M. Barbiers

Nitric oxide synthase immunoreactivity in the enteric nervous system of the developing human digestive tract.

We have investigated indirectly the presence of nitric oxide in the enteric nervous system of the digestive tract of human fetuses and newborns by nitric oxide synthase (NOS) immunocytochemistry and nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry. In the stomach, NOS immunoactivity was confined to the myenteric plexus and nerve fibres in the outer smooth musculature; few immunoreactive nerve cell bodies were found in ganglia of the outer submucous plexus. In the pyloric region, a few nitrergic perikarya were seen in the inner submucous plexus and some immunoreactive fibres were found in the muscularis mucosae. In the small intestine, nitrergic neurons clustered just underneath or above the topographical plane formed by the primary nerve strands of the myenteric plexus up to the 26th week of gestation, after which stage, they occurred throughout the ganglia. Many of their processes contributed to the dense fine-meshed tertiary nerve network of the myenteric plexus and the circular smooth muscle layer. NOS-immunoreactive fibres directed to the circular smooth muscle layer originated from a few NOS-containing perikarya located in the outer submucous plexus. In the colon, caecum and rectum, labelled nerve cells and fibres were numerous in the myenteric plexus; they were also found in the outer submucous plexus. The circular muscle layer had a much denser NOS-immunoreactive innervation than the longitudinally oriented taenia. The marked morphological differences observed between nitrergic neurons within the developing human gastrointestinal tract, together with the typical innervation pattern in the ganglionic and aganglionic nerve networks, support the existenc of distinct subpopulations of NOS-containing enterice neurons acting as interneurons or (inhibitory) motor neurons.

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.

Distribution and morphological features of nitrergic neurons in the porcine large intestine.

The distribution of nitric oxide synthase (NOS), an enzyme involved in the synthesis of the presumed non-adrenergic noncholinergic inhibitory neurotransmitter nitric oxide (NO), was demonstrated in the enteric nervous system of the porcine caecum, colon and rectum. Techniques used were NOS-immunocytochemistry and nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd)-histochemistry. Throughout the entire large intestine, NOS-immunoreactive (IR) and NADPHd-positive neurons were abundant in the myenteric and outer submucous plexus. In the inner submucous plexus, only a small number of positive neurons were found in the caecum and colon, while a moderate number was observed in the rectum. The nitrergic neurons in the porcine enteric nerve plexuses were of a range of sizes and shapes, with a small proportion showing immunostaining for vasoactive intestinal polypeptide. Varicose and non-varicose NOS-IR and NADPHd-positive nerve fibres were present in the ganglia and connecting strands of all three plexuses. Nerve fibres were also numerous in the circular muscle layer, scarce in the longitudinal muscle coat and negligible in the mucosal region. The abundance of NOS/NADPHd in the intrinsic innervation of the caecum, colon and rectum of the pig implicates NO as an important neuronal messenger in these regions of the gastrointestinal tract.

Calcitonin Gene-Related Peptide-Like Immunoreactivity in the Human Small Intestine

Calcitonin-gene-related-peptide (CGRP)-like immunoreactivity was localized in nerve fibres, neuronal somata and in mucosal endocrine cells of the human small intestine. Immunoreactive enteric neurons were more numerous in the submucous plexuses than in the myenteric plexus. Morphologically, they predominantly had the appearance of type II neurons. The majority of the CGRP-like immunoreactive nerve fibres ran within the ganglionic nerve plexuses. Only a small proportion could be observed in the lamina propria, the lamina muscularis mucosae, or the circular and longitudinal outer smooth muscle layer. These findings suggest that within the wall of the human small intestine neuronal CGRP of either extrinsic or intrinsic origin exerts its effect chiefly on other enteric neurons, and might be indirectly involved in the regulatory functions of the human small intestine.

Occurrence, distribution and neurochemical features of small intestinal neurons projecting to the cranial mesenteric ganglion in the pig

The small intestine of the pig has been investigated for its topographical distribution of enteric neurons projecting to the cranial mesenteric ganglion, by using Fast Blue or Fluorogold as a retrogradely transported neuronal tracer. Contrary to the situation in small laboratory animals such as rat and guinea-pig, the intestinofugally projecting neurons in the porcine small intestine were not restricted to the myenteric plexus, but were observed in greater numbers in ganglia of the outer submucous plexus. The inner submucous plexus was devoid of labelled neurons. Retrogradely labelled neurons were mostly found, either singly or in small aggregates, in ganglia located within a narrow border on either side of the mesenteric attachment. For both nerve networks, their number increased from duodenum to ileum. All the retrogradely labelled neurons exhibited a multidendritic uniaxonal appearance. Some of them displayed type-III morphology and stained for serotonin. This study indicates that, in the pig, not only the myenteric plexus but also one submucous nerve network is involved in the afferent component of intestino-sympathico-intestinal reflex pathways. The finding that some of the morphologically defined type-III neurons participate in these reflexes is in accord with the earlier proposal that type-III neurons are supposed to fulfill an interneuronal role, whether intra- or extramurally.

Projections of neurochemically specified neurons in the porcine colon

The intramural projections of nerve cells containing serotonin (5-HT), calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (VIP) and nitric oxide synthase or reduced nicotinamide adenine dinucleotide phosphate diaphorase (NOS/NADPHd) were studied in the ascending colon of 5- to 6-week-old pigs by means of immunocytochemistry and histochemistry in combination with myectomy experiments. In control tissue of untreated animals, positive nerve cells and fibres were common in the myenteric and outer submucous plexus and, except for 5-HT-positive perikarya, immunoreactive cell bodies and fibres were also observed in the inner submucous plexus. VIP- and NOS/NADPHd-positive nerve fibres occurred in the ciruclar muscle layer while VIP was also abundant in nerve fibres of the mucosal layer. 5-HT- and CGRP-positive nerve fibres were virtually absent from the aganglionic nerve networks. In the submucosal layer, numerous paravascular CGRP-immunoreactive (IR) nerve fibres were encountered. Myectomy studies revealed that 5-HT-, CGRP-, VIP- and NOS/NADPHd-positive myenteric neurons all displayed anal projections within the myenteric plexus. In addition, some of the serotonergic myenteric neurons projected anally to the outer submucous plexus, whereas a great number of the VIP-ergic and nitrergic myenteric neurons send their axons towards the circular muscle layer. The possible function of these nerve cells in descending nerve pathways in the porcine colon is discussed in relation to the distribution pattern of their perikarya and processes and some of their morphological characteristics.

Topographical distribution and immunocytochemical features of colonic neurons that project to the cranial mesenteric ganglion in the pig

Using the retrograde neuronal tracers Fast blue and Fluorogold, the topographical distribution and morphological features of porcine colonic neurons projecting to the cranial (superior) mesenteric ganglion have been investigated. Two to four weeks after injection of the tracer into the cranial mesenteric ganglion of immature pigs, labelled neurons were found throughout the colon. In the myenteric and outer submucous plexuses, they were present in ganglia situated to the side of the mesenteric attachment. The highest density of labelled neurons was observed at the end of the ascending colon, which in the pig represents 78-80% of the total colon length. The viscerofugal neurons had a multidendritic appearance and part of them were immunoreactive for calcitonin gene-related peptide or serotonin. This study has revealed similarities but also significant differences in the colono-sympathico-colonic pathways between the pig and small laboratory animals such as the guinea-pig.