Morphology of P2X3‐immunoreactive nerve endings in the rat tracheal mucosa

Abstract

Nerve endings with immunoreactivity for the P2X3 purinoreceptor (P2X3) in the rat tracheal mucosa were examined by immunohistochemistry of whole-mount preparations with confocal scanning laser microscopy. P2X3 immunoreactivity was observed in ramified endings distributed in the whole length of the trachea. The myelinated parent axons of P2X3-immunoreactive nerve endings ramified into several branches that extended two-dimensionally in every direction at the interface between the epithelial layer and lamina propria. The axonal branches of P2X3-immunoreactive endings branched off many twigs located just beneath the epithelium, and continued to intraepithelial axon terminals. The axon terminals of P2X3-immunoreactive endings were beaded, rounded, or club-like in shape and terminated between tracheal epithelial cells. Flat axon terminals sometimes partly ensheathed neuroendocrine cells with immunoreactivity for SNAP25 or CGRP. Some axons and axon terminals with P2X3 immunoreactivity were immunoreactive for P2X2, while some terminals were immunoreactive for vGLUT2. Furthermore, a retrograde tracing method using fast blue (FB) revealed that 88.4% of FB-labeled cells with P2X3 immunoreactivity originated from the nodose ganglion. In conclusion, P2X3-immunoreactive nerve endings in the rat tracheal mucosa have unique morphological characteristics, and these endings may be rapidly adapting receptors and/or irritant receptors that are activated by mucosal irritant stimuli.

Development of a Mouse Reporter Strain for the Purinergic P2X2Receptor

“…Interestingly, the major vagal nerve terminal type found in the ligamentous part of the mouse trachea (termed 'anterolateral segmental array') by Hennel et al (2018) was not labeled in P2X 2 -tdTomato mice, indicating that these probably originate from jugular neurons. Previous tracheal studies have identified A-fiber terminals with complex dendritic arbors innervating the epithelium and subepithelial layers over the cartilage rings in rats (Yamamoto and Nakamuta, 2018) and in the subepithelial layer over the ends of the cartilage rings in guinea pigs (Mazzone et al, 2009). Despite the fact that tracheal A-fibers are likely projected from nodose neurons (Mazzone et al, 2009), they were not detected in either the present study in P2X 2 -tdTomato mice or in the AAV-GFP mouse study by Hennel et al (2018), suggesting species differences in A-fiber innervation of the trachea.…”

Distribution and morphology of P2X3‐immunoreactive subserosal afferent nerve endings in the rat gastric antrum

“…P2X2 immunoreactivity has been observed in the axon terminals of various P2X3‐immunoreactive sensory endings: ramified intraepithelial endings in the laryngeal and tracheal mucosa (Takahashi et al, 2016; Yamamoto & Nakamuta, 2018), ramified endings in lung neuroepithelial bodies (Brouns et al, 2006), IGLEs in the gastrointestinal tract (Wang & Neuhuber, 2003; Xiang & Burnstock, 2004), arterial baroreceptors (Song et al, 2012; Yokoyama et al, 2019), and nerve endings in the taste buds (Ishida et al, 2009). However, P2X2‐immunoreactivity was weak or not observed in P2X3‐immunoreactive subserosal nerve endings.…”

“…An immunoblotting analysis of this antibody labeled a band near 57 kDa in a rat DRG lysate (Xu & Huang, 2002), a 64‐kDa band in a mouse DRG lysate (Cho & Chaban, 2012), and multiple glycosylated forms of P2X3 at 250 and 75 kDa (Hemmings‐Mieszczak et al, 2003). Immunohistochemical analyses of this antibody labeled ramified sensory nerve endings in the rat tracheal mucosa (Yamamoto & Nakamuta, 2018) and sensory nerve endings associated with type I cells in the rat carotid body (Yokoyama et al, 2020). This antibody has also been used for immunohistochemistry to label ganglion neurons in the rat DRG (Huang et al, 2005; Vulchanova et al, 1997) and the rat trigeminal ganglion (Ichikawa & Sugimoto, 2004).…”

“…An immunoblotting analysis showed a 62‐kDa band in HEK cells transfected with a plasmid encoding the P2X2‐FLAG protein or mouse brain tissue (Chaumont et al, 2008). Immunohistochemical analyses of this antibody labeled ramified intraepithelial endings in the laryngeal and tracheal mucosa (Takahashi et al, 2016; Yamamoto & Nakamuta, 2018), and sensory nerve endings associated with type I cells in the rat carotid body (Yokoyama et al, 2020). This antibody has also been used for immunohistochemistry to label cerebellar Purkinje cells and granule cells in the dentate gyrus of the mouse brain (Chaumont et al, 2008).…”

“…In the present study, we examined the distribution and morphology of P2X3‐immunoreactive afferent nerve endings in the rat gastric subserosa using immunohistochemistry with confocal laser scanning microscopy. Since immunohistochemistry with whole‐mount preparations has been widely used in morphological analyses of sensory nerve endings (Pintelon et al, 2007; Song et al, 2012; Suzuki et al, 2012; Takahashi et al, 2016; Yamamoto & Nakamuta, 2018; Yokoyama et al, 2019), we employed this method to demonstrate the morphological characteristics of nerve endings in the gastric subserosa. We also attempted to elucidate the interrelationships between P2X3‐immuhnoreactive nerve endings, visceral smooth muscle cells, and terminal Schwann cells.…”