Calcitonin gene-related peptide: a marker for putative primary afferent neurons in the pig small intestinal myenteric plexus?

For years, calcitonin gene-related peptide (CGRP) has been used as a marker peptide for Dogiel type II neurons, putative intrinsic primary afferent neurons, in the pig enteric nervous system. Recently, some studies showed CGRP-positive neurons displaying distinctly different shapes. The aims of this study were to evaluate (1) the proportion of myenteric type II neurons that contain CGRP and (2) the proportion of myenteric CGRP-positive neurons that display type II vs. non-type II morphologies and to conclude if this peptide could be suited as a marker for type II neurons. For this purpose, nine myenteric whole-mounts (each one from duodenum, jejunum, and ileum, respectively, derived from three pigs) were triple-immunostained for CGRP, neurofilaments (NF), and choline acetyl transferase (ChAT). Each whole-mount was evaluated twice. First, 50 NF-stained type II neurons were selected randomly and their coreactivities for CGRP and ChAT were observed. Second, 50 CGRP-positive neurons were located randomly and their NF morphology and ChAT coreactivity were observed. Altogether, 92% of all type II neurons investigated displayed CGRP immunoreactivity, whereas 94.9% of all CGRP-reactive neurons recorded displayed type II morphology. We observed three further shapes of CGRP-positive neurons: 7 type V neurons (all were ChAT-positive; mainly in the ileal whole-mounts), 6 type I-like neurons (all were ChAT-positive), and 14 type III-like neurons (mostly ChAT-negative; mainly in duodenal and jejunal specimens). We conclude that CGRP-antibodies can be used as markers for type II neurons in the pig small intestinal myenteric plexus in quantitative studies but it should be kept in mind that up to one-tenth of CGRP-reactive neurons may be non-type II neurons. In case of single cell evaluation, CGRP-immunoreactivity alone is not suited as a marker. In such cases additional, morphological analysis is necessary.