The effect of photoreceptor degeneration on ganglion cell morphology

Retinitis pigmentosa refers to a family of inherited photoreceptor degenerations resulting in blindness. During and after photoreceptor loss, neurons of the inner retina are known to undergo plastic changes. Here, we have investigated in detail whether ganglion cells are altered at late stages of degeneration, well after the total loss of photoreceptors. We used mice, rd1‐Thy1, that carry a mutation in the β‐subunit of phosphodiesterase 6 and a fluorescent protein that labels a subset of ganglion cells and B6‐Thy1 control mice. Retinal wholemounts from mice aged 3–11 months were processed for immunohistochemistry and analyzed. Ganglion cells were classified based on soma area, dendritic field size, and branching of dendrites. The dendritic fields of some ganglion cells were further analyzed for their length, area and quantity of branching points. There was a decrease in size and level of branching of A2, B1, and D type ganglion cells in the degenerated retina at 11 months of age. In contrast, C1 ganglion cells remained unchanged. In addition, there was a shift in the proportion of ganglion cells ramifying in the different layers of the inner plexiform layer. Careful analysis of the dendrites of ganglion cells revealed some projecting to new, more distal regions of the inner plexiform layer. We propose that these changes in ganglion cell morphology could impact the function of individual cells as well as the retinal circuitry in the degenerated retina. J. Comp. Neurol. 522:1155–1170, 2014. © 2013 Wiley Periodicals, Inc.     

Retinal organization in the retinal degeneration 10 (rd10) mutant mouse: a morphological and ERG study

Retinal degeneration 10 (rd10) mice are a model of autosomal recessive retinitis pigmentosa (RP), identified by Chang et al. in 2002 (Vision Res. 42:517-525). These mice carry a spontaneous mutation of the rod-phosphodiesterase (PDE) gene, leading to a rod degeneration that starts around P18. Later, cones are also lost. Because photoreceptor degeneration does not overlap with retinal development, and light responses can be recorded for about a month after birth, rd10 mice mimic typical human RP more closely than the well-known rd1 mutants. The aim of this study is to provide a comprehensive analysis of the morphology and function of the rd10 mouse retina during the period of maximum photoreceptor degeneration, thus contributing useful data for exploiting this novel model to study RP. We analyzed the morphology and survival of retinal cells in rd10 mice of various ages with quantitative immunocytochemistry and confocal microscopy; we also studied retinal function with the electroretinogram (ERG), recorded between P18 and P30. We found that photoreceptor death (peaking around P25) is accompanied and followed by dendritic retraction in bipolar and horizontal cells, which eventually undergo secondary degeneration. ERG reveals alterations in the physiology of the inner retina as early as P18 (before any obvious morphological change of inner neurons) and yet consistently with a reduced band amplification by bipolar cells. Thus, changes in the rd10 retina are very similar to what was previously found in rd1 mutants. However, an overall slower decay of retinal structure and function predicts that rd10 mice might become excellent models for rescue approaches.     

Inner retinal preservation in the photoinducible I307N rhodopsin mutant mouse,a model of autosomal dominant retinitis pigmentosa

Rod-cone degenerations, for example, retinitis pigmentosa are leading causes of blindness worldwide. Despite slow disease progression in humans, vision loss is inevitable; therefore, development of vision restoration strategies is crucial. Among others, promising approaches include optogenetics and prosthetic implants, which aim to bypass lost photoreceptors (PRs). Naturally, the efficacy of these therapeutic strategies will depend on inner retinal structural and functional preservation. The present study shows that in photoinducible I307N rhodopsin mice (Translational Vision Research Model 4 [Tvrm4]), a 12k lux light exposure eliminates PRs in the central retina in 1 week, but interneurons and their synapses are maintained for as long as 9 weeks postinduction. Despite bipolar cell dendritic retraction and moderate loss of horizontal cells, the survival rate of various cell types is very high. Significant preservation of conventional synapses and gap junctions in the inner plexiform layer is also observed. We found the number of synaptic ribbons to gradually decline and their ultrastructure to become transiently abnormal, although based on our findings intrinsic retinal architecture is maintained despite complete loss of PRs. Unlike common rodent models of PR degeneration, where the disease phenotype often interferes with retinal development, in Tvrm4 mice, the degenerative process can be induced after retinal development is complete. This time course more closely mimics the timing of disease onset in affected patients. Stability of the inner retina found in these mutants 2 months after PR degeneration suggests moderate, stereotyped remodeling in the early stages of the human disease and represents a promising finding for prompt approaches of vision restoration.     

Aberrant synaptic input to retinal ganglion cells varies with morphology in a mouse model of retinal degeneration

Retinal degeneration describes a group of disorders which lead to progressive photoreceptor cell death, resulting in blindness. As this occurs, retinal ganglion cells (RGCs) begin to develop oscillatory physiological activity. Here we studied the morphological and physiological properties of RGCs in rd1 mice, aged 30–60 days, to determine how this aberrant activity correlates with morphology. Patch‐clamp recordings of excitatory and inhibitory currents were performed, then dendritic structures were visualized by infusion of fluorescent dye. Only RGCs with oscillatory activity were selected for further analysis. Oscillatory frequency and power were calculated using power spectral density analysis of recorded currents. Dendritic arbor stratification, total length, and area were measured from confocal microscope image stacks. These measurements were used to sort RGCs by cluster analysis using Ward's Method. This resulted in a total of 10 clusters, with monostratified and bistratified cells having five clusters each. Both populations exhibited correlations between arbor stratification and aberrant inhibitory input, while excitatory input did not vary with arbor distribution. These findings illustrate the relationship between aberrant activity and RGC morphology at early stages of retinal degeneration. J. Comp. Neurol. 522:4085–4099, 2014. © 2014 Wiley Periodicals, Inc.     

Early remodeling of müller cells in the rd/rd mouse model of retinal dystrophy

We studied the anatomical remodeling and gliosis of retinal Müller cells in the rd/rd mouse model of photoreceptor degeneration. A computational calculation of glutamine synthetase immunoreactivity was developed so we could specifically quantify changes in Müller cell anatomy between control mice (C57Bl/6) and the dystrophic strain. We found no change in the number of Müller cell somata between mice strains, indicating no cell proliferation as a function of development and degeneration. The retinal area occupied by the total Müller cell body (soma and processes) was significantly less in the rd/rd mouse retina compared with control mice. When only the outer retina was considered, we found rd/rd Müller cell processes were dramatically reduced during the cone phase of photoreceptor degeneration. However, at older ages an increase in Müller cell processes was seen. Conversely, glial fibrillary acidic protein (GFAP) expression showed a significant increase during cone degeneration followed by a reduction in older ages. Müller cell electrophysiology, particularly K⁺ currents and membrane potential, was similar between rd/rd and control Müller cells during cone degeneration. Together, these results show that glial remodeling in the rd/rd retina follows separate phases—an initial conservative glial response involving the loss of Müller cells processes, hyperexpression of GFAP, and preservation of normal electrophysiology followed by an active growth of Müller cell processes, glial seal formation, and attenuation of GFAP expression after complete photoreceptor loss. J. Comp. Neurol. 521:2439–2453, 2013. © 2013 Wiley Periodicals, Inc.     

Inner retinal abnormalities in a mouse model of Leber's congenital amaurosis

Leber's congenital amaurosis (LCA) is the earliest and most severe form in the world of genetic retinal dystrophy causing blindness. An animal model of LCA was recently created in which the cone-rod homeobox (crx) gene was disrupted using homologous recombination. Crx-/- mice display abnormal development of photoreceptors followed by their degeneration. We analyzed the morphology of inner retinal cells in crx-/- mice in order to evaluate the effects of abnormal photoreceptor development and death upon other retinal neurons. The identification of a time window during which inner retinal cells are still viable could be very important in view of the possibilities that photoreceptor transplantation or gene therapy might be used to restore vision in LCA. We used a combination of immunocytochemical and confocal microscopy techniques to screen the crx-/- inner retina and verify its morphological integrity after photoreceptor degeneration. We found significant morphological alterations in second-order neurons in crx-/- animals. The appearance of mutant retinas after photoreceptor death is indistinguishable from that of the retinal degeneration (rd/rd) mouse, a different genetic model of a retinal disease characterized by photoreceptor degeneration. However, at early stages of photoreceptor degeneration the morphology of retinal cells in the crx-/- mutant is considerably well preserved. It is likely that different genetic mechanisms that cause abnormal photoreceptor development and/or degeneration lead to a common pathway that determines second-order neuron modifications. The severity of modifications is linked to the timing of onset of the degeneration and appears to increase with time.     

Morphological identification and systematic classification of mammalian retinal ganglion cells. I. Rabbit retinal ganglion cells

Retinal ganglion cells (RGCs) convey visual signals to 50 regions of the brain. For reasons of interest and convenience, they constitute an excellent system for the study of brain structure and function. There is general agreement that, absent a complete “parts list,” understanding how the nervous system processes information will remain an elusive goal. Recent studies indicate that there are 30–50 types of ganglion cell in mouse retina, whereas only a few years ago it was still written that mice and the more visually oriented lagomorphs had less than 20 types of RGC. More than 30 years ago, I estimated that rabbits have about 40 types of RGC. The present study indicates that this number is much too low. I have employed the old but powerful method of Golgi-impregnation to rabbit retina, studying the range of component neurons in this already well-studied retinal system. Close quantitative and qualitative analyses of 1,142 RGCs in 26 retinas take into account cell body and dendritic field size, level(s) of dendritic stratification in the retina's inner plexiform layer, and details of dendritic branching. Ninety-one morphologies are recognized. Of these, at least 32 can be correlated with physiologically studied RGCs, dye-injected for morphological analysis. It is unlikely that rabbits have 91 types of RGC, but is argued here that this number lies between 60 and 70. The present study provides a “yardstick” for measuring the output of future molecular studies that may be more definitive in fixing the number of RGC types in rabbit retina.     

Luteolin delays photoreceptor degeneration in a mouse model of retinitis pigmentosa

Luteolin is neuroprotective for retinal ganglion cells and retinal pigment epithelial cells after oxidative injury,whereby it can inhibit microglial neurotoxicity.Therefore,luteolin holds the potential to be useful for treatment of retinal diseases.The purpose of this study was to investigate whether luteolin exhibits neuroprotective effects on rod cells in rd10 mice,a slow photoreceptor-degenerative model of retinitis pigmentosa.Luteolin(100 mg/kg)intraperitoneally injected daily from postnatal day 14(P14)to P25 significantly enhanced the visual performance and retinal light responses of rd10 mice at P25.Moreover,it increased the survival of photoreceptors and improved retinal structure.Mechanistically,luteolin treatment attenuated increases in reactive oxygen species,photoreceptor apoptosis,and reactive gliosis;increased mRNA levels of anti-inflammatory cytokines while lowering that of pro-inflammatory and chemoattractant cytokines;and lowered the ratio of phospho-JNK/JNK.Application of the JNK inhibitor SP600125 exerted a similar protective effect to luteolin,suggesting that luteolin delays photoreceptor degeneration and functional deterioration in rd10 mice through regulation of retinal oxidation and inflammation by inhibiting the JNK pathway.Therefore,luteolin may be useful as a supplementary treatment for retinitis pigmentosa.This study was approved by the Qualified Ethics Committee of Jinan University,China(approval No.IACUC-20181217-02)on December 17,2018.     

Paradoxical opsin expressing cells in the inner retina that are augmented following retinal degeneration

Here we reveal a population of cells that express cone photoreceptor opsins that are located in the inner retina, distant from outer retinal photoreceptors. These cells are present in rodents and human. They also express a range of key proteins critical in the cone phototransduction cascade and make contact with other retinal neurons. Their opsins are not generally confined to cellular specialized regions but are present throughout the plasma membrane, although their nuclear configurations are similar to those of outer retinal cones. This population is distinct from the ganglion cells that contain melanopsin and which are known to be inner retinal irradiance detectors regulating circadian behaviour. Surprisingly, the size of the population of short wavelength opsin positive cells in the ganglion cell layer is plastic. In normal animals their number declines with age. However, their numbers increase significantly in response to outer retinal photoreceptor loss, probably by drawing on a pool of inner retinal cells that express cone specific markers, but not opsins.     

Central projections of melanopsin-expressing retinal ganglion cells in the mouse

A rare type of ganglion cell in mammalian retina is directly photosensitive. These novel retinal photoreceptors express the photopigment melanopsin. They send axons directly to the suprachiasmatic nucleus (SCN), intergeniculate leaflet (IGL), and olivary pretectal nucleus (OPN), thereby contributing to photic synchronization of circadian rhythms and the pupillary light reflex. Here, we sought to characterize more fully the projections of these cells to the brain. By targeting tau-lacZ to the melanopsin gene locus in mice, ganglion cells that would normally express melanopsin were induced to express, instead, the marker enzyme beta-galactosidase. Their axons were visualized by X-gal histochemistry or anti-beta-galactosidase immunofluorescence. Established targets were confirmed, including the SCN, IGL, OPN, ventral division of the lateral geniculate nucleus (LGv), and preoptic area, but the overall projections were more widespread than previously recognized. Targets included the lateral nucleus, peri-supraoptic nucleus, and subparaventricular zone of the hypothalamus, medial amygdala, margin of the lateral habenula, posterior limitans nucleus, superior colliculus, and periaqueductal gray. There were also weak projections to the margins of the dorsal lateral geniculate nucleus. Co-staining with the cholera toxin B subunit to label all retinal afferents showed that melanopsin ganglion cells provide most of the retinal input to the SCN, IGL, and lateral habenula and much of that to the OPN, but that other ganglion cells do contribute at least some retinal input to these targets. Staining patterns after monocular enucleation revealed that the projections of these cells are overwhelmingly crossed except for the projection to the SCN, which is bilaterally symmetrical.     

Depth segregation of retinal ganglion cells projecting to mouse superior colliculus

The retinotectal projections in the mouse were analyzed with injections of horseradish peroxidase into the superior colliculus and of radioactive amino acids into the eye. At least 70% of the ganglion cells, and possibly all of them, were found to project to the superior colliculus, including ganglion cells of all sizes. Small injections revealed that ganglion cells of different sizes terminate at different levels in the superior colliculus. The small ganglion cells that form the vast majority of all cells project predominantly to the upper stratum griseum superficiale. A small population of mainly medium-sized and large ganglion cells project to the deep stratum griseum superficiale and to the stratum opticum. The ipsilateral projection is restricted to the deep stratum griseum superficiale and stratum opticum and consists predominantly of medium-sized and large ganglion cells.     

Relationships between ganglion cell dendritic structure and retinal topography in the cat

The morphology of ganglion cell dendritic trees varies across the cat retina. Evidence is presented that the variation in two attributes of ganglion cell dendritic structure can be accounted for by specific aspects of the topography of the adult and developing retina. The first attribute considered was the displacement of the center of the dendritic field from the cell body in the plane of the retina. The results of this study provide evidence that most ganglion cell dendritic fields are displaced away from neighboring cells, i.e., down the retinal ganglion cell density gradient. Because of the systematic dendritic displacement locally, the centers of the dendritic fields are arranged in a more precise mosaic than are their cell bodies. The second attribute considered was the elongation and orientation of the dendritic fields. From approximately embryonic day 50 to postnatal day 10 the cat retina undergoes a process of maturation (reviewed by Rapaport and Stone: Neuroscience 11:289-301, '84) that begins at the area centralis and spreads over the retina in a horizontally elongated wave. We found that the elongation and orientation of retinal ganglion cell dendritic fields is significantly correlated with the shape of the wave of maturation. The orientation of a dendritic field is not predicted by the direction of its displacement nor is it directly related to the distribution of neighboring retinal ganglion cells. These results indicate that the displacement of a ganglion cell's dendritic field from its cell body results from mechanisms different from those responsible for the orientation of the dendritic field. Factors that may be responsible for these two attributes of ganglion cell dendritic morphology are discussed.     

APV prevents the elimination of transient dendritic spines on a population of retinal ganglion cells

Blockade of the N-methyl-D-aspartate (NMDA) receptors on retinal ganglion cells (RGCs) during development prevents the elimination of the exuberant spine-like processes in a population of Type I RGCs in hamsters. During the development of RGCs, exuberant dendritic spines have been observed which disappear during maturation. Blocking the NMDA receptors on developing RGCs with the antagonist, DL-2-amino-5-phosphonovaleric acid (APV) and the subsequent retention of some of the normally transient dendritic spines suggest that the morphological development of post-synaptic neurons may be affected by this treatment. Our result further suggests that the elimination of exuberant spines during normal development requires interactions between receptors on the spines and neurotransmitters released by the pre-synaptic inputs.     

Morphological changes in the Royal College of Surgeons rat retina during photoreceptor degeneration and after cell-based therapy

There are concomitant morphological and functional changes in the inner retina during the course of photoreceptor degeneration in a range of animal models of retina degeneration and in humans with eye disease. One concern that has been raised is that the changes occurring in the inner retina might compromise attempts to rescue or restore visual input by various interventional approaches. It is known that cell-based therapy can preserve significant visual capability for many months. In this study, we examine the overall changes in the Royal College of Surgeons (RCS) rat during degeneration and the effects of cell transplantation by means of immunohistochemistry and confocal microscopy. The degenerative changes are complex, and they progress with age. They involve the neurons with which both rods and cones interconnect--retinal second- and third-order neurons underwent dramatic modification, including sprouting, retraction as photoreceptor loss progressed--as well as Müller glia and secondary vascular changes, which were associated at later times with neuronal migration. The pathological vascular changes led to major disruption of inner retina. After introducing a retinal pigment epithelial cell line to the subretinal space early in the progress of photoreceptor degeneration, most inner retinal changes were held in abeyance for up to at least 10 months of age. Given the concern that has been raised regarding whether inner retinal changes might compromise any graft-related benefit, this is an encouraging finding.     

Lutein delays photoreceptor degeneration in a mouse model of retinitis pigmentosa

Retinitis pigmentosa is a retinal disease characterized by photoreceptor degeneration.There is currently no effective treatment for retinitis pigmentosa.Although a mixture of lutein and other antioxidant agents has shown promising effects in protecting the retina from degeneration,the role of lutein alone remains unclear.In this study,we administered intragastric lutein to Pde6brd10 model mice,which display degeneration of retinal photoreceptors,on postnatal days 17(P17)to P25,when rod apoptosis reaches peak.Lutein at the optimal protective dose of 200 mg/kg promoted the survival of photoreceptors compared with vehicle control.Lutein increased rhodopsin expression in rod cells and opsin expression in cone cells,in line with an increased survival rate of photoreceptors.Functionally,lutein improved visual behavior,visual acuity,and retinal electroretinogram responses in Pde6brd10 mice.Mechanistically,lutein reduced the expression of glial fibrillary acidic protein in Müller glial cells.The results of this study confirm the ability of lutein to postpone photoreceptor degeneration by reducing reactive gliosis of Müller cells in the retina and exerting anti-inflammatory effects.This study was approved by the Laboratory Animal Ethics Committee of Jinan University(approval No.LACUC-20181217-02)on December 17,2018.     

Characterization of multiple bistratified retinal ganglion cells in a purkinje cell protein 2‐Cre transgenic mouse line

Retinal ganglion cells are categorized into multiple classes, including multiple types of bistratified ganglion cells (BGCs). The recent use of transgenic mouse lines with specific type(s) of ganglion cells that are labeled by fluorescent markers has facilitated the morphological and physiological studies of BGCs, particularly the directional‐selective BGCs. The most important benefit from using transgenic animals is the capability to perform in vivo gene manipulation. In particular, the Cre/LoxP recombination system has become a powerful tool, allowing gene deletion, overexpression, and ectopic expression in a cell type‐specific and temporally controlled fashion. The key to this tool is the availability of Cre mouse lines with cell or tissue type‐specific expression of Cre recombinase. In this study we characterized the Cre‐positive retinal ganglion cells in a PCP2 (Purkinje cell protein 2)‐cre mouse line. We found that all of the Cre‐positive retinal ganglion cells were BGCs. Based on morphological criteria, we determined that they can be grouped into five types. The On‐ and Off‐dendrites of three of these types stratified outside of the cholinergic bands and differed from directional selective ganglion cells (DSGCs) morphologically. These cells were negative for Brn‐3b and positive for both calretinin and CART retina markers. The remaining two types were identified as putative On‐Off and On‐DSGCs. This Cre mouse line could be useful for further studies of the molecular and functional properties of BGCs in mice. J. Comp. Neurol. 521:2165–2180, 2013. © 2012 Wiley Periodicals, Inc.     

The effect of PKC activation on the survival of rat retinal ganglion cells in culture

Natural cell death is a degenerative phenomenon occurring during the development of the nervous system. Approximately half the neurons initially generated during this period die. The role of trophic molecules produced by target and afferent neurons as well as by glial cells controlling this regressive event has been extensively demonstrated. The aim of this work was to study the role of activated protein kinase C (PKC), an enzyme involved in apoptosis regulation, on the survival of retinal ganglion cells kept "in vitro" for 48 h. For this purpose, we used the phorbol 12-myristate 13-acetate (PMA), a tumor promoter agent that activates PKC. Our results showed that PMA increases the survival of ganglion cells. The effect was dose-dependent and PMA concentrations of 10 or 100 ng/ml produced the maximal effect (a two-fold increase on ganglion cells survival compared with 48 h control). This effect was totally abolished by 1.25 microM chelerythrine chloride (an inhibitor of PKC) and 30 microM genistein (an inhibitor of tyrosine kinase enzymes). Otherwise, PMA was effective only when it was chronically present in the cultures. On the other hand, treatment with 20 microM 5-fluoro-2'-deoxyuridine, an inhibitor of cell proliferation, or 25 microM BAPTA-AM, an intracellular calcium chelator, did not block PMA effect. Our results suggest that the survival of retinal ganglion cells "in vitro" may be mediated by a mechanism that involves PKC activation.     

Target dependence of chick retinal ganglion cells during embryogenesis: cell survival and dendritic development

The survival of retinal ganglion cells and the dendritic development were investigated a) in normal chick embryos, b) in embryos whose primordial optic lobes and adjacent areas were removed (target reduced embryos), and c) in embryos whose optic nerves were transected (target deprived embryos) in order to study the influences of central targets on developing ganglion cells. The ganglion cells were stained postmortem with the carbocyanine dye DiI. Cell body and dendritic field diameters were measured in whole-mounted retinae before and after the period of cell death at embryonic day 10 (E10) and E16. The cell densities within the ganglion cell layer were counted in cresyl violet/thionine stained retinae. The central retinal projection in target reduced embryos was studied with the anterogradely transported fluorescent marker rhodamine-B-isothiocyanate (RITC). In normal embryos, the earliest dendritic processes were observed at E6 in the central retina, whereas at E10 elaborate dendritic branching was found across the retina. Different morphological types of ganglion cells could be identified at E16. In both, target reduced embryos and target deprived embryos, the initial dendritic growth and pattern of ramification were indistinguishable from those of normal embryos up to E10. Cell body diameters, dendritic tree diameters, and cell densities were not significantly different. At the end of the naturally occurring cell death period (E16), the ganglion cell density was strongly reduced in both experimental groups compared to controls. In particular, when the optic nerve was transected, it resulted in the almost complete degeneration of ganglion cells. In target reduced embryos, a small population (about 5% of the normal number) of ganglion cells survived. The proportion of large cells was increased within the total population compared to normal retinae. Displaced ganglion cells were not affected by partial target removal but strongly affected by transection of the optic nerve. Anterograde labelling from the retina revealed that in target reduced embryos the remaining ganglion cells innervated non-tectal primary visual nuclei. The present results suggest the following: a) Before the onset of the cell death period, the growth and ramification of ganglion cell dendrites occur independently of central visual targets. b) In target reduced embryos, a small population of ganglion cells survives, namely, those cells that project to remaining central areas. Complete disconnection from central targets by transecting the optic nerve leads to the degeneration of almost all ganglion cells. c) The surviving ganglion cell population consists mainly of large ganglion cells.