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Freeze-substitution and Postembedding Immunocytochemistry on Rat Taste Buds: G-Proteins, Calcitonin Gene-related Peptide, and Choline Acetyl Transferase

Published online by Cambridge University Press:  31 January 2003

Bert Ph.M. Menco
Affiliation:
Department of Neurobiology and Physiology, O. T. Hogan Hall, Northwestern University, Evanston, IL 60208-3520
Maya P. Yankova
Affiliation:
Department of Neurobiology and Physiology, O. T. Hogan Hall, Northwestern University, Evanston, IL 60208-3520
Sidney A. Simon
Affiliation:
Department of Neurobiology, Duke University, Durham, NC 27710
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Abstract

Abstract: We have explored freeze-substitution combined with low-temperature embedding in rat taste buds for postembedding immunocytochemistry. A major difference in taste bud cells that were rapidly frozen without prior chemical fixation and those that were fixed and cryoprotected before freezing was that electron-dense core granules were virtually absent. The antibodies used in these initial studies were directed against calcitonin gene-related peptide (CGRP), a peptide commonly found in nociceptive neurons; the α-subunits of two G-proteins involved in bitter and sweet taste transduction; and choline acetyl transferase (ChAT), an enzyme involved in the synthesis of acetylcholine. Anti-CGRP immunolabeled a subpopulation of unmyelinated perigemmal neurons; anti-G labeled a larger subpopulation of these neurons and the microvilli of cells that were most likely from Type II vallate taste buds. α-Gustducin was found in cytoplasm of Type II and/or III cells and probably in microvilli of Type I cells of vallate taste buds. The best labeling results were obtained with anti-ChAT, which stained microvilli and lateral membranes of some Type II vallate taste bud cells, and the cytoplasm of some other Type II and/or III vallate cells. In addition, anti-ChAT labeled electron-opaque materials inside taste bud pores of vallate papillae, but, under the same conditions, not granules of Type I cells or most of the vesicles in von Ebner's glands. These data suggest that we can not assume a priori that the contents of the electron-dense core granules of Type I cells, or even of those of von Ebner's glands, contain the precursors of the taste bud pore–dense substances.

Type
Research Article
Copyright
2001 Cambridge University Press

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