Rod Outer Segments Research Articles

Visualization of microbial biofilms in case of digestive disorders in lambs

The paper demonstrates morphometric and densitometric parameters of microbial biofilms recovered from lambs with digestive disorders. Changes of quantitative and species composition of the intestinal microbiocenoses in the lambs with digestive disorders were compared with the ones of the clinically healthy lambs. Light microscopy results demonstrated formation of three-dimensional biofilm structure in the form of dense grid consisting of gram-negative and gram-positive bacteria, yeast cells, hyphas and pseudohyphas surrounded with intracellular polymer matrix. Presence of blastospores aided to the increased number of cells attached to the substrate, and biofilm was formed, which consisted of rod and round cells attached to the microfungi cells. In the process of dispersion that occurred during the destruction of the intercellular matrix and bacterial and yeast cell detachment, branched structures separated from the microcolonies and colonized microorganism- free regions of the substrate. The intensity of biofilm formation by the microorganisms under study was evaluated by optic density measurement in 48 hours of cultivation. Fluorescence microscopy results demonstrated that the dynamics of changes of the viable microbial structures was specified by intermittent periods of increased or decreased biofilm formation intensity. Cells characterized by active growth and replication and forming alternating subpopulations were detected in the examined microbial cultures. When determining the viability of the microorganisms in the biofilms, the viable (green fluorescence) and non-viable (red fluorescence) cells were differentiated.

Rapid and Progressive Loss of Multiple Retinal Cell Types in Cathepsin D-Deficient Mice-An Animal Model of CLN10 Disease.

Vision loss is among the characteristic symptoms of neuronal ceroid lipofuscinosis (NCL), a fatal neurodegenerative lysosomal storage disorder. Here, we performed an in-depth analysis of retinal degeneration at the molecular and cellular levels in mice lacking the lysosomal aspartyl protease cathepsin D, an animal model of congenital CLN10 disease. We observed an early-onset accumulation of storage material as indicated by elevated levels of saposin D and subunit C of the mitochondrial ATP synthase. The accumulation of storage material was accompanied by reactive astrogliosis and microgliosis, elevated expression of the autophagy marker sequestosome 1/p62 and a dysregulated expression of several lysosomal proteins. The number of cone photoreceptor cells was reduced as early as at postnatal day 5. At the end stage of the disease, the outer nuclear layer was almost atrophied, and all cones were lost. A significant loss of rod and cone bipolar cells, amacrine cells and ganglion cells was found at advanced stages of the disease. Results demonstrate that cathepsin D deficiency results in an early-onset and rapidly progressing retinal dystrophy that involves all retinal cell types. Data of the present study will serve as a reference for studies aimed at developing treatments for retinal degeneration in CLN10 disease.

Sildenafil-evoked photoreceptor oxidative stress in vivo is unrelated to impaired visual performance in mice.

PurposeThe phosphodiesterase inhibitor sildenafil is a promising treatment for neurodegenerative disease, but it can cause oxidative stress in photoreceptors ex vivo and degrade visual performance in humans. Here, we test the hypotheses that in wildtype mice sildenafil causes i) wide-spread photoreceptor oxidative stress in vivo that is linked with ii) impaired vision.MethodsIn dark or light-adapted C57BL/6 mice ± sildenafil treatment, the presence of oxidative stress was evaluated in retina laminae in vivo by QUEnch-assiSTed (QUEST) magnetic resonance imaging, in the subretinal space in vivo by QUEST optical coherence tomography, and in freshly excised retina by a dichlorofluorescein assay. Visual performance indices were also evaluated by QUEST optokinetic tracking.ResultsIn light-adapted mice, 1 hr post-sildenafil administration, oxidative stress was most evident in the superior peripheral outer retina on both in vivo and ex vivo examinations; little evidence was noted for central retina oxidative stress in vivo and ex vivo. In dark-adapted mice 1 hr after sildenafil, no evidence for outer retina oxidative stress was found in vivo. Evidence for sildenafil-induced central retina rod cGMP accumulation was suggested as a panretinally thinner, dark-like subretinal space thickness in light-adapted mice at 1 hr but not 5 hr post-sildenafil. Cone-based visual performance was impaired by 5 hr post-sildenafil and not corrected with anti-oxidants; vision was normal at 1 hr and 24 hr post-sildenafil.ConclusionsThe sildenafil-induced spatiotemporal pattern of oxidative stress in photoreceptors dominated by rods was unrelated to impairment of cone-based visual performance in wildtype mice.

Simulation Analysis of the Photoresponse in Retinal Rods

Simulation Analysis of the Photoresponse in Retinal Rods

Protective Effect of Mitochondrially Targeted Peptide Against Oxidant Injury of Cone Photoreceptors Through Preventing Necroptosis Pathway

Retinopathy is an eye disease caused by the death of retinal cells in the macular area and the surrounding choroid. As the retinal rod cell dysfunction and death lead to the loss of night vision, the disease will lead to visual dysfunction and blindness as the disease progresses. Because of the irreversible nature of cell death, gene therapy has become a research hotspot in the field of retinopathy. But the technology is still in animal studies or clinical trials, and more research is needed to prove its feasibility. In this study, oxidative damage cell model was established and divided into a control group, H₂O₂ group, SS31 +NEC1 group, SS31 +H₂O₂ group, and SS31 +NEC1 +H₂O₂ group, for different interventions. The cell survival rate of the H₂O₂ group was significantly increased compared with those of the SS31 + H₂O₂ group, SS31 +NEC1 +H₂O₂ group, and NEC1 +H₂O₂ group. Nec1 combined treatment significantly reduced reactive oxygen species (ROS) production compared with that in the H₂O₂ group. The level of MDA in the SS31 group, Nec-1 group and combined treatment of SS31 +NEC1 group decreased significantly compared with the H₂O₂ group. The proportion of cells with decreased mitochondrial membrane potential in the H₂O₂ group significantly increased, and the rate of positivity for propidium iodide (PI) of 661W cells in the H₂O₂ group and the control group significantly increased. Nine hours after H₂O₂ treatment of 661W cells, the RIP3 expression level began to increase, and peaked at 24 h. The level of RIP3 in the H₂O₂ group was significantly increased, while this level was downregulated in the SS31 and NEC1 treatment groups. Therefore, this study suggests that SS31 has a partial protective effect on 661W cells by inhibiting necrosis, which has certain guiding significance for the treatment of retinal diseases.

The GARP Domain of the Rod CNG Channel's β1-Subunit Contains Distinct Sites for Outer Segment Targeting and Connecting to the Photoreceptor Disk Rim.

Vision begins when light is captured by the outer segment organelle of photoreceptor cells in the retina. Outer segments are modified cilia filled with hundreds of flattened disk-shaped membranes. Disk membranes are separated from the surrounding plasma membrane, and each membrane type has unique protein components. The mechanisms underlying this protein sorting remain entirely unknown. In this study, we investigated the outer segment delivery of the rod cyclic nucleotide-gated (CNG) channel, which is located in the outer segment plasma membrane, where it mediates the electrical response to light. Using Xenopus and mouse models of both sexes, we now show that the targeted delivery of the CNG channel to the outer segment uses the conventional secretory pathway, including protein processing in both ER and Golgi, and requires preassembly of its constituent α1 and β1 subunits. We further demonstrate that the N-terminal glutamic acid-rich protein (GARP) domain of CNGβ1 contains two distinct functional regions. The glutamic acid-rich region encodes specific information targeting the channel to rod outer segments. The adjacent proline-enriched region connects the CNG channel to photoreceptor disk rims, likely through an interaction with peripherin-2. These data reveal fine functional specializations within the structural domains of the CNG channel and suggest that its sequestration to the outer segment plasma membrane requires an interaction with peripherin-2.SIGNIFICANCE STATEMENT Neurons and other differentiated cells have a remarkable ability to deliver and organize signaling proteins at precise subcellular locations. We now report that the CNG channel, mediating the electrical response to light in rod photoreceptors, contains two specialized regions within the N terminus of its β-subunit: one responsible for delivery of this channel to the ciliary outer segment organelle and another for subsequent channel sequestration into the outer segment plasma membrane. These findings expand our understanding of the molecular specializations used by neurons to populate their critical functional compartments.

Olfactory Rod Cells: A Rare Cell Type in the Larval Zebrafish Olfactory Epithelium With a Large Actin-Rich Apical Projection.

We report the presence of a rare cell type, the olfactory rod cell, in the developing zebrafish olfactory epithelium. These cells each bear a single actin-rich rod-like apical projection extending 5–10 μm from the epithelial surface. Live imaging with a ubiquitous Lifeact-RFP label indicates that the olfactory rods can oscillate. Olfactory rods arise within a few hours of the olfactory pit opening, increase in numbers and size during larval stages, and can develop in the absence of olfactory cilia. Olfactory rod cells differ in morphology from the known classes of olfactory sensory neuron, but express reporters driven by neuronal promoters. A sub-population of olfactory rod cells expresses a Lifeact-mRFPruby transgene driven by the sox10 promoter. Mosaic expression of this transgene reveals that olfactory rod cells have rounded cell bodies located apically in the olfactory epithelium and have no detectable axon. We offer speculation on the possible function of these cells in the Discussion.

Leber Congenital Amaurosis Due to GUCY2D Mutations: Longitudinal Analysis of Retinal Structure and Visual Function.

Gene augmentation therapy is being planned for GUCY2D-associated Leber congenital amaurosis (LCA). To increase our understanding of the natural history of GUCY2D-LCA, patients were evaluated twice with an interval of 4 to 7 years between visits using safety and efficacy outcome measures previously determined to be useful for monitoring this disorder. In this group of molecularly-identified LCA patients (n = 10; ages 7–37 years at first visit), optical coherence tomography (OCT) was used to measure foveal cone outer nuclear layer (ONL) thickness and rod ONL at a superior retinal locus. Full-field stimulus testing (FST) with chromatic stimuli in dark- and light-adapted states was used to assay rod and cone vision. Changes in OCT and FST over the interval were mostly attributable to inter-visit variability. There were no major negative changes in structure or function across the cohort and over the intervals studied. Variation in severity of disease expression between patients occurs; however, despite difficulties in quantifying structure and function in such seriously visually impaired individuals with nystagmus, the present work supports the use of OCT as a safety outcome and FST as an efficacy outcome in a clinical trial of GUCY2D-LCA. A wide age spectrum for therapy was confirmed, and there was relative stability of structure and function during a typical time interval for clinical trials.

Rod Photoreceptors Avoid Saturation in Bright Light by the Movement of the G Protein Transducin.

Rod photoreceptors can be saturated by exposure to bright background light, so that no flash superimposed on the background can elicit a detectable response. This phenomenon, called increment saturation, was first demonstrated psychophysically by Aguilar and Stiles and has since been shown in many studies to occur in single rods. Recent experiments indicate, however, that rods may be able to avoid saturation under some conditions of illumination. We now show in ex vivo electroretinogram and single-cell recordings that in continuous and prolonged exposure even to very bright light, the rods of mice from both sexes recover as much as 15% of their dark current and that responses can persist for hours. In parallel to recovery of outer segment current is an ∼10-fold increase in the sensitivity of rod photoresponses. This recovery is decreased in transgenic mice with reduced light-dependent translocation of the G protein transducin. The reduction in outer-segment transducin together with a novel mechanism of visual-pigment regeneration within the rod itself enable rods to remain responsive over the whole of the physiological range of vision. In this way, rods are able to avoid an extended period of transduction channel closure, which is known to cause photoreceptor degeneration.SIGNIFICANCE STATEMENT Rods are initially saturated in bright light so that no flash superimposed on the background can elicit a detectable response. Frederiksen and colleagues show in whole retina and single-cell recordings that, if the background light is prolonged, rods slowly recover and can continue to produce significant responses over the entire physiological range of vision. Response recovery occurs by translocation of the G protein transducin from the rod outer to the inner segment, together with a novel mechanism of visual-pigment regeneration within the rod itself. Avoidance of saturation in bright light may be one of the principal mechanisms the retina uses to keep rod outer-segment channels from ever closing for too long a time, which is known to produce photoreceptor degeneration.

The Heterochromatin Block That Functions as a Rod Cell Microlens in Owl Monkeys Formed within a 15-Myr Time Span.

In rod cells of many nocturnal mammals, heterochromatin localizes to the central region of the nucleus and serves as a lens to send light efficiently to the photoreceptor region. The genus Aotus (owl monkeys) is commonly considered to have undergone a shift from diurnal to nocturnal lifestyle. We recently demonstrated that rod cells of the Aotus species Aotus azarae possess a heterochromatin block at the center of its nucleus. The purpose of the present study was to estimate the time span in which the formation of the heterochromatin block took place. We performed three-dimensional hybridization analysis of the rod cell of another species, Aotus lemurinus. This analysis revealed the presence of a heterochromatin block that consisted of the same DNA components as those in A. azarae. These results indicate that the formation was complete at or before the separation of the two species. Based on the commonly accepted evolutionary history of New World monkeys and specifically of owl monkeys, the time span for the entire formation process was estimated to be 15 Myr at most.

Enterosgel role in neurodegenerative changes in the retina rats under the influence of Cr (VI) - induced retinopathy by study morphological changes in dynamic

Chromium galvanic production have leaded to biosphere pollution. Therefore advisable to study of role in the neurodegenerative development in retinal diseases under experimental conditions. The aim is to study the Enterosgel effect on morphological changes in rats retina with Cr(VI) – induced retinopathy. Materials and methods. An experimental study had carried out on 72 outbred white male rats. The rats had divided into 3 groups: I – control group of intact rats (n = 24). Control rats were received drinking water, II group – rats (n = 24), were received drinking water with Cr (VI) (K2Cr2O7) – 0.02 mol/L, III group – animals (n = 24) were received drinking water with K2Cr2O7– 0.02 mol/L and hydrogel methylsilicic acid (Enterosgel) at a dose of 0.8 mg/kg per day as a corrector. The animals had been decapitated under ether anesthesia. The retina had been studied on days 20, 40 and 60 of the experiment. Morphologically and morphometrically they had analyzed. Results. According to histological studies, it has proved that Cr (VI) causes dystrophic and degenerative changes in all rats retina layers. They increase as the duration of the experiment. The use of Enterosgel as a corrective therapy showed positive results in restoring the morphological structure of rats retina. After Enterosgel 20 days using as a corrector of Cr (VI) exposure, there is a barely noticeable swelling of the outer and inner nuclear layers. Other layers of the retina, morphologically, look undamaged. Forty days Enterosgel treatment have outer and inner nuclear layer edema of retina of animals persists but does not increase. It is easy noticeable swelling of the outer and inner layers of mesh, but no signs of damage processes of cell populations nuclear layers. State ganglionic layer and nerve fiber layer entirely satisfactory. These pathological changes are not critical. After 60 days from the beginning of loading of Cr (VI) and application of Enterosgel in the retina of rats there are initial degenerative changes in the photosensory layer. Cystic dilated outer segments of rods and cones were visible throughout, and areas of their fragmentation were observed. Ganglion neurons are not damaged, but their axons appear somewhat thickened and fluffy. But in general, the typical structure of the retina is preserved. Conclusions. Chromium-induced toxicity in rats is characterized by pronounced histological and morphometric changes and retinal thickness, which appear after 20 days, increase by 40 days and acquire maximum transformations after 60 days of the experiment. The use of Enterosgel improves picture morphological structures of the retina in rats under the influence of Cr (VI). The changes were expressed on days 20 and 40, which indicates the presence of protective properties for the retina

Helical Fasciculation of Bipolar and Horizontal Cell Neurites for Wiring With Photoreceptors in Macaque and Mouse Retinas.

PurposeThe three-dimensional configurations of rod and cone bipolar cell (BC) dendrites and horizontal cell (HC) processes outside rod and cone synaptic terminals have not been fully elucidated. We reveal how these neurites are mutually arranged to coordinate formation and maintenance of the postsynaptic complex of ribbon synapses in mouse and monkey retinas.MethodsSerial section transmission electron microscopy was utilized to reconstruct BC and HC neurites in macaque monkey and mouse, including metabotropic glutamate receptor 6 (mGluR6)-knockout mice.ResultsStarting from sporadically distributed branching points, rod BC and HC neurites (B and H, respectively) took specific paths to rod spherules by gradually adjusting their mutual positions, which resulted in a closed alternating pattern of H‒B‒H‒B neurites at the rod spherule aperture. This order corresponded to the array of elements constituting the postsynaptic complex of ribbon synapses. We identified novel helical coils of HC processes surrounding the rod BC dendrite in both mouse and macaque retinas, and these structures occurred more frequently in mGluR6-knockout than wild-type mouse retinas. Horizontal cell processes also formed hook-like protrusions that encircled cone BC and HC neurites below the cone pedicles in the macaque retina.ConclusionsBipolar and horizontal cell neurites take specific paths to adjust their mutual positions at the rod spherule aperture. Some HC processes are helically coiled around rod BC dendrites or form hook-like protrusions around cone BC dendrites and HC processes. Loss of mGluR6 signaling may be one factor promoting unbalanced neurite growth and compensatory neurite coiling.

The diterpene Manool extracted from Salvia tingitana lowers free radical production in retinal rod outer segments by inhibiting the extramitochondrial F1 Fo ATP synthase.

Uncontrolled oxidative stress production, especially in the outer retina is one of the causes of retinal degenerations. Mitochondria are considered the principal source of oxidative stress. However, a Reactive Oxygen Intermediates (ROI) production in the retinal photoreceptor layer seems to depend also on the expression of an extramitochondrial oxidative phosphorylation (OxPhos) machinery in the rod outer segments (OS). In fact, OS conduct aerobic metabolism, producing ATP through oxygen consumption, although it is devoid of mitochondria. As diterpenes display an antioxidant effect, we have evaluated the effect Manool, extracted from Salvia tingitana, on the extramitochondrial OxPhos and the ROI production in the retinal rod OS. Results confirm that the OxPhos machinery is ectopically expressed in the OS and that F1 Fo -ATP synthase is a target of Manool, which inhibited the OS ATP synthesis, binding the F1 moiety with high affinity, as analysed by molecular docking. Moreover, the overall slowdown of OxPhos metabolism reduced the ROI production elicited in the OS by light exposure, in vitro. In conclusion, data are consistent with the antioxidant properties of Salvia spp., suggesting its ability to lower oxidative stress production, a primary risk factor for degenerative retinal diseases. SIGNIFICANCE OF THE STUDY: Here we show that Manool, a diterpene extracted from Salvia tingitana has the potential to lower the free radical production by light-exposed rod outer segments in vitro, by specifically targeting the rod OS F1 Fo -ATP synthase belonging to the extramitochondrial OxPhos expressed on the disk membrane. The chosen experimental model allowed to show that the rod OS is a primary producer of oxidative stress linked to the pathogenesis of degenerative retinal diseases. Data are also consistent with the antioxidant and anti-inflammatory action of Salvia spp., suggesting a beneficial effect also in vivo.

A hybrid stochastic/deterministic model of single photon response and light adaptation in mouse rods

The phototransduction cascade is paradigmatic for signaling pathways initiated by G protein-coupled receptors and is characterized by a fine regulation of photoreceptor sensitivity and electrical response to a broad range of light stimuli. Here, we present a biochemically comprehensive model of phototransduction in mouse rods based on a hybrid stochastic and deterministic mathematical framework, and a quantitatively accurate description of the rod impedance in the dark. The latter, combined with novel patch clamp recordings from rod outer segments, enables the interconversion of dim flash responses between photovoltage and photocurrent and thus direct comparison with the simulations. The model reproduces the salient features of the experimental photoresponses at very dim and bright stimuli, for both normal photoreceptors and those with genetically modified cascade components. Our modelling approach recapitulates a number of recent findings in vertebrate phototransduction. First, our results are in line with the recently established requirement of dimeric activation of PDE6 by transducin and further show that such conditions can be fulfilled at the expense of a significant excess of G protein activated by rhodopsin. Secondly, simulations suggest a crucial role of the recoverin-mediated Ca2+-feedback on rhodopsin kinase in accelerating the shutoff, when light flashes are delivered in the presence of a light background. Finally, stochastic simulations suggest that transient complexes between dark rhodopsin and transducin formed prior to light stimulation increase the reproducibility of single photon responses. Current limitations of the model are likely associated with the yet unknown mechanisms governing the shutoff of the cascade.

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.

Nocturnal reef residents have deep-sea-like eyes

Swirling with colour, coral reefs teem with vivid life during daylight. But at night, a different crew emerges from the nooks and crannies. ‘The visual systems of teleost fish usually match their habitats and lifestyles’, says Fanny de Busserolles from The University of Queensland (UQ), Australia, explaining that there has been a lot of interest in the vision of the gaudy creatures that populate reefs by day. However, less was known about the vision of their nocturnal cohabitants. ‘I am particularly interested in visual adaptations and capabilities in dim light’, says de Busserolles, who is intrigued by the vision of deep-sea fish. So, when she arrived in subtropical Brisbane to work with Justin Marshall at UQ, the chance to investigate some of the nocturnal creatures residing on the Great Barrier Reef was too good to pass up.‘Holocentrids [squirrelfish and soldierfish] were the ideal family for our study due to their connection to the deep-sea environment’, says de Busserolles, explaining that the fish are linked to her love of the depths; ‘the entire family is thought to have originated in deeper water before most of its representatives migrated up to the reef’, she says. Fishing in the sea around Lizard Island, Australia, with UQ colleagues Fabio Cortesi, Sara Stieb, Martin Luehrmann and Marshall, de Busserolles collected four species of soldierfish and five different squirrelfish. ‘They were pretty hard to catch during the day because they are very shy and tend to hide in holes, caves and crevices’, de Busserolles recalls.Looking at the overall structure of the eyes back in Brisbane, de Busserolles could see that they were large, to collect as much light as possible in the dim conditions. And, when Cortesi extracted the RNA from each retina to find out which light-sensitive genes the fish were expressing, he found that all of the fish expressed one rod opsin – which they produced in high levels in their light-sensitive rod cells. In addition, the fish expressed some colour-sensitive cone opsin genes, but at much lower levels than the rod opsin, with the soldierfish expressing two cone opsins while the squirrelfish expressed three cone opsins. Luehrmann also simulated which shades the colour-sensitive cone opsins detect and it turned out that they pick up shades of green and blue. In addition, Sara Stieb revealed two types of cone in the fishes’ retinas: cones expressing the blue opsin, with a second type expressing one or two green opsin genes. ‘These findings indicate that holocentrids may be able to see colours during the day’, says de Busserolles.However, when she and Lily Fogg (also from UQ), painstakingly investigated the distribution of photoreceptor cells in the fishes’ retinas, it was apparent that their eyes are also extremely well prepared for dim nocturnal conditions. Instead of carrying a single layer of photoreceptors at the back of the eye, the fish's retinas comprised stacks of rod cells layered one on top of another, with up to 17 rod cell layers in the eyes of crimson soldierfish (Myripristis murdjan). Explaining that these ‘multibank’ retinas are mostly found in deep-sea species, de Busserolles says, ‘We do not know what the function of this adaptation is, but possible hypotheses are that they may enhance the sensitivity of the eye and/or they may provide some kind of colour vision in dim light conditions’.So, soldierfish and squirrelfish are well prepared for their nocturnal life on the Great Barrier Reef and de Busserolles and her colleagues are keen to find out just how good the fish's colour vision is, especially in the dimmest conditions.

Interaction of G protein-coupled receptor kinases and recoverin isoforms is determined by localization in zebrafish photoreceptors

The zebrafish retina expresses four recoverin genes (rcv1a, rcv1b, rcv2a and rcv2b) and four opsin kinase genes (grk1a, grk1b, grk7a and grk7b) coding for recoverin and G protein-coupled receptor kinase (opsin kinase) paralogs, respectively. Both protein groups are suggested to form regulatory complexes in rod and cone outer segments, but at present, we lack information about co-localization of recoverin and opsin kinases in zebrafish retinae and which protein-protein interacting pairs form. We analyzed the distribution and co-localization of recoverin and opsin kinase expression in the zebrafish retina. For this purpose, we used custom-tailored monospecific antibodies revealing that the amount of recoverin paralogs in a zebrafish retina can differ by more than one order of magnitude with the highest amount for recoverin 1a and 2b. Further, immunohistochemical labelling showed presence of recoverin 1a in all rod cell compartments, but it only co-localized with opsin kinase 1a in rod outer segments. In contrast, recoverin 2b was only detected in double cones and co-localized with opsin kinases 1b, 7a and 7b. Further, we investigated the interaction between recoverin and opsin kinase variants by surface plasmon resonance spectroscopy indicating interaction of recoverin 1a and recoverin 2b with all opsin kinases. However, binding kinetics for recoverin 1a differed from those observed with recoverin 2b that showed slower association and dissociation processes. Our results indicate diverse recoverin and opsin kinase properties due to differential expression and interaction profiles.

The study of retinal structure in frugivorous bat (Rousettus aegyptiacus) by Light and Electron microscope

Vision is fundamentally important sense in animals for perceiving their environment, but in bat less well known. Due to the importance of vision in the bat as the only mammal that is able to fly, in present study, the retinal tissue of the frugivore bat (Rousettus aegyptiacus) was examined by light and electron microscope. So 5 male bats weighing of 123±0.8g were anesthetized, dissected, and their retina was removed. The sections (1×1mm) were isolated, fixed in 4% glutaradehyde and 2% paraformaldehyde (1hour) and rinsed with sodium cacodylate buffer (0.1). They were post fixed in osmium tetroxid 1%. The semithin sections (0.5μm) were stained with Toluidine blue, and the ultrathin sections (70 nm) were prepared. The retinal cells and layer thickness were measured by Grids-sterolite software. Obtained data were analyzed by ANOVA and t test (p < 0.05). Findings showed that the ratio of the eye’s diameter to the body length was 1:18.83. Retina layers conform to the general mammalian blueprint, but in wavy pattern with 106.61±16.19μm thickness. Inner nuclear layer was the thickest layer (18.52±1.55). Retina is duplex with dominant rod cells especially in centralis [≈25cell/(100μ)2], Cone/ Rod ratios: 1/ 7.95, and four other cell types with different density and distribution were seen. According to obtained electro-micrographs, retinal epithelial layer was thin with poor pigmentation and its surface was covered by microvilli. The arrangement of the photoreceptor parallel to the numerous choroidal papillae, and density of rod cells is higher than cone cells according to obtained results, this species of bat can be active in both dim light and daylight.

Light Adaptation of Retinal Rods, Adaptation Memory, and Afterimages

The GPCR signal cascade in retinal photoreceptor cells – rods and cones – supports powerful signal amplification and allows rods to respond reliably to single light quanta. Rods remain functional at light fluxes down to 105 quanta/sec, which is supported by the highly effective light adaptation system. Light adaptation is based on negative feedback circuits, mostly via changes in intracellular Ca2+ ion concentrations. Three calcium regulation loops have been reliably identified – acceleration of quenching of light-activated rhodopsin, acceleration of cGMP synthesis by guanylate cyclase, and increased affinity of cGMP-controlled ion cannels for nucleotide. However, one or two further highly effective mechanisms of adaptation are known to operate, one of which regulates the lifetime of activated phosphodiesterase; for others, the target of regulation is unknown. The mediators of this regulation are also unknown. Our studies of these mechanisms revealed a novel phenomenon, which has been missed in previous studies. We found that recovery of the dark current of rods after cessation of non-saturating adapting light can take 20–30 sec. Furthermore, after formal return of the membrane current to the dark level, cell sensitivity to test stimuli remains decreased for a further 1–2 min. We termed this phenomenon “adaptation memory.” Adaptation memory is reminiscent of the phenomenology of afterimages. The gradual return of the membrane current to the dark level may correspond to fading of the positive afterimage. Prolonged decreases in photoreceptor sensitivity to additional stimulation can create a negative afterimage. As far as we know, this is the first experimental physiological demonstration of the ability to generate afterimages at the level of single photoreceptors.

Visual adaptation of opsin genes to the aquatic environment in sea snakes

BackgroundEvolutionary transitions from terrestrial to aquatic life history cause drastic changes in sensory systems. Indeed, the drastic changes in vision have been reported in many aquatic amniotes, convergently. Recently, the opsin genes of the full-aquatic sea snakes have been reported. However, those of the amphibious sea snakes have not been examined in detail.ResultsHere, we investigated opsin genes and visual pigments of sea snakes. We determined the sequences of SWS1, LWS, and RH1 genes from one terrestrial, three amphibious and four fully-aquatic elapids. Amino acid replacements at four and one spectra-tuning positions were found in LWS and RH1, respectively. We measured or predicted absorption of LWS and RH1 pigments with A1-derived retinal. During their evolution, blue shifts of LWS pigments have occurred stepwise in amphibious sea snakes and convergently in both amphibious and fully-aquatic species.ConclusionsBlue shifted LWS pigments may have adapted to deep water or open water environments dominated by blue light. The evolution of opsins differs between marine mammals (cetaceans and pinnipeds) and sea snakes in two fundamental ways: (1) pseudogenization of opsins in marine mammals; and (2) large blue shifts of LWS pigments in sea snakes. It may be possible to explain these two differences at the level of photoreceptor cell composition given that cone and rod cells both exist in mammals whereas only cone cells exist in fully-aquatic sea snakes. We hypothesize that the differences in photoreceptor cell compositions may have differentially affected the evolution of opsins in divergent amniote lineages.