Distribution pattern, neurochemical features and projections of nitrergic neurons in the pig small intestine

The presence and topographical distribution of nitrergic neurons in the enteric nervous system (ENS) of the pig small intestine have been investigated by means of nitric oxide synthase (NOS) immunocytochemistry and nicotinamide dinucleotide phosphate diaphorase (NADPHd) histochemistry. Both techniques yielded similar results, thus confirming that within the pig ENS the neuronal isoform of NOS corresponds to NADPHd. Intrinsic nitrergic neurons were not confined to the myenteric plexus; considerable numbers were also present in the outer submucous plexus. In the inner submucous plexus, NOS immunoreactivity or NADPHd staining was restricted to a few nerve fibres and nerve cell bodies. The nitrergic neurons displayed a wide variety in size and shape, but could all be characterized as being multidendritic uniaxonal. Nerve lesion experiments showed that the majority of the myenteric nitrergic neurons project in an anal direction. Evidence is at hand to show that a substantial proportion of these neurons contribute to the dense nitrergic innervation of the tertiary plexus and the circular smooth muscle layer. Some of the nitrergic neurons of the outer submucous plexus were equally found to send their axons towards the circular muscle layer. In some of the nitrergic enteric neurons, VIP, neuropeptide Y, galanin or protein 10 occurred colocalized, but not calbindin or serotonin. The present findings provide morphological evidence for the presence of NOS in a proportion of the enteric neurons in the small intestine of a large omnivorous mammal, i.e. the pig. The topographical features of the staining patterns of NOS and NADPHd are in accord with the results of neuropharmacological studies and argue for the existence of distinct nitrergic subpopulations acting either as interneurons or as motor neurons.