Characterization of proopiomelanocortin mRNA detected by in situ hybridization

Kelsey, J. A.; Watson, S.J.; Burke, Sharon; Akil, Huda; Jl, Roberts

doi:10.1523/jneurosci.06-01-00038.1986

Cited by 39 publications

(20 citation statements)

References 17 publications

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“…These values are close to the value obtained by using a 5'-32P-labeled probe (tm = 690C; Fig. 4), as well as to a theoretically calculated (19,(30)(31)(32) value of 690C. In contrast to these tm values and the characteristic sharp sigmoid melting curve for the intermediate lobe signal, the posterior lobe signal was removed as a linear function of temperature, with the same lower apparent tm value of 520C for the short-and long-tailed probes (Fig.…”

Section: Resultssupporting

confidence: 90%

“…This labeled probe hybridized to the intermediate lobe of pituitary ( Fig. 1), a region shown by previous studies to contain molecules hybridizing to much longer probes derived from POMC cDNA (7,11,14,19). The anatomical specificity of this labeling was indicated by the expected relative absence of label in the posterior lobe.…”

Section: Resultsmentioning

confidence: 64%

“…For example, in rat pituitary, proopiomelanocortin (POMC) mRNA levels are regulated by glucocorticoids only in the anterior lobe corticotrophs and by dopamine only in the intermediate lobe melanotrophs (1)(2)(3)(4)(5)(6)(7). Because of such subtle complexities, investigators have begun to study mRNA regulation at the single-cell level by in situ hybridization with cDNA (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19) or complementary RNA (20) probes. The (21).…”

mentioning

confidence: 99%

“…The tissues were then frozen in liquid N2, and 10-,um-thick sections were cut in a cryostat at -20°C, thaw-mounted onto gelatin-coated slides, and stored at -80°C. Probes The rat POMC cDNA was 32P-labeled by nick-translation as described elsewhere (7,14,19).…”

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confidence: 99%

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Detection of proopiomelanocortin mRNA by in situ hybridization with an oligonucleotide probe.

Lewis

Sherman

Burke

et al. 1986

Proc. Natl. Acad. Sci. U.S.A.

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Synthetic oligonucleotide probes can be easily obtained and used, in contrast to cDNA cloning to develop probes, and thus the present study was carried out to determine whether such probes could also be useful for in situ hybridization. A 24-base synthetic oligonucleotide complementary to part of the a-melanocyte-stimulating hormone (a-MSH) coding region of proopiomelanocortin (POMC) mRNA was 5'-end-labeled by using [y-32P]ATP with T4 polynucleotide kinase or was 3' tailed by using [a-32P] (200-250 g; Charles River Breeding Laboratories) were anesthetized with sodium pentobarbital (150 mg/kg, i.p.) and perfused intracardially with 50 ml of ice-cold saline followed by cold 0.1 M phosphate-buffered 4% formaldehyde for 30 min (21). One group of animals (n = 6) had been treated with haloperidol (2 mg/kg per day, i.p.) or saline for 4 days prior to perfusion. Pituitaries were removed, postfixed in ice-cold buffered formaldehyde for 1 hr, and incubated overnight in 15% sucrose in 0.02 M phosphate-buffered saline (21) at 4°C. The tissues were then frozen in liquid N2, and 10-,um-thick sections were cut in a cryostat at -20°C, thaw-mounted onto gelatin-coated slides, and stored at -80°C.Probes

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Section: Resultssupporting

confidence: 90%

Section: Resultsmentioning

confidence: 64%

mentioning

confidence: 99%

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confidence: 99%

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Detection of proopiomelanocortin mRNA by in situ hybridization with an oligonucleotide probe.

Lewis

Sherman

Burke

et al. 1986

Proc. Natl. Acad. Sci. U.S.A.

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“…The intensity of the hybridization signal was measured as described (23,24). The optical density and area of the hybridization signal on x-ray film over each nucleus were analyzed using an image-analysis system (Loats Associates, Westminster, MD).…”

Section: Methodsmentioning

confidence: 99%

Thyroid hormones regulate levels of thyrotropin-releasing-hormone mRNA in the paraventricular nucleus.

Koller

Wolff

Warden

et al. 1987

Proc. Natl. Acad. Sci. U.S.A.

166

102

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Cellular levels of messenger RNA encoding thyrotropin-releasing hormone (TRH) were measured in the paraventricular nucleus of the hypothalamus and the reticular nucleus of the thalamus in male rats after chemical thyroidectomy and thyroid hormone replacement. TRH mRNA levels were measured by quantitative in situ hybridization histochemistry using a 35S-labeled synthetic 48-base oligodeoxynucleotide probe and quantitative autoradiography. Chemical thyroidectomy, produced by the administration of 6-(n-propyl)-2-thiouracil (PrSur), reduced plasma thyroxine below detection limits and significantly increased TRH mRNA in the paraventricular nucleus. Treatment with exogenous L-triiodothyronine (T3) reduced TRH mRNA to the same level in both hypothyroid and euthyroid animals. Neither PrSur treatment nor T3 replacement influenced TRH mRNA levels in the reticular nucleus of the thalamus. Blot hybridization analysis of electrophoretically fractionated total RNA from pituitaries of these animals indicated that thyrotropin-P mRNA levels were elevated after thyroidectomy and reduced by T3 treatment, showing that the pituitary-thyroid axis was indeed stimulated by PrSur treatment. These results suggest that thyroid hormones are involved, either directly or indirectly, in regulating the biosynthesis of TRH in the thyrotropic center of the hypothalamus.Thyroid hormones [thyroxine (T4) and triiodothyronine (T3)] exert a negative feedback effect on thyrotropin (thyroidstimulating hormone, TSH) secretion from the pituitary (1). However, while thyroliberin (thyrotropin-releasing hormone, TRH) is known to regulate pituitary-thyroid function (2), the effect that thyroid hormones have on TRH has remained unclear. Thyroid hormones do seem to inhibit TRH release, since thyroxine implants in the hypothalamus of the rat and cat inhibit thyroid function (3, 4). Furthermore, thyroxine treatment has been reported to decrease hypothalamic TRH immunoreactivity in intact rats (5) and increase hypothalamic TRH immunoreactivity in thyroidectomized rats (6). However, these findings are not universally accepted (7). Perhaps the discrepant results reported by members of different laboratories are due to the fact that tissue levels of peptides do not necessarily correlate with secretion.Peptide biosynthesis, on the other hand, may provide a reliable index of long-term changes in secretion (8-13). TRH is synthesized from a precursor protein, which is translated from a specific mRNA (14), and we can now measure cellular levels of TRH mRNA by quantitative in situ hybridization histochemistry (15). This technique provides an approach with sufficient sensitivity and neuroanatomical resolution to examine the regulation of TRH biosynthesis in specific subpopulations of TRH-positive cells. Although TRH-containing neurons are widespread throughout the brain (16), the cells involved in regulating thyroid function reside almost exclusively within the paraventricular nucleus (PVN) (17,18). In this report, we describe experiments designed to determine whet...

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Quantitative morphology of the nervous system: Expanding horizons

et al. 1991

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In this review, we show how some of the recent developments in quantitative morphology (QM) are creating exciting new opportunities for studying the structure of the nervous system. We begin with a brief overview of QM, focusing on the problems neurobiologists are likely to encounter when collecting and interpreting data from tissue sections. Many of these problems, which range from selecting a sampling method to learning the latest methods, are being solved by creating a new generation of research tools. We describe several of these new tools and show how they can be used to assemble new quantitative methods for in situ hybridization, immunocytochemistry, and camera lucida drawings. The review includes examples of how QM is being used to study the brain and concludes with a brief discussion of diagnostic pathology and its need for new quantitative approaches.

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Characterization of proopiomelanocortin mRNA detected by in situ hybridization

Cited by 39 publications

References 17 publications

Detection of proopiomelanocortin mRNA by in situ hybridization with an oligonucleotide probe.

Detection of proopiomelanocortin mRNA by in situ hybridization with an oligonucleotide probe.

Thyroid hormones regulate levels of thyrotropin-releasing-hormone mRNA in the paraventricular nucleus.

Quantitative morphology of the nervous system: Expanding horizons

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