Figure 1.
Cross sections of fixed tissue show type 7 cone bipolar cells express GFP in the retinas of age-matched transgenic 357 mice and 357/rd1 mice.
Type 7 bipolar cells (A and B, green, solid arrows) are seen in low power images, whose axonal arbors terminated in the OPL and dendritic arbor (open arrows) stratified in the stratum 4 of the IPL. Solid arrowheads point to faint labeling of rod bipolar cells, whose axonal arbors (A and B, open arrowheads) stratified in the stratum 5 of the IPL. To label the nuclear layers, we stained cell nuclei with diamidinophenylindole (DAPI) (A, blue). The ONL in the retina of 357/rd1 mice was reduced to a single row by P30 (B, Blue). ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bar, 20 µm.
Figure 2.
The correlation of the time course of cone degeneration and type 7 cone bipolar development in rd1 mice.
Counterstaining of retinal sections with antibodies against red/green opsins and Ctbp2 shows cone photoreceptors (blue) and synaptic ribbons (red) in the both OPL and IPL. Type 7 cone bipolar cell dendrites invaded cone terminals to form ribbon synapse complex starting from P8. By P10, a high density of synaptic ribbons were present in the OPL of WT mice (A, red, arrowheads), while few were observed in the OPL of rd1 mice (B, red). Insets illustrate highly magnified image from the boxed regions. Cone photoreceptors did not appear to have a normal morphology by this age (B, blue) compared with their counterparts in WT (A, blue). At P14 (C, D), some cone photoreceptors lost OS and IS, axons and axonal terminals became much smaller in rd1 retinas (D, blue) compared to their counterparts in WT (C, blue). Synaptic ribbons were barely observed in the OPL (D, red, arrowheads). By P21 (E, F), most cones lost OS, IS and axonal terminals, and the absolute number of S cones was decreased (F, blue). Synaptic ribbons were absent from the OPL by this age (F, red, arrewheads). Type 7 cone bipolar cells appeared identical in morphology between WT mice and rd1 mice at each of age-matched retinas (green). ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer; ONH, optic nerve head; OS, outer segment; IS, inner segment. Scale bar, 20 µm.
Figure 3.
Development of dendrites, axon terminals and a regular organization of type 7 bipolar cells in rd1 mice.
A–B, The distributions of somata and the dendritic tilings of type 7 bipolar cells in the flat-mounted retinas of 357 (A) and 357/rd1 (B) mice. The somata of type 7 bipolar cells showed regular distributions, and the dendrites of type 7 bipolar cells tiled the retinal surface with little overlap in both 357 (A) and 357/rd1 (B) retinas. The boundaries of individual dendritic fields are shown by white polygons. Type 7 bipolar cells have similar dendritic and axonal arbors in WT (A, C) and rd1 retinas (B, D). E, Analysis of the spatial distribution of type 7 bipolar cell somata in rd1 retinas: density recovery profile. Three retinas from 3 mice were measured (mean ± s.d.). An exclusion zone is present (arrow), indicating that type 7 bipolar cells are prevented from occupying nearby positions on the retina – they form regular mosaics. Dotted line indicates the random distribution of the same number of cells. F, Histogram showing the average diameters of both dendritic and axonal arbors of type 7 bipolar cells. Twenty cells from 5 mice were measured in each age group (mean ± s.d.). ns, not significant. Scale bar, 5 µm.
Figure 4.
Horizontal views of the dendritic arbors of type 7 bipolar cells in flat-mounted rd1 retinas.
A–B, High-power images show the whole dendritic arbors of individual type 7 cone bipolar cells, illustrating a progressive dendritic retraction with growing age. E, Histogram showing the average diameter of dendritic arbors at different stages of degeneration. Twenty cells from 4 mice were measured in each age group (mean ± s.d.). F, Histogram showing the average diameter of axon arbors at different stages of degeneration. Twenty cells from 4 mice were measured in each age group (mean ± s.d.). G. Gradual loss of synaptic ribbons in the axonal arbors of individual type 7 cone bipolar cells in the IPL of rd1 retinas. Six axonal terminals from 3 mice were measured in each age group (mean ± s.d.). ns, not significant, * = P<0.05, ** = P<0.01, *** = P<0.001, one-way ANOVA. Scale bar, 5 µm.
Figure 5.
Different degeneration patterns of cone photoreceptors and type 7 bipolar cells in rd1 mice.
Counterstaining of retinal sections with an antibody against red/green opsins shows cone photoreceptors (red). A–C, Images were taken from three dorsal regions (M) of 357 mice at 1 month old, illustrating normal densities and morphologies of both type 7 cone bipolar cells (green) and cone photoreceptors (red). D–L, Representative images were taken from three dorsal regions of rd1 retinas at three different ages. Insets illustrate highly magnified image from the boxed regions. Cone photoreceptors degeneration shows a central-to-peripheral progression (from D to F; G to I; J to L). Cone bipolar cell in the same regions, however, did not follow this spatiotemporal degeneration pattern (from D to F; G to I; J to L). ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer; ONH, optic nerve head. Scale bar, 20 µm.
Figure 6.
Uniform loss of type 7 cone bipolar cells across rd1 retinas.
Counterstaining of retinal sections with an antibody against PKCα shows rod bipolar cells (red). A–C, Images were taken from one dorsal region of a WT 357 mouse retina at 1 year old, illustrating normal densities and morphologies of type 7 cone bipolar cells (A) and rod bipolar cells (B). C, The merged image of A and B. Cell nuclei were stained with DAPI (blue). D–L, Representative images were taken from three dorsal regions of rd1 retinal sections (M). Rod bipolar cell degeneration showed a central-to-peripheral progression (from K to H to E). The degeneration of type 7 cone bipolar cell in the same retinal regions, however, did not follow this spatiotemporal pattern (from J to G to D), and cell loss was at the similar rate across three different eccentricities. Cell nuclei were stained with DAPI in merged images (F, I, L, blue). ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer; ONH, optic nerve head. Scale bar, 20 µm.
Figure 7.
Uniform loss of type 3 OFF and 5 ON cone bipolar cells across rd1 retinas over age.
A–B, Calcium-binding protein 5 (Cabp5), is expressed by three subsets of bipolar cells (type 3 OFF and type 5 ON cone bipolar cells, and rod bipolar cells). Their axonal terminals stratified in the stratum 2 (type 3 OFF bipolars) (A, red, blue open arrowheads), the stratum 3 (type 5 ON bipolars) (A, red, red open arrowheads) and the stratum 5 (rod bipolars) (A, red, green open arrowheads) of the IPL, respectively. Cabp5 was not detected in type 7 cone bipolar cells (A, B, arrows), but was detected in rod bipolar cells (A, solid arrowheads). The density of cabp5 positive cells was decreased with age (A, B). C, The graph shows a gradual reduction in the densities of both type 3 OFF and type 5 ON bipolar cells over time. Cells were counted in the dorsal retinas of 8 retinal sections from 8 mice in each age group (mean ± s.d.). D, The graph shows a gradual decline in the densities of type 7 bipolar cells with age (mean ± s.d.). Cells were counted in the dorsal retinas of 8 flat mounted retinas along dorsoventral axe from 8 mice in each age group (mean ± s.d.). OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bar, 10 µm.