Search

Gaseous transmission in the nervous system is considered a separate and unique mechanism of interneuronal communication (Figure 1.1). In contrast to “classical” neurotransmitters, the radical messenger nitric oxide (NO) is synthesized and released without special storage and transfer mechanisms. It is one of the smallest and most diffusible signal molecules known, and freely crosses membrane barriers to exert its effects through direct binding and/or reaction with its target proteins (Stamler et al., 1992). Its diffusion from a point source can affect targets a relatively large distance away from its origin (Wood and Garthwaite, 1994). It becomes apparent that the concept of a NO microenvironment with transient NO gradients would be a more accurate way to describe the actual situation. Nitric oxide is also a highly reactive radical that can give rise to toxic secondary radical species (Stamler et al., 1992). Overproduction of NO contributes to both nonimmune defense mechanisms and numerous pathological conditions (Dawson and Dawson, 1996;Moncada and Higgs, 1995) Figure 1.1.

The anococcygeus muscle was a relatively late addition to the arsenal of smooth muscle preparations utilized in biomedical research, with the first description of the pharmacological properties of the rat anococcygeus being published in 1972, from the laboratories of Professor John Gillespie at the University of Glasgow, Scotland (Gillespie, 1972). Despite its recent utilization, however, the anococcygeus muscle has provided some of the most crucial experimental evidence contributing to the establishment of the concept that nitric oxide (NO) functions as a neurotransmitter in the peripheral nervous system, and to the development of our understanding of the mechanisms underlying such so-called nitrergic neurotransmission (Rand, 1992; Gibson et al., 1995; Rand and Li, 1995a, b). This chapter traces the experimental path that led to the identification of NO as a neurotransmitter in the anococcygeus, and highlights some of the areas of controversy that have developed along the way.

Injection of an Ascaris suum extract (Asc) affects both the humoral and cellular immune responses to unrelated antigens when it is co-administered with these antigens. In the present study we evaluated the effect of Asc on macrophage activation in the early phase of Mycobacterium bovis BCG (Pasteur strain TMCC 1173) infection in C57Bl/6 mice. C57Bl/6 mice were injected intraperitoneally (ip) with 0.1 mg BCG (BCG group) or BCG plus 1 mg Asc (BCG + Asc group). The peritoneal exudates were obtained at 2, 7 and 14 days after infection. The numbers of IFN-gamma-secreting cells were assessed by the ELISPOT assay. Nitric oxide (NO) production was measured by the Griess method and by the evaluation of NADPH diaphorase activity in the peritoneal exudates. The administration of Asc extract increased NADPH diaphorase activity (2 days: control = 0, BCG = 7%, BCG + Asc = 13%, and Asc = 4%; 7 days: control = 4, BCG = 13%, BCG + Asc = 21%, and Asc = 4.5%) and TNF-alpha levels (mean +/- SD; 2 days: control = 0, BCG = 169 +/- 13, BCG + Asc = 202 +/- 37, and Asc = 0; 7 days: control = 0, BCG = 545 +/- 15.5, BCG + Asc = 2206 +/- 160.6, and Asc = 126 +/- 26; 14 days: control = 10 +/- 1.45, BCG = 9 +/- 1.15, BCG + Asc = 126 +/- 18, and Asc = 880 +/- 47.67 pg/ml) in the early phase of BCG infection. Low levels of NO production were detected at 2 and 7 days after BCG infection, increasing at 14 days (mean +/- SD; 2 days: control = 0, BCG = 3.7 +/- 1.59, BCG + Asc = 0.82 +/- 0.005, Asc = 0.48 +/- 0.33; 7 days: control = 0, BCG = 2.78 +/- 1.54, BCG + Asc = 3.07 +/- 1.05, Asc = 0; 14 days: control = 0, BCG = 9.05 +/- 0.53, BCG + Asc = 9.61 +/- 0.81, Asc = 10.5 +/- 0.2 (2 x 10(6)) cells/ml). Furthermore, we also observed that Asc co-injection induced a decrease of BCG-colony-forming units (CFU) in the spleens of BCG-infected mice during the first week of infection (mean +/- SD; 2 days: BCG = 1.13 +/- 0.07 and BCG + Asc = 0.798 +/- 0.305; 7 days: BCG = 1.375 +/- 0. 194 and BCG + Asc = 0.548 +/- 0.0226; 14 days: BCG = 0.473 +/- 0.184 and BCG + Asc = 0.675 +/- 0.065 (x 10(2)) CFU). The present data suggest that Asc induces the enhancement of the immune response in the early phase of BCG infection.

Summary The existence of nitric oxide synthase (NOS) in oligodendroglial cells is still a matter of debate. Therefore, by using immunocytochemistry, NADPH-diaphorase (NADPH-d) histochemistry and in-situ hybridization, NOS was analysed in oligodendroglial cells of rat brain tissue and in tissue cultures. Independent of the method utilized, NOS was found to be absent in oligodendrocytes. An exception was the presence of NADPH-d activity in some oligodendroglial cells when the electron microscopic BSPT-technique was used. However, this activity is probably not related to NOS. The absence of NO-producing capacity in these cells may explain their high vulnerability to radical-mediated toxicity.

The distribution and morphology of nitric oxide-synthesizing elements in the human olfactory bulb were studied using NADPH-diaphorase histochemistry and nitric oxide synthase immunohistochemistry. NADPH-diaphorase was detected in all olfactory fibers and groups of superficial short-axon cells, deep short-axon cells, stellate cells and abundant centrifugal fibers. Similar cell types were nitric oxide synthase immunoreactive but olfactory fibers were immunonegative. The distribution patterns of nitric oxide-synthesizing elements showed significant differences from what has been reported in the olfactory bulb of macrosmatic mammals including rodents and insectivores. These differences are likely to correlate with interspecies differences in the processing of olfactory information.

The expression of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) and nitric oxide synthase (NOS) in spinal ventral horn neurons was studied in the guinea pig after right knee joint immobilisation (RKJI). At 1 wk after RKJI, neurons in the ipsilateral ventral horn from L4 to S1 segments showed a moderate reactivity for NADPH-d staining. At 2 wk, NADPH-d labelled neurons were also observed in the contralateral ventral horn. Ipsilateral NOS immunoreactive cells were not detectable until wk 2. The intensity of NADPH-d and NOS labelled neurons in the bilateral ventral horns was sustained, peaking at the 4th wk after RKJI. In guinea pigs subjected to 4 wk of RKJI and subsequently released from the immobilisation for 2 and 4 wk, NADPH-d and NOS reactivity in ventral horn neurons diminished. The expression of NADPH-d positive neurons differed from that of NOS labelled neurons in terms of time interval, cell number and staining intensity, the latter being later, fewer and weaker. It is suggested that the induction and upregulation of NADPH-d and NOS are attributable to reduced activity of muscles acting on the knee joint after RKJI; the changes are reversible. It is speculated that increased levels of NO production are involved in protective mechanisms against possible neuronal degeneration as a consequence of target dysfunction.

The NADPH-diaphorase histochemical technique was used to visualize the morphological features of the NADPH-diaphorase positive cells and fibres, and their distribution in both parts of the cat's claustrum. Taking into account the size and form of the perikaryon and the dendritic and axonal characteristics, the neurons are grouped in different subclasses: large, medium-sized and small. The present data suggest the occurrence of two populations of NADPH-diaphorase neurons in the claustrum. One population consisting of large and medium-sized positive neurons represents the projection neurons while the other population of small positive neurons corresponds to the local circuit neurons.

In this study, the neuronal nitric oxide synthase (nNOS)-related NADPHd reaction was combined with a silver impregnation method to visualize the morphology of nitrergic and non-nitrergic enteric neurons in the pig. Based on colour combination, NADPHd-positive and impregnated, NADPHd-negative but impregnated and NADPHd-positive but non-impregnated groups of nerve cells could be distinguished in whole-mounts of the small intestine. Neurons of the first two groups could be classified morphologically. NADPHd-positive and impregnated cells showed type III and type VI morphology, the first being located preferably in the upper, the latter in the lower small intestine. Both project largely in an aboral direction. NADPHd-negative but silver impregnated are the orally projecting type I, the adendritic, mostly circumferentially projecting type II, the vertically – to the submucous plexuses – projecting type IV and the aborally projecting type V neurons. Given that NADPHd and nNOS are identical, we conclude that type III and type VI neurons are nitrergic and type I, II, IV and V neurons are non-nitrergic. A considerable number of mostly smaller NADPHd-positive but non-impregnated neurons could not be classified.

To determine the postganglionic targets of NOS-containing preganglionic neurons, we studied the association of NADPH-diaphorase positive preganglionic fibers and retrogradely labeled postganglionic neurons in the superior cervical ganglion (SCG) in rats. Wheat germ agglutinin-horseradish peroxidase solution was applied to the anterior chamber of the eye, middle cerebral artery, subcutaneous layer of the facial skin, or submucosal layer of the inside of the lip. Two days after tracer application, the rats were perfused with fixative solution. Serial sections of the SCG were stained histochemically for NADPH-diaphorase followed by diaminobenzidine reaction. More than 80% of the labeled postganglionic neurons innervating the structures in the subcutaneous or submucosal layer showed close association with NADPH-diaphorase positive preganglionic nerve terminals; approximately one-third of these labeled neurons were encircled by dense baskets of pericellular terminals. On the other hand, most of the postganglionic neurons innervating the iris (69%) or the cerebral artery (90%) did not show a distinct association with NADPH-diaphorase positive terminals. These results suggest that one of the principal roles of the NOS-containing preganglionic neurons may be in controlling the postganglionic neurons which innervate the structures in the subcutaneous or submucosal layer.

The rat nucleus accumbens contains medium-sized, spiny projection neurons and intrinsic, local circuit neurons, or interneurons. Sub-classes of interneurons, revealed by calretinin (CR) or parvalbumin (PV) immunoreactivity or reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry, were compared in the nucleus accumbens core, shell and rostral pole. CR, PV and NADPH-diaphorase-containing neurons are shown to form three non-co-localising populations in these three areas. No significant differences in neuronal population densities were found between the subterritories. NADPH-diaphorase-containing neurons could be further separated morphologically into three sub-groups, but CR- and PV-immunoreactive neurons form homogeneous populations. Ultrastructurally, NADPH-diaphorase-, CR- and PV-containing neurons in the nucleus accumbens all possess nuclear indentations. These are deeper and fewer in neurons immunoreactive for PV than in CR- and NADPH-diaphorase-containing neurons. CR-immunoreactive boutons form asymmetrical and symmetrical synaptic specialisations on spines, dendrites and somata, while PV-immunoreactive boutons make only symmetrical synaptic specialisations. Both CR- and PV-immunoreactive boutons form symmetrical synaptic specialisations with medium-sized spiny neurons and contact other CR- and PV-immunoreactive somata, respectively. A novel non-carcinogenic substrate for the peroxidase reaction (Vector Slate Grey, SG) was found to be characteristically electron-dense and may be distinguishable from the diaminobenzidine reaction product. We conclude that the three markers used in this study are localised in distinct populations of nucleus accumbens interneurons. Our studies of their synaptic connections contribute to an increased understanding of the intrinsic circuitry of this area.

The NADPH-diaphorase reaction was used to determine the distribution of postganglionic autonomic neurons in the branches of the glossopharyngeal and vagus nerves supplying the gill arches of the cod fish, Gadus morhua. Neurons were common in major nerve trunks in all gill arches, especially in the post-trematic rami of the branchial nerves. From about 55% to more than 85% of the neurons in any branchial nerve were reactive for NADPH-diaphorase. The results suggest that the presence of NADPH-diaphorase, and presumably the ability to synthesise nitric oxide, have been a property of cranial parasympathetic neurons from early in the evolution of the vertebrates.

The histochemical NADPH-diaphorase reaction has identified distinct neuronal populations in the nervous system of several species. Considerable evidence suggests that NADPH-d is a neuronal nitric oxide synthase (NOS). We examined spinal cords of adult and metamorphosing Xenopus laevis (XL) for developmental differences in NADPH-d reactivity. In adult XL, labeling was found in all dorsal root ganglia (DRGs) and in their termination sites within the dorsal horn (cutaneous afferent field) and intermediate gray (muscle afferent field). Cell bodies in the intermediate gray regions containing the autonomic preganglionic neurons were labeled in thoracic and sacral sections. Neurons located in the medial (MMC) and lateral motor columns (LMC) of the ventral horn were also stained. In metamorphosing XL, reactivity was detected in neurons in the intermediate gray, in the MMC and in the LMC as in the adult. Additionally, primary motoneurons including those innervating tail musculature were labeled. Neurons in the DRGs were stained at all stages; in the dorsal horn, the density of staining reflected the development of the sensory afferent fields. The conservation of NADPH-d reactivity in adult and metamorphosing XL spinal neurons suggests that NOS may be involved in processes independent of developmental changes occurring in XL spinal cord.

The presence and localization of NADPH-diaphorase in the cerebral ganglion of the crayfish Cambarellus montezumae was shown. The reactivity of this enzyme was found in the deuterocerebrum, mainly in the commissure, in fibers of olfactory and accessory lobes, and in the laterodorsal group of cells. The presence of this enzyme in these cerebral regions suggests that nitric oxide is involved in primary sensory afferents in the crayfish.

1. A large proportion of sympathetic preganglionic neurones contain nitric oxide synthase. The purpose of this study was to determine the effects of facilitation and inhibition of nitric oxide synthesis within the lower thoracic spinal cord (which contains the majority of renal preganglionic neurones) on renal sympathetic nerve activity (rSNA). 2. In anaesthetized rabbits, rSNA was recorded before and after intrathecal injection (50 microliters of 0.5 M solution) of either L-arginine, a precursor of nitric oxide, or N omega-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase, into the lower thoracic spinal cord. Spinal cord sections were also stained for the presence of NADPH diaphorase, a marker of nitric oxide synthesizing neurones. 3. A high density of NADPH diaphorase-containing neurones was found within the intermediolateral cell column of the lower thoracic spinal cord. 4. Intrathecal injection of L-arginine and L-NAME resulted in a large increase (113 +/- 25%) and decrease (43 +/- 8%), respectively, in rSNA. In contrast, injection of the inactive isomers D-arginine and D-NAME had no significant effect on rSNA. 5. The results indicate that endogenous nitric oxide in the lower thoracic spinal cord (1) has a potent excitatory action on renal sympathetic preganglionic neurones, and (2) helps to maintain the tonic activity of renal sympathetic nerves under resting conditions.

The role of nitric oxide (NO) in the pathogenesis of cerebral disorders is poorly understood (Bruhwyler et al., 1993; Choi, 1993). Many reports suggest that brain damage caused by excessive release of excitatory amino acids is mediated by NO formation (Dawson et al., 1991; Nowicki et al., 1991; Buisson et al., 1993). But there are conflicting findings which show that NO can function rather as a protective agent (Yamamoto et al., 1992; Weissmann et al., 1992). Moreover, the contribution of glial cells to the NO production in intact and damaged brain tissue is quite unclear. Choi (1993) has recently summarized the current discussion concluding that “a plethora of variables and the inherent complexity of NO biology hinder present efforts to define the effects of NO upon the injured brain”.

The distribution of the enzyme NADPH diaphorase in the region of the periaqueductal gray (PAG) was studied in the rat, rabbit, cat, monkey and human. In each species, the enzyme labelled a small band of neurones located below the level of the aqueduct and directly above the supraoculomotor gray in the middle of the antero-posterior extent of the PAG. The chemical specificity of this region suggests that it is distinct from the lateral PAG above and the supraoculomotor nucleus (Su3) below. We have named it the supraoculomotor cap (Su3C).

NADPH-diaphorase activity together with nitric oxide synthase- and vasoactive intestinal polypeptide-immunoreactivities were examined in the neurons of the rat pancreas in culture. Nitric oxide synthase (NOS)- and vasoactive intestinal polypeptide (VIP)-immunoreactivities were localized in a subpopulation of neurons which were also NADPH-diaphorase positive. All neurons expressing colocalized activities for NOS/VIP and NADPH-diaphorase were solitary cells in culture. Each of these double-labelled neurons was characterized by a round or oval cell body, with short and long processes emanating from it. Some of these processes traversed several micrometres before terminating on the exocrine acinar and endocrine cells. The present study provides evidence that nitric oxide may act as a regulatory agent in the inhibitory neural control of the secretory functions in the pancreas, thereby maintaining the milieu interieur of the organism.

Nitric oxide synthase has been located in the braini, in several neuronal populations which appear in close contact either with the microvasculature or the external surface of extracerebral vessels. These data provide structural support to the pharmacological evidences of the nitric oxide participation in the regulation of vascular tone of brain blood vessels. [Neurol Res 1994; 16: 113-115]

In this paper we discuss the anatomical localization of NADPH-diaphorase using Nitroblue tetrazolium in perioesophageal ganglia of Helix aspersa. Our results show that the reaction is present in neurons and fibers of the procerebrum, some positive neurons are found in mesocerebrum, and there were positive fibers in the neuropile of postcerebrum and mesocerebrum; likewise, immunopositive fibers were found in the neuropile of pedal, pleural and parietal ganglia. The presence of NADPH-diaphorase in the interneurons of procerebrum suggests the participation of this enzyme in the production of nitric oxide for the processing of the olfactory information, as has been suggested in mammalian olfactory tissue.

The NADPH diaphorase (NADPHd) reaction for nitric oxide synthase (NOS) visualization suffers from the circumstance that the diaphorase activity of NOS represents only part of the total diaphorase activity, and so far all efforts to make the reaction more specific for routine studies failed. The present investigation describes a simple procedure for mouse tissue, which allows the selective staining primarily of neurons, vascular endothelial cells and macula densa cells, those cells where constitutive NOS has been described reliably. In this method unfixed cryosections and 0.5-1% phosphate-buffered formaldehyde containing 0.5 mg NADPH/1 ml and 1 mg nitro BT/1 ml are used. Compared to strong prefixation with formaldehyde after which many additional cells are still positive for NADPHd, presence of formaldehyde in the incubation medium obviously allows the selective reaction of NOS-positive cells. In conclusion, compared with the original technique a more specific method for the visualization of the NADPHd activity of NOS appears to be available now and can be used for NOS studies in all kinds of mammalian species. Acta Histochem

Nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase histochemistry was used to study the morphology of labeled axonal plexuses in the human lateral temporal cortex. Strongly stained non-pyramidal neurons and a dense NADPH diaphorase-positive network of fibers were observed in all cortical layers. Certain stained fibers are found to give rise to basket-like formations. Notably other fibers seem to innervate small blood vessels. In addition, numerous blood vessels show a punctate labeling over their surfaces. These findings provide new morphological and chemical details of the axonal innervation of the human neocortex.

Preganglionic neurons in the spinal cord of the rat were labelled retrogradely with Fluorogold and the spinal cord stained for NADPH diaphorase. The majority of both sympathetic and sacral parasympathetic preganglionic neurons showed staining for NADPH diaphorase. NADPH diaphorasepositive neurons were located more laterally in the intermediate zone than were preganglionic neurons lacking NADPH diaphorase staining. The recent evidence that identifies NADPH diaphorase as nitric oxide synthase raises the possibility that some spinal preganglionic neurons may synthesize nitric oxide.

Coexistence of the calcium binding protein calbindin D-28k and NADPH-diaphorase activity was studied in the magnocellular secretory nuclei of the rat hypothalamus using both immunocytochemical and histochemical techniques. Coexistence was found in all the nuclei considered (supraoptic, paraventricular, circularis and fornicals nuclei) with the exception of the hypothalamic area situated between the supraoptic and the paraventricular nuclei. Since both stainings are reliable markers, not based upon the physiological characteristics at a given moment, our study provides a further characterization of the neurons in the magnocellular neurosecretory nuclei.

Unfixed retinal tissues of adult Bufo marinus were reacted for NADPH-diaphorase histochemistry that resulted in selective staining of populations of bipolar and amacrine cells. The number of stained bipolar cells in the retina was estimated to be 233,600 +/- 38,900 (mean +/- S.D.). These cells were non-uniformly distributed across the retina with the highest density of 4,308 cells/mm2 along the visual streak decreasing to lowest density of 1,378 cells/mm2 at the dorsal and ventral poles of the retina. The stained bipolar cells represented a morphologically uniform population. The number of labelled amacrine cells in the retina amounted to 3,116 +/- 405 including 251 cells that were displaced into the ganglion cell layer. Labelled amacrine cells were also unevenly distributed; with no stained cells in the retinal centre from where the density increased steadily to 72 cells mm2 in the retinal periphery. NADPH-diaphorase positive amacrine cells were classified on the basis of their dendritic arborization pattern into three morphological types: 1) narrow-field cells both at the periphery and the centre of the retina; 2) wide-field amacrine cells, and 3) amacrines with eccentric medium-sized dendritic field. The results of this study provide evidence for the presence of NADPH-diaphorase containing neurons in the anuran retina. Furthermore, this is the first report on the selective staining of bipolar cells for NADPH-diaphorase in the vertebrate retina.

The morphology and distribution of NADPH diaphorase reactive neurons was studied in the normal human hypothalamus. Reactive neurons were divided into three categories on the basis of perikaryal size. Small neurons (8-20 microns) were oval or fusiform, and pale staining. Intermediate neurons (20-30 microns) were fusiform, triangular or pyramidal with a wide range of staining intensity. Large neurons (greater than 30 microns) were triangular or pyramidal with moderate to dark staining. Reactive neurons were found in four major regions: medial preoptic, ventromedial, lateral, and perifornical. Scattered positive neurons were found in several other hypothalamic areas. Reactive fibers were present in the supraoptic decussation, medial forebrain bundle, and stria medullaris thalami. The localization of NADPH diaphorase neurons in hypothalamic nuclei affected by Alzheimer's disease and other degenerative disorders suggests that further studies of this neuronal subset are warranted.

The distribution of NADPH-diaphorase positive cells was examined histochemically in the retinae of the pigeon and frog. In the pigeon, three different types of amacrine cells were identified in the inner nuclear layer (INL) on the basis of cell body size and staining intensity. In the frog two types of NADPH-diaphorase positive amacrine cells have been demonstrated in the INL. Therefore, the NADPH-diaphorase method selectively stains several subtypes of amacrine cells in the retina of lower vertebrates. Although the actual function of NADPH-diaphorase activity is unknown, diaphorase histochemistry provides a convenient method for achieving Golgi-like images of amacrine cells in the retina.

A hitherto unrecognized role for nitric oxide (NO) in innervation of trematodes has been discovered, suggesting that NO is an old signal molecule in evolutionary scale. N. Saha and co-workers found evidence of NO synthesis in the neuronal cell bodies in two cerebral ganglia, the brain commisure and the nerve fibers, in the main nerve cords of the digenetic trematode Fasciolopsis buski [(2001) Parasitol. Int. 50, 157–163]. The innervation of the pharynx, the cirrus sac, the ventral sucker and other nerve tributaries of the general parenchyma also showed evidence of NO synthesis. NADPH diaphorase expression was used as a histochemical marker for NO synthase activity. Biochemically active NO was also detected at the level of the whole organism, suggesting that parasite-derived NO might have consequences for host physiology. SHK

Nitrinergic signal transduction involves two protein components, NO synthase (NOS) types I-III, which form nitrogen oxides (NO) from L-arginine and soluble guanylyl cyclase (GC-S), which is activated by NO and catalyzes the conversion of GTP to cyclic GMP. A polyclonal antiseruzn (6761-8) to rat cerebellar type I NOS and a monoclonal antibody to rat lung GC-S (B4) were generated in order to localize both signal transduction proteins in various rat tissues. NOS was also visualized by its NADPH-diaphorase (NADPH-d) properties through NADPH-dependent nitroblue tetrazolium (NBT) formazan formation.

[no abstract available]