Silver-stained Material Research Articles

Comparative analysis of heteropyknotic chromatin and silver-stained material in Platycarenus umbractulatus (Pentatomidae: Discocephalinae)

The genus Platycarenus is an unusual member of the subfamily Discocephalinae because it does not have a harlequin lobe. To gather information on this genus, a comparative analysis of heteropyknotic chromatin and silver-stained material was carried out in Platycarenus umbractulatus F. (Pentatomidae: Discocephalinae). The testicular lobes were analyzed separately, and meiotic behavior in the seven lobes was found to be similar, i.e., no difference in the meiotic process between the lobes. Major differences were detected during spermiogenesis. Cells of lobes 1, 2, 3, and 7 had similar characteristics, as did lobes 4 and 6, but there were differences between these two groups. The differences between these lobes were in the concentration and location of heteropyknotic chromatin and the amount of silver-stained material. Lobe 5 showed significant differences in comparison with interlobular and intralobular spermatids. Therefore, the results of P. umbractulatus indicate that, although this species does not possess a harlequin lobe, it has differentiated characteristics not yet reported in the literature. Also, a close association of the chromocenter with the nucleolus was demonstrated; it was possible to establish the involvement of sex chromosomes in the formation of the nucleolus. Thus, this study provided insights into the involvement of heteropyknotic chromatin with the nucleolus and also showed the possibility of characterizing the evolution of sperm with a non-fertile function, where more accurate exploration of this theme with other approaches is evident.

Effects of Al3+ on the nucleolus in root tip cells of Allium cepa

The effects of different concentrations of aluminium chloride on the nucleoli in root tip cells of Allium cepa were studied using the silver staining technique. The concentrations used of aluminium chloride (AlCl3) were 1.0 × 10-5, 5.0 × 10 -5, 1.0 × 10 4, 5.0 × 10 4, 1.0 × 10-3, 5.0 × 10-3, 1.0 × 10-2, 5.0 × 10-2, and 1.0 × 10-1 M. After the treatment with Al3+, the nucleolar material was extruded from the nucleus into the cytoplasm. The phenomenon was observed in both the inner root meristem cells and the root cap cells. But there were differences between the two regarding the shape and position of the silver-stained material in the cytoplasm. The present observations with the silver staining technique confirm the phenomenon observed for the first time by FlSKRSJO (1983). The possible mechanism behind this phenomenon is also briefly discussed.

An improved description of Strombidium sulcatum Claparède & Lachmann, 1859 (Ciliophora) from slides of Fauré-Fremiet, and a designation of type material.

Strombidium sulcatum, the type species of the genus, is examined using silver-stained material made by Fauré-Fremiet in the 1950s and 1960s. We found that the stained specimens possessed a prominent protuberance, as previously described; the number of anterior polykinetids (APk) differed between the accounts (15 this study, 12 previous reports); although previously illustrated, no contractile vacuole was observed; and the macronuclear shape, position, and size differed from those previously reported. Our work provides the first detailed re-analysis of the silver-stained material of S. sulcatum. We use data on S. sulcatum to distinguish it from a morphologically similar species, Strombidium inclinatum. Type material for S. sulcatum has been deposited in the Natural History Museum, London.

Effects of Cadmium on Root Growth, Cell Division and Nucleoli in Root Tip Cells of Garlic

The effects of different concentrations (10-7 to 10-2 M) of cadmium chloride on root growth, cell division and nucleoli in root tip cells of Allium sativum L. were investigated. At lower concentrations of Cd2+ (10-7 to 10-6 M), Cd2+ did not influence the root growth, even had a stimulation effects during a short treatment. The results showed that the rate of root growth per day at the treatment groups (10-4 to 10-2 M Cd2+) decreased with increasing duration of the treatment and increasing Cd2+ concentration. Cd2+ induced c-mitosis, anaphase bridges, chromosome stickiness and on nucleoli, causing some particles of similar silver-stained material scattered in the nuclei and making the silver staining reaction at the periphery of the nucleolus weaker.

Heterogeneity of Nigral and Cortical Lewy Bodies Differentiated by Amplified Triple-Labeling for Alpha-Synuclein, Ubiquitin, and Thiazin Red

Alpha-synuclein(α-S) and ubiquitin(Ub) are constituents of the Lewy bodies (LBs), composed of fibrillary structures. To clarify morphological heterogeneity of LBs, we looked for localization of these epitopes in relation to fibrillary structure possibly detectable by a fluorochrome, thiazin red (TR). On the sections of the substantia nigra (SN) and the cingulate gyrus (CG) obtained from Parkinson's disease brains, double amplification by CARD fluorescent immunohistochemistry with anti-α-S monoclonal (LB509) and anti-Ub polyclonal antibodies was performed, followed by staining with TR. These triple-labeled images were captured by a confocal laser microscope and subsequently stained with Campbell–Switzer method, a silver staining specific for LBs. Staining profiles of LBs were different between those in the SN and in the CG. Immunolabeling either with the anti-α-S or anti-Ub antibody was diffuse without halo structure in LBs of CG. In addition to this diffuse staining, a lot of LBs of SN exhibited a halo structure immunopositive for α-S and Ub, probably representing later stages of LB evolution. Irrespective of the presence of this halo structure, the TR signal was always concentrated in the center of LBs, as the silver-stained material was, suggesting that fibrillary components in the central portion of LBs undergo some conformational changes detectable by TR and the silver-staining. This technique reveals different epitopes in relation to LB evolution in vivo. Heterogeneity in staining profile of LBs, as clarified by this method, may represent evolutional changes of LBs, related to conformational states of their constituents.

Effects of Al 3+ on root growth, cell division, and nucleoli in root tip cells of Zea mays L.

The effects of different concentrations (10–5–10–1 M) of aluminum chloride on root growth, cell division, and nucleoli in root tip cells of Zea mays L. were investigated. The results showed that Al inhibited root growth of Zea mays only at 10–2–10–1 M.Al3+ has a marked effect on c-mitosis. Al, at higher concentration, has toxic effects on root tip cells, causing aberration of chromosomal morphology and structure: c-mitosis, anaphase bridges, and chromosome stickiness. Al induces the appearance of some silver-stained particulate material scattered in the nuclei, and extrusion of the nucleolar material from the nucleus into the cytoplasm. Once the nucleolus was affected, and silver-stained nucleolar material was extruded from the nucleus into the cytoplasm, root growth was almost or completely stopped. The possible mechanism of the Al poisoning of root tip cells of Zea mays is also briefly discussed.

EFFECTS OF PB2+ ON ROOT GROWTH, CELL DIVISION, AND NUCLEOLUS OF BRASSICA JUNCEA L.

The effects of different concentrations (10−5-10−2M) of lead nitrate on root growth and nucleoli in root tip cells of Brassica juncea L. were investigated. The results showed that lead nitrate has a stimulatory effect on root growth at lower concentrations, and an inhibitory effect at higher concentrations. Pb has toxic effects on chromosomal morphology, including c-mitosis and anaphase bridges, and on nucleoli, causing some particulate silver-stained material scattered in the nuclei and inducing irregularly shaped nucleoli. Once the nucleolus was affected, the root growth almost or completely stopped.

EVALUATION OF METAL ION TOXICITY ON ROOT TIP CELLS BY THE ALLIUM TEST

The effects of different concentrations of eleven different metals (aluminum chloride, chromium nitrate and potassium dichromate, lead nitrate, copper sulfate, manganous sulfate, cobaltous nitrate, zinc sulfate, magnesium sulfate, nickel sulfate, cadmium chloride, and mercuric chloride) on cell division and nucleoli in root tip cells of Allium cepa were studied. The results showed that the metal ions could, in varying degrees, cause chromosome, nucleus, and nucleolus irregularities, including c-mitosis, chromosome bridges, chromosome stickiness, irregularly shaped nuclei, micronuclei, irregularly shaped nucleoli, some silver-stained material scattered in the nucleus, the weakening of silver-staining reaction at the periphery of the nucleolus, and the release of nucleolar material from the nucleus into the cytoplasm. The Allium test may be useful for the rapid screening of chemicals involved in environmental problems.

Visualization of Ribosomal Gene Activity in Oogenesis of Reticulitermes Iucifugus(Isoptera: Rhinotermitidae) Revealed by Silver Staining.

The Ag-pattern of amplified nucleolar material in various stages of R. lucifugus oogenesis has been followed. At zygotene a nucleolar mass is associated with chromosomal nucleolar organizer regions. At pachytene small brown extra nucleoli are interspersed among the bivalents. At early diplotene the microuncleoli fuse into larger nucleoli. During the late stage of oogenesis large masses of silver stainable material (ribonucleoprotein aggregates) are released in the cytoplasm.

Cytochemical distinction of various nucleolar components in insect cells

The fine structure of the insect Sf9 cell nucleolus has been investigated by means of different cytochemical and immunocytochemical techniques at the electron microscope level. Apart from a few perinucleolar condensed chromatin clumps, the insect cell nucleolus comprises two compartments. The first of these consists of a roundish compact zone formed of fibrillar material. The other is composed of fibrillar and granular structures organized into a network separated by interstitial spaces. But, unlike mammalian cell nucleoli, any fibrillar center has been observed in the Sf9 cell nucleolus, even after actinomycin D treatment. We also show that the compact fibrillar zone of Sf9 cell nucleoli contains silver-stainable material and DNA. In actinomycin D-treated cells, a preferential contact of this compact fibrillar zone with condensed chromatin has been visualized. Finally, silver-stainable material has been found to persist throughout the whole mitosis. These results suggest that the compact fibrillar zone at the insect Sf9 cell nucleolus should, at least partly, correspond to the fibrillar center of mammalian cell nucleoli.

Patterns of activity of nucleolar organizer regions during spermatogenesis and oogenesis in Kalotermes flavicollis Fabr. (Insecta: Isoptera) analyzed by silver staining

The distribution and the behaviour of the nucleolus organizer regions (NORs) were analysed during the spermatogenesis and oogenesis of K. flavicollis with the silver staining method. The Ag-stainability of the NORs increases in growing spermatocytes up to pachytene and is absent during the remainder of the meiotic prophase. During female meiosis the nucleolar material undergoes a more complex transformation. It is active until pachytene; in early diplotene the mass of silver stainable material progressively increases as an effect of rDNA amplification. By the end of meiotic prophase the nucleolar strands disappear and a large nucleolus is rebuilt in the mature oocyte.

A second ascending visual pathway from the optic tectum to the telencephalon in the pigeon (Columba livia).

Previous studies in the pigeon (Karten and Revzin: Brain Res. 2:368-377, '66; Karten and Hodos: J. Comp. Neurol. 140:35-52, '70) have described an ascending tectofugal visual pathway from the optic tectum to the ectostriatum by way of the nucleus rotundus of the thalamus. This present study used anterograde autoradiographic and retrograde horseradish peroxidase pathway-tracing techniques to investigate another ascending tectofugal pathway in the pigeon. Injections of 3H-proline/leucine confirmed a previous report that the optic tectum projects to the nucleus dorsolateralis posterior of the thalamus (DLP). This projection is predominantly ipsilateral and is confined to a large-celled caudal region of the nucleus (DLPc); the rostral region of the nucleus (DLPr) is not tectorecipient. Injections of horseradish peroxidase in DLPc labeled cells predominantly ipsilaterally in layers 8-15 of the optic tectum. Injections of 3H-proline/leucine placed in the DLPc labeled a discrete region of the ipsilateral telencephalon. Similar injections of DLPr labeled a contiguous, but more rostral, region of the neostriatum intermedium. Nissl- and silver-stained material indicated that the region in which DLP terminates is cytoarchitecturally distinct from ventromedial ectostriatal core and belt. Injections of horseradish peroxidase at various locations in the neostriatal DLP terminal field demonstrated a rostrocaudal ordering of the DLP projection upon the neostriatum intermedium. Single-unit recording demonstrated that cells in DLPc respond to whole-field illumination at the same latency as cells in the nucleus rotundus, indicating that the tecto-DLPc-neostriatal pathway transmits visual information to the telencephalon. We suggest that comparable pathways may exist in both reptiles and mammals.

Distribution and immunoreactivity of glia in the retina of the rabbit

Glial markers, namely antibodies to glial fibrillary acidic protein (GFAP), vimentin, galactocerebroside (GC), and 08 antigen, were used to study the occurrence and location of neuroglial cells in adult rabbit retinae. Müllerian glia were vimentin-positive, lacked detectable amounts of GFAP, and were found in all parts of the rabbit retina. The neuronal A-type horizontal cells were labeled by vimentin antibody only in the superior retina and at the medullary rays but not in the inferior retina. They lacked GFAP in all regions. Astroglia showing GFAP and vimentin immunoreactivity were absent from most of the superior and inferior retina, being found only in the myelinated area of the ganglion cell axon bundles, the medullary rays. Thus the rabbit retina differs from the retinae of all mammals studied to date by this restriction of astroglia to just one area. The medullary rays, which are known to be myelinated, were labeled by the antibodies to GC and 08 antigen. Boycott and Hopkins ('84) found, using whole-mounted rabbit retinae stained by the reduced silver method of Richardson, that all neurons in the ganglion cell layer of the rabbit retina have a cilium, while cells that have only a diplosome are either neuroglia or microglia. By using this criterion as a basis to differentiate between neurons and glia, the absence of neuroglia from the nerve fiber layer outside the medullary rays was confirmed in the same silver-stained material. Thus, the data obtained from immunocytochemistry and conventional silver staining agree closely. It has been concluded that, at least in the adult, significant lengths of ganglion cell axons extend without astroglial sheaths.

Electron microscopic demonstration of non-mineralized and hypomineralized areas in dentin and cementum by silver methenamine staining of collagen.

An electron microscopic study on silver methenamine staining of hard dental tissues was made on a material that comprised human permanent teeth and primary tooth germs from human and porcine fetuses. It was demonstrated that silver-stained material consisted of collagen fibrils. In predentin and precementum all collagen fibrils were stained, while collagen fibrils of dentin and cementum were unstained except for some fibrils of minor special areas such as Owen's contour lines, interglobular dentin, Tomes's granular layer, and in cementum small "interglobular-like" areas. It is concluded that silver methenamine visualizes collagen fibrils of hypo- and unmineralized areas in dental hard tissues and therefore may be used to demonstrate abnormal patterns of mineralization. Finally variations of silver methenamine stainability in relation to differences in material and methods were studied and discussed.

Location of neuronal tangles in somatostatin neurones in Alzheimer's disease

Senile dementia of the Alzheimer type is a chronic, progressive neuropsychiatric condition characterized clinically by global intellectual impairment and neuropathologically by the presence of numerous argyrophilic plaques and tangles. Neurochemical investigations have established loss of the cholinergic and aminergic projections to the cerebral cortex and a loss of the content of somatostatin, with preservation of cholecystokinin and vasoactive intestinal polypeptide, neuropeptides also located in cells intrinsic to the cortex. We describe here the relationship between cortical somatostatin immunoreactivity and the plaques and tangles of diseased tissue by immunocytochemical and silver impregnation techniques on paraffin-embedded tissue. In sections of Alzheimer's tissue, cortical somatostatin-immunoreactive perikarya exhibited morphological changes consistent with neuronal degeneration. Silver-stained material immunostained subsequently showed that many neurones containing tangles were also somatostatin positive. No such colocalization was observed using antisera to other neuropeptides. Our findings indicate that a subclass of somatostatin-positive neurones are affected selectively in Alzheimer's disease and that these neurones also contain neuronal tangles. Thus, destruction of somatostatin-containing neurones is an early and perhaps critical event in the disease process.

Behavior of silver stainable material during mitosis inVicia faba

To reveal the behavior of silver stainable material localized mainly in the nucleoli and nucleolar organizing regions (NORs), the somatic cells ofVicia faba were investigated by silver staining throughout the mitotic cell cycle. Nucleoli of interphase and early prophase nuclei were darkly stained. From late prophase to anaphase the secondary constrictions were discriminated as silver stained NORs and many silver grains appeared throughout the cytoplasm. At late prophase the NOR condensed at the same rate as the chromosome arm. Small spherical bodies and two new nucleoli appeared in telophase nuclei and at the same time the cytoplasmic grains disappeared. On the basis of the above observations on the silver stainable material during each mitotic phase, the behavior of silver stainable material is interpreted.

Fiber degeneration associated with hyperphagia-inducing knife cuts in the hypothalamus

The pattern of axonal degeneration associated with hyperphagia-producing hypothalamic knife transections was investigated using the Fink-Heimer method for staining of degenerating axons and their terminal endings. Histological analysis of silver-stained material after parasagittal knife cuts which result in hyperphagia and obesity revealed fiber degeneration coursing longitudinally in the medial forebrain bundle including the perifornical component to reach the nucleus accumbens, the diagonal band, the preoptic-anterior hypothalamic junction, the lateral hypothalamus, the zona incerta, the periventricular thalamus, the parafascicular thalamic nucleus, the substantia nigra pars compacta, the central gray matter, the ventral tegmental area of T'sai and the superior colliculus. The data obtained in the present study lend support to the suggestion that projections coursing in the medial forebrain bundle interconnect the anteriomedial hypothalamus and the midbrain tegmentum and may underlie the hyperphagia and obesity produced by hypothalamic knife cuts.

Localization of the nucleolar organizer by computer-aided analysis of a variant no. 21 in a human isolate.

A variant chromosome no. 21 consisting of two stalks and two satellites in tandem was detected during a survey of a human isolate. The variant segregated in three generations of a large kindred. One male had the variant no. 21, a metacentric Y, and a 47,XXY complement; however, no other evidence of chromosomal nondisjunction was found. Computer-aided analysis of sequentially stained variant no. 21 chromosomes indicated that silver-stained material corresponded to the proximal stalk region (as defined by Giemsa), but often covered both the distal stalk and satellite (also as defined by Giemsa). These data support the hypothesis that human nucleolar organizers are localized to the stalks of acrocentric chromosomes.

Aphagia and adipsia after preferential destruction of nerve cell bodies in hypothalamus.

Microinjections of the excitatory neurotoxin kainic acid into the lateral hypothalamus of rats produced a period aphagia and adipsia. Kainate-treated rats displayed transient motor effects during the first hours after the injection but did not show the persisting sensory-motor and arousal disturbances typically observed in animals with electrolytic lesions in this part of the hypothalamus. Histological examination revealed a significant reduction in the number of nerve cell bodies in the lateral hypothalamus. Silver-stained material indicated no evidence of damage to fiber systems passing through the affected region. Assays of dopamine in hypothalamus, striatum, and telencephalon did not indicate significant differences between experimental and control animals. These results are in agreement with recent reports of the anatomical and biochemical effects of intracerebral kainic acid injections and suggest that the observed effect on feeding behavior is related to the destruction of neurons in the lateral hypothalamus.