Chemical Senses Vol. 29 No. 8 © Oxford University Press
2004; all rights reserved
Laryngeal Chemosensory Clusters
Department of Morphological-Biomedical Sciences, Human Anatomy and Histology Section, University of Verona, Verona, Italy
Correspondence to be sent to: Professor Andrea Sbarbati, DSMB, Human Anatomy and Histology Section,University of Verona, Medical Faculty, Strada Le Grazie 8, 37134, Verona, Italy. e-mail: andrea.sbarbati{at}univr.it
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Abstract |
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The expression of molecules involved in the transductory cascade of the sense of taste (TRs,
-gustducin, PLCß2, IP3R3) has been described in lingual taste
buds or in solitary chemoreceptor cells located in different organs. At the laryngeal
inlet, immunocytochemical staining at the light and electron microscope levels revealed
that -gustducin and PLCß2 are mainly localized in chemosensory clusters (CCs),
which are multicellular organizations differing from taste buds, being generally composed
of two or three chemoreceptor cells. Compared with lingual taste buds, CCs are lower in
height and smaller in diameter. In laryngeal CCs, immunocytochemistry using the two
antibodies identified a similar cell type which appears rather unlike the
-gustducin-immunoreactive (IR) and PLCß2-IR cells visible in lingual taste
buds. The laryngeal IR cells are shorter than the lingual ones, with poorly developed
basal processes and their apical process is shorter and thicker. Some cells show a
flask-like shape due to the presence of a large body and the absence of basal processes.
CCs lack pores and their delimitation from the surrounding epithelium is poorly evident.
The demonstration of the existence of CCs strengthens the hypothesis of a phylogenetic
link between gustatory and solitary chemosensory cells.
Key words: gustducin, neuroendocrine cells, phospholipase, taste, taste receptors
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Introduction |
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In mammals, taste buds are located in the oral cavity, pharynx and larynx. Laryngeal taste buds have been described in several species, including hamsters (Miller and Smith, 1984
), sheep (Bradley et al., 1980), horses
(Yamamoto et al., 2001),
cats (Shin et al., 1995),
monkeys (Khaisman, 1976) and humans
(fetus,
Lalonde and Eglitis, 1961; adult,
Jowett and Shrestha, 1998). These
studies described evident species-related differences, probably linked to feeding habits.
For example, laryngeal taste buds are numerous in herbivorous mammals, particularly in
ruminants (Palmieri et al.,
1983) and are rare in insectivorous animals, while an intermediate number
seems to be found in omnivorous species (Shrestha
et al., 1995).
In rodents, laryngeal taste buds constitute a significant percentage of the total
number of taste buds. In the rat, 103 laryngeal taste buds were found versus only 38
pharyngeal buds (Travers and Nicklas,
1990). In the hamster, 
10% of all taste buds are located in the
larynx (Miller and Smith, 1984).
Other studies provided significantly lower values, but the data may have been influenced
by age-related differences: laryngeal taste buds develop postnatally and their total
number decreases significantly with age (Yamamoto
et al., 2003).
In all the species studied, laryngeal buds are mainly located at the
‘entrance’ of the larynx, primarily on the laryngeal surface of the
epiglottis and aryepiglottal folds (Ide and
Munger, 1980;
Belecky and Smith, 1990;
Travers and Nicklas, 1990;
Shin et al., 1995;
Yamamoto et al., 1997;
Jowett and Shrestha, 1998). In some
species, the taste buds are arranged around the opening of the duct of the epiglottic
glands (Yamamoto et al.,
2001); in other species, accumulations of buds seem to exist at the bases of
the epiglottis.
Most studies did not report any structural differences between laryngeal taste buds
and those located in the oro-pharyngeal cavity, apart from a small number of
-gustducin-immunoreactive (IR) cells (Boughter et al., 1997). Ultrastructural studies
also demonstrated that laryngeal buds are basically similar to oro-pharyngeal taste buds
(Sweazey et al., 1994).
Nevertheless, a great functional difference exists between laryngeal and
oro-pharyngeal buds. Laryngeal taste buds do not play a role in gustation, but are
adapted to detect chemicals that are not saline-like in composition. They are stimulated
by the pH and tonicity of a solution and not by its gustatory properties (Bradley, 2000). Therefore, it is generally thought that
laryngeal taste buds may work as chemosensory detectors to initiate the reflex reaction
to protect the airway from oral substances during swallowing and drinking. Considering
that they are described as quite similar structures, the morphological bases of the
mismatch between laryngeal and oro-pharyngeal taste bud functions are unknown. A new
light on this could be provided by recent developments, which have greatly increased our
knowledge of the transductory machinery present in taste cells (Hoon et al., 1999;
Adler et al., 2000;
Lindemann, 2001;
Margolskee, 2002;
Montmayeur and Matsunami, 2002;
Perez et al., 2003). All the
new data have been obtained from oral taste buds, while information about their laryngeal
counterparts is lacking. The present study focuses on detection in the larynx of
molecules involved in the transductory cascade of taste (i.e. -gustducin and
phospholipase C of the ß2 subtype, PLCß2). The work was undertaken in order to
ascertain whether laryngeal and oral buds are the same from an anatomical and
immunohistochemical point of view in spite of their functional differences, or whether
the chemoreceptor structures of the two areas are differently organized.
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Material and methods |
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Animals
The study was conducted on adult Wistar rats of both sexes (150–200 g; Morini Co., Reggio Emilia, Italy) kept at the departmental animal facility. Animals were anesthetized with ether and handled in accordance with the guidelines for animal experimentation laid down in Italian law.
Immunocytochemistry
Animals were perfused with 4% paraformaldehyde in 0.1 M phosphate buffer, pH
7.4. Larynges were removed and fixed by immersion in the same fixative for 2 h at
4°C. After rinsing in 0.1 M phosphate buffer (PB), specimens were dehydrated through
an ascending series of ethanol, transferred to xylene and embedded in paraffin. Serial 7
µm thick sections were cut on a rotating microtome (Leitz 1512; Leitz, Germany),
collected on polylysine-coated slides and dried overnight at 37°C. The sections were
processed for immunohistochemistry or stained with hematoxylin and eosin. The primary
antibodies were raised in rabbits and directed against -gustducin (1:400; Santa
Cruz Biotechnology Inc.) or PLCß2 (1:2000; Santa Cruz Biotechnology Inc.).
Immunohistochemistry was performed using the avidin–biotin complex (ABC) technique. Sections were deparaffinized and rehydrated through xylene and a descending ethanol series. Endogenous peroxidase was quenched by immersion in 0.3% hydrogen peroxide in methanol for 30 min. After washing in 0.05 M Tris–HCL buffer (pH 7.6), sections were treated with 5% normal swine serum for 20 min. The primary antibody was applied overnight at 4°C. After washes, sections were reacted with biotinylated swine anti-rabbit immunoglobulins (DAKO), diluted 1:400, for 2 h. The immunoreaction was detected using a Vectastain Elite ABC kit (Vector) and visualized by 3,3-diaminobenzidine tetrahydrochloride (DAKO) for 5–10 min. Sections were dehydrated, coverslipped with Entellan and observed in an Olympus BX51 photomicroscope equipped with a KY-F58 CCD camera (JVC). The images were analysed and stored using Image-ProPlus software. Control sections were processed as above, omitting the primary antibodies or after immunoabsorption of the primary antibody with the specific peptide. No immunostaining was observed in control sections.
Transmission electron microscopy (TEM)
For TEM, the specimens were fixed in 2.5% glutaraldehyde in Sorensen’s buffer for 2 h, postfixed in 1% osmium tetroxide in Sorensen buffer for 1 h, dehydrated in graded ethanols, embedded in Epon–Araldite and cut with a Ultracut E ultramicrotome (Reichert-Jung, Wien, Austria). Semithin sections were stained with toluidine blue or PAS. Ultrathin sections were stained with lead citrate and uranyl acetate and observed under an EM10 electron microscope (Zeiss, Oberkochen, Germany).
Immunoelectron microscopy
Animals were perfused with 4% paraformaldehyde in 0.1 M PB, pH 7.4. Larynges
were removed and fixed by immersion in the same fixative for 2 h at 4°C. After
rinsing in 0.1 M PB, specimens were put into 30% sucrose in PB overnight and cut
to 60 µm thickness on a freezing microtome (Reichert-Jung). Free floating sections
were collected in 0.1 M PB, pH 7.4 and processed using the ABC technique. Endogenous
peroxidase was quenched by immersion in a solution of 3% hydrogen peroxide in
H20 for 30 min. After washing in 0.05 M Tris–HCL buffer, pH 7.6,
sections were treated with 10% normal swine serum for 60 min. Subsequently, the
primary antibody (polyclonal antibody anti--gustducin 1:400 or polyclonal antibody
anti-PLCß2 1:2000), diluted with Tris containing 0.02% Triton X100, was
applied overnight. After three washes, sections were then reacted with biotinylated swine
anti-rabbit immunoglobulins (DAKO), diluted 1:400, for 2 h. The immunoreaction was
detected using a Vectastain Elite ABC kit (Vector) and then visualized by
3,3-diaminobenzidine tetrahydrochloride (DAKO) for 5–10 min. Controls for the
specificity of the immunoreactions were performed by omitting the primary antibody.
For ultrastructural examination, after post-fixation in 1% OsO4 in PB for 1 h the tissues were dehydrated in graded concentrations of acetone and embedded in an Epon–Araldite mixture (Electron Microscopic Sciences, Fort Washington, PA). Semithin sections (1 µm thickness) were examined by light microscopy to locate areas containing immunoreactivity. Ultrathin sections were cut at 70 nm thickness on an Ultracut-E ultramicrotome (Reichert-Jung) and observed unstained on an EM 10 electron microscope (Zeiss).
Scanning electron microscopy (SEM)
For SEM, the tissues were fixed with glutaraldehyde 2% in 0.1 M PB, postfixed in 1% OsO4 in the same buffer for 1 h, dehydrated in graded ethanols, critical point dried (CPD 030; Balzers, Vaduz, Liechtenstein), fixed to stubs with colloidal silver, sputtered with gold with an MED 010 coater (Balzers) and examined with a DSM 690 scanning electron microscope (Zeiss).
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Results |
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General morphology
The most rostral portion of the larynx is lined with a pluristratified squamous
epithelium. Caudally to this squamous portion, the laryngeal inlet is lined by the
so-called transitional (intermediate) epithelium, which shows gradations ranging from
stratified squamous through stratified cuboidal to ciliated stratified low-columnar types
(Nakano and Muto, 1987;
Nakano et al., 1989).
According to the classic descriptions of
Smith (1977) and
Lewis and Prentice (1980), this
relatively unusual cuboidal epithelium is present within a band-like transitional area in
the ventrolateral aspect of the larynx at the level of arytenoid projections and in the
ventral pouch.
Light microscopy and immunocytochemistry
In a further step of the study, we tested whether two gustatory markers were expressed
in buds having a morphology similar to those visible in the tongue. Light microscopy and
immunocytochemistry for detection of -gustducin (Figure
1A,B) and PLCß2 (Figure
1C,D) demonstrated the expression of
these proteins in structures at the laryngeal inlet (generally located at the internal
wall of the epiglottis and aryepiglottic folds). In size and shape, the IR structures
appeared quite unlike the lingual taste buds (Figure
1E,F). Small buds were regularly
found, but the most common morphological feature was the ‘chemosensory
cluster’ (CC), composed of two or three IR cells. Precise quantification of the
described structures was difficult because the pattern changed with the level of the
section. Rostrally (at the laryngeal inlet) the buds were more numerous, whereas
caudally, clusters were the commonest structures. Isolated IR cells (solitary
chemosensory cells, SCCs) were also found, but were rare in this portion of the larynx,
being more numerous distally (Sbarbati et
al., 2004). Typical buds composed of more than five or six IR cells were
also rare and restricted to a thin rostral area. With respect to lingual taste buds,
laryngeal buds showed lesser height and diameter, as was evident from a comparison with
control specimens obtained from the vallate papilla (Figure
1E,F). In the taste buds sampled in
the latter, for each of the antibodies used more than five IR cells were usually
visible.
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In laryngeal CCs, delimitation from the surrounding epithelium was poorly evident and pores were lacking. In laryngeal CCs, the two antibodies stained a similar cell type that appeared rather unlike the
-gustducin-IR and PLCß2-IR cells visible in lingual
taste buds. The laryngeal IR cells (Figure
1A–D) were shorter than the
lingual ones and had poorly developed basal processes; in particular, the apical process
was shorter and thicker. Some cells showed a flask-like shape due to the presence of a
large body and to the apparent absence of basal processes. Transmission electron microscopy
Ultrastructural examination of the laryngeal inlet confirmed the presence of elements with a morphology ranging from small buds (Figure 2A) to CCs (Figure 3A,C,D ) and solitary chemosensory cells (SCCs, Figure 3E).
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The laryngeal buds were located at the laryngeal inlet, embedded in a pluristratified epithelium (Figure 2A). The distinction between the bud and the non-receptor epithelium was evident. The cells in the laryngeal buds appeared to be less elongated than those visible in lingual taste buds (Figure 2B). In laryngeal buds, the extremity of the cells often protruded from the free surface (Figure 2C,E) and the dense material in a pore which is visible in lingual taste buds (panel D) was lacking. In general, the apical portion of the laryngeal bud was less complex than the corresponding portion of lingual taste buds (Figure 2D,E).
The cells forming the laryngeal CCs showed the basic features of receptor elements (i.e. apical ends contacting the lumen and innervation). Generally, their microvilli resembled those of type II lingual taste cells (i.e. short, brush-like microvilli). However, a comparative evaluation by ultrastructural methods confirmed the presence of several differences between lingual taste buds and laryngeal CCs. The cells located in a CC were not clearly distinguished from the surrounding epithelium and they were usually shorter than those found in lingual buds (Figures 2B and 3B). In laryngeal CCs, an apical pore was short or absent (Figure 3A). The dense material usually present in the taste pore of lingual buds (Figure 3B) was regularly absent in laryngeal CCs. In the latter, the absence of a true pore and of material covering the cells allowed direct visualization of microvilli by SEM (Figure 3F); this was generally not the case with lingual taste buds.
Ultrastructural immunocytochemistry for -gustducin was used to identify the
submicroscopic characteristics of chemoreceptor elements (Figure
4). The results confirmed the presence
of IR cells disposed in clusters of two or three elements, not organized in buds. These
elements showed nucleo-cytoplasmic characteristics quite different from those of the
surrounding epithelial cells. In particular, the latter showed numerous bundles of
filaments that were virtually absent in IR cells. In transversal sections, the IR cells
appeared as round elements with a variable amount of perinuclear cytoplasm. Associated
elements were usually not visible but, in some cases, the surrounding keratinocytes
wrapped the IR cells with thin cytoplasmic processes (Figure 4).
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Discussion |
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The laryngeal CC
In the present study, we found that the mucosa covering the laryngeal inlet expressed
two molecules involved in taste transduction which are considered to be markers of taste
cells (Rossler et al., 1998;
Perez et al., 2002). These
findings demonstrate an evident link between laryngeal chemoreceptor cells and the taste
cells located in the tongue. However, we also demonstrated that differences exist between
laryngeal chemoreceptors and the ‘classic’ taste buds located in the
oro-pharyngeal cavity. Laryngeal chemoreceptors constitute a polymorphic population. Buds
similar in shape to oral taste buds are few and are generally found in a thin rostral
portion of the larynx, while in most cases laryngeal chemoreceptors are organized in
structures smaller than oral taste buds, i.e. laryngeal CCs. The small size of these
structures may be linked to their location in a thin epithelium, but structural
differences are also evident. In particular, the basal processes of laryngeal
-gustducin-IR cells are poorly developed. This feature is not specific to the
larynx, since similar -gustducin-IR cells have been observed in lingual taste buds
and immunocytochemical studies have described the wide-ranging polymorphism of these
cells (Boughter et al., 1997;
Cho et al., 1998;
Pumplin et al., 1999;
Yang et al., 2000;
Clapp et al., 2001;
Yee et al., 2001).
1Ultrastructural examination confirmed the presence of structural differences between
taste buds and laryngeal CCs. Most CCs display an organization quite different to that of
taste buds and only two or three -gustducin or PLCß2-IR cells can be found in
each CC. The biggest laryngeal CCs resemble the immature taste buds that can be found in
the tongue during development (Sbarbati et
al., 1998, 1999) or post-neurectomy regeneration (Kusakabe et al., 2002).
The new data seem to be in accordance with the old observation by
Khaisman (1976) of the incongruity of
the term ‘taste bud’ in relation to the epiglottis and with observations of
the human epiglottis which found ‘taste’ buds in only three out of five
cases, in which they were restricted to the stratified squamous epithelium, they were
smaller than typical lingual taste buds and it was rare to find a pore (Jowett and Shrestha, 1998).
CCs and SCCs
Laryngeal CCs appear to be a transitional structure between the rostrally located buds
and the SCCs more distally located in specific areas of the larynx (Sbarbati et al., 2004). SCCs are elements which
were described long ago in fishes (Whitear and
Kotrschal, 1988;
Whitear, 1992) and which recently
were also found in mammals (for a review, see
Sbarbati and Osculati, 2003). They
were first seen in the developing gustatory epithelium (Sbarbati et al., 1998) and then their presence was
confirmed in the developing palate. SCCs are also located in the nasal cavity (Zancanaro et al., 1999) and
Finger et al. (2003)
demonstrated in both rats and mice that in this location, SCCs form synaptic contacts
with trigeminal afferent nerve fibers and express T2R ‘bitter-taste’
receptors and -gustducin. Functional studies indicate that bitter substances
applied to the nasal epithelium activate the trigeminal nerve and evoke changes in
respiratory rate. By extending to the surface of the nasal epithelium, these chemosensory
cells serve to expand the repertoire of compounds that can activate trigeminal protective
reflexes (Finger et al.,
2003). On the basis of these findings,
Finger et al. (2003)
hypothesized that trigeminal chemoreceptor cells are likely to be remnants of the
phylogenetically ancient population of SCCs found in the epithelium of all anamniote
aquatic vertebrates.
The absence of a clear delimitation between laryngeal CCs and the surrounding
structures makes them resemble groups of SCCs more than true taste buds. From a
structural point of view, they can be considered analogous to clusters of SCCs present on
the dorsal fin of the rockling fish (Kotrschal
et al., 1993). The presence of such clusters strengthens the analogy
between the chemoreceptorial system of the larynx and that found in fish skin and oral
cavity, which has already been noted (Sbarbati
et al., 2004). In both cases, SCCs or clusters of chemosensory cells
are located in an area rich in intraepithelial axons and not particularly exposed to
drying.
Laryngeal chemoreceptors and neuroepithelial cells
Our work demonstrates that the laryngeal chemoreceptorial system is composed of three
organizations of cells which can be identified by specific molecular markers, i.e. SCCs,
CCs and buds. These structures show a clear topographical pattern with buds, CCs and SCCs
arranged in the supraglottic portion of the larynx in a rostro-caudal sequence. A similar
variable organization has been described for the neuroendocrine elements of the airways
(Sorokin et al., 1997),
which can be present in the form of isolated elements, clusters or more complex
neuroepithelial bodies (NEBs). For these cells, an age-related progression was reported,
with isolated elements proving more numerous in the fetus and NEBs more numerous in the
adult. Further, a rostro-caudal progression was also observed, with a prevalence of
isolated cells proximally and of NEBs distally. For gustatory cells also, a progression
during development from isolated cells to buds was documented in the tongue (Sbarbati et al., 1999). For the larynx,
we have no developmental data to demonstrate a progression from CCs to buds.
Functional considerations
Our data show cells with expression of molecules linked to taste detection in
laryngeal CCs. Previous work demonstrated that SCCs also contain G-protein and other
molecules of the taste cascade. Such structures could be involved in protective action
against exogenous air-borne molecules or material coming from the pharynx. Therefore, the
new data seem to indicate that at the laryngeal inlet also, cells expressing molecules of
the ‘taste machinery’ carry out that sentinel action which has been
demonstrated in other zones of the respiratory apparatus (Finger et al., 2003).
Physiological studies demonstrated the non-gustatory nature of the response elicited
by stimulation of the proximal portion of the larynx (Bradley, 2000), but anatomical studies simply described the
laryngeal chemoreceptors as taste buds. The present work contributes to clarifying this
mismatch by demonstrating that the laryngeal chemosensory system expresses molecules
involved in the taste cascade, but displays three levels of organization: buds, CCs and
SCCs. In the nasal cavities, it was recently reported that chemoreceptors with molecular
markers of taste cells can operate as detectors of irritating stimuli (Finger et al., 2003). It is possible
that the laryngeal chemosensory system has a similar role.
General conclusions
This work represents a step in the definition of a diffuse chemosensory system
composed of taste cell-related elements. In recent years, the identification of cells
with gustatory characteristics located outside the oro-pharyngeal cavity has been
permitted by the discovery of the molecular machinery of taste transduction. This first
allowed the chemical coding of the gustatory cells in taste buds and subsequently allowed
the detection of cells with a similar chemical code in other organs. To date, cells with
similar characteristics have been identified in endodermic derivatives (i.e. the
digestive and respiratory apparatuses), but they always appear in the form of isolated
elements—solitary chemosensory cells or brush cells (Hofer et al., 1996;
Hofer and Drenckhahn, 1998). In this
study, taste cell-related elements were detected in a multicellular form of organization,
the CC. The existence of CCs strengthens the hypothesis of a phylogenetic link between
the gustatory and the SCC system.
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Acknowledgement |
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The authors wish to thank Dr Christine Harris for revising the manuscript.
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Accepted July 30, 2004
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