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.