Innervation of the rat trachea by bilateral cholinergic projections from the nucleus ambiguus and direct motor fibers from the cervical spinal cord: a retrograde and anterograde tracer study

doi:10.1016/j.brainres.2004.10.032

Brain Research

Volume 1031, Issue 1, 7 January 2005, Pages 90-100

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Abstract

A tract–tracer method was employed to examine the innervation of the rat trachea. Cholera toxin β subunit (CTB) was injected into the following locations in separate groups of rats: (1) ventral trachea, (2) lateral trachea, (3) ventral trachea after the excision of the nodose ganglion, and (4) ventral trachea after the transection of C1–C2 spinal nerves. CTB injection in the ventral trachea showed bilateral labeling of neurons in the nucleus ambiguus (NA), medial subnucleus of the nucleus of the solitary nucleus, dorsal motor nucleus of the vagus (DMV), and lamina IX of C1–C6. CTB injection in the lateral trachea showed significant ipsilateral predominance of neuronal labeling in the NA and lamina IX of C1–C2 segments. CTB injection in rats after the excision of the nodose ganglion revealed no labeling in the ipsilateral DMV and NA and a significant reduction of neuronal labeling in C1. CTB injection in rats after the transection of C1–C2 spinal nerves showed a significant decrease in the number of labeled neurons in ipsilateral NA, C1, and C2 and no labeling of fibers in C1–C2. The combination of retrograde fluorogold labeling and choline acetyltransferase (ChAT) immunostaining revealed that all fluorogold-labeled neurons in the NA and lamina IX of C1–C2 colocalized with ChAT. The injection of biotinylated dextran amine in NA produced labeling in axonal terminals on postganglionic neurons, but not in other regions of the trachea. Our findings indicate that the rat trachea is innervated bilaterally by cholinergic motor neurons in NA and C1–C2, while those traveling through the spinal nerves project directly to the trachea.

Introduction

Autonomic innervation of the airway includes the parasympathetic and sympathetic nervous systems, which function to control smooth muscle tension, secretion of mucosal glands, vascular tone, and tracheobronchial reflexes [3], [7]. The preganglionic parasympathetic neurons innervating the trachea originate from the dorsal motor nucleus of the vagus (DMV) and nucleus ambiguus (NA) [5], [8]. Postganglionic parasympathetic neurons are located in the peritracheal ganglion or submucosal plexus [1], [9], [11], [14]. Sensory fibers that are distributed in the tracheal epithelium project to the nucleus of the solitary tract (NTS) through neurons located in the nodose or jugular ganglion.

The innervation pattern of vagal motor neurons to the lung shows species differences: NA in the cat innervates the lung contralaterally [8], whereas in the rat, it innervates the lung bilaterally [12]. The contralateral pathway in bilateral innervation undergoes decussation inside the thorax in the rat [12]. In the trachea, however, the issue of whether the trachea is innervated bilaterally or contralaterally remains to be resolved, although both DMV and NA participate in tracheal innervation [5].

A tract-tracing study using cholera toxin β subunit (CTB) showed that neurons in the dorsomedial part of the ventral horn in C1 and C2 participate in the innervation of the trachea in the dog, ferret, and rat, in addition to NA and DMV [5]. Furthermore, the authors found CTB-labeled fibers in the ventral funiculus of the upper cervical segments in the dog and in the cervical dorsal horn in the ferret. The study suggested that the upper cervical spinal cord participates partially in the control of the trachea.

Sensory or parasympathetic nerve fibers are distributed in the epithelium or lamina submucosa of the trachea of the ferret and rat [4], [9], [13]. Pérez-Fontán and Velloff [11] reported that the injection of CTB in the tracheal lumen resulted in labeling neurons in NA and DMV by transepithelial transport to intra- or subepithelial space, where labeled vagal motor neurons project their axons in the rat. The authors hypothesized that vagal motor neurons in DMV and NA project their axons directly to the epithelium or submucosa without interposition of intrinsic neurons, which are located in the peritracheal parasympathetic ganglion. This hypothesis is contradictory to the classic concept of the airway parasympathetic transmission; preganglionic vagal neurons synapse on postganglionic neurons in the local ganglion, and, in turn, the latter exert their effects on smooth muscles, glandular cells, and epithelial cells [7], [10]. However, their experiment was carried out under the presence of the classic parasympathetic rely [11]. Therefore, the hypothesis of a direct vagal motor innervation to the trachea remains to be confirmed.

The aims of the present study were to determine whether NA and DMV innervate trachea bilaterally and whether the cervical motor neurons directly innervate the trachea. Experiments involved tract tracing using CTB and denervation of the vagus nerve and spinal nerves. In the latter experiment, we chose the cervical spinal cord, where autonomic innervation of both sympathetic and parasympathetic nerves is absent [2]. CTB was injected in the tracheal wall because previous studies reported that retrograde-labeled neurons were more numerous in NA and DMV than those obtained by injection into the tracheal lumen [5], [11].

Section snippets

Materials and methods

A total of 52 Wistar rats (weight 250–300 g, 10–16 weeks, of both sexes) was used in the present study. The experimental protocol was approved by the Ethics Review Committee for Animal Experimentation of Faculty of Agriculture, Gifu University.

Spread of tracers in the trachea

In cases of CTB injections, the tracer was not found in the trachea 4 days after the injections. Therefore, the diffusion of the tracer was examined in 30 min and 2, 24, and 48 h after the injections in the ventral midline of the trachea. Thirty minutes after injection, CTB was seen in the ventral part of the trachea (Fig. 1A). Two and 24 h after injections, CTB extended into the ventral half of the trachea, but 48 h after the injections, it was not yet detected in the trachea. In cases of

Discussion

The present study demonstrated the bilateral innervation of NA and C1–C2 to the trachea and the direct innervation of motor neurons of the cervical spinal cord to the trachea in the rat. The retrograde labeling of motor neurons in DMV and NA and the axonal labeling in NTS in the medulla is in good agreement with those of CTB tract-tracing studies [5], [6]. The combination of fluorogold labeling and ChAT immunostaining showed that ChAT coexists on all fluorogold-labeled neurons in the NA and

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WGA-Alexa 488 demonstrates retrograde transsynaptic labeling following intrapleural injection whereas the previous method of injecting CTB-Alexa 488 only demonstrates retrograde labeling.
Intrapleural injection of WGA-Alexa fluor conjugates is an effective method to transsynaptically label the phrenic motor system providing an alternative for the invasive laparotomy required for intradiaphragmatic injections. Furthermore, the study provides the first anatomical evidence of a direct synaptic relationship between rVRG and select NA cells.
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Research reportInnervation of the rat trachea by bilateral cholinergic projections from the nucleus ambiguus and direct motor fibers from the cervical spinal cord: a retrograde and anterograde tracer study

Abstract

Introduction

Section snippets

Materials and methods

Spread of tracers in the trachea

Discussion

Respir. Physiol.

J. Auton. Nerv. Syst.

Brain Res.

Respir. Physiol.

Respir. Physiol.

The architecture of nerves and ganglia of the ferret trachea as revealed by acetylcholinesterase histochemistry

J. Comp. Neurol.

Peripheral autonomic nervous system

Research report
Innervation of the rat trachea by bilateral cholinergic projections from the nucleus ambiguus and direct motor fibers from the cervical spinal cord: a retrograde and anterograde tracer study