Light and scanning electron microscopic study of the tongue in the estuarine dolphin (Sotalia guianensis van Bénéden, 1864)

The importance of the tongue during feeding, and the limited information on the tongue of most aquatic mammals led us to investigate its morphological aspects in sexually immature and mature Sotalia guianensis. Six tongues were measured and photo-documented after their removal from the oral cavity. The samples were divided into rostral, middle, and caudal regions, and examined using light microscopy and scanning electron microscopy (S.E.M.). Sotalia guianensis tongue presented lateral grooves from the apex to the middle portion, while the anterolateral region presented marginal papillae. Histological characteristics revealed the presence of a keratinized stratified epithelium, salivary glands in the middle and caudal portions of the tongue, and filiform papillae in the caudal region. S.E.M. images revealed the presence of filiform papillae and ducts of salivary glands in the middle and caudal portions of the tongue. We can conclude that the characteristics found in this study may reflect an adaptation to changes in diet after weaning.     

Light and electron microscopic immunocytochemistry on the localization of 3β-hydroxysteroid dehydrogenase/isomerase in the bovine adrenal cortical cells

Summary The localization of 3-hydroxysteroid dehydrogenase/isomerase (3-HSD) was studied in bovine adrenal glands by light as well as electron microscopic immunocytochemistry, using anti-bovine adrenal 3-HSD antibody. With light microscopy the cytoplasm of the glomerulosa cells was weakly immunostained, while that of the fasciculata-reticularis cells was intensely immunostained though both the capsular connective tissue cells and the medullary cells were entirely negative for this reaction. Electron microscopic immunocytochemistry revealed that the positive reaction products for 3-HSD were present on the membrane of smooth endoplasmic reticulum of the cortical cells, especially that of the fasciculata and reticularis cells. Other cell organelles such as mitochondria and Golgi apparatus were entirely negative. The present results indicate that 3-HSD is present in the membrane of smooth endoplasmic reticulum of bovine adrenal cortical cells.Supported by grants from the Ministry of Education Science and Culture, Japan     

Light and electron microscopic detection of (3 Gal β1,4 GlcNAc β1) sequences in asparagine-linked oligosaccharides with the Datura stramonium lectin

Summary The Datura stramonium lectin recognizes with high affinity the disaccharide N-acetyllactosamine (Gal 1,4 GlcNAc). We have developed a highly specific cytochemical affinity technique in which an ovomucoid-gold complex serves as second step reagent for the visualization of this lectin bound to reactive sequences present in tissue sections. The lectin binding sites were detected in semithin and ultrathin sections of aldehyde-fixed and low temperature Lowicryl K4M embedded tissues. For light microscopical labeling the photochemical silver reaction for signal amplification was required. The application of this technique for the detection of N-acetyllactosamine containing asparagine-linked oligosaccharides in various intracellular organelles and the plasma membrane is demonstrated.This study was supported by the Swiss National Science Foundation grant nr. 31-26273.89 (to J.R.) and GM 29470 from the National Institutes of Health (to I.J.G.). Dr. G. Egea was a recipient of a European Molecular Biology Organization long term fellowship.     

Light microscopic distinction between elastin,pseudo-elastica (type III collagen?) and interstitial collagen

Summary Distinction between elastin and collagen in arteriosclerotic lesions is difficult because the so-called elastica stains are bound also by collagen fibers which resemble collagen of premature infants. Investigations of effects of organic solvents on dye binding led to the development of methods for selective demonstration of pseudo-elastica, and for simultaneous visualization of elastin and pseudo-elastica in contrasting colors.Paraffin sections of human autopsy material were stained with solutions of resorcin-fuchsin, orcein or aldehyde fuchsin in absolute ethanol. In other series, sections pretreated with this resorcin-fuchsin solution were counter-stained with tannic acid-phosphomolybdic acid (TP)-dye technics.Solutions of these elastica stains in absolute ethanol colored only pseudo-elastica; elastin, e.g. elastic membranes of aorta, remained unstained. In sections counterstained with TP-dye technics elastin was colored red; pseudo-elastica retained the purplish blue coloration imparted by resorcinfuchsin. Other collagens were stained yellow.A review of the literature showed that until the 1920's elastin was classified as a gelatinoid of the collagen group. Elastic fibers were identified by mechanical properties, not a particular chemical composition. Hence, the elastic fibers of classical histology cannot be equated with the elastin of modern chemistry. Correlation of histochemical observations with chemical data indicates that the collagenous pseudo-elastica corresponds to [1(III)]₃ collagen.     

Light and electron microscopical immunocytochemistry of 5′-nucleotidase in rat cerebellum

Summary 5-Nucleotidase in nervous tissue has so far not been localised at the ultrastructural level using immunocytochemical techniques. We have now applied monoclonal antibodies and a polyclonal antiserum raised against this ecto-enzyme and describe the distribution of 5-nucleotidase antigenicity in rat cerebellum both at the light and electron microscopic levels. Within all cerebellar layers, 5-nucleotidase immunoreactivity was found on plasma membranes of glial elements, i.e. Bergmann glial cell processes crossing the molecular layer, astrocytic end-feet around blood vessels and glial cell extensions surrounding single Purkinje cells. In the granular layer, 5-nucleotidase immunoreactivity was present on glial membranes interposed between granule cells. Neuronal cells or processes were devoid of immunoreactivity. The immunocytochemical results were compared with conventional 5-nucleotidase histochemistry. Both techniques showed the same ecto-localisation of the enzyme and favour the view of 5-nucleotidase being predominantly situated at glial plasma membranes.     

M?ssbauer spectroscopy, electron microscopy and electron diffraction studies of the iron cores in various human and animal haemosiderins

M?ssbauer spectroscopy has indicated significant differences in the iron-containing cores of various haemosiderins. In the present study, haemosiderin was isolated from a number of animal species including man. In addition, haemosiderin was isolated from patients with primary idiopathic haemochromatosis or with secondary (transfusional) iron-overload. The iron cores of the animal and normal human haemosiderin appear to be very similar by M?ssbauer spectroscopy, and the electron diffraction data indicate a ferrihydrite structure similar to that of ferritin cores. The haemosiderin isolated from secondary iron-overload shows anomalous behaviour in its temperature-dependent M?ssbauer spectra. This can be understood in terms of the microcrystalline goethite structure of the cores as indicated by electron diffraction. The haemosiderin cores obtained in the case of primary haemochromatosis have an amorphous Fe(III) oxide structure and show M?ssbauer spectra characteristic of a magnetically disordered material, which only orders at very low temperatures.     

Transitions in functional morphology from “large branchiopods” to Cladocera: Video and confocal microscopic studies of Cyclestheria hislopi (Cyclestherida) and Sida crystallina (Cladocera: Ctenopoda)

Great diversity is found in morphology and functionality of arthropod appendages, both along the body axis of individual animals and between different life-cycle stages. Despite many branchiopod crustaceans being well known for displaying a relatively simple arrangement of many serially post-maxillary appendages (trunk limbs), this taxon also shows an often unappreciated large variation in appendage morphology. Diplostracan branchiopods exhibit generally a division of labor into locomotory antennae and feeding/filtratory post-maxillary appendages (trunk limbs). We here study the functionality and morphology of the swimming antennae and feeding appendages in clam shrimps and cladocerans and analyze the findings in an evolutionary context (e.g., possible progenetic origin of Cladocera). We focus on Cyclestheria hislopi (Cyclestherida), sister species to Cladocera and exhibiting many “large” branchiopod characters (e.g., many serially similar appendages), and Sida crystallina (Cladocera, Ctenopoda), which likely exhibits plesiomorphic cladoceran traits (e.g., six pairs of serially similar appendages). We combine (semi-)high-speed recordings of behavior with confocal laser scanning microscopy analyses of musculature to infer functionality and homologies of locomotory and filtratory appendages in the two groups. Our morphological study shows that the musculature in all trunk limbs (irrespective of limb size) of both C. hislopi and S. crystallina comprises overall similar muscle groups in largely corresponding arrangements. Some differences between C. hislopi and S. crystallina, such as fewer trunk limbs and antennal segments in the latter, may reflect a progenetic origin of Cladocera. Other differences seem related to the appearance of a specialized type of swimming and feeding in Cladocera, where the anterior locomotory system (antennae) and the posterior feeding system (trunk limbs) have become fully separated functionally from each other. This separation is likely one explanation for the omnipresence of cladocerans, which have conquered both freshwater and marine free water masses and a number of other habitats.     

An electron microscopic analysis of notochordal and mesenchymal cell abnormalities in embryos of Danforth’s short-tail (Sd) mice

Abnormalities of notochordal cells and of mesenchymal cells in embryos of Danforth's short-tail (Sd) and C57BL mice were examined by means of electron microscopy and cytochemical staining at 11.0 and 11.5 days of gestation. In abnormal (Sd/+; Sd/Sd) embryos, the notochordal cells were markedly deficient in bundles of filaments and lacked surface protrusions, and the notochordal basal lamina was continuous; in contrast, notochordal cells of normal (+/+) littermates and of C57BL embryos contained numerous bundles of filaments and showed fingerlike surface protrusions and discontinuous basal laminae. The pathologic notochordal cells also lacked the accumulations of glycogen revealed in the normals by means of thiocarbohydrazide cytochemical staining at the electron microscopic level. The mesenchymal cells of abnormals also were deficient in filaments but did stain for glycogen, though less prominently than did normal mesenchymal cells.     

Further studies on branch buds in mosses; “Pseudoparaphyllia” and “scaly leaves”

The development of so-called foliose pseudoparaphyllia in the species of theClimacium-type branch development was studied with a paraffin sectioning method and SEM. Scaly leaves (or scale-like leaves) and “foliose” pseudoparaphyllia proved to originate as leaves by segmentation of an apical cell of a branch initial in the very first stage of development into a branch bud. Branch buds of two species among the six species examined develop linear-lanceolate appendages which serve to protect the buds as well as scaly leaves. These appendages originate in the peripheral part of the epidermal layer of buds, and therefore they can be homologous to trichomes previously reported for species with branch primordia. Emendation of two terms are proposed; every organ originating from buds as leaves that are effective for bud protection should be called scaly leaves, while those which homologous to trichomes should be called pseudoparaphyllia. Both terms are used here in a narrower sense than before: There might befilamentous orfoliose scaly leaves, andfilamentous orfoliose (linear-to broad-lanceolate) pseudoparaphyllia. A scheme is given to show the pattern of branch development and the manner in which branch buds (or primordia) are protected.     

The possible structural changes in the adrenal gland cortex after induction of hepatic ischemia–reperfusion injury in male albino rats: Light and electron microscopic study

The adrenal gland is an important endocrine gland in the body that secrets the adrenal hormones. One of the important clinical issues is the hepatic ischemia–reperfusion (IR) injury. Liver IR injury results in many distant organs dysfunctions such as lung, kidney, intestine, pancreas, and myocardium. The aim of the present study was to investigate the possible remote effects of hepatic IR on the structure of the adrenal cortex. Twenty healthy males, Sprague–Dawley albino rats aged 6–8 weeks were randomly divided into two groups (10 rats each): the sham control group (SC-group) and the ischemia–reperfusion group (IR-group). Sera were estimated for the following: aspartate transaminase (AST), alanine transaminase (ALT), lactic dehydrogenase (LDH), and corticosterone levels. Also oxidative markers such as malondialdehyde (MDA) and tumor necrosis factor-α (TNF-α), and the antioxidative enzyme, catalase were measured. Adrenal glands were processed for light and transmission electron microscopic study. The results showed a significant increase in serum liver enzymes (AST, ALT, and LDH), corticosterone, MDA, and TNF-α levels and a significant decrease in serum levels of catalase in IR-group compared with SC-group. Adrenal cortical tissue of IR-group showed the loss of normal appearance. Some cells of zona glomerulosa and most of the zona fasciculata cells appeared swollen and degenerated with highly vacuolated cytoplasm. Other cells were shrunken with deeply acidophilic cytoplasm and pyknotic nuclei. Degenerated mitochondria with disrupted cristae, lipid droplets were confluent and dilated smooth endoplasmic reticulum were seen. Few zona reticularis cells had the dark nucleus and cytoplasmic vacuolations. In the different zones, blood capillaries were markedly congested and some inflammatory cells infiltrations were observed. Liver IR affected the structure of the adrenal cortex.     

Direct insertion and fluorescence studies of rhodamine-labeled β-adrenergic receptors in cell membranes

Summary Previous studies utilizing the fluorescence of propanolol as a probe for the beta-adrenergic receptor showed that this receptor is motionally constrained. To further study the betaadrenergic receptorin situ we have reinserted rhodamine-labeled beta-receptors into cell membranes. This report presents documentation of their insertion and physiologic viability. Beta-receptors were purified by affinity chromatography (10,000-fold), then fluorescently labeled with tetramethyl rhodamine isothiocyanate, repurified (55,000-fold) and incubated with rat pancreatic islet cells. The binding of³H-dihydroalprenolol by these cells was increased from aB _max of 168±2 to 309±20 fmol/mg protein with no change inK _d . Various treatments which remove peripheral membrane proteins, e.g. NaOH, lithium diiodosalicylate, and trypsinization, did not alter binding by the cells with inserted receptors indicating that the receptors inserted into cell membranes. In islet cells treated with Koshland's reagent I, beta-adrenergic binding was completely abolished, but following incubation with isolated beta-receptors, the cells bound beta-adrenergic radioligand with aB _max of 100 fmol/mg protein, indicating functionality on the part of the inserted receptors. Furthermore, insertion of isolated receptors into frog erythrocytes resulted in increased production of cAMP in response to added isoproterenol. In pancreatic islet cells, incubation with labeled receptors caused the fluorescence to shift in wavelength with increased intensity indicating a shift from an aqueous to a lipid environment, probably into the membrane. Using fluorescence (P), it was found that the inserted receptors became motionally constrained to aP of 0.38 (limitingP _o=0.42) during the first 15 min, remaining so for at least 2 hr. Colchicine (5 g/ml) caused a decrease inP to 0.18 indicating release of constraint. Isoproterenol (10^–5M) caused a rapid decrease toP=0.15. This effect was blocked by propranolol. Propranolol itself (10^–5M) had no effect. These results indicate that the labeled receptors rapidly insert into cell membranes and also support the view that agonist activation of the receptor causes an increase in receptor mobility, presumably due to release of constraint from cytoskeleton elements.     

Effects of starvation on haemolymphatic glucose levels, glycogen contents and nucleotidase activities in different tissues of Helix aspersa (Müller, 1774) (Mollusca, Gastropoda)

In the present study, the glucose concentration in the haemolymph and glycogen levels were determined in the various body parts of the Helix aspersa snail after feeding lettuce ad libitum and after various periods of starvation. To characterize the effect of starvation on nucleotidase activity, enzyme assays were performed on membranes of the nervous ganglia and digestive gland. Results demonstrated the maintenance of the haemolymph glucose concentration for up to 30 days of starvation, probably due to the consumption of glycogen from the mantle. In the nervous ganglia, depletion of glycogen occurs progressively during the different periods of starvation. No significant changes were observed on ATP and ADP hydrolysis in the membranes of nervous ganglia and no alterations in Ca2+ -ATPase and Mg2+ -ATPase occurred in the membranes of the digestive gland of H. aspersa during the different periods of starvation. Although there were no changes in the enzyme activities during starvation, they could be modulated by effectors in situ with concomitant changes in products/reactants during starvation.     

Linkage studies on α-glycerophosphate and isocitrate dehydrogenases in Aedes albopictus (diptera: Culicidae)

Linkage studies were carried out on -glycerophosphate dehydrogenase (-GPDH) and isocitrate dehydrogenase (IDH) in the mosquito Aedes (Stegomyia) albopictus. Only one locus coding for -GPDH was revealed on agar gels by applying adult homogenates. Two loci for IDH were observed using either fourth-instar larvae, pupae, or adults. This study was restricted to the more anodal Idh-2 of the two loci, and -Gpdh. Both -Gpdh and Idh-2 encode dimeric enzymes. Thirteen backcrosses indicated that the -Gpdh and Idh-2 loci are arranged in linkage group 2 in the following order: p (pigmented pupa)—(ca. 2 map units)—Wb (white-body)—(7.5–17.8)—Idh-2—(13.1)—-Gpdh. Females exhibited more recombination than males.This work was supported by a Grant-in-Aid for Co-operative Research from the Ministry of Education, Japan.     

Alpha-adrenergic receptor (α2A ) is colocalized in basal forebrain cholinergic neurons: A light and electron microscopic double immunolabeling study

A variety of data suggest that noradrenaline and acetylcholine may interact in the basal forebrain, however no morphological studies have addressed whether indeed cholinergic neurons express adrenergic receptors. We have investigated the presence of alpha-adrenergic receptor subtype α_2A -AR in cholinergic neurons of the basal forebrain. Cholinergic neurons were identified with an antibody against choline acetyltransferase and the receptor with a polyclonal antibody raised against a 47 amino acid fragment of the third intracellular loop of the α_2A -AR. For double labeling at the light microscopic level the Ni-DAB/DAB technique was used, and for electron microscopy an immunoperoxidase/immunogold method was applied. We detected the α_2A -AR protein in cholinergic as well as in non-cholinergic neurons. Almost half of all cholinergic neurons contained this adrenergic receptor. Double-labeled neurons were distributed throughout the rostro-caudal extent of the basal forebrain cholinergic continuum, including the medial septum, vertical and horizontal diagonal band nuclei, pallidal regions, substantia innominata and the internal capsule. Non-cholinergic neurons that expressed the α_2A -AR outnumbered cholinergic/α_2A -AR neurons by several factors. Electron microscopy confirmed the presence of α_2A -AR in cholinergic neurons in the medial septum, vertical and horizontal diagonal band nuclei. Gold particles (10 nm) indicative of α_2A -AR were diffusely distributed in the cytoplasm and accumulated in cytoplasmic areas near the Golgi complex and cysterns of the endoplasmic reticulum and were associated with the cellular membranes at synaptic and non-synaptic locations. Since many of the α_2A -AR+/non-cholinergic neurons we detected are likely to be GABAergic cells, our data support the hypothesis that noradrenaline may act via basal forebrain cholinergic and non-cholinergic neurons to influence cortical activity.