Light-dependent changes in cytoplasmic aspartate aminotransferase activity in rod spherules of the rat retina. A cytochemical study.

The cytoplasmic activity of the glutamate synthesizing enzyme aspartate aminotransferase (c-AAT) has been investigated on the ultrastructure level in rod spherules of light and dark adapted rat retinae using cytochemistry. Although most rod terminals react negatively, in a subpopulation of rods a weak activity, which is observed in light adapted retinae, is markedly increased under dark conditions. This indicates, that in addition to cones, some rods might use glutamate as their transmitter as well.     

Light-dependent changes in cytoplasmic aspartate aminotransferase activity in rod spherules of the rat retina

Summary The cytoplasmic activity of the glutamate synthesizing enzyme aspartate aminotransferase (c-AAT) has been investigated on the ultrastructure level in rod spherules of light and dark adapted rat retinae using cytochemistry. Although most rod terminals react negatively, in a subpopulation of rods a weak activity, which is observed in light adapted retinae, is markedly increased under dark conditions. This indicates, that in addition to cones, some rods might use glutamate as their transmitter as well.     

Inhibition of cyclic nucleotide phosphodiesterase from the rod outer segments of the frog retina by 3-hydroxypyridine

The influence of 8 analogues of 3-hydroxypyridine upon the phosphodiesterase of rod outer segments of frog retinae has been investigated. It has been shown that the analogues of 3-hydroxypyridine inhibit the enzeme reversely and noncompetitively in case of hydrolysis towards the cAMP and cGMP. The natural analogues of 3-hydroxypyridine (pyridoxol, pyridoxale, pyridoxale-phosphate) do not exert the inhibiting effect. It is suggested that the inhibition of phosphodiesterase from rod outer segments of retinae is caused by the interaction of 3-hydroxypyridines with the hydrophobic microenvironment of the active site of the enzyme.     

The heat-shock response co-inducer arimoclomol protects against retinal degeneration in rhodopsin retinitis pigmentosa

Retinitis pigmentosa (RP) is a group of inherited diseases that cause blindness due to the progressive death of rod and cone photoreceptors in the retina. There are currently no effective treatments for RP. Inherited mutations in rhodopsin, the light-sensing protein of rod photoreceptor cells, are the most common cause of autosomal-dominant RP. The majority of mutations in rhodopsin, including the common P23H substitution, lead to protein misfolding, which is a feature in many neurodegenerative disorders. Previous studies have shown that upregulating molecular chaperone expression can delay disease progression in models of neurodegeneration. Here, we have explored the potential of the heat-shock protein co-inducer arimoclomol to ameliorate rhodopsin RP. In a cell model of P23H rod opsin RP, arimoclomol reduced P23H rod opsin aggregation and improved viability of mutant rhodopsin-expressing cells. In P23H rhodopsin transgenic rat models, pharmacological potentiation of the stress response with arimoclomol improved electroretinogram responses and prolonged photoreceptor survival, as assessed by measuring outer nuclear layer thickness in the retina. Furthermore, treated animal retinae showed improved photoreceptor outer segment structure and reduced rhodopsin aggregation compared with vehicle-treated controls. The heat-shock response (HSR) was activated in P23H retinae, and this was enhanced with arimoclomol treatment. Furthermore, the unfolded protein response (UPR), which is induced in P23H transgenic rats, was also enhanced in the retinae of arimoclomol-treated animals, suggesting that arimoclomol can potentiate the UPR as well as the HSR. These data suggest that pharmacological enhancement of cellular stress responses may be a potential treatment for rhodopsin RP and that arimoclomol could benefit diseases where ER stress is a factor.     

Immuno-Histochemical Analysis of Rod and Cone Reaction to RPE65 Deficiency in the Inferior and Superior Canine Retina

Mutations in the RPE65 gene are associated with autosomal recessive early onset severe retinal dystrophy. Morphological and functional studies indicate early and dramatic loss of rod photoreceptors and early loss of S-cone function, while L and M cones remain initially functional. The Swedish Briard dog is a naturally occurring animal model for this disease. Detailed information about rod and cone reaction to RPE65 deficiency in this model with regard to their location within the retina remains limited. The aim of this study was to analyze morphological parameters of cone and rod viability in young adult RPE65 deficient dogs in different parts of the retina in order to shed light on local disparities in this disease. In retinae of affected dogs, sprouting of rod bipolar cell dendrites and horizontal cell processes was dramatically increased in the inferior peripheral part of affected retinae, while central inferior and both superior parts did not display significantly increased sprouting. This observation was correlated with photoreceptor cell layer thickness. Interestingly, while L/M cone opsin expression was uniformly reduced both in the superior and inferior part of the retina, S-cone opsin expression loss was less severe in the inferior part of the retina. In summary, in retinae of young adult RPE65 deficient dogs, the degree of rod bipolar and horizontal cell sprouting as well as of S-cone opsin expression depends on the location. As the human retinal pigment epithelium (RPE) is pigmented similar to the RPE in the inferior part of the canine retina, and the kinetics of photoreceptor degeneration in humans seems to be similar to what has been observed in the inferior peripheral retina in dogs, this area should be studied in future gene therapy experiments in this model.     

Temperature effects on low-light vision in juvenile rockfish (Genus <Emphasis Type="Italic">Sebastes</Emphasis>) and consequences for habitat utilization

The absolute low-light sensitivity of four congeneric species of rockfish (genus Sebastes) was studied from analysis of electroretinograms measured in living fish. The purpose was: (1) to determine if temperature sensitive noise in rod photoreceptors affects the absolute limit to low-light sensitivity at environmentally realistic temperatures and light levels, and (2) to examine whether interspecific variations in habitat utilization within rockfish communities correlate with differences in visual sensitivity. It was found that the low-light sensitivity of individual retinae is inversely dependent on temperature, decreasing tenfold with a 10°C increase in temperature. While in all four species, temperature had a similar effect on sensitivity, the absolute sensitivity levels were different. The four species could be divided into two groups based on measured sensitivity. Kelp and olive rockfish form a high-sensitivity group capable of responding to light levels approximately 50-fold lower than blue and black rockfish. The sensitivity groups correlated with reported diel activity patterns; the high-sensitivity group forages nocturnally, whereas members of the low-sensitivity group are quiescent during twilight and night and forage during the day.     

Differential modification of phosducin protein in degenerating rd1 retina is associated with constitutively active Ca2+/calmodulin kinase II in rod outer segments

Retinitis pigmentosa comprises a heterogeneous group of incurable progressive blinding diseases with unknown pathogenic mechanisms. The retinal degeneration 1 (rd1) mouse is a retinitis pigmentosa model that carries a mutation in a rod photoreceptor-specific phosphodiesterase gene, leading to rapid degeneration of these cells. Elucidation of the molecular differences between rd1 and healthy retinae is crucial for explaining this degeneration and could assist in suggesting novel therapies. Here we used high resolution proteomics to compare the proteomes of the rd1 mouse retina and its congenic, wild-type counterpart at postnatal day 11 when photoreceptor death is profound. Over 3000 protein spots were consistently resolved by two-dimensional gel electrophoresis and subjected to a rigorous filtering procedure involving computer-based spot analyses. Five proteins were accepted as being differentially expressed in the rd1 model and subsequently identified by mass spectrometry. The difference in one such protein, phosducin, related to an altered modification pattern in the rd1 retina rather than to changed expression levels. Additional experiments showed phosducin in healthy retinae to be highly phosphorylated in the dark- but not in the light-adapted phase. In contrast, rd1 phosducin was highly phosphorylated irrespective of light status, indicating a dysfunctional rd1 light/dark response. The increased rd1 phosducin phosphorylation coincided with increased activation of calcium/calmodulin-activated protein kinase II, which is known to utilize phosducin as a substrate. Given the increased rod calcium levels present in the rd1 mutation, calcium-evoked overactivation of this kinase may be an early and long sought for step in events leading to photoreceptor degeneration in the rd1 mouse.     

Dopaminergic interplexiform cells contact photoreceptor terminals in catfish retina

Summary Dopaminergic interplexiform cells in retinae of glass catfish were investigated using an antiserum against tyrosine hydroxylase and peroxidase-anti-peroxidase (PAP) visualization. In whole-mount preparations, we observed a homogeneous distribution of cell bodies throughout the retina without any indication of regional specializations. At the ultrastructural level, we studied the morphology of labelled telodendria within the outer plexiform layer. Apart from contacts with horizontal cells and bipolar cell dendrites, we report for the first time direct contacts with cone pepdicles and rod spherules. Quantitative evaluation of short series of sections showed that all cone pedicles, and a major part of the rod terminals, were approached in this way. The dopaminergic pathway terminating on photoreceptors is discussed in the context of pharmacological effects of this transmitter in the distal retina during light adaptation, i.e., cone contraction, spinule formation and horizontal cell coupling.     

Light-induced reduction in cyclic GMP of retinal photoreceptor cells in vivo: abnormalities in the degenerative diseases of RCS rats and rd mice.

Abstract— The effect of light on the content of cyclic GMP in degenerative retinae of Royal College of Surgeons (RCS) rats and rd mice was compared with that in control retinae during postnatal maturation. In vivo, the cyclic GMP content of retinae of control rats or mice is light-dependent after photoreceptor outer segments develop. Mature retinae of control animals have high levels of cyclic GMP in the dark which are reduced 40–50% upon illumination. In the rd mouse disorder, a light-induced reduction in cyclic GMP content is observed while the rod outer segments are morphologically intact. The rd photoreceptor cells possess a phosphodiesterase which, when stabilized by freeze-drying, has a K_m similar to that of control photoreceptors, and an apparent V_max that is below normal. It is suggested that developing rd visual cells have an abnormality in cyclic GMP metabolism which results in the accumulation of cyclic GMP within the entire cell but which does not prevent the light-mediated reduction of cyclic GMP in their outer segment organelles. In the RCS dystrophy, a light-induced reduction in cyclic GMP content is observed also during the period when photoreceptor outer segments are present. The cyclic GMP content of dark- or light-adapted RCS retinae is below that of the respective controls. Biochemical and morphological observations show that cyclic GMP levels increase in rd visual cells and that they are reduced in photoreceptor cells of RCS retina before the onset of visual cell degeneration. Until detailed knowledge of the role of cyclic GMP in the visual cells is known, it is suggested that high or low levels of cyclic GMP in rd and RCS photoreceptors, respectively, result from differences in the etiology or histopathology of the mouse and rat diseases.     

Bat Eyes Have Ultraviolet-Sensitive Cone Photoreceptors

Mammalian retinae have rod photoreceptors for night vision and cone photoreceptors for daylight and colour vision. For colour discrimination, most mammals possess two cone populations with two visual pigments (opsins) that have absorption maxima at short wavelengths (blue or ultraviolet light) and long wavelengths (green or red light). Microchiropteran bats, which use echolocation to navigate and forage in complete darkness, have long been considered to have pure rod retinae. Here we use opsin immunohistochemistry to show that two phyllostomid microbats, Glossophaga soricina and Carollia perspicillata, possess a significant population of cones and express two cone opsins, a shortwave-sensitive (S) opsin and a longwave-sensitive (L) opsin. A substantial population of cones expresses S opsin exclusively, whereas the other cones mostly coexpress L and S opsin. S opsin gene analysis suggests ultraviolet (UV, wavelengths <400 nm) sensitivity, and corneal electroretinogram recordings reveal an elevated sensitivity to UV light which is mediated by an S cone visual pigment. Therefore bats have retained the ancestral UV tuning of the S cone pigment. We conclude that bats have the prerequisite for daylight vision, dichromatic colour vision, and UV vision. For bats, the UV-sensitive cones may be advantageous for visual orientation at twilight, predator avoidance, and detection of UV-reflecting flowers for those that feed on nectar.     

Duplex Retina in the Mesopelagic Deep-Sea Teleost Lestidiops affinis (Ege, 1930)

The eye of the deep-sea teleost Lestidiops affinis has been examined primarily by light microscopy and found to possess a duplex retina consisting of two main divisions, a pure-cone and a pure-rod region, with a narrow zone of transition, possessing both cones and rods, joining the two. The pure-cone region is located in the temporal (caudal) part of the retina subserving binocular vision in the rostral direction. It has an area temporalis retinae with particularly long and densely packed single cones arranged in a regular hexagonal mosaic. Joined (double or twin) cones have not been recognized with certainty in the pure-cone region. The pure-rod region, comprising the larger part of the retina, contains rods grouped in bundles separated by retinal pigment epithelium (RPE) processes with pigmented cores. The synaptic endings of the rods are arranged in separate clusters in the outer plexiform layer, there being apparently a separate rod pedicle cluster beneath (vitread to) each rod bundle. Structural comparisons with certain other deep-sea teleosts suggest the likely presence of a retinal tapetum in L. affinis, i.e. each single cone or rod bundle is situated in a reflecting pit formed by the RPE, with a discrete reflector apposed to the tip of each cone outer segment and the tips of the outer segments of each square-cut rod bundle.     

The presence of two major protein components in the bovine photoreceptor disc membrane.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of thoroughly washed rod outer segment membrane preparations from bovine retinae revealed two major membrane-bound components and not one as has been generally thought. The higher molecular weight peak (?38500 molecular weight) contains a carbohydrate component and is covalently bound to the retinylidene chromophore. Moreover, this material is extensively phosphorylated $\text{in vitro}$ upon illumination. Therefore, this component (peak H) is rhodopsin. The nature and function of the other photoreceptor disc membrane component (peak L, ?34500 molecular weight) remains to be determined.     

Purification method of bovine rhodopsin kinase using regeneration of rhodopsin

We report a rapid and high-yield purification method of bovine retinal rhodopsin kinase. According to our method, 500 micrograms of rhodopsin kinase was purified from 100 bovine retinae within 12 h. Rhodopsin kinase bound to bleached rhodopsin was extracted effectively from rod outer segment membranes after regeneration of rhodopsin by the incubation with exogenous 11-cis-retinal. Subsequent DE52 column chromatography further purified the protein to homogeneity on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified rhodopsin kinase had an apparent molecular weight of 68,000 and phosphorylated rhodopsin at the rate of 10 nmol phosphate/min/mg of the enzyme.     

RNA and DNA in developing retinae: comparison of a normal with the degenerating retinae of C3H mice

Abstract— The Nucleic acids were measured in developing retinae of normal (DBA) mice and those afflicted with an inherited degenerative disease (C3H). The content of RNA in normal and C3H retinae increased to a maximum at 5 days of postnatal age. Thereafter, that of C3H retinae declined to a value lower than the normal. The content of DNA in normal and C3H retinae was maximal at 10 and 5 days of postnatal age, respectively. By 20 days, it declined in both retinae to nearly adult values. The DNA/RNA ratio of normal adult retinae was about 3, while that of C3H adult retinae was nearly 1–5. It is proposed that the photoreceptor cells possess a smaller cytoplasmic volume and a larger DNA/RNA ratio than the cells of the inner retina. The loss of DNA in developing normal and C3H retinae appears to result from cellular death. It was calculated that approximately 1 million cells in normal and 6 million cells in C3H retinae disappear during development. Cellular death in C3H retinae may not be restricted to the photoreceptor population.     

Temporal filtering in retinal bipolar cells. Elements of an optimal computation?

Recent experiments indicate that the dark-adapted vertebrate visual system can count photons with a reliability limited by dark noise in the rod photoreceptors themselves. This suggests that subsequent layers of the retina, responsible for signal processing, add little if any excess noise and extract all the available information. Given the signal and noise characteristics of the photoreceptors, what is the structure of such an optimal processor? We show that optimal estimates of time-varying light intensity can be accomplished by a two-stage filter, and we suggest that the first stage should be identified with the filtering which occurs at the first anatomical stage in retinal signal processing, signal transfer from the rod photoreceptor to the bipolar cell. This leads to parameter-free predictions of the bipolar cell response, which are in excellent agreement with experiments comparing rod and bipolar cell dynamics in the same retina. As far as we know this is the first case in which the computationally significant dynamics of a neuron could be predicted rather than modeled.     

Visual cycle: Dependence of retinol production and removal on photoproduct decay and cell morphology

The visual cycle is a chain of biochemical reactions that regenerate visual pigment following exposure to light. Initial steps, the liberation of all-trans retinal and its reduction to all-trans retinol by retinol dehydrogenase (RDH), take place in photoreceptors. We performed comparative microspectrophotometric and microfluorometric measurements on a variety of rod and cone photoreceptors isolated from salamander retinae to correlate the rates of photoproduct decay and retinol production. Metapigment decay rate was spatially uniform within outer segments and 50-70 times faster in the cells that contained cone-type pigment (SWS2 and M/LWS) compared to cells with rod-type pigment (RH1). Retinol production rate was strongly position dependent, fastest at the base of outer segments. Retinol production rate was 10-40 times faster in cones with cone pigments (SWS2 and M/LWS) than in the basal OS of rods containing rod pigment (RH1). Production rate was approximately five times faster in rods containing cone pigment (SWS2) than the rate in basal OS of rods containing the rod pigment (RH1). We show that retinol production is defined either by metapigment decay rate or RDH reaction rate, depending on cell type or outer segment region, whereas retinol removal is defined by the surface-to-volume ratio of the outer segment and the availability of retinoid binding protein (IRBP). The more rapid rates of retinol production in cones compared to rods are consistent with the more rapid operation of the visual cycle in these cells.     

Calcium imaging reveals a network of intrinsically light-sensitive inner-retinal neurons

BACKGROUND: Mice lacking rod and cone photoreceptors (rd/rd cl) are still able to regulate a range of responses to light, including circadian photoentrainment, the pupillary light reflex, and suppression of pineal melatonin by light. These data are consistent with the presence of a novel inner-retinal photoreceptor mediating non-image-forming irradiance detection. RESULTS: We have examined the nature and extent of intrinsic light sensitivity in rd/rd cl retinae by monitoring the effect of light stimulation (470 nm) on intracellular Ca(2+) via FURA-2 imaging. Using this approach, which does not rely on pharmacological or surgical isolation of ganglion cells from the rod and cone photoreceptors, we identified a population of light-sensitive neurons in the ganglion cell layer (GCL). Retinal illumination induced an increase of intracellular Ca(2+) in approximately 2.7% of the neurons. The light-evoked Ca(2+) fluxes were dependent on the intensity and duration of the light stimulus. The light-responsive units formed an extensive network that could be uncoupled by application of the gap junction blocker carbenoxolone. Three types of light-evoked Ca(2+) influx were observed: sustained, transient, and repetitive, which are suggestive of distinct functional classes of GCL photoreceptors. CONCLUSIONS: Collectively, our data reveal a heterogeneous syncytium of intrinsically photosensitive neurons in the GCL coupled to a secondary population of light-driven cells, in the absence of rod and cone inputs.     

Molecular origin of continuous dark noise in rod photoreceptors.

Noise in the rod photoreceptors limits the ability of the dark-adapted visual system to detect dim lights. We investigated the molecular mechanism of the continuous component of the electrical dark noise in toad rods. Membrane current was recorded from intact, isolated rods or truncated, internally dialyzed rod outer segments. The continuous noise was separated from noise due to thermal activation of rhodopsin and to transitions in the cGMP-activated channels. Selectively disabling different elements of the phototransduction cascade allowed examination of their contributions to the continuous noise. These experiments indicate that the noise is generated by spontaneous activation of cGMP phosphodiesterase (PDE) through a process that does not involve transducin. The addition of recombinant gamma, the inhibitory subunit of PDE, did not suppress the noise, indicating that endogenous gamma does not completely dissociate from the catalytic subunit of PDE during spontaneous activation. Quantitative analysis of the noise provided estimates of the rate constants for spontaneous PDE activation and deactivation and the catalytic activity of a single PDE molecule in situ.     

Germinal cells in the goldfish retina that produce rod photoreceptors

Dividing cells and their progeny in retinae of young goldfish were labeled with [3H]thymidine, and selected cells were reconstructed from serial sections processed for electron microscopic autoradiography. Our goals were to characterize the cells that were identified as rod precursors in previous light microscopic autoradiographical studies and to determine their origin and fate. (In fish the population of rods increases several-fold postembryonically by proliferation of rod precursor cells scattered across the retina). Over 200 labeled cells taken from 11 retinas were examined, and 20 of these were reconstructed in their entirety. Some retinas were examined at short intervals (1 to 48 hr) after [3H]thymidine injection in order to study mitotically active cells, and others were examined after longer intervals (9 or 14 days) to discover the nature of the progeny of labeled dividing cells. Previous evidence from thymidine studies in larval goldfish suggested that proliferating cells destined to produce rods appear first in the inner nuclear layer and later in the outer nuclear layer, where they continue to divide and generate new rods (P.R. Johns, (1982) J. Neurosci. 2, 179). The present results provide morphological evidence in support of the suggestion that rod precursors migrate from inner to outer nuclear layer and, furthermore, show that the precursors are closely associated with, and perhaps guided by, the radial processes of Müller glial cells. Examination of EM autoradiographs of labeled cells at 9 and 14 days after a pulse label with thymidine confirms that the differentiated progeny of dividing precursor cells are exclusively rods. To our knowledge, rod precursors are the first example of a neuronal germinal cell in the vertebrate central nervous system that under normal conditions produces only one type of neuron.