Nitric Oxide-producing Neurons Research Articles

Nitric oxide producing neurons in the human colon: an immunohistochemical and histoenzymatical study

The nitric oxide producing neurons of the human colonic myenteric plexus have been studied by using antibodies against cerebellar NO synthase type I (NOS-IR) and NADPH-diaphorase (NAPDH-d) histoenzymatic reaction. The majority of the stained neurons were both NOS-IR and NADPH-d-positive, while a few others were either NADPH-d-positive or NOS-IR only. Among the co-stained neurons, four subpopulations sharing various degrees of staining intensities have been identified. These findings indicate that in the human colon a one-to-one; correlation between NOS-IR and NADPH-d positivity does not exist and thus the NAPDH-d reaction does not delineate with certainty all NO-producing neurons. The degree of staining intensity might account for different intracellular amounts of these two enzymes.

Sympathetic preganglionic neurons contain nitric oxide synthase and project to the superior cervical ganglion: Combined application of retrograde neuronal tracer and NADPH-diaphorase histochemistry

Nitric Oxide (NO), which was initally identified as an endothelium-derived relaxing factor, has recently been demonstrated to be a neuronal messenger in central and peripheral nervous systems. In the present study, we examined the possibility of NO producing neurons in the intermediolateral (IML) cell collum of the thoracic spinal cord (Th) project to the superior cervical ganglion (SCG). First, we observed the NADPH-diaphorase-positive/nitric oxide synthase (NOS)-immunoreactive neurons of the IML and the dorsal part of the central canal at the level of Th1–Th3, and numerous fiber-stainings in the superior cervical ganglion. Second, after injecting WGA-HRP (wheat germ agglutinin-horse radish peroxidase complex), a retrograde neuronal tracer, into the SCG, and developing WGA-immunohistochemistry and the NADPH-diaphorase histochemistry in the same sections, we detected double-labeled neurons in the IML. These findings provide evidence that sympathetic preganglionic NO producing neurons directly innervate to the SCG.

NADPH-diaphorase histochemistry provides evidence for a bilateral, somatotopically inappropriate response to unilateral hindpaw inflammation in the rat

Unilateral hindpaw inflammation produces several neurochemical changes in the ipsilateral spinal dorsal horn that have been interpreted as contributing to the associated hyperalgesia. NADPH-diaphorase histochemical staining was used to examine the response of a population of neurons 1, 2, 6 or 24 h following injection of 6 mg carrageenan into the left hindpaw of the rat. The resulting unilateral hindpaw inflammation produced a bilateral, time-dependent, reversible increase in the number of NADPH-diaphorase stained neurons in the lumbar spinal cord that peaked at 6 h. In laminae I–III, there was a significant increase in the number of NADPH-diaphorase stained neurons both ipsilateral (25.9 ± 2.3) and contralateral (26.3 ± 1.3) to the inflamed hindpaw relative to uninflamed, control animals (18.6 ± 1.7, 17.4 ± 1.7, respectively). A smaller but significant increase was observed in laminae IV–VII and X. Under dark field illumination, an increase in the number of densely stained neurons in laminae I–III was also observed to peak at 6 h. A greater percentage of the neurons observed under bright field illumination were visible under dark field illumination at 6 h (47%) compared to control (18%), suggesting an increase in the enzymatic activity of neurons in laminae I–III in addition to the increase in the number of neurons with threshold levels of NADPH-diaphorase activity. There was no consistent increase in this ratio over time in laminae IV–VII or X. Six hours following carrageenan, there was a bilateral 50% increase in the density of NADPH-diaphorase staining in the neuropil in the medial laminae I–III. Both spinal neurons and primary afferent axons contributed to this bilateral increase in staining as the number of NADPH-diaphorase stained dorsal root ganglion cell bodies increased 47% over control. In addition to the increase in staining in the lumbar spinal cord, at 6 h post carrageenan, there was a bilateral 23% increase over control in the number of stained neurons in the cervical dorsal spinal cord. For comparative purposes, the distribution of Fos-immunoreactive nuclei was compared to NADPH-diaphorase 6 h post carrageenan. The distribution of Fos-immunoreactive nuclei differed from the NADPH-diaphorase stained neurons, suggesting that two separate populations of neurons were stained. Unilateral hindpaw inflammation did not result in an increase in NADPH-diaphorase activity in the periaqueductal gray or the ventroposteriolateral thalamic nucleus. The relationship of NADPH-diaphorase to nitric oxide is discussed and it is concluded that NADPH-diaphorase, while labeling nitric oxide-producing neurons, is not an exclusive marker for nitric oxide synthase following unilateral hindpaw inflammation. A role for NADPH-diaphorase in general excitatory mechanisms in the spinal cord is proposed.

Nitric oxide producing neurons in the monkey and human digestive system.

Nitric oxide has been proposed as an inhibitory transmitter molecule that plays a role in muscle relaxation and vasodilation in the gastrointestinal tract. The present study analyzes the distribution of nitric-oxide-producing neurons in the monkey and human digestive system by means of nicotinamide-adenine-dinucleotide-phosphate-diaphorase histochemistry. This histochemical method is reliable and convenient for the visualization of neuronal nitric-oxide synthase, the enzyme responsible for nitric-oxide generation. In the gastrointestinal tract, nitric-oxide-synthase-related diaphorase activity was present in nerve fibers running throughout the muscular layer (circular > longitudinal) and in numerous ganglion cells and processes in the myenteric plexus of monkeys and humans. Labelled ganglion cells and fibers also were observed in the submucous plexus, although they were much less numerous than those seen in the myenteric plexus. In the submucosa, a few positive fibers were seen around blood vessels. In the mucosa, stained fibers were sparse at the base of the villi and crypts, whereas they were quite abundant in the muscularis mucosae, especially in the small intestine and colon. In the gallbladder (human), labelling was found in ganglion cells and processes of the innermost and outermost ganglionated plexuses. Stained fibers also were distributed to the muscular layer and, less abundantly, to the mucosa and vasculature. Labelled fibers were more abundant in the sphincter of Oddi (human) than in the gallbladder. In the monkey and human pancreas, nicotinamide-adenine-dinucleotide-diaphorase staining was seen mainly in ganglion cells and fibers of intrapancreatic ganglia, and in processes running among acini, around ducts and in the stroma. A moderate density of stained fibers also was distributed to the vasculature, whereas the islets showed few positive processes. Finally, double label experiments performed in the pancreas showed that the vast majority of neurons producing nitric oxide are immunoreactive for vasoactive intestinal peptide.(ABSTRACT TRUNCATED AT 250 WORDS)

The distribution of nitric oxide synthase immunoreactivity in the human brain

Nitric oxide is a free radical which is produced in the brain and is thought to be the first of a new class of neural messenger molecules. It is postulated to act by inducing an increase in cyclic guanosine monophosphate levels in target cells. The neuronal isoform of nitric oxide synthase, the enzyme responsible for the calcium-dependent synthesis of nitric oxide from L-arginine, has been purified from brain homogenate. Using a specific polyclonal antibody, we have localized brain nitric oxide synthase to the cytosol of discrete neuronal subpopulations and glial elements. These include non-pyramidal cells in the cerebral cortex, pyramidal and non-pyramidal cells of the hippocampus, aspiny neurons of the corpus striatum, basket, Purkinje and granule cells in the cerebellum and neurons of various brain stem nuclei. The localization of nitric oxide-producing neurons in morphologically different and neurochemically diverse cell types suggests a widespread neuromodulatory role for nitric oxide in the central nervous system of man.

Immunohistochemical analysis of neurons and their projections in the proximal colon of the guinea-pig.

The arrangement of the enteric nerve plexuses, and the distributions and projections of chemically specified neurons in the proximal colon of the guinea-pig were studied. The neural plexuses were examined using immunoreactivity to neuron specific enolase, and individual subpopulations were studied using antibodies raised against vasoactive intestinal peptide (VIP), substance P (SP), enkephalin, neuropeptide Y (NPY), gastrin releasing peptide (GRP), galanin, somatostatin, calbindin and calretinin. Nitric oxide producing neurons were studied using NADPH diaphorase histochemistry. The myenteric and submucous plexuses were not uniform around the entire circumference; at the mesenteric aspect of the colon there was almost no longitudinal muscle and the circular muscle was unusually thick and cord-like. In this region there was no tertiary plexus of fibres, and the ganglia of the myenteric and submucous plexuses were elongated in the direction of the circular muscle. Neuronal pathways within the antimesenteric aspect of the colon were investigated using nerve lesioning procedures. VIP, GRP, galanin, calbindin and NADPH diaphorase containing neurons lay in anally projecting pathways within the myenteric plexus, while enkephalin and somatostatin appeared in orally projecting nerve pathways. Few NPY immunoreactive nerve cells were found in the myenteric plexus of the proximal colon. The longitudinal muscle was innervated with VIP, SP, enkephalin and NADPH diaphorase containing fibres. The circular muscle was innervated by axons containing all substances investigated except NPY. Galanin, NPY, somatostatin and VIP fibres, all particularly dense in the mucosa, largely arose from nerve cell bodies in the submucous plexus. The results of the present study indicate that chemically specified neuronal populations in the proximal colon of the guinea-pig are more similar to the distal colon than the ileum, but that neuro-chemical and anatomical differences exist between the proximal and distal colon.

Identification of putative nitric oxide producing neurons in the rat amygdala using NADPH-diaphorase histochemistry

Putative nitric oxide-containing neurons in the rat amygdala were studied using reduced nicotinamide adenine dinucleotide phosphate diaphorase histochemistry. All nuclei of the amygdala contained subpopulations of diaphorase-positive neurons, but the staining intensity of different sub-populations varied. Intensely stained neurons exhibited labeling of the cell body and the entire dendritic arborization. The lateral nucleus had the greatest concentration of intensely labeled cells. Many intensely labeled neurons were located along nuclear boundaries and fiber bundles. In addition to neuronal staining, there was a differential staining of the neuropil in different amygdaloid nuclei. In the basolateral and cortical nuclei the diaphorase-positive cells were non-pyramidal neurons that resembled those containing somatostatin and neuropeptide Y. The distribution and neuronal morphology of labeled neurons in the central nucleus and anterior amygdaloid area suggests that diaphorase-positive cells in these areas may be cholinergic. Recent studies have shown that the enzyme responsible for neuronal diaphorase activity is actually the synthetic enzyme for the newly discovered neurotransmitter nitric oxide. Since there is evidence that nitric oxide plays an important role in excitotoxic neuronal degeneration, the neurons identified in the present study may be involved in degenerative diseases of the amygdala.

Cerebral structures involved in transmission and elaboration of noxious stimulation.

Cerebral structures involved in transmission and elaboration of noxious stimulation.