Electron microscopic study of the optic tentacle collar cells of the snail Helix aspersa and immunolocalization of methionine enkephalin-like material in their granular content

An ultrastructural and immunocytological study was carried out on the collar cells of the optic tentacle of Helix aspersa. These cells are supposed to be the source of a reproduction controlling hormone. The immunocytological study was performed using an anti-methionine enkephalin antibody obtained from rabbits in our laboratory. The collar cells are characterized by an enlarged rough endoplasmic reticulum, numerous mitochondria and Golgi bodies surrounded by secretory vesicles, suggesting an intense synthesizing activity. Their principal feature consists of numerous various-sized granules where methionine enkephalin immunoreactivity is localized. No classical neurosecretory granules are observed while synapse-like structures are often encountered. The cells should not be regarded as neurosecretory cells but rather as glandular cells which could ensure different functions, one in relation to reproduction, and another in relation to perception processes, particularly as they contain methionine enkephalin-like material.     

The fine structure of the collar cells in the optic tentacles of Helix aspersa

Summary At the base of the optic tentacular ganglion there is a group of large monopolar cells containing numerous secretory inclusions. These are the collar cells. Secretory material can be seen accumulating in swollen portions of the granular endoplasmic reticulum. It is postulated that this material is transported to the Golgi bodies and thus the limiting membrane of the inclusions is derived from the Golgi membranes. The Golgi bodies appear to be polarized and small vesicles resembling secretory inclusions are associated with one face of these organelles. The secretory inclusions fuse together to form large membrane-bound secretory pools in the perikaryon. The collar-cell processes are packed with secretory inclusions. These processes traverse the digital extensions of the tentacular ganglion and pass into the epithelium covering the tip of the tentacle. The secretory inclusions do not resemble neurosecretory inclusions in other situations. The collar cell processes receive a nerve supply from single axons containing granular and agranular vesicles. The evidence that these cells may be modified neurons is only minimal.This work was supported by the Australian Research Grants Committee.     

Effectors of metabolic depression in an estivating pulmonate snail (Helix aspersa): whole animal and in vitro tissue studies

 We have examined metabolic depression in the land snail (Helix aspersa) during estivation, and have developed a tissue model of metabolic depression using an in vitro mantle preparation. The metabolic rate of H. aspersa is depressed by 84% in vivo within 4 weeks of onset of estivation, and this metabolic depression is accompanied by a decrease in haemolymph PO₂ and pH, and an increase in haemolymph PCO₂. The in vitro mantle preparation has a stable O₂ consumption and energy charge, and an energy charge similar to that of mantle in vivo. The in vitro mantle is an O₂-conforming tissue, with VO₂ varying curvilinearly with PO₂. Consequently, we have developed a mathematical method of calculating tissue VO₂ at any PO₂. These calculations show that under appropriate incubation conditions of pH and PO₂, the mantle from estivating animals shows a stable in vitro metabolic depression of 48% compared to mantle from control snails. The extrinsic effects of PO₂ and pH account for 70% of the total in vitro metabolic depression of mantle tissue; intrinsic effectors contribute a further 30%. Accepted April 26, 1996     

Investigations of the Diurnal Activity of the Endocrine Dorsal bodies in Helix aspersa

Summary

The activity of the endocrine dorsal bodies (DB) of the adult land snail Helix aspersa living in the field shows striking fluctuations during a 24 hr cycle. Quantitative electron microscopical data revealed that the number of secretory granules, the volume of the Golgi apparatus and the number of Golgi saccules containing electron-dense material were maximal at 1 am and minimal at 1 pm. The use of tannic acid indicated the exocytosis of secretory material was intense around 1 pm and only moderate at 1 am. The results suggest that, under natural conditions, the DB have a diurnal rhythm of activity, packaging secretory material into secretory granules mainly during the night and releasing it during the afternoon.     

Evolutionary history of the land snail Helix aspersa in the Western Mediterranean: preliminary results inferred from mitochondrial DNA sequences

Intraspecific phylogeographic methods provide a means of examining the history of genetic exchange among populations. As part of a study of the history of Helix aspersa in the Western Mediterranean, we performed a phylogenetic analysis based on partial sequences of the mitochondrial large ribosomal subunit (16S) gene. Our samples include 31 H. a. aspersa populations from North Africa previously investigated for anatomical and biochemical characters. To clarify subspecific relationships, three individuals of the subspecies H. a. maxima were also studied. The molecular phylogeny inferred agrees largely with previous results, in splitting H. a. aspersa haplotypes into an eastern and a western group. H. a. maxima haplotypes form a third lineage arising before the H. a. aspersa groups. Divergence times estimated between the lineages suggest that dispersal during Pleistocene glaciation and vicariance events due to Pliocene geological changes in the western Mediterranean may both have played a significant part in the establishment of the present range of H. aspersa.     

Surface specializations of the epithelial cells at the tip of the optic tentacle,dorsal surface of the head and ventral surface of the foot in Helix aspersa

Summary Morphologically the surface specializations of the epithelium covering the dorsal head and ventral foot regions in Helix aspersa consists either of cilia or microvilli respectively. The epithelium at the tip of the optic tentacle is a simple one. Each epithelial cell has a number of cilia-like projections from their free surfaces. These projections usually branch at their tips into two or three slender, microvilli-like structures. From the bases of the cilia-like projections arise numerous, tubular processes which form a thick, spongy layer interspersed between these projections. The microvilli-like structures are immersed in a fine, fibrous mat; unlike the fibrous mats on the dorsal head and ventral foot epithelia this material does not autofluoresce. It is suggested that it arises from the collar cells and not from typical mucocytes. The functional relationship between these surface specializations of the optic tentacle epithelium and the abundance of sensory axons in this region is discussed. These epithelial cell projections on the tentacle probably function not only as a protective covering but also to create a fluid trap for odours in the ambient air. The various contacts between epithelial cells serve to maintain the integrity of the epithelium while allowing for stretching due to protrusion of the tentacle.This work has been supported by the Australian Research Grants Committee.     

Electron cytochemical study of carbohydrate components in cultured nerve and glial cells of the snail Helix pomatia

• 1.1. A variety of colloidal gold-labelled lectins with different sugar specificities to determine whether different nerve and glial cells of the snail Helix pomatia cultured in vitro, can be distinguished by the carbohydrates that they express was screened. The analysis of lectin binding has shown substantial differences in the carbohydrate pattern between nerve and glial cells and between the soma of monoaminergic and peptidergic neurons.
• 2.2. The surface of monoaminergic and peptidergic neurons contains N-acetylglucosamine and N-acetyllactosamine determinants, and does not exhibit neuraminic acid and complex branched N-glycosyl chains. Moreover, N-acetylgalactosamine can be detected on peptidergic neuron membranes only.
• 3.3. N-Acetylglucosamine residues are not present on the surface of the glial cells, and the density of the N-acetyllactosamine and/or terminal β-galactose residues is much higher here than on the surface of the nerve cells.
• 4.4. These results suggest that nerve cells in the snail brain can be distinguished from glial cells by the presence of a cell-surface glycoconjugate containing terminal N-acetyl-d-glucosamine residues, whereas peptidergic neurons can be distinguished from monoaminergic neurons by the presence of a surface glycoconjugate containing terminal α-linked N-acetyl-d-galactosamine residues.

     

Nerve cells and synaptic connections in the intestinal nerve of the snail,Helix pomatia L.

Summary The ultrastructure of nerve cells and the finestructural organization of synaptic contacts have been investigated in the intestinal nerve in the snail Helix pomatia. Three types of nerve cells, occurring singly or in groups, can be distinguished on the basis of the ultrastructure of their perikaryon and content of granules. The peripheral output of these nerve cells has been verified by retrograde CoCl₂ and NiCl₂ staining. Both axosomatic and axo-axonic specialized synaptic contacts occur in the intestinal nerve. Presynaptic elements of these synaptic contacts contain 100–120 nm granular vesicles or 120–200 nm neurosecretory-like granules. Following intracellular horseradish peroxidase (HRP) labelling of identified central neurons responsible for peripheral regulatory processes, several labelled axons running toward the periphery can be followed throughout the branches of the intestinal nerve. These labelled axon processes (either primary axon or small collaterals) form specialized synaptic contacts, inside the intestinal nerve, and are always in a postsynaptic position. The occurrence of peripheral axo-somatic and axo-axonic synapses provides a morphological basis for integrative processes taking place in the intestinal nerve (peripheral nervous system) of Helix pomatia.     

Electron microscope studies of the intracellular origin and formation of calcifying granules and calcium spherites in the hepatopancreas of the snail,Helix pomatia L.

Summary It has been previously demonstrated that the hepatopancreas of the snail, Helix pomatia, produce proteinaceous particles (termed b-granules), which are involved in the process of calcification. In calcium cells of this organ calcium spherites arise from these granules. The present study retraces the intracellular development of b-granules from Golgi vesicles and from Golgi saccules encircling the cytoplasmic areas up to the formation of calcium spherites. It is suggested that the mature b-granules are well-defined cytoplasmic units with calcifying capacity. The fine structure of b-granules and calcium spherites is described. The possible function of various cell organelles in the formation of the b-granules and spherites is discussed.This investigation was supported by grants from the Swedish Natural Science Research Council. The assistance of Mrs. I. Rehnberg is gratefully acknowledged.     

Ultrastructural study of the bacillary, granular and mucoid proboscidial gland cells of Riseriellus occultus (Nemertini, Heteronemertini)

Junoy, J., Montalvo, S., Roldán, C. and García‐Corrales, P. 2000. Ultrastructural study of the bacillary, granular and mucoid proboscidial gland cells of Riseriellus occultus (Nemertini, Heteronemertini). — Acta Zoologica (Stockholm) 81 : 235–242. The ultrastructure of six types (G₅‐G₁₀) of proboscidial gland cells whose cell necks emerge independently on the epithelium surface is analysed and compared with data from other nemerteans. These types differ in cytological features, as well as in the morphology of their respective secretory granules. Secretory granules of the types G₅ and G₆ have a bacillary shape, and differ from each other based on their contents and dimensions. Secretory granules of the types G₇ and G₈ are spherical to ovoid; type G₈ gland cells are monociliated, and their secretory granules contain a paracrystalline material. Types G₉ and G₁₀ gland cells are typically goblet‐shaped; secretory granules in the type G₉ have a spherical shape, contain a homogeneous electron dense material and maintain their individuality, whereas those of the G₁₀ type are elongate and have fibrillar contents, showing a tendency to fuse before they are extruded. The mucus sheet of the proboscis is responsible for lubrication of its epithelial surface. Secretion products of type G₁₀ gland cells form the background substance of this mucus, and those of the G₅ type confer stickiness to it. Type G₉ gland cells could provide the toxic component to the mucus, and type G₇ and G₈ gland cells could be concerned with the production of enzymatic secretions.     

Electron microscopic study of the regeneration in vitro of rat peritoneal mast cells after histamine secretion

Summary Regeneration of rat mast cells was studied by TEM from 10 s to 48 h after secretion of histamine induced by compound 48/80. During the first 2 h, small intracellular cavities, formed during compound exocytosis and containing non-membrane-bound remnants of the granules, tended to coalesce, and after 2 h of incubation regeneration started. After 6 h, all the cavities had fused into one large central cavity which contained the remnants of the granules and remained open to the exterior during the entire period. The plasma membrane microfolds which disappeared just after secretion were reformed during regeneration. They were apparently involved in endocytotic-like activity and coated vesicles also appeared beneath the plasmalemma (membrane recycling?). The fate of the granule remnants in the cavity is unknown, as regeneration was not completed after 48 h which is the longest survival time obtained so far in ultrastructural studies of mast cell regeneration in vitro.     

Acute toxicity of cadmium and copper in hepatopancreas cells from the Roman snail (Helix pomatia)

The toxic effects of cadmium (Cd) and copper (Cu) on cellular metabolism and cell morphology were investigated in isolated hepatopancreas cells from the Roman snail (Helix pomatia). Cell viability was unaffected during 1 h of incubation with 100 microM Cd, but was significantly reduced from 93% in controls to 87% and 85% with 100 microM Cu and 500 microM Cd, respectively. The adverse effect of 500 microM Cd on cell viability was not observed in cells isolated from Cd pretreated snails. Oxygen consumption remained constant in the presence of 100 microM Cu but was inhibited by 38% after 1 h of exposure to 500 microM Cd. Hepatopancreas cells showed enhanced formation of reactive oxygen species when exposed to 100 microM Cu, but not in the presence of Cd. Morphologically, an increase in cell volume of Cd-exposed cells was noted, while cell membrane bleb formation was induced by both metals. The latter may have been induced by metal effects on the actin filamentous network of the cells which showed distinct actin-staining within the blebs at the cell surface. Overall, our data indicate that both Cd and Cu are acutely toxic for hepatopancreas cells form the Roman snail with Cu being more toxic than Cd.     

Electron microscopic study of the giant cells in the olfactory bulb of labyrinth fish (Belontiidae,Perciformes)

Summary The giant cells in the olfactory bulb of labyrinth fish, most likely belonging to the nervus terminalis, show ultrastructural features of active synthesis of secretory material. Dense core vesicles (70–100 nm in diameter), found in the perikaryon as well as in the axon, are the possible storage sites of the secretory substance. Its chemical nature is unknown. In some of these vesicles acid phosphatase is demonstrable. Large membrane-bound bodies (up to 2000 nm in diameter), either containing an electron dense matrix or debris of cytoplasmic organelles, are also acid phosphatase-positive, suggesting their lysosomal nature. Some other ultrastructural characteristics of these cells are also described.     

Characterization of neural stem cells and their progeny in the adult zebrafish optic tectum

In the adult teleost brain, proliferating cells are observed in a broad area, while these cells have a restricted distribution in adult mammalian brains. In the adult teleost optic tectum, most of the proliferating cells are distributed in the caudal margin of the periventricular gray zone (PGZ). We found that the PGZ is largely divided into 3 regions: 1 mitotic region and 2 post-mitotic regions—the superficial and deep layers. These regions are distinguished by the differential expression of several marker genes: pcna, sox2, msi1, elavl3, gfap, fabp7a, and s100β. Using transgenic zebrafish Tg (gfap:GFP), we found that the deep layer cells specifically express gfap:GFP and have a radial glial morphology. We noted that bromodeoxyuridine (BrdU)-positive cells in the mitotic region did not exhibit glial properties, but maintained neuroepithelial characteristics. Pulse chase experiments with BrdU-positive cells revealed the presence of self-renewing stem cells within the mitotic region. BrdU-positive cells differentiate into glutamatergic or GABAergic neurons and oligodendrocytes in the superficial layer and into radial glial cells in the deep layer. These results demonstrate that the proliferating cells in the PGZ contribute to neuronal and glial lineages to maintain the structure of the optic tectum in adult zebrafish.     

An electron microscopic study of stellate cells and cavities in the pars distalis of frog pituitary

Stellate cells in the pars distalis of adult Rana ridibunda were observed electron microscopically under normal and experimental conditions (TRH injection). The stellate cells have lengthy processes extending into the intercellular spaces between the secretory cells and scanty cytoplasm surrounding the nucleus. Occasional desmosomes link stellate cells to adjacent secretory cells. In the pars distalis of animals injected with thyrotropic-releasing hormone (TRH), the stellate cells form large cavities (2-6 mum) filled with heterogeneous material. Their cytoplasm contains well-developed Golgi complexes and some lysosomes; these are the principal morphological alterations as compared to those observed in control animals. It is suggested that stellate cells could play an active role in addition to providing a structural framework for the pars distalis.     

Freeze-fracture electron microscopic study of tight junction strands in HEK293 cells and MDCK II cells expressing claudin-1 mutants in the second extracellular loop

Claudins constitute tight junction (TJ) strands. In order to examine the function of the second extracellular loop (ECL2), we constructed 1CLΔFY and 1CLΔPL in which highly conserved amino acids, FY or PL, in the ECL2 of mouse claudin-1 were deleted. They were then tagged with either EGFP at the NH₂-terminus (EGFP1CLΔFY and EGFP1CLΔPL) or the myc-epitope at the COOH-terminus (1CLΔFYmyc and 1CLΔPLmyc). The expression of EGFP1CLΔFY and EGFP1CLΔPL in TJ-free HEK293 cells formed TJ strands resembling those formed by wild-type claudin-1. The expression of 1CLΔPLmyc in TJ-bearing MDCK II cells induced aberrant TJ strands in the lateral plasma membranes whose intramembranous particles were almost equally distributed in the P- and E-face. In contrast, 1CLΔFYmyc formed aggregates of short continuous strands which were frequently associated with vesicle-like structures. Coculture experiments with MDCK II cells showed that 1CLΔPLmyc was localized at heterotypic cell–cell junctions but 1CLΔFYmyc was not. These results suggest that changes in the TJ morphology due to the expression of either 1CLΔFYmyc or 1CLΔPLmyc may be caused by some factors specific to epithelial MDCK II cells including endogenous claudins. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Electron microscope study of the myofibril partial disintegration and recovery in the mitotically dividing cardiac muscle cells

Summary The ultrastructure of the myocyte at all phases of mitosis as well as of early postmitotic cells has been studied in the myocardia of 14- and 18-day rat embryos and 5- and 7-day old rats. The myofibrils remain unchanged up to the late prophase. In prometaphase the majority of Z-disks in embryo myocyte myofibrils and considerable part of these disks in myofibrils of suckling rats are drastically disintegrated.This is followed by a progressive isolation and scattering of the myofilament bundles and of the whole sarcomeres during the subsequent phases of mitosis. Thick myofilaments seem to be unchanged but thin ones become frequently poorly outlined (mainly in embryos). The sarcoplasmic reticulum, including its typically differentiated subsarcolemmal cisternae, exhibits relatively few changes during mitosis. In the early postmitotic period there is a gradual restoration of contrast-rich Z-bands, interconnecting the previously isolated sarcomeres. Patterns of this process have much in common with early stages of myofibrillogenesis (appearance of subsarcolemmal Z-bodies, formation of skeins of thin filaments etc.). The cleavage furrow formation is either absent or considerably retarded up to the postmitotic period.Behaviour of some other organelles during myocyte mitosis has been described. Possible mechanisms and significance of the observed phenomena are discussed.The author is greatly indebted to the late Professor L. N. Zhinkin for his interest in this work. The valuable technical collaboration of N. V. Seina as well as the assistance of V. M. Semenov in operating the electron microscope are gratefully acknowledged.