Intrinsic differences in rod and cone membrane composition: implications for cone degeneration.

PURPOSE: In many retinal pathological conditions, rod and cone degeneration differs. For example, the early-onset maculopathy Stargardts disease type 1 (STGD1) is typified by loss of cones while rods are often less affected. We wanted to examine whether there exist intrinsic membrane differences between rods and cones that might explain such features. METHODS: Abca4 mRNA and protein levels were quantified in rod- and cone-enriched samples from wild-type and Nrl-/- mice retinas; rod- and cone-enriched outer segments (ROS and COS respectively) were prepared from pig retinas, and total lipids were analyzed by flame ionization, chromatography, and tandem mass spectrometry. Immunohistochemical staining of cone-rich rodent Arvicanthis ansorgei retinas was conducted, and ultra-high performance liquid chromatography of lipid species in porcine ROS and COS was performed. RESULTS: Abca4 mRNA and Abca4 protein content was significantly higher (50-300%) in cone compared to rod-enriched samples. ROS and COS displayed dramatic differences in several lipids, including very long chain poly-unsaturated fatty acids (VLC-PUFAs), especially docosahexaenoic acid (DHA, 22:6n-3): ROS 20.6% DHA, COS 3.3% (p < 0.001). VLC-PUFAs (> 50 total carbons) were virtually absent from COS. COS were impoverished (> 6× less) in phosphatidylethanolamine compared to ROS. ELOVL4 ("ELOngation of Very Long chain fatty acids 4") antibody labelled Arvicanthis cones only very weakly compared to rods. Finally, there were large amounts (905 a.u.) of the bisretinoid A2PE in ROS, whereas it was much lower (121 a.u., ~ 7.5-fold less) in COS fractions. In contrast, COS contained fivefold higher amounts of all-trans-retinal dimer (115 a.u. compared to 22 a.u. in rods). CONCLUSIONS: Compared to rods, cones expressed higher levels of Abca4 mRNA and Abca4 protein, were highly impoverished in PUFA (especially DHA) and phosphatidylethanolamine, and contained significant amounts of all-trans-retinal dimer. Based on these and other data, we propose that in contrast to rods, cones are preferentially vulnerable to stress and may die through direct cellular toxicity in pathologies such as STGD1.

Hyperglycemia and circadian disruption lead to retinal dysfunction in a stabilized colony of the fat sand rat Psammomys obesus.

PURPOSE: The Fat Sand Rat (Psammomys obesus) recapitulates several features of human pre-proliferative diabetic retinopathy, but data are restricted to wild animals, incompatible with stringent biomedical research criteria. To overcome this barrier, we characterized retinal changes in a colony of P. obsesus maintained under strictly controlled housing conditions. METHODS: Animals were maintained on low or high caloric energy diets, and raised under either standard (12 h light/12 h dark) or shortened (5 h light/5 h dark) photoperiods. Visual responses were tested by electroretinography, while structural/molecular changes were assayed by immunochemistry and molecular biology (RNAseq and qPCR). RESULTS: Whereas high calorie diet alone did not induce hyperglycemia, coupled with short photoperiod >80 % animals developed severe hyper-insulinemia by 15 weeks, and 16 % animals further developed hyperglycemia. In these groups, electroretinography showed significant declines in visual responses in both hyper-insulinemic and hyperglycemic animals, especially in photopic (cone) responses. Transcriptomics analysis of hyperglycemic compared to low caloric controls revealed major upregulation in pathways involved in glial activation, extracellular matrix remodeling, inflammation, cytokine production, partial ischemic responses and angiogenesis. Western blotting against rhodopsin and cone opsin also showed decreased levels in both groups, overall decreases being greater for cones than rods in hyperglycemic animals. CONCLUSIONS: P. obesus maintained in rigorously monitored captive conditions, albeit showing attenuated responses to dietary overload compared to wild counterparts, nevertheless do develop some retinal features of diabetic retinopathy-like degeneration. Such a colony with known sanitary status opens their broader use for biomedical research.

Diurnal rodents as pertinent animal models of human retinal physiology and pathology.

This presentation will survey the retinal architecture, advantages, and limitations of several lesser-known rodent species that provide a useful diurnal complement to rats and mice. These diurnal rodents also possess unusually cone-rich photoreceptor mosaics that facilitate the study of cone cells and pathways. Species to be presented include principally the Sudanian Unstriped Grass Rat and Nile Rat (Arvicanthis spp.), the Fat Sand Rat (Psammomys obesus), the degu (Octodon degus) and the 13-lined ground squirrel (Ictidomys tridecemlineatus). The retina and optic nerve in several of these species demonstrate unusual resilience in the face of neuronal injury, itself an interesting phenomenon with potential translational value.

An invertebrate-like phototransduction cascade mediates light detection in the chicken retinal ganglion cells.

Prebilaterian animals perceived ambient light through nonvisual rhabdomeric photoreceptors (RPs), which evolved as support of the chordate visual system. In vertebrates, the identity of nonvisual photoreceptors and the phototransduction cascade involved in nonimage forming tasks remain uncertain. We investigated whether chicken retinal ganglion cells (RGCs) could be nonvisual photoreceptors and the nature of the photocascade involved. We found that primary cultures of chicken embryonic RGCs express such RP markers as transcription factors Pax6 and Brn3, photopigment melanopsin, and G-protein q but not markers for ciliary photoreceptors (alpha-transducin and Crx). To investigate the photoreceptive capability of RGCs, we assessed the direct effect of light on 3H-melatonin synthesis in RGC cultures synchronized to 12:12 h light-dark cycles. In constant dark, RGCs displayed a daily variation in 3H-melatonin levels peaking at subjective day, which was significantly inhibited by light. This light effect was further increased by the chromophore all-trans-retinal and suppressed by specific inhibitors of the invertebrate photocascade involving phosphoinositide hydrolysis (100 microM neomycin; 5 microM U73122) and Ca2+ mobilization (10 mM BAPTA; 1 mM lanthanum). The results demonstrate that chicken RGCs are intrinsically photosensitive RPs operating via an invertebrate-like phototransduction cascade, which may be responsible for early detection of light before vision occurs.

Early appearance of nonvisual and circadian markers in the developing inner retinal cells of chicken.

The retina is a key component of the vertebrate circadian system; it is responsible for detecting and transmitting the environmental illumination conditions (day/night cycles) to the brain that synchronize the circadian clock located in the suprachiasmatic nucleus (SCN). For this, retinal ganglion cells (RGCs) project to the SCN and other nonvisual areas. In the chicken, intrinsically photosensitive RGCs (ipRGCs) expressing the photopigment melanopsin (Opn4) transmit photic information and regulate diverse nonvisual tasks. In nonmammalian vertebrates, two genes encode Opn4: the Xenopus (Opn4x) and the mammalian (Opn4m) orthologs. RGCs express both Opn4 genes but are not the only inner retinal cells expressing Opn4x: horizontal cells (HCs) also do so. Here, we further characterize primary cultures of both populations of inner retinal cells (RGCs and HCs) expressing Opn4x. The expression of this nonvisual photopigment, as well as that for different circadian markers such as the clock genes Bmal1, Clock, Per2, and Cry1, and the key melatonin synthesizing enzyme, arylalkylamine N-acetyltransferase (AA-NAT), appears very early in development in both cell populations. The results clearly suggest that nonvisual Opn4 photoreceptors and endogenous clocks converge all together in these inner retinal cells at early developmental stages.

Early onset and differential temporospatial expression of melanopsin isoforms in the developing chicken retina.

PURPOSE: Retinal ganglion cells (RGCs) expressing the photopigment melanopsin (Opn4) display intrinsic photosensitivity. In this study, the presence of nonvisual phototransduction cascade components in the developing chicken retina and primary RGCs cultures was investigated, focusing on the two Opn4 genes: the Xenopus (Opn4x) and the mammalian (Opn4m) orthologs. METHODS: Retinas were dissected at different embryonic (E) and postnatal (P) days, and primary RGC cultures were obtained at E8 and kept for 1 hour to 5 days. Samples were processed for RT-PCR and immunochemistry. RESULTS: Embryonic retinas expressed the master eye gene Pax6, the prospective RGC specification gene Brn3, and components of the nonvisual phototransduction cascade, such as Opn4m and the G protein q (Gq) mRNAs at very early stages (E4-E5). By contrast, expression of photoreceptor cell markers (CRX, red-opsin, rhodopsin, and α-transducin) was observed from E7 to E12. Opn4m protein was visualized in the whole retina as early as E4 and remained elevated from E6 to the postnatal days, whereas Opn4x was weakly detected at E8 and highly expressed after E11. RGC cultures expressed Gq mRNA, as well as both Opn4 mRNAs and proteins. Opn4m was restricted exclusively to the GC layer at all ages, whereas Opn4x was limited to the forming GC layer and optic nerve at E8, but by E15, its expression was mostly in Prox1(+) horizontal cells. CONCLUSIONS: The early expression onset of nonvisual phototransduction molecules could confer premature photosensitivity to RGCs, while the appearance of Opn4x expression in horizontal cells suggests the identification of a novel type of photosensitive cell in birds.

Light activation of the phosphoinositide cycle in intrinsically photosensitive chicken retinal ganglion cells.

PURPOSE: In vertebrates, intrinsically photosensitive retinal ganglion cells (ipRGCs) acting as nonvisual photoreceptors transmit environmental illumination information to the brain, regulating diverse non-image-forming tasks. The phototransduction cascade in chicken ipRGCs has been shown to resemble that of rhabdomeric photoreceptors and involves phospholipase C (PLC) activation. The current work was an investigation of the participation of the phosphoinositide (PIP) cycle in this mechanism and of whether changes in activities of inositol 1,4,5-trisphosphate (IP(3)) and PIP kinase are triggered by light. METHODS: Primary cultures of Thy-1 immunopurified chicken embryonic RGCs were exposed to bright light pulses or kept in the dark, to assess intracellular Ca(2+) mobilization by Fluo-3 AM fluorescence microscopy, IP(3) levels, and enzymatic activities of diacylglycerol, phosphatidylinositol, and phosphatidylinositol phosphate kinases (DAGK, PIK, and PIPK, respectively), by radioactive assays. The presence of different melanopsins (Opn4m and Opn4x) and other photopigments was determined by RT-PCR and immunochemistry. RESULTS: Cultured RGCs expressing different nonvisual photopigments displayed a significant and rapid increase in IP(3) levels (1.3-fold) and Ca(2+) mobilization by light, which was reversed by administration of the PLC inhibitor U73122 (5 µM). Brief light pulses also caused a very rapid and transient activation of DAGK, PIK, and PIPK compared with that in the dark control. CONCLUSIONS: The results indicate for the first time that light stimulation of chicken RGC cultures activates the PIP cycle, causing an increase in intracellular levels of IP(3), changes in levels of phosphatidic acid, PIP, and PIP(2); and mobilization of Ca(2+).

Inner retinal circadian clocks and non-visual photoreceptors: novel players in the circadian system.

Daily and annual changes in ambient illumination serve as specific stimuli that associate light with time and regulate the physiology of the organism through the eye. The eye acts as a dual sense organ linking light and vision, and detecting light that provides specific stimuli for non-classical photoreceptors located in the inner retina. These photoreceptors convey information to the master circadian pacemaker, the hypothalamic suprachiasmatic nuclei (SCN). Responsible for sensing the light that regulates several non-visual functions (i.e. behavior, pupil reflex, sleep, and pineal melatonin production), the retina plays a key role in the temporal symphony orchestra playing the musical score of life: it is intrinsically rhythmic in its physiological and metabolic activities. We discuss here recent evidence in support of the hypothesis that retinal oscillators distributed over different cell populations may act as clocks, inducing changes in the visual and circadian system according to the time of the day. Significant progress has recently been made in identifying photoreceptors/photopigments localized in retinal ganglion cells (RGCs) that set circadian rhythms and modulate non-visual functions. Autonomous retinal and brain oscillators could have a more complex organization than previously recognized, involving a network of "RGC clock/SCN clock cross-talk". The convergence of oscillatory and photoreceptive capacities of retinal cells could deeply impact on the circadian system, which in turn may be severely impaired in different retinal pathologies. The aim of this review is to discuss the state of the art on inner retinal cell involvement in the light and temporal regulation of health and disease.