on the soma and neurites of the RGC subpopulation. Inmunolabeled dendrites stratified in strata S1 and S5 of the IPL. Scale bar: 20 μm. (CF) Examples of the ipRGC types found in whole-mount rat retinas from SD rats. (C) M1 cell, with their soma placed in the GCL and dentrites in the stratum S1 of the IPL. (D) M1 displaced cell (M1d), with their cell body placed in the INL and dendrites in the stratum S1 of the IPL. (E) M2 cell, with their cell body located in the GCL and dendrites in S5 of the IPL. (F) M3 cell, with their cell body placed in the GCL and dendrites in both S1 and S5 of the IPL. Each image is the projection of five to ten focal planes between the GCL and the stratum S1 of the IPL. Scale bar: 100 μm.
ipRGC Types in SD Rat Retinas
Figures 1C through 1F show pictures of the different types of melanopsin-containing ganglion cells, as found in SD rat retinas at 4 months of age. To analyze the morphology of ipRGCs in detail, the stratification patterns of individual cells were manually drawn and digitized from whole-mount rat retinas labeled with immunoperoxidase. Representative stratification patterns, as well as diagrams of the morphologic parameters analyzed, are represented in Figure 2. According to dendritic stratification, we have identified three subtypes of ipRGCs in rat retinas: M1 cells, whose dendrites stratified in the S1 stratum of the IPL (Figs. 1C, 2A); M2 cells, arborizing in the S5 stratum of the IPL (Figs. 1E, 2C); and bistratified M3 cells, with dendrites occupying both S1 and S5 strata of the IPL (Figs. 1F, 2D). M1, M2, and M3 cells had somas located in the GCL. The small group of ipRGCs whose somas were located in the INL exhibited morphologic similarities to M1, including stratification in S1, and therefore they have been considered as displaced M1 cells (M1d; Figs. 1D, 2B). Immunoreactivity of ipRGCs was weaker in M2 cells than in M1 and M3 cells. Although an in-depth calculation of the relative numbers of each cell type in rat retinas was not carried out, the number of M1 cells in all examined stainings was higher than that of M2 and M3 cells. As can be seen in the figures, somatic profiles were approximately circular, with no appreciable differences among cell types. At this age, all four cell types (M1, M1d, M2, and M3) showed similar soma diameter values in SD rats (Fig. 2E). The areas of the measured dendritic profiles were also similar among cell types (Fig. 2F). 
Figure 2
 
Dendritic arborization of melanopsin-containing ganglion cells on the rat retina. (AD) Examples of the soma-dendritic profiles of M1, M1d, M2, and M3 cells. Dendrites in S1 are drawn in blue, and those located in S5 are shown in red. Axons are drawn in black. Somas located in the GCL are drawn in red, whereas those located in the INL (M1d cells) are shown in blue. (E) Quantification of the mean soma diameter of M1, M1d, M2, and M3 cells. Seventy cells from seven retinas were examined for each cell type. (FI) Mean dendritic area (F), number of branch points (G), number of terminal neurite tips per cell (H), and Sholl area (I) of each melanopsin-positive cell type. Thirty cells from seven retinas were examined for each cell type. *P < 0.05, ***P < 0.001; ANOVA. Scale bar: 500 μm.
Figure 2
 
Dendritic arborization of melanopsin-containing ganglion cells on the rat retina. (AD) Examples of the soma-dendritic profiles of M1, M1d, M2, and M3 cells. Dendrites in S1 are drawn in blue, and those located in S5 are shown in red. Axons are drawn in black. Somas located in the GCL are drawn in red, whereas those located in the INL (M1d cells) are shown in blue. (E) Quantification of the mean soma diameter of M1, M1d, M2, and M3 cells. Seventy cells from seven retinas were examined for each cell type. (FI) Mean dendritic area (F), number of branch points (G), number of terminal neurite tips per cell (H), and Sholl area (I) of each melanopsin-positive cell type. Thirty cells from seven retinas were examined for each cell type. *P < 0.05, ***P < 0.001; ANOVA. Scale bar: 500 μm.
A Bonfire analysis was performed to investigate morphologic differences in the dendritic tree among the different cell types in SD rats. As seen in Figure 2, both M1 and M1d cells had higher values than M2 cells in terms of the number of branch points per cell (P < 0.001, in both cases) (Fig. 2G), number of terminal neurite tips per cell (P < 0.001, in both cases) (Fig. 2H), and Sholl area (P < 0.001 and P < 0.05, respectively) (Fig. 2I). The Sholl area in M2 cells was also significantly smaller than that in M3 cells (P < 0.05), indicating that M2 cells had a less complex dendritic arborization. No significant differences in the dendritic parameters analyzed were found among M1, M1d, and M3 cells. 
ipRGC Spatial Distribution and Number in SD Rat Retinas
To characterize the spatial distribution and number of melanopsin-containing ganglion cells on the rat retina, all individual cell bodies were manually traced from whole-mount retinas using a camera lucida attached to the Leica DMR (Leica Microsystems). As seen in Figure 3, ipRGCs were distributed throughout the entire retina, even though some increased density was observed in the upper-temporal part of the retina. No differences in the spatial distribution of ipRGCs were found between SD rats aged 4, 12, and 18 months (Figs. 3A–C, respectively). At 4 months of age, the mean number of melanopsin-containing ganglion cells measured in SD rats was 33.6 ± 1.6 cells/mm2 (2010 ± 112 cells/retina; Figs. 3A, 3G). This density value showed no significant variations when evaluated at 12 and 18 months of age (32.9 ± 3.6 cells/mm2 [2223 ± 194 cells/retina] and 39.5 ± 2.4 cells/mm2 [2505 ± 156 cells/retina], respectively; Figs. 3B, 3C, 3G). 
Figure 3
 
Distribution and number of ipRGCs in SD and P23H rat retinas. (AF) Representative drawings of whole-mount retinas from SD (AC) and P23H (DF) rats at 4, 12, and 18 months of age. Retinas were manually drawn with the aid of a camera lucida, showing the location of individual ipRGCs labeled with immunoperoxidase. The number of ipRGCs represented in the drawings is 2597 (A), 2089 (B), 2783 (C), 1714 (D), 1917 (E), and 491 (F). (G, H) Quantification of the mean ipRGC density (cells/mm2) (G) and the average area of the entire retinas (mm2) (H) in whole-mount retinas from SD and P23H rats at 4, 12, and 18 months of age. Seven retinas (with approximately 2000 cells per retina) were examined on each experimental group. *P < 0.05, ***P < 0.001; ANOVA. Scale bar: 50 μm.
Figure 3
 
Distribution and number of ipRGCs in SD and P23H rat retinas. (AF) Representative drawings of whole-mount retinas from SD (AC) and P23H (DF) rats at 4, 12, and 18 months of age. Retinas were manually drawn with the aid of a camera lucida, showing the location of individual ipRGCs labeled with immunoperoxidase. The number of ipRGCs represented in the drawings is 2597 (A), 2089 (B), 2783 (C), 1714 (D), 1917 (E), and 491 (F). (G, H) Quantification of the mean ipRGC density (cells/mm2) (G) and the average area of the entire retinas (mm2) (H) in whole-mount retinas from SD and P23H rats at 4, 12, and 18 months of age. Seven retinas (with approximately 2000 cells per retina) were examined on each experimental group. *P < 0.05, ***P < 0.001; ANOVA. Scale bar: 50 μm.
Progressive Decrease of ipRGCs in P23H Rats
In 4- and 12-month-old P23H rats, the spatial distribution of melanopsin-containing ganglion cells showed an arrangement similar to that observed in SD rat retinas (Figs. 3D, 3E). However, in 18-month-old P23H rats ipRGCs showed a uniform distribution throughout the retina (Fig. 3F). The mean density of ipRGCs found in P23H rat retinas at 4 months of age (32.6 ± 1.6 cells/mm2 [1700 ± 105 cells/retina]; Figs. 3D, 3G) was similar to that measured at the same age in SD rats (Figs. 3A, 3G), and it was not significantly different from that measured in P23H rats at 12 months of age (32.4 ± 2.7 cells/mm2 [1687 ± 132 cells/retina]; Figs. 3E, 3G). However, a significant decrease in the mean number of ipRGCs was found in 18-month-old P23H rats (10.6 ± 1.6 cells/mm2 [2505 ± 156 cells/retina]; Figs. 3F, 3G), as compared with both 4- and 12-month-old P23H rats and SD rats at any of the three ages analyzed (P < 0.001, n = 7, in all cases). The total area of the P23H rat retinas was smaller (approximately 23% less) than that of SD rats at all three ages tested (Fig. 3H). 
Progressive Degeneration of ipRGC Structure in P23H Rats
Figure 4 shows the reconstruction of the soma and complete dendritic field of any individual ipRGC from a representative region of the central retina located between the superior and nasal quadrants (Figs. 4A, 4C, 4E, 4G), as well as the minimal polygons fitted around the dendritic profile of each fully reconstructed ipRGC in the left panels (Figs. 4B, 4D, 4F, 4H). As can be seen in the figure, the dendritic fields of neighboring ipRGCs overlapped extensively in 4-month-old SD rats (Figs. 4A, 4B). A similar arrangement of dendritic fields was found in 4-month-old P23H rats (Figs. 4C, 4D). At this age, in both SD and P23H rats ipRGC dendrites formed a dense plexus in the S1 stratum of the IPL (Figs. 4A–D). In 12-month-old P23H rats the ipRGC plexus was less complex than that observed in 4-month-old animals, with some overlapping among the dendritic fields in many locations, but also with clear gaps in coverage (Figs. 4E, 4F). At 18 months of age, ipRGCs in P23H rats were few and scattered, with few overlapping and scarce contact between cells (Figs. 4G, 4H). No changes associated with age were found in the ipRGC plexus of SD rats (not shown). 
Figure 4
 
Progressive changes in the morphology and arborization of ipRGCs in P23H rats. (A, C, E, G) Representative drawings of the soma and complete dendritic field of ipRGCs from a region of the central retina (between the superior and nasal quadrants) of a 4-month-old SD rat (A) and P23H rats at 4 (C), 12 (E), and 18 (G) months of age. (B, D, F, H) Minimal polygons fitted around the dendritic profile of each fully reconstructed ipRGC in the left panels. Note the progressive age-dependent degeneration of ipRGCs dendritic field in P23H rats. Scale bar: 100 μm.
Figure 4
 
Progressive changes in the morphology and arborization of ipRGCs in P23H rats. (A, C, E, G) Representative drawings of the soma and complete dendritic field of ipRGCs from a region of the central retina (between the superior and nasal quadrants) of a 4-month-old SD rat (A) and P23H rats at 4 (C), 12 (E), and 18 (G) months of age. (B, D, F, H) Minimal polygons fitted around the dendritic profile of each fully reconstructed ipRGC in the left panels. Note the progressive age-dependent degeneration of ipRGCs dendritic field in P23H rats. Scale bar: 100 μm.
The morphologic parameters quantified on ipRGCs from SD and P23H rats aged 4, 12, and 18 months are shown in Figure 5. No significant differences associated with age were observed in any of the morphologic parameters analyzed in SD rats (Figs. 5A–E), and no significant differences were found between 4-month-old P23H and SD rats (Figs. 5A–E), except for a slightly higher Sholl area in P23H rats with respect to that measured in SD rats (P < 0.05; Fig. 5E). However, a progressive age-dependent decrease in the dendritic area, the number of branch points, the number of terminal neurite tips per cell, and the Sholl area of ipRGCs were observed in P23H rats (Figs. 5B–E). More specifically, at 12 months of age P23H showed a significant decrease in all the dendritic parameters analyzed, with respect to those measured in 12-month-old SD rats (P < 0.01, in all cases). At 18 months of age, the dendritic parameters analyzed in P23H rats were significantly smaller than those obtained in both 18-month-old SD rats (P < 0.001, in all cases) and 12-month-old P23H rats (P < 0.05, for dendritic area and branch points, and P < 0.001, for terminal neurite tips and Sholl area). Figure 5F shows the fall in the Sholl profile in 18-month-old P23H rats, with respect to that observed in 18-month-old SD rats. 
Figure 5
 
Morphologic parameters of ipRGCs in SD and P23H rats. (A) Quantification of the mean soma diameter of ipRGCs in SD and P23H rats aged 4, 12, and 18 months. One hundred cells from seven retinas were examined on each experimental group. (BE) Mean dendritic area (B), number of branch points (C), number of terminal neurite tips per cell (D), and Sholl area (E) of ipRGCs in SD and P23H rats aged 4, 12, and 18 months (35 cells, seven retinas, examined per group). (F) Mean Sholl profiles of ipRGCs from 18-month-old P23H and SD rats, indicating the number of neuritic processes from the cell soma (35 cells, seven retinas). *P < 0.05, **P < 0.01, ***P < 0.001; ANOVA.
Figure 5
 
Morphologic parameters of ipRGCs in SD and P23H rats. (A) Quantification of the mean soma diameter of ipRGCs in SD and P23H rats aged 4, 12, and 18 months. One hundred cells from seven retinas were examined on each experimental group. (BE) Mean dendritic area (B), number of branch points (C), number of terminal neurite tips per cell (D), and Sholl area (E) of ipRGCs in SD and P23H rats aged 4, 12, and 18 months (35 cells, seven retinas, examined per group). (F) Mean Sholl profiles of ipRGCs from 18-month-old P23H and SD rats, indicating the number of neuritic processes from the cell soma (35 cells, seven retinas). *P < 0.05, **P < 0.01, ***P < 0.001; ANOVA.
Figure 6 shows pictures of M1 cells (Figs. 6A–C) and their stratification patterns (Figs. 6D–F) in the retina of P23H rats at 4, 12, and 18 months of age, as well as diagrams of the morphologic parameters analyzed (Figs. 6G–K). In these animals, M1 cells showed a progressive age-related loss of dendrites, indicating a gradual degeneration associated with age (Figs. 6A–F). A progressive decrease in M1 cell diameter was observed in these animals when measured at 12 and 18 months of age (P < 0.05 and P < 0.001, respectively) (Fig. 6G). With regard to the dendritic area of M1 cells, a progressive decrease of dendritic area was observed in P23H rats at 12 and 18 months of age (Fig. 6H). This loss was statistically significant at 18 months of age, as compared with 4 and 12 months of age (P < 0.001 and P < 0.05, respectively). The morphologic Bonfire analysis of the dendritic tree in M1 cells exhibited a progressive decrease in all parameters studied, including the number of branch points (Fig. 6I), the number of terminal neurite tips per cell (Fig. 6J), and the Sholl area (Fig. 6K). In all these parameters, significant differences between 4- and 12-month-old rats (P < 0.05, in all cases) and between 12- and 18-month-old animals (P < 0.01, in all cases) were found. No significant variations were observed with age in SD rats, or between SD and 4-month-old P23H rats, except for an increase in the dendritic area of SD rats between 4- and 18-month-old animals (P < 0.05; Fig. 6H). 
Figure 6
 
Age-related morphologic changes of M1 cells in P23H rats. (AC) Examples of M1 cells in whole-mount rat retinas from P23H rats at 4 (A), 12 (B), and 18 (C) months of age. Each image is the projection of five to ten focal planes between the GCL and the stratum S1 of the IPL. Arrows: axons. Note the progressive loss of dendritic arborization. (DF) Drawings of ipRGCs from P23H rats at 4 (D), 12 (E), and 18 (F) months of age. Cell bodies (red) are located in the GCL. All dendrites (blue) are located in S1. Axons are drawn in black. (G) Quantification of the mean soma diameter of M1 cells in SD and P23H rats aged 4, 12, and 18 months. Sixty cells from seven retinas were examined on each experimental group. (HK) Mean dendritic area (H), number of branch points (I), number of terminal neurite tips per cell (J), and Sholl area (K) of M1 cells in SD and P23H rats aged 4, 12, and 18 months (ten cells, seven retinas, examined per group). *P < 0.05, **P < 0.01, ***P < 0.001; ANOVA. Scale bar: 100 μm (AC), 50 μm (DF).
Figure 6
 
Age-related morphologic changes of M1 cells in P23H rats. (AC) Examples of M1 cells in whole-mount rat retinas from P23H rats at 4 (A), 12 (B), and 18 (C) months of age. Each image is the projection of five to ten focal planes between the GCL and the stratum S1 of the IPL. Arrows: axons. Note the progressive loss of dendritic arborization. (DF) Drawings of ipRGCs from P23H rats at 4 (D), 12 (E), and 18 (F) months of age. Cell bodies (red) are located in the GCL. All dendrites (blue) are located in S1. Axons are drawn in black. (G) Quantification of the mean soma diameter of M1 cells in SD and P23H rats aged 4, 12, and 18 months. Sixty cells from seven retinas were examined on each experimental group. (HK) Mean dendritic area (H), number of branch points (I), number of terminal neurite tips per cell (J), and Sholl area (K) of M1 cells in SD and P23H rats aged 4, 12, and 18 months (ten cells, seven retinas, examined per group). *P < 0.05, **P < 0.01, ***P < 0.001; ANOVA. Scale bar: 100 μm (AC), 50 μm (DF).
In SD rats, M1d cells showed no differences associated with age in any of the morphologic parameters analyzed (Figs. 7G–K), except for a decrease in soma size at 18 months of age, as compared with 4- and 12-month-old animals (P < 0.01, in both cases; Fig. 7G), as well as an increase in the dendritic area at 12 months of age, with respect to that observed in 4-month-old rats (P < 0.05; Fig. 7H). No significant differences were found between M1d cells in SD and 4-month-old P23H rats. However, in P23H rats M1d cells showed a progressive age-associated atrophy of their dendritic tree, observable in both pictures (Figs. 7A–C) and dendritic profiles (Figs. 7D–F). A progressive decrease in all the morphologic parameters analyzed in these cells was observed in P23H rats at 12 and 18 months of age (Figs. 7G–K). Significant differences between 4- and 12-month-old rats were found in soma size (P < 0.05; Fig. 7G), and between 4- and 18-month-old animals in soma size (P < 0.01; Fig. 7G), dendritic area (P < 0.001; Fig. 7H), and Sholl area (P < 0.05; Fig. 7K). 
Figure 7
 
Progressive changes of M1d cells in P23H rats. (AC) Representative images of M1d cells in whole-mount rat retinas from P23H rats at 4 (A), 12 (B), and 18 (C) months of age. Each image is the projection of three to five focal planes between S1 and the INL. Arrow: axon. Note the progressive loss of dendritic arborization. (DF) Stratification patterns of ipRGCs in P23H rats at 4 (D), 12 (E), and 18 (F) months of age. Somas are located in the INL and indicated in blue. All dendrites are located in S1 and drawn in blue. (G) Quantification of the mean soma diameter of M1d cells in SD and P23H rats aged 4, 12, and 18 months. Twenty cells from seven retinas were examined on each experimental group. (HK) Mean dendritic area (H), number of branch points (I), number of terminal neurite tips per cell (J), and Sholl area (K) of M1d cells in SD and P23H rats aged 4, 12, and 18 months (ten cells, seven retinas, analyzed per group). *P < 0.05, **P < 0.01, ***P < 0.001; ANOVA. Scale bar: 100 μm (AC), 50 μm (DF).
Figure 7
 
Progressive changes of M1d cells in P23H rats. (AC) Representative images of M1d cells in whole-mount rat retinas from P23H rats at 4 (A), 12 (B), and 18 (C) months of age. Each image is the projection of three to five focal planes between S1 and the INL. Arrow: axon. Note the progressive loss of dendritic arborization. (DF) Stratification patterns of ipRGCs in P23H rats at 4 (D), 12 (E), and 18 (F) months of age. Somas are located in the INL and indicated in blue. All dendrites are located in S1 and drawn in blue. (G) Quantification of the mean soma diameter of M1d cells in SD and P23H rats aged 4, 12, and 18 months. Twenty cells from seven retinas were examined on each experimental group. (HK) Mean dendritic area (H), number of branch points (I), number of terminal neurite tips per cell (J), and Sholl area (K) of M1d cells in SD and P23H rats aged 4, 12, and 18 months (ten cells, seven retinas, analyzed per group). *P < 0.05, **P < 0.01, ***P < 0.001; ANOVA. Scale bar: 100 μm (AC), 50 μm (DF).
M2 cells in SD animals showed no significant differences with age in cell morphology (Figs. 8G–K), except for a slight increase in soma diameter at 18 months of age (P < 0.01; Fig. 8G). No significant differences were found between SD and 4-month-old P23H rats. However, in P23H animals an age-associated loss in dendritic arborization of M2 cells can be observed, from 4 to 18 months of age in both pictures (Figs. 8A–C) and camera lucida drawings (Figs. 8D–F). Progressive age-dependent decreases in dendritic area, number of branch points and terminal neurite tips per cell, and Sholl area of M2 cells were observed in these animals, with significant differences found between animals at 4 and 18 months of age in dendritic area (P < 0.01; Fig. 8H), number of terminal neurite tips per cell (P < 0.05; Fig. 8J), and Sholl area (P < 0.05; Fig. 8K). At 18 months of age, some cells showed signs of degeneration, with swollen bulbous structures in their dendrites (Fig. 8F, arrows). 
Figure 8
 
Age-related changes of M2 cells in P23H rats. (AC) Representative images of M2 cells in whole-mount rat retinas from P23H rats at 4 (A), 12 (B), and 18 (C) months of age. Each image is the projection of three to five focal planes between the GCL and the stratum S5 of the IPL. Note the progressive loss of dendritic arborization. (DF) Drawings of ipRGCs from P23H rats at 4 (D), 12 (E), and 18 (F) months of age. Note the presence of cells with swollen bulbous structures in their dendrites at 18 months of age ([F], arrows). Cell bodies (red) are located in the GCL. All dendrites are located in S5 and drawn in red. Axons are drawn in black. (GK) Quantification of the mean soma diameter (G), dendritic area (H), number of branch points (I), number of terminal neurite tips per cell (J), and Sholl area (K) of M2 cells in SD and P23H rats aged 4, 12, and 18 months. Ten cells from seven retinas were examined on each experimental group. *P < 0.05, **P < 0.01; ANOVA. Scale bar: 100 μm (AC), 50 μm (DF).
Figure 8
 
Age-related changes of M2 cells in P23H rats. (AC) Representative images of M2 cells in whole-mount rat retinas from P23H rats at 4 (A), 12 (B), and 18 (C) months of age. Each image is the projection of three to five focal planes between the GCL and the stratum S5 of the IPL. Note the progressive loss of dendritic arborization. (DF) Drawings of ipRGCs from P23H rats at 4 (D), 12 (E), and 18 (F) months of age. Note the presence of cells with swollen bulbous structures in their dendrites at 18 months of age ([F], arrows). Cell bodies (red) are located in the GCL. All dendrites are located in S5 and drawn in red. Axons are drawn in black. (GK) Quantification of the mean soma diameter (G), dendritic area (H), number of branch points (I), number of terminal neurite tips per cell (J), and Sholl area (K) of M2 cells in SD and P23H rats aged 4, 12, and 18 months. Ten cells from seven retinas were examined on each experimental group. *P < 0.05, **P < 0.01; ANOVA. Scale bar: 100 μm (AC), 50 μm (DF).
Morphologic analysis of M3 cells in SD rats showed no differences associated with age (see Fig. 9). No significant differences were found between SD and 4-month-old P23H rats. However, from 4 to 18 months of age, P23H animals showed a progressive age-dependent decrease in all dendritic parameters analyzed, including the number of branch points per cell (P < 0.05), the number of terminal neurite tips per cell (P < 0.01), and the Sholl area (P < 0.05) (Figs. 9L–N, respectively), indicating a progressive atrophy of their dendritic field, visible in the pictures taken at the S5 stratum of the IPL (Figs. 9A–C), the pictures taken at the S1 stratum of the IPL (Figs. 9D–F), and the reconstructions of M3 cells' dendritic profiles (Figs. 9G–I). 
Figure 9
 
Age-related morphologic changes of M3 cells in P23H rats. (AC) Representative pictures of M3 cells in whole-mount rat retinas from P23H rats at 4 (A), 12 (B), and 18 (C) months of age. Each image is the projection of three to five focal planes between the GCL and the stratum S5 of the IPL. (DF) Pictures of the M3 cells showed in the top panels taken at the stratum S1. Each image is the projection of three to five focal planes in S1. Arrow: dendrite. Note the progressive loss of dendritic arborization. (GI) Drawings of ipRGCs from P23H rats at 4 (G), 12 (H), and 18 (I) months of age. Cell bodies (red) are located in the GCL. Dendrites in S1 are drawn in blue and those located in S5 are indicated in red. Axons are drawn in black. (JN) Quantification of the mean soma diameter (J), dendritic area (K), number of branch points (L), number of terminal neurite tips per cell (M), and Sholl area (N) of M3 cells in SD and P23H rats aged 4, 12, and 18 months (six cells, seven retinas, analyzed per group). *P < 0.05, **P < 0.01; ANOVA. Scale bar: 100 μm (AC), 50 μm (DF).
Figure 9
 
Age-related morphologic changes of M3 cells in P23H rats. (AC) Representative pictures of M3 cells in whole-mount rat retinas from P23H rats at 4 (A), 12 (B), and 18 (C) months of age. Each image is the projection of three to five focal planes between the GCL and the stratum S5 of the IPL. (DF) Pictures of the M3 cells showed in the top panels taken at the stratum S1. Each image is the projection of three to five focal planes in S1. Arrow: dendrite. Note the progressive loss of dendritic arborization. (GI) Drawings of ipRGCs from P23H rats at 4 (G), 12 (H), and 18 (I) months of age. Cell bodies (red) are located in the GCL. Dendrites in S1 are drawn in blue and those located in S5 are indicated in red. Axons are drawn in black. (JN) Quantification of the mean soma diameter (J), dendritic area (K), number of branch points (L), number of terminal neurite tips per cell (M), and Sholl area (N) of M3 cells in SD and P23H rats aged 4, 12, and 18 months (six cells, seven retinas, analyzed per group). *P < 0.05, **P < 0.01; ANOVA. Scale bar: 100 μm (AC), 50 μm (DF).
Discussion
The present study demonstrates that retinitis pigmentosa is associated in P23H rats with a progressive degeneration of melanopsin-containing retinal ganglion cells. In this work, we evaluated numerical and morphologic changes in ipRGCs of P23H rats, a model of autosomal dominant RP characterized by a slow-pace retinal degeneration. 26,27 We have demonstrated that RP is associated with a progressive loss in density, integrity, and dendritic arborization of melanopsin-containing retinal ganglion cells in advanced stages of the degenerative disease. 
Morphologic analysis of the ipRGCs found in rat retinas demonstrated the presence of at least three subtypes of melanopsin-containing ganglion cells (M1, M2, and M3). M1 cell dendrites were stratified exclusively in the outer margin of the IPL. ipRGCs with a similar pattern of stratification, as well as similar soma size and dendritic area, have previously been described in the retinas of rats, 3,5 mice, 68,43,44 and humans. 45 ipRGCs with somas located in the INL and dendrites stratifying in S1 have also been previously reported in the retinas of mice 7 and marmosets. 46 In these previous studies, displaced melanopsin immunoreactive neurons stratifying in S1 were also considered displaced M1 cells. The dendrites of M2 cells stratified in S5 of the IPL, near the GCL. This is also in line with previous studies, which have described ipRGCs with a similar stratification pattern, soma size, and dendritic area in mouse, 7,44 rat, 47 and human retinas. 45 Bistratified M3 cells, less abundant than M1 and M2 cells, projected their dendrites to both the outer and inner margins of the IPL (S1 and S5, respectively). Once again, previous works have identified bistratified ipRGCs in mouse retinas, 7,43,48 and in relative numbers similar to what we have observed for M3 cells in this study (approximately 10%). 7 However, to our knowledge, this is the first time that M3-type ipRGCs have been described in rats. 
In our study, immunoreactivity of ipRGCs was weaker in M2 cells than that in M1 and M3 cells, which agrees with previous studies in mice. 7 Two isoforms of melanopsin from the mouse Opn4 locus have been identified: a long isoform (Opn4L) and a short isoform (Opn4S). 49 Opn4S and Opn4L are expressed in mouse M1 and M3 ipRGCs, whereas only Opn4L is expressed in M2 ipRGCs. 49 The weaker immunolabeling observed in M2 cells may be due to the expression of only one isoform of melanopsin and, therefore, a lower protein content. ipRGCs were distributed throughout the entire retina, even though some increased density was observed in the upper-temporal part of the retina. This is in agreement with previous results showing that the density of melanopsin-positive cells was slightly higher in the superior and temporal quadrants of the rat retina. 3,5,19,20  
Visual functions decline with age 50 and, thus, aging has been associated with qualitative and quantitative changes in retinal neurons. 5154 Aging has also been associated with alterations in circadian rhythms (amplitude damping, phase shifts, and/or period changes) 55,56 that can be attributed, at least in part, to the aforementioned visual loss. However, some previous experiments show no age-related changes in mechanisms associated with the activity of ipRGCs. In these experiments, age did not reduce, but rather enhanced, pupil responses mediated by ipRGC in humans. 57 During postnatal development no loss of melanopsin-immunoreactive cells occurs in mice. 58 Furthermore, other results have suggested that the retinogeniculate pathways are relatively unaffected by aging in monkeys. 59 In a previous experiment, we showed that body temperature circadian rhythms in 18-month-old SD rats are no different from those of younger animals. 34 Here, we show that ipRGCs in SD rats exhibit no significant differences in density or morphology in connection with aging. There may be several reasons for these discrepancies. First, the complexity of the retinal structure makes detailed structural analysis difficult. Second, the marked heterogeneity of neuronal sizes and morphology can mask changes. Third, the relative age of the subjects used in these studies may not be uniform. Finally, the functional effects of ipRGC deterioration may occur relatively late in relation to the changes caused by degeneration of other retinal cell types. 
Our results demonstrate that mean density of melanopsin-immunoreactive cells progressively decreases in P23H rats. The loss of immunoreactivity can be due to the loss of cells but, also, to a lack of melanopsin expression. The result agrees with previous experiments on Royal College of Surgeons dystrophic rats, 60 showing a loss of ipRGCs as the disease progressed. A reduction in ipRGCs in rodless/coneless (rd/rd cl) mice has also been observed in aged as compared with young animals. 61 Furthermore, in a previous study with P23H rats, we showed that retinal degeneration positively correlates with circadian dysfunctions in transgenic rats. 34 An obvious connection can be drawn with RP patients, who have been shown to exhibit sleep–wake disorders 62,63 and alterations in blood pressure and heart rate circadian rhythms. 64 Finally, it has also been shown that pupil responses to red and blue light stimuli are significantly reduced in patients with RP. 35  
The decrease in the mean number of ipRGCs was significant in 18-month-old P23H rats, in which a significant number of melanopsin-positive cells survived, despite the advanced stage of retinal degeneration. It has been proposed that the morphologic and physiologic properties of ipRGCs, such as having a large soma, long and sparsely branching dendritic fields, and intrinsic photosensitivity, may be factors contributing to their resistance to cell injury. 6567  
Morphologic characterizations of ipRGCs on P23H rats indicates a progressive atrophy of ipRGCs. However, although morphologic changes in the dendritic tree were significant at 12 months of age, the decrease in the dendritic area was not significant until 18 months of age. These results may reflect some form of plasticity and remodeling of ipRGC neurites, and correlate with the abovementioned high resistance to injury of ipRGCs. Retinal remodeling in the IPL has been evidenced in several models of retinal degeneration, 6871 including animal models of RP. 72,73 On the other hand, the observed morphologic changes in the dendritic tree may be accompanied by, and be mechanistically related to, the expression and stabilization of synaptic plasticity. 
The present study reflects that degeneration of photoreceptors and inner retinal neurons, characteristic of RP, has degenerative effects on the number and morphology of ipRGCs. However, it also shows that the impact of this pathology on ipRGCs is relatively belated compared with what is observed in other retinal neurons, including nonmelanopsin RGCs. Both the survival of a significant numbers of ipRGCs and the relative preservation of their dendritic tree in advanced stages of retinal degeneration suggest that a functional population of melanopsin cells may still exist in even the most severe cases of human RP. Future studies will need to be done to determine the relationship between ipRGC degeneration and impairment of circadian rhythms in RP patients. 
Acknowledgments
Supported by the Spanish Ministry of Economy and Competitiveness Grant BFU2012‐36845; Instituto de Salud Carlos III Grant RETICS RD12/0034/0010; Universidad de Alicante Grant UA2010‐48536273; and Organización Nacional de Ciegos Españoles (ONCE). 
Disclosure: G. Esquiva, None; P. Lax, None; N. Cuenca, None 
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Figure 1
 
Melanopsin-positive ganglion cells on the rat retina. (A, B) Representative images of rat retinas vertical sections labeled with anti-melanopsin antibody. Note that melanopsin is present on the soma and neurites of the RGC subpopulation. Inmunolabeled dendrites stratified in strata S1 and S5 of the IPL. Scale bar: 20 μm. (CF) Examples of the ipRGC types found in whole-mount rat retinas from SD rats. (C) M1 cell, with their soma placed in the GCL and dentrites in the stratum S1 of the IPL. (D) M1 displaced cell (M1d), with their cell body placed in the INL and dendrites in the stratum S1 of the IPL. (E) M2 cell, with their cell body located in the GCL and dendrites in S5 of the IPL. (F) M3 cell, with their cell body placed in the GCL and dendrites in both S1 and S5 of the IPL. Each image is the projection of five to ten focal planes between the GCL and the stratum S1 of the IPL. Scale bar: 100 μm.
Figure 1
 
Melanopsin-positive ganglion cells on the rat retina. (A, B) Representative images of rat retinas vertical sections labeled with anti-melanopsin antibody. Note that melanopsin is present on the soma and neurites of the RGC subpopulation. Inmunolabeled dendrites stratified in strata S1 and S5 of the IPL. Scale bar: 20 μm. (CF) Examples of the ipRGC types found in whole-mount rat retinas from SD rats. (C) M1 cell, with their soma placed in the GCL and dentrites in the stratum S1 of the IPL. (D) M1 displaced cell (M1d), with their cell body placed in the INL and dendrites in the stratum S1 of the IPL. (E) M2 cell, with their cell body located in the GCL and dendrites in S5 of the IPL. (F) M3 cell, with their cell body placed in the GCL and dendrites in both S1 and S5 of the IPL. Each image is the projection of five to ten focal planes between the GCL and the stratum S1 of the IPL. Scale bar: 100 μm.
Figure 2
 
Dendritic arborization of melanopsin-containing ganglion cells on the rat retina. (AD) Examples of the soma-dendritic profiles of M1, M1d, M2, and M3 cells. Dendrites in S1 are drawn in blue, and those located in S5 are shown in red. Axons are drawn in black. Somas located in the GCL are drawn in red, whereas those located in the INL (M1d cells) are shown in blue. (E) Quantification of the mean soma diameter of M1, M1d, M2, and M3 cells. Seventy cells from seven retinas were examined for each cell type. (FI) Mean dendritic area (F), number of branch points (G), number of terminal neurite tips per cell (H), and Sholl area (I) of each melanopsin-positive cell type. Thirty cells from seven retinas were examined for each cell type. *P < 0.05, ***P < 0.001; ANOVA. Scale bar: 500 μm.
Figure 2
 
Dendritic arborization of melanopsin-containing ganglion cells on the rat retina. (AD) Examples of the soma-dendritic profiles of M1, M1d, M2, and M3 cells. Dendrites in S1 are drawn in blue, and those located in S5 are shown in red. Axons are drawn in black. Somas located in the GCL are drawn in red, whereas those located in the INL (M1d cells) are shown in blue. (E) Quantification of the mean soma diameter of M1, M1d, M2, and M3 cells. Seventy cells from seven retinas were examined for each cell type. (FI) Mean dendritic area (F), number of branch points (G), number of terminal neurite tips per cell (H), and Sholl area (I) of each melanopsin-positive cell type. Thirty cells from seven retinas were examined for each cell type. *P < 0.05, ***P < 0.001; ANOVA. Scale bar: 500 μm.
Figure 3
 
Distribution and number of ipRGCs in SD and P23H rat retinas. (AF) Representative drawings of whole-mount retinas from SD (AC) and P23H (DF) rats at 4, 12, and 18 months of age. Retinas were manually drawn with the aid of a camera lucida, showing the location of individual ipRGCs labeled with immunoperoxidase. The number of ipRGCs represented in the drawings is 2597 (A), 2089 (B), 2783 (C), 1714 (D), 1917 (E), and 491 (F). (G, H) Quantification of the mean ipRGC density (cells/mm2) (G) and the average area of the entire retinas (mm2) (H) in whole-mount retinas from SD and P23H rats at 4, 12, and 18 months of age. Seven retinas (with approximately 2000 cells per retina) were examined on each experimental group. *P < 0.05, ***P < 0.001; ANOVA. Scale bar: 50 μm.
Figure 3
 
Distribution and number of ipRGCs in SD and P23H rat retinas. (AF) Representative drawings of whole-mount retinas from SD (AC) and P23H (DF) rats at 4, 12, and 18 months of age. Retinas were manually drawn with the aid of a camera lucida, showing the location of individual ipRGCs labeled with immunoperoxidase. The number of ipRGCs represented in the drawings is 2597 (A), 2089 (B), 2783 (C), 1714 (D), 1917 (E), and 491 (F). (G, H) Quantification of the mean ipRGC density (cells/mm2) (G) and the average area of the entire retinas (mm2) (H) in whole-mount retinas from SD and P23H rats at 4, 12, and 18 months of age. Seven retinas (with approximately 2000 cells per retina) were examined on each experimental group. *P < 0.05, ***P < 0.001; ANOVA. Scale bar: 50 μm.
Figure 4
 
Progressive changes in the morphology and arborization of ipRGCs in P23H rats. (A, C, E, G) Representative drawings of the soma and complete dendritic field of ipRGCs from a region of the central retina (between the superior and nasal quadrants) of a 4-month-old SD rat (A) and P23H rats at 4 (C), 12 (E), and 18 (G) months of age. (B, D, F, H) Minimal polygons fitted around the dendritic profile of each fully reconstructed ipRGC in the left panels. Note the progressive age-dependent degeneration of ipRGCs dendritic field in P23H rats. Scale bar: 100 μm.
Figure 4
 
Progressive changes in the morphology and arborization of ipRGCs in P23H rats. (A, C, E, G) Representative drawings of the soma and complete dendritic field of ipRGCs from a region of the central retina (between the superior and nasal quadrants) of a 4-month-old SD rat (A) and P23H rats at 4 (C), 12 (E), and 18 (G) months of age. (B, D, F, H) Minimal polygons fitted around the dendritic profile of each fully reconstructed ipRGC in the left panels. Note the progressive age-dependent degeneration of ipRGCs dendritic field in P23H rats. Scale bar: 100 μm.
Figure 5
 
Morphologic parameters of ipRGCs in SD and P23H rats. (A) Quantification of the mean soma diameter of ipRGCs in SD and P23H rats aged 4, 12, and 18 months. One hundred cells from seven retinas were examined on each experimental group. (BE) Mean dendritic area (B), number of branch points (C), number of terminal neurite tips per cell (D), and Sholl area (E) of ipRGCs in SD and P23H rats aged 4, 12, and 18 months (35 cells, seven retinas, examined per group). (F) Mean Sholl profiles of ipRGCs from 18-month-old P23H and SD rats, indicating the number of neuritic processes from the cell soma (35 cells, seven retinas). *P < 0.05, **P < 0.01, ***P < 0.001; ANOVA.
Figure 5
 
Morphologic parameters of ipRGCs in SD and P23H rats. (A) Quantification of the mean soma diameter of ipRGCs in SD and P23H rats aged 4, 12, and 18 months. One hundred cells from seven retinas were examined on each experimental group. (BE) Mean dendritic area (B), number of branch points (C), number of terminal neurite tips per cell (D), and Sholl area (E) of ipRGCs in SD and P23H rats aged 4, 12, and 18 months (35 cells, seven retinas, examined per group). (F) Mean Sholl profiles of ipRGCs from 18-month-old P23H and SD rats, indicating the number of neuritic processes from the cell soma (35 cells, seven retinas). *P < 0.05, **P < 0.01, ***P < 0.001; ANOVA.
Figure 6
 
Age-related morphologic changes of M1 cells in P23H rats. (AC) Examples of M1 cells in whole-mount rat retinas from P23H rats at 4 (A), 12 (B), and 18 (C) months of age. Each image is the projection of five to ten focal planes between the GCL and the stratum S1 of the IPL. Arrows: axons. Note the progressive loss of dendritic arborization. (DF) Drawings of ipRGCs from P23H rats at 4 (D), 12 (E), and 18 (F) months of age. Cell bodies (red) are located in the GCL. All dendrites (blue) are located in S1. Axons are drawn in black. (G) Quantification of the mean soma diameter of M1 cells in SD and P23H rats aged 4, 12, and 18 months. Sixty cells from seven retinas were examined on each experimental group. (HK) Mean dendritic area (H), number of branch points (I), number of terminal neurite tips per cell (J), and Sholl area (K) of M1 cells in SD and P23H rats aged 4, 12, and 18 months (ten cells, seven retinas, examined per group). *P < 0.05, **P < 0.01, ***P < 0.001; ANOVA. Scale bar: 100 μm (AC), 50 μm (DF).
Figure 6
 
Age-related morphologic changes of M1 cells in P23H rats. (AC) Examples of M1 cells in whole-mount rat retinas from P23H rats at 4 (A), 12 (B), and 18 (C) months of age. Each image is the projection of five to ten focal planes between the GCL and the stratum S1 of the IPL. Arrows: axons. Note the progressive loss of dendritic arborization. (DF) Drawings of ipRGCs from P23H rats at 4 (D), 12 (E), and 18 (F) months of age. Cell bodies (red) are located in the GCL. All dendrites (blue) are located in S1. Axons are drawn in black. (G) Quantification of the mean soma diameter of M1 cells in SD and P23H rats aged 4, 12, and 18 months. Sixty cells from seven retinas were examined on each experimental group. (HK) Mean dendritic area (H), number of branch points (I), number of terminal neurite tips per cell (J), and Sholl area (K) of M1 cells in SD and P23H rats aged 4, 12, and 18 months (ten cells, seven retinas, examined per group). *P < 0.05, **P < 0.01, ***P < 0.001; ANOVA. Scale bar: 100 μm (AC), 50 μm (DF).
Figure 7
 
Progressive changes of M1d cells in P23H rats. (AC) Representative images of M1d cells in whole-mount rat retinas from P23H rats at 4 (A), 12 (B), and 18 (C) months of age. Each image is the projection of three to five focal planes between S1 and the INL. Arrow: axon. Note the progressive loss of dendritic arborization. (DF) Stratification patterns of ipRGCs in P23H rats at 4 (D), 12 (E), and 18 (F) months of age. Somas are located in the INL and indicated in blue. All dendrites are located in S1 and drawn in blue. (G) Quantification of the mean soma diameter of M1d cells in SD and P23H rats aged 4, 12, and 18 months. Twenty cells from seven retinas were examined on each experimental group. (HK) Mean dendritic area (H), number of branch points (I), number of terminal neurite tips per cell (J), and Sholl area (K) of M1d cells in SD and P23H rats aged 4, 12, and 18 months (ten cells, seven retinas, analyzed per group). *P < 0.05, **P < 0.01, ***P < 0.001; ANOVA. Scale bar: 100 μm (AC), 50 μm (DF).
Figure 7
 
Progressive changes of M1d cells in P23H rats. (AC) Representative images of M1d cells in whole-mount rat retinas from P23H rats at 4 (A), 12 (B), and 18 (C) months of age. Each image is the projection of three to five focal planes between S1 and the INL. Arrow: axon. Note the progressive loss of dendritic arborization. (DF) Stratification patterns of ipRGCs in P23H rats at 4 (D), 12 (E), and 18 (F) months of age. Somas are located in the INL and indicated in blue. All dendrites are located in S1 and drawn in blue. (G) Quantification of the mean soma diameter of M1d cells in SD and P23H rats aged 4, 12, and 18 months. Twenty cells from seven retinas were examined on each experimental group. (HK) Mean dendritic area (H), number of branch points (I), number of terminal neurite tips per cell (J), and Sholl area (K) of M1d cells in SD and P23H rats aged 4, 12, and 18 months (ten cells, seven retinas, analyzed per group). *P < 0.05, **P < 0.01, ***P < 0.001; ANOVA. Scale bar: 100 μm (AC), 50 μm (DF).
Figure 8
 
Age-related changes of M2 cells in P23H rats. (AC) Representative images of M2 cells in whole-mount rat retinas from P23H rats at 4 (A), 12 (B), and 18 (C) months of age. Each image is the projection of three to five focal planes between the GCL and the stratum S5 of the IPL. Note the progressive loss of dendritic arborization. (DF) Drawings of ipRGCs from P23H rats at 4 (D), 12 (E), and 18 (F) months of age. Note the presence of cells with swollen bulbous structures in their dendrites at 18 months of age ([F], arrows). Cell bodies (red) are located in the GCL. All dendrites are located in S5 and drawn in red. Axons are drawn in black. (GK) Quantification of the mean soma diameter (G), dendritic area (H), number of branch points (I), number of terminal neurite tips per cell (J), and Sholl area (K) of M2 cells in SD and P23H rats aged 4, 12, and 18 months. Ten cells from seven retinas were examined on each experimental group. *P < 0.05, **P < 0.01; ANOVA. Scale bar: 100 μm (AC), 50 μm (DF).
Figure 8
 
Age-related changes of M2 cells in P23H rats. (AC) Representative images of M2 cells in whole-mount rat retinas from P23H rats at 4 (A), 12 (B), and 18 (C) months of age. Each image is the projection of three to five focal planes between the GCL and the stratum S5 of the IPL. Note the progressive loss of dendritic arborization. (DF) Drawings of ipRGCs from P23H rats at 4 (D), 12 (E), and 18 (F) months of age. Note the presence of cells with swollen bulbous structures in their dendrites at 18 months of age ([F], arrows). Cell bodies (red) are located in the GCL. All dendrites are located in S5 and drawn in red. Axons are drawn in black. (GK) Quantification of the mean soma diameter (G), dendritic area (H), number of branch points (I), number of terminal neurite tips per cell (J), and Sholl area (K) of M2 cells in SD and P23H rats aged 4, 12, and 18 months. Ten cells from seven retinas were examined on each experimental group. *P < 0.05, **P < 0.01; ANOVA. Scale bar: 100 μm (AC), 50 μm (DF).
Figure 9
 
Age-related morphologic changes of M3 cells in P23H rats. (AC) Representative pictures of M3 cells in whole-mount rat retinas from P23H rats at 4 (A), 12 (B), and 18 (C) months of age. Each image is the projection of three to five focal planes between the GCL and the stratum S5 of the IPL. (DF) Pictures of the M3 cells showed in the top panels taken at the stratum S1. Each image is the projection of three to five focal planes in S1. Arrow: dendrite. Note the progressive loss of dendritic arborization. (GI) Drawings of ipRGCs from P23H rats at 4 (G), 12 (H), and 18 (I) months of age. Cell bodies (red) are located in the GCL. Dendrites in S1 are drawn in blue and those located in S5 are indicated in red. Axons are drawn in black. (JN) Quantification of the mean soma diameter (J), dendritic area (K), number of branch points (L), number of terminal neurite tips per cell (M), and Sholl area (N) of M3 cells in SD and P23H rats aged 4, 12, and 18 months (six cells, seven retinas, analyzed per group). *P < 0.05, **P < 0.01; ANOVA. Scale bar: 100 μm (AC), 50 μm (DF).
Figure 9
 
Age-related morphologic changes of M3 cells in P23H rats. (AC) Representative pictures of M3 cells in whole-mount rat retinas from P23H rats at 4 (A), 12 (B), and 18 (C) months of age. Each image is the projection of three to five focal planes between the GCL and the stratum S5 of the IPL. (DF) Pictures of the M3 cells showed in the top panels taken at the stratum S1. Each image is the projection of three to five focal planes in S1. Arrow: dendrite. Note the progressive loss of dendritic arborization. (GI) Drawings of ipRGCs from P23H rats at 4 (G), 12 (H), and 18 (I) months of age. Cell bodies (red) are located in the GCL. Dendrites in S1 are drawn in blue and those located in S5 are indicated in red. Axons are drawn in black. (JN) Quantification of the mean soma diameter (J), dendritic area (K), number of branch points (L), number of terminal neurite tips per cell (M), and Sholl area (N) of M3 cells in SD and P23H rats aged 4, 12, and 18 months (six cells, seven retinas, analyzed per group). *P < 0.05, **P < 0.01; ANOVA. Scale bar: 100 μm (AC), 50 μm (DF).
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