Morphological phenotyping of enteric neurons using neurofilament immunohistochemistry renders chemical phenotyping more precise in porcine ileum

Abstract

The aim of the study was: (1) to test the suitability of neurofilament (NF) immunohistochemistry for representing the shapes of morphologically defined neuron types in the pig ileum myenteric plexus, (2) to estimate the proportions of these neuron types as related to the whole myenteric neuron population and (3) to demonstrate the usefulness of a refined morphological classification of enteric neurons on the paradigm of calcitonin gene-related peptide (CGRP)-immunoreactive neurons. So far, immunoreactivity for this peptide was supposed to be present in the pig enteric nervous system only in type II neurons. Ileal whole mounts of two pigs were stained with the cuprolinic blue (CB) method and, thereafter, incubated with an antibody pool against NF proteins (70, 160 and 210 kDa), visualised with a fluorochrome-tagged secondary antibody. The structural representation of morphologically defined myenteric neuron types typical for pig ileum (Stach I, II, IV, V and VI) was equivalent to their silver impregnated image, as demonstrated in previous studies. Counts of CB-stained neurons revealed between 2,526 and 2,662 neurons per square centimetre in one pig and between 2,027 and 2,763 in the other. As related to these total neuron numbers, the proportions of type I neurons were 1.7% and 1.5%, of type II neurons 7.2% and 7.9%, of type IV neurons 1.9% and 2.4%, of type V neurons 1.1% and 1.5%, and of type VI neurons 1.3% each. These values are generally comparable with those estimated earlier on silver impregnated material. Double labelling for NF and CGRP indicated that CGRP-immunoreactive smooth contoured neurons with long processes could be subdivided into two distinct morphological neuron types, i.e. type II and type V. We conclude that NF immunohistochemistry is an appropriate tool for representation of morphologically defined enteric neuron types in the pig. Combination of this technique with immunohistochemistry for neuroactive substances may be useful for making both morphological and chemical classification schemes mutually more precise.

Chemical Coding of Submucosal Type V Neurons in Porcine Ileum

“…The proportions in both myenteric plexus and ESP, estimated by CB/NF co-immunostaining, are quite comparable. In the myenteric plexus [Brehmer et al, 2002a], we found between 1.1 and 1.5% type V neurons in relation to the number of CB-stained neurons. In the ESP (this study), we estimated between 0.9 and 1.6% type V neurons in relation to all CB-stained neurons.…”

Quantitative estimation of putative primary afferent neurons in the myenteric plexus of human small intestine

“…It differs significantly from the proportion given for the guinea-pig, i.e., between 26% ) and 30% (Costa et al 1996). The difference is smaller between human and pig, in the latter species between 5 and 8% myenteric type II neurons were estimated (Brehmer et al 2002;Brehmer and Stach 1998). These discrepancies may, e.g., be related to differences in the neuronal composition between myenteric and submucosal ganglia in the various species Timmermans et al 2001).…”

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

“…(Stach, 1989;Brehmer et al, 2002aBrehmer et al, , 2002b. The fact that among 50 randomly selected CGRP-reactive neurons, many more were type II neurons in comparison to type V, reflects their quite different proportions in ileal myenteric ganglia: between 7 and 8% of the total population were found to be type II neurons, whereas less than 1.5% were type V neurons (Brehmer et al, 2002b). While type II neurons were generally not surrounded by CGRP-positive boutons, the somata and dendritic processes of type V neurons were regularly outlined by CGRP-positive boutons.…”

“…However, several studies revealed that this peptide, in colocalization with a marker for cholinergic neurons (choline acetyl transferase, ChAT), is present also in neurofilament (NF) reactive, non-type II neurons, indicating that the chemical code NF/CGRP/ChAT, with NF as structural marker, is not specific for type II neurons (Brehmer et al, 2002a,b;Jungbauer et al, 2006). The inclusion of NF into the definition of the chemical code is appropriate because this cytoskeletal protein is present only in subpopulations of enteric neurons (Costa et al, 1996;Brookes, 2001;Brehmer et al, 2002b;Ganns et al, 2006). This study deals only with neurons in the myenteric plexus, because only in this network has the morphology, including the projection pattern of type II neurons and other neuronal populations, been sufficiently investigated.…”