Nitroxidergic Neurons Research Articles

Changes in spatial relationships between catecholamine- and nitroxidergic neurons in the nuclei of the caudal brain stem in the development of hypertension

To obtain the data on the spatial relationships between catecholamine (TH-positive) and nitroxidergic (nNOS-positive) neurons in vasomotor nuclei of the medulla in different periods of hypertension development. The experiment was performed on male Wistar rats (n=45) with induced renovascular hypertension (RVH). TH and nNOS in neurons of solitary tract nuclei, reticular small-and giant cell nuclei were detected using immunohistochemical methods. The most early and severe changes in the intensity of reaction and amount of nNOS-positive neurons were noted in the solitary tract nucleus. Significant changes in the quantitative parameters of TH-positive neurons in RVH were identified only in the reticular giant cell nucleus but they appeared later and were less expressed compared to nNOS-positive cells. This resulted in the changes of spatial relationships between two types of neurons and remodeling of the bulbar region of the cardiovascular center.

Distributions of Cholinergic and Nitroxidergic Neurons in the Spinal Cord of Neonatal and Adult Rats

The aim of the present work was to study the distributions of cholinergic and nitroxidergic neurons in the spinal cord (SC) in neonatal and adult rats. Immunohistochemical detection of choline acetyltransferase (CAT) and nitric oxide synthase (NOS) was performed in the cervical segments of the SC in neonatal (n = 5) and adult (n = 5) Wistar rats. CAT-containing neurons were found to be located in the anterior horns of the SC; occasional cells were located in the posterior horns of the gray matter of the SC, and the central gray matter and at the boundary between Rexed laminae VI and VII. Nitroxidergic neurons were located in the superficial layers of the posterior horns of the gray matter of the SC, the central gray matter, and Rexed laminae VI–VII. The SC of neonatal and adult rats was found to contain NOS-expressing cholinergic neurons. The finding of cells containing both enzymes as early as the fi rst day after birth suggested that they are formed in the rat SC during prenatal ontogeny.

Comparative Study of Catecholaminergic and Nitroxidergic Neurons in the Vasomotor Nuclei of the Caudal Part of the Brainstem in Rats

Immunohistochemical studies in 12 Wistar rats detected tyrosine hydroxylase (TH) and the neuronal form of nitric oxide synthase (nNOS) to address the distribution of catecholaminergic and nitroxidergic neurons, respectively, in the vasomotor nuclei of the medulla oblongata and pons. The highest frequency of TH expression was seen in neurons located in these nuclei and some reticular nuclei (the gigantocellular, paragigantocellular, and caudal nuclei of the pons), though the proportions of immunoreactive neurons in these structures were no greater than 8–14%. In other nuclei (the reticular parvocellular and oral nucleus of the pons, the spinal nucleus of the trigeminal nerve), this proportion ranged from 1 to 3%. Neurons of this type were consistently not detected in a large group of nuclei with confirmed vasomotor functions. Structures with many catecholaminergic neurons generally contained fewer nNOS-positive cells than nuclei with limited numbers of TH-positive neurons.

Norepinephrinergic and nitroxidergic neurons of vasomotor nuclei in hypertensive rats.

The distributions of norepinephrinergic and nitroxidergic neurons in solitary tract nucleus, locus coeruleus, and in the small-celled and gigantocellular reticular nuclei were examined in sham-operated and experimental Wistar rats with induced renovascular hypertension. In control rats, the greatest population of norepinephrinergic cells was revealed in the projections of locus coeruleus and reticular gigantocellular nuclei, while the larger number of nitroxidergic neurons were observed in the solitary tract and the small-celled nuclei. During renovascular hypertension, the earliest and the most pronounced changes in luminescence intensity and the number of nitroxidergic neurons were observed in the solitary tract nucleus, while the changes in locus coeruleus were minimal. The significant changes in similar parameters of norepinephrinergic neurons were documented only in reticular gigantocellular nucleus and locus coeruleus, but they were delayed and less pronounced in comparison with the changes of nitroxidergic neurons.

Distribution of NADPH-Diaphorase and Neuronal NO Synthase in the Nuclei of the Medulla Oblongata in Rats

The distribution of nitroxidergic neurons in the medulla oblongata nuclei in Wistar rats (n = 8) was studied using histochemical (NADPH-diaphorase) and immunohistochemical methods with antisera to the neuronal form of nitric oxide synthase (nNOS). NADPH-diaphorase was found in large and small neurons in the sensory, autonomic, and motor nuclei. The latter were found to contain particularly large numbers of neurons expression this enzyme activity. In contrast to NADPH-diaphorase, nNOS in the corresponding nuclei was always present in smaller quantities of mainly smaller neurons. The sensory nuclei (solitary tract nucleus, reticular parvocellular and lateral nuclei, spinal nucleus of the trigeminal nerve) showed 1.5‐3 times more nNOS neurons than the motor nuclei. In some nuclei (the nucleus ambiguus, the nucleus of the hypoglossal nerve) containing large numbers of NADPH-diaphorase-positive neurons, immunoreactive cells were particularly rare.

Serotoninergic and Nitroxidergic Neurons in the Nuclei of the Medulla Oblongata in the Rat

The aim of the present work was to undertake topochemical mapping of serotoninergic and nitroxidergic neurons in eight medulla oblongata nuclei of the so-called “bulbar vasomotor center” in 12 Wistar rats. The proportion of serotoninergic neurons in the projections of these nuclei was found to average 12–15%, compared with 31–43% in the nuclei of the posterior cervical group. Nitroxidergic neurons were distributed more uniformly, the proportions in the different nuclei ranging from 19% to 49%. Some 2–6% of neurons were located between nuclei and between nuclei and conducting pathways, these cells presumably performing integrative functions in the regulation of hemodynamics.

Enhancement of Rostral Ventrolateral Medulla Neuronal Nitric-Oxide Synthase–Nitric-Oxide Signaling Mediates the Central Cannabinoid Receptor 1-Evoked Pressor Response in Conscious Rats

Our recent studies implicated brainstem GABAergic signaling in the central cannabinoid receptor 1 (CB(1)R)-mediated pressor response in conscious rats. Given the well established link between neuronal nitric-oxide synthase (nNOS)/nitric oxide (NO) signaling and GABAergic transmission in brainstem cardiovascular regulating areas, we elucidated the role of nNOS-generated NO in the central CB(1)R-elicited pressor response. Compared with vehicle, intracisternal (i.c.) microinjection of the CB(1)R agonist (R)-(+)-[2,3-dihydro-5-methyl-3[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-(1-naphthalenyl) methanone mesylate (WIN55212-2) (15 μg/rat) significantly enhanced nNOS phosphorylation as well as the total nitrate and nitrite content in the rostral ventrolateral medulla (RVLM) at 5, 10, and 30 min, which paralleled the elicited pressor response. These findings were corroborated by: 1) the parallel dose-related increases in blood pressure and RVLM-NO levels, measured in real time by in vivo electrochemistry, elicited by intra-RVLM WIN55212-2 (100, 200, or 300 pmol /80 nl; n = 5) in conscious rats; and 2) the significantly higher phosphorylated nNOS (p-nNOS) levels in the WIN55212-2-injected RVLM compared with the contralateral RVLM. Subsequent neurochemical studies showed that WIN55212-2 (15 μg/rat i.c.) significantly increased the number and percentage of neurons immunostained for nNOS (nitroxidergic neurons) and c-Fos (marker of neuronal activity) within the RVLM. The increases in blood pressure and the neurochemical responses elicited by intracisternal WIN55212-2 were attenuated by prior central CB(1)R blockade by N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251; 30 μg/rat i.c.) or selective nNOS inhibition by N(ω)-propyl-(L)-arginine (1 μg/rat i.c.). These findings implicate RVLM p-nNOS/NO signaling as a molecular mechanism in the central CB(1)R-evoked pressor effect in conscious rats.

Gaseous transmitters in the brain of the masu salmon, Oncorhynchus masou (Salmoniformes, Salmonidae)

Distribution of nitroxidergic and H2S-producing neurons in the brain of the masu salmon Oncorhynchus masou was studied by methods of histochemical labeling of NADPH-diaphorase and by immunohistochemical labeling of the neuronal nitric oxide synthase and cystathionine β-synthase (CBS). The established distribution of CBS and nNOS/NADPH-d of neurons and fibers in the masu salmon telencephalon, optic tectum, and cerebellum allows suggesting that the NO- and H2S-producing systems represent individual, non-overlapping neuronal complexes performing specialized functions in the activity of local neuronal networks. In the medullar part, the nNOS-ir and NADPH-d-positive neurons were detected in the composition of viscerosensory (V, VII, and IX–X) and visceromotor (III, IV, and VI) nuclei of craniocerebral nerves, octavolateral afferent complex, reticulospinal neurons, and medial reticular formation. CBS in the masu salmon medulla was revealed in neurons of the nerve X nucleus, reticulospinal neurons, and ventrolateral reticular formation. Distribution of NO-ergic and H2S-producing neurons in the masu salmon medullar nuclei indicates that NO in masu salmon is the predominant neuromodulator of the medullar viscerosensory systems, while H2S seems to modulate only the descending motor systems. The results of the performed study allow suggesting that NO in the masu salmon medulla periventricular area can act as a regulator of postnatal ontogenesis.

Obstructive Jaundice Activates Nitroxidergic Neurons of the Vago-Vagal Neural Circuit That Regulates the Hepatobiliary System in Rabbits

In this study, we investigated the expression of neuronal nitric oxide synthase (nNOS) and nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d), two specific enzymes for nitric oxide (NO) synthesis, in the development of liver fibrosis induced by chronic bile duct ligation (BDL) in the rabbit. We specifically studied the liver-innervated nitroxidergic neurons that originate in the nodose ganglion (NG), nucleus of the solitary tract (NTS) and dorsal motor vagal nucleus (DMV). Our data showed that BDL resulted in overexpression of NADPH-d/nNOS in the NG, NTS and DMV neurons. Using densitometric analysis, we found a significant increase in NADPH-d expression as a result of BDL in the NG, NTS and DMV (72.6, 79.4 and 57.4% increase, respectively). These findings were corroborated by serum biochemistry and hepatic histopathological examination, which were influenced by NADPH-d/nNOS-generated NO in the liver following BDL. Upregulation of NADPH-d/nNOS expression may have important implications, including (1) facilitation of extrahepatic biliary parasympathetic tone that promotes gallbladder emptying of excess stagnant bile; (2) relaxation of smooth muscles of bile canaliculi thus participating in the pathogenesis of cholestasis; (3) dilation of hepatic sinusoids to counter BDL-induced intrahepatic portal hypertension in which endothelia may be damaged, and (4) alterations in hepatic metabolism, such as glycogenesis, bile formation and secretion, and bilirubin clearance.

Age-related changes in nitroxidergic neurons in some nuclei of rat medulla oblongata

The paper presents a comparative study of NO neurons in the solitary tract nucleus, giant-cell, and lateral reticular nuclei in rats at 4, 7, 10, 14, 30, 45, and 60 days old and 3, 6, 12, 18, 24 months old. We determine the active quantitative and qualitative changes that occur in NO-positive neurons in the studied nuclei during the course of postnatal development. A low level of enzyme activity is observed on the first day; it reaches a peak level around the first-third month, then slowly declines. The size and number of nitroxidergic neurons increases, while the relative cell density decreases until the third month of life. We reveal local differences in the ontogenetic development of NO neurons in the studied nuclei. Solitary tract neurons have the highest rate of development, while NO neurons of old animals undergo early and extreme changes as compared to other studied nuclei of rat medulla oblongata.

Nitroxidergic Nerve Fibers of Intracerebral Vessels

Light and electron histochemical methods were used to study the structure and distribution of neurons containing NADPH diaphorase and their processes in the parietal area of the cortex in rats. Most neurons were found to be characterized by tight associations with intracerebral vessels. The smallest distances between the axon plasmalemma and the smooth myocytes of intracerebral arteries in the cerebral cortex were at least 0.3-0.5 microm. Neuron bodies were located at functionally important locations of vessels (sites at which subsidiary vessels branched off, the origins of arterioles), and their processes accompanied vessels, densely entwining the vessels with their branches. Neurons whose dendrites contacted the bodies or process of above- or below-lying neurons often sent nerve conductors to arteries, veins, or capillaries. Thus, nitroxidergic neurons or groups of these neurons may monitor the state of the circulation at different points in the vascular bed, functioning as local nerve centers.

Nitroxidergic Neurons in Nuclei of the Medulla Oblongata in Hypertensive and Normotensive Rats

Distribution of nitroxidergic neurons and neuronal NO-synthase activity in certain medulla oblongata nuclei were studied in normotensive and hypertensive rats with different types of arterial hypertension. In rats with renovascular hypertension, neuronal NO-synthase activity markedly decreased in most nuclei 2 weeks after surgery, while the number of NO-positive cells did not change significantly; after 4 weeks, the percentage of NO-positive neuron markedly decreased and neuronal NO-synthase activity also slightly decreased. No further decrease in neuronal NO-synthase activity was observed 8 week after intervention, but the percentage of NO-neurons decreased compared to that in normotensive rats. In spontaneously hypertensive rats, changes in most nuclei were directed similarly to those observed in 8-week renovascular hypertension, but motor nuclei demonstrated less differentiated reaction to hypertension. In all cases, changes in the parameters observed in sensory nucleus (n. solitarius) appeared earlier and were more pronounced than in nuclei of the reticular formation related to bulbar vasomotor center.

Nitroxidergic neurons in nuclei of rat and human medulla oblongata

Four types of neurons distinguished by NADPH-diaphorase reaction have been identified in seven nuclei of human and rat medulla oblongata. It was found that nitrogen oxide (NO)-positive neurons had similar distributions; neurons with high NO-synthase activity predominated in vasomotor nuclei and, in sensitive nuclei, most neurons had low NO-syntase activity.

NO-Positive Neurons in Some Nuclei of Human Bulbar Vasomotor Center in Arterial Hypertension

The distribution of nitroxidergic neurons and activities of neuronal NO synthase in them in some nuclei of the bulbar vasomotor center were studied in patients with early forms of arterial hypertension. Activity of neuronal NO synthase is reduced significantly in the majority of nuclei in patients with early forms of arterial hypertension, while the content of NO-positive cells was only slightly changed. More pronounced changes in this parameter were detected in the solitary tract nuclei in comparison with the reticular formation nuclei, which had efferent relationships with the intermediate lateral spinal nucleus.

Exposure to aluminium changes the NADPH-diaphorase/ NPY pattern in the rat cerebral cortex

Aluminium (Al) impairs the glutamate-nitric oxide-cGMP pathway and reduces the number of nitroxidergic neurons in the rat somatosensory cortex. To understand better the effect of the time of exposure, we monitored the effect of aluminium administration on nitroxidergic neurons, identified by NADPH-diaphorase (NADPH-d) or by nitric oxide synthase (NOS) staining, after 0.5, 1, 2, 3, 6 and 12 months of aluminium administration. Since neuropeptide Y (NPY) is known to be colocalised with nitric oxide synthase in cortical neurons, the aim of this work was to study the effects of Al administration on the cortical expression of NADPH-d, nNOS, and NPY. NADPH-d or NOS positive neurons were found scattered in the cortex where they constituted about 1% of all neurons. Double staining using NADPH-d and NPY showed that almost all nitroxidergic neurons were co-localised with NPY neurons (NADPH-d/NPY double stained neurons) whereas some neurons were stained only with NPY (NPY single stained neurons) ; these were more numerous than NADPH-d/NPY double stained neurons. Al significantly reduced NADPH-d and nNOS positive neurons in the cerebral cortex time dependently, with the greatest effect appearing after 3 months. Also measured was the integrated optical density (IOD) of nNOS positive neurons showing a significant decrease of NOS immunostaining even in the remaining NOS positive neurons. The double staining experiment exhibited a decrease in NADPH-d/NPY double stained neurons with an apparent increase in NPY single stained neurons; these then decreased after 6-12 months. On the whole, the results confirm that Al impairs nitroxidergic pathways time dependently; moreover, the transient increase in NPY single stained neurons from 1 to 3 months suggests that there is an intraneuronal down-regulation of NOS, without affecting neuronal viability. In addition, the decrease in the NPY system found at 6 and 12 months may indicate that Al affected nitroxidergic and NPY systems at different times.

Nitroxidergic influences on cardiovascular control by NTS: a link with glutamate.

Glutamate (GLU) receptor activation, which is important in cardiovascular reflex transmission through the nucleus tractus solitarii (NTS), leads to release of nitric oxide (NO.) from central nitroxidergic neurons. Therefore, we hypothesized that GLU and NO. are linked in cardiovascular control by NTS. We first sought to determine if NO. released into NTS led to cardiovascular changes like those produced by GLU and found that the nitrosothiol S-nitrosocysteine, but not NO. itself or other NO. donors, elicited such responses in anesthetized rats. The responses were dependent on activation of soluble guanylate cyclase but, not being affected by a scavenger of NO., likely did not depend on release of NO. into the extracellular space. Responses to ionotropic GLU agonists in NTS, like those to S-nitrosocysteine, were inhibited by inhibition of soluble guanylate cyclase. Inhibition of neuronal NO. synthase (nNOS) also inhibited responses to ionotropic GLU agonists. The apparent physiologic link between GLU and NO. mechanisms in NTS was further supported by anatomical studies that demonstrated frequent association between GLU-containing nerve terminals and neurons containing nNOS. Furthermore, GLU receptors were often found on NTS neurons that were immunoreactive for nNOS. The anatomical relationships between GLU and nNOS and GLU receptors and nNOS were more pronounced in some subnuclei of NTS than in others. While seen in subnuclei that are known to receive cardiovascular afferents, the association was even more prominent in subnuclei that receive gastrointestinal afferents. These studies support a role for nitroxidergic neurons in mediating cardiovascular and other visceral reflex responses that result from release of GLU into the NTS.

Chronic exposure to aluminium decreases NADPH-diaphorase positive neurons in the rat cerebral cortex

Aluminium (Al) exposure is neurotoxic and is considered a possible etiological factor for many neurodegenerative disorders. Since it is known that Al impairs the glutamate–nitric oxide–cGMP pathway in neurons, this study was carried out to monitor the expression of NADPH-d in some central nervous system areas of rats after chronic administration of Al in drinking water. We tested three different nervous areas known to contain NADPH-diaphorase positive neurons: two cortical area (somatosensory cerebral cortex and cerebral cortex), a deep brain area (dorsolateral periaqueductal gray matter) and a spinal area (lumbar enlargement of the spinal cord). Our data showed that Al significantly decreased NADPH-d positive neurons in the cerebral cortex and the NADPH-d staining of many granular neurons in the cerebellum. We also found that Al did not cause neuron loss or apoptosis in the cerebral cortex. These findings suggest that the cortical nitroxidergic neurons and granule cells were a specific target of Al neurotoxicity.

Role of Orbital Connective Tissue in the Pathogenesis of Strabismus

Recent studies have shown that each rectus and inferior oblique extraocular muscle passes through a connective tissue sleeve that acts as a pulley, determining muscle pulling direction. Pulleys are condensations of collagen and elastin, suspended from the orbit and from other connective tissues by collagen, elastin, and smooth muscle bands. These soft pulleys function as mechanical origins of the striated extraocular muscles (with respect to muscle pulling directions), and so are active tissues that are clinically relevant and surgically accessible.This paper reviews recent findings regarding the pulleys, including neural control of pulley smooth muscles. Clinical correlations are provided by binocular alignment measures, magnetic resonance images, and binocular, three-dimensional eye movement recordings, in patients with well-defined strabismus syndromes. Computer modeling using the Orbit™ biosimulation program conceptually unifies these data and predicts effects of pulley abnormalities and surgery.Location, structure and composition of extraocular pulleys are very similar across subjects, and comparable across primates. The pulleys are located between the globe equator and the globe-optic nerve junction in primary position, and make only small, but significant, movements with gaze change. Connective tissue bands joining adjacent pulleys are particularly dense between the superior and lateral rectus muscles, and between the medial rectus and the inferior rectus-inferior oblique complex. Smooth muscle is abundant in pulley suspensions, and is innervated by sympathetic, parasympathetic, and nitroxidergic neurons. Extraocular muscle soft pulleys determine the outcome of transposition surgery, and abnormal pulley locations may cause “A” and “V” pattern strabismus.Extraocular soft pulleys, and related connective and smooth muscle tissues, are involved in the etiology and treatment of strabismus. Modern techniques are essential to understanding these complex structures and applying this knowledge to strabismus diagnosis and treatment planning.

Differential projection of cholinergic and nitroxidergic neurons in the myenteric plexus of guinea pig stomach.

The aim of this study was to investigate the organization of myenteric circuits in the guinea pig stomach. Intracellular neurobiotin injections followed by choline acetyltransferase (ChAT) immunohistochemistry and NADPH-diaphorase reaction were used to identify projections of cholinergic and nitroxidergic neurons. Neurons were classified as motor neurons based on varicose endings in the muscle or the occurrence of retraction bulbs, as nonmotor neurons if varicose endings terminated onto other ganglion cells, or as multitargeted neurons. ChAT-positive cells are composed of 64% motor, 27% nonmotor, and 9% multitargeted neurons. The percentages for NADPH-reactive motor, nonmotor, and multitargeted neurons were 57, 39, and 4%, respectively. The majority of ChAT-positive motor (81%) and nonmotor neurons (85%) had ascending projections. In contrast, the majority of NADPH-reactive motor (86%) and nonmotor neurons (86%) had descending projections. Cell bodies of ascending neurons were smaller in size than the descending neurons. The results indicate that ChAT- and NADPH-neurons in the stomach have preferred projections, the former being primarily ascending, the latter mainly descending neurons. This suggests the existence of a basic circuit for polarized reflexes in the myenteric plexus of the stomach, which might mediate descending relaxation and ascending excitation.