Development and maturation of taste buds of the palatal epithelium of the ratHistological and immunohistochemical study.код для вставкиСкачать
THE ANATOMICAL RECORD 263:260 –268 (2001) Development and Maturation of Taste Buds of the Palatal Epithelium of the Rat: Histological and Immunohistochemical Study ASHRAF EL-SHARABY, KATSURA UEDA, KOJIRO KURISU AND SATOSHI WAKISAKA* Department of Oral Anatomy and Developmental Biology, Osaka University Graduate School of Dentistry, Yamadaoka, Suita, Osaka, Japan ABSTRACT Palatal taste buds are intriguing partners in the mediation of taste behavior and their spatial distribution is functionally important for suckling behavior, especially in the neonatal life. Their prenatal development has not been previously elucidated in the rat, and the onset of their maturation remains rather controversial. We delineated the development and frequency distribution of the taste buds as well as the immunohistochemical expression of ␣-gustducin, a G protein closely related to the transduction of taste stimuli, in the nasoincisor papilla (NIP) and soft palate (SP) from the embryonic day 17 (E17) till the postnatal day 70 (PN70). The main findings in the present study were the development of a substantial number of taste pores in the SP of fetal rats (60.3 ⫾ 1.7 out of 122.8 ⫾ 5.5; mean ⫾ SD/animal at E19) and NIP of neonatal rats (9.8 ⫾ 1.0 out of 44.8 ⫾ 2.2 at PN4). ␣-gustducin-like immunoreactivity (-LI) was not expressed in the pored taste buds of either prenatal or newborn rats. The earliest expression of ␣-gustducin-LI was demonstrated at PN1 in the SP (1.5 ⫾ 0.5 cells/taste bud; mean ⫾ SD) and at PN4 in the NIP (1.4 ⫾ 0.5). By age the total counts of pored taste buds continuously increased and their morphological features became quite discernible. They became pear in shape, characterized by distinct pores, long subporal space, and longitudinally oriented cells. Around the second week, a remarkable transient decrease in the total number of taste buds was recorded in the oral epithelium of NIP and SP, which might be correlated with the changes of ingestive behaviors. The total counts of cells showing ␣-gustducin-LI per taste bud gradually increased till the end of our investigation (14.1 ⫾ 2.7 in NIP and 12.4 ⫾ 2.5 in SP at PN70). We conclude that substantial development of taste buds began prenatally in the SP, whereas most developed entirely postnatal in the NIP. The present study provides evidence that the existence of a taste pore which is considered an important criterion for the morphological maturation of taste buds is not enough for the onset of the taste transduction, which necessitates also mature taste cells. Moreover, the earlier maturation of palatal taste buds compared with the contiguous populations in the oral cavity evokes an evidence of their significant role in the transmission of gustatory information, especially in the early life of rat. Anat Rec 263:260 –268, 2001. © 2001 Wiley-Liss, Inc. Key words: taste buds; development; ␣-gustducin; nasoincisor papilla; soft palate; rat Taste transduction is arbitrated by specialized neuroepithelial cells, referred to as gustatory or taste cells that organize into groups forming taste buds of which the vast majority are embedded within the lingual and palatal epithelium. Since Hoffmann (1875) has reported the existence of taste buds on the human palatal mucosa, numerous investigations have been conducted to investigate their spatial distribution rather than gustatory functions. The earliest accounts in the rat were published by Kolmer (1927) and Kaplick (1953) who identified the taste buds in © 2001 WILEY-LISS, INC. the nasoincisor papilla (NIP) and soft palate (SP), respectively. Although the palatal taste buds constitute only *Correspondence to: Satoshi Wakisaka, Department of Oral Anatomy and Developmental Biology, Osaka University Graduate School of Dentistry, 1-8, Yamadaoka, Suita, Osaka 565-0871, Japan. E-mail: email@example.com Received 1 August 2000; Accepted 31 January 2001 Published online 00 Month 2001 DEVELOPMENT AND MATURATION OF PALATAL TASTE BUDS 261 correlation between the onsets of morphological maturation i.e., existence of a taste pore and functional maturation i.e., taste transduction has not previously adopted, particularly in the developing palate. It is commonly believed that the existence of a distinct taste pore is a unique morphological criterion for taste bud maturation. Mistretta and Bradley (1977), however, asserted that the existence of elongated, longitudinally-oriented cells should be additional characteristics of the mature taste buds. Alpha gustducin has been cloned from taste papillae isolated from the lingual tissue of rats (McLaughlin et al., 1992) and microscopic observations suggested that it selectively expressed by cells having the morphological characteristics of matured light cells or type II cells (Tabata et al., 1995; Yang et al., 2000). Moreover, differential expression of ␣-gustducin were significantly more recognized in the taste buds highly sensitive to sweet (palatal) or bitter (foliate and circumvallate) stimuli than fungiform taste buds that are less sensitive to these compounds (Boughter et al., 1997). Thus, this protein is thought to be a suitable marker for matured light cells or type II cells that transduce predominately sweet and bitter stimuli. The present study was undertaken to trace the detailed temporal and spatial distribution of taste buds in the NIP and SP, which may be correlated with the critical changes in ingestive behavior during development. The demonstration of ␣-gustducin in the developing palatal taste buds was also designated to investigate the onset of their functional maturation i.e., taste transduction and its correlation with the morphological maturation and the existence of taste pores. MATERIALS AND METHODS All experiments were reviewed and approved by Osaka University Graduate School of Dentistry Intramural Use and Care Committee before the study. Animals and Tissue Preparation Fig. 1. A: Transverse view of the nasoincisor papilla. The epithelium of the nasoincisor papilla is divided into two regions; nasoincisor ductural epithelium (NID) and oral epithelium (OE), which is further subdivided into epithelium of the papillary dome (DE) and epithelium lateral to the NID (LE). C, cartilage. B: Surface view of the palate. Soft palate (SP) is also divided into two regions; Geschmacksstreifen (GS), the border between SP and hard palate (HP), and posterior palatine field (PPF). T, molar teeth; TR, terminal ridge; NP, nasopharyngeal orifice. 17% of the entire oropharyngeal complement in the adult rat (Miller, 1977), their approximate topography to their fellows in the fungiform, circumvallate and foliate papillae as well as epiglottal surface revealed a curious identity (Miller and Spangler, 1982). Moreover, their spatial distribution is functionally important for the suckling behavior in pre-weanling animals as milk stimulates them much more than the lingual taste buds (Ardran et al., 1958). In the neonatal rat, Harada et al. (2000) assigned that the number of pores in the SP of the rat at birth is significantly higher than the lingual taste bud populations, which reflects the crucial contribution of palatal taste buds in the mediation of taste directly after birth. In contrast to the contiguous taste bud populations, whether those of the palate begin their development prenatally or postnatally has not previously elucidated. Moreover, the Forty-four Sprague–Dawley rats of both genders were used for statistical analysis by ordinary hematoxylineosin (HE) staining in addition to 33 rats for the immunohistochemical investigation for ␣-gustducin. All animals were purchased from Nihon Dobutsu, Osaka, Japan. The day on which a vaginal plug was confirmed was designated as “embryonic day (E) 0.” Pregnant animals at various stages were sacrificed by overdose i.p. injection of chloral hydrate (600 mg/kg) and the fetuses were collected. The whole palates were dissected out and immersed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 3 days. For postnatal development, the day of birth was designated as “postnatal day (PN) 0.” The animals at various postnatal days were deeply anesthetized with i.p. injection of chloral hydrate (600 mg/kg) and perfused transcardially with 0.02 M phosphate-buffered saline (PBS; pH 7.2) followed by 4% paraformaldehyde. The heads were removed and placed in the same fixative for 3– 4 days. Specimens from rats at PN15 and older were decalcified with 7.5% ethylene diamine tetraacetic acid (EDTA) for 1– 6 weeks at 4°C with continuous shaking and weekly change. The palates were removed from the transverse terminal ridge caudally to the nasopharyngeal orifice, whereas the central part rostral to the first antemolar ruga containing nasoincisor ducts was excised. All specimens were dehydrated by an ascending series of ethanol, 262 EL-SHARABY ET AL. Fig. 2. Photomicrographs of the developing palatal taste buds. A: A nonpored taste bud is recognized in the SP at E19. B,C: Pored-taste buds are detected in the SP at E19 (B) and in the NIP at PN4 (C). Cells in these pored taste buds sparsely arranged. Arrows indicate the taste pores. D: Pored-taste bud in the SP at PN7. Cells within the taste buds showed typical pear-shaped characteristics. EP, epithelium; LP, lamina propria. Scale bar ⫽ 30 m. cleared in xylene and embedded in paraffin. The nasoincisor ducts were oriented for sectioning in the transverse plane, whereas the SPs were oriented to cut parasagittally. Complete serial sections cut at 6 –10 m thickness, were mounted on glass slides and stained with routine HE staining for the statistical analysis. For the immunohistochemistry, serial sections were cut at a thickness of 8 –10 m and mounted on ploy-L-lysinesubbed glass slides. They were deparaffinized in xylene and rehydrated through descending series of ethanol. The sections were treated in 0.1 M sodium citrate buffer (pH 6.0) for 10 min in a microwave for antigen retrieval (Cattorretti et al., 1992). Avidin-biotin-complex (ABC) method was applied for demonstration of ␣-gustducin-like immunoreactivity (-LI). The sections were treated with absolute methanol containing 0.3% H2O2 for 30 min to block endogenous peroxidase activity. They were then incubated overnight at room temperature with ␣-gustducin antiserum (1:1,000; Santa Cruz Biotechnology Inc., Santa Cruz, CA). After PBS rinse, the sections were incubated with biotinylated swine anti-rabbit IgG (1:500; Dako, Copenhagen, Denmark), followed by PBS rinse and subsequently with ABC complex (Vector, Burlingame, CA) for 90 min each at room temperature. Horseradish peroxidase activity was detected by incubating the slides with 0.1 M Tris-HCl buffer (pH 7.5) containing 0.04% 3-3⬘ diaminobenzidine trihydrochloride (DAB) and 0.003% H2O2. The DAB reactions were enhanced by 0.08 – 0.1% nickel ammonium sulfate. The immunostained sections were counterstained with methyl green, dehydrated with a graded series of ethanol, cleared in xylene, coverslipped with Permount (Fisher Scientific Inc., Fairlawn, NJ) and examined by light microscope. Photographic images were captured on CCD camera mounted on an Olympus microscope. Images were prepared for printing with Photoshop (Ver. 5.0, Adobe Systems, Inc., San Jose, CA), and printed out by a Fujix Pictrography 3000 digital photographic printer (Fuji Photo Film). Contrast was adjusted as needed. The specificity of the primary antibody was checked by preabsorption test. The sections were incubated with preabsorbed primary antibody with excess amount of antigen (10 g/ml of diluted primary antibody; Santa Cruz Bio- technology Inc.), resulting in no immunoreactions. For control of immunohistochemical procedure, the primary antibody was replaced with nonimmune serum or PBS. In addition, the incubation with secondary antibody or the ABC complex was also omitted. These sections had no immunoreactions. Quantification of the Data We traced the spatial distribution of taste buds in the palate of rats during the prenatal development (E17–20; daily intervals) and at various postnatal ages (PN1, 4, 7, 15, 21, 35 and 70; n ⫽ 4 for each age). Each of the NIP and SP was divided into two areas (Fig. 1A,B). The epithelium of NIP was divided into ductal epithelium (right and left nasoincisor ducts) and oral epithelium (dome of the papilla and adjacent oral epithelium lateral to the two nasoincisor ducts). The SP was divided into a transverse fold, directly posterior to the terminal ridge of hard palate, referred to as Geschmacksstreifen (GS) and a perpendicular central zone referred to as posterior palatine field (PPF). The existence of a taste pore was used as a criterion to distinguish the morphological stage i.e., nonpored and pored taste buds of the palate. For numerical analysis of the taste buds in each region of the palate, every profile of a taste bud, with or without a taste pore in each serial section of the NIP and SP was counted. In the SP, we delineated the zone where the taste buds were distributed at each stage and measuring the distance between the bud and GS or posterior border of the PPF. All taste buds were serially reconstructed on schematic drawings of the palatal structures to ensure that each bud was counted only once. The numbers of all taste buds in the NIP and SP were counted and graphically represented. The immunohistochemical demonstration of ␣-gustducin was used to recognize the onset of functional maturation i.e., taste transduction of the palatal taste buds and its relation with their morphological features or existence of a taste pore. Prenatal ages (E18 –20; daily intervals) and postnatal ages (PN1, 2, 4, 7, 15, 21, 35 and 70; n ⫽ 3 for each age) were used. In each of the NIP and SP, 20 taste buds were randomly selected per animal to count ␣-gustducin-immunoreactive (-IR) cells. Every cell profile containing a nucleus was counted once. The total counts of DEVELOPMENT AND MATURATION OF PALATAL TASTE BUDS 263 Fig. 3. Temporal changes in the number of taste buds at the nasoincisor papilla of the developing rat. Top panel shows the total number of taste buds, and middle and lower panels indicate the number of taste buds in the nasoincisor duct and oral epithelium, respectively. ␣-gustducin-IR cells per taste bud in the NIP and SP were recorded at different ages. RESULTS Morphology of the Developing Palatal Taste Buds The nonpored taste buds were first recognized as spherical masses of columnar cells extended from the basal lamina to a variable distance toward the surface epithelium of the SP and NIP at E18 and E20, respectively (Fig. 2A). These buds acquired a more elongated shape and short neck such that they access the oral cavity via a narrow taste pore (4 – 6 m in diameter) at E19 and PN4, in the SP and NIP, respectively. At these early stages of development, the cells in the pored taste buds were almost sparsely arranged with ovoid nuclei and somewhat eosinophilic cytoplasm and not reached the taste pore (Fig. 2B,C). Toward the end of the first week, the morphological features of pored taste buds became quite discernible. They became pear in shape and characterized by a distinct pore, long subporal space and longitudinally oriented cells pointed toward the pore (Fig. 2D). Fig. 4. Topographic localization of taste buds at the nasoincisor papilla of the rat at E20 (A) and PN 70 (B). One open dot represents one nonpored taste bud, while one closed dot represents one pored taste bud. C, cartilage. Temporo-Spatial Distribution of Taste Buds in the NIP During Development The length of NIP measured about 300 – 450 m at E20 and reached 1.3–1.7 mm at PN70. The orifices of right and left nasoincisor ducts occupied the third quarter of the papilla and measured 120 m at E20 and 500 m at PN70. Figure 3 and 4 show the temporal and spatial distribution of taste buds along the NIP. At E20, 8.5 ⫾ 1.0 taste buds (mean ⫾ SD/animal) were the earliest account along the oral epithelium of the posterior half of the NIP, and all of them belonged to the immature type (nonpored taste buds). They were distributed in the dome region as well as lateral folds of the papilla; medial and lateral to 264 EL-SHARABY ET AL. the orifices of nasoincisor ducts (Fig. 4A). At PN1, 11.0 ⫾ 2.0 buds appeared in the medial aspect of the nasoincisor ducts. Along the first few days, a gradual proliferation was recorded over the whole papilla and the number of nonpored taste buds in the oral epithelium was still higher compared with the nasoincisor ducts i.e., 17.8 ⫾ 3.3 and 16.3 ⫾ 2.1 at PN4, respectively. The earliest existence of taste pores was found simultaneously but higher in the oral epithelium (4.8 ⫾ 0.5) than ductal epithelium populations (3.8 ⫾ 1.0). At PN15, the count of pored taste buds within the nasoincisor ducts exceeded that of the contagious parts of oral epithelium (40.3 ⫾ 8.8 and 19.3 ⫾ 4.6, respectively). A transient decrease in the total number of taste buds was demonstrated in the oral epithelium around the second week. At the end of our investigation, the total count of the taste buds within the nasoincisor ducts was approximately twice that in the oral epithelium i.e., 61.8 ⫾ 1.9 and 27.8 ⫾ 2.5, respectively (Fig. 4B). No significant differences were recorded in the taste bud populations within the right and left nasoincisor ducts. Temporo-Spatial Distribution of Taste Buds in the SP During Development In all investigated specimens, the taste buds were uniquely distributed along T-shaped zone whose transverse part, an incomplete fold known as Geschmacksstreifen (GS), whereas the perpendicular part extended to the nasopharynx known as posterior palatine field (PPF). In the GS whose length was 1.6 –5.6 mm, the taste buds were constantly and densely distributed on both sides of a central zone ranged between 120 –250 m at E18 and PN70, respectively. On the other hand, the buds were variably distributed within a central zone of the PPF whose width were approximately 0.7 and 2.5 mm at E18 and PN70, respectively. Figure 5 and 6 show the temporal and spatial distribution of taste buds along the SP. Proliferation of nonpored taste buds occurred mostly before birth, however, a comparable number was also developed along the first 3 weeks after birth. A total of 16.5 ⫾ 3.1 taste buds in the GS as well as 37.5 ⫾ 6.8 along the middle half of PPF were first recognized at E18 (Fig. 6A). The earliest recognition of taste pores was recorded simultaneously in GS and PPF at E19 (21.0 ⫾ 2.6 and 39.3 ⫾ 2.2, respectively). Although the taste pores concentrated peripherally in GS, they were concentrated more posteriorly in the PPF. At PN1, the number of the taste buds with pores reached 52.0 ⫾ 3.4 and 64.3 ⫾ 3.2 in the GS and PPF, respectively. A gradual increase in the number of pores was traced along the first week with a higher incidence in the GS. At the second week, a transient decrease was recorded in the number of pored buds in both GS and PPF (Fig. 5). Toward the end of our investigation, the majority of taste buds were traced along the posterior 3⁄4 of PPF compared with its anterior 1⁄4 and 266.3 ⫾ 16.5 pored taste buds were recorded on the whole palate, with a double incidence in the PPF compared with GS (Fig. 6B). Immunoreactivity of ␣-Gustducin in the Developing Palatal Taste Buds Figure 7 traces the counts of ␣-gustducin-IR cells per taste bud at different stages of development on both the NIP and SP. The immunoreactivity for ␣-gustducin could not be identified in the nonpored taste buds, which continued proliferation on the palatal epithelium for the first Fig. 5. Temporal changes in the number of taste buds at the soft palate of the developing rat. A: Indicates total number of taste buds. B,C: Indicates the number of taste buds in the Geschmacksstreifen and posterior palatine field, respectively. 3 weeks after birth (Fig. 8A). Moreover, no immunoreactivity could be detected among the pored taste buds developed prenatally or shortly after birth in either the SP or NIP (Fig. 8B). At PN1, ␣-gustducin-LI was recognized in some pored taste buds of the SP (1.5 ⫾ 0.5; mean ⫾ SD), whereas others lacked such immunoreactivity (Fig. 8C). Meanwhile, ␣-gustducin-IR taste cells (1.4 ⫾ 0.5) could be recorded in the NIP at PN4 (Fig. 8D). Occasional taste buds containing only one gustducin-IR cell, however, were recognized in the oral epithelium of the papilla in some PN2 rats. Cells immunoreactive for ␣-gustducin were spindled-shaped, longitudinally-oriented having ovoid nuclei and a smooth regular outline throughout their length with a larger diameter at the nuclear region tapering to a smaller diameter at either ends. A pronounced immunoreactivity was evenly distributed throughout the cytoplasm from the apex where apical processes converged at the taste pore to the basal region of the taste bud. By age, a gradual increase in the counts of ␣-gustducin-IR cells was recognized in the taste buds of the developing NIP and SP (Fig. 8E,F). Significantly higher counts were re- DEVELOPMENT AND MATURATION OF PALATAL TASTE BUDS 265 Fig. 7. Temporal changes in the number of ␣-gustducin-immunoreactive (-IR) cells per taste bud in the nasoincisor papilla (E) and the soft palate (F) of the developing rat. Fig. 6. Topographic localization of taste buds at the soft palate of the rat at E18 (A) and PN70 (B). One open dot represents one nonpored taste bud, and one closed dot represents one pored taste bud. Taste buds are concentrated at the Geschmacksstreifen (GS) and middle portion of the posterior palatine field, forming T-shaped distribution. corded in the NIP as compared with the SP at the third week and older ages. At PN70, 14.4 ⫾ 2.7 and 12.4 ⫾ 2.5 cells were immunoreactive for ␣-gustducin in single taste bud in the NIP and SP, respectively. DISCUSSION Temporal and Spatial Development of the Palatal Taste Buds The prenatal development of the palatal taste buds was traced in mammalian species such as humans, monkeys, sheep, and cats as well as hamsters (Bradley and Stern, 1967; Bradley et al., 1979; Stedman et al., 1983; Zahm and Munger, 1983; Belecky and Smith, 1990). The present study revealed that the development of rat taste buds is almost prenatal in the SP, whereas most entirely postnatal in the NIP. Proliferation of the nonpored taste buds in the whole palatal epithelium was recognized along the first 3 weeks after birth, whereas the counts of pored taste buds continuously increased till the end of our investigation. In the SP, the earliest account of nonpored taste buds was traced at E18, whereas the pored ones developed at E19. A substantial number of pored and nonpored buds were demonstrated at E20 (i.e., about 60% of those recorded in adult; PN70). Harada et al. (2000) reported that 53% of the taste buds in the SP contained a taste pore at birth, which is consistent with the present study. Our findings, however, contrast with Mistretta (1972) who recognized taste buds in the neonatal rats but concluded that they should be morphologically immature at birth. Srivastava and Vyas (1979) reported that they were absent in newborn rats, developed along the first 3 weeks of age, and after this stage there is no taste bud formation. Regarding the taste buds in the NIP, the nonpored buds were recognized at E20 whereas the pored ones developed at PN4. Settembrini (1987) recorded taste bud primordia in the newborn rats but confirmed that these should be regarded as morphologically mature by the second week of postnatal life. Harada et al. (2000) recorded five to seven taste buds within the nasoincisor ducts at birth; one of which contained a taste pore. In the NIP of the mouse, OhtaYamakita et al. (1982) stated that the taste buds appear at 4 or 5 days, matured at 10 days of age and there is a continuous taste bud formation in the papilla over the first 10 weeks of age. Taken together with this evidence, it is concluded that the morphological maturation of the taste buds, i.e., presence of the taste pores, occurs in the SP prenatally and in the NIP at early days after the birth. The present study revealed that the taste buds were scattered between two populations in the NIP, ductal epithelium (medial aspect of the distal part of nasoincisor ducts), and oral epithelium (oral surface of the dome and the epithelium lateral to the papilla). The taste buds in the SP were uniformly distributed within a T-shaped zone 266 EL-SHARABY ET AL. Fig. 8. Photomicrographs of ␣-gustducin-like immunoreactivity in the nonpored (A) and pored taste buds (B–F) of the epithelium of soft palate (A–C,E) and nasoincisor papilla (D,F) of the developing rat. A: No immunoreactivity is detected in the nonpored taste bud in the soft palate at E20. B: A pored taste bud in the soft palate at PN1 has no immunoreactivity. An arrowhead indicates the taste pore. C,D: A pored taste bud has two or three ␣-gustducin-immunoreactive (-IR) cells in the soft palate at PN1 (C) and in the nasoincisor papilla at PN4 (D). E,F: At PN70, taste buds in the soft palate (E) and nasoincisor papilla (F) contain five or more ␣-gustducin-IR cells. Scale bar ⫽ 10 m (A,B,D,E); scale bar ⫽ 50 m (F). composed transversely from the incomplete GS fold and perpendicularly from the PPF at all stages of development. These findings are almost in agreement with the previous investigations (Miller and Spangler, 1982; Ohta- Yamakita et al., 1982; Miller and Smith, 1984; Settembrini, 1987; Harada et al., 1997a), but the detailed topographic distribution of taste buds in the SP, as shown in Figure 6 is different from that reported by Harada et al. DEVELOPMENT AND MATURATION OF PALATAL TASTE BUDS (1997a). We think such difference is due to the variation among the individual animals, as Miller and Spangler (1982) reported the different topographic distribution pattern among the individual animals. Although the topographic distribution pattern of the taste buds in the SP has been reported, the existence of appreciable number of taste buds along the oral epithelium of NIP has not previously taken into detailed consideration. Simultaneous deposition of the taste buds and subsequently their pores were recorded prenatally in the GS and PPF of the SP. Their development was traced most entirely after birth in the NIP where it was earlier in the oral epithelium than the nasoincisor ducts. Such particular deposition indicates the functional role of taste bud populations in the SP and oral epithelium of the NIP compared with that of the nasoincisor ducts along the first week of life. Around the second week, the count of pored buds within the nasoincisor ducts began to exceed that of the contiguous parts of the papilla, whereas a transient decrease was evidently recorded not only in the oral epithelium of NIP but also over the whole SP. Taste Transduction and Morphological Maturation of the Palatal Taste Buds Taste transduction is initiated at the taste pore of a taste bud where microvilli of the mature taste cells make contact with the outside environment. In the present study, the existence of a substantial number of distinct pores in the palate of fetal and newborn rats aroused interesting inquiries about the relationship between taste input and behaviors that depend upon this sensory system. Hall and Bryan (1981) stated that rats as young as 1–3 days of age shows differential responses to oral infusion of sucrose vs. water, which undoubtedly elucidates that functional taste buds should be present to mediate these ingestive responses. Contribution of the palatal taste buds to the taste mechanism in the neonatal rats became conceivably more crucial because Harada et al. (2000) ascertained that 53% of the taste buds in the SP contained a taste pore at birth compared with only 14% within fungiform papillae; whereas no pores were observed within foliate and circumvallate papillae. In the present study, the onset of taste transduction triggered by the earliest ␣-gustducin-IR cells recognized at PN2 (1.6 ⫾ 0.5) and PN4 (1.4 ⫾ 0.5) in the SP and NIP, respectively. Because the morphological observations suggested that ␣-gustducin is selectively expressed by mature taste cells; light or type II cells (Tabata et al., 1995; Yang et al., 2000), these cells should be absent in the all palatal taste buds of fetal and some of the neonatal rats or apparently still immature. Mistretta and Bradley (1977) asserted that the existence of elongated, longitudinally-oriented cells should be additional characteristics of mature taste buds. Moreover, ultrastructural and electrophysiological observations hypothesized the existence of transitional elongated cell type i.e., developing taste cells that had not reached the taste pore (Mackay-Sim et al., 1996). Interpreting with these studies, the present findings disclosed that the taste pore might be no longer a substantive criterion for the maturation of taste buds as it is still commonly believed. Instead, the existence of the pore cooperated by the mature taste cells is essential for the onset of taste transduction. The absence of these cells in the palate of neonatal rats may indicate that feeding stimulation 267 such as suckling is an essential signal for taste transduction. Function of the Palatal Taste Buds During Development Electrophysiological observations on the gustatory stimuli of the peripheral taste fibers argued that the greater superficial petrosal nerve, which innervates the palatal taste buds, is most responsive to sweet-tasting stimuli (Nejad, 1986; Harada et al., 1997b). In support, ␣-gustducin, which is strongly implicated in the mediation of sweet-tasting substances, was strongly expressed in the palatal taste buds (Boughter et al., 1997). The present findings and interpretations on the previous investigations (Miller and Spangler, 1982; Harada et al., 2000) aroused great evidence about the gustatory functions of the palatal taste buds in the neonatal life and their temporal collaboration with contiguous taste bud populations. It is important to emphasize that the onset of sweet transduction in the rat began by the PN1 on the SP and PN4 on the NIP. During the first few days, it is conceivable that the discernible number of soft palatal taste buds collaborated anteriorly by fungiform taste buds accomplish the overall taste behavior, whereas the nasopalatal buds can efficiently share in the taste responses by the end of the first week. Around the second week, a transient decrease in the total counts of taste buds with a relatively insufficient number of ␣-gustducin-IR cells/taste buds may be attributed to the progressive changes in the suckling and behavioral responses to varied food intake (Blass et al., 1979). This might be maintained by the developing foliate and circumvallate taste buds, which achieved a significant maturity around this age (Harada et al., 2000). We conclude that a substantial development of taste buds began prenatally in the SP, whereas most entirely postnatal in the NIP and the existence of both the taste pore and mature taste cells is essential for taste transduction. 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