Retinal degeneration in two lines of transgenic S334ter rats

Abstract

Aim of this study was to examine synaptic connectivity changes in the retina and the location and rate of apoptosis in transgenic S334ter line-3 and line-5 rats with photoreceptor degeneration. Heterozygous S334ter-line-3 and line-5 at P11-13, P30, P60, P90 and several control non-dystrophic rats (Long Evans and Sprague-Dawley) at P60, were studied anatomically by immunohistochemistry for various cell and synaptic markers, and by PNA and TUNEL label.- S334ter line-3 exhibited the fastest rate of degeneration with an early loss of photoreceptors, with 1-2 layers remaining at P30, and only cones left at P60. Line-5 had 4-5 layers left at P30, and very few rods left at P60-90. In both lines, horizontal cell processes (including dendrites and axon) were diminished at P11-13, showing gaps in the outer plexiform layer (OPL) at P60, and at P90, almost no terminal tips could be seen. Bipolar cells showed a retraction of their dendrites forming clusters along the OPL. Synaptic terminals of A-II amacrine cells in the IPL lost most of their parvalbumin-immunoreactivity. The apoptosis rate was different in both lines. Line-3 rats showed many photoreceptors affected at P11, occupying the innermost part of the outer nuclear layer. Line-5 showed a lower number of apoptotic cells within the same location at P13. In summary, the S334ter line-3 rat has a faster progression of degeneration than line-5. The horizontal and bipolar terminals are already affected at P11-P13 in both models. Apoptosis is related to the mutated rhodopsin transgene; the first photoreceptor cells affected are those close to the OPL.

Figure 1. Normal retina controls - Photoreceptors (recoverin, cone transducin, rhodopsin, PNA) and cone bipolar cells (recoverin)

(A–C) Long-Evans (LE) rat (control for S334ter-3). (D–I) Sprague-Dawley (SD) rat (control for S334ter-5). (A,B,D,E): Recoverin (labels photoreceptors and cone bipolar cells). Large arrows point to type 2 OFF cone bipolar cells with large somata; small arrows indicate their axon terminals in sublamina a of the IPL. Arrowheads point to type 8 ON cone bipolar cells with smaller somata; small arrowheads indicate their terminals in sublamina b of the IPL. (B,E): Recoverin (red)/cone transducin (green). (C,F): cone transducin (green; higher magnification). Cone cell bodies are localized close to the outer limiting membrane. Outer segments and cone pedicles are clearly labeled. G,H) rhodopsin (red) stains only rod outer segments in a normal SD rat. H,I) Peanut agglutinin (PNA, green) labels the extracellular matrix around cone outer segments. Magnification bars = 20 μm

Figure 2. Photoreceptor degeneration in S334ter line 3

Left column (A,D,G,J): recoverin (red); middle column (B,E,H,K): cone transducin (green); right column (C,F,I,L): double staining recoverin/cone transducin. (A–C): At P11, the photoreceptor layer shows full thickness, but without rod outer segments (A,C) and only few short cone outer segments (B,C). (C) Double label recoverin (red)/transducin (green). Arrows point to ectopic rod cells outside ONL. Arrowheads indicate cone outer segments. (D–F): At P30 there are approximately 2 layers of nuclei in the ONL, and no outer segments. (E): Cone cell bodies look disorganized without outer segments and are randomly distributed, not lined up close to outer limiting membrane. (F): Remaining rods appear red, remaining cones yellow. (G–I): At P60, the ONL is reduced to one, non-continuous layer of nuclei with only cones which appear yellow in recoverin/transducin double stain. (J–K): Further degeneration at P90: Remaining cones sprout processes at the outermost edge of the retina. Magnification bars = 20 μm

Figure 3. Photoreceptor degeneration in S334ter line 5

Left column (A,D,G,J): recoverin (red); middle column (B,E,H,K): cone transducin (green); right column (C,F,I,L): double staining recoverin/cone transducin. White arrowheads indicate cone outer segments; turquoise arrowheads indicate synapses between cone pedicles and cone bipolar cells; arrows in (I) and (L) indicate remaining rods. (A–C): At P13 the photoreceptor layer is fully developed, with short rod outer segments (A) and prominent cone outer segments (B,C, arrowheads). (B,C): Cone outer segments and pedicles are more strongly stained than in line-3. - (D–F): At P30 there are approximately 4–5 layers of nuclei in ONL, short outer segments. (F): Cones (yellow) are still regularly arranged close to the outer limiting membrane, with stubby outer segments. Remaining rods appear red. - (G–I): At P60, the ONL is reduced to one, non-continuous layer of cone nuclei. Cone cell bodies are oriented parallel to the outer limiting membrane. (J–L): Further degeneration at P90. Magnification bars = 20 μm

Figure 4. Rhodopsin stain in line-3 (A,C,E) and line-5 (B,D,F) retina

A) Line-3 at P11: rod photoreceptor cell bodies stain abnormally for rhodopsin. No developed rod outer segments. B) Line-5 at P13: abnormal rhodopsin stain of rod cell bodies, short rod outer segments. C) Line-3 at P30: few rods left. D) Line-5 at P30: four rows of rods left with distorted outer segments. E) Line-3 at P90: No rods left, except for one cell body without any cell processes. No rod outer segments. F) Line-5 at P90: One row of rods left. No recognizable rod outer segments. Magnification bars = 20 μm

Figure 5. PNA stain in line-3 (A,C,E) and line-5 (B,D,F) retina

A) Line-3 at P11: Very short cone outer segments (arrowheads). B) Line-5 at P13: Cone outer segments appear longer and more numerous than in line-3. C) Line-3 at P30: Reduced number of short cone outer segments (arrowheads). D) Line-5 at P30: Short cone outer segments; density similar to P13. E) Line-3 at P90: no PNA stain. F) Line-5 at P90: PNA staining of distorted short cone outer segments (arrowheads). Magnification bars = 20 μm

Figure 6. Horizontal cells (calbindin)

Normal Long-Evans and Sprague-Dawley rat (A,B) compared with S334ter-3 rats (C,E,G,I) and S334ter-5 rats (D,F,H,J):. Arrowheads in A,B,F,H indicate terminal tips, making synaptic contacts with spherules of rods and pedicles of cones. (C,D) Relatively normal morphology of horizontal cells at P11-13. At later ages (P30: E,F; P60: G,H; and P90: I,J), the morphology of horizontal cell dendrites deteriorates in both transgenic strains. Magnification bars = 20 μm

Figure 7. Rod bipolar cells and A-II amacrine cells of normal SD rat (A,B) compared with S334ter-5 rat (C)

(A) PKC/parvalbumin, Normal SD rat at P60. Arrows point to A-II amacrine - rod bipolar synapses in IPL, close to ganglion cells (sublamina a). (B) Parvalbumin, normal SD rat at P60: arrowheads point to synaptic terminals in IPL sublamina a. (C) Parvalbumin, S334ter-5 at P60: A-II amacrine cells appear shrunken; no synapses labeled in IPL sublamina b, reduced synapses in sublamina a. Magnification bars = 40 μm

Figure 8. Normal Long-Evans rat (LE; A, B) compared with S334ter-3 rat (C-F): Rod bipolar cells (PKC, green) and ribbon synapses (bassoon, red)

(A) Normal LE rat. At high magnification, the interaction of rod bipolar cell dendrites (green) with photoreceptor ribbon synapses (red) can be seen. (B) shows the same image of just the red bassoon channel. (C) Higher magnification of Supplemental Figure S2 C: At P11, there are still numerous photoreceptor ribbon synapses interacting with rod bipolar dendrites. Arrowheads point to bipolar cell dendrites sprouting into the ONL. (D) Higher magnification of Supplemental Figure S2D): at P30, most photoreceptor synaptic ribbons have been lost. There is abnormal sprouting of bipolar cell dendrites (arrow). (E) Few photoreceptor ribbon synapses left at P60. F) Almost complete loss of photoreceptor ribbon synapses at P90. Aberrant retraction of bipolar cell dendrites that are clumped together. Magnification bars = 20 μm

Figure 9. Normal Sprague-Dawley rat (SD; A,B) compared with S334ter-5 rat (C–F): Rod bipolar cells (PKC, green) and synapses (bassoon, red)

(A) At high magnification, the interaction of rod bipolar cell dendrites (green) with photoreceptor ribbon synapses (red) can be seen. (B) shows the same image of just the red bassoon channel. (C) Higher magnification of Supplemental Figure S 3C: at P13, there are still numerous photoreceptor synaptic ribbons interacting with rod bipolar dendrites. Arrowheads point to bipolar cell dendrites sprouting into the ONL. (D) Higher magnification of Supplemental Figure S3 D: at P30, many photoreceptor ribbon synapses have been lost. There is abnormal sprouting of bipolar cell dendrites (arrows). - PKC/bassoon stain at P60 (E) and P90 (F): Further reduction of photoreceptor ribbon synapses, abnormal morphology of rod bipolar cells. Magnification bars = 20 μm

Figure 10. Apoptosis (TUNEL stain, green) in combination with recoverin stain (red), comparison of line-3 (A,C) with line-5 (B,D)

(A) S334ter-3 rat at P11. Numerous TUNEL+ cells in ONL. (B) S334ter-5 rat at P13: Strong TUNEL stain in ONL, but less cells than in line 3. (C) S334ter-3 rat at P30: strong TUNEL stain in remnant of ONL. ONL reduced to 2–3 layers of nuclei. Faint stain of some cells in INL and GCL. (D) S334ter-5 rat at P30: Thicker ONL (5 layers of nuclei) - strong TUNEL stain of cells in ONL. In addition, some cells in GCL are also strongly TUNEL stained. Fainter stain of cells in INL and GCL. Magnification bars = 20 μm