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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

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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. Freefloating 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.

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References

  1. Altar CA, Ryan S, Abood M, Eberwine JH (1989) In situ mRNA hybridization: standard procedures and novel approaches. In: PM Conn (ed) Methods in neuroscience: gene probes. Academic Press, San Diego, pp 238–281

  2. Angerer LM, Stoler MH, Angerer RC (1987) In situ hybridization with RNA probes: an annoted recipe. In: KL Valentino, JH Erberwine, JD Barchas (eds) In situ hybridization: applications to neurobiology, Oxford University Press, London New York, pp 42–70

  3. Baldino F, O'Kane TM, Fitzpatrick-McElligott S, Wolfson B (1988) Coordinate hormonal and synaptic regulation of vasopressin messenger RNA. Science 241:978

  4. Bisset GW, Chowdrey HS (1988) Control of release of vasopressin by neuroendocrine reflexes. Q J Exp Physiol 73:811–872

  5. Brownstein MJ, Mezey E (1986) Multiple chemical messengers in hypothalamic magnocellular neurons. Prog Brain Res 68:161–168

  6. Eckland DJA, Todd K, Lightman SL (1987) Immunoreactive vasopressin and oxytocin in hypothalami-hypophysial portal blood of the Brattleboro and Long-Evans rat: effect of adrenalectomy and dexamethasone. J Endocrinol 117:27–34

  7. Fuller PJ, Clements JA, Funder JW (1985) Localization of arginine-vasopressin-neurophysin II messenger ribonucleic acid in the hypothalamus of control and Brattleboro rats by in situ hybridization histochemistry with a synthetic pentadecamer oligonucleotide probe. Endocrinology 116:2366–2368

  8. Hatton GI (1990) Emerging concepts of structure-function dynamics in adult barin: the hypothalamo-neurophypophysial system. Prog Neurobiol 34:437–504

  9. Herman JP, Schafer MKH, Watson SJ, Sherman TG (1991) Hybridization analysis of arginine vasopressin gene transcription using intron-specific probes. Mol Endocrinol 5:1447–1456

  10. Ichiyama Y, Emson PC, Christodoulou C, Gait MJ, Ruth JL (1989) Simultaneous visualization of vasopressin and oxytocin mRNA-containing neruons in the hypothalamus using non-radioactive in situ hybridization histochemistry. J Neuroendocrinol 1:73–75

  11. Ivell R, Richter D (1984) Structure and comparison of the oxyotcin and vasopressin genes from the rat. Proc Natl Acad Sci USA 81:2006–2010

  12. Jablonski E, Moomaw W, Tullis RH, Ruth JL (1986) Preparation of oligodeoxynuclotide-alkaline phosphatase conjugates and their use as hybridization probes. Nucleic Acid Res 14:6115–6128

  13. Jirikowski GF, Ramalho-Ortigao FJ, Caldwell JD (1991) Transistory coexistence of oxytocin and vasopressin in the hypothalamo neurohypophysial system of parturient rats. Horm Metab Res 23:476–480

  14. Kiyama H, Emson PC (1990) Evidence for the co-expression of oxytocin and vasopressin messenger ribonucleic acids in magnocellular neurosecretory cells: simultaneous demonstration of two neurophysin messenger ribonucleic acids by hybridization histochemistry. J Neuroendocrinol 2:257–259

  15. Larsen PJ, Mikkelsen JD (1992) Vasoactive intestinal peptide (VIP) in magnocellular neurons in the hypothalamo-neurohypophysial system of the mink (Mustela vison) is colocalized with vasopressin or oxytocin. J Comp Neurol 326:180–192

  16. Larsen PJ, Jessop DS, Chowdrey HS, Lightman SL (1992a) Neuropeptide Y messenger ribonucleic acid in the magnocellular system of the rat is increased during osmotic stimulation. Neurosci Lett 138:23–26

  17. Larsen PJ, Sheikh S, Mikkelsen JD (1992b) Evidence for neuropeptide Y in the magnocellular hypothalamo-neurohypophysial pathway is involved in the regulation of water homeostasis. Brain Res 573:181–189

  18. Levey AI, Bolam JP, Rye DB, Hallanger AE, Demuth RM, Mesulam MM, Wainer BH (1986) A light and electron microscopic procedure for sequential double antigen localization using diaminobenzidine dihydrochloride. J Histochem Cytochem 34:1449–1457

  19. Lightman SL, Young WS (1987) Vasopressin, oxytocin, dynorphin, enkephalin and corticotrophin-releasing factor mRNA stimulation in the rat. J Physiol 394:23–39

  20. Meister B, Cortés R, Villar MJ, Schalling M, Hökfelt T (1990a) Peptides and transmitter enzymes in hypothalamic magnocellular neurons after administration of hyperosmotic stimuli: comparison between messenger RNA and peptide/protein leveels. Cell Tissue Res 260:279–297

  21. Meister B, Villar MJ, Ceccatelli S, Hökfelt T (1990b) Localization of chemical messengers in magnocellular neurons of the hypothalamic supraoptic and paraventricular nuclei: an immunohistochemical study using experimental manipulations. Neuroscience 37:603–633

  22. Mezey E, Kiss JZ (1991) Coexpression of vasopressin and oxytocin in hypothalamic supraoptic neurons of lactating rats. Endocrinology 129:1814

  23. Mikkelsen JD, O'Hare MMT (1991) An immunohistochemical and chromatographic analysis of the distribution and processing of proneuropetide Y in the rat suprachiasmatic nucleus. Peptides 12:177–185

  24. Penschow JD, Haralambidis J, Pownall S, Coghan JP (1989) Location of gene expression in tissue sections by hybridization histochemistry using oligodeoxyribonucleotide probes. In: PM Conn (ed) Methods in neuroscience: gene probes. Academic Press, San Diego, pp 222–238

  25. Rehfeld J (1978) Immunochemical studies on cholecystokinin I. Development of sequence-specific radioimmunoassays for porcine tritiacontapeptide cholecystokinin. J Biol Chem 253:4016–4021

  26. Schalling M, Hökfelt T, Wallace B, Goldstein M, Filer D, Yamin C, Schlesinger DH (1986) Tyrosine 3-hydroxylase in rat brain and adrenal medulla: hybridization histochemistry and immunohistochemistry combined with retrograde tracing. Proc Natl Acad Sci USA 83:6208–6212

  27. Shivers BD, Harlan BD, Pfaff DW, Schachter BS (1986) Combination of immunocytochemistry and in situ hybridization in the same tissue section of rat pituitary. J Histochem Cytochem 34:39–43

  28. Singer RH, Lawrence BJ, Rashtchian RN (1987) Toward a rapid and sensitive in situ hybridization methodology using isotopic and nonisotopic probes. In: KL Valentino, JH Eberwine, JD Barchas (eds) In situ hybridization: application to neurobiology. Oxford University Press, New York, pp 71–96

  29. Sternberger LA (1979) The unlabeled antibody peroxidase-antiperoxidase (PAP) method. In: LA Sternberger (eds) Immunocytochemistry. Wiley & Sons, New York, pp 104–169

  30. Tecott LH, Eberwine JH, Barcas JD, Valentino KL (1994) Methodological considerations in the utilization of in situ hybridization. In: JH Eberwine, KL Valention, JD Barchas (eds) In situ hybridization in neurobilogy. Advances in methodology, Oxford University Press, New York Oxford, pp 3–24

  31. Trembleau A, Roche D, Calas A (1993) 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 41:489–498

  32. Uhl GR, Zingg HH, Habener JF (1985) Vasopressin mRNA in situ hybridization: localization and regulation studied with oligonucleotide cDNA probes in normal and Brattleboro rat hypothalamus. Proc Natl Acad Sci USA 82:5555–5559

  33. Vanderhaeghen JJ, Lotstra F, Vandesande F, Dierickxs K (1981) Coexistence of cholecystokinin and oxytocin-neurophysin in some magnocellular hypothalamo-neurohypophyseal neurons. Cell Tissue Res 221:227–231

  34. Vandesande F, Dierickx K (1979) The activated hypothalamic magnocellular neurosecretory system and the one neuron-one neurohypophysial hormone concept. Cell Tissue Res 200:29–33

  35. Watson SJ, Akil H, Fischli W, Goldstein A, Zimmerman EA, Nilaver G, Wimersma Gredanius TB van (1982) Dynorphin and vasopressin: common localization in magnocellular neurons. Science 216:85–87

  36. Watts AG, Swanson LW (1989) Combination of in situ hybridization with immunocytochemistry and retrograde tract tracing. In: PM Conn (ed) Methods in neuroscience: gene probes. Academic Press, San Diego, pp 127–136

  37. Wolfson B, Manning B, Davis LG, Arentzen R, Baldino F (1985) Co-localization of corticotropin releasing factor and vasopressin mRNA in neurones after adrenalectomy. Nature 315:59–61

  38. Young WS (1989) Simultaneous use of digoxigenin and radiolabeled oligodeoxyribonucleotide probes for hybridization histochemistry. Neuropeptides 13:271–275

  39. Young WS, Mezey E, Siegel RE (1986) Vasopressin and oxytocin mRNAs in adrenalectomized and Bratleboro rats: analysis by quantitative in situ hybridization histochemistry. Mol Brain Res 1:231–241

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Correspondence to P. J. Larsen.

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Larsen, P.J., Mikkelsen, J.D. 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. Histochemistry 102, 415–423 (1994). https://doi.org/10.1007/BF00269572

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Keywords

  • Vasopressin
  • Oxytocin
  • Cholecystokinin
  • Immunohistochemical Detection
  • Benzidine