Identification of functional intramuscular rectal mechanoreceptors in aganglionic rectal smooth muscle from piebald lethal mice

The mechanosensitive endings of low-threshold, slowly adapting pelvic afferents that innervate the rectum have been previously identified as rectal intraganglionic laminar endings (rIGLEs) that lie within myenteric ganglia. We tested whether the aganglionic rectum of piebald-lethal (sl/sl) mice lacks rIGLEs and whether this could explain impaired distension-evoked reflexes from this region. Extracellular recordings were made from fine rectal nerves in C57BL/6 wild-type and sl/sl mice, combined with anterograde labeling. In C57BL/6 mice, graded circumferential stretch applied to the rectum activated graded increases in firing of slowly adapting rectal mechanoreceptors. In sl/sl mice, graded stretch of the aganglionic rectum activated similar graded increases in rectal afferent firing. Stretch-sensitive afferents responded at low mechanical thresholds and fired more intensely at noxious levels of stretch. They could also be activated by probing their receptive fields with von Frey hairs and by muscle contraction. Anterograde labeling from recorded rectal nerves identified the mechanoreceptors of muscular afferents in the aganglionic rectal smooth muscle. A population of afferents were also recorded in both C57BL/6 and sl/sl mice that were activated by von Frey hair probing, but not stretch. In summary, the aganglionic rectum is innervated by a population of stretch-sensitive rectal afferent mechanoreceptor which develops and functions in the absence of any enteric ganglia. These results suggest that in patients with Hirschsprung's disease the inability to activate extrinsic distension reflexes from the aganglionic rectum is unlikely to be due to the absence of stretch-sensitive extrinsic mechanoreceptors.

Number and Distribution of Intraganglionic Laminar Endings in the Mouse Esophagus as Demonstrated with Two Different Immunohistochemical Markers

Intraganglionic laminar endings (IGLEs) represent the only vagal mechanosensory terminals in the tunica muscularis of the esophagus. Two specific markers for IGLEs were recently described in mouse: the purinergic P2X2 receptor and the vesicular glutamate transporter 2 (VGLUT2). This study aimed at comparing both markers with respect to their suitability for quantitative analysis. We counted IGLEs immunostained for VGLUT2 and P2X2, respectively, and mapped their distribution in esophageal wholemounts of C57Bl/6 mice. Numbers and distribution of IGLEs were compared with those of myenteric ganglia as demonstrated by cuprolinic blue histochemistry. Whereas the distribution of VGLUT2-immunopositive IGLEs closely matched that of myenteric ganglia, P2X2-immunopositive IGLEs were rarely found in upper and middle esophagus but increasingly in its lower parts. P2X2 stained only half the number of IGLEs found with VGLUT2 immunostaining. We also investigated the correlation between anterograde tracing and immunohistochemistry for identifying IGLEs. Confocal microscopy revealed colocalization of all three markers in ∼50% of IGLEs. The remaining IGLEs showed only tracer and VGLUT2 labeling but no P2X2 immunoreactivity. Thus, VGLUT2 and P2X2 represent two specific markers for qualitative demonstration of esophageal IGLEs. However, VGLUT2 may be superior to P2X2 as a quantitative marker for IGLEs in the esophagus of C57Bl/6 mice.