Calcitonin Gene‐Related Peptide: A Marker for Putative Primary Afferent Neurons in the Pig Small Intestinal Myenteric Plexus?

Abstract

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 nontype 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.

The Influence of Low Doses of Zearalenone and T-2 Toxin on Calcitonin Gene Related Peptide-Like Immunoreactive (CGRP-LI) Neurons in the ENS of the Porcine Descending Colon

“…First of all, this peptide takes part in the conduction of sensory and pain impulses [19,20], but it also increases a blood flow in mesenteric vessels [21], inhibits gastric acid secretion, and regulates the absorption of nutrients from the gut [22]. Other important functions of CGRP are the ability to stimulate the secretion of other neurotransmitters, including somatostatin and nitric oxide [38], and the protection of endothelial cells against damage [24].…”

“…One of the substances expressed by enteric nerve cells is calcitonin gene related peptide (CGRP), which to date has been found in the ENS and extrinsic innervation of the GI tract of several mammal species, including humans [15,16,17,18]. Within the intestine CGRP is regarded as the key neurotransmitter and/or neuromodulator of the ENS participating in sensory and nociceptive transmission and a marker of the intrinsic primary afferent neurons [19,20]. It is also known that this substance inhibits gastric acid secretion, increases mesenteric blood flow, protects intestinal mucosa and, possibly, exerts relaxatory effects on the gastrointestinal muscle layer [21,22,23].…”

“…Due to the significant sensitivity of the ENS to the presence of harmful agents in the GI tract [28] and the variety of functions that CGRP exerts on the intestinal regulatory processes [20,21,22], these changes may represent the first subclinical symptoms of the damage caused by the mycotoxins studied. The obtained results will enable the development of knowledge about the actions of low doses of ZEN and T-2 toxin on living organisms this study is of particular interest, given that the pig is considered to be an excellent animal model of human physiology [31].…”

Neurovascular Interface in Porcine Small Intestine: Specific for Nitrergic rather than Nonnitrergic Neurons

“…Thus, it was tempting to test the hypothesis that the approached neurons were nitrergic and that, by applying NF costaining, they display type III morphology. The neurons not preferentially approached by capillaries, among them the morphologically striking, nondendritic type II neurons, were shown to be nonnitrergic but cholinergic [Brehmer et al, 2004;Wolf et al, 2007]. It can be assumed that additional neuroactive substances are colocalized with nNOS in the respective myenteric and submucosal type III neurons [Timmermans et al, 2001;Kapp et al, 2006;Wojtkiewicz et al, 2012;Petto et al, 2015].…”

High- and medium-molecular-weight neurofilament proteins define specific neuron types in the guinea-pig enteric nervous system

“…In the enteric nervous system, there is also differential staining of neurons revealed by anti-neurofilament antibodies (Barbiers et al 1995;Costa et al 1996;Brehmer et al 2002Brehmer et al , 2004Hu et al 2002;Sayegh and Ritter 2003;Ganns et al 2006). Type II neurons, which are readily distinguished by their large round or oval cell bodies and multiple axons (Brehmer et al 1999;Furness 2006) are characteristically revealed by anti-NF-H antibodies in the human and pig small intestine, where this is one of the best ways to demonstrate type II neurons (Brehmer et al 2002;Ganns et al 2006;Brehmer 2007;Wolf et al 2007). Anti-NF antibodies are also ideal for the localisation of type II neurons in cattle (Balemba et al 1999), sheep (Mazzuoli et al 2007) and mice (Qu et al 2008).…”