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

Abstract

The present study investigated the morphological characteristics of subserosal afferent nerve endings with immunoreactivity for the P2X3 purinoceptor (P2X3) in the rat stomach by immunohistochemistry of whole-mount preparations using confocal scanning laser microscopy. P2X3 immunoreactivity was observed in subserosal nerve endings proximal and lateral to the gastric sling muscles in the distal antrum of the lesser curvature. Parent axons ramified into several lamellar processes to form netlike complex structures that extended two-dimensionally in every direction on the surface of the longitudinal smooth muscle layer. The axon terminals in the periphery of P2X3-immunoreactive net-like structures were flat and looped or leaf-like in shape. Some net-like lamellar structures and their axon terminals with P2X3 immunoreactivity were also immunoreactive for P2X2. P2X3-immunoreactive nerve fibers forming net-like terminal structures were closely surrounded by S100Bimmunoreactive terminal Schwann cells, whereas axon terminals twined around these cells and extended club-, knob-, or thread-like protrusions in the surrounding tissues. Furthermore, a retrograde tracing method using fast blue dye indicated that most of these nerve endings originated from the nodose ganglia of the vagus nerve. These results suggest that P2X3-immunoreactive subserosal nerve endings have morphological characteristics of mechanoreceptors and contribute to sensation of a mechanical deformation of the distal antral wall associated with antral peristalsis.

Morphology of P2X3‐immunoreactive basket‐like afferent nerve endings surrounding serosal ganglia and close relationship with vesicular nucleotide transporter‐immunoreactive nerve fibers in the rat gastric antrum

“…The procedures for preparing whole‐mount preparations of the serosa with the smooth muscle layer of the gastric antrum were conducted as described in our previous study (Hirakawa et al, 2021). Thirty‐nine rats were transcardially perfused with Ringer's solution (200 ml) under deep anesthesia by an intraperitoneal pentobarbital injection (150 mg/kg).…”

“…An immunoblotting analysis of this antibody labeled a band near 57‐kDa in a rat dorsal root ganglion lysate (Xu & Huang, 2002), a 64‐kDa band in a mouse dorsal root ganglion 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), sensory nerve endings associated with chemoreceptor type I cells in the rat carotid body (Yokoyama et al, 2020), and subserosal nerve endings in the rat antral lesser curvature (Hirakawa et al, 2021). In our experiment, no P2X3‐immunoreactive products were observed in any region of subserosal tissue in the antral lesser curvature when a preabsorbed antibody was used (2 μg/μl; Figure not shown).…”

“…An immunoblotting analysis labeled a single band of 42–43 kDa in human gingival fibroblast cultures (Çomut et al, 2000) and ovine fetal kidneys (Yang et al, 2001). This antibody has also been used for immunohistochemical analyses of smooth muscle cells in the rat carotid sinus (Yokoyama et al, 2019) and smooth muscle layers of the rat gastric antrum (Hirakawa et al, 2021).…”

“…In the rat gastric wall, our previous study revealed that P2X3‐immunoreactive vagal nerve endings were distributed in subserosal tissue of the rat distal antral lesser curvature (Hirakawa et al, 2021). Subserosal nerve endings had large web‐like complex structures with pleomorphic flattened axon terminals, and therefore may be a subtype of vagal mechanoreceptors activated by mechanical deformation of the gastric antral wall associated with antral peristalsis.…”

“…In the present study, we examined the morphological relationship between P2X3‐immunoreactive afferent nerve endings and serosal ganglia in the rat gastric antrum using immunohistochemistry with confocal scanning laser microscopy. Since immunohistochemistry with whole‐mount preparations has been widely used in morphological analyses of sensory nerve endings (Hirakawa et al, 2021; Pintelon et al, 2007; 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 antrum. We also performed multilabeling immunofluorescence in order to elucidate the spatial relationships between P2X3‐immunoreactive nerve endings, ganglionic cellular components, and autonomic nerves in the serosal ganglia.…”