Rod photoreceptors protect from cone degeneration-induced retinal remodeling and restore visual responses in zebrafish

Abstract

Humans are largely dependent upon cone-mediated vision. However, death or dysfunction of rods, the predominant photoreceptor subtype, results in secondary loss of cones, remodeling of retinal circuitry, and blindness. The changes in circuitry may contribute to the vision deficit and undermine attempts at restoring sight. We exploit zebrafish larvae as a genetic model to specifically characterize changes associated with photoreceptor degenerations in a cone-dominated retina. Photoreceptors form synapses with two types of second-order neurons, bipolar cells, and horizontal cells. Using cell-specific reporter gene expression and immunolabeling for postsynaptic glutamate receptors, significant remodeling is observed following cone degeneration in the pde6c(w59) larval retina but not rod degeneration in the Xops:mCFP(q13) line. In adults, rods and cones are present in approximately equal numbers, and in pde6c(w59) mutants glutamate receptor expression and synaptic structures in the outer plexiform layer are preserved, and visual responses are gained in these once blind fish. We propose that the abundance of rods in the adult protects the retina from cone degeneration-induced remodeling. We test this hypothesis by genetically manipulating the number of rods in larvae. We show that an increased number and uniform distribution of rods in lor/tbx2b(p25bbtl) or six7 morpholino-injected larvae protect from pde6c(w59)-induced secondary changes. The observations that remodeling is a common consequence of photoreceptor death across species, and that in zebrafish a small number of surviving photoreceptors afford protection from degeneration-induced changes, provides a model for systematic analysis of factors that slow or even prevent the secondary deteriorations associated with neural degenerative disease.

Figure 1.

Development of ON bipolar cell contacts with rod and cone photoreceptors. YFP expression by ON bipolar cells (green) at 3 dpf (a, b) and 4 dpf (c, d), zpr-1 immunolabeling for cones (red; a, c), and 4c12 immunolabeling for rods (red; b, d). a, b, At 3 dpf, association of the dendritic arbor of individual bipolar cells with cone and rod terminals can be observed. c, At 4 dpf, cone-bipolar cell invaginating synapses form lock and key configurations that span the OPL (arrowheads). d, Mixed ON bipolar cell dendrites also contact rod spherules (open arrowheads). Scale bar, 5 μm.

Figure 2.

Dendritic remodeling in pde6c^w59 larvae. YFP expression by ON bipolar cells (green) at 6 dpf (a–d), zpr-1 immunolabeling for cones (red; a, b), and 4c12 immunolabeling for rods (red; c, d). a, c, The uniform arrangement of cone (a) and rod (c) bipolar cell synapses is evident in WT retinas. b, OPL is remodeled in pde6c^w59 retinas. Ectopic dendritic processes project into the ONL (open arrowheads). d, OPL organization is not altered with rod loss in Xops:mCFP^q13 retinas. e, Number of ectopic dendrites per retinal section is significantly greater in pde6c^w59 versus WT retinas 5–8 dpf. f, Length of ectopic dendrites in pde6c^w59 retinas is significantly longer than in WT retinas only at 5 dpf. WT (n = 4), pde6c^w59 (n = 8), Xops:mCFP^q13 (n = 7–8). *p < 0.05; X̄ ± SD. Scale bar, 5 μm.

Figure 3.

Aberrant axon projections in pde6c^w59 larval retinas. YFP expression by ON bipolar cell axons (green; a–f) and Syto61 nuclear labeling of the GCL (red; a–f). WT (a, b) and Xops:mCFP^q13 (e, f) retinas maintain well defined IPL lamination at 6 (a, e) and 8 (b, f) dpf. c, d, Axon terminals of pde6c^w59 retinas extend ectopic processes into the GCL (arrowheads) shown here at 6 and 8 dpf, respectively. g, Number of ectopic axonal processes per retinal section is significantly greater in pde6c^w59 versus WT retinas consistently through 5–8 dpf. No significant difference is obtained between Xops:mCFP^q13 and WT ectopic processes. h, Ectopic processes in pde6c^w59 retinas are significantly longer than those in WT retinas at 8 dpf. No differences are detected between WT and Xops:mCFP^q13 retinas. WT (n = 4), pde6c^w59 (n = 8), Xops:mCFP^q13 (n = 7–8). *p < 0.05; X̄ ± SD. Scale bar, 5 μm.

Figure 4.

Altered glutamate receptor distribution in larval pde6c^w59 retinas. Merged images of YFP expression by ON bipolar cells (green; a–f), GluR4 immunolabeling on OFF bipolar cells (red; a–c), and GluR2 labeling on horizontal cell dendrites (red; d–f). a, GluR4 puncta cluster at the proximal portions of ON bipolar cell processes in the WT OPL (arrowhead) and their distribution is disrupted and decreased in pde6c^w59 retinas (b). c, Rod loss elicits no change in GluR4 labeling (arrowhead). d, In WT larvae, GluR2 immunolabeling appears as rosettes distal to the bipolar cell process (arrowhead). e, pde6c^w59 retinas display decreased amounts of rosettes and gaps in immunolabeling. f, Xops:mCFP^q13 shows uniformly distributed rosettes throughout the OPL comparable to the WT GluR2 distribution (arrowhead). Insets show individual labels. Scale bar, 2 μm.

Figure 5.

Preservation of photoreceptor-bipolar cell synapses in the pde6c^w59 and Xops:mCFP^q13 adults. YFP expression by mixed ON bipolar cells (green; a, c, e; gray; b, d, f) and zpr-1 immunolabeling of cones (red; a, c, e). a, In WT retinas, YFP expression and zpr-1-labeled cones form contacts (arrowhead). b, Image of dendritic arbor of an individual bipolar cell with longer dendrites that putatively contact rod photoreceptors (a, arrows) resulting in a bushy appearance. c, Cone-ON bipolar cell invaginating synapses are maintained in pde6c^w59 retinas (arrowheads) and arbors exhibit a bushy appearance. d, Individual arbor from a pde6c^w59 retina showing preservation of dendritic processes as well as collateral processes. e, ON bipolar dendritic knobs invaginate cone pedicles throughout the Xops:mCFP^q13 OPL (arrowhead). f, Loss of rod-bipolar synapses confines branching of the Xops:mCFP^q13 ON bipolar dendritic tree. Scale bars: (in e) a, c, e, 10 μm; (in f) b, d, f, 5 μm.

Figure 6.

Preservation of photoreceptor-horizontal cell synapses in pde6c^w59 and Xops:mCFP^q13 adults. Ribeye-b labels photoreceptor terminals (green). GluR2 labels horizontal cell processes (red). a, In the WT OPL, colabeling for GluR2 and Ribeye-b reveals isolated 1 μm puncta at rod spherules (arrowheads) and 2.5 μm rosette structures at cone pedicles (arrows). b, Rod-horizontal cell synapses are maintained in the pde6c^w59 OPL (arrowheads). c, The Xops:mCFP^q13 OPL only contains rosette synaptic complexes representing cone-horizontal cell synapses (arrow). Scale bar, 5 μm.

Figure 7.

Preservation of visual responses in pde6c^w59 adults. WT and pde6c^w59 adults show robust phototaxis (73.9 and 79.6%, respectively). Adults exhibiting rod and cone degeneration (pde6c^w59/Xops:mCFP^q13, 51.3%) or following optic nerve transection (ON, 46.8%) show no preference. Light preferences of the WT and pde6c^w59 groups do not significantly differ from one another, but are significantly greater than those of pde6c^w59/Xops:mCFP^q13 and ON animals. Shown are the data for one of the two trial days. WT (n = 8), pde6c^w59 (n = 4), pde6c^w59/Xops:mCFP^q13 (n = 8), ON (n = 3). *p < 0.05; X̄ ± SEM.

Figure 8.

Protection of mixed ON bipolar morphology in pde6c^w59/six7-MO larvae. Syto61 nuclear label (gray), 4c12 immunolabeling for rods (red), and YFP expression by ON bipolar cells (green). a, Syto61 labeling in WT larvae shows the lamination of the outer retina. Few rods are present in the WT central retina, and YFP distribution highlights the continuous OPL and knob-like processes. b, Rod photoreceptor cell number is increased but laminar organization is maintained in six7 morphants. c, ONL is lost and lamination is disrupted in pde6c^w59 larvae; the remaining rods are dysmorphic and ON bipolar dendritic processes are disorganized. d, An ONL forms from a single layer of rods and lamination is preserved in pde6c^w59/six7-MO larvae. e, Number of ectopic dendrites (white bars) (p < 0.001) and ectopic axon terminal processes (black bars) (p < 0.001) is significantly different between WT and pde6c^w59 retinas. The number of ectopic processes is significantly reduced in pde6c^w59/six7-MO compared with pde6c^w59 retinas. f, No significant differences are observed in the length of ectopic processes among any of the experimental groups. All groups n = 4. *p < 0.05; X̄ ± SD. Scale bar, 5 μm.