Morphological Development and Expression of Neurotrophin Receptors in the Laryngeal Sensory Corpuscles.код для вставкиСкачать
THE ANATOMICAL RECORD 294:694–705 (2011) Morphological Development and Expression of Neurotrophin Receptors in the Laryngeal Sensory Corpuscles YOSHIO YAMAMOTO,* MOMO HASHIGUCHI, AND MISUZU YAMAGUCHI-YAMADA Laboratory of Veterinary Biochemistry and Cell Biology, Faculty of Agriculture, Department of Veterinary Sciences, Iwate University, Morioka, Japan ABSTRACT Morphological development of sensory structures in the laryngeal mucosa of postnatal rats was observed by use of immunohistochemistry for protein gene-product 9.5 (PGP9.5). Moreover, expression changes of high afﬁnity neurotrophin receptors, TrkA, TrkB and TrkC, and low afﬁnity neurotrophin receptor p75NTR were examined to elucidate the relationship to morphogenesis. Intraepithelial nerve endings and parent axons of the laminar endings with immunoreactivity for PGP9.5 have already appeared in the rat on embryonic day 18 (E18) as well as solitary chemoreceptor cells in the glottic cleft. According to neurotrophin receptors, TrkA immunoreactivity were observed on and after postnatal week 3 (3W) in the nervous sensory structures, that is, free nerve endings, laminar endings and sub- and intragemmal plexuses of the taste buds. In the laminar endings, TrkC immunoreactivity was also observed on and after 3W. According to the laryngeal sensory cells, the solitary chemoreceptor cells were immunoreactive to TrkA, TrkB, and TrkC on and after postnatal day 3 (P3). In the taste buds in arytenoid region, taste cells were immunoreactive for TrkA, TrkB, and TrkC on and after 3W, P14, and 3W, respectively. Immunoreactivity for p75NTR was observed on the surface of taste cells on and after P9. The results of the present study suggest that sensory structures in the laryngeal mucosa were developed on perinatal days to involve respiratory reﬂex, and that neurotrophin receptors may take part in the regulation and maintenance of sensory structures. Anat C 2011 Wiley-Liss, Inc. Rec, 294:694–705, 2011. V Key words: sensory corpuscles; neurotrophin development; larynx; respiration receptors; INTRODUCTION In the respiratory tract of adult mammals, several classes of sensory receptors exist to sense chemical irritants, airﬂow, and various endogenous stimulants (Widdicombe, 1998, 2001). In the larynx, ﬁve types of sensory receptors are electrophysiologically categorized by appropriate stimuli (Sant’Ambrogio et al., 1995; Widdicombe, 2001): (1) pressure receptors for laryngeal pressure changes, (2) drive receptors for laryngeal muscle contraction, (3) cold receptors for declining temperature of laryngeal cavity, (4) irritant receptors for acid, water, C 2011 WILEY-LISS, INC. V Grant sponsor: Japan Society for the Promotion of Science (JSPS), Japan; Grant number: 19658108. *Correspondence to: Yoshio Yamamoto, Laboratory of Veterinary Biochemistry and Cell Biology, Faculty of Agriculture, Iwate University, 18-8 Ueda 3-chome, Morioka, Iwate 020-8550, Japan. Tel./Fax: þ81-19-621-6273. E-mail: firstname.lastname@example.org Received 9 July 2010; Accepted 30 September 2010 DOI 10.1002/ar.21344 Published online 2 March 2011 in Wiley Online Library (wileyonlinelibrary.com). DEVELOPMENT OF LARYNGEAL SENSORY CORPUSCLES and touch stimuli, and (5) C-ﬁber nociceptors for chemical irritants including capsaicin. Morphologically, several sensory structures have also been reported in the laryngeal cavity: (1) intraepithelial free nerve endings (Domeij et al., 1991; Yamamoto et al., 1998b), (2) subepithelial laminar nerve endings (Yamamoto et al., 1998a, 2000a, b), (3) solitary chemoreceptor cells (Yu et al., 1996; Yamamoto et al., 2000b), and (4) taste buds (Nishijima and Atoji, 2004; Sbarbati et al., 2004b; Yamamoto et al., 1997). Although the correlation between physiological classiﬁcation and structure has not been well speciﬁed, laminar nerve endings, taste buds, and intraepithelial free nerve endings are thought to be pressure, irritant, and C-ﬁber receptors, respectively. These receptors are involved in respiratory regulation for normal breathing and/or defense reﬂex. In neonatal animals, respiration is signiﬁcantly developed from the fetal stage but is still immature (Abu-Shaweesh, 2004). In addition to central respiratory output, it has been suggested that the property of peripheral afferent inputs in response to CO2 and acidity is not mature (Abu-Shaweesh, 2004; Praud and Reix, 2005). However, the development of laryngeal sensory receptors, in terms of morphology and physiology, is not well known. It is well known that neurotrophins and their receptors are involved in the development and differentiation of sensory neurons (Ernfors, 2001) and mediate intracellular signaling pathways (Kaplan and Miller, 2000; PataTABLE 1. Number of rats used in this study Age Number E18 E20 P0 P3 P5 P7 P9 P11 P14 3W 5W 8W 13 12 9 11 11 11 10 7 7 7 10 10 E, Embryonic day; P, Postnatal day; W, Postnatal week. 695 poutian and Reichardt, 2001). Neurotrophin receptors can be divided into high- and low- afﬁnity receptors (Reichardt, 2006). The high-afﬁnity neurotrophin receptors are three subtypes of tyrosine kinase, TrkA, TrkB, and TrkC, and the low-afﬁnity neurotrophin receptor is the p75NTR. TrkA, TrkB, and TrkC can bind to nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin 3 (NT3), respectively. Furthermore, these receptors can also bind to neurotrophin 4/5 (NT4/5). p75NTR can bind to all types of neurotrophin. In the respiratory organs, neurotrophins and their receptors were also found to be expressed in both neuronal and non-neuronal tissue. The neurotrophins and their receptors are widely distributed in the nasal mucosa (Wu et al., 2006) and lung (Ricci et al., 2004). In the nasal mucosa, immunoreactivities for NGF, TrkA, and p75NTR were found to be distributed in epithelial cells and subepithelial gland (Wu et al., 2006). It was also demonstrated that the nerve ﬁbers were immunoreactive for TrkA and p75NTR. In the lung, Ricci et al. (2004) reported that immunoreactivities for NGF and BDNF were found in the alveolar epithelial cells, while TrkA, TrkB, TrkC, and p75NTR were distributed in the nerve ﬁbers. Furthermore, it has been suggested that BDNF and glial cell line-derived growth factor (GDNF) are required for the development of respiratory chemoafferent neurons (Katz, 2003). Erickson et al. (1996) reported that BDNF-knockout mice lack afferent nodose ganglion neurons, which leads to severe respiratory abnormalities, such as depressed and irregular breathing, and reduced chemosensory drive. Thus, the distribution of neurotrophin receptors in the developing laryngeal sensory structures is informative for understanding of respiration in neonatal animals. In this study, we examined morphogenesis of laryngeal sensory structures, that is, intraepithelial free nerve endings, subepithelial laminar endings, solitary chemoreceptor cells, and taste buds, by use of immunohistochemistry for a pan-neuronal marker, protein geneproduct 9.5 (PGP9.5). Furthermore, the distributions of neurotrophin receptors, TrkA, TrkB, TrkC, and p75NTR, were examined in the laryngeal mucosa in neonatal to adult rats to elucidate the relationship between morphogenesis and expression of neurotrophin receptors in the laryngeal sensory structures. TABLE 2. Antibodies used in this study Primary antibodies PGP9.5 TrkA TrkB TrkC p75NTR Calretinin GFAP GFAP Secondary antibodies Biotinylated anti-rabbit IgG Biotinylated anti-mouse IgG Alexa ﬂuor 488 labeled anti-rabbit IgG Alexa ﬂuor 488 labeled anti-mouse IgG TRITC labeled anti-goat IgG TRITC labeled anti-mouse IgG Code Host Dilution Source RA95101 SC-118 SC-12 SC-117 AN-170 AB1550 MS-280-P Z0334 Rabbit Rabbit Rabbit Rabbit Mouse Goat Mouse Rabbit 1:5,000 1:100 1:100 1:100 1:5,000 1:5,000 1:200 1:2,000 UltraClone Santa Cruz Biotech. Santa Cruz Biotech. Santa Cruz Biotech. Alomone Chemicon Lab Vision DAKO Cytomation Isle of Wight, UK Santa Cruz, CA Santa Cruz, CA Santa Cruz, CA Jerusalem, Israel Temecula, CA Fremont, CA Glostrup, Denmark 711-065-152 715-065-151 A-21202 A-21204 705-025-147 715-025-151 Donkey Donkey Donkey Donkey Donkey Donkey 1:100 1:100 1:200 1:200 1:100 1:100 Jackson Immunoresearch Jackson Immunoresearch Invitrogen Invitrogen Jackson Immunoresearch Jackson Immunoresearch West Grove, PA West Grove, PA Tokyo, Japan Tokyo, Japan West Grove, PA West Grove, PA 696 YAMAMOTO ET AL. TABLE 3. Combinations of antibodies for double immunoﬂuorescence Primary antibody 1 TrkA TrkA TrkB TrkB TrkC TrkC p75NTR p75NTR Secondary antibody 1 Primary antibody 2 Secondary antibody 2 a a a a a a d c Calretinin GFAP (MS-280-P) Calretinin GFAP (MS-280-P) Calretinin GFAP (MS-280-P) Calretinin GFAP (Z0334) b c b c b c b a a, Alexa ﬂuor 488 labeled anti-rabbit IgG; c, TRITC labeled anti-mouse IgG; b, TRITC labeled anti-goat IgG; d, Alexa ﬂuor 488 labeled anti-mouse IgG. MATERIALS AND METHODS All animal experiments in this study were approved by the local ethics committee of Iwate University. Materials Male Wistar rats and mated female rats were purchased from Japan SLC, Inc. (Slc: Wistar, Japan SLC, Hamamatsu, Japan). Embryonic day 18 (E18) and E20 fetuses were obtained by cesarean section from timemated rats killed on the expected day of gestation. Postnatal day 1 (P1) was set as the day of birth. Animals at P1, P3, P5, P7, P9, P11, and P14 were killed by decapitation. Number of animals in each stage was tabulated in Table 1. The larynges were dissected out and ﬁxed with Zamboni’s ﬁxative (4% paraformaldehyde and 0.5% picric acid in 0.1 M phosphate buffer; pH 7.4). The animals at postnatal week 3 (3W), 5W, and 8W were anesthetized by intraperitoneal injection of pentobarbital (15 mg/kg) and transcardially perfused with Ringer’s solution (500 mL) followed by Zamboni’s ﬁxative (500 mL); the larynges were further ﬁxed with the same ﬁxative overnight. The tissues were soaked with 30% sucrose in phosphate-buffered saline (PBS; pH 7.4) and frozen in deep freezer at 80 C. At least three rats were used at each stage. Immunohistochemistry The larynges were serially sectioned at 10 or 20 lm. The sections were mounted on glass slides coated with chrome alum-gelatin. The sections were immersed in 0.3% hydrogen peroxide in methanol for 30 min to block intrinsic peroxidase activity and incubated for 30 min with non-immune donkey serum (1:50). Then, the sections were incubated overnight at 4 C with rabbit polyclonal antisera against protein gene product 9.5 to reveal postnatal development of sensory structures in the laryngeal mucosa. Meanwhile, antibodies to TrkA, TrkB, TrkC, and p75NTR were applied to adjacent sections. Details of the antibodies used in the present study are summarized in Table 2. After incubation, the sections were treated with biotinylated donkey antibody against rabbit IgG or mouse IgG for 30 min at room temperature. Finally, the sections were treated with 0.02% 3-30 -diaminobenzidine tetrahydrochloride (DAB) and 0.006% H2O2 in 0.05 M Tris-HCl buffer (pH 7.4) to visualize immunoreaction sites. The sections were dehy- drated with graded series of ethanol, cleared with xylene, and coverslipped. Negative controls were incubated with preabsorbed antibody (1 lg antigen/1 lg antibody) or 0.05 M phosphate-buffered saline (pH 7.4; PBS) instead of primary antisera. The sections were examined with a light microscope (BX-50, Olympus, Tokyo, Japan). Observation was focused on intraepithelial free nerve endings and subepithelial laminar endings in the epiglottis, solitary chemoreceptor cells in the glottic cleft, and taste buds in the arytenoid region because these structures were shown to be densely distributed in each region (Yamamoto et al., 1997, 1998a, b, 2000a, b). Double Immunoﬂuorescence To identify immunoreactive structures in the subepithelial nerve endings, frozen sections were also stained by double immunoﬂuorescence for neurotrophin receptors with calretinin as a marker of subepithelial laminar endings (Yamamoto et al., 1998a) and with glial ﬁbrillary acidic protein (GFAP) as a glial marker. Details of antibody combinations are summarized in Table 3. After incubation with normal donkey serum, sections were incubated with a mixture of primary antibodies for 12 hr at 4 C. After rinsing with PBS, the sections were incubated with a mixture of secondary antibodies for 90 min at 25 C. The sections were coverslipped with Fluoromount (Diagnostic Biosystems, Pleasanton, CA), and examined with an epiﬂuorescence microscope (E-600, Nikon, Tokyo, Japan). RESULTS The results in this study are tabulated in Table 4 to show the appearance of laryngeal sensory structures and the expression of neurotrophin receptors. No immunoreactivity was found in the sections stained with preabsorbed antibodies. Intraepithelial Free Nerve Endings On E18 and E20, a few subepithelial varicose nerve ﬁbers immunoreactive to PGP9.5 were observed in the subepithelial layer of the epiglottis, and no intraepithelial free nerve endings were observed (Fig. 1A). Some epithelial cells with PGP9.5 immunoreactivity were observed on E20. On P1, subepithelial nerve ﬁbers were increased in number and subepithelial plexus appeared (Fig. 1B). Several varicose nerve endings were observed TABLE 4. Immunoreactivity for the components of the laryngeal sensory structures Immunopositive. Immunonegative. Chemoreceptor cells were not distinguished because many ciliated epithelial cells were positive. 698 YAMAMOTO ET AL. Fig. 1. Intraepithelial free nerve endings in developing rats. A–F, PGP9.5 immunoreactive nerve ﬁbers in the epiglottic mucosa. On E20 (A), a few nerve ﬁbers were found under the epithelial layer (E). On P1 (B), numerous immunoreactive nerve ﬁbers were found in the subepithelial plexus, and some varicose nerve ﬁbers intruded into the epithelial layer. Some epithelial cells were also immunoreactive to PGP9.5 (arrowheads). On P3 (C), free nerve endings reached the laryngeal lumen (arrow). On P5 (D), P14 (E), and 8W (F), intraepithelial free nerve endings with PGP9.5 immunoreactivity gradually increased in number. G–I, TrkA immunoreactivity in the epiglottic mucosa. On P1 (G) and P5 (H), TrkA immunoreactivity was only observed in the epithelial cells. On 5W (I), some subepithelial nerve ﬁbers were immunoreactive for TrkA (arrows). J–L, TrkB immunoreactive nervous elements in the epiglottic mucosa. On P5 (J), P11 (K), and 8W (L), subepithelial nerve ﬁbers were immunoreactive to TrkB (arrows). A few intraepithelial nerve endings were also observed on 8W (arrowheads). M-O, p75NTR immunoreactivity in the epiglottic mucosa. On P5 (M) and P11 (N), subepithelial connective tissues were diffusively immunoreactive for p75NTR (asterisks). On P11 and 8W (O), the surface of basal cells (double arrows) in epithelia was also immunoreactive. in the lower third of the epithelial layer, and a few nerve endings reached the laryngeal lumen at this stage. On P3, the subepithelial nerve plexus was developed, and a few intraepithelial free nerve endings reached the laryngeal lumen (Fig. 1C). On and after P5, the intraepithe- lial free nerve endings were increased in number, and most of them reached the laryngeal lumen (Fig. 1D–F). No TrkA immunoreactivity was observed in the epiglottic mucosa except for epithelial cells in the spinous layer of squamous stratiﬁed epithelium before P14 (Fig. DEVELOPMENT OF LARYNGEAL SENSORY CORPUSCLES 699 Fig. 2. Subepithelial laminar nerve endings in the epiglottic mucosa of developing rats. A–F, PGP9.5 immunoreactive laminar endings. On E20 (A), branched thick nerve ﬁbers were observed under the epithelial layer (arrow). On P3 (B) and P5 (C), the thick nerve ﬁbers (arrows) with subepithelial terminal swellings (arrowheads) were immunoreactive for PGP9.5. On P11 (D), 3W (E), and 8W (F), PGP9.5 immunoreactive terminal swellings gradually developed in size and increased in number. G–I, Distribution of TrkA immunoreactivity in subepithelial laminar nerve endings. On P9 (G), no immunoreactivity was found. On 3W (H) and 5W (I), terminal swellings (arrowheads) and some parent axons (arrows) were immunoreactive for TrkA. J–L, TrkB immunoreactivity in laminar endings. The thick parent axons to laminar endings (arrows) were immunoreactive to TrkB on P9 (J), 3W (K), and 8W (L). The terminal swellings were also weakly immunoreactive on 3W and 8W (arrows). M–O, Distribution of TrkC immunoreactivity in laminar endings. On P9 (M), immunoreactivity for TrkC was observed on a few epithelial cells in the epiglottis, but not nervous elements. On 3W (N) and 8W (O), terminal swellings (arrowheads) and some parent axons (arrows) were immunoreactive to TrkC. 1G, H). On and after 3W, some nerve ﬁbers in the subepithelial nerve plexus were immunoreactive to TrkA (Fig. 1I). In some part of the epiglottic epithelium, epi- thelial cells were immunoreactive to TrkA to various degrees at all developmental stages. In the case of TrkB immunoreactivity, subepithelial nerve ﬁbers were 700 YAMAMOTO ET AL. Fig. 3. Double immunoﬂuorescence for neurotrophin receptors with calretinin and GFAP. A, Punctate labelings for TrkA (green) in the subepithelial region were identical to regions immunoreactive for calretinin (red), and revealed as a yellowish color (arrows). Epithelial layer (E) was also immunoreactive for TrkA. B, Region of TrkB immunoreactivity (green) was also immunoreactive to calretinin (red), as shown in yellow in the merged ﬁgure (arrowheads). C, GFAP immunoreactivity (red) was not colocalized to TrkB immunoreactivity (green). D, Similar to TrkA, punctate labelings for TrkC (green) in the subepithelial region were identical to the region immunoreactive for calretinin (red) and showed a yellowish color (arrowheads). E, F, p75NTR immunoreactivity (green in E, red in F) was localized with neither calretinin immunoreactive nerve endings (red in E) nor GFAP immunoreactive Schwann cells (green in F). positive on and after P3 (Fig. 1J, K), and the intraepithelial free nerve endings were immunoreactive after 3W (Fig. 1L). TrkC immunoreactivity was not found in the free nerve endings and subepithelial nerve plexus but was observed in some epithelial cells. p75NTR immunoreactivity appeared in the subepithelial connective tissues before P14 (Fig. 1M, N). In some regions, the basal cells in the epiglottic epithelium were immunoreactive for p75NTR in all developmental stages (Fig. 1N, O). but not for GFAP (Fig. 3C). Similar to TrkA, the laminar endings were immunoreactive for TrkC on and after 3W (Fig. 2M–O). Stainings of the terminal swellings and the parent axon were intense and weak, respectively (Fig. 3D). p75NTR immunoreactivity did not coexist with immunoreactivities for calretinin and GFAP and was found in the perineuronal cells around the parent axon of laminar endings (Fig. 3E, F). Solitary Chemoreceptor Cells Subepithelial Laminar Nerve Endings On E18 and E20, a few branched thick nerve ﬁbers with PGP9.5 immunoreactivity were observed just below the epithelial layer in the epiglottis (Fig. 2A), but terminal swelling was not distinguished at these stages. On P3, some ﬂattened laminar structures appeared at some tips of the branched nerve ﬁbers (Fig. 2B). The laminar terminations were gradually increased in number from P5 to P14 (Fig. 2C, D). After P14, subepithelial laminar nerve endings consisted of a thick branched parent axon with numerous laminar terminations (Fig. 2E, F). Immunoreactivity for TrkA was not observed before 3W (Fig. 2G). On 3W, 5W, and 8W, the thick parent axon and laminar terminations were immunoreactive to TrkA (Fig. 2H, I). The terminal swellings were intensely stained for TrkA but the parent axons were only weakly stained at all stages. In double immunoﬂuorescence, the laminar terminal swellings with TrkA immunoreactivity were also immunoreactive to calretinin (Fig. 3A). Parent axons to laminar nerve endings with TrkB immunoreactivity were found in the larynx on P5 and at subsequent stages. The laminar terminations were weakly immunostained for TrkB on and after P9 (Fig. 2J–L). The parent axon and the terminal swellings were also immunoreactive to calretinin in double immunoﬂuorescence (Fig. 3B) Flask-shaped solitary chemoreceptor cells immunoreactive to PGP9.5 were found in the mucosa of glottic cleft of rats on E18 and E20 although nerve ﬁbers attached to chemoreceptor cells were not observed (Fig. 4A). On P1, a few nerve ﬁbers immunoreactive to PGP9.5 were associated with the chemoreceptor cells (Fig. 4B). PGP9.5 immunoreactive varicose nerve ﬁbers gradually increased by P11 (Fig. 4C–E), and numerous nerve ﬁbers associated with the chemoreceptor cells were observed in the glottic cleft of the rats at 3W, 5W, and 8W (Fig. 4F). Solitary chemoreceptor cells were also immunoreactive for TrkA, TrkB, and TrkC (Fig. 4G–L). Immunoreactivity for TrkB was found in the solitary chemoreceptor cells on and after P3 (Fig. 4I). Immunoreactivities for TrkA and TrkC were also found in the chemoreceptor cells on P3 to P9 (Fig. 4G, J). On P7 and P9, some ciliated epithelial cells in the glottic cleft were weakly immunoreactive for TrkA and TrkC. On and after P11, the chemoreceptor cells immunoreactive for TrkA and TrkC were not distinguished because most of the ciliated epithelial cells were intensely immunoreactive (Fig. 4H, K). Nerve ﬁbers associated with chemoreceptor cells were not immunoreactive for TrkA, TrkB, and TrkC at all developmental stages examined. No immunoreactivity for DEVELOPMENT OF LARYNGEAL SENSORY CORPUSCLES 701 Fig. 4. Solitary chemoreceptor cells and their associated nerve ﬁbers on the glottic cleft. A–F, Solitary chemoreceptor cells with PGP9.5 immunoreactivity. On E20 (A), the chemoreceptor cells appeared within the epithelial layer of rat at E20 (arrows). On P1 (B), varicose nerve ﬁbers with PGP9.5 immunoreactivity were observed around the cells (arrows in inset). After P1, the chemoreceptor cells and nerve ﬁbers gradually increased in number (C, P3; D, P5; E, P11; F, 3W). G–K, Immunoreactivity for TrkA (G, H), TrkB (I), TrkC (J, K) and p75NTR (L) in the laryngeal epithelium. Immunoreactivities for TrkA (G), TrkB (H), and TrkC (I) were observed in the solitary chemoreceptor cells on P5 rats (arrows in panels G, H and J). On 3W, solitary chemoreceptor cells immunoreactive for TrkA and TrkC were not distinguished because numerous ciliated cells were immunoreactive (H, K). No chemoreceptor cell was identiﬁed on the section stained for p75NTR (L). P75NTR was identiﬁed in the chemoreceptor cells at any stage (Fig. 4L). taste cells. On P9 and P11, PGP9.5 immunoreactivity was found in the intragemmal nerves and in the taste cells, respectively (Fig. 5A, B). After P11, the subgemmal nerve ﬁbers, intragemmal plexus, and taste cells were immunoreactive for PGP9.5 (Fig. 5C). Immunoreactivities for TrkA and TrkB were found in both nerve ﬁbers and taste cells (TrkA, Fig. 5D–F; TrkB, Fig. 5G–I). Some nerve ﬁbers were immunoreactive to Taste Buds On P7, taste buds were found in the laryngeal mucosa, and PGP9.5 immunoreactivity was detected in the subgemmal plexus but not in the intragemmal plexus and 702 YAMAMOTO ET AL. Fig. 5. DEVELOPMENT OF LARYNGEAL SENSORY CORPUSCLES TrkA or TrkB in the subgemmal plexus on P9 (Fig. 5D), and in both the intragemmal nerve ﬁbers and the subgemmal plexus on P11 and P14 (Fig. 5D, E, G). On and after 3W, some taste cells were immunoreactive to TrkA and TrkB in addition to the nerve ﬁbers (Fig. 5F, I). On the other hand, TrkC immunoreactivity was not found in the nervous elements at any developmental stage (Fig. 5J–L). In the taste cells, TrkC immunoreactivity was detected on and after P14 (Fig. 5K, L). Weak immunoreactivity for p75NTR was observed in the taste cells on and after P9, but not in the nerve ﬁbers associated with taste buds at any stage (Fig. 5M–O). Around the taste buds, the cell surface of the basal cells of squamous stratiﬁed epithelium was also immunoreactive for p75NTR as well as the epiglottic mucosa (Fig. 5M–O). DISCUSSION As shown in Table 4, morphogenesis of sensory structures in the laryngeal mucosa was mainly developed on perinatal days and was different in each sensory structure. Furthermore, immunoreactivities for neurotrophin receptors were appeared during development of sensory structures. On the other hand, immunoreactivity for p75NTR were not found in the sensory structures expect for taste cells. Intraepithelial Free Nerve Endings Previous immunohistochemical studies demonstrated that some laryngeal intraepithelial free nerve endings were immunoreactive to TRPV1, the sensory molecule activated by acidity, higher temperature, and capsaicin (Uno et al., 2004; Yamamoto and Taniguchi, 2005). These reports suggest that part of the intraepithelial free nerve endings comprises nociceptors elicited respiratory reﬂexes like apnea, bronchoconstriction, and cough (Sant’Ambrogio et al., 1995). In sheep at birth, nerve impulses in the superior laryngeal nerve were found to be increased by the application of capsaicin to the laryngeal mucosa (Roulier et al., 2003). Because the laryngeal free nerve endings with PGP9.5 immunoreactivity were present even on embryonic days, nociceptive function in laryngeal mucosa may already be present, even at birth. However, the neurotrophin receptors were not observed by P3. Thus, TrkA and/or TrkB may not participate in the differentiation of the endings, and they seem to play a role in the regulation and maintenance of the laryngeal intraepithelial free nerve endings. 703 endings are activated by the epithelial tension that is caused by pressure changes in the laryngeal cavity (Yamamoto et al., 1998, 2000a, b). In the tracheobronchial tree, Hering-Breuel reﬂex, which is induced by extension of smooth muscle of trachea and bronchi, develops until P4 in rat (Merazzi and Mortola, 1999). The existence of subepithelial laminar endings in rat at P3 suggests that pressure changes also occurred in the larynx at an early postnatal stage, namely, P3 or P4. The morphology of the subepithelial laminar endings resembles Rufﬁni’s endings, as previously discussed (Yamamoto et al., 1998a). In BDNF/TrkB double-knockout mice, Rufﬁni’s endings in the periodontal ligaments decreased in number (Hoshino et al., 2003). This report seems to indicate that signaling pathways using BDNF and TrkB are important for the differentiation of periodontal Rufﬁni’s endings. Moreover, Meissner’s corpuscles were diminished in both BDNF- and TrkBknockout mice (González-Martı́nez et al., 2004, 2005) and increased in BDNF-overexpressed mice (LeMaster et al., 1999). These reports suggest that interaction between BDNF and TrkB is important for the morphogenesis of Meissner’s corpuscles. In this study, immunoreactivity for TrkB was observed in P5, but those of TrkA and TrkC appeared after 3W. Therefore, TrkB may take part in the early development of the laminar endings, as well as Rufﬁni’s endings and Meissner’s corpuscles, while TrkA and TrkC seem to maintain mature endings after 3W. Because immunoreactivity for Trk receptors was not observed earlier than P3, another developmental mechanism associated with Trk receptors may be important in the early development of laryngeal laminar endings. p75NTR has been suggested to be an important factor for axon development and myelination (Bentley and Lee, 2000; Cosgaya et al., 2002). In this study, immunoreactivity for p75NTR was only observed in the epithelial cells in the basal layer and the perineurial cells but not in the Schwann cells. p75NTR in the epithelial cells may induce axon enlargement in immature laminar endings. However, p75NTR in the perineurial cells may guide the course of the parent axon of the endings by epithelial cells, and involve myelination of Schwann cells by perineurial cells. Solitary Chemoreceptor Cells On the basis of morphological observations, it has been suggested that the subepithelial laminar nerve Solitary chemoreceptor cells are brush cells with numerous innervations (Yu et al., 1996). They contain molecules for sensory signaling pathways, such as phospholipase C b2 and a-gustducin (Sbarbati et al., 2004a; Merigo et al., 2005), and are increased in number under hypercapnic hypoxia (Yamamoto et al., 2000a, b). In this Fig. 5. Taste buds in the laryngeal mucosa-covered arytenoid cartilage. A–C, PGP9.5 immunoreactivity in the arytenoid taste buds in rats. The PGP9.5 immunoreactivity was observed in intragemmal and subgemmal nerve ﬁbers on P9 (A). On P11 (B), some taste cells were also immunoreactive to PGP9.5 in addition to the nerve ﬁbers. In 8W (C), numerous taste cells were immuoreactive to PGP9.5, and welldeveloped subgemmal plexus was shown. D-F, TrkA immunoreactivity in the taste buds. At P11 (D) and P14 (E), a few subgemmal nerve ﬁbers were immunoreactive to TrkA (arrowheads). Several taste cells were immunoreactive at 3W (arrow in F). G–I, TrkB immunoreactivity in the taste buds. TrkB immunoreactivity was mainly distributed in the intragemmal and subgemmal nerve ﬁbers at all developmental stages (G, P9; H, P14; I, 3W). On 3W, some taste cells were also immunoreactive to TrkB (arrow in I). J–L, TrkC immunoreactivity in the taste buds. No immunoreactivity for TrkC was observed at the taste buds before 3W (J). Some taste cells were immunoreactive to TrkC, but the nerve ﬁbers were not observed on 3W (K) and 5W (L). M–O, p75NTR immunoreactivity in the taste buds. Taste cells were weakly immunoreactive to p75NTR during all developing stages examined in the present study (M, P9; N, 3W; O, 5W). Surface of epithelial cells in the basal layer of squamous stratiﬁed epithelium was also immunoreactive to p75NTR. Subepithelial Laminar Endings 704 YAMAMOTO ET AL. study, PGP9.5 immunoreactive cells were already observed at birth, gradually increased in number, and were innervated. These results suggest that laryngeal mucosa may have a chemosensory function for environmental gases at birth, and that mucosal chemosensory function develops with age. Because TrkA, TrkB, and TrkC immunoreactivities were observed after P5, the Trk receptors are considered to be related to differentiation of chemosensory function at postnatal stages. Moreover, TrkA and TrkC were widely distributed in the epithelial layer after P11. Thus, TrkA and TrkC molecules may regulate epithelial cells other than chemoreceptor cells at juvenile to adult stages. It has been reported that ventilation volume per minute and frequency of respiration decreased after 5% hypercapnic stimulation at P16-17 and P41-42 but were sustained at P5 (Abu-Shaweesh et al., 2007). CO2 is mainly sensed in the sensory neurons in medulla oblongata and glomus cells in carotid body (Lahiri and Forster, 2003), but the chemoreceptor cells in the laryngeal mucosa may also sense CO2 for local regulation during the neonatal period. Taste Buds In the lingual gustatory papillae, it has been reported that morphological development of the taste buds occurs postnatally (Mistretta and Liu, 2006). Similarly, the results of this study indicated that the laryngeal taste buds are also developed after birth. Functionally, it is suggested that laryngeal taste buds sense low osmolarity of the laryngeal mucosal surface (Bradley, 2000; Hanamori, 2001). Additionally, prolonged apnea and arousal were found to be the predominant responses to laryngeal chemoreﬂex in newborn puppies, and generally diminished after the ﬁrst week (Boggs and Bartlett, 1982). Maturation of laryngeal taste buds after birth may be related to the change of laryngeal chemoreﬂex. In the taste buds in the circumvallate papillae, it has been reported that BDNF were expressed in type II and III taste cells but not in type I taste cells and basal cells (Yee et al., 2003). Immunoreactivities for NGF, TrkA, and TrkB were shown in these cells, and it has been suggested that the signaling pathways of NGF/TrkA and BDNF/TrkB play important roles in the maturation of type II and type III taste cells, respectively (Yee et al., 2005). It has also been reported that TrkB immunoreactivity was colocalized with BDNF and NT3 in the taste cells, and these were thought to be the most important molecules in the neurotrophin receptors for taste bud survival on the basis of denervation study of glossopharyngeal nerve (Ganchrow et al., 2003). However, in the laryngeal taste buds, the immunoreactivities for TrkA and TrkB were restricted in the nerve ﬁbers at early developmental stages before 3 weeks after birth. In contrast to TrkA and TrkB, TrkC in the taste cells was immunohistochemically detected at P14. In laryngeal taste buds, it is speculated that TrkA and TrkB participate in differentiation of taste ﬁbers, while TrkC may play an important role in the maturation of taste cells. On the other hand, p75NTR immunoreactive epithelial cells were observed around developmental circumvallate taste buds (Oakley and Witt, 2004). The distribution pattern of p75NTR in the laryngeal taste buds was similar to that in the lingual taste buds. Moreover, the numbers of taste buds in circumvallate and fungiform papillae were reduced in p75NTR-knockout mice, which suggested that p75NTR is required for taste bud development (Krimm, 2006). p75NTR may be involved in the maturation of laryngeal taste buds as suggested for lingual taste buds. Neurotrophin Receptors in the Epithelial Cells In this study, neurotrophin receptors were distributed in the epithelial cells and connective tissue in the laryngeal mucosa of developing rats. Immunoreactivities for high afﬁnity neurotrophin receptors, TrkA, TrkB, and TrkC have been reported in many nonneuronal tissues including various epithelial cells of human (Shibayama and Koizumi, 1996). In the epidermis, they reported the basal cells immunoreactive for TrkA, TrkB, and TrkC. In squamous stratiﬁed epithelium in the epiglottis and arytenoid region, epithelial cells immunoreacitve TrkA and TrkC may contribute to regulate cellular function. Furthermore, it has been reported that p75NTR could be used as a stem cell marker on human esophagus (Okumura et al., 2003). Because immunoreactivity for p75NTR was restricted in basal cells of the epiglottic and arytenoid mucosa, p75NTR may play a role on maturation of epithelial cells. On the other hand, the ciliated epithelium cells were immunoreactive for TrkA in the nasal mucosa of horse and human (Garcia-Suarez et al., 1997; Wu et al., 2006). Basal cells in the nasal mucosa were also immunoreactive for p75NTR in human (Wu et al., 2006). In the human lung, immunoreactivity for neurotrophin receptors were not identiﬁed in bronchial ciliated cells but immunoreactivities for TrkA and TrkC were found in the alveoli (Ricci et al., 2004). The ciliated cells immunoreactive for TrkA and TrkC in the glottic cleft seem to be similar to the nasal epithelium, although immunoreactivity for p75NTR was not identiﬁed in this study. Increase of immunoreactive epithelial cells in later period of development may indicate neurotrophin involve maintain of mature cells rather than cellular development. CONCLUSION In conclusion, the results of this study suggest that sensory structures in the laryngeal mucosa were developed on perinatal days to involve respiratory reﬂex. 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