Airway-related vagal preganglionic neurons express multiple nicotinic acetylcholine receptor subunits

Abstract

Nicotine acting centrally increases bronchomotor tone and airway secretion, suggesting that airway-related vagal preganglionic neurons (AVPNs) within the rostral nucleus ambiguus (rNA) express nicotinic acetylcholine receptors (nAChRs). In the present study, we examined the three main functionally characterized subtypes of nAChRs in the CNS, the alpha7 homomeric and alpha4beta2 heteromeric receptors. First, we characterized the expression of these subunits at the message (mRNA) and protein levels in brain tissues taken from the rNA region, the site where AVPNs are located. In addition, double labeling fluorescent immunohistochemistry and confocal laser microscopy were used to define the presence of alpha7, alpha4, and beta2 nAChRs on AVPNs that were retrogradely labeled with cholera toxin beta subunit (CTb), injected into the upper lung lobe (n=4) or extrathoracic trachea (n=4). Our results revealed expression of all three studied subunits at mRNA and protein levels within the rNA region. Furthermore, virtually all identified AVPNs innervating intrapulmonary airways express alpha7 and alpha4 nAChR subunits. Similarly, a majority of labeled AVPNs projecting to extrathoracic trachea contain alpha7 and beta2 subunits, but less than half of them show detectable alpha4 nAChR traits. These results suggest that AVPNs express three major nAChR subunits (alpha7, alpha4, and beta2) that could assemble into functional homologous or heterologous pentameric receptors, mediating fast and sustained nicotinic effects on cholinergic outflow to the airways.

Fig. 1

RT-PCR and Western blot analysis of nicotinic acetylcholine receptor mRNA and protein expression in the rostral nucleus ambiguus (rNA) where airway-related vagal preganglionic neurons are located. (A) Schematic representation of a coronal section (Paxinos and Watson, 1986) and (B) the distribution of CTb-labeled AVPNs visualized with rhodamine indicating the location of AVPN within the rNA regions (C), RT-PCR analysis of nicotinic acetylcholine receptor (nAChR) subunits: α4, α7, β2, and β-actin seen at 483, 510, 507, and 638bp, respectively and Western blot analysis of α4, α7, β2, and β-actin, seen at 69, 55, 55, and 41kDa, respectively (Scale bar: 100μm).

Fig. 2

(A) An example of a confocal image of the α7-nAChR subunit expressed by neurons within the rostral nucleus ambiguus region (rNA). The α7-nAChR like-immunoreactive product is localized on the cell membrane and dendrites. However, it was also observed in the cytoplasm, suggesting internalization of receptor protein. (B) After omission of primary antibody no labeling of neurons or the fibers was observed (Scale bar: 20μm).

Fig. 3

Confocal images of (A), retrogradely labeled airway-related vagal preganglionic neurons (AVPNs) within the rNA, following microinjection of cholera toxin β subunit (CTb) tracer into the upper right lung lobe; (B) α7-nAChR subunit immunoreactive neurons in the same field. (C) The merged (M) image showing the CTb colocalized with an α7-nAChR traits. (D) Total number of counted CTb labeled neurons and the number of the double labeled cells (CTb and α7-nAChR). Scale bar: 20μm.

Fig. 4

Confocal images of (A), retrogradely labeled airway-related vagal preganglionic neurons (AVPNs) within the rNA, following microinjection of cholera toxin β subunit (CTb) tracer into the upper right lung lobe; (B) α4-nAChR subunit immunoreactive neurons in the same field. (C) The merged (M) image showing the CTb colocalized with an α4-nAChR traits. (D) Total number of counted CTb labeled neurons and the number of the double labeled cells (CTb and α4-nAChR). Scale bar: 20μm.

Fig. 5

Confocal images of (A), retrogradely labeled airway-related vagal preganglionic neurons (AVPNs) within the rNA, following microinjection of cholera toxin β subunit (CTb) tracer into the extrathoracic trachea; (B) β2-nAChR subunit immunoreactive neurons in the same field. (C) The merged (M) image showing the CTb colocalized with a β2-nAChR traits. (D) Total number of counted CTb labeled neurons and the number of the double labeled cells (CTb and β2-nAChR). Scale bar: 20μm.

Fig. 6

Summary diagram. Possible involvement of acetylcholine (ACh)-nicotinic acetylcholine receptors (nAChRs) signaling pathway in regulating the excitability of AVPNs. ACh released from passing fibers or from AVPNs could reach perisynaptic nAChRs (a7 homologous, α7β2, and/or α4β2 heterologous pentameric channels) causing activation of the cells, or it could activate α7 homomeric receptor expressed by axon terminals innervating AVPNs and facilitate glutamate release and further augment cholinergic outflow to the airways. Nicotine and nicotine-like substances use the same receptors to influence activity of AVPNs.