Mouse Mandibular Retromolar Taste Buds Associated With a Mucus Salivary Gland

Abstract

We have characterized a recently rediscovered chemosensory structure at the rear of the mandibular mucosa in the mouse oral cavity originally reported in the 1980s. This consists of unorganized taste buds, not contained within troughs, associated with the ducts of an underlying minor salivary gland. Using whole-mount preparations of transgenic mice expressing green fluorescent protein under the promoter of taste-signaling-specific genes, we determined that the structure contains taste bud clusters and salivary gland orifices at the rear of each mandible, distal to the last molar and anterior to the ascending ramus. Immunohistochemical analysis shows in the retromolar taste buds expression of the taste receptors Tas2R131 and T1R3 and taste cascade molecules TrpM5, PLCβ2, and GNAT3, consistent with type II taste cells, and expression of GAD1, consistent with type III taste cells. Furthermore, the neuronal marker, calcitonin gene-related peptide, in retromolar mucosa tissue wrapping around TrpM5+ taste buds was observed. RT-PCR showed that retromolar taste buds express all 3 mouse tas1r genes, 28 of the 35 tas2r genes, and taste transduction signaling genes gnat3, plcb2, and trpm5, making the retromolar taste buds similar to other lingual and palate taste buds. Finally, histochemistry demonstrated that the mandibular retromolar secretory gland is a minor salivary gland of mucous type. The mandibular retromolar taste structure may thus play a role in taste sensation and represent a potential novel pharmacological target for taste disorders.

Keywords: chemosensory; minor salivary gland; retromolar; taste buds; taste receptors.

Figure 1.

Location of the retromolar chemosensory structure in the oral cavity. (Left) Schematic drawing of the mouse oral cavity. At the top is the palate and maxillary oral epithelium; at the bottom is the tongue and mandibular mucosa. Green dots represent the location of taste buds throughout the oral cavity. The retromolar chemosensory structure, posterior to the third molar (M3), is composed of taste buds (TBs) and secretory ducts from an underlying salivary gland. (Center) Whole-mount preparations of palate and retromolar mucosa from TrpM5-GFP mice (areas shown by red boxes at left) showing the taste bud (GFP-expressing) from a top view. (Right) Magnified areas of the palate and retromolar mucosa (locations indicated by blue boxes at left) both showing taste buds in the vicinity (palate) or surrounding (retromolar chemosensory structure) a ductal orifice (yellow asterisks) of the secretory gland laying below the mucosa. Scale bars = 100 µm.

Figure 2.

Transversal serial sections of tongue and surrounding buccal mucosa. (Top left) Micro-CT picture image of the mouse mandible with a schematic representation of the retromolar chemosensory structure, showing the location of TrpM5-GFP+ taste buds (green) and associated minor salivary gland (magenta). The red box represents the area sectioned, starting posterior of the third molar and extending distally through the tongue (blue arrow). (Top right) Micrograph of the retromolar mucosa with schematic of serial sectioning (dashed lines, A–K) of the chemosensory structure with taste buds (GFP) and ductal orifice (yellow asterisk). Scale bar = 50 µm. (A–P) Serial sections of the retromolar chemosensory structure. Red arrows indicate locations of taste buds in the mucosal epithelium (B–H, K). Taste buds first appear distal to the third molar and surround the ductal orifice and secretory duct (C–G) of the underlying salivary gland (C–P). The minor salivary gland, indicated by yellow arrows, is first visible in panel C and terminates in (P). Muscle, connective tissue, and the gland show a certain level of autofluorescence that is also present in wild-type mice (see Supplementary Figure 1). Scale bar = 100 µm.

Figure 3.

Quantification of taste buds of the retromolar chemosensory structure and immunohistochemistry for markers of the taste signaling transduction cascade. (Top) Quantification of the retromolar taste buds in transgenic mice expressing GFP under the promoter for gnat3, trpm5, tas1r3, or tas2r131 (mean ± SD). Representative fluorescent micrographs are shown for each GFP-transgenic strain. Scale bar = 100 µm. (Bottom) Immunohistochemical analysis of chemosensory markers in transgenic mice expressing GFP under the promoter for trpm5, tas1r3, tas2r131 (all markers for type II taste cells), and gad1 (marker for type III taste cells). Sections are stained with primary antibodies against Gnat3 or PLCβ2 as markers for type II taste cells and CGRP as marker of peptidergic nociceptive nerve fibers. PLCβ2 immunoreactivity (red) is visible in TrpM5-GFP+ taste bud cells (A). Gnat3 (Gustd; red) co-stains most of the T1R3-GFP+ cells (B and C) and all the Tas2R131-GFP-positive cells (F–F’) but does not colocalize with GAD1-GFP+ cells (E). TrpM5-GFP+ taste buds show perigemmal CGRP-immunoreactive (red) peptidergic innervations (D–D’). (D’) and (F’) are magnified micrographs of (D) and (F). Scale bars = B, 50 µm for panels B, C, E, D’, F’; 100 µm in A, D, and F.

Figure 4.

Expression analysis of the retromolar chemosensory structure for sweet, umami, and bitter taste receptor and downstream signaling effector genes. The retromolar taste buds (RM) express all 3 mouse tas1r genes, as well as the taste transduction signaling cascade genes gnat3, plcb2, and trpm5. Unlike in the circumvallate papilla (CV), only 28 of the 35 tas2r genes are expressed in the retromolar taste mucosa tissue. The CV was used as positive control for all the genes. RT+ and RT− = RNA samples transcribed in the presence or absence (negative control) of reverse transcription enzyme, respectively. actb = β-actin as housekeeping gene. Table 3 lists band sizes and primer sequences.

Figure 5.

H&E and PAS histochemical staining of the retromolar mucous salivary gland and salivary duct. Micrographs of salivary gland tissue sections from posterior lingual tissue and retromolar mucosa with H&E and PAS histochemical staining. (A–C) The acinus cells of the salivary gland and the salivary secretions in the ducts are composed of mucin glycoprotein, as evidenced by the magenta color, a subproduct of the histochemical reaction of the PAS staining methodology. (A’–C’) Serial sections of the retromolar chemosensory structure stained with H&E, confirming that the retromolar salivary gland is of mucous type. Acini comprise light-stained mucous cells with nuclei positioned at the base, compared with the darker staining typical of the serous von Ebner gland cells (Supplementary Figure 2). Visible in (A) and (A’) the secretory duct ending in an epithelial orifice located between the taste buds of the mandibular retromolar taste structure. (A), (B), (A’), and (B’) are at 10× magnification, whereas C and C’ are at 40×. The seriate sections of the entire retromolar salivary gland are available in Supplementary Figure 3. Scale bars = 100µm.