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PubMed1988

Demonstration by staining and radioautography of cyclical distributions of protein at the enamel surface in rat incisors.

Staining patterns in the enamel during the maturation stage of amelogenesis reflect the banded distribution of ruffle-ended and smooth-ended ameloblasts. This study investigated the possibility that proteins at the enamel surface may be distributed cyclically according to cyclical changes in ameloblast morphology. Dissected lower rat incisors were wiped free of their enamel organs and immediately immersed in fixative containing one of the following heavy metal and histological stains: uranyl acetate, lead citrate, Coomassie blue, alcian blue and ruthenium red. Other animals were injected with [35S]methionine to label newly-formed enamel proteins. Their incisors were dissected, the enamel organs were wiped from the enamel surface, and the teeth were processed as whole mounts for radioautography. Teeth stained by heavy metals were also viewed by back-scattered electron imaging. The in-situ staining revealed that proteins were distributed in bands and stripes across maturing enamel. Radioautography revealed that the proteins in the stripes were newly-synthesized and secreted into the enamel by certain maturation ameloblasts. We conclude that the enamel organ expresses cyclical activity in part through secretion of proteins.

Ameloblasts↗

PubMed1976

Multipotentiality of Schwann cells in cross-anastomosed and grafted myelinated and unmyelinated nerves: quantitative microscopy and radioautography.

Cross-anastomoses and autogenous grafts of unmyelinated and myelinated nerves were examined by electron microscopy and radioautography to determine if Schwann cells are multipotential with regard to their capacity to produce myelin or to assume the configuration seen in unmyelinated fibres. Two groups of adult white mice were studied. (A) In one group, the myelinated phrenic nerve and the unmyelinated cervical sympathetic trunk (CST) were cross-anastomosed in the neck. From 2 to 6 months after anastomosis, previously unmyelinated distal stumps contained many myelinated fibres while phrenic nerves joined to proximal CSTs became largely unmyelinated. Radioautography of distal stumps indicated that proliferation of Schwann cells occurred mainly in the first few days after anastomosis but was also present to a similar extent in isolated stumps. (B) In other mice, CSTs were grafted to the myelinated sural nerves in the leg. One month later, the unmyelinated CSTs became myelinated and there was no radioautographic indication of Schwann cell migration from the sural nerve stump to the CST grafts. Thus, Schwann cell proliferation in distal stumps is an early local response independent of axonal influence. At later stages, axons from the proximal stumps cause indigenous Schwann cells in distal stumps from the previously unmyelinated nerves to produce myelin while Schwann cells from the previously unmyelinated nerves to produce myelin while Schwann cells from the previously myelinated nerves become associated with unmyelinated fibres. Consequently, the regenerated distal nerve resembled the proximal stump. It is suggested that this change is possible because Schwann cells which divide after nerve injury reacquire the developmental multipotentiality which permits them to respond to aoxonal influences.

Animals↗

PubMed1984

Histogenesis, cytodifferentiation, and its subcellular steroidogenic sites in the virilizing ovarian Leydig cell tumor: light microscopic dry-mounting radioautography for [3H]cholesterol and electron microscopic cytochemistry for 3 beta-hydroxysteroid dehydrogenase activity.

A case of a virilizing ovarian Leydig cell tumor in a 38-year-old woman with a marked elevated plasma testosterone level was investigated using light microscopic dry-mounting radioautography and electron microscopic cytochemistry. Following a total abdominal hysterectomy and bilateral salpingo-oophorectomy, the plasma testosterone level decreased abruptly. Light microscopic dry-mounting radioautography for [3H]cholesterol showed silver grains localized mainly over the cytoplasm of the neoplastic Leydig cells which were of three different cell types: fibroblast-like cells, Leydig cells (steroid-secreting cells), and transitional cells (partially or incompletely differentiated Leydig cells). Reaction products for 3 beta-hydroxysteroid dehydrogenase activity were localized on the tubular or lamellar cristae and inner membranes of the mitochondria, and on the membranes of smooth endoplasmic reticulum in the transitional cell as well as in the Leydig cell. From these observations, it is suggested that the Leydig cell tumor is derived from the fibroblast-like cell and from the transitional cell, morphologically falling between the fibroblast-like cell and the Leydig cell, has fine structural evidence of steroidogenic activity, and has an ability to secrete testosterone.

3-Hydroxysteroid Dehydrogenases↗

PubMed1983

Perinatal development of estrogen receptors in mouse brain assessed by radioautography, nuclear isolation and receptor assay.

The development of estrogen receptors was investigated in vivo in the brains of fetal and neonatal mice 2 h after administering [3H]moxestrol to the pregnant mothers or neonates. Moxestrol bypasses the alpha-fetoprotein 'protective barrier' and gains access to estrogen receptors. Analysis of [3H]moxestrol uptake by radioautography and by cell nuclear isolation and counting of radioactivity revealed a marked increase in the number of estrogen receptors and estrophilic cells in the brain during late fetal and early postnatal development. Assays of cytosol estrogen receptors were conducted in parallel and revealed a comparable pattern of development. The increase in estrogen receptors and labeling was especially great from embryonic day (E) 15 to E18. Cytosol assays revealed a low level of receptors in the whole brain on E13. Radioautography revealed that clearly labeled cells in the hypothalamus and preoptic area were virtually absent on E13 but were evident on E15, with marked increases occurring between E15 and E18, both in number of labeled cells and in intensity of labeling per cell. Within the cerebral cortex the dorsal cingulate cortex was the most extensively labeled area; however, clearcut labeling was not evident on E13 or E15. Thus, the development of cortical estrogen receptors occurs somewhat later than that in the hypothalamus and preoptic area. The perinatal increase in estrogen receptors usually begins several days after the birthdates of neurons in these estrophilic regions of the brain, and corresponds to the early responsiveness of these neurons to the organizational and activational influences of estrogen.

Animals↗

PubMed1994

Increased monoamine oxidase B activity in plaque-associated astrocytes of Alzheimer brains revealed by quantitative enzyme radioautography.

The aetiology and pathogenesis of Alzheimer's disease are currently poorly understood, but symptomatic disease is associated with amyloid plaques, neurofibrillary tangles, neuronal loss and numerous alterations of neurotransmitter systems in the CNS. Monoamine oxidase type B is known to be increased in Alzheimer diseased brains. The distribution and abundance of catalytic sites for monoamine oxidases A and B in post mortem human brains of 11 Alzheimer disease cases and five age-matched controls were investigated by quantitative enzyme radioautography. Using tritiated monoamine oxidase inhibitors (Ro41-1049 and lazabemide)--as high affinity substrates selective for monoamine oxidases A and B, respectively--it was found that monoamine oxidase B activity increased up to three-fold exclusively in temporal, parietal and frontal cortices of Alzheimer disease cases compared with controls. This increase was restricted to discrete patches (approximately 185 microns in diameter) which occupied approximately 12% of the cortical areas examined. In other brain regions (hippocampal formation >> caudate-putamen > cerebellum), patches of [3H]lazabemide-enriched binding were less abundant. [3H]Ro41-1049 binding (i.e. monoamine oxidase A) was unchanged in all tissues of diseased versus control brains. The monoamine oxidase B-enriched patches in all cortical regions correlated, in their distribution and frequency, with glial fibrillary acidic protein-immunoreactive clusters of astrocytes. Diffuse and mature beta-amyloid-immunoreactive senile plaques as well as patches of high density binding of [3H]PK-11195--a high-affinity ligand for peripheral-type (mitochondrial) benzodiazepine binding sites in microglia/macrophages--were found throughout Alzheimer diseased cortices. The up-regulation of monoamine oxidase B in plaque-associated astrocytes in Alzheimer's disease--in analogy to its proposed role in neurodegenerative disorders such as Parkinson's disease--might, indirectly, be a potential source of cytotoxic free radicals. Lazabemide, a selective reversible monoamine oxidase B inhibitor, is currently under clinical evaluation for the treatment of Parkinson's and Alzheimer's diseases. We conclude that enzyme radioautography with [3H]lazabemide is a reliable high resolution assay for plaque-associated astroglioses in Alzheimer's disease. Its clinical diagnostic utility for positron emission tomography or single photon emission computer tomography studies is being investigated.

Aged↗

PubMed1996

Molecular neuroanatomy of human monoamine oxidases A and B revealed by quantitative enzyme radioautography and in situ hybridization histochemistry.

Monoamine oxidases are key enzymes in the metabolism of amine neurotransmitters and neuromodulators and are targets for drug therapy in depression, Parkinson's and Alzheimer's diseases. Knowledge of their distribution in the brain is essential to understand their physiological role. To study the regional distribution and abundance of monoamine oxidases A and B in human brain, pituitary and superior cervical ganglion, we used quantitative enzyme radioautography with radioligands [3H]Ro41-1049 and [3H]lazabemide, respectively. Furthermore, 35S-labelled oligonucleotides complementary to isoenzyme messengerRNAs were used to map the cellular location of the respective transcripts in adjacent sections by in situ hybridization histochemistry. A markedly different pattern of distribution of the isoenzymes was observed. Highest levels of monoamine oxidase A were measured in the superior cervical ganglion, locus coeruleus, interpeduncular nucleus and ventromedial hypothalamic nucleus. The corresponding messengerRNA was detected only in the noradrenergic neurons of the superior cervical ganglion and locus coeruleus. In contrast to rat brain, monoamine oxidase B was much more abundant in most human brain regions investigated. Highest levels were measured in the ependyma of ventricles, stria terminalis and in individual hypothalamic neurons. Monoamine oxidase B transcripts were detected in serotoninergic raphe neurons, histaminergic hypothalamic neurons and in dentate gyrus granule cells of the hippocampal formation. We conclude that [3H]Ro41-1049 and [3H]azabemide are extremely useful radioligands for high-resolution analyses of the abundance and distribution of catalytic sites of monoamine oxidases A and B, respectively, in human brain sections. From levels of messenger RNA detected, the cellular sites of synthesis of the isoenzymes are the noradrenergic neurons of the locus coeruleus (for monoamine oxidase A) and the serotoninergic and histaminergic neurons of the raphe and posterior hypothalamus, respectively (for monoamine oxidase B). The combination of quantitative enzyme radioautography with in situ hybridization histochemistry is a useful approach to study, with high resolution, both the physiology and pathophysiology of monoamine oxidases in human brain.

Aged↗

PubMed1961

Electron microscopic radioautography of thin sections: the Golgi zone as a site of protein concentration in pancreatic acinar cells.

Electron microscopic radioautographs of guinea pig pancreatic exocrine cells were obtained by covering thin sections ( approximately 600 A) of OsO(4)-fixed, methacrylate-embedded tissue with thin layers of Ilford K-5 nuclear research emulsion. After an exposure of 13 days at 4 degrees C., the preparations were photographically processed, stained with uranyl acetate, and examined in an electron microscope. The label used was leucine-H(3) injected intravenously 20 minutes before collection of the specimens. Conventional radioautographs of thicker sections (0.4 micron) were also examined in a phase contrast microscope. The advantages obtained from electron microscopic radioautography are: the higher radioautographic resolution (of the order of 0.3 micron) due to the thinness of the emulsion and the specimen, and a high optical resolution permitting a clear identification of the labeled structure. In the guinea pig pancreas this technique demonstrated that, at the time studied, newly synthesized proteins were concentrated in the structures of the Golgi complex and especially in large vacuoles partially filled with a dense material. The vacuoles are probably a precursor to the secretion granules (zymogen granules) in which the label becomes segregated at a later time. These observations demonstrate directly the role of the Golgi complex in the secretion process. They also illustrate the possibilities of this method for radioautography at the intracellular level.

Acinar Cells↗

PubMed1968

The incorporation of radioactive proline into cultured cells. Interpretations based on radioautography and electron microscopy.

Cultured carrot explants, stimulated to grow rapidly in a medium containing coconut milk, were labeled with radioactive proline. After an initial period of absorption (8 hr for proline-(3)H; 24 hr for proline-(14)C) the tissue was allowed to grow for a further period of 6 days in a similar medium free from the radioactivity. Samples were prepared for electron microscopy and radioautography at the end of the absorption period and also after the further growth. The distribution of the products from the radioactive proline in the cells is shown by high-resolution radioautography and is rendered quantitative for the different regions of the cells. The results show that the combined label, which was present in the form of proline and the hydroxyproline derived from it, was all in the protoplasm, not in the cell walls. Any combined label that appeared to be over the cell walls is shown to be due to scatter from adjacent cytoplasmic sites. Initially the radioactivity was concentrated in nuclei, even more so in nucleoli, but it subsequently appeared throughout the ground cytoplasm and was also concentrated in the plastids. The significance of these observations for the general concept of a plant cell wall protein and for the special problem of growth induction in otherwise quiescent cells is discussed.

Autoradiography↗

PubMed1969

A study of nucleolar vacuoles in cultured tobacco cells using radioautography, actinomycin D, and electron microscopy.

Previously it has been found that in tobacco callus cells nucleolar vacuoles repeatedly form and contract. In this study, nucleolar vacuoles were investigated by using radioautography, actinomycin D, and electron microscopy. It was found, from grain counts of nucleoli labeled with uridine-(3)H, that nucleoli containing vacuoles had more than three times as many grains/micro(2) of nucleolar substance as did nucleolei without vacuoles. Treatment of tobacco callus cells with various concentrations of actinomycin D caused the percentage of cells containing nucleolar vacuoles to decrease; with the highest concentration the percentage of these cells dropped from the normal level of about 70% to less than 10%. However, after removal of actinomycin D the cells regained nucleolar vacuoles up to the control level. When radioautography was used with actinomycin D, it was found that the actinomycin D inhibited the uptake of uridine-(3)H, i.e. inhibited RNA synthesis, in those nucleoli which lost their nucleolar vacuoles. In addition, after removal of the cells from actinomycin D, it was found that as the cells regained nucleolar vacuoles the nucleoli also began to incorporate uridine-(3)H. Electron micrographs showed the nucleoli to be composed of a compact, finely fibrous central portion surrounded by a layer of dense particles 100-150 A in diameter. Nucleolar vacuoles occurred in the fibrous central portion. Dense particles similar to those in the outer layer of the nucleoli were found scattered throughout the vacuoles and in a dense layer at their outer edge. These data suggest that in cultured tobacco callus cells the formation and contraction of nucleolar vacuoles is closely related to RNA synthesis in the nucleolus.

Autoradiography↗

PubMed1980

Direct in vivo demonstration by radioautography of specific binding sites for calcitonin in skeletal and renal tissues of the rat.

An in vivo binding assay using radioautography was employed to visualize calcitonin receptors in rat tissues. At 2 min after intravenous injection of biologically active 125I-salmon calcitonin, free hormone was separated from bound hormone by intracardiac perfusion with lactated Ringer's followed by fixation with 2.5% glutaraldehyde. Various tissues were removed and processed for light and electron microscope radioautography. These were compared to tissues removed from animals that received identical amounts of labeled hormone with a large excess of unlabeled calcitonin. Among the tissues investigated, kidney and bone demonstrated labeling. In kidney, most silver grains were located over vesicles below the brush border of cells of theproximal convoluted tubules. These grains were still present after simultaneous injection of excess unlabeled hormone and most likely represented binding to sites involved with ingestion and degradation of hormone from the urinary filtrate. In contrast, grains localized to the basal surfaces of distal convoluted tubule cells were significantly reduced in number in control animals and represented sites of saturable, specific hormone binding. In bone, specific binding sites were found only at the periphery of osteoclasts. These labeled cells were located at resorption sites examined in tibia, humerus, and alveolar bone. This demonstration of the localization of 124I-calcitonin in situ provides a new approach for study the interaction of calcium-regulating hormones with their target cells.

Animals↗

PubMed1984

Acetylcholine-synthesizing amacrine cells: identification and selective staining by using radioautography and fluorescent markers.

The fluorescent DNA stain 4,6,diamidino-2-phenylindole (DAPI) was applied to the cut axons of the rabbit optic tract, from which it was retrogradely transported to the retinal ganglion cell bodies. The labelled retinas were isolated from the eye and maintained in vitro in the presence of [3H]choline. They were then quick-frozen, freeze-dried, vacuum-embedded, and radioautographed on dry emulsion for identification of the acetylcholine-synthesizing cells. Inspection of the radioautographs by fluorescence microscopy showed the two labels not to co-exist: the cells that contained the transported fluorescence did not contain radioactive acetylcholine. In other animals the optic nerve was sectioned, causing retrograde degeneration of a large fraction of the ganglion cells. A population of small, round neurons in the ganglion cell layer was spared. These retinas synthesized [3H]acetylcholine at the same rate as control tissues; and radioautography showed an identical distribution of the acetylcholine-synthesizing cells. We conclude that the acetylcholine-synthesizing neurons of the ganglion cell layer are displaced amacrine cells. When DAPI was injected intraocularly instead of being applied to the optic tract, a regular mosaic of neurons in the ganglion cell layer was selectively stained, and two bands of fluorescence were observed in the inner plexiform layer, at the level where two bands of radioactive acetylcholine were observed in radioautographs. Quantitative analysis showed that the DAPI-stained cells were the same size as those that survive optic nerve section. Like the acetylcholine-synthesizing cells, they appear to be displaced amacrines; when wheatgerm agglutinin labelled by Evans blue was applied to the optic tract and DAPI was injected intraocularly, the red fluorescence of Evans blue and the blue fluorescence of DAPI accumulated in different cells. When DAPI was injected intraocularly and radioautography for acetylcholine was carried out, the cells brightly labelled by DAPI were found to have synthesized acetylcholine. We conclude that topically applied DAPI selectively labels the acetylcholine-synthesizing neurons of the ganglion cell layer. The distribution of the acetylcholine-synthesizing cells was established by counting the DAPI-labelled cells in whole-mounts.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetylcholine↗

PubMed1972

Radioautography of the optic tectum of the goldfish after intraocular injection of ( 3 H)proline.

Radioautography of the optic tectum of the goldfish, performed after injection of [(3)H]proline into the contralateral eye, effectively resolves several distinct layers of retinal synapses. Silver grains are found unilaterally over nerve tracts containing efferent fibers from the tectum, a result that suggests intercellular migration of labeled molecules. The low background and high specific grain density obtained with [(3)H]proline radioautography indicate the usefulness of this technique for the elucidation of neuroanatomical connections in the visual system.

Animals↗

PubMed1965

ELECTRON MICROSCOPIC RADIOAUTOGRAPHY OF HELA CELLS INFECTED WITH ADENOVIRUS.

Harford, Carl G. (Washington University, St. Louis, Mo.), and Alice Hamlin. Electron microscopic radioautography of HeLa cells infected with adenovirus. J. Bacteriol. 89:1540-1547. 1965.-Because intracellular particles of adenovirus are not seen by light microscopy, radioautography with tritiated thymidine was carried out with an electron microscope. Reacted grains were present over many nuclei containing viral aggregates, but only 16% of these grains were over the aggregates. Control observations with tritium-labeled bacteria indicated that this low incidence of labeling of viral aggregates could not be attributed to errors of the method. We think that deoxyribonucleic acid (DNA) labeled with tritiated thymidine in these experiments was mainly the excess viral DNA that is known to accumulate in adenovirus-infected cells. We did not exclude the possibility that some cellular DNA was labeled.

Adenoviridae↗

PubMed1983

Identification of ending containing dopamine and vasopressin in the rat posterior pituitary by a combination of radioautography and immunocytochemistry at the ultrastructural level.

In order to study the morphological relationship between the dopaminergic and the vasopressinergic endings in the rat posterior pituitary, a combination of radioautography and immunocytochemistry techniques was applied to the same ultrathin section of the posterior pituitary. Immunocytochemical localization (peroxidase-antiperoxidase technique) of vasopressin was first applied, followed by the radioautography detection of [3H] dopamine that had been injected 30 and 60 min before fixation. This double labeling technique has established that the dopaminergic endings that contain both dense core vesicles (60-100 nm in diameter) and small clear vesicles are always found in close proximity to vasopressinergic neurosecretory endings, suggesting an interaction between these two systems. This relatively easy approach should be very useful to study the connections between different neurotransmitter systems in the nervous system.

Animals↗

PubMedDate not supplied

Study of the effects of aging on macromolecular synthesis in mouse steroid secreting cells using microscopic radioautography.

The effects of aging on DNA, RNA and protein synthesis in adrenal gland cortical cells and testicular Leydig cells of ddY mice at various ages (from prenatal day 19 to postnatal days 1, 3, 7, 14, months 1 and 6 and 1 and 2 years after birth) were examined using light and electron microscopic (EM) radioautography after labeling with [3H]-thymidine, [3H]-uridine and [3H]-leucine. The percentage of the labeled cells in the adrenal glands after [3H]-thymidine injection was greatest in the zona glomerulosa of the cortex and the medulla on embryonic day 19, in the zona fasciculata and zona reticularis of the cortex on postnatal day 1 and gradually decreased with aging. EM radioautography revealed that well developed cell organelles such as smooth surfaced endoplasmic reticulum, Golgi apparatus, mitochondria with tubular cristae and lipid droplets were more frequently observed in the cytoplasm of unlabeled cells as compared to cells labeled with [3H]-thymidine in the three zones of the cortex. The effects of aging on RNA synthesis in adrenal glands after [3H]-uridine injection revealed that all types of cells of the adrenal gland were labeled. In each cell, silver grains were localized over the nuclei and cytoplasm and were more dense in the nuclei than the cytoplasm. Grain counts were highest in the cortex and medulla on fetal day 19 and then gradually decreased with aging from postnatal day 14 to 1 year after birth. In the cortex, the number of silver grains was higher in the zona glomerulosa than in the other zones from fetal day 19 to 1 year and grain counts were higher in the medulla in the embryonic stage as compared to the postnatal stages. However, the number of labeled mitochondria and the mitochondrial labeling index increased with aging. The [3H]-thymidine labeling index in the Leydig cells of the testis was low at embryonic and early postnatal stages, increased slightly at 6 months and reached a peak at 9 months which was maintained at a relatively high level in senescence. The number of the silver grains over the nuclei and cytoplasm of Leydig cells due to [3H]-uridine labeling was observed from embryonic day 19 and increased from month 3 onwards. From adult to senescence, [3H]-uridine incorporation was maintained at high levels in the nuclei and was relatively low in the cytoplasm. The effects of aging on [3H]-leucine incorporation in Leydig cells were also examined. The labeling indices between embryonic and early postnatal stages showed no obvious differences although the number of the silver grains in both cytoplasm and nucleus increased from 6 months onwards and was maintained at high levels until senescence. From these results, it was concluded that the effects of aging on DNA, RNA and protein synthesis in steroid secreting cells such as adrenal gland cortical cells and testicular Leydig cells of mice correlated with the hormonal changes observed with aging.

Adrenal Cortex↗

PubMed1987

The use of slotted grids for electron microscopic radioautography.

A method for electron microscopic radioautography on slotted grids is presented which allows examination of the distribution of silver grains over sections of entire structural units without interference by grid bars. Tissue sections of a size such as to fit the opening of the grid slot are placed on slides coated with a Formvar film of sufficient strength to permit transfer of the completed radioautograph onto the grid and to support it over the slot. Sections are block stained prior to radioautography to minimize the risk of loss of the radioautograph during the procedure.

Autoradiography↗

PubMed2001

Rabbit tissue distribution of 3H-ouabain by digital radioautography.

3H-ouabain is useful to evaluate the tissue localization of Na,K-ATPase. In this work we determined the distribution of 3H-ouabain in rabbit tissue by digital radioautography. Using this method, we were able to obtain a comparison of various organs in a relatively short time (6.5 days), while with traditional radioautography, only the kidney was detectable after seven months of film exposure. The kidney has the highest intensity (concentration of 3H-ouabain), followed by the heart, liver, muscle, lung, spleen and finally brain, which was almost undetectable. In kidney the activity was higher in the cortex, while the other tissues displayed a more uniform intensity, suggesting that Na,K-ATPase was evenly distributed.

Animals↗

PubMed1990

Study of RNA synthesis in the livers of aging mice by means of electron microscopic radioautography.

The application of 3H-uridine radioautography results in labeling of the liver cells in which RNA is synthesized at various ages of the mouse. Quantitative changes of RNA synthesis in the hepatocytes of aging mice were studied by electron microscopic radioautography. The silver grains were mainly located in the nucleoli and nuclei and a few in the mitochondria and rough surfaced endoplasmic reticulum of almost all of the cell populations at various ages. The number of silver grains in the hepatocyte gradually increased after birth, reached the maximum at 14 days of postnatal age, then decreased to 24 months with aging. The number of silver grains of the euchromatin was more than those of the heterochromatin of the hepatocyte nuclei at various ages. The number of silver grains of the granular components was more than those of the fibrillar components of the hepatocyte nucleoli at various ages. However, the ratio of silver grains among euchromatin, heterochromatin, granular components and fibrillar components remained approximately constant.

Aging↗