Ganglion cells and topographically related nerves in the vallate papilla/von Ebner gland complex.

Ganglion cells and topographically related nerves in the vallate papilla/von Ebner gland complex were investigated in rat tongue by cytochemical, immunocytochemical, and ultrastructural methods to evaluate the possible presence of different neuronal subpopulations. Immunostaining for neurofilaments and protein gene product 9.5 revealed ganglionic cell bodies and nerve fibers. A large part of the neurons were positive at immunostaining for neuronal nitric oxide synthase (NOS), vesicular acetylcholine transporter (VAChT), or vasoactive intestinal peptide (VIP). A small subset of nerve fibers revealed immunoreactivity for cholecystokinin. Axons traveling under the lingual epithelium were evidenced by their content of calcitonin gene-related peptide (CGRP) or substance P (SP). Cell bodies positive for SP or CGRP were not detected. Using methods of co-localization, three different neuronal classes were detected. The main population was composed of AChE/NADPH-diaphorase (NADPHd)-positive cells. Small groups of acetylcholine esterase (AChE)-positive/NADPHd-negative cells were visible. Isolated neurons were AChE-negative/NADPHd-positive. The results of co-localization experiments for VAChT/NOS were consistent with those obtained by cytochemical co-localization of AChE and NADPHd. Experiments of co-localization for peptidergic and nitrergic structures revealed CGRP- and SP-immunoreactive fibers in the vallate papilla/von Ebner gland ganglion. In conclusion, the results demonstrated in the VP/VEG complex peptidergic, cholinergic, and nitrergic neurons. The presence of different neuronal subclasses suggests that a certain degree of functional specialization may exist.     

Nitrergic innervation and nitrergic cells in arteriovenous anastomoses

Nitrergic innervation and nitrergic epithelioid cells were studied in arteriovenous anastomoses of the tongue, ear, eye, and glomus organ of the finger in different species (rat, rabbit, dog, and man), by means of immunohistochemistry for nitric oxide synthase and enzyme histochemistry utilizing the catalytic activity of this enzyme (the NADPH-diaphorase reaction). Nitrergic perivascular fibers of the tongue were concentrated along the arterial tree and were maximal at the arteriovenous anastomoses in all species. Generally, fewer fibers were located around comparable segments of the episcleral eye vasculature. Only a few nitrergic fibers were found in the canine and rabbit ear, and in the glomus organ of the human finger; however, epithelioid cells in the tunica media of arteriovenous anastomoses of these organs were NADPH-diaphorase-positive and were moderately immunoreactive for nitric oxide synthase. In the epithelioid cells, the reaction product of the NADPH-diaphorase could also be demonstrated by transmission electron microscopy. The epithelioid cells were negative for the panneural and neuroendocrine marker PGP 9.5 confirming the myocytotic nature of these nitrergic cells. Thus, nitric oxide might play a role in mediating the vessel tone of arteriovenous anastomoses via nitrergic nerves or epithelioid cells.     

Quantitative distribution of NADPH-diaphorase-positive myenteric neurons in different segments of the developing chicken small intestine and colon

NADPH-diaphorase (NADPH-d) was used as a marker for neuronal nitric oxide synthase in order to investigate the nitrergic neurons of the developing myenteric ganglia on whole-mount preparations in the proximal and distal segments of the small intestine and in the colon of the chicken embryo, between incubation days 12 and 19. Neurons that were positive for NADPH-d were counted in randomly selected myenteric ganglia. The data obtained from each area and each age group were subjected to two-way analysis of variance (ANOVA) and the Student–Newman–Keuls test. Between incubation days 12 and 19, the originally narrow-meshed myenteric plexus with its high ganglionic density progressively became wide-meshed and the ganglionic density decreased significantly. Quantitative analysis further revealed a significant decrease in the NADPH-d-positive nerve cell density with age. At the same time, the constant or even increasing number of nitrergic cells per ganglion may indicate that the decreasing cell density may be a result of the growth of the bowel with decreasing ganglion density rather than a decrease in the total number of myenteric nitrergic cells. Regional differences in the dynamics of the quantitative changes were revealed. A significant decrease in the nitrergic cell number appeared earlier in the proximal than in the distal segments of the small intestine or in the colon. In contrast, the significant decline of the ganglionic density was first noticed in the colon at the same time.     

Neuronal and endothelial nitric oxide synthase immunoreactivity and NADPH-diaphorase staining in rat and human pancreas: influence of fixation

In this study, we wished to clarify the distribution and co-localization of nitric oxide synthase and NADPH-diaphorase (NADPH-d) in nerve cells, nerve fibres and parenchymal cells in exocrine and endocrine pancreas, and to assess the influence of fixation on the staining pattern obtained. For this purpose, we applied nitric oxide synthase immunocytochemistry and NADPH-d histochemistry to rat and human pancreas under different fixation conditions. Antibodies to neuronal and endothelial nitric oxide synthase were similarly applied. We found complete co-localization of neuronal nitric oxide synthase and NADPH-d in ganglion cells, and in nerve fibres around acini, excretory ducts, blood vessels and in islets of Langerhans of rat and human pancreas. Immunoreactivity for endothelial nitric oxide synthase was co-localized with NADPH-d in endothelial cells. However, in NADPH-d reactive islet and ductal epithelial cells we could detect neither brain nor endothelial nitric oxide synthase immunoreactivity with any fixation protocol applied. There were marked differences in NADPH-d staining of both neurons and parenchymal cells under different fixation conditions. These results indicate the existence of different types of NADPH-d, which are associated or not associated with nitric oxide synthase(s), and which are differently influenced by various fixation procedures in rat and human pancreas.     

Nitric oxide synthase immunoreactive nerves in rat and ferret salivary glands, and effects of denervation

Nitric oxide has been implicated in mechanisms mediating nerve-evoked vasodilatory and secretory responses in salivary glands. In the present study, the occurrence and distribution of nitric oxide synthase (NOS)-immunoreactive nerves in ferret and rat salivary glands were investigated using immunocytochemistry with rabbit and sheep NOS antisera, and using NADPH-diaphorase enzyme histochemistry. In the parotid, submandibular and sublingual glands of the rat and the ferret, NOS-immunoreactive varicose terminals encircled acini and arteries of various sizes. In the ferret, collecting ducts were also supplied with NOS-immunoreactive fibres. In the rat, only the granular ducts of the submandibular gland were supplied with such fibres. The NOS-immunoreactive innervation of acinar cells was more abundant in the rat than in the ferret, whereas the opposite was true for the innervation of blood vessels. No NOS immunoreactivity was observed in the vascular endothelium. In both species, NOS-positive ganglionic cell bodies were found in the hilar regions of the submandibular and sublingual glands, whereas none could be detected in the parotid glands. NADPH-diaphorase reactivity had the same neuronal distribution as NOS immunoreactivity and, in addition, NADPH-diaphorase reactivity was expressed in ductal epithelium. Neither sympathetic denervation (by removal of the superior cervical ganglion) nor treatment with the sensory neurotoxin capsaicin reduced the NOS-immunoreactive innervation of the parotid gland. However, parasympathetic denervation (by cutting the auriculo-temporal nerve) caused an almost total disappearance of the NOS-immunoreactive innervation. The present findings provide a morphological background to the suggested role of nitric oxide in parasympathetic secretory and vascular responses of salivary glands. This revised version was published online in November 2006 with corrections to the Cover Date.     

Histochemical differentiation between nitric oxide synthase-related and -unrelated diaphorase activity in the rat olfactory bulb

The widely used NADPH-diaphorase reaction for demonstrating neuronal nitric oxide synthase is not as specific as previously thought, as it visualizes both a nitric oxide synthase-related activity and a nitric oxide synthase-unrelated diaphorase. In the present study, we used the rat olfactory bulb as a model to characterize the NADPH-diaphorase activity of neuronal nitric oxide synthase histochemically in comparison with neuronal nitric oxide-unrelated diaphorase activity. The NADPH-diaphorase activity of nitric oxide synthase peaked at pH 8 and at Triton X-100 concentrations of 1--2.5%. It was stable in an acidic environment but was reduced in the presence of Triton X-100 and was inactivated by the flavoprotein inhibitor, diphenyleneiodonium. It preferred beta-NADPH as the co-substrate to alpha-NADPH and alpha-NADH. In contrast, nitric oxide synthase-unrelated diaphorase peaked at pH 10, displayed a Triton X-100 optimum at a concentration of 1%, was unstable in an acidic environment and used beta-NADPH, alpha-NADPH and alpha-NADH to similar extents. Differences in the characteristics between neuronal nitric oxide synthase-related and nitric oxide synthase-unrelated NADPH-diaphorase can be used to increase the specificity of the histochemical nitric oxide synthase marker reaction. © Chapman & Hall     

The protective effect of aminoguanidine on cerebral ischemic damage in the rat brain

The NADPH-diaphorase (NADPH-d) histochemical technique is commonly used to localize the nitric oxide (NO) produced by the enzyme nitric oxide synthase (NOS) in neural tissue. The expression of inducible nitric oxide synthase (iNOS) is induced in the late stage of cerebral ischemia, and NO produced by iNOS contributes to the delay in recovery from brain neuronal damage. The present study was performed to investigate whether the increase in nitric oxide production via inducible nitric oxide synthase was suppressed by the administration of aminoguanidine, a selective iNOS inhibitor, as it follows a decrease of NADPH-diaphorase activity (a marker for NOS) after four-vessel occlusion used as an ischemic model. The administration of aminoguanidine (100 mg/kg i.p., twice per day up to 3 days immediately after the ischemic insult) reduced the number of NADPH-diaphorase positive cells to control levels. Our results indicated that aminoguanidine suppressed NADPH-diaphorase activity, and also decreased the number of NADPH-diaphorase positive cells in the CA1 region of the hippocampus following ischemic brain injury.     

河北环毛蚓神经系统 一氧化氮合酶的组织化学定位

用依赖还原型辅酶Ⅱ的黄酶组织化学方法,研究了环节动物门寡毛纲种类河北环毛蚓（Pheretima tschiliensis)神经系统k 一氧化氮合酶(NOS)阳性细胞及阳性纤维的分布,结果表明,河北环毛蚓神经系统中脑神经节背侧有大量细胞呈现NO强阳性反应,胞体和突起染色明显。咽下神经中偶尔能见少数染色较浅的神经元。在脑神经节腹内侧、围咽神经、 咽下神经节外侧部及腹神经链中都有一氧化氮合酶阳性纤维存在脸染色很深,实验结果表明,在环节动物中作为信息分子的一氧化氮已广泛存在于神经系统中。     

Vascular distribution of nitric oxide synthase and vasodilation in the Australian lungfish, Neoceratodus forsteri

The presence of nitric oxide synthase (NOS) and role of nitric oxide (NO) in vascular regulation was investigated in the Australian lungfish, Neoceratodus forsteri. No evidence was found for NOS in the endothelium of large and small blood vessels following processing for NADPH-diaphorase histochemistry. However, both NADPH-diaphorase histochemistry and neural NOS immunohistochemistry demonstrated a sparse network of nitrergic nerves in the dorsal aorta, hepatic artery, and branchial arteries, but there were no nitrergic nerves in small blood vessels in tissues. In contrast, nitrergic nerves were found in non-vascular tissues of the lung, gut and kidney. Dual-wire myography was used to determine if NO signalling occurred in the branchial artery of N. forsteri. Both SNP and SIN-1 had no effect on the pre-constricted branchial artery, but the particulate guanylyl cyclase (GC) activator, C-type natriuretic peptide, always caused vasodilation. Nicotine mediated a dilation that was not inhibited by the soluble GC inhibitor, ODQ, or the NOS inhibitor, L-NNA, but was blocked by the cyclooxygenase inhibitor, indomethacin. These data suggest that NO control of the branchial artery is lacking, but that prostaglandins could be endothelial relaxing factors in the vasculature of lungfish.     

Immunohistochemical Evidence for the Presence of Synaptic Connections of Nitrergic Neurons in the Rat Rostral Migratory Stream

The rostral migratory stream (RMS) is a migration route for neuroblasts originating in the richest neurogenic niche of the adult mammalian brain—the subventricular zone. Most studies are focused on cellular dynamics of migrating neuroblasts and interactions between neuroblasts and astrocytes which both represent the major cellular component of the RMS. Our previous experiments have brought evidence about the existence of a small population of mature neurons in the adult rat RMS with capacity to produce nitric oxide (NO). In order to further support functional significance of nitrergic cells, the aim of the present study was to determine whether NO producing neurons could form synapses. Sagittal sections from the adult rat brain were processed for simultaneous immunohistochemical detection of neuronal nitric oxide synthase (nNOS), the enzyme present in NO producing cells and synaptophysin, a glycoprotein found in synaptic vesicles. Synaptophysin positivity in the RMS was significantly lower in comparison with other brain areas, but its colocalization with nNOS-positive neurons was obvious. Our results suggest that nitrergic neurons in the RMS could be involved in a neuronal circuitry with potential impact on regulation of neurogenesis in the RMS.     

A large proportion of pelvic neurons innervating the corpora cavernosa of the rat penis exhibit NADPH-diaphorase activity

Nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry, which indicates the presence of neural nitric oxide synthase, the enzyme responsible for the generation of nitric oxide, was used in combination with retrograde labelling methods to determine, in whole-mounts and sections of rat major pelvic ganglia, whether neurons destined for the penile corpora cavernosa were able to produce nitric oxide. In whole-mount preparations of pelvic ganglia, among the 607±106 retrogradely labelled neurons innervating the penile corpora cavernosa, 84±7% were NADPH-diaphorase-positive, 30±7% of which were intensely histochemically stained. In serial sections of pelvic ganglia, out of a mean count of 451 retrogradely labelled neurons, 65% stained positively for NADPH-diaphorase. An average of 1879±363 NADPH-diaphorase positive cell bodies was counted in the pelvic ganglion. In the major pelvic ganglion, neurons both fluorescent for Fluorogold or Fast Blue and intensely stained for NADPH-diaphorase were consistently observed in the dorso-caudal part of the ganglia in the area close to the exit of the cavernous nerve and within this nerve. This co-existence was much less constant in other parts of the ganglion. In the rat penis, many NADPH-diaphorase-positive fibres and varicose terminals were observed surrounding the penile arteries and running within the wall of the cavernous spaces. This distribution of NADPH-diaphorase-positive nerve cells and terminals is consistent with the idea that the relaxation of the smooth muscles of the corpora cavernosa and the dilation of the penile arterial bed mediated by postganglionic parasympathetic neurons is attributable to the release of nitric oxide and that nitric oxide plays a crucial role in penile erection. Moreover, the existence in the pelvic ganglion of a large number of NADPH-diaphorase-positive neurons that are not destined for the corpora cavernosa suggests that nitric oxide is probably also involved in the function of other pelvic tissues.     

Distribution and colocalization of nitric oxide synthase and NADPH-diaphorase in adrenal gland of developing,adult and aging Sprague-Dawley rats

The distribution and colocalization of nitric oxide synthase and nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-diaphorase) was investigated in the adrenal gland of developing, adult and aging rats with the use of immunohistochemical and histochemical techniques. Nitric oxide synthase-immunoreactive neurons within the adrenal gland were found from the 20th day of gestation onwards. During early development the neurons were found as small clusters of smaller-size cells compared to those observed in the adult gland. Their number reached that of adult level by the 4th day after birth, and in the glands from aging rats a 28.6% increase was observed. Whilst no immunofluorescence was seen in chromaffin cells during early development, some cells from glands of aging rats showed nitric oxide synthase-immunoreactivity with varying intensity. The immunoreactive neurons from postnatal rat adrenals were also positive for NADPH-diaphorase, whilst those in prenatal rats were negative or lightly stained. Nitric oxide synthase-immunoreactive nerve fibres were present in all adrenal glands examined from the 16th day of gestation onwards. A considerable degree of variation in the distribution of immunoreactive fibres both in medulla and outer region of cortex at the different age groups was observed and described. Most, but not all, nitric oxide synthase-immunoreactive nerve fibres also showed NADPH-diaphorase staining.     

Nitric oxide synthase in the rat carotid body and carotid sinus

The participation of nitric oxide synthase (NOS) in the innervation of the rat carotid body and carotid sinus was investigated by means of NADPH-diaphorase histochemistry and NOS immunohistochemistry using antisera raised against purified neuronal NOS and a synthetic tridecapeptide. NOS was detected in 23% of neurons at the periphery of the carotid bodies. Some negative neurons were surrounded by NOS-positive terminals. NOS-containing varicose nerve fibres innervated the arterial vascular bed and, to a lesser extent, the islands of glomus cells. These fibres persisted after transection of the carotid sinus nerve and are probably derived from intrinsic neurons. Large NOS-positive axonal swellings in the wall of the carotid sinus were absent after transection of the sinus nerve, indicating their sensory origin. The results suggest a neuronal nitrergic control of blood flow, neuronal activity and chemoreception in the carotid body, and an intrinsic role of NO in the process of arterial baroreception.     

Induction of cytosolic NADPH-diaphorase/nitric oxide synthase in reactive microglia/macrophages after quinolinic acid lesions in the rat striatum: an electron and light microscopical study

Induction of nitric oxide synthase and increased production of nitric oxide in microglia may play a crucial role in neuronal damage and neurodegenerative disorders. In the present study we have used light and electron microscopical NADPH-diaphorase histochemistry as the visualization procedure for nitric oxide synthase to investigate the time-course and subcellular patterns of NADPH-diaphorase expression in microglia/macrophages of quinolinic acid-lesioned rat striatum. For light microscopy, NADPH-diaphorase histochemistry sections were stained with nitroblue tetrazolium, while for ultrastructural analysis the tetrazolium salt 2-(2-benzothiazolyl)-5-styryl-3(4-phthalhydrazidyl) tetrazolium chloride (BSPT) was applied. Light microscopical inspection revealed a progressively increasing number of positive cells with increasing intensity of NADPH-diaphorase staining in microglia/macrophages from day 1 after quinolinic acid injection onward. Electron microscopical examination revealed a membrane bound NADPH-diaphorase in quiescent microglia as well as in activated microglia/macrophages through all stages of the lesion studied. Predominantly membranes of the nuclear envelope and the endoplasmic reticulum were labeled with BSPT-formazan, while in advanced stages selective membrane portions of mitochondria, Golgi apparatus and plasmalemma were also stained. From day 5 onward after lesion induction, a very distinctive type of NADPH-diaphorase was observed, forming accumulations of electron-dense grains that were distributed differentially throughout cytoplasmic areas and phagocytic vacuoles. Dynamics of expression, unique cytosolic localization and occurrence exclusively in activated microglia/macrophages suggest that this particular NADPH-diaphorase activity probably reflects the inducible isoform of nitric oxide synthase, whereas the membrane-bound precipitate may represent the neuronal and/or the endothelial isoform of the enzyme.     

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.     

Nitric oxide synthase in human salivary glands

The distribution of the three nitric oxide synthase (NOS) isoforms was determined immunohistochemically in the human minor and major salivary glands with comparison to that of rat salivary glands. In contrast to rat glands, which contained a dense plexus of neuronal NOS-immunoreactive nerve fibers, only a minority of the nerve fibers in human glands showed neuronal NOS immunoreactivity. Human labial and submandibular glands contained sparse NOS-immunoreactive fibers, while only occasional nerve fibers in the parotid or sublingual glands were stained. Furthermore, in contrast to the animal glands, most duct epithelial cells in all human salivary glands were immunoreactive for neuronal NOS. No specific immunoreactivity for inducible or endothelial NOS were observed in the nerve fibers or duct epithelium. We provide evidence to suggest that the role of nitric oxide in the regulation of salivary gland function is different in human as compared to experimental animals. Nitricergic innervation in human tissue is very sparse and thus nitric oxide is probably of minor importance as a neural regulator of salivary glands. Instead, NOS localized in duct epithelial cells suggests that nitric oxide might directly regulate saliva secretion and it is a putative source of nitrates previously reportedly secreted into the saliva.     

NADPH-diaphorase activity in the nociceptive pathways of land snail Megalobulimus abbreviatus: the involvement of pedal ganglia

NADPH-diaphorase (NADPH-d) is a histochemical marker for nitric oxide synthase (NOS), widely used to identify nitric oxide (NO) producing cells in the nervous system of both vertebrates and invertebrates. Using NADPH-d histochemistry and semi-quantitative optical densitometry, we characterized the NO-producing neurons in the pedal ganglia of young and adult Megalobulimus abbreviatus, subjected to aversive thermal stimulus. The animals were killed at different times (3, 6, 12 and 24 h) following stimulus. The enzymatic activity was detected in different cellular subsets and neuronal processes. In all the studied pedal ganglia subregions, the optical density of positive neurons (P < 0.05) and neuropilar area 1 (P < 0.01) was significantly different in treated animals when compared to controls. The increase in nitrergic activity induced by nociceptive stimulus suggests the involvement of NO in the nociceptive circuit of M. abbreviatus, which is well maintained throughout evolution, and could be helpful in drawing cellular homologies with other gastropods.     

Age-Related Changes of NADPH-Diaphorase Positivity in the Rat Rostral Migratory Stream

Summary Accumulating evidence confirms that nitric oxide (NO), a versatile diffusible signaling molecule, contributes to controling of adult neurogenesis. We have previously shown the timing of NADPH-diaphorase (NADPH-d) positivity within the rat rostral migratory stream (RMS) during the first postnatal month. The present study was designed to describe further age-related changes of NO presence in this neurogenic region. The presence of NO synthesizing cells in the RMS was shown by NADPH-d histochemistry and neuronal nitric oxide synthase (nNOS) immunohistochemistry. The phenotypic identity of nitrergic cells was examined by double labeling with GFAP and NeuN. Systematic qualitative and quantitative analysis of NADPH-d-positive cells was performed in the neonatal (P14), adult(5 months) and aging (20 months) rat RMS. 1. Nitrergic cells with different distribution pattern and morphological characteristics were present in the RMS at all ages examined. In neonatal animals, small, moderately stained NADPH-d-positive cells were identified in the RMS vertical arm and in the RMS elbow. In adult and aging rats a few labeled cells could be also detected in the RMS horizontal arm. NADPH-d-positive cells in adult and aging rats were characterized by long varicose processes and displayed dark labeling in comparison to the neonatal group. 2. Double immunolabeling has revealed that nNOS-immunoreactivity co-localized with that of NeuN. This indicates that nitrergic cells within the RMS are neurons. 3. Quantitative analysis showed that the number of NADPH-d-positive cells increases with advancing age. The presence of NO producing cells in the RMS of neonatal adult and aging rats indicates, that this proliferating and migratory area is under the influence of NO throughout the entire life of the animals.     

Correlation between NADPH-diaphorase and iNOS in bank vole Leydig cells in vitro and in testicular sections with the use of histochemistry and immunocytochemistry

Histochemistry for NADPH-diaphorase detects an enzymatic activity associated with nitric oxide synthase while immunohistochemistry detects the nitric oxide synthase molecule. NADPH-diaphorase and inducible isoform of nitric oxide synthase in Leydig cells in vitro and in testis sections of the bank vole were demonstrated histochemically and immunocytochemically. Histochemical studies revealed localization of NADPH-diaphorase reaction product in the cytoplasm of cultured Leydig cells as well as in the interstitial area, mainly in Leydig cells and in vascular endothelium. Distribution pattern of NADPH-diaphorase was different in Leydig cell cytoplasm of individual cells. Using immunocytochemistry, the immunoreactivity for nitric oxide synthase was observed both in cultured Leydig cells and testis sections. Moreover, a co-localization of positively immunostained cells with those histochemically detected was noticed. Addition of hCG to the cultured medium or injections in vivo resulted in a small decrease in reaction intensity in Leydig cells. Treatment with N omega-nitro-L-arginine methyl ester resulted in distinctly weaker reactivity of the enzymes studied which was correlated with a higher testosterone and estradiol levels in Leydig cells measured radioimmunologically. The results have indicated that nitric oxide synthase is able to act directly within the male gonad regulating androgen secretion by Leydig cells.     

Distribution of NADPH-diaphorase and nitric oxide synthase in the trigeminal ganglion and mesencephalic trigeminal nucleus of the cat. A histochemical and immunohistochemical study

The trigeminal ganglion (TrG) and mesencephalic trigeminal nucleus (MTN) neurons are involved in the transmission of orofacial sensory information. The presence of nitric oxide (NO), a putative neurotransmitter substance in the nervous system, was examined in the cat TrG and MTN using nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry and nitric oxide synthase (NOS) immunohistochemistry. In the TrG, where the majority of the trigeminal primary afferent perikarya are located, most of the intensely NADPH-d/ NOS-stained cells were small in size and distributed randomly throughout the ganglion. The medium-sized neurons were moderately stained. A plexus of pericellular varicose arborizations around large unstained ganglion cells and densely stained fibers in-between could also be observed. In the caudal part of the MTN, both NADPH-d activity and NOS immunoreactivity was present in MTN neurons. In addition, a few scattered NADPH-d/NOS-containing neurons were found in the mesencephalic-pontine junction part of the nucleus. In contrast, only nerve fibers and their terminals were present at a more rostral level in the mid- and rostral MTN. MTN neuronal perikarya were enveloped in fine basket-like NADPH-d/ NOS-positive networks. Differential expression patterns of NOS and its marker NADPH-d suggest that trigeminal sensory information processing in the cat MTN is controlled by nitrergic input through different mechanisms. We introduce the concept that NO can act as a neurotransmitter in mediating nociceptive and proprioceptive information from periodontal mechanoreceptors but may also participate in modulating the activity of jaw-closing muscle afferent MTN neurons.