Membrane turnover in crab photoreceptors studied by high-resolution scanning electron microscopy and by a new technique of thick-section transmission electron microscopy

Summary Crab photoreceptors were examined after treatment by the osmium-DMSO-osmium method for high-resolution scanning electron microscopy. This technique of specimen preparation was also adapted for transmission electron microscopy, enabling sections up to 1 urn thick to be viewed in a conventional microscope at 75 kV. With appropriate pretreatment, some cytoskeletal elements can be visualised by both techniques. The methods were then used to investigate some of the daily changes known to occur in photoreceptor cell structure. Striking differences were found in the structure of Golgi bodies present in retinula cells during the synthesis and breakdown phases of the daily cycle of photoreceptor membrane turnover. Cyclic changes were also noticed in the mitochondria of retinula cells, and additional evidence was found for a previously proposed model of rhabdomeral microvillus formation.     

Volume and surface changes of smooth endoplasmic reticulum in crayfish retinula cells upon light- and dark-adaptation

Summary Adaptation to light and darkness involves major transformations of the smooth endoplasmic reticulum (SER) in the retinula cells of the crayfish and other invertebrates, the mechanisms of which are unknown. This paper presents measurements of SER stereological parameters at three levels along the main axis of light-adapted and dark-adapted retinula cells of the crayfish. Both the volume density and the surface density of SER in the perinuclear region of dark-adapted cells are more than double the values found in light-adapted cells. This relationship is inverted in the axoplasm above and below the basement membrane, where SER volume and surface in dark-adapted cells are approximately half of the quantities measured in light-adapted cells. Although this proportional correspondence between changes in separate regions of the cells might suggest merely an intracellular shifting of SER membrane, calculation of the approximate absolute amounts of membrane involved makes this possibility very unlikely. It is concluded that the vast increase of SER in the perinuclear region of dark-adapted cells implies a large input of membrane, which is subsequently removed during light-adaptation.Abbreviation SER smooth endoplasmic reticulum     

The retina of the phalangid,Opilio ravennae,with particular reference to arhabdomeric cells

Summary The retina of the phalangid, Opilio ravennae, consists of retinula cells with distal rhabdomeres, arhabdomeric cells, and sheath cells. The receptive segment of retinula cells shows a clear separation into a Proximal rhabdom, organized into distinct rhabdom units formed by three or four retinula cells, and a Distal rhabdom, consisting of an uniterrupted layer of contiguous rhabdomeres. One of the cells comprising a retinula unit, the so-called distal retinula cell (DRC), has two or three branches that pass laterally alongside the rhabdom, thereby separating the two or three principal retinula cells of a unit. The two morphologically distinct layers of the receptive segment differ with respect to the cellular origin of rhabdomeral microvilli: DRC-branches contribute very few microvilli to the proximal rhabdom and develop extremely large rhabdomeres in the distal rhabdom only, causing the rhabdom units to fuse. Principal retinula cells, on the other hand, comprise the majority of microvilli of the proximal rhabdom, but their rhabdomeres diminish in the distal rhabdom. It is argued that proximal and distal rhabdoms serve different functions in relation to the intensity of incident light.In animals fixed 4 h after sunset, pigment granules retreat from the distal two thirds of the receptive segment. A comparison of retinae of day- and night-adapted animals shows that there is a slight (approximately 15%) increase in the cross-sectional area of rhabdomeral microvilli in dark-adapted animals, which in volume corresponds to the loss of pigment granules from the receptive segment. The length of the receptive segment as well as the pattern and shape of rhabdom units, however, remain unchanged.Each retinula unit is associated with one arhabdomeric cell. Their cell bodies are located close to those of retinula cells, but are much smaller and do not contain pigment granules. The most remarkable feature is a long, slender distal dendrite that extends up to the base of the fused rhabdom where it increases in diameter and develops a number of lateral processes interdigitating with microvilli of the rhabdom. The most distal dendrite portion extends through the center of the fused rhabdom and has again a smooth outline. All dendrites end in the distal third of the proximal rhabdom and are never present in the layer of the contiguous distal rhabdom. Arhabdomeric cells are of essentially the same morphology in day- and night-adapted animals. They are interpreted as photoinsensitive secondary neurons involved in visual information-processing that channel current collected from retinula cells of the proximal rhabdom along the optic nerve. A comparison is made with morphological equivalents of these cells in other chelicerate species.     

Calmodulin associated with rhabdomeral photoreceptor microvilli of arthropods and squid

Summary A monoclonal antibody against pea-leaf calmodulin was used to localise this calcium-binding protein on frozen sections of compound eyes of several arthropod species and on nitrocellulose replicas of electrophoretically separated peptides of isolated photoreceptor membrane from crayfish, fly, and squid. We report the presence of immunochemically detectable amounts of calmodulin specifically associated with the photoreceptor microvilli of rhabdomeral photoreceptors. A weak immunofluorescent signal was also observed in the cytoplasm of retinula cells. The presence of calmodulin in rhabdomeral microvilli is discussed in view of its possible implication in phototransduction and/or involvement in cytoskeletal structures associated with photoreceptor membranes in invertebrates.     

Electrophysiological evidence for interaction between retinula cells in the flesh-fly

Summary In the electrical response of retinula cells to polarized light in the flesh-flyBoettcherisca peregrina, the polarization plane which showed the maximum sensitivity (Pol_max phase) to illumination by a small spot of light just large enough to cover only one retinula cell was found to differ from that with illumination by a larger spot of light which included adjacent cells. There was a difference of about 30°.This difference in Pol_max phase was assumed to indicate the occurrence of interaction between retinula cells even in the fly photoreceptor having rhabdom of the open type. This assumption was confirmed by the following experiments. (1) Under selective adaptation by a large spot of polarized light so as to eliminate the interaction effect, the Pol_max phase was found to be the same as that measured by a small spot even though the measurement had been made using a large spot of light. (2) The responses to polarized light illuminated from along some restricted off-axes showed a 60° shift in the Pol_max phase in comparison to those obtained from along the other axes. (3) The spectral sensitivity curves to illumination from along off-axes were almost all the same and were for the peripheral retinula cells. (4) The receptor potentials were found to increase in amplitude in a certain limited off-axis area that corresponded to the specific off-axis direction of illumination which had resulted in a shift of the Pol_max phase.It is concluded from these results that the peripheral retinula cells in the flesh-fly demonstrate interaction between certain two adjacent retinula cells. This interaction is positive but not a simple algebraic sum of the activity of two cells.This work was partly supported by a grant from the Japan Education MinistryI wish to thank the Department of Biology, Faculty of Sciences, Kyushu University (Prof. H. Morita and Prof. H. Tateda) for the constant supply of flesh-flies.     

Monoclonal antibodies to crayfish rhodopsin. I. Biochemical characterization and cross-reactivity

Five antibody secreting cell lines were selected on the basis of specific binding to photoreceptive structures from a fusion of myeloma cells with spleen cells from BALB/c mice immunized with photoreceptor membrane from crayfish compound eyes. On Western blots derived from one- and two-dimensional polyacrylamide gels of purified photoreceptor membrane the antibodies bound strongly to the major 35 kDa peptide and are therefore specific for the visual pigment, rhodopsin. Four antibodies also recognized a minor 24 kDa peptide probably representing a breakdown product generated in vivo by the action of lysosomal hydrolases. Epitope characterization of the antibodies using peptide maps of opsin after protease treatment revealed three grossly different specificities. Three antibodies recognize a major antigenic site located within the large proteolytic fragment of about 24 kDa, possibly derived from the aminoterminus of the molecule. Antibodies applied to lightly fixed frozen sections or semi-thin sections of crayfish retina embedded in Lowicryl or polyethyleneglycol specifically bound to the rhabdomeral structure formed by receptor cells R1-R7, but failed to show significant cross-reaction with R8, the blue receptor, proving significant differences in the primary structure of the apoproteins of visual pigments involved in crayfish colour vision. None of the antibodies revealed any cross-reactivity with Drosophila or squid rhodopsin, corroborating this finding. The antibodies also recognized granular material in the vicinity of the rhabdoms at sites occupied by secondary lysosomes containing degraded rhabdomeral membrane. No significant binding was observed to the outer plasma membrane of the retinula cells, or in any other part of the retina.     

The role of reflecting pigment cells in the turnover of crayfish photoreceptors

Summary The photoreceptors of the crayfish Procambarus clarkii undergo an extensive cycle of turnover in the late afternoon. Quantitative light and electron microscopy reveal a sharp increase in the fractional volume (i.e., density) of reflecting-pigment-cell granules and vacuoles shortly following late-afternoon photoreceptor turnover. The reflecting pigment cells (RPCs), which permanently reside within the crayfish retina, are shown to serve much the same function as the vertebrate pigment epithelium. The RPCs phagocytose partially digested photoreceptive microvilli and the ingested debris is degraded further into the granules and vacuoles which characterize these cells. Phagosome degradation appears to be mediated by Golgi complexes. Acid phosphatase appears to be involved in the initial rhabdom breakdown but not in the final reduction of RPC granules.     

Extracellular shedding of photoreceptor membrane in the open rhabdom of a tipulid fly

Summary The compound eyes of the Australian tipulid fly, Ptilogyna, shed the bulk of their rhabdomeral membrane to extracellular space during turnover. The rhabdomeres of the retinulae lie in a common extracellular space (ECS), which is subdivided in the proximal retina. Before dawn, a distal region of the microvilli in each rhabdomere differentiates and becomes less electron-dense after conventional fixation. The differentiated region then dilates and develops an irregular profile. A few hours after dawn, the transformed tips break off and form a detritus in the ECS. The degraded membrane is internalised back into the retinula cells by mass endocytosis. Retinulae develop pseudopodia at sites bordering the ECS and engulf the membrane detritus, which comes to lie first of all in vacuoles within the receptor cells and then forms very large multivesicular bodies. The latter transform to multilamellar and residual bodies and are, presumably, lysed. Surrounding these secondary lysosomes are rough endoplasmic reticulum and smooth tubular systems, tentatively considered on comparative grounds to provide hydrolases. The literature concerning the ultrastructure of compound eyes offers a small number of instances where extracellular shedding can be suspected for morphological reasons. Attention is drawn to analogies with the shedding of photoreceptor membranes in vertebrate retinae.     

双翅目昆虫复眼性特化光感受器的比较研究

雄性双翅目昆虫,包括家蝇Musca domestica、丽蝇Calliphora erythrocephala、华虻Tabanus mandarinus和憎黄虻Atylotus miser Szilady,其复眼性特化光感受器中央小网膜细胞R₇的分布从背区扩展到腹区。在雄性家蝇、华虻和憎黄虻复眼中,性特化光感受器中央小网膜细胞R₇的感杆延伸到基底膜,并同中央小网膜细胞R₈的感杆并列排列。但在雄性丽蝇复眼中,性特化光感受器中央小网膜细胞R₇的感杆不延伸到基底膜。在雌性双翅目昆虫复眼中,性特化光感受器中央小网膜细胞R₇仅仅分布在复眼的腹区,其数量比中央小网膜细胞R₈少得多。     

Variability and the nature of the insect electroretinogram

A study was made of the electroretinogram (ERG) waveform of over eighty species of diurnal Lepidoptera. In all, the two major components were found to be a cornea-negative potential originating in the retinula cells, and a cornea-positive component originating deep in the retina. These components can be shown to be physiologically independent of each other on the basis of the selective action of drugs, various inorganic ions, etc. They are different from each other in terms of flicker fusion frequency, latency, spectral sensitivity, and the effect of light adaptation. It is proposed that the cornea-positive component originates in the eccentric retinula cell, located close to the basement membrane. These cells appear to function as a photopic photoreceptor. The nature of the response of such cells varies as a result of small shifts in inorganic (Na⁺) ion concentration, thereby accounting for the observed variability of the insect ERG. It is suggested that such cells are associated with inhibitory events occurring in the optic lobe.     

Identificaton of UV,green and red receptors,and their projection to lamina in the cabbage butterfly,Pieris rapae

Summary The photoreceptors in the compound eye of a cabbage butterfly, Pieris rapae, were examined by conventional and intracellular-labeling electron microscopy by the use of the cobalt(III)-lysine complex as an ionized marker. Five types of spectral sensitivity were recorded intracellularly in electrophysiological experiments. They peaked at about 340, 380, 480, 560 and 620 nm, respectively. One of the distal retinula cells (R2) was a UV receptor, whereas the R4 distal retinula cell was a green receptor. The basal retinula cell, R9, was found to be a red receptor; it was localized near the basement membrane, having a bilobed cell body with an individual nucleus in each lobe. A small number of rhabdomere microvilli were present in a narrow cytoplasmic bridge connecting the two lobes. The axons of six retinula cells (R3–R8) in each ommatidium terminated at the cartridge in the lamina (short visual fiber), whereas those of the other three retinula cells, R1, R2 and R9, extended to the medulla (long visual fiber). The information from the UV and red receptors is therefore probably delivered directly to the medulla neurons, independent of that from the other spectral receptor types.     

Description of intracerebral ocelli in two species of North American crayfish: Orconectes limosus (Cambaridae) and Pacifastacus leniusculus (Astacidae)

Abstract. Among malacostracan crustaceans, intracerebral ocelli were first discovered in Isopoda, but they have been more recently reported from a crayfish ( Cherax destructor ) and a sandhopper ( Talitrus saltator ). This electron microscopic study increases the number of crayfish taxa in which intracerebral ocelli are now known to occur by two: Astacidae and Cambaridae. These photoreceptors are always integrated into the anteromedio-dorsal part of the brain and are not visible externally. Each ocellus is made up of 4–5 photoreceptor cells and is characterized by the presence of a fused rhabdom. The occurrence of different kinds of lysosomes in the cytoplasm is indicative of metabolic activity and perhaps membrane turnover. One typical feature of crayfish ocelli is their extraordinary variability in number. This trait is exemplified by individuals of Pacifastacus leniusculus , where as many as 14 ocelli were identified in a single brain. The arrangement of the ocelli is often not symmetrical with regard to the brain's midline and the ocelli always lack dioptric structures. Thus, it is difficult to see how they are involved in image formation. However, further research is needed to determine the precise role of these "hidden" receptors.     

Membrane maintenance and electrical properties of photoreceptors of wild-type andrpa (receptor potential absent) mutant blowflies (Calliphora erythrocephala)

Summary The maintenance of photoreceptor cell membranes in the blowfly was investigated in relation to the diurnal cycle, age, and therpa (receptor potential absent) phototransduction mutation. The effect of disturbed membrane assembly on the electrical membrane properties was examined using single-electrode discontinuous current-clamp techniques. In wild-type flies the cross-sectional dimensions of the rhabdomeres were markedly reduced with age, and the quantity of synthetic organelles decreased concurrently, whereas no correlation was found between the diurnal cycle and membrane turnover. Therpa mutation is thought to block the visual transduction cascade in photoreceptor cells and to lead to degeneration of the photoreceptor cell bodies. The volume of rhabdomeres decreased markedly inrpa mutants and the quantity of synthetic organelles was reduced significantly, indicating an imbalance between photoreceptive membrane renewal and degradation. Also, the plasma membrane underwent degenerative changes. The passive electrical properties of photoreceptor cells — resting membrane voltages and input resistances — were only slightly changed from those of wild-type flies, although the photoreceptive membrane did not depolarize in response to light. This indicates no apparent disturbance in the function of the ionic channels in these membranes. Taken together, these results suggest that the photoreceptor cells need a functional phototransduction cascade with its feedback controls to maintain continuous renewal of rhabdomeres, but that the plasma membrane maintains its normal electrochemical properties despite extreme morphological degeneration of photoreceptor cell.     

Selective illumination of single photoreceptors in the house fly retina: local membrane turnover and uptake of extracellular horseradish peroxidase (HRP) and Lucifer Yellow

Summary Single photoreceptor cells in the compound eye of the housefly Musca domestica were selectively illuminated and subsequently compared electron-microscopically with the unilluminated photoreceptors in the immediate surroundings. The rhabdomeres of the illuminated cells remain largely unaffected, but the cells show an increase in the number of coated pits, various types of vesicles, and degradative organelles; some of the latter organelles are described for the first time in fly photoreceptors. Coated pits are found not only at the bases of the microvilli, but also in other parts of the plasma membrane. Degradative organelles, endoplasmic reticulum (ER) and mitochondria aggregate in the perinuclear region. The rough ER and smooth ER are more elaborate, the number of Golgi stacks, free ribosomes and polysomes is increased, and the shape and distribution of heterochromatin within the nuclei are altered. Illuminated photoreceptors also interdigitate extensively with their neighbouring secondary pigment cells. These structural changes in illuminated fly photoreceptor cells indicate an increase in membrane turnover and cellular metabolism. When applied to the eye, Lucifer Yellow spreads into the extracellular space and is taken up only by the illuminated photoreceptor cells. These cells show the same structural modifications as above. Horseradish peroxidase applied in the same way is observed in pinocytotic vesicles and degradative organelles of the illuminated cells. Hence, the light-induced uptake of extracellular compounds takes place in vivo at least partially as a result of an increase in pinocytosis.     

The compound eye of<Emphasis Type="Italic"> Scutigera coleoptrata</Emphasis> (Linnaeus, 1758) (Chilopoda: Notostigmophora): an ultrastructural reinvestigation that adds support to the Mandibulata concept

The lateral compound eye of Scutigera coleoptrata was examined by electron microscopy. Each ommatidium consists of a dioptric apparatus, formed by a cornea and a multipartite eucone crystalline cone, a bilayered retinula and a surrounding sheath of primary pigment and interommatidial pigment cells. With reference to the median eye region, each cone is made up of eight cone segments belonging to four cone cells. The nuclei of the cone cells are located proximally outside the cone near the transition area between distal and proximal retinula cells. The connection between nuclear region and cone segment is via a narrow cytoplasmic strand, which splits into two distal cytoplasmic processes. Additionally, from the nuclear region of each cone cell a single cytoplasmic process runs in a proximal direction to the basement membrane. The bilayered rhabdom is usually made up of the rhabdomeres of 9–12 distal retinula cells and four proximal retinula cell. The pigment shield is composed of primary pigment cells (which most likely secrete the corneal lens) and interommatidial pigment cells. The primary pigment cells underlie the cornea and surround, more or less, the upper third of the crystalline cone. By giving rise to the cornea and by functioning as part of the pigment shield these pigment cells serve a double function. Interommatidial pigment cells extend from the cornea to the basement membrane and stabilise the ommatidium. In particular, the presence of cone cells, primary pigment cells as well as interommatidial pigment cells in the compound eye of S. coleoptrata is seen as an important morphological support for the Mandibulata concept. Furthermore, the phylogenetic significance of these cell types is discussed with respect to the Tetraconata.     

Evidence for putative photoreceptor axon terminals in the medulla externa of the crayfish

Summary Within the medulla externa of the crayfish compound eye a class of axonal endings with similar characteristics to the photoreceptor terminals of the lamina ganglionaris were studied with light and electron microscopic techniques. These terminals are restricted to the superficial layers of the medulla externa and each is marked by a rod-shaped inclusion selectively impregnated with reduced silver methods.Electron microscopy of the medullary terminals confirms the presence of a rod inclusion composed of fine regularly arranged filaments. These inclusions are often closely associated with mitochondria and glycogen deposits within the endings. Synaptic contacts made by these terminals are characterized by a presynaptic ribbon density which is in contact with two or three postsynaptic elements. Often one postsynaptic element participates in more than one synaptic complex. Numerous invaginated processes, microtubules, synaptic vesicles, and ER cisternae are also present in the medullary terminals.The eighth retinula cell in the retina of the crayfish studied here resembles that previously observed by Nässel (1976). The similarity of the medullary terminals to the photoreceptor endings in the lamina suggest that they may belong to one of the eight photoreceptor cells forming an ommatidium.This work was supported in part by grants from the National Science Foundation (BNS77-15803) and National Institute of Health (NS08964)The authors wish to acknowledge the technical assistance of Ms. Georgia Hammond-Soltis     

The diurnal pattern of protein and photopigment synthesis in the retina of the crayfish,Procambarus clarkii

Summary The interrelationship between the diurnal cycle of membrane loss and synthesis of new rhabdom components remains a key element in forming a complete picture of the turnover of photopigment-containing membrane in the crayfish photoreceptor cell. In order to examine this aspect of the turnover process, the diurnal pattern of photopigment synthesis was examined using an in vitro incubation system for incorporation of³H-leucine into photoreceptor protein. The incorporation of³H-leucine into total protein and photopigment specifically was measured in photoreceptors isolated from incubated retinas. The results indicate that for both total protein and photopigment there is no significant variation in the rate of synthesis during the 12-12 light-dark cycle. These data combined with earlier data on diurnal membrane loss from the rhabdom suggest that light-stimulated rhabdom membrane loss is superimposed on a diurnally constant level of synthesis and assembly of new rhabdom constituents.Abbreviations dpm disintegration per minute - LRB lysosome related body - TCA trichloroacetic acid     

Fine structural organization of the lateral ocelli in two species of Scolopendra (Chilopoda: Pleurostigmophora): an evolutionary evaluation

The lateral ocelli of Scolopendra cingulata and Scolopendra oraniensis were examined by electron microscopy. A pigmented ocellar field with four eyes arranged in a rhomboid configuration is present frontolaterally on both sides of the head. Each lateral ocellus is cup-shaped and consists of a deeply set biconvex corneal lens, which is formed by 230–2,240 cornea-secreting epithelial cells. A crystalline cone is not developed. Two kinds of photoreceptive cells are present in the retinula. 561–1,026 cylindrical retinula cells with circumapically developed microvilli form a large distal rhabdom. Arranged in 13–18 horizontal rings, the distal retinula cells display a multilayered appearance. Each cell layer forms an axial ring of maximally 75 rhabdomeres. In addition, 71–127 club-shaped proximal retinula cells make up uni- or bidirectional rhabdomeres, whose microvilli interdigitate. 150–250 sheath cells are located at the periphery of the eye. Radial sheath cell processes encompass the soma of all retinula cells. Outside the eye cup there are several thin layers of external pigment cells, which not only ensheath the ocelli but also underlie the entire ocellar field, causing its darkly pigmented. The cornea-secreting epithelial cells, sheath cells and external pigment cells form a part of the basal matrix extending around the entire eye cup. Scolopendromorph lateral ocelli differ remarkably with respect to the eyes of other chilopods. The dual type retinula in scolopendromorph eyes supports the hypothesis of its homology with scutigeromorph ommatidia. Other features (e.g. cup-shaped profile of the eye, horizontally multilayered distal retinula cells, interdigitating proximal rhabdomeres, lack of a crystalline cone, presence of external pigment and sheath cells enveloping the entire retinula) do not have any equivalents in scutigeromorph ommatidia and would, therefore, not directly support homology. In fact, most of them (except the external pigment cells) might be interpreted as autapomorphies defining the Pleurostigmophora. Certain structures (e.g. sheath cells, interdigitating proximal rhabdomeres, discontinuous layer of cornea-secreting epithelial cells) are similar to those found in some lithobiid ocelli (e.g. Lithobius). The external pigment cells in Scolopendra species, however, must presently be regarded as an autapomorphy of the Scolopendromorpha.     

The superposition eye of Cloeon dipterum: The organization of the lamina ganglionaris

Summary The lamina ganglionaris of the superposition eye of Cloeon dipterum is composed of separate optic cartridges arranged in a hexagonal pattern. Each optic cartridge consists of one central, radially branched monopolar cell (Li) surrounded by a crown of seven retinula cell terminals and two more unilaterally branched monopolar cells (La1/La2) situated close together outside the cartridge. Projections to neighbouring cartridges have not been observed.In most cases, synaptic contacts could be seen between a presynaptic retinula cell and more than two other postsynaptic profiles, which belong to monopolar cells or sometimes to glial cells.Seven retinula cell fibers of one ommatidium pass in a bundle through the basement membrane, run into their respective cartridges without changing orientation and terminate at approximately equal levels in the lamina. Long visual fibers with endings in the medulla are not visible in the superposition eye lamina, but are present in the lateral apposition eye. The relationship between the behaviour of the animal, optic mechanisms of the superposition eye and the structure of the lamina is discussed.     

Rhabdom changes in the shrimp,Palaemonetes

The fine structure of the principal compound eye of the shrimp, Palaemonetes, was studied under conditions of light and dark adaptation. Ommatidium the situation in other decapod crustaceans. Light and dark adapted eyes differ in that the rhabdom changes its shape; morphological evidence suggests a possible sequence of events involving production, utilization, and degradation of photoreceptor membrane, a discontinuous process occurring only during changes from light to dark and dark to light. A hypothesis of membrane turnover is proposed.