Presynaptic tubular structures in photoreceptor cells

Summary After the application of fixatives including phosphotungstic acid or a mixture of osmium tetroxide and zinc iodide, complex tubular structures are evident in the presynaptic side of the synapses between photoreceptor and bipolar cells of the rat's retina. In the first case only the limiting membranes are visualized, while in the second only the content of the tubules is stained. These tubules seem to be related, on a morphological ground, with the formation of synaptic vesicles. These tubular structures are not observed when fixation is done with osmium tetroxide or glutaraldehyde-osmium tetroxide.This work has been supported by grants from the Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina, and from National Institutes of Health, U.S.A., (5 RO1 NS 06953-05 NEUA).We want to express our gratitude to Mrs. Haydée Agoff de Zimman and Mr. Alberto Saénz for their skillful technical assistance.     

Mitochondrial structure revealed by high-resolution scanning electron microscopy

Mitochondrial structure has been examined in three dimensions using high-resolution scanning electron microscopy in cells from rat liver, retina (photoreceptors and retinal pigment epithelium), and kidney (proximal convoluted tubular cells and podocytes). Tissues were prepared by aldehyde-osmium fixation and freeze cleavage using a cryoprotectant, followed by removal of the cytosol by immersion in a dilute osmium tetroxide solution. The microscope used (Hitachi S-570) was equipped with a secondary electron detector located in the column above the specimen, situated within the objective lens. Mitochondria in all tissues examined were found to have only tubular cristae, which in some instances could be seen to span the entire diameter of the organelle. The walls of the tubular cristae, when unfractured, were in contact with the inner mitochondrial membrane; and their lumens were open to the intermembranous space. We hypothesize that in cells of many, perhaps most tissues, mitochondrial cristae are not shelf-like but are, in fact, tubes which span the mitochondrial matrix and are continuous with the inner mitochondrial membrane at both ends.     

Studies on microperoxisomes. VII. Pigment epithelial cells and other cell types in the retina of rodents

The pigment epithelial cell of the retina actively participates in two aspects of lipid metabolism: (a) the fatty acid esterification of vitamin A and its storage and transport to the photoreceptors, and (b) the phagocytosis and degradation of the lipoprotein membrane disks shed from the photoreceptor cells. Study of the pigment epithelial cells of adult albino and pigmented rodents has revealed the abundance of an organelle, microperoxisomes, not previously known to exist in this cell type. The metabolism, transport, and storage of lipids are major functions of other cell types which possess large numbers of microperoxisomes associated with a highly developed smooth endoplasmic reticulum. Microperoxisomes were encountered, but relatively rarely, in Müller cells and vascular endothelial cells. A tubular system in photoreceptor terminals is reactive in the cytochemical procedure used to visualize microperoxisomes.     

THE FINE STRUCTURE OF CHONDROCOCCUS COLUMNARIS : I. Structure and Formation of Mesosomes

Cells of Chondrococcus columnaris were sectioned and examined in the electron microscope after fixation by two different methods. After fixation with osmium tetroxide alone, the surface layers of the cells consisted of a plasma membrane, a dense layer (mucopeptide layer), and an outer unit membrane. The outer membrane appeared distorted and was widely separated from the rest of the cell. The intracytoplasmic membranes (mesosomes) appeared as convoluted tubules packaged up within the cytoplasm by a unit membrane. The unit membrane surrounding the tubules was continuous with the plasma membrane. When the cells were fixed with glutaraldehyde prior to fixation with osmium tetroxide, the outer membrane was not distorted and separated from the rest of the cell, structural elements (peripheral fibrils) were seen situated between the outer membrane and dense layer, and the mesosomes appeared as highly organized structures produced by the invagination and proliferation of the plasma membrane. The mesosomes were made up of a series of compound membranes bounded by unit membranes. The compound membranes were formed by the union of two unit membranes along their cytoplasmic surfaces.     

Fine structure of the segmental ocelli of Armandia brevis (Polychaeta: Opheliidae)

Summary Armandia brevis responds negatively to light during the benthic phase and positively to light during the epitokous phase of its life history. In addition to the prostomial photoreceptors this slender translucent marine worm possesses eleven pairs of ocelli arranged serially from the 7th to the 17th segments.Each ocellus is located at the inner edge of the epidermis slightly in front of the parapodium and contains a single photoreceptor cell which gives off approximately 15 sensory processes. These processes are composed of a central core of neurofibrils surrounded by a mitochondrial layer and a compact array of microvilli. The sensory processes project into and nearly fill the ocellar cavity which is lined by squamous glial cells.The pigment cup enclosing the photoreceptor is composed of about 30 cuboidal cells packed with brown granules. The pigment cells form a mesothelium, being in direct continuity with the coelom. The cup is separated from the glial cells by a basal lamina which separates the epidermal tissues from the mesodermal derivatives of the body wall. Slender muscle fibers traverse the coelom and pass between the cells of the pigment cup.The prostomial photoreceptors were re-examined and found in this material to be composed of microvilli rather than of folds containing labyrinthine tubular infoldings of the cell surface as previously reported.The author thanks Dr. Richard M. Eakin for support and criticism. This investigation was financed by a postdoctoral fellowship, number 1-F2-GM-20, and grant number GM 10292 from the National Institute of General Medical Science.     

Light dependence of osmium reactivity in mouse photoreceptor cells.

Mouse photoreceptor cells exhibit local accumulations of osmium deposits after prolonged osmic staining at slightly elevated temperatures. Deposits were evident along the membranes of outer segment lamellae, Golgi cisternae and vesicles, nuclear envelopes, and synaptic vesicles. Other membranes within the photoreceptor cells were unreactive. No osmium reactivity was seen in other cells of the retina except for osmiophilic outer segment material which had been phagocytized by the pigment epithelium. In the outer segments, inner segments, and synaptic regions of the photoreceptor cells, the amount of osmium reactivity was increased by light stimulation and decreased following extended dark adaptation. The possible significance of the localized osmium reactivity is discussed.     

Retinal Pigment Epithelium–Calycal Processes Association in the Catfish Clarias batrachus(Linnaeus, 1758)

Abstract In the duplex retina of the catfish Clarias batrachus(Linnaeus, 1758), the apical processes of the pigment epithelial cells have been found by transmission electron microscopy to be in intimate contact with the calycal processes around the basal portion of the photoreceptor outer segments. It is hypothesized that the retinal pigment epithelium effectively transports synthesized products and metabolites to the photoreceptor inner segments via the anatomical zone of the apical–calycal processes interface in this species.     

Sieve-plate pores in leaf veins of Hordeum vulgare

Summary The sieve-plate pores of sieve elements in leaf veins of Hordeum vulgare, fixed in glutaraldehyde with postfixation in osmium tetroxide, were lined by the plasmalemma and variable amounts of callose. All pores were filled with endoplasmic reticulum, which was continuous from cell to cell. Mature sieve elements lacked P-protein.     

ELECTRON MICROSCOPY OF THE OXYNTIC CELL IN THE GASTRIC GLANDS OF THE BULLFROG, RANA CATESBIANA : II. The Acid-Secreting Gastric Mucosa

The oxyntic cell in the gastric glands of the bullfrog was examined in lead hydroxide-stained sections of gastric mucosae fixed in buffered osmium tetroxide and embedded in n-butyl methacrylate. During gastric acid secretion (pH 1–2) induced by histamine administration in cannulated frogs, the pattern of fine structure in the oxyntic cell differs strikingly from that in the oxyntic cell of the non-acid-secreting stomach. The relative number of smooth surfaced profiles decreases and a greater concentration of these elements is associated with the apical region of the oxyntic cell facing the lumen of the gastric gland. Similar concentrations of these elements are found in those regions of cytoplasm surrounding intercellular canaliculi which lie between adjacent cells and communicate with the lumen of a gastric gland. In these regions, the smooth surfaced profiles (35 to 65 mµ in width) characteristically form a tubular network. The membrane-bounded contents appear to be continuous with the extracellular medium, both on the capillary side and at the apical surface of the cell adjoining the lumen of the gastric gland. Mitochondria are distributed randomly in the cytoplasmic matrix of the oxyntic cell.     

Pigment,abnormal mitochondria and laminar bodies in human sympathetic neurons

Summary A study was made of the fine structure of neurons in lumbar sympathetic ganglia which were surgically removed from a 52 year old male patient. Some samples were fixed by direct immersion into osmium tetroxide, others were first prefixed with an aldehyde solution. Large vesiculated pigment granules were described, compared with similar particles, previously seen in human and animal autonomic neurons, and their glycogen and lipid-soluble components were tentatively identified. These granules increase numerically during aging, are probably derivatives of lysosomes, and may incorporate both lipofuscin and melanin in the same particle. Mitochondrial changes (enlargement, disappearance of cristae and deposit of opaque central material) in sympathetic perikarya occurred under all conditions of preservation, and were interpreted as hypoxia-induced structures which developed during life. Abnormal aggregates of mitochondria were seen in nerve processes. Laminar bodies, chiefly associated with mitochondria were present, especially, in aldehyde prefixed material. It remained undecided whether these structures occurred during life or were artifacts. Compared with direct osmium tetroxide fixation, prefixation with aldehyde, followed by osmium tetroxide proved to be a superior, but not entirely adequate method of preserving deep layers of neuronal tissue. This investigation was supported (in whole) by United States Public Health Service Grant NB 1879-09, Institute for Nervous Diseases and Blindness.The author is greatly indebted to Dr. Jere W. Lord, Jr., Department of Surgery, New York University Medical Center, for the surgically removed tissues used in this study; he is grateful to Dr. Sanford L. Palay, Department of Anatomy, Harvard Medical School, for suggesting the use of the then unpublished method of prefixation with the aldehyde solution. It is a pleasure to acknowledge the expert skill with which Miss Carmen DeLemos and Mr. Arnlod Cianella assisted in completing this work.     

An electron microscopic study of retinal degeneration in Sprague-Dawley rats

Retinal degeneration in untreated, female Sprague-Dawley rats was studied by electron microscopy and horseradish peroxidase tracer technique. The degeneration appeared to have started at a very young age. The severity of the defect varied from a decrease of photoreceptor nuclei to total loss of receptor cells and the pigment epithelium. In mild degeneration some regions of the retinal pigment epithelium became bilayered and the basal plasma membrane became flattened or formed elaborate infoldings. Breaks in Bruch's membrane occurred in severe degeneration. Degeneration of the pigment epithelium allowed permeation of tracer material from the choroid into the retina.     

Differentiation of the membrane system in the cells of imaginai discs

Summary Imaginal discs from developing larvae of the fly Calliphora erythrocephala fixed in permanganate or osmium tetroxide and embedded in Epon 812 were observed by electron microscopy. When the larval growth ceases, the differentiation manifests itself through an enlargement of the endoplasmic reticulum, by which continuous membrane contact is established between all cell organelles. During the same time mitochondria swell up and transform into lipid granules and the intercellular contacts weaken.I am indebted to Mrs Mariann Carleson and Miss Brita Nilsson for technical aid.     

An Organotypic Culture of the Newt Retina together with Other Tissues of the Posterior Eye Segment as a Model for Studying the Effects of Cell Adhesion Glycoproteins

The adult newt retina explanted together with the posterior eye wall and cultivated for a short time in a serum-free medium was tested as an experimental model by several criteria, including the expression of protein markers of the main retinal cell types. Some differences in the expression of specific photoreceptor, interneuron, and glial cell proteins as well as the localization of acetylcholinesterase activity were found during in vitro cultivation. Using this model, preliminary tests of new cell adhesion glycoproteins from the bovine retina and pigment epithelium were conducted, and the role of pigment epithelial cell proteins in improving cell viability in the cultivated newt retina was revealed. Moreover, the fraction of basic adhesion proteins from the bovine pigment epithelium improved the survival potential of the macroglial (Muller) cell population, compared to that in the control.     

Glutaraldehyde fixation of central nervous tissue: an electron microscopic evaluation in the isolated rabbit retina

The isolated rabbit retina was studied electron microscopically after fixation with a 3% solution of glutaraldehyde in a 0.05 M S?rensen's phosphate buffer. In radial sections, the inner segments, nuclei, and synapses of the photoreceptor cells seemed similar in size to those from retinas that had been fixed in an isotonic solution containing 1 % crystalline osmium tetroxide in the incubating medium used for the isolation procedure. However, when the number of comparable structures was greatly increased by viewing them in tangential sections, the cellular shrinkage and mitochondrial swelling produced by this widely used, hypertonic, glutaraldehyde fixative were obvious.     

The homology of spindle tubules and neuro-tubules in the chick embryo retina

Summary An ultrastructural study of the 8 day chick embryo retina has been performed using gluteraldehyde and osmium tetroxide fixation. It has been noted that the centrioles of ganglion cells show clavate extensions similar to those seen in the basal bodies of photoreceptors and that these appear to give rise to the neurotubules. The neurotubules thus are shown to originate from the same organelle and have the same morphology as the spindle tubules in the mitotic cell, although the mode of origin is quite different. Some implications of these observations have been discussed.Supported by Grants NB-02255, 2M-6418, and GM 11558-01 from the National Institutes of Health.     

Ultrastructural localization of glycogen in the granulocytes of normal rabbit bone marrow.

The glycogen of rabbit granulocytes has been studied in glutaraldehyde and osmium tetroxide fixed bone marrow by the periodic acid-thiocarbohydrazide-silver proteinate procedure (PA-TCH-SP). The PA-TCH-SP procedure involved the staining of intracytoplasmic glycogen more densely than the routine lead citrate staining. The PA-TCH-SP procedure demonstrated the intracytoplasmic glycogen in all three kinds of granulocytes. Though a sequence of intensity was observed in each stage of cell maturation, intracytoplasmic glycogen increased generally in accordance with cell maturation in the granulocytes. Functional significance of the glycogen in the granulocytes was discussed in relation to its staining. A very weak reaction in the granules of the granulocytes was described in relation to their contents.     

Autoradiographic identification of acetylcholine in the rabbit retina

Rabbit retinas were studied in vitro under conditions known to maintain their physiological function. Retinas incubated in the presence of [3H]choline synthesized substantial amounts of both [3H]phosphorylcholine and [3H]acetylcholine. With time, [3H]phosphorylcholine proceeded into phospholipids, primarily phosphatidylcholine. Retinas pulse-labeled by a 15-min exposure to 0.3 microM [3H]choline were incubated for a subsequent hour under chase conditions designed either to retain newly synthesized acetylcholine within synapses or to promote its release. At the end of this time the two groups of retinas were found to contain equal amounts of radioactivity in the phospholipid pathway, but only the retinas incubated under the acetylcholine-protecting conditions contained [3H]acetylcholine. Freeze-dried, vacuum-embedded tissue from each retina was autoradiographed on dry emulsion. All retinas showed silver grains over the photoreceptor cells and faint labeling of all ganglion cells. In the retinas that contained [3H]acetylcholine, silver grains also accumulated densely over a few cells with the position of amacrine cells, over a subset of the cells of the ganglion cell layer, and in two bands over the inner plexiform layer. Fixation of the retina with aqueous osmium tetroxide retained only the radioactive compounds located in the photoreceptor and ganglion cells. Sections from freeze-dried tissue lost their water-soluble choline metabolites when exposed to water, and autoradiography of such sections again revealed radioactivity primarily in the photoreceptor and ganglion cells. Radioactive compounds extracted from the sections were found to faithfully reflect those present in the tissue before processing; analysis of the compounds eluted from sections microdissected along the outer plexiform layer showed [3H]acetylcholine to have been synthesized only by cells of the inner retina. Taken together, these results indicate that the photoreceptor and ganglion cells are distinguished by a rapid synthesis of choline-containing phospholipids, while acetylcholine synthesis is restricted to a few cells at both margins of the inner plexiform layer. They imply that the only neurons to release acetylcholine within the rabbit retina are a small group of probable amacrine cells.     

The anatomy of the median ocellus of Limulus

Summary The median ocellus of Limulus consists of irregular groups of large photoreceptor cells which form a cup-shaped retina around the ocellar lens. Each group is surrounded and penetrated by guanophores and glia. The photoreceptor cells have extensive rhabdomeric regions, both along infoldings of cell membranes and between cells. Five-layered junctions occur between rhabdomeric microvilli. An occasional arhabdomeric (AR) cell is associated with a group of photoreceptors. Fine dendritic branches of the AR cell penetrate the rhabdomeric regions and form five-layered junctions with photoreceptor rhabdomeres. Axons of photoreceptor cells, and of at least some AR cells, gather at the proximal side of the cup to form an optic nerve.Supported in part by NIH EY00312 and EY00377.We would like to thank Dr. W. K. Stell, Dr. A. C. Bell, and Dr. W. H. Fahrenbach for their helpful discussions.     

Effects of alkaline phosphatase inhibitors on chick neural retinal cell differentiation in vitro: ultracytochemical studies

In the matured chick retina, alkaline phosphatase (ALPase) activity is specifically localized in the outer plexiform layer and in horizontal and Müller cells. In the developing chick retina, ALPase activity is first recognized in growing neurites from horizontal cells during the 13th day of incubation, when synaptogenesis begins in the outer plexiform layer. Intraocular administration of ALPase inhibitors to developing chick embryos resulted in developmental disturbances in differentiation of the outer plexiform layer and also of photoreceptor cells. We have now extended these studies to an in vitro system. ALPase activity was studied by ultracytochemistry in cultured retinal cells from chick embryos, and the effects of specific ALPase inhibitor on retinal development were also analyzed. Two cell types showed intense ALPase activity: 1) flat glial cell, which formed a multi-layered epithelial sheet and 2) neuronal cell found within cell aggregates. Some cellular processes forming a neuropil-like structure within these aggregates also showed ALPase activity. When the ALPase inhibitor bromotetramisole was present in the culture medium, there was delay in aggregate formation and the development of neuritic processes was also affected. Moreover, this treatment also caused a considerable reduction in the number of photoreceptor cells present in the culture. The present results indicate that ALPase activity plays a significant role in retinal cell differentiation.     

Ultrastructural localization of glycogen in the granulocytes of normal rabbit bone marrow

Summary The glycogen of rabbit granulocytes has been studied in glutaraldehyde and osmium tetroxide fixed bone marrow by the periodic acid-thiocarbohydrazide-silver proteinate procedure (PA-TCH-SP). The PA-TCH-SP procedure involved the staining of intracytoplasmic glycogen more densely than the routine lead citrate staining. The PA-TCH-SP procedure demonstrated the intracytoplasmic glycogen in all three kinds of granulocytes. Though a sequence of intensity was observed in each stage of cell maturation, intracytoplasmic glycogen increased generally in accordance with cell maturation in the granulocytes. Functional significance of the glycogen in the granulocytes was discussed in relation to its staining. A very weak reaction in the granules of the granulocytes was described in relation to their contents.