Cellular localization of 3H-oestradiol in the hypothalamus

Summary The hypothalamus of male and female rats, given 0.3 g/100 g body weight of 6.7-³H-oestradiol-17 and killed 1 hour after the injection, was examined by autoradiography in order to 1) localize the areas and the cells involved in the uptake of the hormone, and 2) study the intracellular localization of the labelled material.Only nerve cells contained radioactive material while glial and ependymal cells were not significantly labelled. In the anterior hypothalamus, labelled nerve cells were concentrated in areas corresponding to nucleus preopticus medialis and nucleus preopticus, pars suprachiasmatica. The nucleus supraopticus was unlabelled. In the medial basal hypothalamus, neurons corresponding to the nucleus arcuatus and the lateral part of the nucleus ventromedialis showed marked labelling. No significant labelling was observed in the nucleus paraventricularis, pars magnocellularis.Although the individual nerve cells varied in their extent of labelling, the major proportion of the silver grains were consistently concentrated over the nuclei. Castration was not found to influence the results. The findings were essentially the same in male and female rats and appear to suggest that oestradiol exerts a direct effect on nerve cells in certain hypothalamic areas.This work was supported by grants from the Norwegian Cancer Society, Nordisk Insulinfond and Anders Jahres Fond. The skilful assistance of Miss Helga Friedl and Mrs. Jane Larsen is gratefully acknowledged.     

Cellular and subcellular localization of 3H-diethylstilboestrol in the central nervous system

Summary The cellular and subcellular localization of radioactivity in the brain of immature female rats was determined by dry-mount autoradiography 2 h after iv injection of 1.0 g of (monethyl-³H) diethylstilboestrol per 100 g body weight. A specific topographic pattern of nuclear concentration of the synthetic oestrogen was obtained similar to that for ³H-oestradiol-17 in specific neurons of the basal hypothalamus, preoptic region and amygdala. In competition experiments, the nuclear concentration of radioactivity in all areas studied was inhibited by unlabeled oestradiol, while unlabeled testosterone had no effect. These data suggest that although oestradiol can bind to androgen receptors, the oestrogen receptor itself can account for the localization seen after the injection of ³H-oestradiol.This research was supported in part by US PHS Grant No. NS12933NIH Career Development Awardee No. NS00164The expert technical assistance of Ms. Riki Ison and Ms. Linda Furr is gratefully acknowledged.     

Ultrastructure of the male rat hypophysis chronically grafted under the kidney capsule

Summary Male rats were divided in two experimental groups. In group I two partes distales of the hypophysis were grafted under the kidney capsule and in group II two complete hypophyses were transplanted. Animals were killed 5 to 22 months after the operation. The grafted tissue was excised and processed for light and electron microscopy.The transplanted pars distalis tissue showed a well developed vascularisation in contrast to the pars intermedia which appeared poorly vascularised. Six different cell types were observed in grafted pars distalis. They correspond to the different types of cells found in the rat pars distalis in situ. The predominant cell type in the graft displayed all the morphological characteristics of stimulated prolactotrophs. Pars intermedia cells appeared hypertrophied resembling the MSH cells under stimulation. Two types of syncytial formations were frequently seen. One of them appeared to originate from prolactotrophs and the other from MSH cells. Bodian impregnated fibres and structures resembling either growth cones of axons or typical nerve endings were observed in the pars intermedia of long-term grafted hypophyses. Pituicytes remained as isolated clusters of cells. Canaliculi lined by two or more pituicytes were observed. Saccular formations resembling the hypophyseal cleft appeared in all grafts studied. The present findings suggest that in the male rat the chronically grafted pituitary gland is capable of synthesising most or all the hormones which are known to be produced by the gland in situ. Furthermore, prolactin and MSH seem to be the predominant secretion of the transplanted pituitary.Supported by the National Research Council of Argentina. Grant 7315/74Members of the Research Career of Conicet, Argentina     

Evaluation of the effects of sensory denervation on osteoblasts by 3H-proline autoradiography

Summary The inferior alveolar nerve was unilaterally resected in 30-day-old mice; other animals were unilaterally sham-operated. At 15, 30, 60, 90, or 150 days after surgery, the mice were injected with 2Ci of ³H-proline (sp. act. 1.0 Ci/mM) per g of body weight and killed 15, 30, or 60 min later. Autoradiographs were prepared from 5m decalcified sagittal sections of mandibles and grain counts made over periosteal osteoblasts mesial to the first molar. In denervated mandibles, osteoblasts incorporated less isotope compared to controls with differences being maximal at the early intervals. These differences became attenuated with time, possibly due to an intrinsic compensatory mechanism, secondary to neurotrophic regulation.Supported in part by NSF grant GU-3566     

Autoradiographic assessment of 3H-proline uptake by osteoblasts following guanethidine-induced sympathectomy in the rat

Summary Sympathectomy was carried out in rats by injections of guanethidine-sulfate from birth to 14 days of age. At 45 days of age, the activity of osteoblastic cells was monitored by ³H-proline autoradiography. Effectiveness of sympathectomy was verified by light-microscopic examination of superior cervical and celiac ganglia. Grain counts over periosteal osteoblasts of the femoral diaphysis and osteoblasts mesial to the first molar in the mandible demonstrated a significantly reduced uptake of ³H-proline in the sympathectomized rats. The data provide direct evidence of sympathetic influence on osteoblastic activity and suggest that sympathectomy may result in the loss of a trophic influence which is important in the regulation of osteogenesis.Supported by N.I.D.R. grant DEO 4557 (R.M.K.), N.I.H. grant 5-SO1RR-5373 to K.U.M.C., and N.I.H. grant RR-05332 to N.Y.U. (U.S.). We thank Charles A. Brownley of CIBA-Geigy, Summit, N.J. for the guanethidine sulfate     

Incorporation of 3H-thymidine in the nephron of Gasterosteus aculeatus L. and its stimulation by methyltestosterone

Summary The rate of ³H-thymidine incorporation into different parts of the renal proximal tubule of female sticklebacks treated with methyltestosterone was investigated using high-speed scintillation autoradiography. The results are compared with those from normal males before or after mucous transformation of the kidney. Labelled cells are observed in all parts of the proximal tubule, with marked variations from one segment to another. They are numerous in part 2 of the proximal tubule, particularly in the distal region. Male sex hormones affect the labelling rate in all parts of the nephron, especially in the distal region of part 2 of the proximal tubule. In that particular area, new tubule formation by budding is observed in some individuals, but this process does not appear to be a general one. Correlation between the frequency of these figures and the time of treatment could not be established. Comparing the action of sex hormones in females with that in males reveals a difference in reactivity in the proximal zone of part 2 of the proximal tubule, where methyltestosterone has a strong action in females; in contrast, in mature and immature males, only a few labelled cells are present in this region.It is concluded that kidney enlargement during the breeding season does not result only from a swelling of cells belonging to part 2 of the proximal tubule, as was generally believed, but also from a lengthening or even a proliferation of the proximal tubules, induced by an increase in mitotic activity controlled by male sex hormones.

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Résumé L'incorporation de thymidine tritiée dans les tubules proximaux du rein est étudiée par autoradiographie rapide chez des Epinoches femelles préalablement traitées par la méthyltestostérone. Les résultats sont comparés avec ceux obtenus chez des mâles normaux ayant ou non développé un rein muqueux. Des cellules marquées sont présentes à tous les niveaux des tubules proximaux, mais leur nombre varie considérablement d'un segment à l'autre. Elles sont les plus nombreuses dans la seconde partie du tubule proximal, particulièrement dans sa région distale. Le taux de marquage est modifié dans toutes les régions du néphron, mais les variations sont les plus intenses dans la région distale de la seconde partie du tubule proximal, où l'on observe parfois la formation de nouveaux tubules par bourgeonnement. La comparaison des résultats obtenus dans les deux sexes fait apparaître une différence de réactivité au niveau de la zone proximale de la seconde partie du tubule proximal, où la méthyltestostérone agit fortement chez les femelles, alors qu'elle n'exerce pas d'effet notable chez les mâles.L'augmentation de volume du rein chez le mâle lors de la période de reproduction ne résulte donc pas uniquement d'un gonflement des cellules de la seconde partie du tubule proximal, comme on le pensait généralement. Des phénomènes mitotiques interviennent également dans ce processus, sous l'influence des hormones sexuelles mâles, qui conduisent à un allongement, voire à une multiplication des tubules proximaux.

     

Light and electron microscopic autoradiography of substantia nigra of rat after intraventricular administration of tritium labelled norepinephrine,dopamine, serotonin and the precursors

Summary In autoradiographies of substantia nigra in rat, it has been observed that after intraventricular injections of ³H-dopamine and ³H-norepinephrine respectively the silvergrains are accumulated in nigra neurons and their dendritic branches. The incorporation was more pronounced in the case of ³H-norepinephrine than ³H-dopamine. This seems to indicate that exogenous norepinephrine may have stronger affinity to nigra neurons and their dendrites than exogenous dopamine. In addition, some ³H-dopamine and ³H-norepinephrine labelled nerve terminals were observed in axo-dendritic synapses. In contrast to these data, ³H-5HTP and ³H-5HT administration showed almost all silver grains accumulated in the neuropil when observed in light microscopic autoradiography. Electron micrographs further reveal that the incorporation of ³H-5HTP and ³H-5HT was mostly within axo-dendritic boutons with more frequent dense core vesicles. These data again strongly suggest that substantia nigra receives a large number of serotoninergic fibres forming axo-dendritic synapses which may play an important role in modulation of substantia nigra function.Dr. Parizek was on leave of absence from the Charles University, Faculty of Medicine, Hradec Králové, Czechoslovakia.     

Intercellular channels in the pars tuberalis of the rat hypophysis and their relationship to the subarachnoid space

Summary A system of intercellular channels is described in the pars tuberalis (PT) of the female rat. These spaces are lined by all types of cells found in the PT and are not sealed off by tight junctions. Ventrally and dorsally, the intercellular spaces open toward the basement membranes separating the PT from (i) the subarachnoid space, and (ii) the perivascular space of the portal capillaries, respectively. These intercellular channels differ from the follicles, which are also found in the PT, being lined by a particular type of cell.In a second group of female rats an epoxy mixture was injected into the third ventricle; 10 min thereafter horseradish peroxidase was infused into the cisterna magna. After processing the brain for the demonstration of exogenous peroxidase, it was found that the tracer had reached the subarachnoid space adjacent to the hypothalamus and entered into all ventricular cavities with the exception of the infundibular recess. Under these experimental conditions it was found that the tracer fills all intercellular channels of the PT, thus indicating that there is no barrier between the subarachnoid space and the PT. It is suggested that the subarachnoid space should be regarded as a probable route for the transport of trophic factor(s) and/or secretory product(s) of the PT.Supported by Grant S-80-13 from Directión de Investigaciones, Universidad Austral de Chile     

Quantitative electron microscopic autoradiography on the mouse thyroid gland after administration of 3H-L-DOPA

Summary The cellular and subcellular distribution of radioactivity in the mouse thyroid gland different times (20 min — 8 hours) after intravenous administration of ³H-L-DOPA was studied by means of quantitative electron microscopic autoradiography.High concentrations of autoradiographic silver grains occur over parafollicular cells and adrenergic nerves while the labelling of follicular cells and lumina is low or absent and similar to the labelling of connective tissue cells at all observation times.Over the parafollicular cells high levels of radioactivity can be recorded already 20 min after administration of the labelled amino acid. The grain counts are highest at 1 hour and decrease then at 2.5 and 8 hours.The intracellular distribution of label is similar at all observation times; thus, the concentration of silver grains over the typical cytoplasmic granules of the parafollicular cells is 4–5 times higher compared to the concentration over the remainder of the cytoplasm and the nucleus.Treatment with a decarboxylase inhibitor prior to the injection of ³H-L-DOPA results in a low and uniform labelling of all thyroid cells. This finding, taken together with the observation that also pretreatment with reserpine abolishes the autoradiographic reaction over the cytoplasmic granules, gives strong support to the idea that the great majority of silver grains observed over parafollicular cells represents dopamine formed by decarboxylation of the labelled precursor.This study was supported by grant K71-12X-3352-01 from the Swedish Medical Research Council. The author wishes to express his gratitude to Mrs. Gunnel Bokhede and Miss Dala Sjögren for expert technical assistance.     

In vitro binding of 125I-HCG to the rat ovary from birth to puberty

Summary Neither homogenates nor frozen ovarian sections of rats aged 1, 5 and 7 days demonstrated specific binding of ¹²⁵I-HCG. However, from day 10 to day 35, the homogenates could bind specifically, with an equilibrium association constant in the range of 0.9–2.7 × 10¹⁰ M^–1. The binding capacity increased from 5.8 fmol/mg tissue at day 10 to 15.7 fmol/mg tissue at day 35.As revealed by autoradiography, the frozen ovarian sections from 10-day-old rats showed ¹²⁵I-HCG binding localized over the interstitial and thecal tissues and, in the older age groups, also in the granulosa cells of follicles larger than 500 m in diameter.These results indicate that LH/HCG receptors appear in the rat ovary at the beginning of the second postnatal week, and that the interstitial cells are the main site of action of LH before puberty.     

Specific localization of 2′,3′-cyclic nucleotide 3′-phosphohydrolase, (Ca/Mg)-ATPase, and acetylcholinesterase in human erythyrocyte membrane

A procedure was developed for the detection of 2′,3′-cyclic nucleotide 3′-phosphohydrolase in myelin. This assay was sufficiently sensitive to detect the low levels of 2′,3′-cyclic nucleotide 3′-phosphohydrolase in human erythrocytes. The 2′,3′-cyclic nucleotide 3′-phosphohydrolase of human erythrocytes was determined to be exclusively associated with the inner (cytosolic) side of the membrane. Leaky ghostsand resealed ghosts were assayed for 2′,3′-cyclic nucleotide 3′-phosphohydrolase, (Ca²⁺/Mg²⁺-ATPase, and acetylcholinesterase activity, and the 2′,3′-cyclic nucleotide 3′-phosphohydrolase profile is the same as that of the (Ca²⁺/Mg²⁺)-ATPase, an established inner membrane maker.     

Immunohistochemical localization of taurine in various tissues of the mouse

Summary The localization of taurine was investigated in several tissues of the mouse. Immunohistochemical methods using a polyclonal antibody for taurine derived from rabbits was used in these studies. This method was used since it is a simple procedure and the results are clear and reliable. Tissues were fixed with paraformaldehyde, embedded in paraffin and treated in a microwave oven before using an avidin-biotin-complex method (ABC method). Control staining was accomplished by employing absorption staining using various amino acids: taurine, arginine, cysteine, hypotaurine and others. For purposes of comparison, radioautography (RAG) with³H-taurine was performed to confirm the reliability of the immunohistochemical staining compared with the localization of the³H-taurine incorporation in endothelial cells of the blood vessels of several tissues. In this investigation, immunoreactivity was broadly observed in many tissues: Purkinje cells of the cerebellum, glia cells of brain tissue, cardiac muscle cells, matrices of the bone, mucus granules of goblet cells of the intestines, and brown adipose cells of the fetus. Although the meaning of this widespread localization of taurine can not be explained completely, we surmise that taurine may have a different function in each of the tissues. In addition, taurine reactivity was observed in cell nuclei which was evidence of the presence of taurine in the nuclei.     

Autoradiographic localization of 3H-5-HTP and 3H-5-HT in the pineal organ and circumventricular areas in the rainbow trout,Salmo gairdneri Richardson

Summary The time course of incorporation and cellular localization of ³H-5-hydroxytryptophan (³H-5-HTP) and ³H-5-hydroxytryptamine (³H-5-HT) in the pineal and some brain regions in rainbow trout, Salmo gairdneri, were studied by quantitative and qualitative autoradiography.Among the tissues examined, the pineal shows the highest and most rapid uptake of the two isotopes. Maximum incorporation of ³H-5-HTP is achieved by 2 h and that of ³H-5-HT by 20 minutes post injection. At the end of the six-hour experimental period, a significantly high amount of radioactivity is still detectable in the pineal. The results indicate a much slower turnover of the two indoles, especially 5-HTP, in the trout than is known for mammalian tissues.Both the ependymal supporting cells and the receptor cells of the pineal localize these isotopes. In contrast, the intrapineal neurons remain unlabeled. This is taken to suggest lack of capacity of these cells to metabolize 5-HTP and 5-HT.In the circumventricular regions, the two indoles occur in the ependyma of the recessus lateralis and the recessus praeopticus. The label is also localized in the neuropil and the neurons of the nuclei recessus lateralis and praeopticus. Semiquantitative estimates reveal a significant labeling of these areas only 20 minutes after injection, although a weak but inconsistent labeling of the ependyma is evident at 5 minutes. The significance of these results is discussed in regard to (a) normal capacity of circumventricular areas to metabolize indoles and (b) a possible chemical interaction between the pineal and the brain involving a direct pineal-cerebrospinal fluid pathway.Supported in part by a grant to the senior author from the University of Kentucky Research FoundationThe authors wish to thank the Department of Fish and Game, Kentucky, for supplying rainbow trout. The technical assistance of Mrs. Munira Nasser is gratefully acknowledged     

Cellular localization of a putative Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchanger 3 during ontogeny in the pronephros and mesonephros of the Japanese black salamander (<Emphasis Type="Italic">Hynobius nigrescens</Emphasis> Stejneger)

The cloning of cDNA and an examination of the tissue distribution of Na⁺/H⁺ exchanger 3 (NHE3) were carried out in the Japanese black salamander, Hynobius nigrescens. The cellular localization of Hynobius NHE3 was examined by in situ hybridization and immunohistochemistry during ontogeny in the nephron of the pronephros and mesonephros of the salamander. The partial amino acid sequence of Hynobius NHE3 was 81% and 72% identical to rat NHE3 and stingray NHE3, respectively. Hynobius NHE3 mRNA and protein were exclusively expressed along the late portion of the distal tubule to the anterior part of the pronephric duct of premetamorphic larvae (IY stages 43–50). NHE3 mRNA was expressed in the pronephros but not in the external gills in the larvae at the digit differentiation stage (IY stage 50). In the adult, mRNA was strongly expressed in the mesonephros but not in the ventral and dorsal skin. In juvenile and adult specimens, NHE3 immunoreactivity was observed at the apical membrane of the initial parts of the distal tubules of the mesonephric kidney. Immunohistochemical and in situ hybridization studies suggested that Na⁺ absorption coupled with H⁺ secretion via NHE3 occurred in the distal nephron of the pronephros and mesonephros. This is the first study to indicate NHE3 expression during ontogeny in amphibians. This work was supported in part by a research grant (a priority project in Science Faculty) from the University of Toyama to M.U.     

Cellular localization of cannabinoid receptors and activated G-proteins in rat anterior cingulate cortex

Cannabinoid receptors are found in moderate density throughout the cerebral cortex. The anterior cingulate cortex (ACC) is of particular interest due its high level of cannabinoid receptors and role in behaviors known to be modulated by cannabinoids. These studies were conducted to determine the cellular localization of cannabinoid receptors and to compare the level of cannabinoid receptor binding with receptor-mediated G-protein activity in the rat ACC. Either ibotenic acid or undercut lesions were made in ACC, and brains were processed for [3H]WIN 55,212-2 and WIN 55,212-2-stimulated [35S]GTPgammaS autoradiography. Both cannabinoid receptors and receptor-activated G-proteins were highest in laminae I and VI of ACC in control tissue. Although similar levels of receptor binding were found in these laminae, significantly higher levels of receptor-activated G-proteins were found in lamina VI. Ibotenic acid lesions that destroyed ACC neurons decreased [3H]WIN 55,212-2 binding by 60-70% and eliminated WIN 55,212-2-stimulated [35S]GTPgammaS binding. In contrast, deafferentation of the ACC with undercut lesions had no significant effect on cannabinoid receptor binding or G-protein activation. These results indicate that cannabinoid receptors in laminae I and VI of the ACC are located on somatodendritic elements or axons intrinsic to the ACC. In addition, differences in the relative levels of cannabinoid binding sites and activated G-proteins between cortical laminae indicate that the efficiency of cannabinoid receptors for G-protein activation may vary within a specific brain region.     

Estradiol receptor binding to the epithelium of uterine lumen and glands: region- and time-related changes during preimplantation and periimplantation periods studied by autoradiography

The presence and changes of estradiol nuclear binding and related functions in uterine luminal and glandular epithelium were studied before and after blastocyst implantation using receptor autoradiography with ³H-estradiol-17 in association with ³H-thymidine incorporation and immunocytochemical binding of antibody to estrogen receptor ER-. ³H-estradiol nuclear binding is present but variable during days 1.5–7.5 of pregnancy. Sites of strong nuclear binding of ³H-estradiol exhibit strong immunocytochemical staining with ER- antibody. Qualitative and quantitative evaluation of autoradiograms reveal that there is a general increase of nuclear ³H-estradiol binding during the first 3 days after fertilization in both luminal and glandular epithelium. The binding of estradiol is stronger in glandular epithelium from day 2.5 to day 7.5, paralleled by a rise in ³H-thymidine incorporation on day 2.5. By comparison, in the epithelium of the uterine lumen ³H-estradiol nuclear binding is low, but relatively high in epithelial cells at lateral branching of the lumen where the increase in ³H-estradiol binding corresponds to an increased labeling index with ³H-thymidine. A highly differentiated binding of ³H-estradiol to luminal and glandular epithelium was demonstrated with region- and time-specific changes of related effects on cell proliferation, differentiation, and secretion, probably involving involution and remodeling. The strong ³H-estradiol binding to glandular epithelium suggests that estradiol exerts pronounced effects on glandular activities in the periimplantation period.     

Neophobia and cortical and subcortical binding of the dopamine D2 receptor antagonist [3H]-raclopride

The potential role of dopamine system in response to novelty was analysed using the selective dopamine D2 receptor antagonist, raclopride, in behavioral and biochemical assays, in rats (the open field test, and specific binding of [3H]-raclopride, within different brain structures measured with autoradiography). It was found that raclopride at a low dose (50 microg/kg, IP) caused anxiolytic-like effect (increased the anti-thigmotactic index), whereas at a higher dose (500 microg/kg, IP) produced general inhibitory influence, and decreased the anti-thigmotactic index. Analysis of the behavioral and biochemical results of the experiment revealed a significant negative correlation between the ligand binding in the substantia nigra pars reticulata (SNR), and the number of entries into the central sector of the open field (r=-0.48, p<0.05), as well as the positive correlation between time spent in the central sector of the open field and [3H]-raclopride binding within nucleus accumbens septi (r=0.57, p<0.05). Factor analysis revealed a Factor 1 (eigenvalue=3.361) grouping parameters of central entries into the open field and [3H]-raclopride binding in the SNR (factor loadings are 0.814 and 0.703 respectively), indicating that both phenomena are under control of a similar central process. The above data are discussed in relation to the structure dependent dopamine D2 receptor mechanisms in a rat response to novelty.     

Cellular and transepithelial responses of goldfish intestinal epithelium to chloride substitutions

Summary In goldfish intestine chloride was substituted by large inorganic anions (gluconate or glucuronate) either mucosally, serosally or bilaterally. Changes in intracellular activities of chloride (a _i Cl^–), sodium (a _i Na⁺) and potassium (a _i K⁺), pH_i, relative volume, membrane and transepithelial potentials, transepithelial resistance and voltage divider ratio were measured. Control values were:a _i Cl^–=35 meq/liter, a _i Na⁺=11 meq/liter and a _i K⁺=95 meq/liter. During bilateral substitution the latter two did not change while a _i Cl^– dropped to virtually zero.Mucosal membrane potentials (_ms) were: control,-53 mV; serosal substitution,-51 mV; bilateral substitution,-66 mV; while during mucosal substitution a transient depolarization occurred and the final steady state _ms was-66 mV.During control and bilateral substitution the transepithelial potentials (_ms) did not differ from zero. During unilateral substitutions _ms was small, in the order of magnitude of the errors in the liquid junction potentials near the measuring salt bridges.During bilateral substitution pH _i increased 0.4 pH units. Cellular volume decreased during mucosal substitution to 88% in 40 min; after serosal substitution it transiently increased, but the new steady-state value was not significantly above its control value.Three minutes after mucosal substitution ana _i Cl^– of approx. 10 meq/liter was measured.Chemical concentrations of Na, K and Cl were determined under control conditions and bilateral substitution. Cl concentrations were also measured as a function of time after unilateral substitutions.The data indicate an electrically silent chloride influx mechanism in the brush border membrane and an electrodiffusional chloride efflux in the basolateral membrane. A substantial bicarbonate permeability is present in the basolateral membrane. The results are in agreement with the observed changes in membrane resistances, volume changes and pH changes.