Progression of neuronal and synaptic remodeling in the rd10 mouse model of retinitis pigmentosa

Abstract

The Pde6b(rd10) (rd10) mouse has a moderate rate of photoreceptor degeneration and serves as a valuable model for human autosomal recessive retinitis pigmentosa (RP). We evaluated the progression of neuronal remodeling of second- and third-order retinal cells and their synaptic terminals in retinas from Pde6b(rd10) (rd10) mice at varying stages of degeneration ranging from postnatal day 30 (P30) to postnatal month 9.5 (PNM9.5) using immunolabeling for well-known cell- and synapse-specific markers. Following photoreceptor loss, changes occurred progressively from outer to inner retina. Horizontal cells and rod and cone bipolar cells underwent morphological remodeling that included loss of dendrites, cell body migration, and the sprouting of ectopic processes. Gliosis, characterized by translocation of Müller cell bodies to the outer retina and thickening of their processes, was evident by P30 and became more pronounced as degeneration progressed. Following rod degeneration, continued expression of VGluT1 in the outer retina was associated with survival and expression of synaptic proteins by nearby second-order neurons. Rod bipolar cell terminals showed a progressive reduction in size and ectopic bipolar cell processes extended into the inner nuclear layer and ganglion cell layer by PNM3.5. Putative ectopic conventional synapses, likely arising from amacrine cells, were present in the inner nuclear layer by PNM9.5. Despite these changes, the laminar organization of bipolar and amacrine cells and the ON-OFF organization in the inner plexiform layer was largely preserved. Surviving cone and bipolar cell terminals continued to express the appropriate cell-specific presynaptic proteins needed for synaptic function up to PNM9.5.

Figure 1. VGluT1 expression in C57BL/6 and rd10 retina

A, D: In the retinas from wild-type C57BL/6 (c57) mice at postnatal day 30 (P30, panel A) and postnatal month 9.5 (PNM9.5, panel D), VGluT1 antibodies label rod and cone terminals in the outer plexiform layer (OPL) (arrowhead) and bipolar cell terminals in the inner plexiform layer (IPL). Large, lobular rod bipolar cell terminals in the innermost IPL are prominently labeled (arrow). B, C, E, F: VGluT1 expression in the rd10 retina from P30 to PNM9.5. B. At P30, prominent gaps between VGluT1-positive photoreceptor terminals are present in OPL. Rod bipolar cell terminals in the IPL (arrow) are less distinctive than in the wild-type retina. C. At P50, VGluT1 staining in the OPL is reduced (arrowheads). The spacing and morphology of labeled terminals indicates that primarily cone pedicles remain at this time point. E. At PNM3.5, VGluT1 staining in the rd10 OPL labels the few surviving cone terminals (arrowheads). In the IPL, no morphologically distinct rod bipolar cell terminals are discernable. F. At PNM9.5, little to no expression of VGluT1 is detected in the OPL; VGluT1 is still present in the IPL. Scale bar = 20 μm, all panels.

Figure 2. PKCα and VGluT1 double labeling of C57BL/6 and rd10 retinas

A, C, E: C57BL/6 (c57) retina at PNM3.5. A. Rod bipolar cells labeled with antibodies against protein kinase C-α (PKC) show normal morphology. C. VGluT1 labels photoreceptor terminals in the OPL (arrow) and bipolar cell terminals in the IPL. E. Merged image of PKC and VGluT1 labeling. PKC-positive rod bipolar cell dendrites extend into the plexus of VGluT1-positive photoreceptor terminals in the OPL. Rod bipolar cell terminals in the IPL show strong labeling for VGluT1. B, D, F: rd10 retina at PNM3.5. B. In the rd10 retina, rod bipolar cells have retracted their dendrites and have disorganized axon terminals, with some ectopic processes (arrowheads). Surviving rod bipolar cell bodies are found in clusters (arrows). D. Scattered VGluT1-positive cone terminals remain in the OPL at PNM3.5. F. Merged image of PKC and VGluT1 labeling. Rod bipolar cell bodies cluster near the remaining VGluT1-positive cone terminals in the OPL (arrows). Rod bipolar cell terminals in the inner IPL show reduced labeling for VGluT1, with ectopic processes lacking VGluT1 staining (arrowheads). Scale bar = 20 μm, all panels.

Figure 3. Immunostaining of ON-cone and rod bipolar cells in C57BL/6 and rd10 retinas

A–C: C57BL/6 (c57) retina at P30. A. Gγ13 labeling of dendrites, cell bodies and axon terminals of all ON-bipolar cells (rod and cone). Large, lobular rod bipolar cell terminals in the innermost IPL are intensely labeled. B. Antibodies against vesicular glutamate transporter 1 (VGluT1) label the axon terminals of rod and cone photoreceptors in the OPL and both ON and OFF bipolar cells in the IPL. C. Overlay of panels A and B showing ON-OFF stratification in the IPL. Terminals of OFF-cone bipolar cells in the distal IPL are VGluT1-positive, Gγ13-negative. Terminals of ON-cone and rod bipolar cells are VGluT1-positive and Gγ13-positive and can be distinguished by size and position in the inner IPL. D–F: C57BL/6 retina at PNM9.5. The labeling patterns for Gγ13 (D) and VGluT1 (E) remains unchanged. F. Overlay of D and E. G–I: rd10 retina at P30. G. Labeling for Gγ13 shows abnormally thickened dendritic processes in the OPL (arrow). ON bipolar cell terminals stratify in the correct sublamina of the innermost IPL. H. A discontinuous band of VGluT1 immunoreactivity is still present in OPL. VGluT1-positive bipolar cell terminals are present throughout the IPL but the rod bipolar terminals are less distinctive than in the wild-type retina. I. Overlay of Gγ13 and VGluT1 labeling shows proper lamination of ON and OFF bipolar cell terminals in the IPL. J–L: rd10 retina at PNM9.5. J. Gγ13-positive dendritic processes are generally absent, although some dense areas of staining persist (arrow). Most ON bipolar cell terminals stratify properly within the ON sublamina of the IPL, although some ectopic processes are present at the INL/IPL border and extend into the ganglion cell layer (GCL) (arrowheads). K. Few sites of VGluT1 labeling remain in the OPL (arrow). Bipolar cell terminals throughout the IPL show VGluT1 labeling but rod bipolar cell terminals are not morphologically identifiable. L. Overlay of Gγ13 and VGluT1 labeling. In the OPL, areas of dense Gγ13 staining are associated with remaining VGluT1-positive puncta (arrow). In the IPL, Gγ13 and VGluT1 co-localize in ON bipolar cell terminals and the ON and OFF stratification remains largely intact. Many ectopic Gγ13-positive processes (arrowheads) also show VGluT1 labeling. M–O: Retinas from C57BL/6 at PNM9.5 immunostained for (M) Gγ13, showing all ON bipolar cells and (N) PKC, showing rod bipolar cells. O. An overlay of M and N distinguishes rod and ON cone bipolar cells. P–R: Retinas from rd10 mice at PNM9.5 immunostained for (P) Gγ13, and (Q) PKC. Ectopic processes in the INL show strong Gγ13 labeling and faint PKC labeling (arrows). R. Overlay of P and Q showing clusters of rod and ON-cone bipolar cells in the INL. The appropriate stratification of rod bipolar and ON-cone bipolar cell terminals in the IPL is still apparent. Scale bar = 20 μm, all panels.

Figure 4. Calbindin immunostaining in C57BL/6 and rd10 retinas

A, D: C57BL/6 (c57) retina at P30 (A) and PNM9.5 (D). Antibodies against calbindin label horizontal cells and their fine processes associated with the terminals of photoreceptors (arrowheads) in the OPL and a subset of amacrine cells and their terminals in the INL and IPL. B, C, E, F: rd10 mouse retina. B. At P30, during early stages of degeneration, puncta associated with horizontal cell dendrites and axon terminals are reduced but still present (arrowhead). Some horizontal cells sprout ectopic processes (arrows). C. At P50, the puncta associated with fine processes of horizontal cells are no longer detected. E. At PNM3.5, staining of horizontal cell lateral processes in OPL is greatly reduced. F. At PNM9.5, gaps are visible in the calbindin-positive horizontal cell plexus in the OPL. Ectopic processes from horizontal cells are still detected (arrows). Amacrine cells show minimal changes and the stratified organization of amacrine cell processes in the IPL is preserved. Scale bar = 20 μm, all panels.

Figure 5. Immunostaining for calbindin, syntaxin 4 and VGluT1 in the C57BL/6 and rd10 retinas at late stages of degeneration

A, D, G, J, M: C57BL/6 (c57) retina at PNM9.5. A. Calbindin labeling. D. Syntaxin 4 labeling of the fine dendrites of horizontal cells and a sparse plexus in the IPL. G. VGluT1 labeling. J. Overlay of panels D and G showing the direct apposition of presynaptic VGluT1 expression with postsynaptic syntaxin 4 expression in the OPL. M. Overlay of panels A, D, and G showing the normal relationship of presynaptic VGluT1 and syntaxin 4 in fine horizontal cell processes and the remainder of the horizontal cell plexus in the OPL. B, E, H, K, N: rd10 retina at PNM3.5. B. Calbindin labeling reveals horizontal cell process loss. E. Syntaxin 4 labeling in the OPL is restricted to small, widely spaced patches (arrows), but remains essentially normal in the IPL. H. Restricted sites of VGluT1 labeling in the OPL (arrows). K. Overlay of panels E and H showing direct apposition of syntaxin 4 and VGluT1 labeling in the OPL. N. Overlay of panels B, E, and H, shows the relationship of presynaptic VGluT1, syntaxin 4 and the horizontal cell plexus in the OPL. C, F, I, L, O: rd10 retina at PNM9.5. C. Calbindin labeling reveals a severe reduction of horizontal cell processes at PNM9.5. F. Patches of syntaxin 4 labeling in the OPL become smaller at PNM9.5 in the rd10 retina (arrows). I. Surviving cone terminals in the PNM9.5 rd10 OPL express VGluT1. L. Overlay of panels F and I showing juxtaposition of syntaxin 4 and VGluT1 labeling in the OPL. O. Overlay of panels C, F, and I, showing the close physical relationship between syntaxin 4 expression, the horizontal cell plexus and surviving terminals of cones in the OPL at late stages of degeneration. Scale bar = 20 μm, all panels.

Figure 6. Stratification of amacrine cell processes in the plexiform layers of C57BL/6 and rd10 retinas at PNM9.5

A. C57BL/6 (c57) retina immunostained with antibodies against the vesicular glutamate transporter 3 (VGluT3) reveals stratification of putative glutamatergic amacrine cells in the middle of the IPL. B. In the rd10 retina, VGluT3-positive amacrine cells retain normal stratification in the middle of the IPL. C. In the c57 retina, antibodies against the 65kDa isoform of glutamic acid decarboxylase (GAD-65) label 3 distinct strata containing the terminals of GABAergic amacrine cells. Arrowheads indicate the location of the GAD-65-negative processes of starburst amacrine cells. D. In the rd10 retina, the normal stratification of GAD-65-positive terminals in the IPL is present at PNM9.5. E. Overlay of VGluT3 (A) and GAD-65 (C) immunolabeling in the wild-type c57 retina shows stratification of the VGluT3 amacrine cells in the IPL (arrow) between the GAD-65-negative, starburst amacrine cell strata (arrowheads). F. Overlay of VGluT3 (B) and GAD-65 (D) immunolabeling in the rd10 retina showing the continued stratification of VGluT3 amacrine cell processes in the IPL at PNM9.5. G. Wild-type c57 mouse retina at PNM9.5, immunostained with antibodies against tyrosine hydroxylase (TH, green), and calretinin (magenta). Antibodies against TH label dopaminergic amacrine cells and their processes along the INL/IPL border (arrow). Antibodies against calretinin labels amacrine cells and their processes and reveal three distinct strata in the IPL (arrowheads). The upper and lower strata correspond to the processes of the OFF and ON starburst amacrine cells, respectively. Calretinin labeling also is present in cells in the ganglion cell layer and ganglion cell axons. H. Double labeling for TH and calretinin in the rd10 retina at PNM9.5. Despite some disorganization, the TH-positive processes of the dopaminergic amacrine cells are still located along the INL/IPL border (arrow). Three strata of calretinin-positive amacrine cell processes are still present in the IPL. However, the OFF starburst amacrine cell stratum appears more diffuse. Labeling of cells and axons in the GCL appears normal. Scale bars = 20 μm, all panels.

Figure 7. Immunostaining for PKC, SNAP-25 and VGluT1 in the C57BL/6 and rd10 retina

A, C, E, G: Wild-type C57BL/6 (c57) retina at PNM9.5. A. Antibodies against protein kinase C-α (PKC) identify rod bipolar cells, which show normal morphology and stratification in the IPL. C. In c57 retina, the presynaptic SNARE protein SNAP-25 is found in the conventional and ribbon synapses of the OPL and IPL. E. VGluT1 expression in the C57BL/6 retina. G. Overlay of panels A, C and E. B, D, F, H: rd10 retina at PNM9.5. B. Although many rod bipolar cells are lost by PNM9.5 and most surviving rod bipolar cells have retracted their dendrites, rod bipolar cells located near surviving cone terminals extend abnormal, thickened dendrites (arrow) toward the surviving cone terminals (asterisk, see panel F). Many ectopic rod bipolar cell bodies (small arrowheads) and processes (circle) are present. Note the sparse plexus of rod bipolar cell terminals in the IPL. D. SNAP-25 labeling is highly elevated in the OPL but appears unchanged in the IPL. The cellular origin of the highly SNAP-25-positive processes in the outer retina is uncertain. F. A single VGluT1-positive terminal remains in this region of the OPL (asterisk). H. Overlay of panels B, D and F. Abnormal PKC-positive rod bipolar cell dendrites extend toward the surviving VGluT1-positive terminal (asterisk). Scale bar = 20 μm, all panels.

Figure 8

Glutamate receptors are progressively lost from horizontal cells in the rd10 retina. A-C: GluR4 localizes to the tips of horizontal cell dendrites in the wildtype retina at PNM9.5. A. GluR4 receptor expression in the OPL in the wildtype C57BL/6 retina. B. Calbindin labeling in horizontal cells in the wildtype C57BL/6 retina. C. Overlay of GluR4 and calbindin labeling. GluR4 often localizes to the fine dendritic tips of horizontal cells. D-F: GluR4 localization in the rd10 OPL at PN50. D. GluR4 expression in the rd10 retina at PN50. At this age, GluR4 receptors are decreased in number and often appear in discreet clusters (arrows) that likely correspond to the location of cone terminals. E. The fine dendritic tips of horizontal cells have largely disappeared in the rd10 retina at PN50, but the thicker processes within the proximal OPL remain. F. Overlay of GluR4 and calbindin labeling. The clusters of remaining GluR4 receptors are localized to the same region of the OPL occupied by the thick processes of the horizontal cells. G-I: GluR4 localization in the rd10 OPL at PNM9.5. G.GluR4 expression is drastically reduced in the OPL of the rd10 retina at PNM9.5. The remaining GluR4 are found in small aggregates (arrow). H. Horizontal cell processes are dramatically reduced in the rd10 retina at PNM9.5. I. The remaining GluR4 expression corresponds to some of the remaining horizontal cell processes (arrow). Scale bar = 20 μm, all panels.

Figure 9

Surviving photoreceptors promote local rod bipolar cell survival and remodeling of rod bipolar cell dendrites. A, C, E, G: Triple labeling for PKCα, SNAP-25 (a ubiquitous synaptic SNARE protein) and VGluT1 in the wildtype C57BL/6 retina at PNM9.5. A. PKCα expression identifies rod bipolar cells, which show normal morphology and stratification in the IPL. C. In the wildtype C57BL/6 retina, the presynaptic SNARE protein SNAP-25 is found in conventional and ribbon synapses in both plexiform layers. E. VGluT1 expression in the C57BL/6 retina. G. Overlay of labeling for PKCα, SNAP-25 and VGluT1 in the wildtype C57BL/6 retina. B, D, F, H: Triple labeling for PKCα, SNAP-25, and VGluT1 in the rd10 retina at PNM9.5. B. Although many rod bipolar cells are lost by PNM9.5 and most surviving rod bipolar cells have retracted their dendrites, those rod bipolar cells located near surviving cone terminals extend abnormal, thickened dendrites (arrow) toward the surviving cone terminals (asterisk, see panel F). Many ectopic cell bodies (arrowheads) and axon terminals are also present (circle). Note the sparse plexus of rod bipolar cell terminals in the IPL. D. SNAP-25 labeling appears normal in the IPL, but is highly elevated in the OPL of the rd10 retina at PNM9.5. F. A single surviving cone terminal expressing VGluT1 is present in this region of the OPL (asterisk). Labeling for VGlut1 is present in bipolar cell terminals in the ON and OFF sublaminae of the IPL. H. Overlay of labeling for PKCα, SNAP-25 and VGluT1 in the rd10 retina reveals abnormal rod bipolar cell dendrites extending toward the surviving cone terminal (asterisk). The cellular origin of the highly SNAP-25 positive processes in the outer retina is unclear. Given the dearth of photoreceptors at this late stage of degeneration, the labeling does not arise from photoreceptor terminals. Comparison of SNAP-25 and PKCα labeling indicates that elevated SNAP-25 is not associated specifically with the abnormal rod bipolar cell dendrites. Scale bar = 20 μm, all panels.

Figure 10

Co-localization of Syntaxin 3, a ribbon synapse-specific SNARE protein, and VGluT1 is progressively reduced in photoreceptor terminals but is largely retained in bipolar cell terminals in the rd10 retina. A-C: Expression of synaptic proteins associated with the glutamatergic ribbon synaptic terminals of photoreceptors and bipolar cells in the wildtype C57BL/6 mouse retina at PNM9.5. A. Syntaxin 3 in the wildtype retina is expressed exclusively in the ynaptic terminals of photoreceptors in the OPL and bipolar cells in the IPL. B. Similarly, VGluT1 in the wildtype retina is expressed exclusively in the synaptic terminals of photoreceptors in the OPL and bipolar cells in the IPL. C. Overlay of panels A and B. D-F: Localization of ribbon synapse associated proteins in the rd10 mouse retina at PN30. D. Syntaxin 3 expression in the rd10 retina at PN30. Surviving photoreceptor terminals (arrow) continue to express syntaxin 3, although the loss of terminals from the OPL is already apparent even at this early stage of degeneration. E. Surviving photoreceptor terminals also continue to express VGluT1. F. Overlay of panels D and E. The two proteins show a high degree of co-localization in both the OPL and IPL (arrow). G-I: Localization of ribbon synapse associated proteins in the rd10 mouse retina at PNM9.5. G. Syntaxin 3 expression persists in surviving cone terminals in the OPL and bipolar cell terminals in the IPL (arrowheads). H. VGluT1 expression also persists in surviving cone terminals in the OPL and bipolar cell terminals in the IPL. I. The merged image shows co-localization of syntaxin 3 and VGluT1 labeling in surviving cone terminals, however, VGLUT1 expression is reduced compared to syntaxin 3. Bipolar cell terminals continue to show extensive co-localization of syntaxin 3 and VGluT1 in the IPL. Scale bar = 20 μm, all panels.