The distribution of transient receptor potential melastatin-8 in the rat soft palate, epiglottis, and pharynx

Abstract

Immunohistochemistry for transient receptor potential melastatin-8 (TRPM8), the cold and menthol receptor, was performed on the rat soft palate, epiglottis and pharynx. TRPM8-immunoreactive (IR) nerve fibers were located beneath the mucous epithelium, and occasionally penetrated the epithelium. These nerve fibers were abundant in the posterior portion of the soft palate and at the border region of naso-oral and laryngeal parts of the pharynx. The epiglottis was free from such nerve fibers. The double immunofluorescence method demonstrated that TRPM8-IR nerve fibers in the pharynx and soft palate were mostly devoid of calcitonin gene-related peptide-immunoreactivity (CGRP-IR). The retrograde tracing method also demonstrated that 30.1 and 8.7 % of sensory neurons in the jugular and petrosal ganglia innervating the pharynx contained TRPM8-IR, respectively. Among these neurons, the co-expression of TRPM8 and CGRP-IR was very rare. In the nodose ganglion, however, pharyngeal neurons were devoid of TRPM8-IR. Taste bud-like structures in the soft palate and pharynx contained 4-9 TRPM8-IR cells. In the epiglottis, the mucous epithelium on the laryngeal side had numerous TRPM8-IR cells. The present study suggests that TRPM8 can respond to cold stimulation when food and drinks pass through oral and pharyngeal cavities.

Fig. 1

Schematic drawings of cranial and cervical regions (a, b). Panel b indicates the gray square in panel a at a higher magnification. In panel b, gray area shows the territory of the pharynx. TRPM8-IR nerve fibers are rich in the posterior portion of the soft palate and at the border region between naso-oral and laryngeal parts of the pharynx (red area in b). The nasal mucosa, epiglottis, and larynx also have TRPM8-IR epithelia (yellow lines in b). In addition, the soft palate and pharynx contain TRPM8-IR taste bud-like structures (blue ovals in b). Double immunofluorescent microphotographs of TRPM8 (c, f, i, l), CGRP (d, g, j, m) and both (e, h, k, n) in the posterior part of the soft palate (c–k) and epiglottis (l–n). Panels c–e, f–h, i–k, and l–n show the same fields of view, respectively. Many TRPM8 and CGRP-IR nerve fibers are located beneath the mucous epithelium in the soft palate (arrows in c–e, i–k). Epithelial cells in the mucous duct also express TRPM8-IR but not CGRP-IR (c–e). In addition, one taste bud-like structure in f–h has TRPM8-IR cells but not CGRP-IR cells (arrowheads). CGRP-IR is detected in nerve fibers beneath and within the structure (g, h). However, the co-expression of TRPM8 and CGRP-IR is very rare in the soft palate (c–n). Bars = 50 μm (c), 20 μm (f), 100 μm (i), and 100 μm (l). Panels c–e, f–h, i–k, and l–n are at the same magnifications, respectively. BP border region of naso-oral (NOP) and laryngeal (LP) parts of the pharynx, ES esophagus, EP epiglottis, LA larynx, LEP laryngeal side of the epiglottis, M mandible, NC nasal cavity, OC oral cavity, PA palate, PEP pharyngeal side of the epiglottis, SPA soft palate, T tongue, EP epithelium, and DU duct. (For interpretation of the references to color in the figure caption, the reader is referred to the web version of the article.)

Fig. 2

Double immunofluorescent microphotographs of FG (a, d), TRPM8 (b, e), and CGRP (c, f) in the JG (a–c) and PG (d–f), and the histograms of bar graphs for cell size spectra of TRPM8-positive and/or CGRP-positive JG (g) and PG (h) neurons innervating the pharynx. Panels a–c and d–f show the same fields of view, respectively. Sensory neurons in the JG (a–c) and PG (d–f) were retrogradely labeled from the pharynx with FG. In the JG, two FG-positive neurons express TRPM8-IR but not CGRP-IR (arrows in a–c). Arrowheads in a–c point to one FG-positive neuron in the JG which is devoid of TRPM8 and CGRP-IR. One FG-positive neuron in the PG is immunoreactive for both TRPM8 and CGRP (double arrowheads in d–f). The data for histograms were obtained from 111 JG and 94 PG neurons in 7 ganglia from 4 animals. Bars 100 μm (a), 50 μm (d). Panels a–c and d–f are at the same magnifications, respectively