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

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Abstract

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.

Introduction

The cholinergic system in the larynx has been described mainly by a number of physiological studies. The cholinergic special visceral efferent fibers play roles in the contraction of the intrinsic laryngeal muscles, and in the control of both phonation and respiration (Ohyama et al., 1972, Iwasaki et al., 1994, Igarashi and Iwasaki, 1995, Inagi et al., 1998a). The cholinergic general visceral efferent fibers act in a stimulatory way on the laryngeal gland secretion (Haxhiu et al., 1991, Hejal et al., 1993) and on the blood flow (Shin et al., 1970, Matran et al., 1989, Matran, 1991).

Neuroanatomical investigation of cholinergic nerves in the larynx, however, has been hampered by the lack of suitable means. There is no histochemical technique for visualizing acetylcholine itself. Acetylcholinesterase (AChE), a catalyzing enzyme of acetylcholine, has been widely used as a histochemical marker for cholinergic nerves in the larynx. For example, positive staining for AChE activity has been found in neuronal cell bodies of the intralaryngeal ganglia (Domeij et al., 1991a, Domeij et al., 1991b, Yoshida et al., 1993), and nerve fibers around the laryngeal glands and blood vessels (Domeij et al., 1991a). Such positive staining for AChE activity, however, may not be a reliable marker for cholinergic structures. For example, the special visceral efferent fibers, believed to be cholinergic and to innervate the intrinsic laryngeal muscles, have never been demonstrated by AChE histochemistry. Moreover, AChE-positive structures have been shown to be located at the subneural apparatus of the neuromuscular junction of the larynx (Kanda et al., 1983, Yoshihara et al., 1984). In the brain, AChE activity has also been found in non-cholinergic, dopaminergic cells of the substantia nigra and serotoninergic cells of the dorsal raphe nucleus (Levey et al., 1983, Mesulam et al., 1984).

So far, immunohistochemistry for choline acetyltransferase (ChAT), a synthesizing enzyme for acetylcholine, has been thought to be the most reliable histochemical technique for visualizing cholinergic structures. However, most of available antibodies against ChAT have often failed to show peripheral cholinergic nerves, although they are powerful in revealing central cholinergic structures clearly. Indeed, there is no information concerning the distribution of ChAT immunoreactivity in the larynx. This fact indicates that a difference may exist in the molecular form of ChAT between the central and peripheral nervous systems.

Recently, Tooyama and Kimura (1999) cloned a short form of ChAT cDNA from the rat pterygopalatine ganglion. They termed its product as choline acetyltransferase of a peripheral type (pChAT). The mRNA of pChAT lacks the exons 6–9 of the reported ChAT gene (Hahn et al., 1992), suggesting its formation by alternative splicing. A rabbit antiserum against pChAT has been raised using an immunogen consisting of a 41 amino acid polypeptide, which is translated from a nucleotide sequence spanning over the splice joint between exons 5 and 10. This antiserum clearly stained many neurons of presumed peripheral cholinergic cells including the pterygopalatine ganglion, while it failed to detect positive neurons in the rat forebrain. Hence, it is strongly suggested that the pChAT antiserum recognizes the variant ChAT protein which is preferentially expressed in peripheral cholinergic cells.

The present study was undertaken, using this novel antiserum, to evaluate the immunohistochemical distribution of pChAT-positive structures in the larynx of the rat. We further carried out double immunohistochemical staining and denervation experiments, in order to clarify and characterize the trajectory of pChAT-positive fibers in the larynx.

Section snippets

Tissue preparation of the larynx

Twenty young adult male Wistar rats (Clea Japan, Japan), weighing 200–300 g, were used. Under deep anesthesia with sodium pentobarbital (80 mg/kg), the animals were perfused through the ascending aorta with 10 mM phosphate-buffered saline (pH 7.4), followed by an ice-cold fixative of 4% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.4). The larynx, extending from the top of the epiglottis to the lower border of the cricoid cartilage, was excised together with parts of the pharynx and

Distribution of pChAT-immunoreactive neuronal cell bodies in the larynx

Neuronal somata positive for pChAT were found in clusters throughout the larynx extending from the supraglottic to subglottic areas. In the supraglottic area (Fig. 1A and B), a dense cluster of intensely stained somata was observed immediately medial to the thyroid cartilage. There were also intensely stained fiber bundles, probably of the internal superior laryngeal nerve, and the positive cell clusters were located along one of these bundles (Fig. 1A). Such a cluster was composed of positive

Discussion

The nerve supply of the larynx is from the internal and external laryngeal branches of the superior laryngeal and from the recurrent laryngeal and sympathetic nerves. These nerves contain fibers of, in different combinations, special visceral efferent, special visceral afferent, and sympathetic and parasympathetic general visceral efferents. The internal laryngeal nerve, for example, is almost entirely sensory and autonomic, with a few participating special visceral motor fibers. Among these

Conclusion

As summarized in Fig. 6, immunohistochemical localization of pChAT, a probable novel marker for cholinergic structures, has been studied in the larynx. Structures positive for pChAT come in three types: (1) special visceral efferents to the intrinsic laryngeal muscles, (2) postganglionic parasympathetic efferents, and (3) afferents to the laryngeal mucosa. The results appear to provide the first morphological evidence supporting the physiological concept that both special and general visceral

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