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

Abstract

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.

Fig. 1

Co-immunoreactivity for leu-enkephalin (ENK) of neurofilament (NF)-stained type I neurons (a–d; arrows = axons). Most of the type I neurons were positive for ENK (a′,b′; filled arrowheads) whereas a minority were negative (c′,d′; empty arrowheads). NF-stained neurons in both (a) and (b) displayed frayed outlines of their somata and most of their short dendrites had similarly frayed endings. The NF-stained type I neurons in (c) and (d) display sharply contoured somal and dendritic outlines. Arrowed scale bars (25 µm) point orally.

Fig. 4

Co-immunoreactivity of neurofilament (NF)-stained type VI neurons for neuronal nitric oxide synthase (nNOS) and vasoactive intestinal peptide (VIP; a), as well as for galanin (GAL) and VIP (b). In (a), two type VI neurons (filled arrowheads; arrows = axons; axonal dendrites are seen in between both markings) are co-reactive for both nNOS (a′) and VIP (a″; filled arrowheads). In (b), three type VI neurons (filled arrowheads; arrows = axons, one axon is beneath another type VI neuronal soma) are co-reactive for both GAL and VIP (b′,b″; filled arrowheads). A fourth neuron (left side) without distinct axonal dendrites is also co-reactive for both GAL and VIP. Arrowed scale bars (50 µm) point orally.

Fig. 2

Co-immunoreactivity for calcitonin gene-related peptide (CGRP) of neurofilament (NF)-stained type I neurons (a–d; arrows = axons). Most of the type I neurons were negative for CGRP (a′,b′; empty arrowheads), whereas a minority were positive (c′,d′; filled arrowheads). Most type I neurons negative for CGRP had sharply outlined somata and dendrites (a), whereas most CGRP-positive neurons had frayed contours (d). Arrowed scale bars (25 µm) point orally.

Fig. 3

Co-immunoreactivity for substance P (SP) of neurofilament (NF)-stained type I neurons (a–d; arrows = axons). Most of the type I neurons were negative for SP (a′,b′; empty arrowheads), whereas a minority were positive (c′,d′; filled arrowheads). Most type I neurons negative for SP had sharply outlined somata and dendrites (a), whereas most SP-positive neurons had frayed contours (d). Arrowed scale bars (25 µm) point orally.

Fig. 5

Immunoreactivity for vasoactive intestinal peptide (VIP), galanin (GAL), leucine-enkephalin (ENK), substance P (SP) and calcitonin gene-related peptide (CGRP) in nerve fibres of the secondary (a–e) and tertiary (a′–e′) components of the myenteric plexus. The longitudinal axis of the wholemount is orientated horizontally (according to the scale bar = 100 µm). Secondary strands are situated on the inner (circular muscle) side of the main plexus (‘Pr’ = primary strands of the myenteric plexus) and run perpendicularly, and the tertiary strands are located on the outer (longitudinal muscle) side and run horizontally. VIP- and GAL-immunoreactive fibres are abundant in both the secondary (a,b; arrows) and the tertiary component (a′,b′; arrows) of the myenteric plexus. By contrast, ENK and SP immunoreactivity were found in fibres of the secondary (c,d; arrows) but only scarcely in the tertiary component (c′,d′; arrows). In (e), CGRP is seen in some fibres of the secondary strands (arrows) but only exceptionally in tertiary fibres (e′, arrow).