Sensory Innervation of the Human Soft Palate.

The human soft palate plays an important role in respiration, swallowing, and speech. These motor activities depend on reflexes mediated by sensory nerve endings. To date, the details of human sensory innervation to the soft palate have not been demonstrated. In this study, eight adult human whole-mount (soft palate-tongue-pharynx-larynx-upper esophagus) specimens were obtained from autopsy. Each specimen was bisected in the midline, forming two equal and symmetrical halves. Eight hemi-specimens were processed with Sihler's stain, a whole-mount nerve staining technique. The remaining eight hemi-soft palates were used for immunohistochemical study. The soft palatal mucosa was dissected from the oral and nasal sides and prepared for neurofilament staining. Our results showed that the sensory nerve fibers formed a dense nerve plexus in the lamina propria of the soft palatal mucosa. There was a significant difference in the innervation density between both sides. Specifically, the oral side had higher density of sensory nerve fibers than the nasal side of the soft palate. The mean number and percent area of the sensory nerve fibers in the mucosa of the nasal side was 78% and 72% of those in the mucosa of the oral side, respectively (P < 0.0001). The data presented here could be helpful for further investigating the morphological and quantitative alterations in the sensory nerves in certain upper airway disorders involving the soft palate such as obstructive sleep apnea (OSA) and for designing effective therapeutic strategies to treat OSA. Anat Rec, 301:1861-1870, 2018. © 2018 Wiley Periodicals, Inc.

Immunohistochemical study of TAFII250 in the rat laryngeal nervous system.

The cause of spasmodic dysphonia, a dystonic disorder of the larynx, remains unclear. Recently, TAFII250, TATA-box binding protein associated factor, was suggested to be involved in dystonia parkinsonism. There is a possibility that TAFII250 is involved in spasmodic dysphonia, but little information is available about the expression of TAFII250 in the laryngeal nervous system. In this study, we investigated the localization of TAFII250 protein in the rat laryngeal nervous system by immunohistochemistry. TAFII250-immunoreactivity was detected in the nodose ganglion and superior cervical ganglion. In these nuclei, TAFII250 was localized in the nucleus of NeuroTrace-positive neurons but not in GFAP-positive glial cells. No positive cells were detected in the motor and parasympathetic nervous system. TAFII250-immunoreactivity was sustained between 3 and 7 days after vagotomy, but at 14 days expression was down-regulated in the distal part of the nodose ganglion. These findings suggest that TAFII250 plays an important role in the laryngeal innervation of the sensory and sympathetic nervous systems.

Serotonin inputs to inspiratory laryngeal motoneurons in the rat.

Serotonergic neurons are distributed widely throughout the central nervous system and exert a tonic influence on a range of activities in relation to the sleep-wake cycle. Previous morphologic and functional studies have indicated a role for serotonin in control of laryngeal motoneurons. In the present study, we used a combination of intracellular recording, dye-filling, and immunocytochemistry in rats to demonstrate close appositions between serotonin immunoreactive boutons and posterior cricoarytenoid (PCA) and cricothyroid (CT) motoneurons, both of which are located in the nucleus ambiguus and exhibit phasic inspiratory activity. PCA motoneurons received 29 +/- 5 close appositions/neuron (mean +/- SD, n = 6), with the close appositions distributed more frequently on the distal dendrites, less frequently on the proximal dendrites, and sparsely on the axons and somata. CT motoneurons received 56 +/- 15 (n = 6), with close appositions found on both the somata and dendrites, especially proximal dendrites. Close appositions on the axons were only seen on one CT motoneuron. These results demonstrate a significant serotonin input to inspiratory laryngeal motoneurons, which is more prominent on CT compared with PCA motoneurons, and may reflect the different functional role of the muscles that they innervate during the sleep-wake cycle.

Local differences in vagal afferent innervation of the rat esophagus are reflected by neurochemical differences at the level of the sensory ganglia and by different brainstem projections.

The objective of the present study was to characterize further the vagal afferent fibers in the rat esophagus, particularly those in its uppermost part, their cell bodies in vagal sensory ganglia, and their central projections. We applied immunohistochemistry for calretinin, calbindin, and calcitonin gene-related peptide (CGRP); retrograde tracing with FluoroGold; and transganglionic tracing with wheat germ agglutinin-horseradish peroxidase in combination with neurectomies. Vagal terminal structures in the muscularis propria of the whole esophagus consisted of calretinin-immunoreactive intraganglionic laminar endings that were linked to cervical vagal and recurrent laryngeal nerve pathways. The mucosa of the uppermost esophagus was innervated by a very dense net of longitudinally arranged, calretinin-positive fibers that were depleted by section of the superior laryngeal nerve. Distal to this area, the mucosa was virtually devoid of calretinin-immunoreactive vagal afferents. Calretinin-positive mucosal fibers in the upper cervical esophagus were classified into four types. One type, the finger-like endings, was sometimes immunoreactive also for CGRP. About one-third of cell bodies in vagal sensory ganglia retrogradely labeled from the upper cervical esophagus expressed CGRP, whereas two-thirds coexpressed calretinin and calbindin but not CGRP. In addition to the central subnucleus of the nucleus of the solitary tract, vagal afferents from the upper cervical esophagus also projected heavily to the interstitial subnucleus. This additional projection was attributed to mucosal afferents traveling through the superior laryngeal nerve. The present study provides a possible morphological basis for bronchopulmonary and aversive reflexes elicited upon stimulation of the esophagus.

The distribution of nerve fibres immunoreactive for vasoactive intestinal peptide, calcitonin gene-related peptide, substance P and dopamine beta-hydroxylase in the normal equine larynx.

The autonomic innervation of the mammalian respiratory system is complex, and involves a wide variety of peptide and non-peptide neurotransmitters which will have an important role in normal laryngeal function and the response to disease. This innervation has been partially described in the horse airway and lung, but there is no information on the equine larynx. This paper describes the expression and distribution of nerve fibres immunoreactive for vasoactive intestinal peptide (VIP), calcitonin gene-related peptide (CGRP), substance P (SP) and the adrenergic enzymatic marker dopamine beta-hydroxylase (DBetaH) in the mucosa of the equine larynx. The overall relative density of nerve fibres immunoreactive for the different antigens was VIP>>CGRP>SP>>DBetaH. There were differences in the distribution of nerve fibre types, although each antigen was found in nerve fibres adjacent to blood vessels and mucous glands. VIP -like immunoreactivity (VIP -Li) was particularly extensive in association with mucous glands. SP - and CGRP -like immunoreactivity (SP -Li, CGRP -Li) were also seen close to the epithelium, with occasional nerve fibres coursing beneath and between the epithelial cells. Fragments of SP -Li and CGRP -Li fibres were also present in large nerve fibre bundles and ganglionic cell clusters, but not in the neurons themselves. The density of nerve fibres immunoreactive for DBetaH was very low and restricted to blood vessels and mucous glands. There was marked variation in the density of nerve fibres at the different sites, with the greatest density, particularly for VIP, over the arytenoid cartilage. Immunoreactive nerve fibres were less plentiful over the epiglottis, and the density of all types of nerve fibres was low over the cricoid cartilage. Overall VIP -Li nerve fibres were the most plentiful.

CGRP and substance P in intraepithelial neuronal structures of the human upper respiratory system.

The distribution of intraepithelial nerve fibres and neuroendocrine cells within the surface and glandular epithelium of human nasal mucosa and larynx was examined using immunohistochemical techniques. Neuronal structures were immunostained for the general neuroendocrine marker protein gene-product (PGP) 9.5, and the two neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) using immunofluorescence and streptavidin-biotin-peroxidase complex (S-ABC) methods. Intraepithelial nerve fibres with free nerve endings contained PGP 9.5 and were found within the respiratory surface epithelium of the nasal mucosa and the squamous epithelium of the larynx. A subpopulation of these nerve fibres showed positive immunoreactivties with antibodies against SP and CGRP. Nerve fibres within the ductal epithelium of subepithelial excretory ducts passing the basal membrane and reaching the luminal part were detected. These nerve fibres showed CGRP-like immunoreactivity but not for SP. A dense network of nerve fibres within the squamous surface epithelium was detected in the subglottic and epiglottic region containing CGRP and SP in a small subpopulation of nerve fibres. Single intraepithelial taste buds in the epiglottic region and neuroendocrine cells within the subglottic epithelium expressed PGP 9.5.

Similar distribution of mast cells and substance P- and calcitonin gene-related peptide-immunoreactive nerve fibers in the adult human larynx.

The mechanisms causing supraglottic and subglottic edema in the human larynx are not fully understood. Substance P (SP)- and calcitonin gene-related peptide (CGRP)-containing nerve fibers and mast cells have been suggested to induce inflammation and edema in other parts of the body. In this study of the adult human larynx the distribution of mast cells was studied in relation to SP- and CGRP-containing nerve fibers. Substance P- and CGRP-immunoreactive nerve fibers and numerous mast cells were found in the epiglottis and in the subglottic region of the larynx. Only occasional mast cells and no nerve fibers showing SP- or CGRP-like immunoreactivity were found in the vocal cords. In conclusion, the present study has shown that the distribution of nerve fibers showing SP- and CGRP-like immunoreactivity and mast cells has a similar regional variability. As the highest density of SP- and CGRP-containing nerve fibers and mast cells was present in the regions of the larynx where edema occurs, SP, CGRP, and/or mast cells might be involved in the pathogenesis of edema.

Nitrergic innervation of the rat larynx measured by nitric oxide synthase immunohistochemistry and NADPH-diaphorase histochemistry.

We evaluated the involvement of nitric oxide (NO) in the laryngeal innervation of rats using NADPH-diaphorase (NADPH-d) histochemistry and neuronal nitric oxide synthase (nNOS) immunohistochemistry. The findings obtained by NADPH-d histochemistry were identical with those obtained by nNOS immunohistochemistry, indicating that NADPH-d is nNOS in the laryngeal innervation system. We found NADPH-d-positive nerve fibers in every region of the larynx. In the epithelia of the mucosa, a small number of NADPH-d-positive nerve fibers were detected. The plexus of NADPH-d-positive nerve fibers was commonly found in the lamina propria, and some of these fibers were clearly associated with blood vessels. We also noted NADPH-d-positive nerve fibers in the region of laryngeal glands. Some of these fibers appeared to terminate in the glandular cells. We found NADPH-d-positive nerve fibers with varicosities in the intrinsic laryngeal muscle and free-ending nerve fibers on the muscle fiber. Motor end plate-like structures were positive for NADPH-d histochemistry. The NADPH-d-positive nerve fibers appeared to terminate at motor end plate-like structures in two of nine rats examined. A cluster of NADPH-d-positive neurons were occasionally present in the lamina propria of the laryngeal mucosa, in the connective tissue between the thyroid cartilage and intrinsic laryngeal muscle, and in the connective tissue near the cricoarytenoid joint. The present findings suggest that NO participates in the autonomic, sensory, and motor innervation of the larynx.

Immunocytochemical survey of the neuroepithelial endocrine system in the respiratory tract of the Tokyo salamander, Hynobius nebulosus tokyoensis TAgo.

The epithelial lining of the respiratory tract of urodeles has been shown to harbor an innervated system of neuroepithelial endocrine (NEE) cells. Even between phylogenetically closely related species, large differences have been reported in the appearance and chemical coding of the NEE system. Although urodeles are well suited for the purpose, none of the prior studies have provided an immunocytochemical survey of the NEE system in all parts of the respiratory tract. In the present study, many bioactive substances and a general marker were immunocytochemically demonstrated in serial sections of the entire respiratory tract of the Tokyo salamander, Hynobius nebulosus tokyoensis, a species in which neuroepithelial bodies (NEBs) were previously characterized at the electron microscopic level. In the current study, serotonin-immunoreactive solitary NEE cells were observed in variable numbers in the larynx, in all parts of the trachea, and in areas of the lungs covered with ciliomucous epithelium. Serotonin-containing NEBs, however, were detected in small cranial areas of the lung only. Solitary NEE cells were seen in the trachea and lungs of H. nebulosus tokyoensis by immunocytochemical staining for somatostatin, calcitonin, calcitonin gene-related peptide, and bombesin, but the number, localization, and appearance of the labeled NEE cells differed considerably. Only calcitonin-like immunoreactivity was also noted in some NEB-like cell clusters in the cranial parts of the lungs. Unlike many other vertebrates, neuron specific enolase was found to be a poor marker for the NEE system in the salamander species used in this investigation. It may be concluded that the NEE system of H. nebulosus tokyoensis contains at least five different bioactive substances.(ABSTRACT TRUNCATED AT 250 WORDS)

Vagal afferent nerve endings in the trachealis muscle of the dog.

Nerve endings in the canine tracheal muscle were examined by light microscopy after immunohistochemical staining with an antibody against neurofilament protein, and by transmission electron microscopy. The endings were found to consist of accumulations of ramified axon terminals. The endings ranged from 100-400 microns in maximal length to 30-80 microns in minimal length. Most of the endings were arranged parallel to the smooth muscle strands, with 80-90% located in the membranous wall and densely distributed in the cranial region of the trachea. The endings were covered with a connective tissue sheath that contained fine elastic fibers. Ultrastructural examination revealed that axon terminals were derived from myelinated axons, and were located in a connective tissue sheath among the smooth muscle cells. Each axon terminal contained large numbers of mitochondria, lysosomes, neurofilaments, glycogen granules and synaptic vesicle. Incomplete coverage by Schwann cells and multiple layers of basal lamina were observed around the axon terminals. Surgical denervation revealed that the endings were of vagal origins. The access pathway to the endings in about one-third of the cranial region of the trachea appeared to be the cranial laryngeal nerve, while in about two-thirds of the caudal region, this appeared to be the tracheal branches derived from the recurrent laryngeal nerve and/or from the vagosympathetic trunk.

CGRP-immunoreactive cells supplying laryngeal sensory nerve fibres in the cat's nodose ganglion.

Through a combination of retrograde staining by wheat germ agglutinin (WGA) and immunohistochemistry, calcitonin gene-related peptide (CGRP)-reactive sensory neurons projecting from the laryngeal mucosa were detected in the feline nodose ganglion. The size of the CGRP-immunoreactive cell which was regarded as a laryngeal sensory neuron, was about 60 microns in diameter: the shape of the immunoreactive laryngeal sensory neuron was unipolar. CGRP-reacted laryngeal sensory cells were found in the rostral part of the nodose ganglion extending to the middle part. They aggregated in the most rostral part, were sparse in other parts and were approximately 50 per cent of WGA-reactive laryngeal sensory neurons in number. Our results suggest that this neurotransmitter might play an important role in laryngeal peripheral sensory innervation.

Distribution of SP- and CGRP-immunoreactivity in the cat's larynx.

The distribution of substance P (SP) and calcitonin gene-related peptide (CGRP) immunoreactive (ir) fibres in the cat's larynx was investigated utilizing immunohistochemistry. Many SP- and CGRP-ir fibres with varicosities were found within and below the epithelium and along the basement membrane of the mucosa of all different regions except in the membranous portion of the vocal fold. In the subepithelium, some SP- and CGRP-ir nerve bundles and nerve fibres were recognized around the vessels and glands. In the mucosa, the pattern of distribution and the density of SR-ir fibres were similar to those of CGRP-ir fibres. These reactive fibres were denser in the supraglottic region than in the subglottic region. In the taste bud-like structures, only SP-ir fibres appeared, whereas in the motor endplates, CGRP-reaction was found exclusively. The present findings suggest that the regional distribution of SP- and CGRP-immunoreactivity might be related with sensory and autonomic innervation in the larynx.

Regulatory peptides in the human larynx and recurrent nerves.

We studied the peptide-innervation of the human larynx (vocal cord, ventricular folds, epiglottis, subglottic region and the recurrent nerves) using immunocytochemical and radioimmunological methods. In the tissues of the larynx investigated, the following regulatory peptides were detected: vasoactive intestinal polypeptide (VIP), peptide histidine methionine (PHM), helospectin, neuropeptide Y (NPY), C-flanking peptide of NPY (C-PON), calcitonin gene-related peptide (CGRP), substance P and neurokinin. In the recurrent nerves only small numbers of peptide-immunoreactive nerve fibers were found. Most of them showed positive immunoreactivities with antibodies to PHM, NPY and C-PON, but only rare and scattered nerve fibers were positive for VIP-, CGRP- and substance P.

Distributions of the calcitonin gene-related peptide and substance P in the monkey larynx.

The distribution of calcitonin gene-related peptide (CGRP)- and substance P (SP)-like immunoreactivity in the monkey larynx was studied with light microscopy using the immunofluorescence method. We divided the larynx into the following six regions: epiglottis, arytenoid region, false cords, ventricle, vocal cords and subglottis. The distribution of immunoreactivity in the epithelium and subepithelial layer was determined. CGRP- and SP-like immunoreactive nerve fibers were observed in all regions of the laryngeal epithelium except in the vocal cords. In the epithelium of the epiglottis and arytenoid region, numerous taste buds containing several CGRP- and SP-like immunoreactive nerve fibers were observed. In the subepithelial layer, CRGP- and SP-like immunoreactive nerve fibers were observed in all regions of the larynx. In order of decreasing density, these fibers were found in the arytenoid region (especially the corniculate tubercle), the epiglottis, the false cords, the subglottis, the ventricle, and the vocal cords. In the corniculate tubercle, CGRP- and SP-like immunoreactive nerve fibers formed a network, whereas around the cuneiform tubercle, dense CRPG- and SP-like immunoreactive nerve fibers were noted parallel to the basement membrane. SP-like immunoreactive nerve fibers showed a very similar distribution to the CRPG fibers in the epithelium and the subepithelial layer, but SP-like fibers were sparser in all regions of the larynx. These results, together with previous findings indicate that the arytenoid region and the epiglottis of the monkey larynx play very important roles in airway protection, swallowing, and respiration.

Distribution of calcium binding proteins in sensory organs of the ear, nose and throat.

Distributions of spot-35 protein (S-35), calbindin (CaB), and parvalbumin (PaV), three types of calcium-binding protein, were examined immunohistochemically in sensory organs of the ear, nose and throat in guinea pigs and rats. Immunoreactivity of S-35 and CaB was found in the outer hair cells and in vestibular sensory cells situated at the top of the ampulla, and in some cells in the macula. Microvillar cells in the olfactory epithelium, periglomerular cells, and small numbers of cells in the mitral cell layer in the olfactory bulb reacted to anti-S-35 and anti-CaB antisera. In taste buds, most gustatory receptor cells reacted to anti-CaB, although a few reacted to anti-S-35 antiserum. Neuron-like cells in the upper respiratory tract reacted similarly to these antisera. No PaV-immunoreactivity was found in any region. These results indicate that S-35 and CaB play important roles in the special kinds of mechanoreceptor and chemoreceptor cells found in the otolaryngeal area.

Long-term hypoxia induces changes in the substance P immunoreactivity pattern in laryngeal nerve paraganglia.

We previously showed that long-term hypoxia increases the dopamine content in rat laryngeal nerve paraganglia. In the present study paraganglia of rats exposed to hypoxia (10 +/- 0.5% O2) for 14 days were examined immunohistochemically to detect changes in the expression of neuropeptides and catecholamine-synthesizing enzymes. Hypoxia induced an intense cellular substance P (SP)-like immunoreactivity (LI) in some paraganglia and an increase in the number of stromal nerve fibers showing SP-LI in others. The patterns of tyrosine hydroxylase-, dopamine-beta-hydroxylase-, phenylethanolamine-N-methyltransferase-, vasoactive intestinal polypeptide-, neuropeptide-Y and calcitonin gene-related peptide-LI were not changed in response to hypoxia. The results show that hypoxia induces changes in the pattern of SP immunoreactivity in laryngeal nerve paraganglia and may indicate that SP plays a role in the regulation of catecholamine metabolism in this tissue.

Immunohistochemical study of intralaryngeal ganglia in the cat.

To study the mechanism of autonomic regulation in the larynx, intralaryngeal local ganglia of the cat were investigated using immunohistochemical techniques. Small intralaryngeal ganglia were found in the peripheral portions of internal branches of the superior laryngeal nerve. Ninety-one percent of the ganglionic neurons were immunoreactive (IR) to vasoactive intestinal polypeptide (VIP), and 10% of the VIP-IR cells were also immunoreactive to enkephalin (ENK) and/or substance P (SP). The immunoreactivity of neuronal cell bodies remained unchanged even after denervation of the bilateral superior and recurrent laryngeal nerves. A dense distribution of calcitonin gene-related peptide (CGRP)-IR nerve fibers was found around almost all neuronal cells in the intralaryngeal ganglia. A few VIP-IR, ENK-IR, and SP-IR nerve fibers were also observed. Only the CGRP-IR fibers disappeared after the denervation experiments. In the laryngeal glands and mucosal arterioles, VIP-IR nerve terminals were found that were also immunoreactive to ENK and/or SP. However, these immunoreactive nerve endings in the glands and arterioles remained after the denervation experiments. The results of our study indicate that laryngeal exocrine secretion and blood flow are regulated by postganglionic autonomic parasympathetic fibers from intralaryngeal ganglia that contain VIP alone or VIP with ENK and/or SP, and that these ganglionic neurons may be innervated by CGRP-IR extrinsic nerve fibers.

Distribution of calcitonin gene-related peptide nerve fibers in the canine larynx.

Immunohistochemistry was used to investigate the distribution pattern of calcitonin gene-related peptide (CGRP) nerve fibers in the laryngeal mucosa, glands and intrinsic muscles of the dog. CGRP immunoreactive nerve fibers were found more frequently than substance P immunoreactive nerve fibers in every region of the larynx. In the epithelia, CGRP nerve fibers were mainly found in the epiglottis, arytenoid region and subglottis. Many taste buds were observed in the arytenoid region and were densely innervated by the CGRP nerve fibers. In the lamina propria, the plexus of CGRP nerve fibers was present, with some of these fibers associated with blood vessels. Laryngeal glands were also innervated by a few CGRP nerve fibers. In the intrinsic laryngeal muscles, abundant immunoreactivity was observed and many motor end-plate-like structures were found with CGRP immunoreactivity. These findings strongly suggest that CGRP plays an important role in all of the sensory, motor and autonomic nervous systems of the larynx.

Distribution of neuropeptide-like immunoreactive nerve fibers in the canine larynx.

The distribution of neuropeptide immunoreactive nerve fibers in the canine larynx was examined. In the epithelium of supra- and subglottic regions, a dense distribution of substance P (SP)- and calcitonin gene-related polypeptide (CGRP)-immunoreactive (IR) nerve fibers was observed. Some vasoactive intestinal polypeptide (VIP)-IR intraepithelial nerve fibers were also seen in the subglottic region. In the laryngeal glands, a dense distribution of VIP-IR nerve fibers with a few SP- and enkephalin (ENK)-IR nerve fibers were found around the acini. In the walls of arteries in the lamina propria, many VIP-, SP-, and CGRP-IR nerve fibers were seen, whereas neuropeptide Y-, ENK-, and VIP-IR nerve fibers were predominantly distributed around the arteries in the vocal muscle. In the free edge of the vocal cord, few immunoreactive nerve fibers were detected within the epithelium and around the arteries in the lamina propria. These results suggest that there are regional differences in the occurrence of peptides in nerve fibers innervating the epithelium and the blood vessels in the larynx and that the perception mechanism of the epithelium and the regulatory system of local blood flow are varied according to their location in the larynx.