|Molecular Vision 2002;
Received 2 July 2002 | Accepted 24 August 2002 | Published 27 August 2002
Cone neurite sprouting: An early onset abnormality of the cone photoreceptors in the retinal degeneration mouse
Department of Ophthalmology and Visual Science, Yale University School of Medicine, New Haven, CT
Correspondence to: Yijian Fei, MD, Department of Ophthalmology and Visual Science, Yale University School of Medicine, BML 233, P. O. Box 208061, New Haven, CT, 06520; Phone: (203) 785-6044; FAX: (203) 785-7068; email: email@example.com
Purpose: Mutations in many rod genes can cause inherited blinding neurodegeneration in the retina characterized by sequential death of rod and cone photoreceptors. This study was to examine the morphological changes of the cone photoreceptors in retinal degeneration (rd1) mice caused by rod-specific cGMP phosphodiesterase beta-subunit gene mutation and to gain insights into the early cellular events underlying the secondary cone death.
Methods: Transgenic mice that have their living cones labeled by the green fluorescent protein (GFP) transgene and carry the homozygous rd1 mutation were generated, and identified by PCR analysis of the mouse tail DNA and PCR coupled Dde I digestion. The morphology of cone cells in live and fixed retinas from developing and adult mice was examined with fluorescence and scanning laser confocal microscopy. Some fixed mouse retinas were also examined by immunocytochemical staining. Volume images from the confocal three-dimensional (3D) data sets were processed with IMARIS software for 3D view of the detailed cone cell morphology.
Results: The cone photoreceptors in the rd1 retinas exhibited a novel process of neurite sprouting, in addition to the general pathological changes of cone degeneration such as shortening and loss of cone outer and inner segments, and loss and death of the cones. The cones gave rise to prominent neurite outgrowth from their axons and synaptic pedicles as well. Most neurites had beaded varicosities along their length and some terminated as bulbous structures. Some cone pedicles showed abnormally elongating and branching processes. The degenerating cones were disorganized, and migrated into the inner nuclear layer. Some cone neurites extended horizontally and appeared to contact the rod bipolar cells, while others projected into the inner plexiform layer. The aberrant cone sprouting started from P8 when rod degeneration generally began, and became evident by P10. In contrast, this abnormal cone neurite sprouting was not observed in the examined control mice that did not carry the rd1 mutation. Double-labeling with cone cell-specific peanut agglutinin confirmed that the fluorescent cells expressing the GFP in the rd1 retinas were indeed the cone photoreceptors.
Conclusions: Cone photoreceptors in the rd1 mice underwent a remarkable process of neurite sprouting that appeared to start before the onset of cone cell death and persisted throughout the course of cone degeneration. This novel process of cone neurite sprouting may be a part of the early cellular events leading to the cone photoreceptor death in retinal degeneration of the rd1 mice.
Retinitis pigmentosa (RP) is a heterogeneous group of inherited human retinal degeneration characterized by sequential rod and cone photoreceptor dysfunction and death. It is a common cause of inherited blindness worldwide . As yet, no cure is available. Current understanding of the cellular and molecular mechanisms underlying photoreceptor death is still quite limited. Most of the genetic defects causing RP have been located in the rods (RetNet). Nevertheless, dysfunction and death of cone photoreceptors occur following rod degeneration, and abnormalities of other retinal neurons also appear in the later stage of degeneration [2-6]. It is the death of cone photoreceptors that directly cause the devastating blindness. One long-standing fundamental question is why the genetically normal cones die after the rods carrying the defective gene product degenerate. There appear to be two hypothetical models available for the secondary cone death. One is the direct bystander effect model, where the cone cells die either due to the loss of normal rod-cone interactions, particularly rod-derived trophic factors [7,8], or due to direct exposure to toxic molecules  from the surrounding degenerating rods, or a combination of both. The other is the abnormal synaptic target model, where loss of synaptic targets resulting from the dysfunction and degeneration of the second order retinal neurons in the inner retina [10-12] may cause the cone death. However, the precise mechanisms are unknown.
The retinal degeneration (rd1) mouse has an early occurring, rapid degeneration of rods followed by a slowly progressive loss of cones as a result of a spontaneous mutation in the rod-specific cGMP phosphodiesterase (PDE) beta-subunit gene [9,13-15]. The rd1 mouse is a model system for exploring the mechanisms of rod gene mutation-induced cone death. One problem with studying the early cellular and molecular events leading to the cone death is that there are few early-appearing, cone-specific markers that can reveal the detailed morphology of the entire cone cell. The relative lack of the adequate cone cell markers constrains our understanding of the cellular and molecular mechanisms underlying the cone death. To overcome this problem, transgenic mice, in which cone cells can be readily identified through their expression of green fluorescent protein (GFP), were created . To better unravel the consequences of rod degeneration on the cones and to gain insights into the early cellular events leading to the cone death, in the present study, the GFP transgene was moved into the rd1 mice so that the detailed morphology of the cone cells could be examined during rod degeneration. The transgenic GFP marker selectively labels the entire cone cell beginning from the early developmental stage, making it possible to visualize the details of the fine cell structures and early pathological alterations of cones during rod degeneration.
Generation and identification of GFP transgenic rd1 mice
The creation and characterization of the transgenic mice with the GFP expression in the cone cells was described previously . A 6.8 Kb 5' sequence of the human red opsin gene that contains the promoter and the red/green opsin gene locus control region was isolated from the pR6.5 lacZ clone  (a generous gift from Dr. Jeremy Nathans, Johns Hopkins University), and used to drive the GFP expression in the mouse cone photoreceptors. The founders were SJL x C57BL/6J mice. Transgenic mice were identified by polymerase chain reaction (PCR) analysis of mouse tail DNA using the human red opsin promoter and the GFP transgene-specific primers. Positive founder mice were bred with C57BL/6J mice to produce the first generation transgenic mice (F1). Positive transgenic mice carrying the rd1 allele inherited from the background SJL strain were detected by PCR coupled with Dde I (New England BioLabs, Inc., Beverly, MA) restriction fragment length polymorphism (RFLP) analysis of the rd1 gene using the same primers and protocols described by Pittler and Baehr . Homozygous rd1 mice carrying the GFP reporter were generated through inbreeding mice carrying one allele of the rd1 mutation. Age-matched wild type GFP transgenic mice that did not carry the rd1 allele were used as controls. Animals used in this study were housed and cared for in accordance with the Yale Animal care and Use Committee guidelines, and all experimental procedures conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.
Preparation of retinal wholemounts and cross sections
At each age, 3 to 4 mice were killed by inhalation of an overdose of Isofluorane (Fort Dodge Animal Health, Fort Dodge, Iowa). The mouse eyes were marked for orientation by burning the cornea at 12 o'clock limbus with a surgical cautery, and then enucleated. To examine the live cone cells, one retina from each mouse was freshly dissected in ice-cold Hanks' buffer (Ca2+ and Mg2+ free) under a dissection microscope (Olympus, Tokyo, Japan), flat-mounted on a glass slide with the photoreceptor side up, coverslipped with Hanks buffer, and examined immediately with microscopy. The other eye of each mouse was fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS, pH 7.4) for 1-2 h. The fixed mouse eyes were rinsed in PBS for 3-4 h. Then, the neural retinas were dissected free and flat-mounted on glass slides for microscopy. Some fixed retinal samples were embedded in agarose. Cross sections (50 to 100 μm) were made along the dorsal-ventral meridian with a vibratome (Electon Microscopy Sciences, Fort Washington, PA).
Double-labeling of retinal wholemounts with peanut agglutinin
Peanut Agglutinin (PNA), a cone cell-specific marker , was used to stain the wholemount preparation of the retina to reveal the specificity of GFP labeling of the mouse cone cells. The fixed retinal wholemounts were first incubated in PBS containing 10% normal goat serum and 0.2% Triton X-100 for 3 h at room temperature, then rinsed with PBS and reacted with rhodamine-conjugated PNA (Vector Laboratories, Burlingame, CA) overnight at 1:10 dilution with PBS containing 1 mM CaCl2 and 1 mM MgCl2. PNA-stained retinas were rinsed with PBS three times, each for 30-40 min, and mounted on a glass slide with 80% glycerol in PBS containing 0.4% phenylendiamine.
Counterstaining of retinal sections with propidium iodide and anti-PKC-alpha antibody
In order to view the cellular layers of the retinal structure, the nuclei of retinal cells in some sections were counterstained with propidium iodide (at a final concentration of 2 μg/ml) or Topro-3 (10 μM; Molecular Probes, Eugene, OR) for 1 h and rinsed in PBS for 30-60 min. Rod bipolar cells in the mouse retina were identified with rabbit polyclonal anti-protein kinase C-alpha antibody (Biomol, Plymouth Meeting, PA). To block nonspecific binding, retinal sections were first incubated in blocking buffer solution containing 10% normal goat serum and 0.2% Triton X-100 in PBS for 2 h at room temperature with shaking, then reacted with the primary antibodies (1:100 anti-PKC-alpha antibody) in the same buffer at 4 °C overnight. The reacted sections were washed with the blocking buffer solution for 30 min, and then incubated with secondary antibodies (1:300 Texas Red-conjugated goat-anti rabbit IgG, Jackson ImmunoResearch Laboratories, Inc, West Grove, PA) at room temperature for 2-3 h with shaking, followed by 3 washes in PBS for 10 min each. A control for non-specific staining by the secondary antibody was performed in parallel using the same procedures except for omitting the primary antibody.
Epifluorescence and scanning laser confocal microscopy
The retinal wholemounts and retinal cross sections were first examined by epifluorescence microscopy with a Zeiss microscope equipped with a Micromax CCD camera (Princeton Instruments, Trenton, NJ) and standard HQ FITC/Texas Red filter sets (Chroma, Brattleboro, VT). A Biorad MRC 600 scanning laser confocal microscope (Hercules, CA) fitted with standard FITC and rhodamine filter sets was then used to collect three-dimensional (3D) image sets. The digital images from both epifluorescence and confocal microscopy were analyzed with IPLabs software (Scanalytics Inc., Fairfax, VA). Some volume images from the confocal 3D data sets were processed with IMARIS software (Bitplane AG, Zurich, Switzerland) on a SGI silicon graphics octane 2 work station (Silicon Graphics, Inc., Mountain View, CA) for 3D view of the cone cell morphology. Image manipulations included adjusting the brightness and contrast, and adding scale bars and labels. The final figures were composed with Adobe Photoshop 5.5.
Degenerating cones have abnormal neurite sprouting in adult rd1 retina
The homozygous rd1 mice that show retinal degeneration and carry the GFP transgene can be readily distinguished from the phenotypically normal heterozygous rd1 and the wild type transgenic mice by PCR coupled RFLP analysis (Figure 1A-C). In the 2-month old adult rd1 mouse retinas, most of the cones had markedly shortened or absent cone outer segments, some even lost their inner segments and axons and left only dead, shrunk cell bodies that had diminished fluorescence (Figure 2A, fluorescence microscopic image from live, unfixed rd1 retinal wholemount, compared with the control in Figure 2B). Loss of cones in the thinning outer nuclear layer (ONL) was evident as a consequence of cone death. The remaining cones were disorganized (Figure 2C, fluorescence microscopic image from fixed rd1 retinal section, compared with the control in Figure 2D) and some migrated toward the inner retina. These general pathological changes of cones are consistent with previous observations from light and electron microscopic studies of the rd1 mice [9,19-21]. However, a previously unidentified striking abnormality is that the cone photoreceptors underwent an abnormal process of neurite sprouting. The sprouted cone neurite, a beaded neural process with a bulb-shaped terminal, was readily identifiable in both live (Figure 2A; arrowhead) and fixed (Figure 2C; arrowheads) adult retinas of the rd1 mice aged 2 months. The abnormal cone neurite sprouting, present in both the central (Figure 2E,G) and the peripheral (Figure 2F) regions of the rd1 mouse retinas, was more evident in 3D view of the cones (Figure 2E-G). The vitreally-oriented cone axon gave rise to multiple branching neurites (Figure 2E; arrowheads). Some cone pedicles extended abnormally long philopodia-like processes (Figure 2F,G; arrowheads), which are likely another form of neurite sprouting. These abnormal cone neurites were observed in all adult rd1 mice examined, but not in the corresponding controls (Figure 2B,D,H) where typical morphology of cones showed apparent outer and inner segments, somata, axons and large, flattened pedicles with much shorter basal processes.
Counterstaining of the retinal sections from adult rd1 mice with propidium iodide revealed the loss of cones, markedly reduced thickness of the ONL, migration of the remaining cone somata into the inner nuclear layer (INL) and the sprouted cone neurites extending toward different directions (Figure 3A). While in the control retina, the cone somata were restricted within the ONL and the cone pedicles within the OPL (Figure 3B). These observations were further confirmed by immunostaining of rod bipolar cells (RBC), one of the major cell types in the inner retina, with anti-PKC-alpha antibody (Figure 3C,D). The degenerating cones migrated into the INL and mixed with the RBC somata. Some cone neurites extended horizontally along the surface of RBC dendrites (Figure 3C; arrowheads) and appeared to contact the RBCs, while others extended vitreally past the bipolar cell somata (Figure 3C, arrows), and entered the inner plexiform layer (IPL). These abnormalities did not appear in the control retina immunostained with the same antibody (Figure 3D). The immunostaining background control where the anti-PKC-alpha antibody was omitted did not show bipolar cell labeling.
Timing of the cone neurite sprouting in developing rd1 retina
To determine the timing of the aberrant cone neurite sprouting in the rd1 mice, retinas from the rd1 and the control mice starting from postnatal day (P) 6 were examined. At P6, the photoreceptor cGMP level in the rd1 mice began to rise as a consequence of the genetic defect in rod cGMP PDE-beta gene [15,22]. By P7, the cone cells in both live (Figure 4A) and fixed (Figure 4C) retinas of the rd1 mice appeared to be morphologically normal compared to the controls (Figure 4B,D). Although some cone pedicles had prominent processes in the rd1 retinas, these basal processes of cone pedicles were also observed in the control retinas. On P8, however, when rod degeneration started [9,13,15], noticeable cone neurite outgrowth began to develop in both live (Figure 5A, arrowhead) and fixed (Figure 5C, arrowhead) rd1 mouse retinas. In contrast, these abnormal cone neurite sprouting were not observed in the controls (Figure 5B,D), although spike-like fine projections of the cone axons could occasionally be noticed in the control as well as the rd1 retinas during earlier developmental stage. This early onset of the abnormal cone neurite outgrowth in the rd1 retina indicates that the cones, like the rods, are also affected in the early stage of retinal degeneration, although cones degenerate more slowly than rods. All rd1 mice examined starting from P8 showed abnormal cone neurite outgrowth, although the extent of neurite growth appeared to vary with the mouse age. By P10, when photoreceptor death generally began in rd1 mice, as evidenced by the presence of pyknotic photoreceptor nuclei , neurite sprouting of the cones in the rd1 retina became more evident. There was a robust cone neurite outgrowth, branching and elongation (Figure 6A,B; arrowheads). Some pedicles projected abnormally long and branching processes. The cones in the control retina did not show these abnormalities (Figure 6C). Some cones seemed to have a normal contact with their neighboring pedicles (Figure 6B, CP), and the neurites appeared to extend from the sites of a pedicle that did not seem to make direct contact with the neighboring pedicle. Even though some cones sprouted apparent neurites, the overall morphology of the cone outer and inner segments appeared to be normal(Figure 6B, OS and IN).
Specificity of GFP labeling of cone photoreceptor cells
To examine whether the pathological environment in the degenerating retinas altered the cone cell-specific expression pattern of the GFP transgene observed in the normal retinas , retinal wholemounts from the adult and developing rd1 mice starting from P7 were double labeled with the cone cell-specific PNA marker . This staining showed that all green fluorescent cone cells in the developing (Figure 7A,C,E) and adult (Figure 7B,D,F) rd1 retinas were stained by the PNA marker (arrows) although not all cones expressed the GFP (Figure 7E,F; arrowheads), which confirms that retinal degeneration does not change the specificity of GFP expression in the developing and mature cone photoreceptors, and that all green fluorescent cells in the rd1 retinas are indeed the cone photoreceptors.
The devastating blindness in retinal degeneration is the direct consequence of cone cell death. The pathophysiology of the cones, however, is not well studied. The relative lack of early appearing, cone-specific markers that can readily reveal the entire morphology of the cone cell constrains our understanding of the early cellular and molecular events leading to the cone cell death. In this study, the cone cell morphology of the rd1 mice carrying a transgenic cone marker was first examined in the mature retinas, and it was found that the cones underwent a novel process of neurite sprouting in response to the rod gene mutation-caused retinal degeneration. Then, the timing of cone neurite sprouting in the developing rd1 retinas was examined. Interestingly, the cones in the developing rd1 mice extend neurites at the time when rod degeneration generally begins. Further, cone sprouting could occur even when a cone still appeared to preserve otherwise a grossly normal morphology. These findings indicate that cone neurite sprouting, an early abnormality of the cones in the rd1 mouse, appears to occur well before the cones die. Thus, this novel process of cone neurite outgrowth could be a part of the early cellular events leading to cone death in retinal degeneration of the rd1 mice.
Previous immunocytochemical studies on human RP have shown that the rods in the peripheral retinas had neurite sprouting [23-25]. Rod neurites were also noticed in cats with rod/cone dysplasia , but not in mice carrying the retinal degeneration slow (rds) mutation . In transgenic pigs carrying a rhodopsin mutation, filopodia-like projections were seen in the synaptic terminals of rods, and were thought to be the early stage of neurite formation . In addition to rods, other retinal neurons, such as the horizontal cells in RCS rats  and the horizontal and amacrine cells in human RP , also showed similar neurites. On the other hand, enlarged, distorted cone pedicles and thickening of cone axons were recently found in human cone-rod dystrophy . However, neurite sprouting from cone cells have not been previously observed either in human RP or in animal models of retinal degeneration. It is possible that this type of cone abnormality may be unique to the rd1 mice. Alternatively, previous immunocytochemical studies might have failed to reveal potentially abnormal cone neurites as a result of using conventional cone markers that have limited resolution in visualizing the entire morphology of the cone cell.
What is the significance of cone neurite sprouting in response to the rod degeneration in the rd1 mice? The abnormal cone sprouting may have implications for understanding the cone cell death caused by malfunction of rod-specific genes. Although the precise mechanisms of the secondary cone death remain unknown, two models, the direct bystander effect [7-9,30,31] and the abnormal postsynaptic neurons, have been proposed [10-12]. Little is actually known about what really happens in the inner retina during rod death, but a systematic study revealed dramatic abnormalities and developmental failure in bipolar and horizontal cells of the rd1 mice . In addition, ectopic synaptogenesis between cones and rod bipolar cells was reported . Further, a detailed electrophysiological study in transgenic pigs carrying a rhodopsin mutation showed abnormal maturation of the cone circuitry . Although it is possible that the loss of synaptic or neurotrophic inputs from rods could cause the cone death, given these early abnormalities of the inner retinal neurons accompanying the rod degeneration and a previous observation that the basic fibroblast growth factor capable of rescuing photoreceptor degeneration [32,33] is upregulated in the rd1 mouse retinas , the findings of cone sprouting, neurite outgrowth into the inner retina as well as migration of cones to the INL appear to be more consistent with the model where loss of the postsynaptic contacts might cause the cone sprouting and subsequent death. It will be interesting to further characterize the cone neurite sprouting quantitatively during the course of photoreceptor degeneration and to examine simultaneously the cone neurite outgrowth, rod degeneration and inner retinal alterations in the same retinal samples to test these possibilities.
Cone sprouting may also have practical implications. The presence of abnormal cone neurites might interfere with the inner retinal function and restrain current therapeutic attempts in photoreceptor transplantation [35-37] and electrical stimulation  for restoring the function of the degenerated retinas.
What triggers the process of cone neurite sprouting in the rd1 retina? One obvious possibility is the loss of synaptic targets [39,40], which might activate certain signal pathways to modulate sprouting-related certain molecular cues such as neurotrophic factors , adhesion molecules [42,43] and target-derived chemoattractants . It was noticed that some neurites appeared to extend from the sites of a cone pedicle that did not seem to make direct contact with the neighboring pedicle. These sites could be the contact sites of cones with rods or other inner retinal neurons that might be altered as a result of the degeneration. Another possibility is the loss of repulsive cues present in the inner retina. It was shown that cultured adult salamander cones and rods were capable of regenerating neurites and synaptic processes , and that the preferential synaptic targets of the regenerating photoreceptors were amacrine and ganglion cells . This indicates that there may be certain repulsive cues in the inner retina that prevent the photoreceptor processes from growing into the inner retina. Alternatively, cone neurite sprouting may be triggered by the abnormal cyclic nucleotides in the degenerating photoreceptors. The cGMP level in rd1 retina begins to rise from P6 and reaches peak at about P14 [15,22]. The cAMP level also increases when the degeneration begins and persists in the adult rd1 retina . It has been demonstrated that cAMP controls the capacity of neurons to regenerate axons, and modulates neurite outgrowth of retinal ganglion cells during development . In addition, the repulsion or attraction response of neural growth cones to their guidance cues can be reversed by cyclic nucleotides [48-50]. It would be interesting to examine whether application of cyclic nucleotides, growth factors or blockers, could alter the pattern of cone neurite growth.
Sprouting of cones in the adult rd1 retinas also indicates the mature retinal neurons posses the potential of morphological plasticity in response to disease or injury. The precise mechanisms mediating the neurite sprouting process remain to be elucidated, and the functional significance of these cone neurites also awaits further study. Nevertheless, this study provides the first in vivo evidence of aberrant neurite sprouting of the cone photoreceptors in the rd1 model of human RP. Examination of other RP mouse models carrying this GFP transgene would be interesting to test if the cone neurite sprouting is a unique feature of the rd1 mouse. Further studies to identify the factors and signaling pathways initiating the cone neurite sprouting may shed light on the molecular and cellular events leading to the death of cone photoreceptors in retinal degeneration.
This work was supported by NEI grant EY08362 and The Matilda Zeigler Foundation awarded to Dr. Thomas. E. Hughes. I am grateful to Dr. Hughes for his generous support of this project and critical reading the initial drafts of this manuscript. I am greatly indebted to Drs. Jeremy Nathans and Yanshu Wang for kindly providing the pR6.5 lacZ clone.
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