Vagal and spinal afferent innervation of the rat esophagus: A combined retrograde tracing and immunocytochemical study with special emphasis on calcium-binding proteins

Abstract

Vagal afferent neurons contain a variety of neurochemical markers and neuroactive substances, most of which are present also in dorsal root ganglion cells. To test for the suitability of the calcium-binding protein calretinin as a specific marker for vagal afferent fibers in the periphery, immunocytochemistry for this protein was combined with retrograde tracing. Nerve fibers in the rat esophagus, as well as vagal and spinal sensory neurons innervating the esophagus, were investigated for co-localization of calretinin with calbindin, calcitonin gene-related peptide, and NADPH diaphorase. The results indicated that calretinin immunocytochemistry demonstrates neuronal structures known as vagal afferent from other studies, in particular intraganglionic laminar endings. A few enteric neurons whose distribution was unrelated to intraganglionic laminar endings also stained for calretinin. Strikingly, calretinin immunoreactivity was absent from spinal afferent neurons innervating the rat esophagus. In intraganglionic laminar endings and nodose ganglion cells calretinin was highly co-localized with calbindin but not with calcitonin gene-related peptide. On the other hand, calbindin was also found in spinal afferents to the esophagus where it was co-localized with calcitonin gene-related peptide. Vagal afferent neurons innervating the esophagus were never positive for NADPH diaphorase. Thus, calretinin appears to be a more specific marker for vagal afferent structures in the esophagus than calbindin, which is expressed by both vagal and spinal sensory neurons. Calretinin immunocytochemistry may be utilized as a valuable tool for investigations of subpopulations of vagal afferents in certain viscera.

Cytoplasmic, but not nuclear, expression of the neuronal nuclei (NeuN) antibody is an exclusive feature of Dogiel type II neurons in the guinea-pig gastrointestinal tract

“…Similar to observations made in the rat esophagus (Du¨tsch et al 1998;Kuramoto and Kuwano 1994), this study revealed that also the myenteric plexus of the guinea-pig esophagus harbours CB-ir neurons. In the cervical and thoracic regions of the esophagus, approximately 5% of all intrinsic neurons expressed CB, while no CB-positive neurons were present in the abdominal region.…”

Intraganglionic laminar endings and their relationships with neuronal and glial structures of myenteric ganglia in the esophagus of rat and mouse

“…SP/VGLUT2 co-staining in varicose fibers could also be of spinal primary afferent origin. However, as most if not all afferent SP neurons contain CGRP (Dütsch et al 1998;Holzer 1988;Maggi 1995), and CGRP/ VGLUT2 fiber co-staining was never observed in our previous study (Raab and Neuhuber 2003), SP/VGLUT2 colocalization as observed in the present study was unlikely to be ascribed to spinal primary afferents.…”

“…The colocalization mismatch of VGLUT2-ir and calretinin-ir or tracer, respectively, found in our previous study (Raab and Neuhuber 2003), could therefore best be explained by a less complete demonstration of IGLEs by both anterograde tracing and calretinin-ir. Thus, VGLUT2-ir may be used as a selective and even more efficient and reliable marker than calretinin (Dütsch et al 1998) and anterograde tracing for IGLEs in the esophagus of rat and mouse, respectively, and may be used as an alternative to P2X2 which was shown to label IGLEs in both mouse (Castelucci et al 2003) and rat (Wang and Neuhuber 2003).…”

Distribution of P2X3 receptor immunoreactivity in myenteric ganglia of the mouse esophagus

“…7b). In all myenteric ganglia examined (n = 12) no CGRP-ir could be detected within neuronal perikarya resembling the pattern previously described (Dütsch et al 1998;Sang and Young 1998). Analysis in merged two-channel single optical sections revealed no spots of colocalization within the same proWle but close apposition of P2X 3 -iry and CGRP-ir varicosities (yellow) at the site of contact, indicating proximity beyond the resolution of the confocal technique (arrows in Fig.…”