Chem. Senses 24: 469-472,
1999
© Oxford University Press 1999
-Gustducin-immunoreactive Solitary Chemosensory Cells in the Developing Chemoreceptorial Epithelium of the Rat Vallate Papilla
Institute of Human Anatomy and Histology, University of Verona, Verona, Italy
Correspondence to be sent to: Andrea Sbarbati, Institute of Anatomy and Histology, University of Verona, Medical Faculty, Strada Le Grazie 8, I-37134, Verona, Italy. e-mail:sbarbati{at}borgoroma.univ.it
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Abstract |
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The presence of solitary chemosensory cells was studied in rat vallate papillae during the first week of post-natal life by
-gustducin immunocytochemistry. In 1- to 3-day-old
rats, isolated -gustducin-immunoreactive cells were found within the epithelium of the
vallate papilla. These cells, mainly located in the basal layer, were scattered among keratocytes
and wrapped in -gustducin-negative epithelial cells in a glia-like fashion. The
-gustducin-immunoreactive cells were usually round and some of them gave rise to short,
large processes directed towards the lumen of the oral cavity or the basal lamina. Rarely, some
cells showed an evident bipolar shape. Small taste buds containing either
-gustducin-immunoreactive or -gustducinnegative cells appeared in the vallate
papillae of 4-day-old rats in which isolated, bipolar-shaped -gustducin-immunoreactive
cells were also found. After the first week of post-natal life, the taste buds appeared basically
similar to those of adult animals. In conclusion, the present study demonstrates that the presence
of epithelial cells with characteristics of solitary chemosensory cells precedes the development of
the taste buds. |
Introduction |
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TopAbstractIntroductionMaterial and methodsResultsConclusionReferences |
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In a previous study, we demonstrated that during the first days of post-natal life the epithelia of rat vallate papillae contain isolated cells with a bipolar shape, nerve contacts and neuroendocrine-type granules (Sbarbati et al., 1999
). On the
basis of ultrastructural characteristics, we suggested that these elements could be homologous to
the solitary chemosensory cells (SCCs) described in aquatic vertebrates. In these latter animals,
feeding and exploration of the chemical environment is performed using, in addition to the
olfactory and to the gustatory systems, a further kind of epithelium-nerve organization based on
the presence of SCCs. The elementary unity of this chemoreceptorial structure differs from the
taste buds of the gustatory apparatus, and consists of a single epithelial cell contacted by nerves
and lacking a specialized connective bed. In fishes, SCCs form a system of differentiated sensory
epithelial cells which is not organized into discrete end organs and may occur in the epithelia of
the oropharynx, the gills and the skin (Whitear, 1992). In the lamprey, the
corresponding cells have been called oligovillous cells (Whitear and Lane, 1983). To date, SCCs are considered to be typical for aquatic vertebrates and seem to be
absent in the skin of mammals, although some data suggest that cells with characteristics of
SCCs may be present in the digestive apparatus of rodents (Hofer and Drenckhahn,
1996; Hofer et al., 1996; Luciano and
Reale, 1997).
In the rat, a demonstration of elements with the morphological characteristics of SCCs in an
early stage of development of the gustatory epithelium may be relevant to understanding the
ontogeny of the chemoreceptive system of the oral cavity. We have not found a description of
similar cells in previous studies on taste bud development (Farbman, 1965; Miller and Chaudhry, 1974; Hoseley and
Oakley, 1987; Miller and Smith, 1988; Farbman
and Mbiene, 1991; Oakley et al., 1991; Barlow et al., 1996; Wakisaka et al., 1996;
Witt and Reutter, 1996; Northcutt and Barlow, 1998). Therefore, further data are necessary to assess the origin, eventual functional role
and fate of these cells.
To further characterize these cells, we have studied their immunoreactivity for
-gustducin during early post-natal development. Taste-specific G protein was first
demonstrated in rats (McLaughlin et al., 1992) and then
confirmed in man (Takami et al., 1994). -Gustducin is
expressed specifically in taste cells of the circumvallate, foliate and fungiform papillae of rat
lingual tissue. In rat vallate taste buds, -gustducin has been found in cells with
characteristics of Type II (light) cells (Boughter et al., 1997).
Other authors have suggested that -gustducin is also expressed in the cytoplasm of Type
III cells and probably in microvilli of Type I cells of the vallate taste buds (Menco et al., 1997). Therefore, -gustducin is considered to be a potent
marker of cells which are chemosensitive, and the visualization of an eventual -gustducin
immunoreactivity (IR) in the developing gustatory epithelium could provide information about
the relationship between SCCs and the taste organs.
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Material and methods |
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TopAbstractIntroductionMaterial and methodsResultsConclusionReferences |
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The study was performed on Wistar rats at different ages, ranging from immediately after birth to 11 days old, and maintained at the departmental animal facility. Animals were anesthetized with ether and killed by dislocation of cervical vertebrae. Ten rats were perfused i.a. with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Following the perfusion fixation, the lingual tissue was postfixed by immersion for 1 h in the fixative. Afterwards, the tissue was transferred in a 10% sucrose solution in phosphate buffer. Blocks of lingual tissue containing the vallate papillae, as well as adjacent epithelium, muscles and glands, were dissected. For immunohistochemistry, tissue blocks were sectioned on a sliding, freezing microtome. Parallel free-floating sections (30 mm) were collected in phosphate buffered saline (PBS) (pH 7.4) and washed in several changes of this solution before further processing.
In another seven rats the tongues were removed after death and fixed by immersion in 4% neutral buffered formalin for 2–6 h at 4°C, rapidly dehydrated by alcohol steps, transferred to xylene and embedded in paraffin (melting point 52°C; Merck, Darmstadt, Germany). Paraffin sections (6–8 µm) were cut and stretched at 45°C, allowed to dry and stored at 4°C until use.
For the immunohistochemical experiments free-floating sections were processed without pre-treatment, while paraffin sections were deparaffinized in xylene and dehydrated in a graded series of ethanol.
Free-floating as well as paraffin sections were incubated for 10 min with 3% hydrogen
peroxide in methanol to inhibit endogenous peroxidases. Sections were then incubated for
15–20 min with 3% normal swine serum diluted in PBS/bovine albumin serum
(PBS/BSA) 1.5% (pH 7.4). Afterwards, the sections were incubated for 2 h with the primary
antibody anti--gustducin (Santa Cruz Biotechnology Inc., Heidelberg, Germany, Cat. No.
sc-395, dilution 1:200). Sections were washed in PBS and then incubated with a secondary
antibody (biotinylated swine anti-rabbit, Code No. E353) (Dako, Glostrup, Denmark, dilution
1:400). An avidin–biotin complex (ABC) technique was used to reveal sites of
antigen–antibody reaction. For the ABC method a commercial kit (ABComplex/HRP
code no. K0355, Dako) was used. Kit instructions were followed with regard to dilution and
incubation times.
Peroxidase activity was revealed by diaminobenzidine (Sigma; St Louis, MO). The sections were then dehydrated through an ethanol series, cleared in xylene and coverslipped with Entellan.
Parallel to the above immunohistochemical procedures, controls were performed by replacing the primary antibody with 10% non-immune serum or with PBS/BSA 1.5%. Further controls were carried out by omitting the secondary antibody.
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Results |
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TopAbstractIntroductionMaterial and methodsResultsConclusionReferences |
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In 1-to 3-day-old rats, vallate papillae were already evident. The epithelium of the vallum was rather thin and, indeed, was composed of five or six layers of cells. In this epithelium, isolated a-gustducin-immunoreactive (IR) cells were found (Figure 1a,b). These cells, mainly located in the basal layer of the epithelium, were scattered among keratocytes and were often in contact with the basal lamina (Figure 1a,b). They were wrapped in
-gustducin-negative cells in glia-like manner. A clear extracellular space was often
visible around the latter. The -gustducin-IR cells were round, but often gave rise to short,
large processes directed toward the lumen or the basal lamina (Figure 1a).
Rarely, a bipolar shape was evident.
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During the first 3 days after birth, we rarely found more than one
-gustducin-IR cell
in the same bud-like structure. In 4- to 5-day-old rats, the pattern of the chemosensory epithelium
was more pleomorphic, and we observed taste buds composed of both -gustducin-IR and
-gustducinnegative cells (Figure 1c,d). Isolated, bipolar-shaped
-gustducin-IR cells were also found.
After the first week of post-natal life, the taste buds appeared basically similar to those of
adult animals, being composed of numerous -gustducin-IR cells separated by
-gustducin-negative cells (Figure 1e,f).
The controls were always negative.
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Conclusion |
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TopAbstractIntroductionMaterial and methodsResultsConclusionReferences |
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The present work demonstrates that single cells present in the vallate papillae of the rat during the first days of extrauterine life are
-gustducin-IR, similar to clustered taste cells
located in the buds of adult rodents (McLaughlin et al., 1992; Boughter et al., 1997). Studies in -gustducin-knockout
mice implicate this protein subunit in the transduction of both bitter- and sweet-tasting
substances (Wong et al., 1996) and therefore the present work is
consistent with the hypothesis that these elements are related to the chemoceptive cell line. This
result is also consistent with our previously reported electron microscopic findings which
demonstrated nerve fibers contacting isolated bipolar cells in the developing chemoreceptive
epithelium (Sbarbati et al., 1999). The present work also
provides further data on the morphology and location of these cells, as we have demonstrated that
during the first days of post-natal life, these cells are round elements which do not seem to reach
the free surface of the epithelium. A relationship with the basal membrane does not seem to be
constant, although it is difficult to evaluate this relationship at the light microscopic level. The
finding that in the posterior portion of the tongue, the presence of -gustducin-IR cells is
limited to the vallate papilla suggests that the future gustatory epithelium is determined before
the appearance of the taste buds. The immunocytochemical and ultrastructural features
(tonofilaments, desmosomes) of the -gustducin-IR cells demonstrate that they are
epithelial cells. This conclusion seems to be in agreement with recent studies on the ontogeny of
the gustatory epithelium (Stone et al., 1995; Barlow et al., 1996). In addition, the present work demonstrates that the cells
wrapping the putative chemoceptive cells in a glia-like manner are -gustducin-negative.
Further studies seem to be necessary to evaluate an eventual relationship between these cells and
-gustducin negative cells present in taste buds.
Taken together with our previous ultrastructural data, the present immunocytochemical
findings suggest that SCCs may be present in the oral cavity of mammals. This may be a parallel
to the findings in aquatic vertebrates. Further data are necessary to determine if a relationship
exists between the SCCs present in the developing gustatory epithelium and the
-gustducin-IR isolated cells which are located in other portions of the digestive system,
such as the epithelia of the pancreatic ducts or gallbladder (Hofer and Drenckhahn,
1996; Hofer et al., 1996). It has been suggested that
these latter cells may be also solitary chemoreceptors. At the present phase of study, to speculate
about possible functional roles of the cells that we have described in the vallate papilla is
difficult. However, in our opinion, the demonstration of the presence of these cells could be
important because it suggests that oral chemoreception in the suckling rats could be mediated by
an SCC system. The cells that we have described precede the development of taste buds and,
since similar elements have not been described in adult mammals, it is possible that their
importance is limited to a short period of post-natal life. In the following days, the SCCs could be
removed by apoptosis or be incorporated in the taste buds. The latter hypothesis seems to be in
accordance with the suggestion that taste buds may be compound sensory organs containing
several cell types, one of which may be an SCC (Sbarbati et al., 1990; Osculati and Sbarbati, 1995; Finger, 1997).
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Accepted May 17, 1999
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