Chemical coding of myenteric neurons with different axonal projection patterns in the porcine ileum

The aim of this study was to perform an immunohistochemical characterization of two different myenteric neuron types of the pig displaying opposite axonal projections. These were type I neurons equipped with lamellar dendrites that projected mainly orally, and type VI neurons that displayed typical axonal dendrites and projected anally. Double immunostainings of longitudinal muscle/myenteric plexus wholemounts from ileal segments of four pigs were performed to visualize neurofilaments (NF) in combination with calcitonin gene-related peptide (CGRP), leu-enkephalin (ENK) and substance P (SP), respectively. Triple immunostainings of wholemounts, using antibodies against neuronal nitric oxide synthase (nNOS) and vasoactive intestinal peptide (VIP) as well as against VIP and galanin (GAL), were performed. We found that 78% of type I neurons immunoreacted to ENK, 21% to CGRP and 24% to SP. The NF-positive type I neurons co-reactive for one of the three above markers displayed mostly frayed outlines of both their somal contours and their broadened dendritic endings. By contrast, most of the non-coreactive type I neurons displayed rather sharply outlined somata and dendrites. No type I neuron immunoreacted to nNOS, VIP or GAL and none of the type VI NF-reactive neurons reacted to CGRP, ENK or SP. All type VI neurons investigated displayed immunoreactivity for nNOS, 92% of which were co-reactive for VIP. Co-reactivity for VIP and GAL was found in 69% of type VI neurons, 21% were positive for VIP but negative for GAL, 9% were negative for both GAL and VIP, and 1% were positive for GAL but negative for VIP. We conclude that there are two subpopulations of morphological type I neurons. One of these displays mainly oral projections and could not be further characterized in this study. The other, which may correspond to neurons innervating the longitudinal and circular muscle layers, were partly immunoreactive for ENK, CGRP and/or SP. Type VI neurons are immunoreactive for nNOS frequently co-localized with VIP and, partly, also GAL. These may be inhibitory motor neurons and are different from VIP/GAL-coreactive minineurons described earlier.