Density Of Silver Grains Research Articles

Loading and transport of assimilates in different maize leaf bundles

The loading and transport functions of vascular bundles in maize (Zea mays L.) leaf strips were investigated by microautoradiography after application of (14)CO2. The concentrations of (14)C-contents in thin-walled sieve tubes of individual bundles in the loading and transport regions were determined by digital image analysis of silver-grain density over the sieve tubes and compared. In the loading region, relatively high concentrations of (14)C-contents were found in the thin-walled sieve tubes of small bundles and in the small, thin-walled sieve tubes of the intermediate bundles; the concentration of (14)C-label in large bundles was very low. In the transport region, at a transport distance of 2 cm, all of the small bundles contained (14)C-assimilates, but generally less than the same bundles did in the loading region; by comparison, at that distance intermediate and large bundles contained two-to threefold more (14)C-assimilates than the same bundles in the loading region. The lateral transfer of assimilates from smaller to larger bundles via transverse veins could be demonstrated directly in microautoradiographs. A reverse transport from larger to smaller bundles was not found. At a transport distance of 4 cm, all large and intermediate bundles were (14)C-labeled, but many of the small bundles were not. Although all longitudinal bundles were able to transport (14)C-asimilates longitudinally down the blade, it was the large bundles that were primarily involved with longitudinal transport and the small bundles that were primarily involved with loading.

The effect of CuIUD on estradiol receptor in the endometrium of rat--an autoradiographic study.

Autoradiography (ARG) and scanning microspectrophotometry were applied to study the changes of eosinophilic type II estradiol-binding sites in the endometrium of Sprague-Dawley rats with regular 4 -day estrous cycle, after 30 days of bearing a copper intrauterine device (CuIUD; 40.8mm2) in uterus.3H™estradiol™l7β (3H™E2) labelled eosinophils were mainly distributed in the deep endometrial stroma and varied in quantity with the estrous cycle. In comparison with the contralateral uterine horn, the number of3H-E2 labelled cells was significantly reduced in the CuIUD-bearing horn at estrus and diestrus The mean density of silver grains in the ARG positive eosinophils in the endometria exposed to CuIUD was decreased at diestrus, as compared with that in contralateral horns, and at proesttus with that in contralateral and control-group horns. The results suggested that the decrease of type II estradiol-binding sites induced by CuIUD may be closely related to the contraceptive action of copper.

Complementary distribution of carbamoylphosphate synthetase (ammonia) and glutamine synthetase in rat liver acinus is regulated at a pretranslational level.

We studied the distribution of the mRNAs for carbamoylphosphate synthetase (ammonia) and glutamine synthetase in frozen sections of adult rat liver by in situ hybridization to [35S]-labeled cDNA probes. The density of silver grains resulting from hybridization to the labeled cDNA probe for carbamoylphosphate synthetase is highest around the portal venules, decreases towards the central venule, and is virtually absent from an area two to three cells wide that lines the central venules in which mRNA for glutamine synthetase is predominantly localized. Therefore, both mRNAs show the same complementary distribution within the liver acinus that was found for the proteins they encode, demonstrating that compartmentalization of the expression of these enzymes is controlled at a pretranslational level. In addition, we found that carbamoylphosphate synthetase mRNA is present mainly in the epithelium of the crypts of the proximal part of the small intestine, whereas carbamoylphosphate synthetase protein is present in the epithelium of both crypts and villi.

Cell-surface enrichment of fucosylated glycoconjugates in the 8- to 16-cell mouse embryo : An autoradiographic study.

The destination of 3H-fucose was examined in preimplantation mouse embryos at the 8- to 16-cell stage by autoradiography of semi-thin sections. At the end of a 16-18 h incubation in medium containing 200 μCi/ml L-[6-3H] fucose the density of silver grains over the combined surface of cells where they faced their neighbours was 40% greater than that over the outer polar cell surface and 80% greater than that over the cytoplasm. These differences were statistically significant (p<0.01). The outer cell surface also had a grain density above that found over the cytoplasm (p<0.01). There was a rapid decrease of 18-24% in the incorporated radioactivity during the first 1.5 to 3 h in 'chase' medium containing 10 mM 'cold' fucose. Following this the grain density remained relatively stable until it decreased further between 24 and 48 h. By 48 h there was no significant difference between the density of grains over cytoplasm, apposed cell surfaces and outer cell surface: all were reduced compared to the initial values, to 29% in the case of the surfaces and 10% in the case of the cytoplasm. These results indicate that there is an enrichment in the incorporation of 3H-fucose into macromolecules at the cell surfaces where neighbouring cells confront one another relative to the outer polar cell surface, as well as the cytoplasm, at the 8- to 16-cell stage. Conclusions about the relative density of fucoconjugates per unit length of apposed and polar membrane cannot be reached from such a light microscopic study. Fucoconjugates appear to belong to two groups: the first are turned over relatively rapidly (within 3 h after removal from radioactive medium) while the second are more stable, decaying between 24 and 48 h after removal from 3H-fucose.

Calcitonin binding site distribution in the cat central nervous system: A wider insight of the peptide involvement in brain functions

Calcitonin (CT) binding site distribution has been studied in the cat CNS. The autoradiographic analyses of | 125I|-eelCT (ECT) binding showed high density of silver grains in the mesencephalic PAG, in the raphe nuclei and in the dorsal horns, laminae I, IV, V, and VI, where ECT may act to inhibit nociceptive transmission. Other binding-rich areas included the caudatus, the amygdala, the hypothalamus, the substantia nigra, the locus coeruleus and the formatio reticularis mesencephalica. Medium to low density was seen, amongst other areas in the cortex piriformis, the hippocampus, the medial and intralaminar thalamus and the tractus spino-thalamicus. ECT binding site distribution revealed essentially homologous locations in the cat and rat CNS. At difference, the presence of binding in the piriform cortex and in discrete thalamic nuclei suggests a widespread involvement of ECT in a variety of central functions in addition to what already demonstrated.

The role of secretory granules in the transport of basement membrane components: radioautographic studies of rat parietal yolk sac employing 3H-proline as a precursor of type IV collagen.

Biosynthesis of type IV collagen in the parietal endodermal cells of 12 day gestant Sherman rats was examined following intraconceptal injection of 3H-proline. The concepti were removed at times varying from 2 minutes to 24 hours after the injection. The parietal wall of the yolk sac, including endodermal cells and the associated basement membrane known as Reichert's membrane were processed for electron microscopic radioautography. Silver grains were counted over the organelles of endodermal cells as well as over Reichert's membrane. Radioactivity was high in endodermal cells during the first 2 hr after 3H-proline injection and later dropped to some extent, while radioactivity rose in Reichert's membrane. Examination of endodermal cell organelles showed some early labeling over rER and Golgi apparatus without a clear-cut trend, except for a drop in Golgi label at late times after 3H-proline injection. The density of silver grains over secretory granules rose significantly by 40 min, reached a high peak by 4 hr and then declined at the time when radioactivity increased over Reichert's membrane. Furthermore, the radioactively-labeled secretory granules were localized mainly at the trans Golgi face soon after injection and near the cell surface adjacent to Reichert's membrane at later times. Biochemical reports indicate that a substantial amount of the proline taken up by the 12-14.5 day rat embryo endodermal cells is incorporated into type IV collagen. Since there is high labeling of the secretory granules from 40 min to 4 hr and the labeled granules are associated with the Golgi apparatus at early times, it is proposed that collagen precursors are processed through rER and Golgi apparatus, packaged into secretory granules and then transported to the cell surface where type IV collagen or its precursors are released and subsequently deposited into Reichert's membrane.

Cytological approach to the nucleolar functions detected by silver staining

The pattern of nucleolar silver staining was determined in the interphase and mitosis of plant cells using two cytological techniques, the selectivity of which was tested densitometrically. For complete accuracy, silver grains were removed and the sections were then observed under uranyl and lead staining. During interphase, the highest density of silver grains occurred in the portions of the dense fibrillar component nearest to the fibrillar centres; the latter were also stained except for the condensed chromatin inclusions typical of the heterogeneous type. During mitosis, not only the nucleolar organiser region (NOR) was stained, but also the prenucleolar material in anaphase and telophase. Two different proteins or sets of proteins are suggested to be responsible for this staining pattern; one of them is associated with NOR chromatin (which is not contained in the mitotic prenucleolar material) and the other with early ribosomal precursors (which have never been found in fibrillar centres). From these two proteins, the “Ag-NOR” reveals either transcriptionally active or potentially active chromatin, characterized by an uncoiled, extended structure, regardless of the state of transcription. This is the structural feature detected by Ag-NOR staining.

Penetration of various molecular-weight proteins into the enamel organ and enamel of the rat incisor

During enamel maturation, most of the organic matrix is removed as the mineral content increases; it is postulated that proteolytic enzymes within enamel break down large proteins into more mobile fragments. To predict how such fragments might leave the enamel, the entry and penetration of various proteins into it was examined. Rats (100 g) were injected via the external jugular vein with 125I-iodinated calcitonin (3600), insulin (5700), epidermal growth factor (EGF; 6100) and albumin (68,000). They were killed after 10 min and radioautographs made to visualize these molecules in the incisor enamel organ and enamel. In addition, dissected incisors were wiped free of their enamel organs, dipped in the iodinated protein solutions for 10 min, and processed for radioautography. In all dipped teeth, except those exposed to albumin, there was a gradient of silver-grain density over the entire thickness of enamel in both the secretion and maturation zones. In all injected animals, enamel labelling in the secretion zone was only slightly above background. In the maturation zone of animals injected with calcitonin and insulin, many grains were over enamel adjacent to smooth-ended ameloblasts but not ruffle-ended ones. Animals injected with EGF and albumin had no labelled enamel in the maturation zone. Thus dipped rat incisor enamel was permeable to proteins with molecular weights as high as 6100. Localization of injected proteins indicates that the enamel organ restricts their passage into enamel, but proteins with molecular weights as high as 5700 may pass into enamel through or between smooth-ended ameloblasts. As exogenous proteins readily diffused into the enamel, it seems likely that enamel proteins of similar size can leave enamel by a similar route.

Adrenergic beta receptors are not uniformly distributed in the cerebellar cortex.

The noradrenergic (NE) innervation of the cerebellar cortex is sparse, forming a broad plexus of radially oriented axons distributing throughout the granular and molecular layers. Autoradiographic studies of beta-adrenergic receptor distribution in the rat show the greatest density of silver grains in the molecular layer (Palacios and Kuhar, '82). In the course of studies of NE hyperinnervated structures, we found that beta receptors are nonhomogeneously distributed in the Purkinje cell layer, where they occur in "patches" overlying small groups of Purkinje cell somata. Tissue sections were incubated in 10 pM 125iodocyanopindolol (ICYP), which binds equally to beta1 and beta2 adrenergic receptors. Nonspecific binding was determined in sections incubated in 125ICYP and 1 microM dl-propranolol. Beta-adrenergic receptor patches are of irregular size and are most prominent in the vermis of lobules I-IX, although the medial cerebellar hemispheres also show areas of increased silver grains over Purkinje cells. In order to determine the subtype of beta receptors, adjacent sections were incubated with either 125ICYP and the beta 2-selective antagonist IPS-339, or 125ICYP and the beta 1-selective antagonist practolol. Patches were observed after each incubation procedure, indicating that they are composed of both beta1 and beta2 receptors. Patches are observed in normal animals and also in rats in which cerebellar NE content was increased 165% by neonatal treatment with 6-hydroxydopamine. This treatment does not alter the density of beta receptors. The cerebellar elements in which the beta receptors are located is not known. While silver grains accumulate over small groups of Purkinje cell somata, they are not coextensive with these cell bodies. The distribution of beta-adrenergic receptors does not parallel the arrangement of noradrenergic varicosities in the rat cerebellar cortex.

Characterization of [3H]5-hydroxytryptamine uptake within rat cerebrovascular tree

The in vitro uptake of tritiated serotonin ([3H]5HT) was studied in a preparation of rat extracerebral arteries. The uptake of [3H]5HT was time- and temperature-dependent and of high affinity; linear regression analysis gave a Km value of 6.48 × 10−7 M for the specific uptake. Bilateral superior cervical ganglionectomy was without effect on [3H]5HT uptake while it significantly reduced the uptake of tritiated norepinephrine by the preparation of rat extracerebral arteries. The serotonergic neurotoxin 5,7-dihydroxytryptamine and lesions to both the medial and the dorsal raphe nuclei caused a marked loss of [3H]5HT uptake but did not change the uptake of tritiated norepinephrine. Competition studies with norepinephrine, desimipramine (a noradrenergic uptake blocker), nomifensine (a dopaminergic uptake blocker) and fluoxetine (a 5HT uptake blocker) confirmed the specificity of the [3H]5HT uptake mechanism. Histoautoradiographic studies showed the highest density of silver grains at the level of the adventitial-medial border of the basilar artery. Fluoxetine inhibited the accumulation of silver grains within the adventitial-medial border in the blood vessel studied. The present data further support the view that a neuronal serotonergic system may play a role in the control of blood flow in the cerebrovascular tree.

Autoradiographic localization of calcium channel antagonist receptors in rat brain with [ 3H]nitrendipine

In vitro autoradiographic techniques have been used to localize [ 3H]nitrendipine binding sites in the rat brain. The superficial cerebral cortex, the ventral, lateral and posterior nuclei of the thalamus, the molecular layer of the dentate gyrus, the substantia nigra and the external plexiform layer of the olfactory bulb, all contain high densities of silver grains. The level of binding sites are greatly reduced in areas low in synaptic connections. The corpus callosum, the alveus hippocampi and the dorsal commissure of the fornix all lack specific grains, as does the lateral olfactory tract of theolfactory bulb. Specific silver grains are not found in the habenula or the hypothalamus. Grains are not associated with blood vessels profiles. The discrete localizations of [ 3H]nitrendipine bindings sites suggest a specific synaptic role.

Autoradiographic localization of benzodiazepine receptors in the rat kidney.

The localization of benzodiazepine (BZD) receptors in the rat kidney was studied by autoradiography after in vitro labeling of kidney slices with flunitrazepam. The affinity, density, and rank order of displacement of [3H]-flunitrazepam by several BZDs (RO 5-4864 greater than diazepam greater than clonazepam) demonstrated that binding was to BZD receptors of the peripheral type. In autoradiograms obtained with tritium-sensitive film, a high density of silver grains was obtained in the outer medulla, with lower densities in the cortex. Binding was absent from the inner medulla (papilla). In higher resolution autoradiograms obtained with an emulsion-coated cover slip procedure, silver grains were seen to be concentrated over a tubular element in both outer medulla and cortex, identifiable by morphology and distribution as the thick ascending limb of the loop of Henle and the distal convoluted tubule. The identity of the labeled tubules was confirmed by immunofluorescent localization in adjacent slices of Tamm-Horsfall protein, a specific marker for these segments of tubules. Investigation of the effects of peripherally specific BZDs such as RO 5-4864 on distal tubule function is indicated.

Study of the mechanism of release of [3H]GABA from a teleost retina in vitro.

gamma-Aminobutyric acid (GABA) is thought to be a neurotransmitter in the vetebrate retina. We studied the voltage and Ca2+ dependency of the process of release of [3H]GABA from the retina of the teleost Eugerres plumieri, using a microsuperfusion technique. Two depolarizing agents, veratridine and high potassium, produced a concentration-dependent release of [3H]GABA. The veratridine effect was inhibited in Na+-free solution, but was not affected by 1 microM tetrodotoxin. A substantial inhibition (about 75%) of the veratridine- and potassium-stimulated release of [3H]GABA occurred in Ca2+-free medium. Inhibitors of the Ca2+ channel, such as Mg2+ (20 mM), La3+ (0.1 mM), and methoxy-verapamil (4 microM-0.4 mM), inhibited the veratridine- and K+-stimulated release. However, Co2+ and Cd2+ caused a potentiation and no change of the K+- and veratridine-stimulated release, respectively. This release process is apparently specific, since both depolarizing agents were unable to release [3H]methionine, a nontransmitter amino acid, under the same experimental conditions. Autoradiographic studies with [3H]GABA, using the same incubation conditions as for the release experiments, showed a high density of silver grains over the horizontal cells with almost no accumulation by amacrine cells and Müller cells. beta-Alanine and nipecotic acid were used as two relative specific inhibitors of the glial and neuronal GABA uptake mechanisms, respectively. Only a small heteroexchange with [3H]GABA was found with beta-alanine, and no inhibition of the subsequent veratridine-stimulated release. On the other hand, nipecotic acid produced a strong heteroexchange with [3H]GABA and lacked the capacity to induce the veratridine-stimulated release of [3H]GABA. These results suggest a voltage- and Ca2+-dependent neuronal release of [3H]GABA from retina.

Differential distribution of diagonal band afferents to subnuclei of the interpeduncular nucleus in rats

Previous research has demonstrated a subnuclear organization within the interpeduncular nucleus (IPN) based upon cytoarchitecture, synaptology, and the distribution of biogenic amines and peptides. To determine whether individual subnuclei of IPN can be further differentiated with regard to their afferents, we investigated the distribution of inputs from the nucleus of the diagonal band. Autoradiographic analyses demonstrated a diagonal band projection to IPN which is not homogeneously distributed among individual subnuclei. The greatest density of silver grains was located over the dorsal subnucleus, followed in order of diminishing density by the rostral, central, intermediate and lateral subnuclei. These findings confirm the existence of a projection from the nucleus of the diagonal band to the IPN, and support the concept that individual subnuclei within the IPN may be further differentiated on the basis of their afferent input.

Radioautographic visualization and biochemical identification of O-phosphoserine- and O-phosphothreonine-containing phosphoproteins in mineralizing embryonic chick bone.

We injected NaH2(33)PO4 into normal 14-d-old embryonic chicks and examined the long bones by both radioautography and biochemical analyses from 10 to 240 min after the injection was completed. At 30 min, determination of the radiographic grain density revealed that 33P was concentrated principally in fibroblasts, preosteoblasts, and osteoblasts. With time, there was a progressive increase in the density of silver grains located over both the osteogenic cells and the regions of uncalcified (osteoid) and calcified extracellular organic matrices. Biochemical analyses identified 33P-O-phosphoserine as the major 33P component in glutaraldehyde-treated whole demineralized bone tissue and in EDTA-soluble, nondiffusible proteins extracted from the bones, both at the same time periods that 33P-induced silver grains were visualized by radioautography. 33P-O-phosphothreonine was also identified in experiments using a dosage of 10 mCi per embryo. The results provide the first combined direct biochemical and radioautographic identification that phosphoproteins are synthesized in bone and are located morphologically at the sites of mineralization. The data provide further evidence that phosphoproteins play a critical role in the biological calcification of vertebrate tissues.

Retrograde labeling, enrichment, and characterization of retinal ganglion cells from the neonatal rat

We have developed a method for labeling retinal ganglion cells in neonatal rats by retrograde transport of the fluorescent dye, True Blue (TB), injected into the optic chiasm. Following proteolytic dissociation of labeled retinas into single cells, the labeled cells could be enriched 50- to 100-fold by centrifugation in a 5%/10% metrizamide gradient. When plated in Ham's F-10 medium in the presence of fetal calf serum and chick optic tectum-conditioned medium, the labeled cells could be maintained in vitro up to 48 hr. In these cultures, the ganglion cells (GCS) constituted 50 to 70% of the total cell population. When GC-rich fractions or GC cultures were stained with a monoclonal antibody to Thy-1 antigen, greater than 90% of the TB-labeled cells were reactive. In order to localize voltage-sensitive sodium channels, GC-rich cultures were reacted with 125I-scorpion toxin. Analysis of the autoradiograms showed that the density of silver grains was about 10-fold higher on TB-labeled cells than on nonfluorescent cells, or in controls which contained excess of unlabeled toxin. When GC cultures were incubated with micromolar concentrations of putative GC transmitters, aspartate and glutamate, the amino acids were accumulated by 15 to 20% of labeled cells. Several lectin receptors were also localized on TB-labeled cells in situ. Whereas the lectins wheat germ agglutinin, concanavalin A, peanut agglutinin, Dolichos biflorus agglutinin, and Limulus polyphemus agglutinin bound to TB-labeled cells, others such as Ricinus communis agglutinin I, Ulex, and Lotus lectins showed no binding. The lectin binding was specific since preincubation with the appropriate hapten sugar blocked lectin binding.

Light- and cytidine-dependent phosphatidylinositol synthesis in photoreceptor cells of the rat.

Incorporation of [3H]inositol into phosphatidylinositol (Pl) in isolated rat retinas is enhanced by light and by the addition of cytidine to the incubation media. In retinas preincubated with [3H]inositol in dark, [3H]inositol was chased into Pl in light by addition of unlabeled cytidine and was chased out of Pl in light by addition of unlabeled cytidine plus inositol. Autoradiograms of retinas show a heavy density of silver grains over photoreceptor cell inner segments (with chase-in) and a loss of labeling (with chase-out). Exogenous cytidine and inositol were shown to enhance not only the turnover of Pl within photoreceptor cells but the synthesis of Pl as well; in media supplemented with these precursors, approximately 50% of [14C]glycerol and 25% of [32Pi]incorporated into lipid in light were associated with Pl. These results suggest that availability of both cytidine and inositol may play a role in the light-dependent changes in Pl metabolism within photoreceptor cells.

Autoradiographical studies on the localization of metallothionein in proximal tubular cells of the rat kidney.

The renal distribution of 125I-labelled metallothionein (125I-MT), administered as a tracer dose to female Wistar rats, has been investigated by light and electron microscopical autoradiography. 125I-MT was absorbed rapidly and 80% of the administered dose was observed in the kidney within 60 min after dosing. At 30 min, a high density of silver grains due to 125I, was observed by light microscopy in the convoluted proximal tubules. Electron microscopical autoradiography showed that silver grains were located in large apical vacuoles and cytoplasmic bodies (heterolysosomes) of the endocytotic system in the apical or middle regions of the cells lining convoluted proximal tubules. The results suggest that endocytotic systems are associated with 125I-MT absorption by epithelial cells lining the renal convoluted proximal tubules. The apparent inconsistency on the distribution pattern of silver grains due to 109Cd-labelled MT and 125I-labelled MT in these is also discussed.

The noradrenergic system in cultured aggregates of fetal rat brain cells: Morphology of the aggregates and pharmacological indices of noradrenergic neurons

Spherical aggregates formed rapidly in culture by re-aggregation of trypsin-dissociated brain cells from the 17-day-old fetal rat. Over about 10 days in initially random distribution of cells evolved into a 3-layered arrangement; cells with characteristics of neurons were found largely in the intermediate layer. The survival of neuronal and glial cell types was evaluated histologically and verified by electron microscopy, which revealed synaptic and myelin structures that rapidly increased in number after 18 days in culture. Levels of norepinephrine (NE) and dopamine (DA) reached peaks of 9.5 and 4.4 ng/mg protein, respectively, at culture day 21. Uptake of [3H]NE paralleled these amine levels and was blocked by desipramine or pretreatment with either reserpine or 6-OH-DA. Autoradiographs of aggregates labeled with [3H]NE showed a high density of silver grains over cells, apparently neurons, with branching processes traced for 120 micrometer. Previously accumulated [3H]NE was released under depolarizing conditions (high [K+] or vertridine) only in the presence of Ca2+. Release was induced to a lesser extent by kainic greater than glutamic acid. Thus, such aggregates appear to contain catecholaminergic neurons capable of synthesis, uptake and release of NE. The time course of development of these functions supports suggestions that aggregate preparations might be useful in studying neurochemical or morphological aspects of brain development and function in vitro.

Timing and localization of the stimulatory effect of follicle-stimulating hormone on uridine incorporation in the mouse testis in vivo.

Human pituitary FSH increased the incorporation of [5-(3)H]uridine into RNA in vivo in the testes of intact 9-day-old mice and of hypophysectomized adults. In both groups the effect was greatest 8 h after administration of 5--7.5 i.u. FSH. Autoradiographs were prepared from the testes of hypophysectomized adult mice given subcutaneous injections of FSH or of 0.9% saline 6 h, and [5-(3)H]uridine 1.5 h, before death. Treatment with FSH caused statistically significant increases in the density of silver grains over the nuclei of Sertoli cells, type A and intermediate spermatogonia, and preleptotene and mid-pachytene primary spermatocytes. It was concluded that FSH has a generalized stimulatory action on RNA synthesis in the nuclei of Sertoli cells and those types of germinal cell which synthesize RNA most actively.