Localization Of NADPH-diaphorase Research Articles

Fine Structure and Synaptology of the Nitrergic Neurons in Medial Septum of the Rat Brain

The nitrergic neuron population and certain aspects of their connectivity (peptidergic inputs, co-localization with GABA, synaptic target distribution) were studied in the medial septum of the rat brain. The histochemical localization of NADPH diaphorase and immunohistochemical identification of nNOS at light and electron microscopic level was applied. Double-labeling experiments with galanin and leucine enkephalin, moreover the postembedding GABA immunogold staining was also carried out. NADPH diaphorase- and nNOS-immunopositive neurons could be identified inside the borders of medial septum. Out of their peptidergic inputs galanin- and leucine enkephaline-immunopositive varicose fibers were found in close apposition with nNOS-immunopositive neurons. Based on fine structural characteristics (large indented nucleus, thin cytoplasmic rim, lack of axosomatic synapses) the nitrergic neurons are suggested to be identical with the septal cholinergic nerve cells. Their boutons established asymmetrical synapses mainly on dendritic shafts and spines, some of which were also nNOS-immunopositive. A lower amount of nNOS-immunopositive boutons of presumably extrinsic origin were found to be GABAergic.

Ultrastructural localization of NADPH diaphorase and nitric oxide synthase in the neuropils of the snail CNS

Comparative studies on the nervous system revealed that nitric oxide (NO) retains its function through the evolution. In vertebrates NO can act in different ways: it is released solely or as a co-transmitter, released from presynaptic or postsynaptic site, spreads as a volumetric signal or targets synaptic proteins. In invertebrates, however, the possible sites of NO release have not yet been identified. Therefore, in the present study, the subcellular distribution of the NO synthase (NOS) was examined in the central nervous system (CNS) of two gastropod species, the terrestrial snail, Helix pomatia and the pond snail, Lymnaea stagnalis, which are model species in comparative neurobiology. For the visualization of NOS NADPH-diaphorase histochemistry and an immunohistochemical procedure using a universal anti-NOS antibody were applied. At light microscopic level both techniques labeled identical structures in sensory tracts ramifying in the neuropils of central ganglia and cell bodies of the Lymnaea and Helix CNS. At ultrastructural level NADPH-d reactive/NOS-immunoreactive materials were localized on the nuclear envelope and membrane segments of the rough and smooth endoplasmic reticulum, as well as the cell membrane and axolemma of positive perikarya. NADPH-d reactive and NOS-immunoreactive varicosities connected to neighboring neurons with both unspecialized and specialized synaptic contacts. In the varicosities, the majority of the NADPH-d reactive/NOS-immunoreactive membrane segments were detected in round and pleomorph agranular vesicles of small size (50–200nm). However, only a small portion (16%) of the vesicles displayed the NADPH-d reactivity/NOS-immunoreactivity. No evidence for the postsynaptic location of NOS was found. Our results suggest that the localization of NADPH-diaphorase and NOS is identical in the snail nervous system. In contrast to vertebrates, however, NO seems to act exclusively in an anterograde way possibly released from membrane segments of the presynaptic transmitter vesicle surface. Based on the subcellular distribution of NOS, NO could be both a volume and a synaptic mediator, in addition NO may function as a co-transmitter.

NADPH-Diaphorase-Positive Structures in the Spinal Cord and Spinal Ganglia

Using the histochemical reaction with the various incubation medium pH, the localization of NADPH-diaphorase was studied in the spinal cord and spinal ganglia of the adult rat. The data obtained indicate, that the application of the incubation medium with pH 7.4 allows to demonstrate the neurons with various NADPH-diaphorase activity, to define not only the density of enzyme distribution, but also to calculate the numbers of NADPH-diaphorase-positive neurons. It was established, that both in the spinal cord and spinal ganglia, NADPH-diaphorase activity was found not in all the neurons: high activity was found in small and medium neurons of the spinal ganglia, while low activity was detected in motoneurons. Other cellular structures of the spinal cord also possessed NADPH-diaphorase activity and were found to be the interneurons.

Neuroplastic and neuropathological changes in the central nervous system of the Gray mussel Crenomytilus grayanus (Dunker) under environmental stress

We studied here neuron ultrastructure, synaptic plasticity and subcellular localization of NADPH-diaphorase (NADPH-d), a cytochemical marker for nitric oxide syntase, in the pedal ganglia of the Gray mussel Crenomytilus grayanus sampled from the polluted and reference sites in Amursky Bay (Sea of Japan) at lower and higher water temperature (in the beginning and the end of August, respectively). At lower temperature, neuroplastic changes in mussel ganglia prevailed: a sharp increase in the number of cytosomes in NADPH-d-positive neurons and a sharp decrease in the number of mitochondria in both NADPH-d-positive and NADPH-d-negative neurons. At higher temperature, neurodegenerative changes prevailed: disruption of a part of NADPH-d-negative axons and interneuronal contacts, formation of concentric lamellar structures in the neuropils, and accumulation of autophagosomes in NADPH-d negative neurons. The results suggest that the stress-induced production of nitric oxide in cytosomes of mussel neurons and plasticity of gap junctions have a neuroprotective effect.

Localization and correlation between NADPH-diaphorase and nitric oxide synthase isoforms in the porcine uterus during the estrous cycle

Nitric oxide (NO), a highly reactive free radical is involved in vasodilation, neurotransmission, hormone secretion, and reproduction. Since all known nitric oxide synthase (NOS) isoforms possess NADPH-diaphorase (NADPH-d) activity, NADPH-d histochemistry was used as a commonly accepted procedure for NOS identification. The aim of our study was to determine the cellular localization of NADPH-d, eNOS, and iNOS in the porcine uterus and the correlation between NADPH-d and NOS activity in the early, middle, late luteal, and follicular phase of the estrous cycle. Light-microscopic observations of the sections revealed the differential expression of the NADPH-d in the analyzed stages of the estrous cycle. The most intense staining was observed in the luminal epithelium in the late luteal phase and in some groups of the endometrial glands in all studied stages. Positive reaction was also found in the endothelial cells of blood vessels and in the myometrium itself. Immunostaining for eNOS was observed in the luminal and glandular epithelium in all studied stages, but no clear fluctuations were observed. The endothelium of both endometrial and myometrial blood vessels displayed pronounced eNOS immunostaining. Strong iNOS staining was observed in the luminal epithelium in the late luteal and follicular phase and in selected groups of endometrial glands. Thus, only NADPH-d and iNOS undergo cyclic changes in the studied stages of the estrous cycle. The differential expression of NADPH-d/NOS in the porcine uterine horn during the estrous cycle suggests a role for NO in modulating uterine function.

Histochemical localization of NADPH-diaphorase in neurons of the pheasant ileum

Localization patterns of NADPH-diaphorase-positive neurons in the pheasant ileum were investigated using an enzyme histochemical method. NADPH-diaphorase activity in the pheasant ileum was demonstrated in neuronal cells bodies and nerve fibres. The NADPH-diaphorase-positive nerve cells showed a polygonal shape and were present solitary or arranged in groups in the submucosal and muscular layers. Nerve fibres penetrated the wall of the ileum at its serosal surface, frequently in the vicinity of ileal arterial branches. They were abundantly present in muscular and submucosal layers of the ileum forming thicker nerves. Some nerve fibres traversed the submucosa into the lamina propria mucosae to form dense nerve plexuses. Fine nerve fibres were found to penetrate into intestinal villi encompassing the crypts underneath the epithelium. We conclude that the pheasant ileum is characterized by abundance of NADPH-diaphorase-positive nerve structures which may play a significant functional role in the small intestine of the pheasant.

The influence of chronic moderate ethanol administration on NADPH-diaphorase (nitric oxide synthase) activity in rat brain.

Nitric oxide synthase (NOS), the enzyme with reduced nicotinamide-adenine dinucleotide phosphate (NADPH)-diaphorase activity, generates nitric oxide (NO) which is an important bioregulatory molecule in the nervous, immune, and cardiovascular systems. NOS is linked to non-adrenergic non-cholinergic (NANC) neuronal pathways and modulation of the N-methyl-D-aspartate receptors, yet its modification by ethanol has been little explored. A possible modification by chronic ethanol administration of activity and/or localization of NADPH-diaphorase (NO-synthase) in rat brain may thus provide the pathogenic basis of alcohol-induced brain injury. When female Wistar rats were treated chronically with ethanol for 50 days, the NADPH-diaphorase staining of granular neurons and neurons located in the molecular layer of the cerebral cortex was significantly reduced. Chronic ethanol consumption led to a significant reduction in NADPH-diaphorase staining in the superficial layers of the superior colliculus. The number of NADPH-diaphorase-positive neurons was significantly reduced (P < 0.001) in the stratum zonale and stratum griseum superficiale (by 42.3-65.6% of control values). This could alter synaptic processes in the highly organized structures involved in oculomotor and somatic motor coordination and thus contribute to the motor disturbances which are associated with alcohol abuse.

Localization of the origin of retinal efferents in the turtle brain and the involvement of nitric oxide synthase

Previous studies have used selective neurochemical markers or retrograde tracers to localize the cells in the brain giving rise to efferents to the turtle retina. Because of the relative selectivity of the neurochemical markers or the lack of sensitivity of the previously employed retrograde tracers, these studies did not locate all the efferent cell bodies, or they could not describe the anatomy of the efferent cells. In the present study, cholera toxin B was used as a highly sensitive retrograde tracer to investigate the distribution, number, and morphology of the retinal efferent or centrifugal cell system in turtle brain. Previous studies of the turtle retina have indicated that nitric oxide synthase may be found in some retinal efferents. Therefore, we also did colocalization studies of the retrograde tracer with reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry to investigate nitric oxide as a possible transmitter used by efferent fibers and to localize these NADPH-diaphorase-positive efferent cell bodies in the turtle brain. We found that each eye received projections from approximately 40 efferent cell bodies that were located primarily in the contralateral midbrain. The majority of efferent cell bodies were centered in the isthmic tegmentum; other efferent cells extended more rostrally into the substantia nigra, and some efferent cells extended more caudally into the nucleus raphes superior. The double-label results showed that 30% of the cholera toxin B-like immunoreactive cells were also positive for NADPH-diaphorase. The location of these double-labeled cells around the locus coeruleus corresponded to the NADPH-diaphorase-positive efferent cells in the avian isthmo-optic field. The localization of NADPH-diaphorase in these efferents indicated that they may use nitric oxide to modulate retinal function. J. Comp. Neurol. 393:185–195, 1998. © 1998 Wiley-Liss, Inc.

Localization of the origin of retinal efferents in the turtle brain and the involvement of nitric oxide synthase

Previous studies have used selective neurochemical markers or retrograde tracers to localize the cells in the brain giving rise to efferents to the turtle retina. Because of the relative selectivity of the neurochemical markers or the lack of sensitivity of the previously employed retrograde tracers, these studies did not locate all the efferent cell bodies, or they could not describe the anatomy of the efferent cells. In the present study, cholera toxin B was used as a highly sensitive retrograde tracer to investigate the distribution, number, and morphology of the retinal efferent or centrifugal cell system in turtle brain. Previous studies of the turtle retina have indicated that nitric oxide synthase may be found in some retinal efferents. Therefore, we also did colocalization studies of the retrograde tracer with reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry to investigate nitric oxide as a possible transmitter used by efferent fibers and to localize these NADPH-diaphorase-positive efferent cell bodies in the turtle brain. We found that each eye received projections from approximately 40 efferent cell bodies that were located primarily in the contralateral midbrain. The majority of efferent cell bodies were centered in the isthmic tegmentum; other efferent cells extended more rostrally into the substantia nigra, and some efferent cells extended more caudally into the nucleus raphes superior. The double-label results showed that 30% of the cholera toxin B-like immunoreactive cells were also positive for NADPH-diaphorase. The location of these double-labeled cells around the locus coeruleus corresponded to the NADPH-diaphorase-positive efferent cells in the avian isthmo-optic field. The localization of NADPH-diaphorase in these efferents indicated that they may use nitric oxide to modulate retinal function.

Localization of NADPH diaphorase in the thoracolumbar and sacrococcygeal spinal cord of the dog

The distribution of NADPH-d activity and NOS-immunoreactivity in the spinal cord of the dog was studied to evaluate the role of nitric oxide in lumbosacral afferent and spinal autonomic pathways. At all levels of the spinal cord examined, NADPH-d staining and NOS-immunoreactivity were present in neurons and fibers in the superficial dorsal horn, dorsal commissure and in neurons around the central canal. Sympathetic preganglionic neurons in the rostral lumbar segments identified by choline acetyl transferase (ChAT) immunoreactivity exhibited prominent NADPH-d and NOS-immunoreactive staining; whereas the ChAT-immunoreactive parasympathetic preganglionic neurons in the sacral segments were not stained. The most prominent NADPH-d activity in the sacral segments occurred in fibers extending from Lissauer's tract through lamina I along the lateral edge of the dorsal horn to the region of the sacral parasympathetic nucleus. These fibers were prominent in the S1–S3 segments but not in adjacent segments (L5–L7 and Cx1) or in thoracolumbar segments. The NADPH-d fibers were not NOS-immunoreactive, but did overlap with a prominent fiber bundle containing vasoactive intestinal polypeptide immunoreactivity in the sacral spinal cord. These results indicate that nitric oxide may function as a transmitter in thoracolumbar sympathetic preganglionic neurons, but not in sacral parasympathetic preganglionic neurons. The functional significance of the NADPH-d positive, NOS-negative fiber bundle on the lateral edge of the sacral dorsal horn remains to be determined. However, based on anatomical studies in other species it seems reasonable to speculate that the fiber tract represents, in part, visceral afferent projections to the sacral parasympathetic nucleus.

Localization of NADPH Diaphorase and Vasoactive Intestinal Polypeptide-Containing Neurons in the Efferent Pathway to the Rat Corpus cavernosum

We examined the coexistence of nitric oxide synthase (NOS) and vasoactive intestinal peptide (VIP) in the efferent pathway to the rat corpus cavernosum. We used NADPH diaphorase (ND) histochemical staining, a specific marker of neuronal NOS, in combination with retrograde axonal transport of True Blue (TB) and VIP immunohistochemistry. Many neurons were labeled by TB in the cavernous nerve and the body of the major pelvic ganglion (MPG), and fewer neurons in the pelvic nerve. In the cavernous nerve, about 90% of TB-labeled efferent neurons were ND positive, and about 80% in the body of the MPG and pelvic nerve. Besides, 60-80% of TB-labeled efferent neurons projecting to the corpus cavernosum showed VIP immunoreactivity. VIP was colocalized with NOS in 70-80% of neurons. These findings suggest that most of the ND-positive nerves and terminals in the corpus cavernosum come from the MPG via the cavernous nerve and that NO plays an important role; it may act in combination with NO because most NOS-containing neurons simultaneously showed VIP immunoreactivity.

Enteric innervation in idiopathic megarectum and megacolon.

We have studied the resection specimens from 5 patients with idiopathic megarectum and megacolon and 10 control subjects with non-obstructing colonic cancer. Histological staining with haematoxylin and eosin, and immunocytochemical staining for protein gene product 9.5 (PGP9.5), S100 protein, vasoactive intestinal polypeptide (VIP) and calcitonin gene-related peptide (CGRP), and histochemical localization of NADPH diaphorase was performed. The amount of VIP and CGRP present in samples was measured using an enzyme-linked immunosorbent assay. Patients with idiopathic megarectum and megacolon showed hypertrophy of the muscularis mucosae and muscularis externa. The architecture of the innervation as assessed by immunoreactivity for PGP9.5 and S100 protein appeared normal. There was a decrease in the density of innervation of the longitudinal muscle in rectal tissue from patients with idiopathic megarectum, with fewer VIP- and NADPH-diaphorase-containing nerves. In the muscularis mucosae and lamina propria of the rectal samples of patients with idiopathic megarectum, VIP immunoreactivity was higher and more NADPH-diaphorase-containing nerves were seen. CGRP-immunoreactive nerve fibres were only seen in the myenteric plexus. No CGRP-immunoreactive cell bodies were seen. In summary, there is an increase in VIP and nitric oxide containing fibres in the muscularis mucosae and lamina propria and a decrease in the longitudinal muscle in rectal tissue of patients with idiopathic megarectum. Both are NANC (nonadrenergic noncholinergic) inhibitory transmitters in the gut and the possible relationship of the changes in their density with gut function is discussed.

Electron microscopic localization and experimental modification of NADPH-diaphorase activity in crustacean sensory axons

The origin and ultrastructural localization of NADPH-diaphorase (NADPH-d) in the olfactory afferent pathway of the crayfishPacifastacus leniusculus was investigated by means of histochemical techniques. Sensory axons in the antennular nerve and the olfactory lobe glomeruli of normal animals expressed NADPH-d staining properties. The NADPH-d staining of each glomerulus was regionalized showing pronounced staining in the apical cap-region. Following ablation of the chemosensory input for 30 days, the staining properties of the antennular nerve and the glomeruli were reduced. At the electron microscopic level, the NADPH-d precipitate was found to be distributed on various membranes in neuronal profiles and glial cells. Stained neuronal profiles were frequently observed in the glomeruli, whereas the number of positive glial cells was low. Almost all NADPH-d positive profiles in the neuropil had an intraglomerular localization. The present findings suggest that NADPH-d in the crayfish olfactory lobe neuropil is localized to terminals of olfactory sensory axons.

The ultrastructural localization of NADPH-diaphorase in the cerebral arteries of rats.

The ultrastructural localization of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d), which has been considered to be a neuronal nitric oxide synthase (NOS), was explored in the vascular endothelial cells and perivascular nerves of the cerebral arteries in the rat. In order to detect NADPH-d activity, 2-(2'-benzothiazolyl)-5-styryl-3-4-(4'-phthalhydrazidyl) tetrazolium chloride was utilized as a substrate for NADPH-d histochemistry at the electron microscopic level. In vascular endothelial cells, NADPH-d positive deposits were observed on the nuclear envelope and the endoplasmic reticulum (smooth or rough surfaced). Positive deposits were seen on distinct membrane portions of the endoplasmic recticulum (ER) in the perivascular nerves (axons), but no positive materials were observed either in the cytoplasm of the endothelial cells or in the axoplasm of the perivascular nerves. It was concluded that NOS is located on the membranes of the ER and the nuclear envelope, and that NOS may play substantial roles in the regulation of the cerebral vessels.

Ultrastructural localization of NADPH-diaphorase in the goldfish brain

The ultrastructural localization of NADPH-diaphorase was studied in the goldfish brain by means of the tetrazolium salt technique. The reaction product was found to be bound to the endoplasmic membranes of neurons in different brain areas. In the synaptic structures both pre and post-synaptic profiles showed the reaction product. Furthermore non-neuronal structures were intensely labeled. Endothelial cells revealed the membranous localization of NADPH-diaphorase and the glial cells of the hypothalamic nuclei and of the paraventricular organ were labeled. In some cases the reaction product was seen also in oligodendrocytes and in microglial cells.

Histochemical methods for detecting nitric oxide synthase

The three isoforms of nitric oxide synthase (NOS), neuronal (nNOS), endothelial (eNOS), and inducible (iNOS), can be visualized in cells and tissues by NADPH-diaphorase (NADPH-d) histochemistry, immunocytochemistry and in situ hybridization. Histochemical demonstration of NADPH-d shows the formazan final reaction product as a solid blue deposit. The ultrastructural localization of NADPH-d in the rat hippocampus showed an electron-dense deposit on membranes predominantly of the endoplasmic reticulum. The immunohistochemical demonstration of nNOS, using the nickel enhancement technique, shows positive reaction product over the dendrites and the soma of the nerve cell in the rat brain. Ultrastructural localization of nNOS in whole mount preparations of myenteric plexus and circular smooth muscle from guinea-pig ileum shows that NOS immunoreactivity was patchily distributed in myenteric neurones and was not specifically associated with any intracellular organelles or with plasma membranes. In situ hybridization, using radio-labelled probes, was used to study nNOS mRNA in lumbar dorsal root ganglia after peripheral transection of the sciatic nerve in rats. Labelling of the NOS mRNA-positive neurones is observed as a series of dense granules over the entire cell. NADPH-d histochemistry, immunocytochemistry and in situ hybridization each have a significant role to play in the localization of NOS. NADPH-d detects an enzyme associated with the NOS molecule, immunocytochemistry detects the NOS molecule, and in situ hybridization detects mRNA for NOS. Therefore, if each of these techniques is applied in carefully controlled experiments, consideration of the accumulated data should be valuable in revealing insights into the biology of NOS.

Localization of NADPH-diaphorase in the goldfish brain

The distribution of NADPH-diaphorase positive neurons was studied by using the enzyme histochemical method. Numerous neurons were labeled in different brain areas of the goldfish and their distribution showed some differences in comparison with other studied teleosts, indicating a species-specific pattern of NADPH-diaphorase distribution as observed in mammals. The localization of NADPH-diaphorase in the thalamic nuclei, in the paraventricular organ, in the inferior hypothalamic lobe, in the periventricular neurons of the optic tectum, in the nucleus isthmi and in the mesencephalic reticular formation was comparable to the one observed in other teleosts. In addition in the goldfish the telencephalic neurons of the pars centralis and lateralis of the area dorsalis, the habenular neurons, the bipolar neurons of the central grey layer of the optic tectum and the motor neurons of the hypertrophied vagal lobe were labeled. The localization of NADPH-diaphorase positive neurons, compared to the distribution of cholinergic neurons described in fish, indicated that the production of nitric oxide was prevailing in the brain areas where cholinergic circuits are active.

Histochemical distribution of NADPH-diaphorase in the cerebral ganglion of the crayfish Cambarellus montezumae

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.

Choline acetyltransferase and NADPH-diaphorase localization in the goldfish habenulo-interpeduncular system

The habenular nuclei are the major sources of projections to the interpeduncular nucleus. The habenular neurons of the goldfish are ChAT and NADPH-diaphorase positive. The localization of these two enzymes in the habenulae suggests the involvement of acetylcholine and nitric oxide (the product of NADPH-diaphorase activity in the nervous tissue) in the habenulo-interpeduncular connection. This finding is supported by the presence of ChAT and NADPH-diaphorase activity in the neuropilar area of the interpeduncular nucleus. This activity was depleted 12 days after habenular ablation. The overlap of ChAT and NADPH-diaphorase localization in goldfish habenular neurons is consistent with previous observations on the co-localization of these two enzymes in some brainstem neurons of other vertebrates. It is, however, not identical to the localization of NADPH-diaphorase in the habenulae and in the interpeduncular nucleus of the rat.

A developmental study of the localization of NADPH-diaphorase in the ganglionated plexus of the guinea-pig gallbladder

A histochemical investigation of age-related changes that occur with respect to the localization of NADPH-diaphorase in the ganglionated plexus of the guinea-pig gallbladder was carried out. In all age groups examined (embryonic stages day 34 and 52, 2 to 4-day old, 6-month old and 2-year old), the mean percentage of NADPH-diaphorase-positive neurons per ganglion was obtained by taking the number of neurons that were immunoreactive to protein gene product 9.5 (a general neuronal marker) as 100%. In addition, the possible co-existence of NADPH-diaphorase and nitric oxide synthase in the ganglionated plexus of 2 to 4-day old and 6-month old guinea-pig gallbladder was investigated. NADPH-diaphorase was not present in the ganglionated plexus of the gallbladder at embryonic day 34. At embryonic day 52, all the protein gene product 9.5-immunoreactive neurons showed positive staining to NADPH-diaphorase; this dropped to a minimum at 2-4 days (26.7%), rose slightly at 6 months (33.6%), and finally returned close to the 100% value at 2 years. In the gallbladders of 2-year old guinea-pigs, some (3 out of 10) ganglia were devoid of protein gene product 9.5-immunoreactive neurons, but NADPH-diaphorase-stained granules were found within the ganglia. However, all those neurons that were immunopositive to protein gene product 9.5 also expressed NADPH-diaphorase. Moreover, NADPH-diaphorase-positive neurons in the gallbladder of 2 to 4-day-old and 6-month-old guinea-pigs were found to express nitric oxide synthase.