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Morphological Development and Expression of Neurotrophin Receptors in the Laryngeal Sensory Corpuscles.

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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: yyoshio@iwate-u.ac.jp
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. In
addition, neurotrophin receptors may take part in the
regulation and maintenance of sensory structures rather
than morphogenesis.
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