4. Discussion
4.1 The rd10 mouse: a good model of human RP
We have shown that in the rd10 mouse, photoreceptors start to die past the late phases of retinal synaptogenesis (Sharma et al., 2003) and die out more slowly in comparison to the well known rd1 mutant. Rd10 rods degenerate maximally between P20 and P25, in a clear centre-to-periphery gradient. Secondary degeneration of cones proceeds in parallel at a slower rate; a scattered population of aberrant cones persists for up to at least 9 months of age. These findings agree with, and significantly extend, previous studies based on this mutant (Chang et al., 2002; Otani et al., 2004). Besides photoreceptors, our work focussed on secondary changes affecting inner retinal cells, starting from bipolar and horizontal cells to include ganglion cells, thus comprising the main neuronal path followed by the visual signal to exit the retina.
With respect to second order neurons, our data show that in the rd10 retina several morphological modifications are already detectable at around 1 month of age and become widespread at 2 months. Changes affect mostly rod bipolar and horizontal cells. Their dendritic complement becomes progressively scant and disorganized and eventually regresses completely. Cone bipolar cells follow, at a slower rate. Dendritic retraction in bipolar cells that carry mGluR6 receptors is preceded by a decrease in the immunoreactivity for this marker and by its displacement to the cell bodies and bipolar cell axons in the INL (Gargini et al., 2007).
At present, retraction of dendrites from the OPL has been proved to occur in rd10 and rd1 mutants, in crx-null mice, and in other rodent models of retinal degeneration (Jones et al., 2003; Cuenca et al., 2004; Pignatelli et al., 2004;
Wang et al., 2005). Preliminary results by our group suggest that this occurs in human RP as well (Strettoi et al., 2003 and unpublished data). Thus, this form of remodeling can be considered as a hallmark of the secondary effects of photoreceptor death, independent of the underlying genetic defect and the affected species; it is a true stereotyped consequence of photoreceptor degeneration.
Similarly to what is observed in the rd1 mutant, the morphological changes in bipolar and horizontal cells occurring in the rd10 retina are followed by the death
of these neurons: their survival rate drops right after the peak of photoreceptor degeneration and decreases slowly over the following months. Furthermore, we have found a gradient in rod bipolar and horizontal cell death: rod bipolars maximally die following a front that starts from the dorsal-nasal quadrants of the retina; horizontal cells, instead, start to die from the dorsal-temporal quadrant. It reminds to be established whether this asymmetric profile of degeneration correlates to known asymmetries in the degeneration of rods and cones, described for the rd1 mutant (Garcia-Fernandez et al., 1995) and not yet studied in the rd10 mouse.
Subsequently to the death of second order neurons, the INL disorganizes, and typical rosettes appear. Other cell types, studied but not shown here, such as AII and cholinergic amacrine cells, do not seem to be affected in the first 5 months of life.
Throughout the period of active photoreceptor degeneration, and particularly between P20 and P30, as well as at later stages, GFAP is upregulated in Müller cells (Gargini et al., 2007). As in much other retinal pathology, including diabetic retinopathy and human RP, a glial reaction with intermediate-filament hypertrophy accompanies and follows the death of photoreceptors (reviewed in Marc et al., 2003b).
All together, the rd10 mutation appears milder compared to the aggressive rd1, as the rd10 inner retina is more resistant to remodeling; however, ultimately, secondary modifications are remarkably similar in the two mutants and recall patterns of alterations described in other models of photoreceptor disease (Milam et al., 1998; Jones et al., 2003; Cuenca et al., 2004; Wang et al., 2005). Several morphological changes described here bear a clear physiological correlate. The death of rods that occurs in the rd10 mutants between P20 and P30 is reflected in the drastic reduction of the scotopic a-wave of the ERG occurring at the same time, also showing a concomitant decrease in the responsiveness of the residual rods.
The results on rod bipolar cells are consistent with the idea that rod degeneration is associated with functional changes in bipolar cells as well, as shown with single-cell electrophysiological experiments (Varela et al., 2003). It is interesting to note that alterations in the shape and relative amplitude of the b-wave of the ERG (indicative of bipolar cell activity) are detected as early as P18; at this age, no major morphological modifications occur in the inner retina. Only the active
degeneration of photoreceptors is evident. Thus, ERG changes in the b-wave precede the detection of main morphological abnormalities in the inner retina and occur well before the extensive loss of dendrites from the OPL (Gargini et al., 2007).
The fact that a different mutation in the pde6b gene also mutated in the rd1 mouse results in slower and milder change in the inner retina of rd10 mice demonstrates critical factors determining the overall outcome of the pathology: the slower phenotype is likely to be caused by a delayed death of photoreceptors. As for other mutations of the pde6b gene (Hart et al., 2005), the missense rd10 mutation ensures retention of a residual PDE activity. Thus, it is conceivable that cyclic guanosine monophosphate accumulation and calcium rise, both associated with a defective PDE (Bowes et al., 1989 and 1990), reach toxic levels over a longer time period. Also, the metabolic overload taking place in mutated photoreceptors to extrude the excess calcium by active pumping is likely to be less intense in rd10 retinas. Hence, photoreceptor death starts later and persists for a longer period compared with the rd1 retina.
Overall, the rd10 mutant is a good model of typical RP in humans and represents an excellent background for rescue studies. Indeed, rd10 mice have been used for stem cell transplantation and pharmacological treatments (Otani et al., 2004;
Rex et al., 2004).
An important indication confirmed from the present study is that, given the relatively slow occurrence of secondary events in the inner retina of the rd10 mutant, it would be significant to use this model to test restoration strategies after the end of rod degeneration; this would mimic the human conditions more closely, as a possible intervention in human patients is usually considered when large fractions of photoreceptors have already degenerated.
4.2 Single retinal ganglion cell study in rd10/Thy1-GFP mice
Investigations of the structural and functional organization of the vertebrate retina using live preparations have been facilitated by improvements in cell labeling methods and by microscopy techniques that permit high-resolution of cells in vitro and in vivo. Specifically, the generation of transgenic animals with fluorescently labelled retinal cells has made possible real-time visualization of cell generation, migration, differentiation and growth in the developing and adult retina. In
particular, the Thy-1 and other GFP-transgenic lines have gained immense popularity: these mutants are useful for studies requiring labeling of cells in live tissues and for studies that require rapid screening of cellular morphology in mutant or diseased backgrounds (Feng et al 2000).
This is the first study in which transgenics with fluorescent retinal cells have been applied to study RP degenerative effects on the fine morphology of RGCs at single cell level. We created ad hoc mutants crossing the rd10 mouse (after systematic characterization of its retinal degeneration pattern) with the Thy1-GFP transgenic mouse.
Other methods to visualize RGCs are available, including ballistic labeling with fluorescent dyes or antibody staining. These methods, rather than staining single cells, label many cells simultaneously, with dendritic overlapping. On the other hand, many antibodies (i.e. melanopsin) are specific for a too small population of RGCs, whereas others are specific for a compartment of the cell (axon, or dendritic tree). So, none of these methods is actually suitable to study dendritic tree complexity in a systematic manner.
The use of the new rd10/Thy1-GFP strain turned out to be a very good approach to study in detail single ganglion cells, to assess the possibility that these neurons are affected by the degeneration of photoreceptors, possibly by a transynaptic propagation of the negative remodelling and death of second order neurons.
Up to now, studies on ganglion cells in both human RP and animal models of this disease are sparse and contradictory, providing a confused scenario. This is possibly caused by the large genetic heterogeneity of the pathology, by differences in species, strains and age, in the layers and regions of the retina studied; and by different methodological approaches. For example, three major papers based on morphometric analysis of inner neurons (mainly RGCs) from human RP eye donors (Santos et al., 1997; Stone et al., 1992; Humayun et al., 1999b), conclude that there is “a substantial preservation of RGCs” in this disease, despite a reported rate of degeneration varying from 20% to 80%, depending on the RP (moderate or severe).
Other studies about effects of photoreceptor degeneration upon inner retinal cells are concentrated on two principal RP rodent models: the rd1 mouse and the Royal College Surgeon rat. They differ completely in terms of mutation type and progression of the pathology (Graftein et al., 1972; Eisenfeld et al., 1984). In the rd1, a non sense mutation of beta subunit of rod PDE leads to rod death from P8.
In this model of severe human arRP, there are two principal events: early rod degeneration and failure of bipolar cell wiring in the outer retina. Secondary changes, likely to occurr in RGCs, could be triggered by either one of these events, or by their combination. Results are complicated by the fact that the genetic background of the animal can affect the severity of the phenotype.
Graftein et al., (1972), for example, studied rd1 mice on a C57Bl/6J background.
They found that RGC morphology was “quite normal” at the electron microscopy level suggestive of a regular protein metabolism in these cells. It is to note that these authors also found a 20% decrease in the mean size of the largest ganglion cells. The RCS rat has a retinal pigment epithelium cell defect which causes a progressive loss of rods occurring primarily over the first few months of life. Here, photoreceptors die because of an incorrect recycle of visual pigments. Eisenfeld et al., (1984) reported no changes in ganglion cell size, synapses and axonal transport in RGCs of RCS rats.
Here, we analyse the fine structure of RGCs and their survival rate in a model of RP, establishing a conceptual link with observations of inner retinal remodeling and survival of the same mutant, thus comparing events in neurons directly connected to photoreceptors to events occurring in retinofugal cells, of key importance for the device of epiretinal prostheses.
We demonstrate in a definitive manner that in the rd10 mutant mouse the fine dendritic tree morphology and complexity of RGCs is retained during the progression of the pathology (3, 7 and 9 months of age). This analysis has been performed over a sample of 572 RGCs, divided according to Sun et al., (2002a) in 17 types. The analysis comprises the evaluation of the dendritic tree arborization and the cell body area. The analysis of dendritic trees shows that there are no appreciable differences in the area and pattern of stratification with time. Data of cell body areas demonstrate that individual types of RGC, at different ages, do not show signs of shrinkage or swelling, suggestive of active degeneration processes (apoptosis or necrosis, respectively). Eight types of RGCs, (164 cells in total), were chosen for evaluating the total dendritic length and the number of branching points, to gain indications of the complexity of single ganglion cell arborizations.
The 8 types include the A group RGCs, homologous to α cells of other vertebrates, the best known exemplar of GC from the time of Golgi and Cajal (Peichl, 1991). We also analysed some types belonging to B and C groups: B1, B3 outer and C2 outer cells that stratify in the OFF sublamina of the IPL and then
B3 inner and C2 inner cells that stratify in the ON sublamina. These 5 types are shown as the most frequently RGCs present in mouse retina (Sun et al., 2002a).
The dendritic tree complexity of all these 8 types of RGCs does not appear to change over 9 months of age.
All these data are accompanied and reinforced by the evidence that the survival rate of the cells in the GCL of rd10/Thy1-GFP mice at 9 months is identical to that of the wt counterpart at the same age. In general, the GCL contains approximately equal numbers of RGCs and displaced amacrine cells (Jeon et al., 1998; Farah et al., 2006 for a review). The fact that we find the same number of cells in the layer of mutants and control mice means that both populations of cells remain unchanged in the time period examined. This also implicates that displaced amacrine cells, including 20% of cholinergic amacrine, plus other subsets that express GABA, CD-15, tyrosine hydroxylase, remain constant in their total number during all this time in the mutant retina.
The evidence of excellent survival of RGCs is contrary to some literature on the human disease (Santos et al., 1997; Stone et al., 1992 and Humayun et al., 1999b). Graftein also finds a loss of 20% RGCs in C57Bl6/J -rd1 mice (Graftein et al. 1972). Our results agree instead with the work of Eisenfeld and co-workers (1984) on C57Bl6/J - rd1, which report no changes in area, perimeter, diameter and density of RGCs in animals of age generically comprised between 3 and 6 months; since the animal age is a crucial issue, further analysis on the rd1 RGC morphology and survival remains necessary to definitely clarify this point.
The characterization of degeneration events happening to retinal neurons in the rd10 mutant shows a quite catastrophic scenario because no photoreceptors remain viable (there is a row of aberrant cones) and cells that make directly contact with them (rod bipolar, cone bipolar and horizontal cells) undergo negative remodeling eventually culminating with their death.
Since rod bipolar cells do not make directly synaptic contact with RGCs, but ON and OFF cone bipolar do, we would expect to find signs of transynaptic alterations (atrophy or sprouting, for example), also in the dendrites of RGCs. Surprising, our data show the conservation of the fine morphology of dendritic tree arborizations and a full survival of these neurons. Thus, the progression of degeneration remains compartmentalised to second order neurons and does not proceed beyond the first retinal synapses. Indeed, also amacrine cells do not seem affected, since their number in the GCL remains unchanged and many of them do
not show negative signs of remodeling at least up to 5 months of age (Gargini et al., 2007).
A recent study by Marc and co-workers (2007) on two experimental models of RP, demonstrates a loss of expression of a metabotropic glutamate receptors in the inner retina (like we do), while rod bipolar cells transitorily express ionotropic glutamate receptors. Contemporary to rod death, rod bipolar cells make ectopic synapses with remaining cones. If in a normal retina rod bipolar cells excite the ON cone pathway and inhibit the OFF cone pathway by means of AII amacrine cells, the ectopic expression of ionotropic receptors would cause a shift in rod bipolars, that would result in a decreased excitation of ON cone bipolars and a decreased inhibition of OFF cone bipolars. The Authors call this novel network a corruptive circuit. While we cannot exclude the occurrence of such a circuit in the young rd10 retina, it is important to note that this consists of a transient remodeling that can persist only as long as patches of cones remain functional.
In rd10 mutants aged 9 months there are no residual patches of cones. Any remodeling occurring in the inner retina sooner than that age, does not leave traces on the morphology of RGCs, practically intact before and long after the peak of rod bipolar cell death.
Functional correlations to our morphological study can be inferred from a recent electrophysiological investigation on rd1 mice. RGCs remain electrically excitable by exogenous current injections up to 4 months, long after the age at which an ERG could be recorded and 3 months after complete photoreceptor loss (Stasheff, 2008; Suzuki et al., 2004). However, a significantly higher threshold stimulation charge is necessary to stimulate rd1 ganglion cells, compared to their wt counterpart. This result is consistent with electrical stimulation tests in humans in which it has been demonstrated that retinal areas of more severe damage, either caused by RP or laser, have a higher electrical stimulation threshold (Humayun et al., 1996 and 1999a). This is a known limitation factor to the application of epiretinal prostheses, as it is known that high current densities, necessary to stimulate inner retinal cells, might be highly toxic to neurons. One possibility is that the high threshold of inner retinal cells is caused by the formation of a glial scar around them, acting as an electrical insulator. Given the milder nature of the rd10 mutation compared to the rd1, it is possible that the threshold effects are shifted in time.
The retention of RGC morphological features and survival in the rd10 mutant differ sensibly from electrophysiological data of Pu et al., 2006, who profiled response properties of RGCs of RCS rats, at P30 and P100. Concentrating on α- cells, the Authors found that RGCs alter slowly over the course of photoreceptor degeneration, but with significant decreases in apparent receptive field size, contrast and threshold sensitivity by the first month of life. Furthermore, spatial frequency tuning and contrast responses appear extremely weak, paralleled by a progressive decline in signal to noise ratio. This decline is not only a simple loss of responsiveness but also a decrease in the signal, as background firing rates increased substantially over time; and, whereas wt retinas are dominated by ON- centre cells, dystrophic animals were dominated by OFF-centre cells. Further, they show that there is progressive loss of RGCs counted by retrograde transport partly due to variable constriction of trapped axons by ingrowing vessels and other extensive alterations such as cell death, migration, and rewiring (Rollag et al., 2003).
We do not find morphological differences either among ON or OFF RGC types of the rd10 mutant mouse. Albeit a specific electrophysiological profiling should be provided for this mouse model as well, some functional indications (besides excellent morphology and survival) can be obtain by the retention of anterograde axonal transport. This shows intact projections of RGCs to superior colliculus and dorsal lateral geniculate nucleus at 9 months of age. The marker we use to trace anterograde projections, cholera toxin, is more than a good neuronal tracer. In fact, in contrast to Horseradish Peroxidalse (HRP) that is passively taken up by neurons, cholera-toxin (a bacterial toxin produced by Vibrio cholerae) binds specifically to surface receptors of neurons and is actively taken up and transported by the axons (Luppi et al., 1987). This means that large patches of ganglion cells incapable of transporting centripetally are absent in the mutant.
This is important in view of the fact that in various forms of human RP there is atrophy of RGC axons, damage of optic nerves and of the optic chiasm (Newman et al., 1987). Evidently, the rd10 mutant is an example of mild, negative effects caused by RP, both intraretinally and in higher visual centres. Indeed, human RP patients with pde6b mutations show a slow decay in visual function.
In the past, axonal transport rates in RGCs have been evaluated with amino acids incorporation using C57Bl6/J -rd1 mutants (Graftein et al., 1972; Eisenfeld et al., 1984), with contradictory results. While the older study reports a 35% decrease in
slow axonal transport in the mutants, the work of 1984 reports no changes in both the rd1 and the RCS rat.
Atrophic changes in retinal vasculature are consequences of RP in humans and in animal models. While it is thought that this atrophy is secondary to diminished metabolic demand in the face of retinal degeneration, Otani et al. (2004) were able of successful photoreceptor rescue with adult bone marrow-derived stem cells, thus implicating that preserving the normal vasculature of the deep plexus results into preservation of cones in the ONL.
We have investigated if this is also true for the vasculature of superficial and intermediate plexa irrorating respectively RGCs bodies and dendritic arborizations; before our study, very little was known about preservation of the blood vessels network subserving the inner retina in this and in other mutants.
Since that RGC morphology and survival are preserved at late ages in rd10 mice, we would have expected a hypertrophy or, at minimum, a maintenance of the vascular network irrorating the GCL, as a compensatory effort to ensure blood supply to inner retinal neurons. RGCs require oxygen in a special way, because of their strong electrical activity and the necessity to sustain a long axon.
Our results show that inner retinal blood vessels are significantly less complex (12%) in the rd10 compared to the wt just, already at P20, before the peak of rod degeneration. The decrement in complexity enhances during the progression of the pathology, reaching 16% at P30, and 30% at 9 months. These results confirm an old notion, and namely that photoreceptor degeneration and loss of blood vessels are two parallel events that are both present since the beginning of the pathology. On the other hand, loss of vascular complexity means a decrement of part of the trophic support to RGCs and other inner retinal cells (Yourey et al., 2000; Kilic et al., 2006). In view of the findings that RGCs morphology and survival have not been affected, it is interesting to address the issue on the possible sources of trophic factors for these neurons.
A possibility to explain the good retention of RGC morphology notwithstanding blood vessels atrophy is that choroidal oxygenation provides some compensation in the mutant (reviewed in Yu and Cringle, 2001). In fact, choriocapillaries, the blood vessels that supply nutrients to the outer retina, have the highest per unit volume of blood in the human body (Weiland et al., 2005). It is likely that, in the rd10 mutant, the reduction in retinal thickness caused by photoreceptor
degeneration makes oxygen, nourishment and neurotrophic factors of choroid capillaries accessible to inner retinal cells, enhancing their resistance.
Another explanation may be the relative maturity of the retina at the time of the photoreceptor loss. Perhaps, the relatively normal visual input that the inner retina has received for several weeks before most of the photoreceptors are lost confers a resistance to transneuronal degeneration. In fact, if we compare with rd1 mutant in which synaptogenesis is not still finished when photoreceptors start to die, rd10 retina is almost fully mature before there are any signs of degeneration. It is likely that a well-established architecture in a particular phase of retinal development could be crucial for the future events.
Another factor that must be considered is the possibility that the inner retina of rd10 mice is stimulated sufficiently to maintain it in a normal state. On this account, Müller glial cell activation typical of retinal degeneration could itself represent a response leading to neurotrophic factor secretion. Armson et al.
(1987) have shown that in early development, during the period of programmed cell death, culture medium conditioned with Müller cell is a powerful enhancer of RGC survival. It is possible to speculate that Müller cells retain their trophic potential when cell death is caused by a mutation, dramatically reducing the survival of photoreceptors. Müller cells have been shown to be endowed with remarkable plasticity in recent studies (Fisher and Reh, 2001; Reh and Fischer, 2001; Vetter and Moore, 2001) and now there is evidence that they have stem cell properties in the retina (Insua et al., 2008).
The retrograde transport of a combination of neurotrophic molecules normally derived by the cells of the central visual pathways could also represent a source of survival factors for RGCs. Since central connections are still intact in the adult rd10, it is possible that trophic factors, such as BDNF, CTNF, IGF-1 from the central visual targets support RGC survival. In addition, an intra-retinal source of BDNF has been hypothesized (for a review, Yip and So, 2000).
In parallel to the classical visual pathways, neurotrophic factors and other molecules, such as serotonin (Mikkelsen, 1988), could reach the retina from the few retinopetal neurons (centrifugal visual system) located in different brain regions (for a review, Reperant et al., 2006). The centrifugal visual fibres are unmyelinated or myelinated and, in some cases, their neurons form part of a feedback loop, relaying information from a primary visual centre back to the retina.
For example, it appears that serotonin from the dorsal raphe nucleus might be
involved in retinal melatonin synthesis and coordination of circadian rhythms (Mikkelsen, 1988).
The morphological preservation and survival of RGCs in the rd10 mutant could be supported also by events happening before eye-opening. In this period, developmental refinement of retinal circuits and central projections occur. It is known that retinal spontaneous activity affects the formation and/or alteration of central connections (Torborg and Feller, 2005). It is possible to speculate that the spontaneous activity might preserve the intra-retinal architecture of ganglion cells.
It is known that even blind mice, such as the rd1, have normal circadian rhythms, as fully sighted animals. This is explained by the existence of photosensitive RGCs (melanopsin ganglion cells, or pRGCs) that act as photoreceptors, in the sense that they carry a photopigment (Foster and Hankins, 2007). Studies about pRGCs monitoring the effects of light stimulation on intracellular calcium reveal that there are three types of light-evoked calcium influxes: sustained, transient, and repetitive, suggestive of distinct functional types of RGCs. These studies show the existence of an extensive and heterogeneous syncytium of intrinsically photosensitive neurons in the GCL, which can be uncoupled by application of gap-junctional blockers (Sekaran et al., 2003). These cells are not de-afferented by photoreceptor degeneration and it is possible to speculate that their connections with other retinal neurons are sufficient, or contributes to, the preservation of the overall population of RGCs.
Whatever the intrinsic or exogenous source of positive factors altogether contributing to the survival and preservation of retinal ganglion cells in the rd10 mutant mouse might be, our study demonstrate that this mutant is an excellent model to attempt restoration of vision in RP based on epiretinal prostheses, cellular transplantation beyond the photoreceptor level and direct stimulation of the ganglion cell layer. We also conclude that, given the similarities between the rd10 mutant and the disease progression in human RP patients with a phosphodiesterase mutation, this mouse should be preferred to the rd1 model in studies of the cell biology of Retinitis Pigmentosa.
5. Concluding remarks and future directions
This study has characterized extensively a new mouse model of typical moderate human RP, the rd10 mutant mouse. With this mutant, we have analyzed a number of retinal events correlated to photoreceptor death caused by a single nucleotide mutation. Particular attention has been devoted to understand and characterize quantitatively secondary effects upon inner retinal neurons (rod bipolar and horizontal cells) and verify the possibility of a transneuronal occurrence of remodeling and death signals to ganglion cells, the only neurons with an exit from the retina.
Despite the occurrence of remodelling and slow degeneration of all second order neurons making synapses with rods and despite blood vessels attenuation contemporary to photoreceptor death, RGCs remain preserved in their finest morphological features and number for a long period of the animal life-span (9 months of age). In addition, the RGC projection to the LGN and CS are well preserved at the same age.
There are at least two therapeutically significant consequences emerging from this study.
The first confirms the importance of an early RP diagnosis based upon ERG recordings, from which changes in the b-wave can be detected (Fleischhauer et al., 2005). In humans, RP is diagnosed in late adolescence. In general, when this happens, rod and cone degeneration has already fairly progressed and second order neurons remodeling presumably started. It is important to detect early signs of remodeling if inner retinal cells have to be used for restoration therapies.
The other consequence points out to RGCs as a favourable biological substrate for a disease treatment. Epiretinal prostheses, electronic or coupled with the delivery of neurotransmitters, could represent a broad-range type of treatment, also showing the advantage of being mutation-independent.
On the other hand, our study confirms that many aspects of the downstream effects of rod degeneration have yet to be investigated. A true correlation between morphology and function has not yet been achieved for the rd10 mutant, and our analysis of RGCs should be paired to a detailed electrophysiological approach, already performed for the rd1 mutant (Stasheff, 2008; Suzuki et al., 2004) and on RCS rats (Pu et al., 2006). This approach could have some important
consequences on epiretinal prosthesis design. On this account, the knowledge of synaptic architecture, i.e. synaptic receptor and channel distribution upon the dendrites of single ganglion cells can help designing epiretinal prosthesis in which neurotransmitter are locally released.
At present, two types of methodological approaches are used on the normal mouse (C57Bl6/J and Thy1-GFP-M) investigating the distribution of GABA-A and glutamate receptors on individual ganglion cells. The first is a screening of the expression of ionotropic glutamate receptor subunits in different populations of inner retinal neurons by molecular biology techniques (Jacobs et al., 2007); the second is a morphological study of the same receptors (Masland, European Retina Meeting, 2007 discussion). A similar analysis in RP mutant mice would reveal the exact localization of important neurotransmitter receptors and help the design of chemically-based electronic prostheses.
Studying morphological events related to photoreceptor degeneration in this and other mouse model, we found that stereotyped events occur in the inner retina after rod degeneration; the stereotype seem to include human RP subjects as well (Strettoi et al., 2003; Pignatelli et al., 2004; Strettoi, unpublished data).
Stereotyped remodelling culminating with cell death is suggestive of conserved cellular mechanisms of reaction to injury, possibly leading to common cellular responses in inner retinal neurons.
This is not unlikely, since common degeneration pathway exists for rods and cones independently from the underlying genetic mutation. For instance, calpain activity is markedly increased in rd1 photoreceptor cells and co-localizes with markers of cell death (Paquet-Durand et al., 2006). Since calpains are proteases which act downstream calcium signals and ultimately triggering cell death, these data correlate their increase in the rd1 to the effect of a pde6b mutation (stationary opening of calcium channels controlled by c-GMP). This finding is probably true for all the known pde6b mutations, as well as for other genetic defects leading to an increased calcium influx in photoreceptors.
A major, secondary effect of rod apoptosis is the degeneration of cones, independently from the primary genetic defect triggering rod death. The present view is that, among others, rods promote cone viability by secreting a factor known as Rod-derived Cone Viability Factor (Leveillard et al., 2004a, b).
Altogether, this study represents a fairly complete starting point toward future investigations for RP treatment based upon data from different disciplines, including ophthalmology, molecular biology, electrophysiology, bio-engineering and biophysics.
