Ultrastructure of carotenoid deprivation in photoreceptors of Manduca sexta: myeloid bodies and intracellular microvilli

Summary The ultrastructural effects of carotenoid (vitamin A) deprivation were studied in the adult photoreceptors of the tobacco hornworm moth Manduca sexta. Moths were reared on a deprived diet, which lacked the carotenoid sources of the photopigment chromophore, 3-hydroxy retinal, or on a control, fortified diet, containing ample carotenoid. The latter supported normal levels of visual function, whereas visual pigment and sensitivity were reduced by at least 3 log units in moths reared on the deprived diet. Myeloid bodies, massed cisternae of hypertrophied smooth endomembrane, filled the cytoplasm in the receptors of deprived animals. The myeloid bodies assumed various configurations that included lamellate stacks of parallel cisternae, and tubular networks in a paracrystalline form. Freeze-fracture preparations of myeloid membranes revealed a high density of P-face particles. Vacuoles containing microvilli similar to those of the rhabdomere were also present in deprived photoreceptors. We suggest that the elaboration of smooth endoplasmic reticulum as myeloid bodies in chromophore-deprived photoreceptors may stem from the hypertrophy of a biochemical system for processing the chromophore or the interruption of the intracellular pathway that normally carries visual pigment to the rhabdomere.     

Ultrastructural study of the retinal pigment epithelium during metamorphosis in the sea lamprey (Petromyzon marinus L.)

Summary The sequence of morphological changes in the retinal pigment epithelium during the metamorphic period of the sea lamprey Petromyzon marinus L. has been investigated using electron microscopy. At early metamorphic stages (stages I and II), photoreceptors are present in a small zone of the retina. During these stages, the lateral surface of the epithelial cells shows zonulae occludentes and adhaerentes. The degree of cell differentiation varies throughout the retinal pigment epithelium. Cells covering the differentiated photoreceptors in the central retina have phagosomes, whereas pigment granules appear only in the retinal pigment epithelium dorsal to the optic nerve head. Most epithelial cells have myeloid bodies; their morphology is more complex around the optic nerve head. At stage III, when photoreceptors develop over the whole retina, the distribution of cytoplasmic organelles is almost homogeneous in the retinal pigment epithelium. Subsequently, the basal plasma membrane of the epithelial cells becomes progressively folded and their apical processes enlarged. In addition, extensive gap junctions develop between retinal pigment cells. In late metamorphic stages, noticeable growth of myeloid bodies occurs and consequently the retinal pigment epithelium resembles that of the adult. This study also describes, for the first time, the presence of wandering phagocytes in the retinal pigment epithelium of lampreys; their role in melanosome degradation is discussed.     

Biogenesis of myeloid bodies in regenerating newt (Notophthalmus viridescens) retinal pigment epithelium

Summary Myeloid bodies are believed to be differentiated areas of smooth endoplasmic reticulum membranes, and they are found within the retinal pigment epithelium in a number of lower vertebrates. Previous studies demonstrated a correlation between phagocytosis of outer segment disc membranes and myeloid body numbers in the retinal pigment epithelium of the newt. To test the hypothesis that myeloid bodies are directly involved in outer segment lipid metabolism and to further characterize the origin and functional significance of these organelles, we examined the effects on myeloid bodies of eliminating the source of outer segment membrane lipids (neural retina removal) and of the subsequent return of outer segments (retinal regeneration) in the newt Notophthalmus viridescens. Light- and electron-microscopic analysis demonstrated that myeloid bodies disappeared from the pigment epithelium within six days of neural retina removal. By week 6 of regeneration, rudimentary photoreceptor outer segments were present but myeloid bodies were still absent. However, at this time, the smooth endoplasmic reticulum in some areas of the retinal pigment epithelial cells had become flattened, giving rise to small (0.5 m long), two-to-four layer-thick lamellar units, which are myeloid body precursors. Small myeloid bodies were first observed one week later at week 7 of retinal regeneration. This study revealed that newt myeloid bodies are specialized areas of smooth endoplasmic reticulum. It also showed that a contact between functional photoreceptors and the retinal pigment epithelium is essential to the presence of myeloid bodies in the epithelial cells.     

Effects of light and dark on photoreceptors in the polychaete annelid Nereis limnicola

Summary The effects of light and dark on photoreceptors of the brackish-water polychaete annelid Nereis Hmnicola were studied by electron microscopy. Animals dark-adapted for one or two days exhibited well-formed straight microvilli (rhabdomeres) on the sensory cell processes. Continuous illumination of worms for one or two days caused extensive breakdown of the microvilli into vesicles and debris. Thirty minutes to three h of exposure of dark-adapted animals to light produced increasing severity of degradation of photoreceptoral microvilli. Light-adapted worms placed in darkness for one-half to three h showed progressive restoration of the microvilli to the dark-adapted condition. The products of degradation were internalized by both sensory and pigmented supportive cells by phagocytosis and pinocytosis.     

Rhabdom size and photoreceptor membrane turnover in a muscoid fly

Summary The cross-sectional area of rhabdomeres in the compound eye of the blowfly, Lucilia, was found to remain constant under 12 h light/12 h dark cyclic lighting, and 10 days constant light or darkness. It was reduced only slightly during 3 h light after 10 days darkness (by 21%), or on exposure to 2h darkness + 1.5 h light after 10 days light (by 10%). Morphological evidence indicates that shedding of photoreceptor membrane during turnover is achieved by an extracellular route, and by pinocytosis from the bases of the microvilli. The photoreceptor membrane shed by both mechanisms appears to accumulate in multivesicular bodies. The amount of photoreceptor membrane shedding, as indicated by numbers of multivesicular bodies, is constant throughout the day and night on cyclic lighting, decreases in constant darkness, but returns to its normal level after an exposure to 3 h light subsequent to 10 days darkness.     

Use of gain-of-function study to delineate the roles of<Emphasis Type="Italic">crumbs</Emphasis> in<Emphasis Type="Italic">Drosophila</Emphasis> eye development

The cell polarity gene,crumbs (crb), has been shown to participate in the development and degeneration of theDrosophila retina. Mutations inCRB1, the human homologue ofDrosophila crb, also result in retinitis pigmentosa and Leber congential amaurosis. In this study, we used the gain-of-function approach to delineate the roles ofcrb in developingDrosophila eye. In the third-instar larval stage, eye development is initiated with photoreceptor differentiation and positioning of photoreceptor nuclei in the apical cellular compartment of retinal epithelium. In the pupal stage, differentiated photoreceptors begin to form the photosensitive structures, the rhabdomeres, at their apical surface. UsingGMR-Gal4 to drive overexpression of the Crb protein at the third-instar eye disc, we found that differentiation of photoreceptors was disrupted and the nuclei of differentiated photoreceptors failed to occupy the apical compartment. Usinghs-Gal4 to drive Crb overexpression in pupal eyes resulted in interference with extension of the adherens junctions and construction of the rhabdomeres, and these defects were stage-dependent. This gain-of-function study has enabled us to delineate the roles of Crb at selective stages of eye development inDrosophila.     

Rhabdomeric membrane and smooth endoplasmic reticulum in photoreceptors of Manduca sexta: modulations associated with the diurnal light/dark cycle and effects of chromophore deprivation

Summary Aberrations of photoreceptor ultrastructure resulting from carotenoid/retinoid (vitamin A) deprivation were studied in the retina of Manduca sexta. The syndrome of chromophore deficiency included hypertrophy of smooth endoplasmic reticulum, variable dilation of rhabdomeric microvilli, the insertion of endomembrane fingers into such enlarged microvilli, and the formation of rhabdomeric vacuoles, intracellular compartments containing microvilli similar to those of the rhabdomere. Retinas were processed either with conventional procedures employing preliminary aldehyde fixation followed by heavy metal postfixation, or by fixation and incubation in unbuffered OsO₄. The latter method deposits osmium throughout the endomembrane system, within the rhabdomeric vacuoles, and in the extracellular space of the rhabdom. However, the intravillous fingers were rarely impregnated with osmium, despite their continuity with densely stained cisternae of the smooth endoplasmic reticulum. We suggest that the insertion of endomembrane fingers into dilated microvilli results from a cytoskeleton-mediated link between cisternae of the smooth endoplasmic reticulum and the rhabdomeric membrane, an association that may be important in the turnover of photoreceptor membrane. We interpret endomembrane hypertrophy and development of rhabdomeric vacuoles as symptoms of disturbance in the pathway leading to the assembly of the rhabdomere resulting from reduced synthesis of visual pigment.     

Mutation that selectively affects rhodopsin concentration in the peripheral photoreceptors of Drosophila melanogaster

A Drosophila mutant (ninaAP228) that is low in rhodopsin concentration but identical to the wild-type fly in photoreceptor morphology has been isolated. R1-6 photoreceptors of the mutant differ from those of wild type in that (a) the prolonged depolarizing afterpotential (PDA) is absent, (b) concentrations of rhodopsin and opsin are substantially reduced, and (c) intramembrane particle density in the membranes of the rhabdomeres is low. Each of these traits is mimicked by depriving wild- type flies of vitamin A. The ninaAP228 mutation differs from vitamin A deprivation in that in the mutant (a) the rhabdomeric membrane particle density is reduced only in the R1-6 photoreceptors and not in R7 or R8, (b) the PDA can be elicited from the R7 photoreceptors, and (c) photoconversion of R1-6 rhodopsin to metarhodopsin by ultraviolet (UV) light is considerably more efficient than in vitamin A-deprived flies. The absorption properties of the mutant rhodopsin in the R1-6 photoreceptors appear to be identical to those of wild type as judged from rhodopsin difference spectra. The results suggest that the mutation affects the opsin, rather than the chromophore, component of rhodopsin molecules in the R1-6 photoreceptors. The interaction between the chromophore and R1-6 opsin, however, appears to be normal.     

Vitamin a deficiency reduces the concentration of visual pigment protein within blowfly photoreceptor membranes

Visual pigment extracts prepared from rhabdomeric membranes of vitamin A deficient blowflies contain a 5–10 times lower concentration of rhodopsin than extracts from flies which were raised on a vitamin A rich diet. Spectrophotometry showed that digitonin-solubilized rhodopsin from blowfly photoreceptors R_1–6 has an absorbance maximum at about 490 nm, but no unusually enhanced β-band in the ultraviolet. The extracts did not contain detectable concentrations of other visual pigments nor was there any evidence for the presence of photostable vitamin A derivatives.Sodium dodecyl sulfate polyacrylamide gel electrophoresis demonstrated that the concentration of opsin in the rhabdomeric membrane is significantly reduced in vitamin A deficient flies compared to normal flies. The results indicate that the synthesis of opsin or its incorporation into the photoreceptor membrane is regulated by the chromophore concentration in the receptor cell. Furthermore, our findings open up the possibility that differences in the spectral absorption and excitability of photoreceptors from normal and vitamin A deficient flies result from the differing opsin content of the rhabdomeres.     

Vitamin A deficiency reduces the concentration of visual pigment protein within blowfly photoreceptor membranes.

Visual pigment extracts prepared from rhabdomeric membranes of vitamin A deficient blowflies contain a 5-10 times lower concentration of rhodopsin than extracts from flies which were raised on a vitamin A rich diet. Spectrophotometry showed that digitonin-solubilized rhodopsin from blowfly photoreceptors R1-6 has an absorbance maximum at about 490 nm, but no unusually enhanced beta-band in the ultraviolet. The extracts did not contain detectable concentrations of other visual pigments nor was there any evidence for the presence of photostable vitamin A derivatives. Sodium dodecyl sulfate polyacrylamide gel electrophoresis demonstrated that the concentration of opsin in the rhabdomeric membrane is significantly reduced in vitamin A deficient flies compared to normal flies. The results indicate that the synthesis of opsin or its incorporation into the photoreceptor membrane is regulated by the chromophore concentration in the receptor cell. Furthermore, our findings open up the possibility that differences in the spectral absorption and excitability of photoreceptors from normal and vitamin A deficient flies result from the differing opsin content of the rhabdomeres.     

Studies of fluorescence inDrosophila compound eyes: changes induced by intense light and vitamin A deprivation

Summary Ultraviolet light excites a red fluorescence fromDrosophila R1–6 rhabdomeres which is superimposed on a blue background emission. Metarhodopsin (M570) pigment generates some or all of the vitamin A dependent red emission. However, the excitation spectrum for red emission peaks in the UV. This suggests that the pigment which sensitizes R1–6's visual pigment to UV light (sensitizing pigment) absorbs the UV light, sensitizing metarhodopsin's fluorescence by energy transfer. Blue emission is neither from sensitizing pigment nor from visual pigment as shown by vitamin A deprivation studies.Very intense UV or blue stimulation causes these changes: (1) conversion of visual pigment into a fluorescent product; (2) destruction of this fluorescent product; (3) a decrease in the blue background fluorescence (even in vitamin A deprived flies); and (4) a permanent destruction of visual pigment and retinal degeneration. The first effect requires intensities 3 log units brighter than needed to interconvert rhodopsin and metarhodopsin 1/2 way to photoequilibrium. UV light is about 5 times as effective as blue light for the conversion of visual pigment into fluorescent product.     

Photic regulation of pineal function. Analogies between retinal and pineal photoreception

Light absorbed by a photopigment in a photoreceptor cell causes a photochemical reaction converting the 11-cis retinal chromophore into the all-trans configuration. These changes lead to a series of events that causes cGMP hydrolysis, a following decrease of cGMP in the cytoplasm of the photoreceptor outer segment and a closure of cGMP-gated cationic channels. As a consequence of these processes the membrane hyperpolarizes. In pineal photoreceptor cells of lower vertebrates these processes are only partly investigated. Molecules involved in the phototransduction process and the desensitization, like opsin, vitamin A, α-transducin and arrestin, have been immunocytochemically localized in pineal photoreceptors and also electrophysiological studies have shown that phototransduction mechanisms in pineal photoreceptors might be very similar to those found in retinal photoreceptors. This review will summarize some of the current knowledge on pineal photoreception and compare it with retinal processes.     

Molecular and evolutionary aspects of microbial sensory rhodopsins

Retinal proteins (~ rhodopsins) are photochemically reactive membrane-embedded proteins, with seven transmembrane α-helices which bind the chromophore retinal (vitamin A aldehyde). They are widely distributed through all three biological kingdoms, eukarya, bacteria and archaea, indicating the biological significance of the retinal proteins. Light absorption by the retinal proteins triggers a photoisomerization of the chromophore, leading to the biological function, light-energy conversion or light-signal transduction. This article reviews molecular and evolutionary aspects of the light-signal transduction by microbial sensory receptors and their related proteins. This article is part of a Special Issue entitled: Retinal Proteins - You can teach an old dog new tricks.     

Circadian clock function in isolated eyestalk tissue of crayfish.

Electrical mass response of crayfish photoreceptors (electroretinogram) was recorded continuously for up to seven days in isolated preparations that consisted of the retina and lamina ganglionaris. Electroretinogram amplitude varied in a circadian manner with a nocturnal acrophase and a period of 22-23 h in preparations kept in darkness. Acclimatization of animals to reversed light/dark cycles resulted in a phase reversal of the rhythm in vitro. The per (period) gene of Drosophila has been implicated in the genesis of rhythms in insects and in vertebrates. Immunocytochemical staining with an antibody against the PER gene product revealed immunoreactivity in the retinal photoreceptors, as well as in cell bodies in the lamina ganglionaris. Labelled axons run distally towards the photoreceptors and proximally to other areas of the lamina.     

Chemistry and biology of vision

Visual perception in humans occurs through absorption of electromagnetic radiation from 400 to 780 nm by photoreceptors in the retina. A photon of visible light carries a sufficient amount of energy to cause, when absorbed, a cis,trans-geometric isomerization of the 11-cis-retinal chromophore, a vitamin A derivative bound to rhodopsin and cone opsins of retinal photoreceptors. The unique biochemistry of these complexes allows us to reliably and reproducibly collect continuous visual information about our environment. Moreover, other nonconventional retinal opsins such as the circadian rhythm regulator melanopsin also initiate light-activated signaling based on similar photochemistry.     

The function of photostable pigments in fly photoreceptors

The photoreceptors in the fly's ommatidia contain a bistable visual pigment, which can be shifted back and forth by means of light of appropriate wavelengths. The situation is complicated, however, by the presence of photostable pigments. One of them (located in rhabdomeres no. 1–6) absorbs in the UV, another one (in rhabdomeres no. 7y) in the blue spectral range. Such pigments act as (dichroic) colour filters that modify the spectral and polarisation sensitivity of the photoreceptors by means of absorption. It could be shown furthermore that such pigments can also act as sensitizing pigments that modify spectral sensitivities due to sensitization.Based on material presented at the European Neurosciences Meeting, Florence, September 1978     

Influence of light on the morphological changes that take place during the development of the <Emphasis Type="Italic">Flammulina velutipes</Emphasis> fruit body

We show that fruit bodies of Flammulina velutipes can be induced in complete darkness after a sharp temperature reduction (23° to 16°C). However, the fruit bodies that form in complete darkness have a long stipe with an undeveloped pileus on the top (pinhead fruit bodies) and are thinner and whiter than the normal fruit bodies which are formed in the light. This finding suggests that F. velutipes fruit bodies cannot mature in complete darkness. However, when we irradiated the fruit bodies that had formed in complete darkness, a pileus developed immediately, and 4 days later the separation between the stipe and the pileus could be observed. Immediately after light exposure, the stipe also thickened and became increasingly pigmented. The stipe elongation was inhibited until 8 days after light exposure, although stipe elongation progressed very quickly thereafter. Basidospores were also visible in the gills 8 days after light exposure. We consider that the basidiospore development is involved in this rapid stipe elongation, which aids the effective dispersal of basidiospores.     

Localization of visual pigments within rhabdoms of the compound eye of Spodoptera exempta (Insecta,Noctuidae)

Summary In the noctuid moth Spodoptera exempta, the distribution of visual pigments within the fused rhabdoms of the compound eyes was investigated by electron microscopy. Each ommatidium regularly contains eight receptor cells belonging to three morphological types: one distal, six medial, and one basal cell (Meinecke 1981); four different visual pigments — absorption maxima at approximately 355, 465, 515, and 560 nm — are known to occur within the eye (Langer et al. 1979). The compound eyes were illuminated in situ by use of monochromatic light of different wavelengths. This illumination produced a wide scale of structural changes in the microvilli of the rhabdomeres of individual cells. Preparation of eyes by freeze-substitution revealed the structural changes in the rhabdomeres to be effects of light occurring in vivo.The degree of structural changes may be considerably different in rhabdomeres within the same ommatidium; it was found to depend on the wavelength and the duration of illumination, the intensity received by the ommatidia as well as the spectral sensitivity of the receptor cells. Therefore, it was possible to estimate the spectral sensitivities of the morphological types of receptor cells. Generally, all medial cells are green receptors and all basal cells red receptors; distal cells are blue receptors in about two-thirds of the ommatidia, while in the remaining third of them distal cells are sensitive to ultraviolet light.Supported by Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 114 (Bionach)     

Light exposure during embryonic and yolk-sac alevin development of Chinook salmon Oncorhynchus tshawytscha does not alter the spectral phenotype of photoreceptors

Colour vision is mediated by the expression of different visual pigments in photoreceptors of the vertebrate retina. Each visual pigment is a complex of a protein (opsin) and a vitamin A chromophore; alterations to either component affects visual pigment absorbance and, potentially, the visual capabilities of an animal. Many species of fish undergo changes in opsin expression during retinal development. In the case of salmonid fishes the single cone photoreceptors undergo a switch in opsin expression from SWS1 (ultraviolet sensitive) to SWS2 (blue-light sensitive) starting at the yolk-sac alevin stage, around the time when they first experience light. Whether light may initiate this event or produce a plastic response in the various photoreceptors is unknown. In this study, Chinook salmon Oncorhynchus tshawytscha were exposed to light from the embryonic (5 days prior to hatching) into the yolk sac alevin (25 days post hatching) stage and the spectral phenotype of photoreceptors assessed with respect to that of unexposed controls by in situ hybridization with opsin riboprobes. Light exposure did not change the spectral phenotype of photoreceptors, their overall morphology or spatial arrangement. These results concur with those from a variety of fish species and suggest that plasticity in photoreceptor spectral phenotype via changes in opsin expression may not be a widespread occurrence among teleosts.     

Effects of continuous light and darkness on the eyes of the troglobitic salamander Typhlotriton spelaeus

Larval Typhlotriton spelaeus collected from five caves in Pulaski Co., Missouri, were kept as larvae or induced to transform in darkness or continuous fluorescent illumination. Larvae maintained in darkness for 215 and 279 days had smaller eyes, smaller rod inner and outer segments, and fewer metaphase figures in the genninative zone of the neural retina than comparable larvae maintained in light (258 lux). Except for visual cell size, differences were small and for each characteristic exceptions were observed. One larva kept in light showed early retinal degeneration comparable to that in transformed adults of T. spelaeus. All larvae exhibited optomotor behavior both before and after the experiment. Among animals induced to transform by L-thyroxin and maintained in darkness 111 to 366 days, visual cell and pigment epithelium degeneration was more extensive and more frequent than in animals kept for the same length of time in light (237-298 lux). In darkness the frequency of animals with retinal degeneration increased between 111 and 366 days. In light some animals exhibited pigment epithelium reduction with normal visual cells, and others had free, pigmented cells in the subretinal space. These effects were not comparable to degeneration in darkness. Eyelids covered the eyes of only a few animals in both light and dark treatments. The extent of eyelid encroachment over the eye was greater in darkness than in light. Most animals exhibited optomotor responses after experiments, but responses of animals kept in darkness were impaired in comparison to those of animals kept in light.