Correlated morphological and chemical phenotyping in myenteric type V neurons of porcine ileum

Abstract

The study was aimed at the immunohistochemical characterization of myenteric Stach type V neurons of the pig ileum that were not included in the widely used Dogiel classification. So far, this conspicuous population has been defined morphologically on the basis of silver-impregnated specimens only. By using neurofilament immunohistochemistry, type V neurons that occur singly or in aggregates could be identified unequivocally and could be distinguished from other smoothly contoured myenteric neurons, i.e., type II and type IV. Double-labeling immunohistochemistry revealed a number of potentially neuroactive substances or their synthesizing enzymes to be present in type V neurons. Choline acetyltransferase immunoreactivity (-ir) was found in all type V neurons, whereas neuronal nitric oxide synthase was detected in none. Leu-enkephalin-ir was found within 92.3%, somatostatin (SOM)-ir within 91.1%, calcitonin gene-related peptide (CGRP)-ir within 80.6% and metenkephalin-ir within 74.7% of type V neurons. Triple-labeling immunohistochemistry was applied to address the question of a specific chemical coding for myenteric type V neurons. In contrast to other combinations of neuroactive substances/enzymes that were found in both type V and other, nontype V neurons, SOM/CGRP-ir was the only combination observed exclusively within type V neurons. Both substances were colocalized in 79.3% of type V neurons. This colocalization discriminates four-fifths of the type V neurons chemically from both type II neurons (CGRP positive, SOM negative) and type IV neurons (CGRP negative, SOM positive), which both share, at first glance, a similar morphology with type V neurons. These results further support the concept of a close correlation between morphologically defined neuronal type and chemical coding and, it is likely, also function in the enteric nervous system of larger mammals.

Chemical Coding of Submucosal Type V Neurons in Porcine Ileum

“…Moreover, the coreactivity for CGRP and SOM could be shown to occur exclusively in myenteric type V neurons. Since we also found numerous CGRP/SOM-reactive boutons closely apposing type V neurons, we suggested that these neurons may be descending interneurons provided that there are no submucosal or extrinsic CGRP/SOM-coreactive neurons which project onto type V neurons [Brehmer et al, 2002b].…”

“…Myenteric type V neurons, projecting mainly anally, are immunopositive for two different forms of choline acetyltransferase (ChAT; the common variant cChAT and the peripheral variant pChAT) [Tooyama and Kimura, 2000] and also display immunoreactivities for enkephalin (ENK), calcitonin gene-related peptide (CGRP) and somatostatin (SOM) [Brehmer et al, 2002b[Brehmer et al, , 2004b. Moreover, the coreactivity for CGRP and SOM could be shown to occur exclusively in myenteric type V neurons.…”

“…Some ESP neuron populations seem to be identical with myenteric populations, e.g., motoneurons innervating the circular or the longitudinal musculature [Timmermans et al, 2001]. Another, morphologically defined ESP neuron population, namely type V neurons [Stach, 1985[Stach, , 1989, was characterized chemically by using neurofilament (NF) antibodies as structural markers in the myenteric [Brehmer et al, 2002b] but not in the submucosal plexus. Both myenteric and ESP type V neurons are uniaxonal, dendritic neurons and display an eccentric nucleus; they occur either as single cells or clustered within aggregates.…”

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

“…Scale bar 5 50 mm. (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).…”

“…However, the same observation (type V neurons outlined by CGRP-reactive boutons, type II neurons not outlined) was made in ultrastructural studies without colchicine pretreatment (Brehmer, 2006). The close topographical vicinity between CGRP-reactive boutons and type V neurons may indicate an interneuronal role for this population (Brehmer et al, 2002a). That we have found no aggregated type V neurons among the 150 ileal CGRP-reactive neurons selected is surely a mere coincidence.…”