Nobuhisa Tadaki

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.

Neuropeptide Participation in Canine Laryngeal Sensory Innervation: Immunohistochemistry and Retrograde Labeling

We investigated the quantitative participation of calcitonin gene-related peptide (CGRP), substance P (SP), and leu-enkephalin (ENK) in canine laryngeal sensory innervation by immunohistochemistry in combination with retrograde labeling using the recently introduced retrograde tracer cholera toxin subunit B—conjugated gold (CTBG). In the nodose ganglion, neurons labeled from the internal branch of the superior laryngeal nerve with CTBG were investigated immunohistochemically by means of antisera against CGRP, SP, and ENK. The percentages of neurons immunoreactive to each neuropeptide were as follows: CGRP 81.5%, SP 24.5%, and ENK 7.0%. These results suggest that CGRP is the main sensory neurotransmitter in canine laryngeal sensory innervation.

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

SummaryImmunohistochemistry 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 immunoreative 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.

Nitrergic neurons in the canine intrinsic laryngeal muscle

Nitrergic ganglionic cells located in the canine intrinsic laryngeal muscle were studied by NADPH-diaphorase (NADPH-d) histochemistry and neuronal nitric oxide synthase (nNOS) immunohistochemistry. Cells intensely stained by NADPH-d histochemistry were found between the striated muscle fibers of the intrinsic laryngeal muscle. Most of these cells were bipolar or pseudounipolar in form. Some NADPH-d negative cells were observed to be enveloped in a mesh by varicose NADPH-d positive nerve fibers. The findings obtained by nNOS immunohistochemistry corresponded well with those obtained by NADPH-d histochemistry, indicating that NADPH-d activity in the ganglion in the intrinsic laryngeal muscle is nNOS. The present findings clearly indicate that some of the ganglion cells located in the canine intrinsic laryngeal muscle are nitrergic, and that the ganglionic cells synapse together with the participation of nitric oxide in integrating ganglionic cells.

NADPH-diaphorase and nitric oxide synthase in the canine superior cervical ganglion

By means of NADPH-diaphorase (NADPH-d) histochemistry and nitric oxide synthase (NOS) immunohistochemistry, we demonstrate that considerable numbers of NADPH-d-positive neurons are distributed throughout the canine superior cervical ganglion (SCG). These neurons also show NOS immunoreactivity. This finding indicates that NADPH-d histochemistry, a simple and reliable technique, can be used as a reliable marker of NOS activity in the sympathetic innervation of canine head and neck. The present findings suggest that the participation of nitric oxide in the SCG differs greatly between species.

Neurotransmitters and neuromodulators involved in laryngeal innervation

The distribution and role of neurotransmitters and neuromodulators in laryngeal innervation are reviewed, and our recent findings regarding the nitrergic innervation of the larynx are demonstrated for the better understanding of the complexity of the laryngeal innervation system. Noradrenergic innervation of the larynx was studied with fluorescence histochemistry and electron microscopy after application of 5-hydroxydopamine. These studies confirmed the existence of noradrenergic innervation for the submucosal glands and blood vessels, and the origin and course of noradrenergic nerve fibers contained in the laryngeal nerves and their destinations in the larynx. Cholinergic innervation of the larynx has not been clarified in detail. Many kinds of neuropeptides have been demonstrated to be involved in laryngeal innervation. Vasoactive intestinal polypeptide originating from intralaryngeal ganglionic neurons participates in laryngeal vasodilation and reduction of laryngeal seromucous secretion. Neuropeptide Y nerve fibers are few in the larynx, and most originate from the superior cervical ganglion. They are distributed around the large or medium-sized blood vessels, especially arteries. They are also associated with excretory structures. Substance P was the first neuropeptide found to be a sensory neurotransmitter in the laryngeal afferent system. It is also involved in regulation of laryngeal blood flow and secretion. Calcitonin gene—related peptide is associated with the sensory, autonomic, and motor innervation of the larynx. The majority of enkephalin nerve fibers are located close to excretory structures, although no information on the physiological significance of enkephalin is available. In addition to the above neuropeptides, the peptides histidine isoleucine, histidine methionine, and helospectin have been shown to exist in the larynx. The nitrergic innervation of the larynx has been recently studied with NADPH-diaphorase histochemistry and immunohistochemistry using antiserum against nitric oxide synthase. Nitric oxide originates from the neurons in the intralaryngeal ganglia and is believed to modulate blood flow and secretion of the larynx. It controls the laryngeal exocrine secretion in cooperation with intrinsic vasoactive intestinal polypeptide and/or extrinsic calcitonin gene—related peptide. Nitric oxide from the nodose ganglion may modulate nociception of the larynx. The existence of nitrergic neurons located in the intrinsic laryngeal muscles has been demonstrated. Many of them are bipolar or pseudounipolar, so they might be sensory in nature. The effect of injury of the recurrent laryngeal nerve on the induction of nitric oxide synthase in the laryngeal motoneurons is also discussed.

Calcitonin Gene-related Peptide-like Immunoreactive Motoneurons Innervating the Canine Inferior Pharyngeal Constrictor Muscle

The percentage of the calcitonin gene-related peptide-immunoreactive motoneurons (Mn) in the nucleus ambiguus (NA) innervating the canine inferior pharyngeal constrictor muscle was examined using the cholera toxin B subunit-gold (CTBG) as a retrograde tracer and by immunohistochemistry. Labeled neurons with CTBG (CTBG MNs) from the thyropharyngeal muscle (TP) were located in the dorsomedial division of the rostral part of the NA and the average percentage of neurons with CGRP immunoreactivity was 70.3%. CTBG MNs from the cricopharyngeal muscle (CP) were located in the dorsal division of the rostral part of the NA and the average percentage of neurons with CGRP immunoreactivity was 21.2%. This low percentage of CGRP immunoreactivity is noteworthy and supports the idea that the CP is a particular striated muscle in function and innervation manner.

Neurotransmitters for the canine inferior pharyngeal constrictor muscle

The inferior pharyngeal constrictor muscle plays an important role at the pharyngeal phase of deglutition and is anatomically composed of the thyropharyngeal muscle and cricopharyngeal muscle. In this study we investigated the distribution pattern of neuropeptidergic and catecholaminergic nerve fibers in the thyropharyngeal muscle and cricopharyngeal muscle of seven puppies by immunohistochemistry. Some of the calcitonin gene-related peptide-, substance P-, vasoactive intestinal polypeptide-, and tyrosine hydroxylase-immunoreactive nerve fibers were found to lie parallel to the muscle fibers in both the thyropharyngeal muscle and cricopharyngeal muscle. Nerve fibers with immunoreactivity to all substances examined were found to be associated with blood vessels in both the thyropharyngeal muscle and cricopharyngeal muscle, and the number of calcitonin gene-related peptide, neuropeptide Y, and tyrosine hydroxylase nerve fibers was higher than the number of substance P, vasoactive intestinal polypeptide, and galanin nerve fibers. Motor end plate-like structures with calcitonin gene-related peptide immunoreactivity were found in both the thyropharyngeal muscle and cricopharyngeal muscle. These structures in the cricopharyngeal muscle were clearly less than those in the thyropharyngeal muscle. Some clusters of neurons were detected only in the cricopharyngeal muscle of all dogs examined. Substance P-, vasoactive intestinal polypeptide-, galanin-, and neuropeptide Y-immunoreactive neurons were found in this ganglion, and the vasoactive intestinal polypeptide-immunoreactive neurons were the most abundant. Abundant calcitonin gene-related peptide- and vasoactive intestinal polypeptide-immunoreactive nerve fibers, and some substance P- and galanin-immunoreactive nerve fibers were distributed in the ganglion.(ABSTRACT TRUNCATED AT 250 WORDS)

Tyrosine hydroxylase-immunoreactive cells in the nodose ganglion for the canine larynx.

Several substances have been reported as candidates for the neurotransmitter in the laryngeal afferent system. In the present study we demonstrated that catecholamine is also a candidate neurotransmitter in the canine laryngeal afferent system using tyrosine hydroxylase (TH) immunochemistry in combination with retrograde labelling with cholera toxin B in subunit-conjugated gold (CTBG). A few cells in the nodose ganglion labelled by application of CTBG to the internal branch of the superior laryngeal nerve were also TH-immunoreactive. These cells were also labelled following application of CTBG to the nucleus of the solitary tract. These results indicate that some of the TH-IR cells in the nodose ganglion could be primary afferent neurones for the canine larynx.

Coexistence of calcitonin gene-related peptide and NADPH-diaphorase in the canine superior cervical ganglion

By means of double staining technique of NADPH-diaphorase (NADPH-d) histochemistry and calcitonin gene-related peptide (CGRP) immunohistochemistry, we investigated the coexistence of NADPH-d reactivity and CGRP immunoreactivity in the canine superior cervical ganglion (SCG). Most of NADPH-d reactivity and CGRP immunoreactivity were coexisted in the principal postganglionic neurons. These neurons were distributed throughout the ganglion without specific localization. The present findings suggest the intimate role of CGRP and nitric oxide in postganglionic neurons of the canine SCG.