Simultaneous Detection of Neuropeptides and Messenger RNA in the Magnocellular Hypothalamo-neurohypophysial System by a Combination of Non-radioactive in Situ Hybridization Histochemistry and Immunohistochemistry

Overview

Journal Histochemistry

Specialty Biochemistry

Date 1994 Dec 1

PMID 7698898

Authors

P J Larsen

J D Mikkelsen

Affiliations

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Abstract

A protocol was developed combining non-radioactive in situ hybridization histochemistry with enzyme based immunohistochemistry, detect the expression of mRNA in phenotypically defined neurons. Free-floating brain sections were hybridized with the oligonucleotide probes which have been 3'-end labelled with biotin-11-dUTP. The hybridized probe was visualized by a combined avidin-biotin bridge method, anti-avidin immunohistochemistry, and horseradish peroxidase detection using diaminobenzidine as a substrate. The in situ hybridization step yielded a very stable reaction product enabling subsequent immunohistochemical reactions using horseradish peroxidase and benzidine dihydrochloride as a chromogen. Magnocellular neurons of the hypothalamo-neurophypophysial system synthesize either vasopressin or oxytocin; water deprivation and chronic saline ingestion are potent stimuli for the expression of both of the genes encoding these neuropeptides. A number of other neuropeptides with putative transmitter action are synthesized in magnocellular neurons during such stimulation. Experiments were performed to explore whether neuropeptide Y immunoreactivity is present within magnocellular vasopressin mRNA-expressing neurons of the hypothalamo-neurophypophysial system. The results clearly demonstrated that neuropeptide Y-immunoreactive elements were present within a number of magnocellular vasopressin mRNA-containing cells. In addition, immunohistochemical detection of the neuropeptides ocytocin and cholecystokinin was carried out on sections hybridized non-radioactively for vasopressin; as expected vasopressin mRNA did not co-exist with cholecystokinin, whereas a few oxytocin immunoreactive neurons in osmotically stimulated animals also contained vasopressin mRNA. The developed method makes possible the immunohistochemical detection of intracellular antigens with concomitant detection of intracellular mRNA.

References

Young 3rd W . Simultaneous use of digoxigenin- and radiolabeled oligodeoxyribonucleotide probes for hybridization histochemistry. Neuropeptides. 1989; 13(4):271-5. DOI: 10.1016/0143-4179(89)90081-4. View

Jablonski E, Moomaw E, TULLIS R, Ruth J . Preparation of oligodeoxynucleotide-alkaline phosphatase conjugates and their use as hybridization probes. Nucleic Acids Res. 1986; 14(15):6115-28. PMC: 311625. DOI: 10.1093/nar/14.15.6115. View

Eckland D, Todd K, Lightman S . Immunoreactive vasopressin and oxytocin in hypothalamo-hypophysial portal blood of the Brattleboro and Long-Evans rat: effect of adrenalectomy and dexamethasone. J Endocrinol. 1988; 117(1):27-34. DOI: 10.1677/joe.0.1170027. View

Brownstein M, Mezey E . Multiple chemical messengers in hypothalamic magnocellular neurons. Prog Brain Res. 1986; 68:161-8. DOI: 10.1016/s0079-6123(08)60237-x. View

Ivell R, Richter D . Structure and comparison of the oxytocin and vasopressin genes from rat. Proc Natl Acad Sci U S A. 1984; 81(7):2006-10. PMC: 345425. DOI: 10.1073/pnas.81.7.2006. View

Schalling M, Hokfelt T, Wallace B, Goldstein M, Filer D, Yamin C . Tyrosine 3-hydroxylase in rat brain and adrenal medulla: hybridization histochemistry and immunohistochemistry combined with retrograde tracing. Proc Natl Acad Sci U S A. 1986; 83(16):6208-12. PMC: 386469. DOI: 10.1073/pnas.83.16.6208. View

Watson S, Akil H, Fischli W, Goldstein A, Zimmerman E, Nilaver G . Dynorphin and vasopressin: common localization in magnocellular neurons. Science. 1982; 216(4541):85-7. DOI: 10.1126/science.6121376. View

Fuller P, Clements J, Funder J . Localization of arginine vasopressin-neurophysin II messenger ribonucleic acid in the hypothalamus of control and Brattleboro rats by hybridization histochemistry with a synthetic pentadecamer oligonucleotide probe. Endocrinology. 1985; 116(6):2366-8. DOI: 10.1210/endo-116-6-2366. View

Trembleau A, Roche D, Calas A . Combination of non-radioactive and radioactive in situ hybridization with immunohistochemistry: a new method allowing the simultaneous detection of two mRNAs and one antigen in the same brain tissue section. J Histochem Cytochem. 1993; 41(4):489-98. DOI: 10.1177/41.4.8095508. View

10.

Larsen P, Mikkelsen J . Vasoactive intestinal peptide (VIP) in magnocellular neurons of the hypothalamo-neurohypophysial system of the mink (Mustela vision) is co-localized with vasopressin or oxytocin. J Comp Neurol. 1992; 326(2):180-92. DOI: 10.1002/cne.903260203. View

11.

Baldino Jr F, OKane T, Wolfson B . Coordinate hormonal and synaptic regulation of vasopressin messenger RNA. Science. 1988; 241(4868):978-81. DOI: 10.1126/science.3406747. View

12.

Shivers B, Harlan R, Pfaff D, Schachter B . Combination of immunocytochemistry and in situ hybridization in the same tissue section of rat pituitary. J Histochem Cytochem. 1986; 34(1):39-43. DOI: 10.1177/34.1.3510246. View

13.

Uhl G, Zingg H, Habener J . Vasopressin mRNA in situ hybridization: localization and regulation studied with oligonucleotide cDNA probes in normal and Brattleboro rat hypothalamus. Proc Natl Acad Sci U S A. 1985; 82(16):5555-9. PMC: 391161. DOI: 10.1073/pnas.82.16.5555. View

14.

Larsen P, Sheikh S, Mikkelsen J . Osmotic regulation of neuropeptide Y and its binding sites in the magnocellular hypothalamo-neurohypophysial pathway. Brain Res. 1992; 573(2):181-9. DOI: 10.1016/0006-8993(92)90761-w. View

15.

Meister B, Villar M, Ceccatelli S, Hokfelt T . Localization of chemical messengers in magnocellular neurons of the hypothalamic supraoptic and paraventricular nuclei: an immunohistochemical study using experimental manipulations. Neuroscience. 1990; 37(3):603-33. DOI: 10.1016/0306-4522(90)90094-k. View

16.

Hatton G . Emerging concepts of structure-function dynamics in adult brain: the hypothalamo-neurohypophysial system. Prog Neurobiol. 1990; 34(6):437-504. DOI: 10.1016/0301-0082(90)90017-b. View

17.

Rehfeld J . Immunochemical studies on cholecystokinin. I. Development of sequence-specific radioimmunoassays for porcine triacontatriapeptide cholecystokinin. J Biol Chem. 1978; 253(11):4016-21. View

18.

Young 3rd W, Mezey E, Siegel R . Vasopressin and oxytocin mRNAs in adrenalectomized and Brattleboro rats: analysis by quantitative in situ hybridization histochemistry. Brain Res. 1986; 387(3):231-41. DOI: 10.1016/0169-328x(86)90029-x. View

19.

Mezey E, Kiss J . Coexpression of vasopressin and oxytocin in hypothalamic supraoptic neurons of lactating rats. Endocrinology. 1991; 129(4):1814-20. DOI: 10.1210/endo-129-4-1814. View

20.

Kiyama H, Emson P . Evidence for the co-expression of oxytocin and vasopressin messenger ribonucleic acids in magnocellular neurosecretory cells: simultaneous demonstration of two neurohypophysin messenger ribonucleic acids by hybridization histochemistry. J Neuroendocrinol. 2009; 2(3):257-9. DOI: 10.1111/j.1365-2826.1990.tb00401.x. View