journal article
LitStream Collection
Home
doi: 10.1177/29.1A_SUPPL.7288149pmid: 28054884
Examples of the multiple applicability of the double labeling method with 3H- and 14C-TdR are demonstrated. Double labeling with 3H- and 14C-TdR makes it possible to determine the cycle and its phases with high precision by modifying the usual percent labeled mitoses method with a single injection of 3H-TdR. This results in a considerable improvement of the resolution of the percent labeled mitoses curve. In addition, data is provided on the variances of the transit times of the cells through the cycle phases. For example, in the case of the jejunal crypt cells of the mouse, the transit times through successive cycle phases are uncorrelated. In the case of glial cells the double labeling method provides cell kinetic parameters despite the paucity of proliferating glial cells. In the adult untreated animal, glial cell mitoses are so rare that the percent labeled mitoses method can not be utilized. However, the S-phase duration can be measured by double labeling and the cycle time can be determined by the so-called method of labeled S phases. With the latter method the passage through the next S phase of the 3H-TdR-labeled S phase cells can be registered by injecting 14C-TdR at different time intervals following 3H-TdR application. In this way an S-phase duration of about 10 hr and a cycle time of about 20 hr was found for glial cells in the adult untreated mouse. An exchange of glial cells between the growth fraction and the nongrowth fraction has also been shown by double labeling. A quite different application of the double labeling method with 3H- and 14C-TdR is the in vivo study of the cell cycle phase-specific effect of drugs used in chemotherapy of tumors. Up to now studies of the effect of cytotoxic drugs on cells in different cycle phases were confined to in vitro experiments, since such studies need synchronized cells. However, the double labeling method, which leads to well-defined subpopulations of differently labeled cells in different phases of the cycle, allows such studies to be carried out under in vivo conditions. The effect of vincristine on these cells has been studied. Vincristine affects cells in S and G2 in such a manner that they are arrested during the next metaphase and subsequently become necrotic. It has no effect on G1 cells.
doi: 10.1177/29.1A_SUPPL.7288150pmid: 28054878
The major advantages and disadvantages of the autoradiographic method for tracing pathways in the central nervous system have become clear in the decade since its introduction. Attention is focused here on two major problems associated with the interpretation of autoradiographic experiments. First, it is often difficult to determine the effective size of an injection site, which may be different for different projections in the same experiment. Quantitative evidence presented here also shows that the apparent size of injection sites in the hippocampus decreases considerably in diameter 1 to 2 weeks after the injections are made. And second, the morphology of labeled pathways must be inferred from a pattern of silver grains lying over the tissue section. Several examples are used to illustrate the point that the autoradiographic method should be used in conjunction with retrograde transport, immunohistochemical, and electron microscopic techniques to investigate the origin, course, and synaptic relationships of individual pathways in the brain and spinal cord.
Wamsley, J.K.; Palacios, J.M.; Young, W.S.; Kuhar, M.J.
doi: 10.1177/29.1A_SUPPL.6270206pmid: 28054889
Light microscopic autoradiographical localization of drug and neurotransmitter receptors provides investigators with a tool to determine the sites of drug action with a high degree of anatomical resolution. In the cortex, where there is a distinct laminar organization, the differential distribution of receptors can be determined. We have found that differences in the density of specific receptors exist between layers of the cerebral cortex. Our observations include the localization of muscarinic cholinergic, benzodiazepine, opiate, neurotensin, histamine-H1, alpha-adrenergic, beta-adrenergic, and high-affinity GABA receptors in the cerebral cortex. Of this list of neurotransmitters only significant concentrations of benzodiazepine, GABA, and beta-adrenergic receptors were found in the cerebellar cortex. Receptor localizations such as these can be coupled with information from immunohistochemical studies to provide the basis for future experimentation in order to define neuro-transmitter-specific pathways in the cerebral and cerebellar cortices.
doi: 10.1177/29.1A_SUPPL.6270207pmid: 28054888
Recent advances in autoradiographic technique make it possible to localize quite a diverse class of soluble and/or diffusible substances. However since the molecules in question could move or be displaced at any time during preparation it is necessary to employ chemical or physiological tests to prove the validity of the localization. For two classes of compounds this can be done quite readily. 3H-ouabain, a cardiac glycoside that binds specifically to the Na pump, can be monitored for its effect on Na transport and the binding itself can be controlled by manipulating specific ligands known to modulate the ouabain-Na pump interaction. Acetylcholine can be readily analyzed by chemical means and its solubility characteristics allow one to extract it even from osmium-fixed embedded sections. Thus it is possible to monitor for the presence of acetylcholine in tissue throughout the processing for autoradiography and even chemically characterize small areas of tissue by microdissection of sectioned material.
Mizuhira, Vinci; Shiihashi, Michiko; Futaesaku, Yutaka
doi: 10.1177/29.1A_SUPPL.7288151pmid: 28054877
Three important factors are necessary for successful electron microscope autoradiography (EM-ARG): good resolution, proper preparation of the radioactive isotope (RI) labeled diffusible compounds, and shortened exposure time for ARG. The resolution problem is fundamental to EM-ARG. However, unless the diffusible RI compounds have been fixed correctly in the tissues during preparation, good resolution is useless. It is also necessary to shorten the exposure time for ARG. As yet, a high-speed ARG method for electron microscopy has not been reported, although scintillation ARG methods have been applied to macro- and micro-ARG since 1960. High specific activity, a large amount of radioactivity per unit exposure for radio incorporation (incubation), and careful selection of labeled compounds that concentrate in the DNA or RNA of cell organelles may increase the sensitivity of the emulsion and shorten the exposure time for ARG. For example, labeled thymidine accumulates in nuclear DNA, 3H-SPG (Schizophyllan-produced polyglucan) is incorporated into lysosomal granules, and labeled iodine concentrates in thyroid follicles, often increasing the sensitivity of the emulsion and shortening the exposure time. High-speed ARG yields good data in a very short time, but high-resolution ARG continues to be necessary, even though it requires 4 weeks or more of exposure time. Scintillation autoradiography using tritium seems unstable. We propose a new way to shorten exposure time for EM-ARG, by combining overdevelopment with coating both sides of the grid with emulsion. This method is approximately 100 times more sensitive than the conventional method, and only 4 days of exposure time are required, in contrast to the 1 month usually needed.
Sar, Madhabananda; Stumpf, Walter E.
doi: 10.1177/29.1A_SUPPL.6895229pmid: 28054880
A combined technique of autoradiography and immunohistochemistry is described for localization of radioactively labeled ligands and antibodies to neuropeptides, protein hormones and neurotransmitter synthesizing enzymes in the brain. This method permits the simultaneous visualization of radioactively labeled cells and neuropeptide-producing cells in the same tissue preparation. Autoradiograms are fixed with weak paraformaldehyde solution prior to photographic processing for subsequent immunoperoxidase staining. With this procedure 3H-estradiol is localized in neurophysin I and arginine vasopressin-producing cells of the mouse supraoptic and paraventricular nuclei, neurophysin-producing cells of the guinea pig hypothalamus, and dopamine-β-hydroxylase-containing neurons of the rat lower brainstem; 3H-dihydrotestosterone in pituitary gonadotropes and thyrotropes; and 3H-1,25-(OH)2 vitamin D3 in pituitary thyrotropes and pancreatic B-cells.
doi: 10.1177/29.1A_SUPPL.7288152pmid: 28054876
A refined version of the combined dry-mount auto-radiographic-immunocytochemical technique (Keefer DA, Stumpf WE, Petrusz P: Cell Tissue Res 166:25, 1976) is described in detail. In vivo nuclear estrogen uptake is measured by silver grain counting in immunocytochemically stained gonadotropes (G), somatotropes (S), lactotropes (L), corticotropes (C), and thyrotropes (T) of male rats. In rats 1 day after orchidectomy and adrenalectomy, the order of nuclear estrogen uptake was S = L G = C T, with T concentrating less than half as much radioactivity as L or S. Fifteen percent of anterior pituitary cells neither concentrated estrogen nor stained immunocytochemically. Estrogen uptake was examined in gonadotropes 1, 14, and 50 days after orchidectomy and was found to be identical at all three times. Estrogen uptake in gonadotropes of rats 14 days following orchidectomy and treatment with progesterone was reduced significantly. Guidelines for reliable quantification of the autoradiographic data is discussed.
Grant, Lester D.; Stumpf, Walter E.
doi: 10.1177/29.1A_SUPPL.7053241pmid: 28054875
A combined technique of formaldehyde-induced fluorescence (FIF) and autoradiography is described for the localization of radioactively labeled substances in relation to monoamine neurons. This method permits the simultaneous visualization of 3H-labeled steroid hormone or drug uptake sites and fluorescing monoamine neural elements (cell bodies, fiber projections, terminals) in the same tissue section. Thin frozen sections cut in a cryostat are freeze-dried, exposed to formaldehyde vapor at 80°C, and carried through dry-mount autoradiography processing steps before fluorescence microscopy screening. Subsequent histological staining of sections and light microscopy are employed for conventional autoradiogram screening. With this procedure, 3H-estradiol and 3H-dihydro-testosterone are localized in various catecholamine (CA) neurons in the diencephalon and lower brain stem of the rat. Also, catecholaminergic as well as noncatechol-aminergic sex steroid target neurons are seen to be innervated by CA terminals in various rat brain regions.
doi: 10.1177/29.1A_SUPPL.7288153pmid: 28054883
An autoradiographic technique adapted to diffusible compounds was used to localize steroid receptor sites in various tissues of the chicken embryo. In this article are presented results obtained on the gonads, the reproductive tract, the bursa of Fabricius, and the anterior pituitary after injection of either 3H-estradiol or 3H-dihydrotes-tosterone into 5 1/2- to 15-day-old chicken embryos. Target cells for steroid hormones, either estrogen or androgen, or both, are present in these organs early in the development. The precocity of the receptors suggests that sex steroids may influence embryonic differentiation earlier than currently recognized. The presence of the receptors in a variety of organs or tissues, known or unsuspected target for steroids, emphasizes the very diversified roles of sex steroid hormones in the differentiation processes. Conditions for these receptors to be active are discussed with respect to particularities of the embryonic system.
Showing 1 to 10 of 16 Articles