Abstract
The medulla of the adrenal gland is a neuroendocrine tissue in which catecholamine-storing chromaffin cells exist. The chromaffin cells are derived from neural crest cells and distinctly differentiated into two types of cells, epinephrine (E) (adrenaline)-storing and norepinephrine (NE) (noradrenaline)-storing cells. Using histochemical or immunostaining methods, the two types of chromaffin cells have been differentially distinguished. However, difficulties and/or drawbacks of the procedures have somewhat restricted the progress of research in differential functions of E-storing and NE-storing cells. Here, we show a new method for the differential demonstration of these two cell types. We found that mouse and rat adrenomedullary cells are heterogeneously stained with Harris hematoxylin after treatment with citrate buffer at pH 6. The cell clusters stained with hematoxylin were positive for tyrosine hydroxylase, which is an enzyme involved in catecholamine biosynthesis. Furthermore, the cell clusters were negative for phenylethanolamine-N-methyl transferase, which is an enzyme responsible for the conversion from NE to E and expresses in E-storing chromaffin cells. Moreover, we found that the cell clusters stained with hematoxylin can also be stained with nitroblue tetrazolium at pH 11, using Hopsu and Mäkinen’s method by which NE-storing chromaffin cells are stained. These observations indicate that the cytoplasm of NE-storing chromaffin cells is specifically stained with hematoxylin after treatment with citrate buffer at pH 6. This method will allow us to facilitate cell-type specific research of chromaffin cells. Indeed, this method revealed that α-synuclein selectively expresses in E-storing chromaffin cells, but not in NE-storing chromaffin cells.
Similar content being viewed by others
Abbreviations
- E:
-
Epinephrine
- NE:
-
Norepinephrine
- PNMT:
-
Phenylethanolamine-N-methyl transferase
- TH:
-
Tyrosine hydroxylase
- ChAT:
-
Choline acetyltransferase
- PFA:
-
Paraformaldehyde
- DAB:
-
Diaminobenzidine
- PB:
-
Phosphate buffer
- PBS:
-
Phosphate-buffered saline
- NBT:
-
p-Nitroblue tetrazolium chloride
- Ach:
-
Acetylcholine
- H&E:
-
Hematoxylin and eosin
References
Anglade P, Larabi-Godinot Y (2010) Historical landmarks in the histochemistry of the cholinergic synapse: perspectives for future researches. Biomed Res 31:1–12
Auluck PK, Caraveo G, Lindquist S (2010) α-Synuclein: membrane interactions and toxicity in Parkinson’s disease. Annu Rev Cell Dev Biol 26:211–233
Aunis D, Langley K (1999) Physiological aspects of exocytosis in chromaffin cells of the adrenal medulla. Acta Physiol Scand 167:89–97
Chang WW, Bencosme SA (1968) Selective staining of secretory granules of adrenal medullary cells by silver methenamine: a light and electron microscopic study. Can J Physiol Pharmacol 46:745–747
Coupland RE (1989) The natural history of the chromaffin cell—twenty-five years on the beginning. Arch Histol Cytol 52(Suppl):331–341
Coupland RE, Hopwood D (1966) The mechanism of the differential staining reaction for adrenaline- and noradrenaline-storing granules in tissues fixed in glutaraldehyde. J Anat 100:227–243
Cuevas EC, Bateman AC, Wilkins BS, Johnson PA, Williams JH, Lee AH, Jones DB, Wright DH (1994) Microwave antigen retrieval in immunocytochemistry: a study of 80 antibodies. J Clin Pathol 47:448–452
Goldstein M, Fuxe K, Hokfelt T, Joh TH (1971) Immunohistochemical studies on phenylethanolamine-N-methyltransferase, dopa-decarboxylase and dopamine-hydroxylase. Experientia 27:951–952
Hokfelt T, Fuxe K, Goldstein M, Joh TH (1973) Immunohistochemical localization of three catecholamine synthesizing enzymes: aspects on methodology. Histochemie 33:231–254
Honore LH (1971) A light microscopic method for the differentiation of noradrenaline- and adrenaline-producing cells of the rat adrenal medulla. J Histochem Cytochem 19:483–486
Hopsu V, Mäkinen EO (1966) Two methods for the demonstration of noradrenaline-containing adrenal medullary cells. J Histochem Cytochem 14:434–435
Hou XE, Dahlstrom A (1996) Synaptic vesicle proteins in cells of the sympathoadrenal lineage. J Auton Nerv Syst 61:301–312
Huber K, Kalcheim C, Unsicker K (2009) The development of the chromaffin cell lineage from the neural crest. Auton Neurosci 151:10–16
Kober AK, Aoyama M, Sugita S (2010) Immunohistochemical localization of catecholamine biosynthetic enzymes in the adrenal gland of the domestic fowl (Gallus domesticus). Poult Sci 89:1709–1715
Lee BR, Kamitani T (2011) Improved immunodetection of endogenous α-synuclein. PLoS ONE 6:e23939
Lloyd RV, Sisson JC, Shapiro B, Verhofstad AA (1986) Immunohistochemical localization of epinephrine, norepinephrine, catecholamine-synthesizing enzymes, and chromogranin in neuroendocrine cells and tumors. Am J Pathol 125:45–54
Maroteaux L, Campanelli JT, Scheller RH (1988) Synuclein: a neuron-specific protein localized to the nucleus and presynaptic nerve terminal. J Neurosci 8:2804–2815
Matsuo Y, Kamitani T (2010) Parkinson’s disease-related protein, α-synuclein, in malignant melanoma. PLoS ONE 5:e10481
Mravec B (2005) A new focus on interoceptive properties of adrenal medulla. Auton Neurosci 120:10–17
Odagiri S, Tanji K, Mori F, Kakita A, Takahashi H, Kamitani T, Wakabayashi K (2012) Immunohistochemical analysis of Marinesco bodies, using antibodies against proteins implicated in the ubiquitin–proteasome system, autophagy and aggresome formation. Neuropathology (in press)
Phillips JK, Dubey R, Sesiashvilvi E, Takeda M, Christie DL, Lipski J (2001) Differential expression of the noradrenaline transporter in adrenergic chromaffin cells, ganglion cells and nerve fibres of the rat adrenal medulla. J Chem Neuroanat 21:95–104
Shi SR, Chaiwun B, Young L, Cote RJ, Taylor CR (1993) Antigen retrieval technique utilizing citrate buffer or urea solution for immunohistochemical demonstration of androgen receptor in formalin-fixed paraffin sections. J Histochem Cytochem 41:1599–1604
Solcia E, Sampietro R, Capella C (1969) Differential staining of catecholamines, 5-hydroxytryptamine and related compounds in aldehyde-fixed tissues. Histochemie 17:273–283
Strack AM, Sawyer WB, Marubio LM, Loewy AD (1988) Spinal origin of sympathetic preganglionic neurons in the rat. Brain Res 455:187–191
Tanji K, Kamitani T, Mori F, Kakita A, Takahashi H, Wakabayashi K (2010) TRIM9, a novel brain-specific E3 ubiquitin ligase, is repressed in the brain of Parkinson’s disease and dementia with Lewy bodies. Neurobiol Dis 38:210–218
Tramezzani JH, Chiocchio S, Wassermann GF (1964) A technique for light and electron microscopic identification of adrenalin- and noradrenalin-storing cells. J Histochem Cytochem 12:890–899
Ubink R, Lange W, Verhofstad A (1995) Simultaneous immunoenzymatic staining of catecholamines, catecholamine-biosynthesizing enzymes, and bromodeoxyuridine in adrenal medullary cells of the rat. J Histochem Cytochem 43:39–46
Verhofstad AA, Steinbusch HW, Penke B, Varga J, Joosten HW (1980) Use of antibodies to norepinephrine and epinephrine in immunohistochemistry. Adv Biochem Psychopharmacol 25:185–193
Acknowledgments
We thank Dr. William E. Rainey for helpful discussion and Dr. Gregory Liou for kindly providing rat adrenal glands. This work was supported in part by National Institutes of Health Grant R01AG024497 (to T.K.).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Khan, M.B., Lee, B.R. & Kamitani, T. A simple and sensitive method for the demonstration of norepinephrine-storing adrenomedullary chromaffin cells. Histochem Cell Biol 138, 155–165 (2012). https://doi.org/10.1007/s00418-012-0942-3
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00418-012-0942-3