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Retinitis Pigmentosa (RP), a family of inherited disorders characterized by progressive photoreceptor death, is a leading cause of blindness with no available cure. Despite the genetic heterogeneity underlying the disease, recent data on animal models show that the degeneration of photoreceptors triggers stereotyped remodeling among their postsynaptic partners. In particular, bipolar and horizontal cells might undergo dendritic atrophy and secondary death. The aim of this study was to investigate whether or not concomitant changes also occur in ganglion cells (RGCs), the only retinal projection neurons to the brain and the proposed substrate for various therapeutic approaches for RP.
We assessed the retention of morphology, overall architecture and survival of RGCs in a mouse model of RP at various stages of the disease. To study the morphology of single RGCs , we generated a new mouse line by crossing Thy1-GFP-M mice (Feng et al., 2000), which express GFP in a small number of heterogeneous RGCs types, and rd10 mutants, a model of autosomal recessive RP, which exhibit a typical rod-cone degeneration (Chang et al., 2002). We show remarkable preservation of RGC structure, survival and projections to higher visual centers in the time span from 3 to 9 months of life, well beyond the death of photoreceptors. Thus, unlike second order neurons, RGCs appear as a considerably stable population of cells, potentially constituting a favorable substrate for restoring vision in RP individuals by means of electronic prostheses or direct expression of photosensitive proteins.
Many common causes of blindness involve the death of retinal photoreceptors: in Retinitis Pigmentosa (RP) mutations, typically in photoreceptor specific genes, lead to progressive degeneration of rods and cones and gradual loss all useful sight. A strategy for improving vision in RP is the development of prostheses based upon retinal implants designed to electrically stimulate deep retinal layers and thereby taking over the function of lost photoreceptors (Zrenner, 2002; Loewenstein et al., 2004; Rizzo et al., 2007). Both subretinal and epiretinal electronic prostheses have the capability to stimulate retinal ganglion cells (RGCs) directly (i.e. Jensen et al., 2005; Jensen and Rizzo, 2006). Recently a newer strategy has led to recovery of some visual function in animal models of RP by transgenic expression of photosensitive molecules directly in bipolar and RGCs (Bi et al., 2006; Lagali et al., 2008; Lin et al, 2008).These strategies equally depend upon the viability and survival of inner retinal cells following photoreceptor death.
In both humans and animal models of RP, the inner retina remodels upon photoreceptor loss, undergoing regressive changes and secondary degeneration (Milam et al., 1998; Strettoi and Pignatelli, 2000; Strettoi et al., 2002, 2003; Marc et al., 2003; Jones et al., 2003; Cuenca et al., 2004; Jones and Marc, 2005; Gargini et al., 2007). The time course and sequence of events affecting bipolar, horizontal cells and retinal glia have been studied in some detail; however, effects on RGCs, the only retinal exit to the brain, are poorly understood, and few studies are available at the single cell level. Estimates of RGC survival in animal models have lead to somewhat contradictory results, while interpretation of human RP data is still difficult (Grafstein et al.,1972; Stone et al., 1992; Eisenfeld et al., 1984; Santos et al., 1997; Walia and Fishman, 2008). Functional studies have shown that RGCs might retain glutamate excitability even in the presence of alterations in input neurons implicating loss of glutamate-coupled depolarization mechanisms in cone bipolar cells (Marc et al., 2007). In a classical paradigm of RP, the rd1 mutant mouse, RGCs exhibit paroxysmal electrical activity after photoreceptor death (Stasheff, 2008). The aberrant activity seems generated by abnormal synaptic input, because the intrinsic membrane and firing properties of at least some types of RGCs appear preserved (Margolis et al., 2008).
Here, we report a longitudinal study of RGC structure and survival in the C57Bl/6J-Pde6brd10/J mouse, commonly known as rd10 mutant, which carries a nonsense mutation of the beta subunit of the rod-specific phosphodiesterase gene (Chang et al., 2002). Humans with similar mutations display autosomal recessive RP and this mouse can be considered a faithful model of the typical form of the disease (McLaughlin et al., 1993). Using a single cell approach, we show that all types of RGCs studied retain their dendritic architecture and overall viability, even after complete photoreceptor degeneration and regressive remodeling of the retina's second-order neurons.
Experimental procedures were in accordance with institutional guidelines and with the ARVO statement for the use of animals in research. All mice were kept in a local facility with water and food ad libitum, in a 12-hour light/dark cycle, with illumination levels below 60 lux.
C57Bl/6J-Pde6brd10/J mutants (from now on, rd10 mice) and C57Bl/6J wild-type controls (wt) were obtained from the Jackson Laboratories, Bar Harbor, ME, USA. Mice of the B6.Cg-Tg(Thy1-GFP-M)JRS/RHM strain were a kind gift from R.H. Masland, Boston, MA, USA and were homozygous for the Thy1-GFP allele. These were derived by breeding from the B6.Cg-Tg(thy1-YFP)/J strain originally devised by J. Sanes (Feng et al., 2000) and will be referred to as Thy1-GFP-M from now on. In these homozygous animals (Thy1-GFP/Thy1-GFP), a small number of RGCs (50-70 cells/retina) strongly express GFP. A total of 75 mice were used for this study. Transgenic mice of the Thy1-GFP-M strain, aged 3-9 months, were used as a set of founders. A new line of mice, Pde6brd10/rd10/Tg(Thy1-GFP-M)Jrs, from now on named rd10/Thy1-GFP-M, was obtained by crossing rd10 with Thy1-GFP-M mice. Thy1-GFP-M mice were first crossed with homozygous rd10/rd10 animals. Individuals obtained from the first generation (F1) were backcrossed with rd10/rd10 animals obtaining the F2. Genotyping was performed by PCR on tail-extracted DNA of F2 individuals to identify Thy1-GFP positive animals. The following primers were used:
Thy1-GFP F (AAGTTCATCTGCACCACCG) and Thy2-GFP R (TCCTTGAAGAAGATGGTGCG), following a protocol recommended by the Jackson Laboratories. The PCR amplification of the corresponding 173 base pair fragment was performed in 35 cycles by denaturation at 94 °C for 1.5 min; annealing at 94, 61 and 72 °C, respectively, for 30 s, 1 min and 1 min; and elongation at 72 °C for 2 min.
To identify mice homozygous for the rd10 mutation among Thy1-GFP-M positive individuals, a second PCR was performed. In this case, the primers were:
RD10-F (CTTTCTATTCTCTGTCAGCAAAGC) and RD10-R (CATGAGTAGGGTAAACATGGTCTG). The corresponding PCR amplification was performed in 30 cycles by denaturation at 94 °C for 3 min; annealing at 94, 60 and 72 °C, respectively, for 1′, 30″ and 1′ and elongation at 72 °C for 7′. The product obtained was purified and digested with the enzyme HhaI (New England BioLabs, Ipswich, MA, USA), whose restriction site is not included in the rd10 mutant DNA. The recognized sequence is: 5′…G/CGC…3′ and 3′…CGC/G…5′ (Chang et al., 2007). After 2-hrs incubation in the enzyme at 37 °C, the digested DNA was run upon a Metaphor agarose (Cambrex, NJ, USA) for separation of short DNA fragments. The homozygous rd10 mutation is revealed by the presence of a single band having a size of 97 base pairs.
rd10/Thy1-GFP-M mice, aged 3, 7 and 9 months and Thy1-GFP, control mice, aged 8-9 months, were anesthetized with intraperitoneal injections of Avertin (0,1ml/5g) and euthanized with an overdose of anesthetic upon eye removal. Eyes were quickly enucleated, a reference on the dorsal pole was taken with a lab marker and a small cut was made at the corneal margin prior to immersion in fixative (4% paraformaldheyde, PAF, in 0.1 M phosphate buffer, PB, pH 7.4) for 1 hr, at 4°C. Subsequently, the anterior segment of the eye was removed and the retina separated from the pigment epithelium (still maintaining a reference at the dorsal pole) and flattened by making 4 radial cuts toward the optic nerve head. Routinely, retinas were infiltrated for several hours in 30% sucrose in 0.1 M PB, frozen in OCT Tissue-Tek (Sakura, NL) and stored at −20 °C. Upon use, retinal samples were brought to room temperature, washed extensively in 0.1 M PB and left overnight in a solution with 0,5% Triton X-100, 10% rabbit serum, 5% BSA in phosphate-buffered saline (PBS; pH 7.4, Sigma-Aldrich, St. Louis, MO, USA) at 4°C. Then, retinas were incubated in a 1:500 solution of rabbit anti-GFP-Alexa Fluor 488 (Molecular Probes, Invitrogen, Milan, Italy), with 0,1% Triton X-100, 1% rabbit serum, 1% BSA in PBS, for two days at 4°C, to enhance the GFP signal. Retinal specimens were then rinsed 3 × 15′ in PBS and incubated in a solution of RNAse A (Invitrogen) (1:1000 in PBS) at 37°C for 1h. After rinsing in PBS, retinas were stained with 2 micromolar Ethidium homodimer-1 (Molecular Probes, Invitrogen) for 1h at room temperature on a rotary shaker. This allowed fluorescent staining of nuclei necessary to locate the boundaries between retinal layers. Finally, retinas were rinsed extensively in PBS and mounted on glass slides with Vectashield (H1000, Vector Laboratories, Burlingame, CA, USA), “ganglion cells up”. Retinal preparations were coverslipped, sealed with nail polish and inspected with a Zeiss Axioplan fluorescence microscope (Carl Zeiss, Milan, Italy), using 5x or 10x objectives. GFP-positive RGCs were localized; when necessary for cell retrieval, low power images of the whole retinas were taken with a Zeiss Axiocam color camera. Subsequently, well-isolated RGCs were scanned with a Leica TCS-NT confocal microscope (Leica Microsystem, Milan, Italy) equipped with an Argon-Krypton laser, at resolutions of 1024×1024 or 512×512 pixels. Images were obtained using a 25x PL FLUOTAR 0.75 oil or a 40x HCX PL APO 1.25 oil objectives. Z-stacks were obtained encompassing the optic fiber, GC , inner plexiform and innermost part of the inner nuclear layers. The distance between adjacent focal planes was set at a constant value (1,013 μm). Image files were saved in export format and analyzed off-line with Metamorph (v. 5.0r1 Metamorph, Universal Imaging, Inc., Downingtown, PA, USA), to perform 3D reconstructions of single cells and to measure their dendritic tree and body areas. A total number of 50 retinas from different animals were analyzed (see Table 1).
RGCs were classified following Sun et al. (2002). Accordingly, the parameters used were 1) the diameter of the dendritic tree. This was obtained measuring with Metamorph the area of the smallest 2D convex polygon traced along each dendritic tip on a projection of the dendritic arborization when collapsed along the z-axis. This measure was repeated 3 times for each cell; the average was taken as the area of the cell dendritic tree and then used to calculate the diameter, assuming a circular shape of the tree. 2) The diameter of the RGC body, measured after tracing the contour of the projection of the cell body, obtained from optimal, non saturated confocal images, usually separate from those used for 3-D reconstruction of the cell. 3) The mean stratification depth of the GC dendritic arborization within the inner plexiform layer (IPL), measured on orthogonal projections of the cells obtained from confocal z series, as reported in Badea and Nathans, 2004. 4) The shape of the dendritic arbor, according to the description of Sun et al. (2002), as well as following Lin et al., 2004 and Kong et al., 2005. This feature constitutes a blueprint of a cell type and allows the distinction among cells sharing some morphometric parameters. RGC types belonging to the most numerous groups and originating from retinas of different animals were selected for contour tracing and drawn along the whole z-stack with Neurolucida software (3.2 version, MicroBright Field, Inc. Williston, VT, USA). At least 4-6 cells from each type were traced. Neurolucida data were further analyzed with NeuroExplorer. To evaluate whether modifications in the dendritic complexity occurred as a function of the disease progression, 3 additional morphological parameters were considered: total dendritic length, total number of nodes and total dendritic tree area. Data were statistically evaluated with Origin (v.7SR1, OriginLab Corporation, MA, 01060, USA). A total of 595 RGCs were classified; of these, 165 were chosen for Neurolucida drawing. They cover 8 different types, of the 17 totally identified by Sun et al., 2002. A summary of the samples examined and of the total number of cells analyzed is reported in Table 1.
Retinal whole mounts obtained as above and counterstained with Ethidium homodimer-1 were used to estimate survival of cells in the GC layer (GCL) of rd10/Thy1-GFP-M and wt/Thy1-GFP-M aged 9 months. Four retinas from different animals were used for rd10/Thy1-GFP-M and 3 for control mice. Confocal microscopy was used to obtain serial optical sections at 1,013 μm intervals encompassing the thickness of the entire GCL, using a 40x objective as above. Sampling areas were 16 fields (250×250 micrometer) per retina, regularly spaced along the dorsal-ventral and nasal-temporal retinal meridians. Counts of cells were performed on extended-focus images of the GCL, covering an average thickness of 20 μm on the z plane. Endothelial and peri-vascular cells were excluded from the counts on the basis of their characteristic shape and high intensity of staining. Total numbers of cells per retina were obtained multiplying average cellular densities by corresponding retinal areas. These were measured on low magnification images obtained at the Zeiss light microscope with a Zeiss Axiocam camera. Statistical analysis (one-way ANOVA) on cell counts was performed with Origin 7.0.
Additional counts were performed on retinas from rd10/Thy1-GFP-M, rd10, and C57Bl6J animals aged 9 months (n=3 retinas for each strain). These were incubated as explained above but with goat polyclonal antibodies against Brn3b (Millipore Billerica, MA, USA), a transcription factor selectively expressed in a large fraction of adult RGCs.. Images of the GCL were obtained at the confocal microscope at peripheral and central retinal locations, using a 40x oil immersion objective. Cellular densities were estimated for each retinal sample and compared statistically by one way ANOVA.
This was studied in RGCs by injection of fluorescent B subunit of cholera toxin into the eyes of 7 rd10, 3 rd10/Thy1-GFP-M and 3 C57Bl6J wt mice aged 9 months. Animals were anesthetized as above; 0.5 μl of biotinilated cholera toxin B fragment (10mg/ml) (Sigma-Aldrich) were injected into the vitreous of the left eye; 0.5 μl of FITC-cholera toxin (1mg/ml, Alexa Fluor-488 conjugate, Invitrogen) were injected into the right eye. Injections were done under a high power dissecting microscope using a glass micropipette with a tip diameter of about 0.5 μm (Clark Electromedical Instruments, Pangbourne, GB), driven by a hydraulic micromanipulator. 24 hours after the injection, mice were perfused transcardially with 4% PAF in 0.1 M PB, pH 7.4. The brains were dissected and post-fixed in 4% PAF for 2 hours. After rinsing in buffer, brains were infiltrated overnight in 30% sucrose and frozen in dry ice. Coronal brain sections (40 μm thick) were obtained with a freezing sliding microtome. Selected sections containing the lateral geniculate nucleus (LGN) or the superior colliculus (SC) were incubated overnight in Cy3 conjugated avidin (1:500, Molecular Probes, Invitrogen), rinsed in PBS 3 times × 15′, mounted in glycerol and visualized at the confocal microscope. Acquisitions were done using 5x (N-PLAN 0.12) and 10x (N-PLAN 0.25) dry objectives. Brightness and contrast of images were adjusted with Adobe Photoshop CS.
There are 12-17 types of RGCs in the retina of the mouse: as in all mammals, these neurons can be distinguished on the basis of their morphological and functional features (Sun et al., 2002; Carcieri et al. 2003; Badea and Nathans, 2004; Kong et al., 2005; Coombs et al., 2006). To characterize the effects of retinal degeneration upon individual RGCs, we took advantage of the M-line of Thy1-GFP transgenic mice, in which the GFP transgene, controlled by thy-1 cassette (Caroni, 1997), is expressed in a small number of RGCs, so that these neurons are entirely labeled (axon, soma and dendrites) but rarely overlap (Feng et al., 2000). As previously reported, GFP labeling of RGCs is semi-random, in the sense that some cell types are encountered more frequently, irrespectively to their density in the retina.
We crossed homozygous Thy-1/GFP-M transgenics with rd10 mutant mice, a model of autosomal recessive human RP. rd10/Thy1-GFP-M mice displayed the same temporal and spatial pattern of photoreceptor degeneration described for the parent rd10 strain (Chang wet al., 2002; Gargini et al., 2007). Secondary changes detected in bipolar and horizontal cells also adhered strictly to what found in the original rd10 mutant. Photoreceptor degeneration and second order neuron alterations are summarized in supplemental Figure 1.
As shown before (Lin et al., 2004), different types of RGCs are encountered at various rates in the Thy1-GFP-M line, with no single retina exhibiting every existing type of RGC labeled. In the rd10/Thy1-GFP-M strain, the number of GFP-positive RGCs is lower that in the parent strain (approximately 30 cells/retina). While the origin of such variability is not clear, it appears that only the analysis of numerous retinal samples can give an account of most of the existing types of RGCs (Table 1).
The analysis of 469 RGCs from rd10/Thy1-GFP-M mutant mice belonging to the 3 age groups selected (3, 7 and 9 months of age) did not reveal obvious morphological abnormalities in any of them. Each cell could be appropriately assigned to a formerly described type as deduced by morphology, depth of stratification, dendritic and cell body area. In total, we studied the morphology of 8 types of RGCs (A1, A2 inner and outer, B1, B3 inner and outer, and C2 inner and outer). These include 4 types of cells ramified in the outer part of the IPL (functionally defined as “OFF” varieties) (Figure 1) and 4 types ramified in the inner part of the IPL (“ON” varieties) (Figure 2). Large, medium and small sized RGCs are comprised in the groups studied and some of these have homologues in other mammalian retinas. Their distinctive features are summarized in supplemental Tables 1 and 2, using the system of names provided by Sun et al., 2002.
A set of morphological aberrations have previously been described for bipolar and horizontal cells of the rd10 mutant (Gargini et al., 2007; Barhoum et al., 2008), as well as for RGCs of rodents with other retinal and systemic diseases (i.e. glaucoma and diabetes). These include: retraction and atrophy of dendrites (“pruning”), cell body shrinkage or hypertrophy, loss of laminar organization and degeneration (Jakobs et al., 2005; Gastinger et al., 2008; Kern and Barber, 2008). None of these were detected in any of the cells observed. Aberrant elongation of dendrites, axonal ectopic branching (“sprouting”), also observed in RGCs and other neurons of various disease models (Cheng et al., 1998; Phokeo et al., 2002; Pignatelli et al., 2004) were not detectable in the RGCs of the strain under study. All the cells examined were undistinguishable from those of mice without photoreceptor degeneration, as judged from comparisons with 126 RGCs labeled in adult Thy1-GFP-M mice that did not carry the rd10 mutation.
From all the RGCs labeled in rd10/Thy1-GFP-M mice, we selected a pool of 123 neurons belonging to the 8 types listed above for a computational analysis of the dendritic morphology and complexity as a function of age. Parameters for complexity assessment were the area of the dendritic tree, the total dendritic length and the total number of nodes. For each cell type, at each age, parameters were obtained from Neurolucida tracings and compared to those of corresponding RGCs (n=42) from adult, Thy1-GFP-M mouse retinas. All three indicators of dendritic tree complexity are highly sensitive to alterations in the architecture of arborizations: either sprouting or pruning is immediately reflected into changes in the parameters measured, commonly used to assess neuronal responses to injury, disease or environmental conditions (i.e. Alpàr et al., 2003). Fixing confidence interval at level of 0.01, statistic analysis by one-way ANOVA revealed that there were no significant differences in morphometric parameters among mutants of various age groups and between mutant and wt (p value for each analysis is reported in Supplemental Table 3). Measures remained identical to corresponding parameters of RGCs of the same types from adult retinas from Thy1-GFP-M mice. In turn, these conformed entirely to measurements from mouse RGCs of identical types available from the literature (Sun et al., 2002; Badea and Nathans, 2004; Coombs et al., 2006) (Figures (Figures33 and and4).4). Correlated parameters, such as dendritic density, defined as the ratio of the number of nodes to the dendritic tree area, were also maintained in each cell. For additional examples of RGCs classified for this study see Supplemental Figures 2, 3 and 4.
We conclude that RGCs in the rd10 mutant mouse retain their characteristic morphology and fine dendritic geometry well beyond the complete death of photoreceptors and despite major alterations of other retinal cell classes (Gargini et al., 2007).
We identified numerous RGCs with dendrites branching in both the inner and outer halves of the IPL; these are the well-known bistratified RGCs (n=32).They were assigned to either the D1 or D2 types (Table 1) following Sun et al. (2002). We could not assess the dendritic tree complexity because they show enormous heterogeneity in the size of both arborizations and in their branching depth in the IPL. However, no major deviations from known morphometric and morphological features were evident in these cells in the rd10/Thy1-GFP-M mutant retinas (see supplemental Figure 3).
Displaced RGCs were encountered in almost all the rd10/Thy1-GFP-M and control retinas examined, particularly in the nasal quadrant. For these rare neurons, Sun et al., (2002) did not provide a classification. Our sample included members of the monostratified, displaced (ON or OFF) types and, more rarely, exemplars of the bistratified type (ON-OFF). Their morphology appeared undistinguishable from their counterparts observed in Thy1-GFP-M retinas (see supplemental Figure 4). Finally, several cells defined by Sun et al., 2002 as “unclassified” were also encountered in Thy1-GFP-M and retinal degeneration mice of various ages (Table 1 and supplemental Figure 4). None of these cells showed signs of morphological abnormality.
We counted the nuclei of all the neurons in the GCL of animals aged 9 months, with the rationale that the oldest age would increase the chance of detecting secondary degeneration of RGCs. The number of cells in the GCL of rd10 mice (both rd10 and rd10/Thy1-GFP-M mutants) did not change in time and was statistically identical to that of wt, control animals (Figure 5, A-C). This finding is quite consistent, as we sampled a large fraction of the retinal surface (i.e. a total of 4mm2/ retina, or, approximately 28% of the total retinal area) and we took into account all the eccentricities. Since we this method we could not distinguish between amacrine and GCs, we stained rd10, rd10/Thy1-GFP-M and wt retinas, aged 9 months, with antibodies against Brn3b, a transcription factor expressed in the majority of adult RGCs (de Melo et al., 2003). Counting of Brn3b-positive profiles in retinal whole mounts returned statistically identical densities (α=0.01) of RGCs in mutant and wt retinas (Figure 5, D-G).
We concluded that survival and detailed morphology are excellent in RGCs of both rd10 and rd10/Thy1-GFP-M mutants
Axons of RGCs fasciculate along the optic nerves and tracts to reach higher visual centers. Almost all RGC axons project to the SC and about 30% send collaterals that reach the dorsal LGN. Central projections can be visualized using B subunit of cholera toxin, a commonly employed anterograde neuronal tracer that is actively taken up and transported by neurons. After injecting in the eyes of rd10, rd10/Thy1-GFP-M and control mice, aged 9 months, fluorescent or biotinilated cholera toxin, we recovered a normal pattern of labeling in both the LGN (Figure 6) and the SC (supplemental Figure 5) thus obtaining evidence of preserved anterograde axonal transport from RGCs to central visual targets. The pattern of retinofugal projections of RGCs in rd10 mutant mice appeared also undistinguishable from that of wt mice (Figure 6 and supplemental Figure 5)
By crossing a transgenic expressing GFP in RGCs with a phosphodiesterase mutant with a typical rod-cone degeneration, we could demonstrate remarkable preservation of fine dendritic architecture, complete survival, retention of anterograde axonal transport and maintenance of a normal projection pattern in RGCs of a mouse model of typical RP. The individual cell analysis includes both ON and OFF functional varieties of RGCs, as well as some of the best characterized RGCs types in mammals (i.e. alpha RGCs). These data strongly suggest overall RGC viability in this model of typical, autosomal recessive, RP.
Many signs of pathology are evident in RGCs of animal models of various retinal diseases, typically glaucoma and diabetes. These can be divided into regressive changes (i.e. reduction in size of cell body and dendritic arborization), loss of fine dendritic branches (pruning), and, eventually, cell death; and more plastic alterations, comprising abnormal lamination and dendritic and axonal elongation (sprouting). We found no signs of such pathological changes in the RGCs of rd10/Thy1-GFP-M mice after analyzing almost 600 cells of various types. Yet, in the same retinas, we confirmed regressive remodeling of bipolar and horizontal cells, already reported for this mutant by us and by other Authors (Gargini et al., 2007; Barhoum et al., 2008) Moreover, morphometric evaluation of 8 out of all the types of RGCs described for the mouse retina confirmed preservation of fine dendritic architecture in these neurons even at 9 months of age, well beyond the complete loss of all photoreceptors.
Obviously, GFP-positive ganglion cells in the Thy1-GFP-M mouse were identical to RGCs of the mouse retina described by others using different methods, including the use of an alkaline phosphatase reporter in retinal neurons (Badea and Nathans, 2004), Lucifer Yellow injections (Sun et al., 2002; Coombs et al., 2006), DiI diolistic labeling and GFP transgenic expression. The preservation of fundamental morphological features detected in RGCs of the rd10/Thy1-GFP-M mutant was confirmed also when the same neurons were separated into discrete types using a different taxonomy from that introduced by Sun et al. (2002). For instance, most RGCs belonging to the A1 inner and A2 inner types of the present study fall into cluster n.8 of Kong et al., 2005. “A type” RGCs of the rd10/Thy1-GFP-M retina falling in cluster n.8 at 3 months of age also maintain the parameters typical of that cluster at 7 and 9 months, confirming retention of their fundamental morphological properties. Thus, morphological preservation of RGCs in the rd10/Thy1-GFP-M mutant, which exactly match RGC types of “normal”, wild type mice, is confirmed also when unsupervised classification criteria are used.
We and others have shown that in mutants with inherited photoreceptor degenerations inner retinal cells undergo various degrees of remodeling. This complex process occurs at different rates and with variable aggressiveness partly as a function of the mutation causing the disease and the time of onset of the primary death of rods. Stereotyped aspects of remodeling involve Müller glial reactivity and abnormalities in rod bipolar and horizontal cells, the neurons directly postsynaptic to degenerating rods. Dendritic atrophy, complex phenotypic deconstruction and secondary death of these neurons have been shown in various animal models and appear as a consistent finding (i.e. Marc et al., 2003).
Dendritic atrophy and secondary death are typical reaction to synaptic deafferentation described elsewhere in the CNS, for instance among LGN neurons when the optic nerve is severed (Somogyi J et al., 1987) or in deafferented neurons of the nucleus laminaris maintained in vitro (Sorensen and Rubel, 2006). One would have expected that, ultimately, the consequences of progressive transynaptic degeneration initiated by the primary loss of photoreceptors would need to be propagated to the RGCs. This does not appear to be the case, at least based on the finding on rd10 mutants described here. Dendritic atrophy, degeneration and loss of molecular markers, reported for outer retinal cells (supplemental Figure 1), are not detectable at all in RGCs of the rd10/Thy1-GFP-M mutant mouse, in the 3-9 months time window. Virtually no functional photoreceptors are left in the retina at that time, and most bipolar cells (both rod and cone bipolars) have remodeled extensively. Glial reactivity and atrophy of blood vessels are a part of the common response to damage or remodeling (Otani et al., 2004). Yet, RGCs appear stable in fine morphology and number. Systemic conditions, including diabetes and hypertension, have been reported to affect the morphology of RGCs, exhibiting irregularly swollen and beaded dendrites, reduction in arborization size and branching frequency (Meyer-Rüsenberg et al., 2007). Ultimately, these cells undergo apoptotic death. If this is the eventual fate of RGCs in the rd10/Thy1-GFP-M mutant as well, then those changes occur at a very slow rate, totally separated in time from photoreceptor death.
Other kinds of evidence suggest that retention of function in RGCs might also be a common aspect of inherited photoreceptor degeneration. Morphological studies aimed at detecting responsiveness to glutamate in RGCs of various RP mutants have indicated that these neurons are extremely active through ionotropic glutamatergic receptors even when their input neurons (cone bipolar cells) cannot generate glutamatergic signals (Marc et al., 2007). Indeed multielectrode recording s from the retina of rd1 mutant mice, a largely used model of early-onset recessive RP, demonstrate increased excitability in RGCs, persisting well after the disappearance of any functioning photoreceptors (Stasheff, 2008). Very recently, single cell recordings from different types of ON and OFF RGCs demonstrated retention of highly distinctive membrane and firing properties, overall suggestive of inherent stability of these neurons during retinal degeneration (Margolis et al., 2008). A detailed study of RGCs morphology in the rd1 mutant is in progress in our laboratory; whatever the findings might be, it can be concluded already that activity itself does not seem necessary to RGC survival: In nob2 mutant mice, a case in which a mutation in a calcium channel of photoreceptors causes impaired transmission of signals from the outer to the inner retina, but without physical degeneration of the photoreceptors, RGCs appear to retain their basic center-surround organization (Chang et al., 2006). It could be the case that mutations occurring in the outer retina have limited effects upon RGCs.
Several aspects could contribute to the better preservation of RGCs than of rod bipolar and horizontal cells inherited photoreceptor degeneration. First of all, both rod bipolar and horizontal cells loose all of their synaptic inputs upon photoreceptor demise. Neither one of these second-order neurons is directly connected to RGCs, which receive their input from cone bipolar and amacrine cells. As remodeling of cone bipolar cells is relatively slower (Strettoi et al., 2002; Cuenca et al., 2004) one should then expect a proportionately longer time before the effects of de-afferentation have an impact onto RGCs.
Intra-retinal circuitry generating aberrant synaptic input and causing persistent excitation of RGCs might also contribute to their viability. While the source of retinal generated activity is still a matter of debate, it might be speculated that activity itself could contribute to viability of RGCs, as it is well known that silenced neurons in vitro and in vivo suffer from progressive atrophy and death (Ramakers and Boer, 1991; Catsicas et al., 1992). Melanopsin RGCs, intrinsically responsive to light, are known to survive and to retain spiking activity in degenerated retinas (Semo et al., 2003; Zhu et al., 2007); it is possible that melanopsin-initiated electrical signals invade other retinal neurons as well, although the pathway used for such a propagation needs to be clarified.
RGC survival could also be supported by their central targets as well, by means of synaptic and trophic interactions. Finally, astrocytes of the retina and optic nerve could play a supportive role in the long run: these cells are known to release trophic factors and might respond to retinal degeneration by exerting a protective action, similarly to what has been described in various examples of CNS injury (Sofroniew, 2005). Additional trophic support could be exerted also by exogenous devices including electronic prostheses implanted to restore vision (DeMarco, 2007).
This is one of the few single-cell studies illustrating the effects of inherited photoreceptor degeneration upon RGCs of various types, in a time window encompassing both early and late stages after rod and cone death. Whatever the mechanism supporting RGC viability might be, the findings reported here have consequences for the treatment of the family of disorders collectively known as RP . RGCs are the only retinofugal neurons, and are also the cells stimulated directly by epiretinal prostheses and indirectly by certain types of subretinal electronic implants (Winter et al., 2007). Their long term viability in an animal model which closely resembles recessive RP suggests that RGCs may be a better target of activation than the bipolar cells for restoring vision in humans with similar phenotypes. Although there are certain advantages to prosthetic or molecular stimulation of bipolar cells, their greater propensity to degenerate may make the ganglion cells a more desirable target in attempts to restore vision after photoreceptor degeneration.
Whole mount images and descriptions of main morphological parameters for 4 types of RGCs from the normal mouse retina stratified in the outer half of the IPL, classified according to Sun et al., 2002. The same 4 types were analyzed in the retina of rd10/Thy1-GFP M mice.
Whole mount images and descriptions of main morphological parameters of 4 types of RGCs from the normal mouse retina stratified in the inner half of the IPL, classified according to Sun et al., 2002. The same 4 types were analyzed in the retina of rd10/Thy1-GFP M mice.
Top: Statistical analysis onto morphometric parameters of RGC types stratified in the inner half of the IPL from the rd10/Thy1-GFP mouse, compared to counterparts from the adult wt retina.
Bottom: Statistical analysis onto morphometric parameters of RGC types stratified in the outer half from the rd10/Thy1-GFP-M mouse, compared to counterparts from the adult wt retina.
Whole mount retinal preparations showing correspondence of retinal degeneration of parent rd10 and rd10/Thy1-GFP-M retinas. A and B: in both strains, photoreceptor degeneration peaks at P24. Many bright, pycnotic nuclei of dying rods are visible in the outer nuclear layer of both retinas at this age. C and D: horizontal cells, stained by Calbindin D antibodies, show irregularity of their mosaics and loss of fine dendritic ramifications after one moth of age. E and F: remodeling of rod bipolar cells. By P45, dendritic retraction in rod bipolar cells, stained by PKC antibodies, becomes apparent. No dendritic plexus in visible in the outer plexiform layer. G -N: at later stages, dendrites become even more attenuated and cellular loss is apparent among rod bipolar cells (in red) and horizontal cells (in green). Bar in L applies to horizontal cells only. Bar in N applies to all the other images.
additional examples of RGCs belonging to the A and B groups encountered in the adult rd10/Thy1-GFP-M retina. In this and the following illustrations, the topmost panel represents a whole mount view of an individual cell projected in a single plane; the bottom panel is the orthogonal view on the same cells obtained by computer rotation, also showing ethidium nuclear staining (red signal). Nomenclature according to Sun et al., 2002, is reported on the left. Some cells (i.e. A2 outer and B1) have dendritic arborizations ramified in the outer half of the IPL (outer cells, or OFF types); other cells (i.e. A1, A2 inner and B3 inner) send their dendrites in the inner half of the IPL (ON types).
Examples of group C RGCs encountered in the adult rd10/Thy1-GFP-M retina.
Additional RGC types from the adult rd10/Thy1-GFP-M retina. A1-A2: two examples of D type (bistratified) RGCs. A3-A4: members of the so-called “unclassified” group are also shown, with dendrites in the outer and inner half of the IPL, respectively. A5: one displaced RGCs. These cells have their cell body located in the innermost row of cells of the inner nuclear layer.
Coronal sections of the Superior Colliculus (SC) from wt and rd10 mutant mice aged 9 months, after injections in the two eyes of cholera toxin conjugated to different fluorophores. Each pairs of images shows complementary red/green fields, comprising a large projection from the contralateral eye and a small field, receiving the ipsilateral projection.
This work was supported by the Italian CNR and by the NEY grant R01-12654. We are indebted to Shegang He for helpful suggestions on ganglion cell classification, to Richard H Masland for the generous gift of homozygous Thy1/GFP-M mice, to MariaCristina Cenni for helpful comments on the manuscript and to Giulio Cesare Cappagli for excellent technical support.
E. Strettoi dedicates this paper to the memory of Ramon F. Dacheux, unforgettable retinal neuroscientist and mentor.