Retinal organization in the retinal degeneration 10 (rd10) mutant mouse: a morphological and ERG study

Abstract

Retinal degeneration 10 (rd10) mice are a model of autosomal recessive retinitis pigmentosa (RP), identified by Chang et al. in 2002 (Vision Res. 42:517-525). These mice carry a spontaneous mutation of the rod-phosphodiesterase (PDE) gene, leading to a rod degeneration that starts around P18. Later, cones are also lost. Because photoreceptor degeneration does not overlap with retinal development, and light responses can be recorded for about a month after birth, rd10 mice mimic typical human RP more closely than the well-known rd1 mutants. The aim of this study is to provide a comprehensive analysis of the morphology and function of the rd10 mouse retina during the period of maximum photoreceptor degeneration, thus contributing useful data for exploiting this novel model to study RP. We analyzed the morphology and survival of retinal cells in rd10 mice of various ages with quantitative immunocytochemistry and confocal microscopy; we also studied retinal function with the electroretinogram (ERG), recorded between P18 and P30. We found that photoreceptor death (peaking around P25) is accompanied and followed by dendritic retraction in bipolar and horizontal cells, which eventually undergo secondary degeneration. ERG reveals alterations in the physiology of the inner retina as early as P18 (before any obvious morphological change of inner neurons) and yet consistently with a reduced band amplification by bipolar cells. Thus, changes in the rd10 retina are very similar to what was previously found in rd1 mutants. However, an overall slower decay of retinal structure and function predicts that rd10 mice might become excellent models for rescue approaches.

Figure 1

Vertical sections of the rd10 mouse retina, illustrating the progressive degeneration of photoreceptors. Red signal: ethidium nuclear staining; green signal: PNA staining of cone inner segments and synaptic endings in the opl. A. At P20, many picnotic nuclei are visible in the central retina, where photoreceptors are actively dying (arrows). At the same age, photoreceptor death at the periphery is slightly behind (arrows in B). C and D: In the following 10 days, photoreceptor number drops continuously; the onl is reduced to 3 rows in the central (C) and to 5–6 rows in the peripheral retina (D). Cones are still clearly visible, though their outer and inner segments appear short and distorted. E and F: late stages of photoreceptor degeneration; PNA staining at this late stage is inadequate to reveal cones. At P45, only one row of nuclei persists in the onl (E). At 9 months, the row is discontinuous (F). Bar is 20 micrometers.

Figure 2

Apoptotic markers in the photoreceptor layer. A: Fluorojade-positive profiles are scattered in the outermost portion of the retina at P20. B: photoreceptors stain positively with the TUNEL method at P25. Apoptotic profiles are clearly visible (inset: 4x enlargement of the onl, similar to that shown in B). Bars are 20 micrometers.

Figure 3

In the retina of rd10 mice, rod bipolar cells show a clear reduction in their complement of dendrites at P30 (arrows in A), compared to their wild-type counterparts (arrows in B). Bar is 20 micrometers.

Figure 4

PKC staining (green) of rod bipolar cells shows the progressive retraction of dendrites, that are completely lost between P30 (A) and P45 (B, arrowhead). mGluR6 aggregates (red labeling) also decrease with time and become displaced to the cell body membrane and axons of bipolar cells (arrow). Residual clusters in the opl are mostly localized onto the dendritic tips of cone bipolar cells. Bars are 20 micrometers.

Figure 5

Dendritic loss from cone bipolar cells at late stages of the rd10 degeneration. A: NK3-R staining of cone bipolar cells with axonal arbors in the outer half of the ipl shows normal dendritic morphology. B: In animals 9 months and older, cone bipolar cells of the same type, labeled with recoverin antibodies (red) have totally lost the dendrites; cell bodies appear completely smooth (left arrow). Green signal: Goalpha labeling of bipolar neurons show virtual absence of processes in the opl (double arrows). Bar is 20 micrometers.

Figure 6

Calbindin (red signal) and neurofilament (green) staining highlight the loss of complexity of the dendrites and axonal arborizations of horizontal cells in the rd10 mouse, at P30 (A), compared to the wt counterpart (B). The fine dendritic branches of the wt horizontal cells (arrows), are completely absent in the mutant. Bar is 20 micrometers.

Figure 7

Calbindin staining of retinal whole mounts shows the massive loss of horizontal cell processes in the retina of rd10 mice (A), compared to a wt type (B). Age (7 months), eccentricity (dorsal periphery) and thickness of the projection images are matched. Bar is 20 micrometers.

Figure 8

Up-regulation of GFAP in Müller cells of rd10 retinas aged P20 (A) and P60 (B). Note the reduction in the retinal thickness from A to B. Bar is 20 micrometers.

Figure 9

Histograms illustrating the survival of rod bipolar and horizontal cells in rd10 retinas aged 1.5, 3.5, 7 and 9 months, respectively. Columns are average total number of cells/retina, with standard errors. Asterisks indicate significative differences between the number of cells of each age group and that found at 1.5 months. Data were obtained by 4 independent experiments performed in triplicate and analyzed with one way ANOVA (** is for p<0.05 and *** for p<0.01).

Figure 10

Isodensity maps of rod bipolar (left) and horizontal cells (right) of the rd10 retina, at 1.5 (top) and 3.5 (bottom) months of age. Each map is based on the average densities of 4 different, age-matched retinas. Legends are cells/mm². Note the net decrement in the density of rod bipolar cells in the nasal retina; horizontal cells, instead, decrease more rapidly in the temporal retina. At 3.5 months, the survival maps for the two cell types are almost symmetric along the dorsoventral axis.

Figure 11

Flash responses to three different luminances in scotopic conditions, from rd10 mice (P18: blue line; P26: red line) and from P25 wild type mice (black line). (Φ= photoisomerizations rod ⁻¹ per flash; dim: Φ=40; intermediate: Φ = 15000; bright: Φ=38000).

Figure 12

Amplitude and time-to-peak of scotopic ERG b-wave components, plotted as a function of stimulus intensity. Illustrated are: P25 wt mice (black line; n=8); P18 rd10 mice (blue line; n=5); P26 rd10 mice (red line; n=5). Data were analyzed with one way ANOVA (* P <0.05; ** P<0.001).

Figure 12

Amplitude and time-to-peak of scotopic ERG b-wave components, plotted as a function of stimulus intensity. Illustrated are: P25 wt mice (black line; n=8); P18 rd10 mice (blue line; n=5); P26 rd10 mice (red line; n=5). Data were analyzed with one way ANOVA (* P <0.05; ** P<0.001).

Figure 13

Recordings from wt mice (A) and from P21 rd10 mice (B), normalized to the saturating a-wave response. The dotted red line, superposed on the response traces, has been obtained by fitting the leading edge of the a-wave according to the equations reported in Methods. Stimulus intensities are Φ= 2.400; 15.000; 38.000; 143.000 for wt animals; and Φ= 2.400; 15.000; 38.000 for rd10 mice.

Figure 14

Average values for two parameters of the a-wave: the amplification parameter (A) and the effective delay (t_eff), measured at 15.000 photoisomerization rod ⁻¹. For both wt and rd10 mice, measurements were performed on 5 animals. (** P<0.001). Bars are S.E.M.

Figure 15

Sinusoidal ERG. Blue and red lines refer to rd10 mice, aged P18 and P26, respectively. Black lines refer to wt, age-matched animals. Gray lines represent the sinusoidal light stimulus. Left panel: sinusoidal ERG responses to 4 different temporal frequencies. Right panel: Kinetic analysis of ERG responses, obtained from rd10 mice aged P18 (A; n=8) and P26 (B; n=8) and from age-matched wt mice (n=8, black lines), using light stimuli sinusoidally modulated in time, from 0.3 to 30 Hz. The amplitude of the response, plotted as a function of the temporal frequency of the stimulus (attenuation characteristics), appears suppressed in the frequency range of 2–10 Hz.