Expression of nNOS in the human larynx.

Although intrinsic laryngeal neurons and ganglia have been studied in various species, they have been overlooked in humans. We aimed to investigate the presence of intrinsic laryngeal neurons in humans and, if present, to analyze their neuronal nitric oxide synthase (nNOS) expression. An immunohistochemical study using anti-nNOS antibodies was performed on samples obtained from four cadavers. Intrinsic laryngeal nNOS+ neurons were assessed in the submucosal layer, but nNOS+ nerves were found in all histological layers of the larynx. nNOS expression was also found in striated muscle fibers of larynx. This might reveal the anatomical basis of an upwards extension of the nonadrenergic noncholinergic system in human airways, but further experiments are needed to assess an exact role of NO influence on neural transmission and muscular functions of human larynx.

Tyrosine hydroxylase-immunoreactive fibers in the human vagus nerve.

Sympathetic catecholaminergic fibers in the vagus nerve were immunohistochemically examined in formalin-fixed human cadavers using an antibody against the noradrenalin-synthetic enzyme tyrosine hydroxylase (TH). TH-positive fibers were extensively distributed in the vagal nerve components, including the superior and inferior ganglia, the main trunk and the branches (superior and recurrent laryngeal, superior and inferior cardiac, and pulmonary branches). The inferior ganglion and its continuous cervical main trunk contained numerous TH-positive fibers with focal or diffuse distribution patterns in each nerve bundle. From these findings, we conclude that sympathetic fibers are consistently included in the human vagus nerve, a main source of parasympathetic preganglionic fibers to the cervical, thoracic and abdominal visceral organs.

Immunohistochemical localization of choline acetyltransferase of a peripheral type in the rat larynx.

As shown in the accompanying paper, choline acetyltransferase, so far the best histochemical marker for identifying cholinergic structures, has at least one alternative splice variant. The variant, termed pChAT because of its preferential expression in peripheral organs, encouraged us to study peripheral, probably cholinergic, cells and fibers by immunohistochemistry using an antiserum against a peptide specific for pChAT. We chose the larynx of the rat, since cholinergic innervation in this organ has been well established by physiological studies, but not sufficiently by chemical neuroanatomy. Neuronal somata positive for pChAT were found in the intralaryngeal ganglia. Our double staining study indicated that these somata always possessed acetylcholinesterase activity, while the reverse did not hold true. Nerve fibers positive for pChAT were distributed widely in the intrinsic laryngeal muscles, laryngeal glands, blood vessels and laryngeal mucosa. In the intrinsic laryngeal muscles, pChAT-positive terminals were apposed closely to motor end-plates which were stained positively for acetylcholinesterase activity. Denervation experiments revealed that there were three types of pChAT-positive fibers in the larynx: (1) special visceral efferent fibers to the intrinsic laryngeal muscles, which decreased dramatically in number after vagotomy; (2) parasympathetic postganglionic fibers near the laryngeal glands and blood vessels, which appeared unaffected after vagotomy or cervical sympathectomy: and (3) afferent fibers innervating the laryngeal mucosa, which reduced markedly in number after vagotomy performed distal, but not proximal, to the nodose ganglion. Such afferent fibers remained unchanged following the neonatal capsaicin treatment, suggesting their independence from those containing substance P.

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.

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.

Expression of catecholamine-synthesizing enzymes in paraganglionic and ganglionic cells in the laryngeal nerves of the rat.

The rat recurrent and superior laryngeal nerves with adjacent connective tissue were examined by immunohistochemical techniques for localization of the catecholamine-synthesizing enzymes tyrosine hydroxylase, dopamine-beta-hydroxylase and phenylethanolamine-N-methyltransferase. Most of the cells in the paraganglia of the recurrent and superior laryngeal nerves showed an intense tyrosine hydroxylase-like immunoreactivity. A few paraganglionic cells exhibited dopamine-beta-hydroxylase-like immunoreactivity while none of the cells displayed phenylethanolamine-N-methyltransferase-like immunoreactivity. Some of the ganglionic cells in the recurrent and superior laryngeal nerves showed dopamine-beta-hydroxylase-like immunoreactivity whilst these cells never showed tyrosine hydroxylase- or phenylethanolamine-N-methyltransferase-like immunoreactivity. The arterioles were supplied with plexuses of nerve fibres showing tyrosine hydroxylase- and dopamine-beta-hydroxylase-like immunoreactivity. The results indicate that dopamine is the major catecholamine located in the laryngeal nerve paraganglia and show that ganglionic cells in the recurrent and superior laryngeal nerves show immunolabelling for one of the enzymes in the catecholamine synthetic pathway, dopamine-beta-hydroxylase.

Enkephalin-like immunoreactivity in ganglionic cells in the larynx and superior cervical ganglion of the rat.

The distribution of enkephalin-like immunoreactivity (ENK-LI) in the larynx, the superior cervical ganglion (SCG) and the nodose ganglion of adult rats was examined in the present study. A substantial number of the local acetylcholinesterase (AChE)-positive, presumably parasympathetic, ganglionic cells in the larynx displayed ENK-LI. These cells also exhibited neuropeptide Y (NPY)- and vasoactive intestinal polypeptide (VIP)-LI. Varicose nerve fibers showing ENK-LI were observed close to the acini and ducts of the glands, in the perichondrium and in the lamina propria. The varicosities exhibiting ENK-LI frequently displayed NPY- and VIP-LI. The ENK-LI was detected in a subpopulation of AChE-positive nerve fibers in the laryngeal tissue. In the SCG, only a small number of the ganglionic cells displayed ENK-LI. These cells, in contrast to other ganglionic cells of the SCG, did not show NPY-LI. None of the ganglionic cells of the nodose ganglion exhibited ENK-LI. Sympathectomy and vagotomy affected neither the number nor the distribution of fibers showing ENK-LI in the larynx. In conclusion, ENK appears to be present together with NPY and VIP in the parasympathetic innervation of the larynx and in a very limited number of the ganglionic cells of a sympathetic ganglion, the SCG, of the adult rat.

[Auxiliary motor innervation of the laryngeal muscles via the internal branch of the superior laryngeal nerve]. / Untersuchungen zur zusätzlichen motorischen Innervation der Kehlkopfmuskulatur über den Ramus internus des Nervus laryngeus superior.

According to our present knowledge of the neuromuscular innervation of the intrinsic laryngeal muscles, the cricothyroid muscle is innervated by the external branch of the superior laryngeal nerve (NLS), whereas all other remaining muscles get their supply from the inferior laryngeal (recurrent) nerve. Mainly in the phoniatric literature, however, opinions differ concerning an additional motoric laryngeal innervation. In human larynges, excised for large unilateral carcinoma, horseradish peroxidase (HRP) was injected into the internal branch of the NLS. Anterograde labelling of axons was demonstrated histochemically. In adjacent sections of the different muscles, end plates and axons were stained histochemically with silver impregnation and acetylcholinesterase. Evidence is presented of motor innervation of the internal branch of the NLS in some laryngeal muscles. With retrograde HRP-tracing in sheep, motoneurons were detected in the nucleus ambiguus, although the recurrent nerve and the external branch had been divided and excised. Thus, histologically an additional neuromuscular supply via the internal branch of the NLS is demonstrated.