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PubMed1979

[Electron microscopic autoradiography on semithin sections (author's transl)].

The present study applies semithin sections for electron microscopic autoradiography. Due to (a) the decrease of exposition time, and (b) lower intensity of isotope labelling, low power electron microscopic autoradiography on semithin sections is favoured in comparison with conventional (ultrathin section high power) electron microscopic autoradiography, and due to (c) more precise localization of silver granules, and (d) ease of quantitative evaluation of autoradiography, it is superior to ordinary (thick section) light microscopic autoradiography.

Animals↗
PubMed2004

[Transferrin receptor expression of human mesenchymal stem cells and in vitro tracking by autoradiography after transplantation in spinal cord].

OBJECTIVE: To determine transferrin receptor (TfR) expression of human mesenchymal stem cells (MSCs) in vitro and after transplantation in rabbit spinal cord,and to detect implanted MSCs by in vitro autoradiography. METHODS: Human mesenchymal stem cells (hMSCs) were isolated from fetal blood. Flow cytometry assay, immuno-fluorescent staining and receptor binding assay were used to determine TfR expression of hMSCs. Radioiodinated transferrin saturated with iron [(125)I-Tf(Fe)(2)] was used as tracer. The hMSCs transplanted in rabbit spinal cord was tracked by in vitro autoradiography. Diffusion of (125)I-Tf(Fe)(2) in spinal cord was examined with autoradiography. RESULTS: TfR expression of MSCs was demonstrated by flow cytometry assay, immuno-fluorescent staining and receptor binding assay in vitro. (125)I-Tf(Fe)(2) bound to hMSCs with a equilibrium dissociation constant (KD) of (0.98+/-0.12) nmol/L and a maximal density of binding sites (B(max)) of (107 702+/-6 226) sites per cell. Immuno-fluorescent staining showed that TfRs were expressed on hMSCs on the 2nd day but not be expressed on the 10th day post transplantation. Autoradiography showed distinct accumulation of (125)I-Tf(Fe)(2) but not (125)I-HSA at hMSCs implantation sites of spinal cord sections on the 2nd day post transplantation. (125)I-Tf(Fe)(2) had diffused into spinal cord 16 hours after incubation. CONCLUSION: Implanted hMSCs could be detected by in vitro autoradiography with (125)I-Tf(Fe)(2) on the 2nd day after being transplanted in spinal cord. To track implanted hMSCs with radionuclide imaging techniques in vivo, TfR was a suitable target for imaging and radioiodinated Tf(Fe)(2) was a feasible tracer.

Animals↗
PubMed1993

Synthesis of Mallory body, intermediate filament, and microfilament proteins in liver cell primary cultures. An electron microscopic autoradiography assay.

BACKGROUND: The synthesis of Mallory body (MB) and cytoskeletal proteins in hepatocytes have not been studied in vitro. To visualize the incorporation of radiolabeled amino acid into MBs and cytoskeletal filaments separately in the same cell, we developed a new technique using high resolution electron microscopic autoradiography. EXPERIMENTAL DESIGN: Primary cultures of hepatocytes isolated from griseofulvin (GF)-fed mice and control mice were used. The hepatocytes labeled with [35S]methionine were extracted with detergent. The detergent-resistant residual cytoskeleton was characterized by polyacrylamide gel electrophoresis and autoradiography. For light microscopic and electron microscopic autoradiography, the hepatocytes were labeled with [3H]methionine or [3H]leucine, then extracted with detergent and then embedded in plastic. To obtain high resolution of the labeled cytoskeleton, exposed ultrathin sections were treated by the gold latensification and physical developing methods and then examined by electron microscopy. RESULTS: Autoradiography of polyacrylamide gel electrophoresis showed [35S]methionine was incorporated into three major bands: cytokeratin 55, cytokeratin 49, and actin in control and GF hepatocytes. In GF hepatocytes, high molecular weight proteins that remained near the top of the gel (MB-derived proteins) were also labeled. The electron microscopic autoradiography revealed that the intermediate filaments, microfilaments, MBs, and nuclear matrix were labeled and the resolution of the labeling was high enough to localize the labeled amino acids incorporated into the filaments. The background was very low. The labeling using [3H]methionine was greater than [3H]leucine, but the pattern of labeling was the same. In control mice, hepatocytes intermediate filaments and microfilaments were heavily labeled at the cell border. In GF-fed mice, the MBs were heavily labeled compared with the intermediate filaments at the cell borders within the same cells. The intensity of labeling varied from cell to cell. CONCLUSIONS: These results suggested that the newly synthesized cytokeratin monomers were incorporated into the already polymerized filamentous network as well as the MB filaments in cell culture.

Actin Cytoskeleton↗
PubMed1987

Regulatory peptide receptors: visualization by autoradiography.

The receptors for regulatory peptides have been extensively characterized using radioligand binding techniques. By combining these binding techniques with autoradiography it is possible to visualize at the light and electron microscopic levels the anatomical and cellular localization of these receptors. In this review we discuss the procedures used to label peptide receptors for autoradiography and the peculiarities of peptides as ligands. The utilization of autoradiography in mapping peptide receptors in brain and peripheral tissues, some of the new insights revealed by these studies particularly the problem of 'mismatch' between endogenous peptides and receptors, the existence of multiple receptors for a given peptide family and the use of peptide receptor autoradiography in human tissues are also reviewed.

Animals↗
PubMed2004

Radioprotection of the murine submandibular gland by isoproterenol: autoradiography study with 3H-leucine.

Irradiation of the salivary glands results in the generation of free radicals from metal ions present in the secretory granules of acinar cells, a process that is believed to exacerbate radiation damage to the salivary glands. We therefore conducted a comparative investigation of radiation damage to the acinar cells of murine submaxillary glands in which granule secretion had been induced, and used autoradiography to visualize the pathological changes. Male BALB/c mice, at 8 weeks of age, were divided into four groups: a no-isoproterenol (IPR) and no-irradiation group (group I), a no-IPR, irradiated group (group II), an IPR, no-irradiation group (group III), and an IPR, irradiated group (group IV). Intraperitoneal injections of IPR were used, and 3 h later, the submaxillary region was irradiated with X-rays at a dose of 10 Gy. Three days after the irradiation, 3H-leucine was administered, and submaxillary glands were removed at predetermined times. Thin sections were prepared, and light- and electron-microscope autoradiography was performed. The number of reduced silver particles per unit acinar cell area was determined by light-microscopic autoradiography, and the proportion of reduced silver particles in the rough endoplasmic reticulum, the Golgi apparatus, and secretion granules was determined by electron-microscopic autoradiography. The result indicated that the effects of the radiation on the secretory potential of the submaxillary glands were diminished in acinar cells with a higher secretory granule content.

Adrenergic beta-Agonists↗
PubMed1995

Comparison of autoradiography, liquid scintillation counting and immunoenzymatic staining of 5-bromo-2'-deoxyuridine for measurement of unscheduled DNA synthesis and replicative DNA synthesis in rat liver.

Different methods for evaluating unscheduled DNA synthesis (UDS) and replicative DNA synthesis (RDS) were studied in hepatocytes of F344 rats exposed in vivo to dimethylnitrosamine (DMN) or CCl4. Hepatocytes were isolated and incubated in Williams' medium E supplemented with either [3H]thymidine for autoradiography or 5-bromo-2'-deoxyuridine for immunoenzymatic staining. In the method of liquid scintillation counting, the cells were incubated with [3H]thymidine with or without hydroxyurea. The nuclear fraction was isolated and the incorporation of [3H]thymidine into nuclear DNA was determined by a liquid scintillation counter. DMN at doses of 0.625-5 mg/kg body weight induced UDS of 1.6-37.9 (0 dose; -6.9) net grains/nucleus measured by autoradiography and 337-1377 (0 dose; 177) dpm/microgram DNA in the presence of hydroxyurea measured by a liquid scintillation counter. CCl4 at doses of 50-400 mg/kg body weight induced RDS in 1.5-12.1% (0 dose; 0.12%) and 1.8-14.6% (0 dose; 0.16%) of cells with the methods of autoradiography and immunoenzymatic staining, respectively, and of 2991-24256 (0 dose; 324) dpm/microgram DNA in the absence of hydroxyurea with the method of liquid scintillation counting. Similar dose-dependent induction of UDS and RDS was observed with these methods. These results suggest that the methods of liquid scintillation counting and immunoenzymatic staining have almost the same sensitivity for measuring UDS and RDS as that of autoradiography.

Animals↗
PubMed1983

Gamma-aminobutyrate as an autonomic neurotransmitter: release and uptake of [3H]gamma-aminobutyrate in guinea pig large intestine and cultured enteric neurons using physiological methods and electron microscopic autoradiography.

To provide further evidence that some enteric neurons use gamma-aminobutyrate (GABA) as a neurotransmitter, we have demonstrated a depolarization-induced release of [3H]GABA from isolated myenteric ganglia in culture, and from segments of large intestine containing the myenteric plexus. In addition, light and electron microscopic autoradiography has been employed to visualize the putative GABAergic neurons and their projections, both in cultured ganglia and in sections from the gut wall. Explant cultures of the guinea-pig myenteric plexus, containing only neurons and glia intrinsic to the gut, were incubated with 0.14 microM [3H]GABA, washed and then repeatedly depolarized by 62 mM K+. The depolarizations always evoked release of [3H]GABA. The evoked release was reversibly blocked by 5 mM Co2+, suggesting a dependence on Ca2+ influx, a characteristic of neuronal transmitter release. Strips of the guinea-pig taenia coli, containing the myenteric plexus, were incubated with 0.14 or 0.7 microM [3H]GABA, washed and subjected to electrical field stimulation. This caused release of [3H]GABA, which could be evoked successively on repeated stimulation. The release was of neuronal origin and Ca2+ dependent, since it was abolished by 3 microM tetrodotoxin and reversibly blocked by 10 mM Co2+. By combined electrophoresis and chromatography the released tritium was identified as being attached to GABA. Autoradiography, following incubation with low concentrations of [3H]GABA, was used to identify specifically putative GABAergic neurons. Light microscopic autoradiography of cultured ganglia, and electron microscopic autoradiography of sections from the taenia coli including the myenteric plexus, were in good agreement, showing a selective and heavy labelling over a sub-population of neurons, and light labelling over glial cells. The majority of neurons and the non-neural cells were unlabelled. The electron microscopic autoradiographs also showed heavy labelling over some, but not all, axons in the fine axon bundles that innervate the longitudinal muscle of the taenia. These results strongly support our previous suggestion that a population of myenteric neurons are GABAergic, although more work is needed before this is fully established. Our data also suggest that some of the putative GABAergic neurons project out to the gut musculature, where their axons, together with axons from other neurons, run in bundles to form the intramuscular nerves. We therefore propose that one of the roles of these neurons in the gut is the presynaptic modulation of transmitter released from neighbouring axons in these nerves.

Animals↗
PubMed1987

Pharmacological characteristics and anatomical distribution of [3H]oxytocin-binding sites in the Wistar rat brain studied by autoradiography.

Oxytocin-binding sites were detected by autoradiography on rat brain sections incubated in the presence of the [3H]oxytocin. These sites were characterized pharmacologically using quantitative autoradiography. High pressure liquid chromatography controls of the incubation media indicated that labelling was due to the intact [3H]oxytocin molecule. Pharmacological analysis of different locations (central amygdaloid nucleus, ventral subiculum and ventromedial hypothalamic nucleus) showed that the sites detected had a high affinity for oxytocin and also for arginine-vasopressin. In contrast, some areas known to bind vasopressin intensely, such as suprachiasmatic and lateral septum nuclei, had little or no affinity for oxytocin. Autoradiographs revealed [3H]oxytocin-binding sites in already known brain areas (olfactory centres, ventral subiculum, central amygdaloid nucleus, bed nucleus of the stria terminalis) albeit with more extensive labelling of some of these formations, in particular, the amygdaloid complex. In addition, specific [3H]oxytocin-binding sites were found in areas not yet reported to bind oxytocin, such as the paraventricular thalamic and caudate nuclei. In the hypothalamus, specific binding sites were not detected in the supraoptic and paraventricular nuclei: the only structure labelled was the ventrolateral part of the ventromedial nucleus. Discrepancies between the concentrations of [3H]oxytocin-binding sites, the known distribution of oxytocin-containing endings and electrophysiological data indicate that autoradiography, under our conditions, apparently only reveals some of the oxytocin receptors in the brain. Thus, in the hypothalamus, no relationship can be established between the known effect of oxytocin on oxytocinergic magnocellular neurons and detection of specific [3H]oxytocin-binding sites. Autoradiography may reveal mainly oxytocin-binding sites in areas receiving diverse "parasynaptic" information, where oxytocin might play a modulatory role rather than exerting rapid, short-term effects of the neurotransmitter type.

Amygdala↗
PubMed2005

Effect of intratumoral heterogeneity in oxygenation status on FMISO PET, autoradiography, and electrode Po2 measurements in murine tumors.

PURPOSE: To explore conflicting results obtained when tumor hypoxia is assessed with Eppendorf electrode Po(2) measurements and with positron emission tomography (PET) by use of [(18)F]fluoromisonidazole (FMISO). METHODS AND MATERIALS: We compared the 2 methods in conjunction with 2-[(18)F]fluoro-2-deoxy-D-glucose (FDG) PET, dual-tracer ex vivo autoradiography (FMISO and 2-deoxy-D-[1-(14)C]glucose (2DG)), and histology in 2 murine tumor models, the C3H mammary carcinoma and the SCCVII squamous cell carcinoma. RESULTS: 2-[(18)F]fluoro-2-deoxy-D-glucose (FDG)-PET showed tumor-to-reference tissue ratios of 3.5 in both tumor models after 2 hours. C3H mammary carcinoma reached an FMISO PET ratio of 11 after 3.5 hours. Autoradiography showed large confluent areas of FMISO and 2DG uptake. Median Po(2) was 7 mm Hg and necrotic fraction was 10% to 30%. SCCVII squamous-cell carcinoma reached an FMISO PET tumor-to-reference tissue ratio of 2 after 2.5 hours. Autoradiography showed homogeneous 2DG uptake and scattered foci of high FMISO uptake. Median Po(2) was 1 mm Hg and necrotic fraction was below 5%. CONCLUSIONS: Ex vivo dual-tracer autoradiography documented the ability of in vivo FMISO PET to distinguish between confluent areas of either viable tissue or necrosis. Electrode Po(2) measurements could not be ascribed to specific areas in the tumors. Less uptake of FMISO in SCCVII squamous-cell carcinoma than in C3H mammary carcinoma could be caused by scattered foci versus confluent areas of viable hypoxic tissue in the 2 tumors, respectively.

Animals↗
PubMedDate not supplied

Autoradiography with positron emitting isotopes in positron emission tomography tracer discovery.

Rapid development of labeling chemistry and generation of new chemical entities for biologic interactions via combinatorial chemistry and high throughput screening gives great potential for the development of new positron emission tomography (PET) tracer candidates. It must, however, be realized that a large fraction of these candidates will fail to characterize the desired biochemistry in vivo due to undesirable properties that are not relevant to providing a specific signal in vitro. A full characterization of a PET tracer is a lengthy and expensive procedure, and it is necessary to establish confidence via a number of assays that a tracer will provide useful data in the target species (generally human). These assays should also serve as a background to choose or to reject a tracer at an early time point, conserving valuable resources and time. Autoradiography performed as an ex vivo binding technique or as ex vivo recording of in vivo tracer distribution are, in this respect, very important tools. Autoradiography binding methods allow a range of frozen tissues to be sectioned and incubated with the PET tracer, and with due caution and controls with selective blockade of binding, quantitative values can be obtained with respect to tracer binding and its regional distribution. The method is easy to learn and set up, and should be included in all PET research and development labs. Ex vivo autoradiography of selected organs or whole animals gives qualitative images of a tracer's distribution with high resolution and is especially valid for 18F-labeled tracers. When tracer administration is not limited by mass due to specific radioactivity, 11C-tracers can also be readily used. This method is attractive to use as a complement to small animal imaging due to its high resolution and anatomical correlate. Living slice autoradiography is a more cumbersome method, but has an advantage of utilizing live tissue that retains certain metabolic functions. The use of these different methods in the validation of tracers and for supplying complementary information is illustrated.

Animals↗
PubMed1992

A new method to localize active renin in tissues by autoradiography: application to dog kidney.

A method was developed to localize active renin in dog kidney sections using autoradiography to detect in vitro binding of the radiolabeled renin inhibitor, 125I-H77. Light fixation by prior perfusion of the kidney with paraformaldehyde was used to immobilize renin without denaturing its binding activity. Snap frozen sections were cut on a cryostat and incubated with 125I-H77. Dry film autoradiography revealed discrete binding of 125I-H77 to the vascular pole of glomerulus as well as diffuse binding to the outer medulla and to the cortex. Binding of 125I-H77 to other aspartyl proteases in the latter two regions was then suppressed by addition of the aspartyl protease inhibitor, N-acetyl-pepstatin (1 microM). This revealed only the juxtaglomerular binding and successfully suppressed binding of the radioligand to other sites. Light microscopic emulsion autoradiography revealed highly selective discrete labelling of the juxtaglomerular apparatus. Competition for this 125I-H77 binding by a series of structurally different renin inhibitors showed a close correspondence between their reported inhibitory potency for renin and potency in the binding system. This strongly suggests that the radioligand binds to the active site of renin immobilized in the kidney. These results demonstrate a new method to localize active renin in tissues using in vitro autoradiography and radioinhibitor binding. The method shows promise for localization and quantitation of tissue renin in extra renal tissues.

Animals↗
PubMed1998

Guanosine 5'-(gamma-[35S]thio)triphosphate autoradiography allows selective detection of histamine H3 receptor-dependent G protein activation in rat brain tissue sections.

Histamine elicits its biological effects via three distinct G protein-coupled receptors, termed H1, H2, and H3. We have used guanosine 5'-(gamma-[35S]thio)triphosphate (GTPgamma[35S]) autoradiography to localize histamine receptor-dependent G protein activation in rat brain tissue sections. Initial studies revealed that in basal conditions, adenosine was present in tissue sections in sufficient concentrations to generate an adenosine A1 receptor-dependent GTPgamma[35S] signal in several brain regions. All further incubations therefore contained 8-cyclopentyl-1,3-dipropylxanthine (10 microM), a selective A1 receptor antagonist. Histamine elicited dose-dependent increments in GTPgamma[35S] binding to discrete anatomical structures, most notably the caudate putamen, cerebral cortex, and substantia nigra. The overall anatomical pattern of the histamine-evoked binding response closely reflects the known distribution of H3 binding sites and was faithfully mimicked by N(alpha)-methylhistamine, (R)-alpha-methylhistamine, and immepip, three H3-selective agonists. In all regions examined, the GTPgamma[35S] signal was reversed with thioperamide and clobenpropit, two potent H3-selective antagonists, whereas mepyramine, a specific H1 antagonist, and cimetidine, a prototypic H2 antagonist, proved ineffective. These data indicate that in rat brain tissue sections, GTPgamma[35S] autoradiography selectively detects H3 receptor-dependent signaling in response to histamine stimulation. As the existing evidence suggests that GTPgamma[35S] autoradiography preferentially reveals responses to G(i/o)-coupled receptors, our data indicate that most, if not all, central H3 binding sites represent functional receptors coupling to G(i/o), the inhibitory class of G proteins. Besides allowing more detailed studies on H3 receptor signaling within anatomically restricted regions of the CNS, GTPgamma[35S] autoradiography offers a novel approach for functional in vitro screening of H3 ligands.

Adenosine↗
PubMed1964

A STUDY OF THE ULTRASTRUCTURAL LOCALIZATION OF HAIR KERATIN SYNTHESIS UTILIZING ELECTRON MICROSCOPIC AUTORADIOGRAPHY IN A MAGNETIC FIELD.

The sites of the incorporation of labeled cystine into keratinizing structures were studied in electron microscopic autoradiographs. The tracer used was cystine labeled with S(35) emitting long-range ionizing particles. During exposure for 1 to 2 months, according to our method of electron microscopic autoradiography, emulsion-coated specimens were exposed to a static magnetic field which appeared to result in a marked increase in the number of reacted silver grains. In young Swiss mice receiving intraperitoneal injections at 1, 3, and 6 hours before biopsy, conventional autoradiography demonstrated that S(35)-cystine was intensely localized in the keratogenous zone of anagen hair follicles, and that the radioactivity there increased in intensity progressively with time while the radioactivity in the hair bulb always remained very low. Our observations with electron microscopic autoradiography in a magnetic field appeared to indicate that at 3 and 6 hours after injection the S(35)-cystine was directly and specifically incorporated into tonofibrils in the hair cortex and into amorphous keratin granules of the hair cuticle layer, possibly without any particular concentration of this substance in the other cellular components. There seemed to be an appreciable concentration of cystine in tonofibrils of the cuticle of the inner root sheath. However, trichohyalin granules in the hair medulla and inner root sheath failed to show any evidence of cystine concentration. The improved sensitivity of the electron microscopic autoradiography with S(35)-cystine appeared to be partly due to the application of a static magnetic field. However, the reason for this could not be explained theoretically.

Animals↗
PubMed2000

The performance of a CCD digital autoradiography imaging system.

Autoradiography is a widely used technique for imaging trace quantities of radioactivity within biological samples, conventionally using photographic film. This method produces images with high spatial resolution, but it suffers from very low sensitivity and poor dynamic range. Digital autoradiography systems with greatly improved sensitivity and linearity are commercially available, but the spatial resolution is usually much less than that achieved using film. We report here the design, construction and characterization of a novel digital autoradiography system based on scientific-grade charged coupled devices (CCDs). Images of x-ray and beta emissions from radionuclides commonly used in autoradiography show that the system can perform high-speed quantitative imaging with a spatial resolution of approximately 30, microm. Using a frame by frame acquisition method the dynamic range is shown to be at least three orders of magnitude. The absolute detection efficiency is comparable to the best of the currently available digital systems. CCD images of 125I and 14C radioisotope distributions in tissue samples are superior to the equivalent film images and have been acquired in 1-10% of the time.

Animals↗
PubMed1994

Localization and quantitation of active renin in monkey kidney by radioinhibitor binding and in vitro autoradiography.

We developed an in vitro autoradiographic method to localize and quantify active renin in primate tissues. Active renin in monkey kidney sections was labeled with the primate specific renin inhibitor, 3H-CGP29287, and quantitated with autoradiography and computerized densitometry. Microscopic emulsion autoradiography was carried out to clarify the detailed localization of the binding. Non-specific binding to aspartyl proteases other than renin was blocked using 1 mumol/L of N-acetyl-pepstatin. To assess the usefulness of this procedure, binding of 3H-CGP29287 was examined both by film and emulsion autoradiography in the kidneys of monkeys (Macaca fuscata) that were given chronically either an angiotensin converting enzyme inhibitor (trandolapril), an angiotensin II receptor antagonist (E4177), or vehicle. 3H-CGP29287 was found to bind very selectively to the juxtaglomerular apparatus (JGA) under control conditions. In monkeys treated with trandolapril or E4177, 3H-CGP29287 binding was increased in proportion to the increase in renal renin concentration determined enzymatically; in these kidneys, emulsion autoradiography revealed radioinhibitor binding extending far from the JGA. The potency of a series of unlabeled renin inhibitor in competing for 3H-CGP29287 binding in the autoradiographic system closely paralleled their potencies, as determined in inhibiting renin by an enzymatic assay. This technique permits specific labeling of the catalytic site of renin in the monkey kidney sections.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1983

Thymidine plaque autoradiography of thymidine kinase-positive and thymidine kinase-negative herpesviruses.

Plaques formed by herpes simplex virus (HSV), pseudorabies virus, and varicella-zoster virus were studied by plaque autoradiography after [14C]thymidine labeling. Standard thymidine kinase-positive (TK+) viruses and TK- mutants of HSV types 1 and 2 and pseudorabies virus were studied, including cell cultured viruses and viruses isolated from animals. Autoradiography was performed with X-ray film with an exposure time of 5 days. After development of films, TK+ plaques showed dark rims due to isotope incorporation, whereas TK- plaques were minimally labeled. Plaque autoradiography of stock TK- viruses showed reversion frequencies to the TK+ phenotype of less than 10(-3). Autoradiography indicated that TK- virus retained the TK- phenotype after replication in vivo. In addition, it was shown that TK- HSV could be isolated from mouse trigeminal ganglion tissue after corneal inoculation of TK- HSV together with TK+ HSV. The plaque autoradiographic procedure was very useful to evaluate proportions of TK+ and TK- virus present in TK+-TK- virus mixtures.

Animals↗
PubMed1988

Distribution of progestin-concentrating cells in rat brain: colocalization of [3H]ORG.2058, a synthetic progestin, and antibodies to tyrosine hydroxylase in hypothalamus by combined autoradiography and immunocytochemistry.

The anatomical distribution of progestin-concentrating cells in rat brain was investigated by thaw-mount autoradiography. Fifteen 23-day-old ovariectomized and adrenalectomized female Holtzman rats were injected sc with 5 micrograms 17 beta-estradiol in oil/100 g BW daily for 4 days. On the fifth day, all animals were injected iv with 1 microgram/100 g BW [3H]ORG.2058, a synthetic progestin. The animals were killed after 30 or 60 min, and the brains were frozen and processed for autoradiography. Nuclear concentration of radioactivity was found in certain cells of the forebrain and midbrain and was prevented by prior injection (15 min) of excess unlabeled ORG.2058. In the preoptic-septal region, progestin-labeled neurons were observed in the nucleus (n.) septi lateralis, n. interstitialis striae terminalis, n. preopticus medialis, n. preopticus lateralis, n. preopticus periventricularis, and n. preopticus suprachiasmaticus. In the hypothalamus, labeled neurons were seen in n. periventricularis hypothalami, n. arcuatus hypothalami, n. ventromedialis hypothalami, n. dorsomedialis hypothalami, n. premammillaris ventralis, n. premammillaris dorsalis, and lateral hypothalamus. In the extrahypothalamic region, a few labeled cells were found in the central, medial, and cortical nuclei of the amygdala, the organum subforniculare, the lateral geniculate nucleus, the parietal and entorhinal cortex, and the central gray of the midbrain. Some of the progestin-concentrating cells were characterized with combined autoradiography and immunocytochemistry. Tyrosine hydroxylase-containing cells of the arcuate nucleus and the hypothalamic periventricular nucleus (group A12) showed nuclear concentration of radioactivity. Tyrosine hydroxylase cells in groups A11, A13, and A14; the substantia nigra (group A9); and the ventral tegmental area (group A10) did not show nuclear concentration of [3H]ORG.2058. The autoradiographic study demonstrates specific progestin-binding sites in nuclei of neurons in select areas of the rat brain. The results of combined autoradiography and immunocytochemistry suggest, for the first time, a direct action of progestin on tuberoinfundibular dopaminergic neurons.

Animals↗
PubMed1996

Measurement of cell adhesion and migration using phosphor-screen autoradiography.

This study demonstrates the use of phosphor-screen autoradiography as a means of measuring cell adhesion and cell expansion on polymer surfaces. The method has particular merit in cases where a specific substrate may be opaque or biochemically incompatible with colorimetric assay methodologies. With the phosphor-screen autoradiography method, there was a linear relationship between cell number and quantitated radioactivity. The technique has also been validated by comparison with a colorimetric assay of adhesion conducted for attachment to conventional culture substrata. The data supported the view that the use of phosphor-screen autoradiography was a valid method for detecting cell attachment, and it gave equivalent results to the colorimetric assay. Furthermore, a comparison between phosphor-screen autoradiography and a dye-staining method showed that this technique can be used as a means of quantifying cellular expansion over surfaces.

Autoradiography↗