Key Specifications Table
|Species Reactivity||Key Applications||Host||Format||Antibody Type|
|H, M, Mk, R, Sh||IHC, WB||Rb||Serum||Polyclonal Antibody|
|Description||Anti-Nitric Oxide Synthase I Antibody|
|Presentation||Rabbit Serum. Liquid containing 0.05% sodium azide.|
|Safety Information according to GHS|
|Material Size||50 µL|
References | 29 Available | See All References
|Reference overview||Application||Species||Pub Med ID|
|Prolonged acetylsalicylic-acid-supplementation-induced gastritis affects the chemical coding of the stomach innervating vagal efferent neurons in the porcine dorsal motor vagal nucleus (DMX). |
Gańko, M; Całka, J
Journal of molecular neuroscience : MN 54 188-98 2014
The main goal of our research was to study the possible alterations of the chemical coding of the dorsal motor vagal nucleus (DMX) neurons projecting to the porcine stomach prepyloric region following prolonged acetylsalicylic acid supplementation. Fast Blue (FB) was injected into the studied area of the stomach. Since the seventh day following the FB injection, acetylsalicylic acid (ASA) was given orally to the experimental gilts. All animals were euthanized on the 28th day after FB injection. Medulla oblongata sections were then processed for double-labeling immunofluorescence for choline acetyltransferase (ChAT), pituitary adenylate cyclase-activating peptide (PACAP), vasoactive intestinal polypeptide (VIP), nitric oxide synthase (NOS), galanin (GAL), substance P (SP), leu enkephalin (LENK), and cocaine- and amphetamine-regulated transcript (CART). In the control DMX, only PACAP was observed in 30.08 ± 1.97 % of the FB-positive neurons, while VIP, NOS, GAL, SP, LENK, and CART were found exclusively in neuronal processes running between FB-labeled perikarya. In the ASA DMX, PACAP was revealed in 49.53 ± 5.73 % of traced vagal perikarya. Moreover, we found de novo expression of VIP in 40.32 ± 7.84 %, NOS in 25.02 ± 6.08 %, and GAL in 3.37 ± 0.85 % of the FB-labeled neurons. Our results suggest that neuronal PACAP, VIP, NOS, and GAL are mediators of neural response to aspirin-induced stomach inflammatory state.
|Characterization of neuronal populations in the human trigeminal ganglion and their association with latent herpes simplex virus-1 infection. |
Flowerdew, SE; Wick, D; Himmelein, S; Horn, AK; Sinicina, I; Strupp, M; Brandt, T; Theil, D; Hüfner, K
PloS one 8 e83603 2013
Following primary infection Herpes simplex virus-1 (HSV-1) establishes lifelong latency in the neurons of human sensory ganglia. Upon reactivation HSV-1 can cause neurological diseases such as facial palsy, vestibular neuritis or encephalitis. Certain populations of sensory neurons have been shown to be more susceptible to latent infection in the animal model, but this has not been addressed in human tissue. In the present study, trigeminal ganglion (TG) neurons expressing six neuronal marker proteins were characterized, based on staining with antibodies against the GDNF family ligand receptor Ret, the high-affinity nerve growth factor receptor TrkA, neuronal nitric oxide synthase (nNOS), the antibody RT97 against 200 kDa neurofilament, calcitonin gene-related peptide and peripherin. The frequencies of marker-positive neurons and their average neuronal sizes were assessed, with TrkA-positive (61.82%) neurons being the most abundant, and Ret-positive (26.93%) the least prevalent. Neurons positive with the antibody RT97 (1253 µm(2)) were the largest, and those stained against peripherin (884 µm(2)) were the smallest. Dual immunofluorescence revealed at least a 4.5% overlap for every tested marker combination, with overlap for the combinations TrkA/Ret, TrkA/RT97 and Ret/nNOS lower, and the overlap between Ret/CGRP being higher than would be expected by chance. With respect to latent HSV-1 infection, latency associated transcripts (LAT) were detected using in situ hybridization (ISH) in neurons expressing each of the marker proteins. In contrast to the mouse model, co-localization with neuronal markers Ret or CGRP mirrored the magnitude of these neuron populations, whereas for the other four neuronal markers fewer marker-positive cells were also LAT-ISH+. Ret and CGRP are both known to label neurons related to pain signaling.
|Morphological, immunocytochemical, and functional characterization of esophageal enteric neurons in primary culture. |
Dong, H; Jiang, Y; Srinivasan, S; Mittal, RK
American journal of physiology. Gastrointestinal and liver physiology 305 G129-38 2013
The enteric nervous system of the esophagus plays an important role in its sensory and motor functions. Although the esophagus contains enteric neurons, they have never been isolated and characterized in primary culture. We isolated and cultured enteric neurons of the rat esophagus and determined their morphological appearance, chemical coding for neurotransmitters, and functional characteristics. After primary culture for 2 wk, dendrites and axons appeared in the enteric neurons, which usually have one axon and several dendrites. Although the size of neuronal bodies varied from Dogiel type I to type II, their average size was 39 ± 1.8 μm in length and 23 ± 1.4 μm in width. Immmunocytochemical studies revealed that over 95% of these cells were positively stained for two general neuronal markers, PGP 9.5 or Milli-Mark Fluoro. Chemical coding showed that the neurons were positively stained for choline acetyltransferease (53 ± 6%) or nNOS (66 ± 13%). In functional studies, membrane depolarization and stimulation of several G protein-coupled receptors (GPCRs) induced Ca²⁺ signaling in the esophageal enteric neurons. The GPCR stimulation was found to induce both intracellular Ca²⁺ release and extracellular Ca²⁺ entry. The functional expressions of Ca²⁺ channels (voltage-gated Ca²⁺ channels and store-operated channels) and Ca²⁺ pump (sarcoplasmic reticulum Ca²⁺-ATPase) were also demonstrated on these neurons. We have grown, for the first time, esophageal enteric neurons in primary culture, and these contain excitatory and inhibitory neurotransmitters. The functional integrity of GPCRs, Ca²⁺ channels, and Ca²⁺ pump in these neurons makes them a useful cell model for further studies.
|Mice overexpressing wild-type human alpha-synuclein display alterations in colonic myenteric ganglia and defecation. |
Wang, L; Magen, I; Yuan, PQ; Subramaniam, SR; Richter, F; Chesselet, MF; Taché, Y
Neurogastroenterology and motility : the official journal of the European Gastrointestinal Motility Society 24 e425-36 2012
Prevalent non-motor symptoms of Parkinson's disease (PD) include gastrointestinal motor impairments and advanced stage PD displays pathological aggregates of α-synuclein in colonic enteric neurons. We previously showed that 12 months old mice overexpressing human wild type (WT) α-synuclein under the Thy1 promoter (Thy1-aSyn) displayed colonic motor dysfunction. We investigated functional gut alterations at earlier ages and histological correlates.Defecation, gastric emptying (GE), and immunostaining for α-synuclein, peripheral choline acetyltransferase (pChAT), tyrosine hydroxylase (TH), neuronal nitric oxide synthase (nNOS), and vasoactive intestinal peptide (VIP) in distal colon myenteric plexuses were assessed in male Thy1-aSyn compared to littermate WT mice.Thy1-aSyn mice aged 2.5-3 or 7-8 months old had 81% and 55% reduction in fecal pellet output, respectively, in the first 15 min of exposure to a novel environment. The reduction remained significant in the older group for 2-h, and subsequent refeeding resulted also in a 60% and 69% reduction of defecation in the first hour, respectively. Thy1-aSyn mice (8-10 months) displayed increased α-synuclein in the myenteric plexuses with abundant varicose terminals surrounding pChAT-immunoreactive (ir) neurons, and only a few, nNOS-ir neurons. There were no conspicuous changes in pChAT- and nNOS-ir neurons, or TH- and VIP-ir nerve fibers. Thy1-aSyn mice aged 4-18 months had normal GE.The occurrence of over-production of pre-synaptic α-synuclein in colonic myenteric ganglia several months before the loss of striatal dopamine may provide an anatomical basis for interference with cholinergic neuronal activation, causing an early impairment in defecation to stimuli.
|Reticular groove of the domestic ruminants: histochemical and immunocytochemical study. |
G Scala,L Maruccio
Anatomia, histologia, embryologia 41 2012
The reticular groove mucosa of adult cattle, buffalo and sheep was investigated by histochemical and immunocytochemical techniques. Intense NADPH-d staining was observed in the folds of the epithelium mucosa and at the bottom of the reticular groove in all domestic ruminants studied. The NADPH-d staining showed that the innervations of the tunica muscularis of the reticular groove lip were composed of nerve corpuscles, nerve fibres and nerve cells of the mucosa epithelium. SEM analysis showed an intense nitric oxide synthase (NOS) I immunoreactivity in deep and medium cellular layers. It is interesting to note that the same morphologies were observed in samples of the mucosa epithelium, and of the tunica muscularis processed by NADPH-d and in those processed by immunogold techniques. This study has demonstrated that nitric oxide (NO) is involved in the rumination activity and that it plays a double role in this activity in the reticular groove of all domestic ruminants studied: (1) NO plays a role similar to the one it has in the mucosa epithelium of all the other compartments of the ruminant forestomach, (2) The lip sections of the reticular groove has shown abundant innervations that may indirectly coordinate and control the forestomach motility through the direct activation of the nitrergic (nitroxidergic) nerve cells and nerve fibres.
|Role of neural NO synthase (nNOS) uncoupling in the dysfunctional nitrergic vasorelaxation of penile arteries from insulin-resistant obese Zucker rats. |
Sánchez, A; Contreras, C; Martínez, MP; Climent, B; Benedito, S; García-Sacristán, A; Hernández, M; Prieto, D
PloS one 7 e36027 2012
Erectile dysfunction (ED) is considered as an early sign of vascular disease due to its high prevalence in patients with cardiovascular risk factors. Endothelial and neural dysfunction involving nitric oxide (NO) are usually implicated in the pathophysiology of the diabetic ED, but the underlying mechanisms are unclear. The present study assessed the role of oxidative stress in the dysfunctional neural vasodilator responses of penile arteries in the obese Zucker rat (OZR), an experimental model of metabolic syndrome/prediabetes.Electrical field stimulation (EFS) under non-adrenergic non-cholinergic (NANC) conditions evoked relaxations that were significantly reduced in penile arteries of OZR compared with those of lean Zucker rats (LZR). Blockade of NO synthase (NOS) inhibited neural relaxations in both LZR and OZR, while saturating concentrations of the NOS substrate L-arginine reversed the inhibition and restored relaxations in OZR to levels in arteries from LZR. nNOS expression was unchanged in arteries from OZR compared to LZR and nNOS selective inhibition decreased the EFS relaxations in LZR but not in OZR, while endothelium removal did not alter these responses in either strain. Superoxide anion production and nitro-tyrosine immunostaining were elevated in the erectile tissue from OZR. Treatment with the NADPH oxidase inhibitor apocynin or acute incubation with the NOS cofactor tetrahydrobiopterin (BH4) restored neural relaxations in OZR to levels in control arteries, while inhibition of the enzyme of BH4 synthesis GTP-cyclohydrolase (GCH) reduced neural relaxations in arteries from LZR but not OZR. The NO donor SNAP induced decreases in intracellular calcium that were impaired in arteries from OZR compared to controls.The present study demonstrates nitrergic dysfunction and impaired neural NO signalling due to oxidative stress and nNOS uncoupling in penile arteries under conditions of insulin resistance. This dysfunction likely contributes to the metabolic syndrome-associated ED, along with the endothelial dysfunction also involving altered NO signalling.
|Effects of nitric oxide synthase-3 overexpression on post-translational modifications and cell survival in HepG2 cells. |
P Aguilar-Melero,G Ferrín,J Muntané
Journal of proteomics 75 2012
Hepatocarcinoma is the fifth most common neoplasm and the third cause of cancer-related death. The development of genetic- and/or molecular-based therapies is urgently required. The administration of high doses of nitric oxide (NO) promotes cell death in hepatocytes. NO contributes to cell signaling by inducing oxidative/nitrosative-dependent post-translational modifications. The aim of the present study was to investigate protein modifications and its relation with alteration of cell proliferation and death in hepatoma cells. Increased intracellular NO production was achieved by stable nitric oxide synthase-3 (NOS-3) overexpression in HepG2 cells. We assessed the pattern of nitration, nitrosylation and carbonylation of proteins by proteomic analysis. The results showed that NOS-3 cell overexpression increased oxidative stress, which affected proteins mainly involved in cell protein folding. Carbonylation also altered metabolism, as well as immune and antioxidant responses. The interaction of nitrosative and oxidative stress generated tyrosine nitration, which affected the tumor marker Serpin B3, ATP synthesis and cytoskeleton. All these effects were associated with a decrease in chaperone activity, a reduction in cell proliferation and an increased cell death. Our study showed that alteration of nitration, nitrosylation and carbonylation pattern of proteins by NO-dependent oxidative/nitrosative stress was related to a reduction of cell survival in a hepatoma cell line.
|Immunohistochemical characterization of superior cervical ganglion neurons supplying porcine parotid salivary gland. |
Joanna Wojtkiewicz,Judyta K Juranek,Ireneusz Kowalski,Marek Bladowski,Jaros?aw Ca?ka,Mariusz Majewski
Neuroscience letters 500 2011
The main goal of our study was to investigate the chemical coding of the superior cervical ganglion (SCG) sympathetic neurons supplying the porcine parotid gland. Additionally, the chemical nature of the vicinal nerve fibers surrounding the parotid SCG perikarya was investigated. Fast blue (FB) retrograde tracing of the parotid gland and immunofluorescent labelling of SCG neurons were studied in juvenile female pigs. Microscopic analysis revealed that only ipsilateral SCG neurons were retrogradely labelled. The labelled neurons formed a discrete cluster in the middle and caudal region of the ganglion. Immunofluorescent labelling revealed that virtually all of the FB-positive parotid gland neurons were immunoreactive to tyrosine hydroxylase (TH), confirming their sympathetic nature. In addition to TH, the majority of the FB-positive neurons were found to be immunoreactive to calbindin (CB) and to a lesser extent for neuropeptide Y (NPY), leu-enkephalin (LENK) and galanin (GAL). In the close proximity of the FB-traced perikarya, a large number of immunoreactive (IR) vasoactive intestinal peptide (VIP-IR), pituitary adenylate cyclase-activating polypeptide (PACAP-IR), nitric oxide synthase (NOS-IR) processes were identified. Moreover, calcitonin gene related peptide-immunoreactive (CGRP-IR), substance P-immunoreactive (SP-IR), vesicular acetylcholine transporter (VAChT-IR), calretinin (CRT-IR), GAL-IR, LENK-IR and CB-IR protrusions were observed. The results of the present study provide a detailed characteristic of the location and neurochemical coding of sympathetic SCG neurons innervating the parotid salivary gland of the pig and lay ground for more advanced, clinical studies on salivary gland innervations.
|Neurochemical phenotypes of myenteric neurons in the rhesus monkey. |
Ali Reza Noorian,Georgia M Taylor,Dana M Annerino,James G Greene
The Journal of comparative neurology 519 2011
Understanding the neurochemical composition of the enteric nervous system (ENS) is critical for elucidating neurological function in the gastrointestinal (GI) tract in health and disease. Despite their status as the closest models of human neurological systems, relatively little is known about enteric neurochemistry in nonhuman primates. We describe neurochemical coding of the enteric nervous system, specifically the myenteric plexus, of the rhesus monkey (Macaca mulatta) by immunohistochemistry and directly compare it to human tissues. There are considerable differences in the myenteric plexus along different segments of the monkey GI tract. While acetylcholine neurons make up the majority of myenteric neurons in the stomach (70%), they are a minority in the rectum (47%). Conversely, only 22% of gastric myenteric neurons express nitric oxide synthase (NOS) compared to 52% in the rectum. Vasoactive intestinal peptide (VIP) is more prominent in the stomach (37%) versus the rest of the GI tract (≈10%), and catecholamine neurons are rare (≈1%). There is significant coexpression of NOS and VIP in myenteric neurons that is more prominent in the proximal GI tract. Taken as a whole, these data provide insight into the neurochemical anatomy underlying GI motility. While overall similarity to other mammalian species is clear, there are some notable differences between the ENS of rhesus monkeys, humans, and other species that will be important to take into account when evaluating models of human diseases in animals.
|Tetrodotoxin- and resiniferatoxin-induced changes in paracervical ganglion ChAT- and nNOS-IR neurons supplying the urinary bladder in female pigs. |
Piotr Józef Burli?ski,S?awomir Gonkowski,Jaros?aw Ca?ka
Acta veterinaria Hungarica 59 2011
The aim of the present study was to establish the effect of intravesical administration of resiniferatoxin (RTX) and tetrodotoxin (TTX) on the chemical coding of paracervical ganglion (PCG) neurons supplying the urinary bladder in the pig. In order to identify the PCG neurons innervating the bladder, retrograde tracer Fast Blue was injected into the bladder wall prior to intravesical RTX or TTX administration. Consequent application of immunocytochemical methods revealed that in the control group 76.82% of Fast Blue positive PCG neurons contain nitric oxide synthetase (nNOS), and 66.92% contain acetylcholine transferase (ChAT). Intravesical infusion of RTX resulted in a reduction of the nNOS-IR neurons to 57.74% and ChAT-IR to 57.05%. Alternative administration of TTX induced an increase of nNOS-IR neurons up to 79.29% and a reduction of the ChAT-IR population down to 3.73% of the Fast Blue positive PCG cells. Our data show that both neurotoxins affect the chemical coding of PCG cells supplying the porcine urinary bladder, but the effects of their action are different. Moreover, these results shed light on the possible involvement of NO-ergic and cholinergic neurons in the mechanisms of therapeutic action exerted by RTX and TTX in curing the overactive bladder disorder.
|A blueprint for the spatiotemporal origins of mouse hippocampal interneuron diversity. |
Tricoire, L; Pelkey, KA; Erkkila, BE; Jeffries, BW; Yuan, X; McBain, CJ
The Journal of neuroscience : the official journal of the Society for Neuroscience 31 10948-70 2011
Although vastly outnumbered, inhibitory interneurons critically pace and synchronize excitatory principal cell populations to coordinate cortical information processing. Precision in this control relies upon a remarkable diversity of interneurons primarily determined during embryogenesis by genetic restriction of neuronal potential at the progenitor stage. Like their neocortical counterparts, hippocampal interneurons arise from medial and caudal ganglionic eminence (MGE and CGE) precursors. However, while studies of the early specification of neocortical interneurons are rapidly advancing, similar lineage analyses of hippocampal interneurons have lagged. A "hippocampocentric" investigation is necessary as several hippocampal interneuron subtypes remain poorly represented in the neocortical literature. Thus, we investigated the spatiotemporal origins of hippocampal interneurons using transgenic mice that specifically report MGE- and CGE-derived interneurons either constitutively or inducibly. We found that hippocampal interneurons are produced in two neurogenic waves between E9-E12 and E12-E16 from MGE and CGE, respectively, and invade the hippocampus by E14. In the mature hippocampus, CGE-derived interneurons primarily localize to superficial layers in strata lacunosum moleculare and deep radiatum, while MGE-derived interneurons readily populate all layers with preference for strata pyramidale and oriens. Combined molecular, anatomical, and electrophysiological interrogation of MGE/CGE-derived interneurons revealed that MGE produces parvalbumin-, somatostatin-, and nitric oxide synthase-expressing interneurons including fast-spiking basket, bistratified, axo-axonic, oriens-lacunosum moleculare, neurogliaform, and ivy cells. In contrast, CGE-derived interneurons contain cholecystokinin, calretinin, vasoactive intestinal peptide, and reelin including non-fast-spiking basket, Schaffer collateral-associated, mossy fiber-associated, trilaminar, and additional neurogliaform cells. Our findings provide a basic blueprint of the developmental origins of hippocampal interneuron diversity.
|Neurotransmitters in airway parasympathetic neurons altered by neurotrophin-3 and repeated allergen challenge. |
Pan, J; Rhode, HK; Undem, BJ; Myers, AC
American journal of respiratory cell and molecular biology 43 452-7 2010
Changes in airway nerves associated with chronic inflammation may underlie the pathogenesis and symptoms of lower airway diseases, such as asthma. The molecules most likely causing such alterations are neurotrophins (NTs) and/or related neurokines. In several species, including humans, lower airway parasympathetic postganglionic neurons that project axons to airway smooth muscle are either cholinergic or nonadrenergic noncholinergic (NANC), the latter synthesizing vasoactive intestinal peptide and nitric oxide, but not acetylcholine. In guinea pig trachealis smooth muscle, cholinergic nerve terminals arise from ganglionic neurons located near the tracheal smooth muscle, whereas the source of NANC nerve fibers is from neurons in ganglia located in the adjacent myenteric plexus of the esophagus, making this an ideal species to study regulation of parasympathetic neurotransmitter phenotypes. In the present study, we determined that, 48 hours after repeated allergen challenge, the NANC phenotype of airway parasympathetic ganglionic neurons changed to a cholinergic phenotype, and NT-3 mimicked this change. Nerve growth factor, brain-derived neurotrophic factor, leukemia inhibitory factor, or IL-1β had no effect on either phenotype, and they did not induce these neurons to synthesize substance P or tyrosine hydroxylase. These results indicate a role for inflammation and NT-3 in regulating biochemical and anatomical characteristics of principal neurons in adult airway parasympathetic ganglia.
|Age-related changes in nitric oxide synthase in the lateral geniculate nucleus of rats. |
Seung-Jun Hwang,Youngbuhm Huh
Journal of molecular histology 41 2010
Age-related changes in nitric oxide production in the visual system have not been well characterized. Therefore, we used staining and image-processing approaches to describe changes in levels of neuronal nitric oxide synthase (nNOS), the NADPH-diaphorase (NADPH-d) histochemical marker, and 3-nitrotyrosine in the lateral geniculate nucleus (LGN) of young and aged rats. The LGN plays an important role in the visual system, as it acts as a visual relay nucleus. Quantitative analysis of NADPH-d-positive and nNOS-immunoreactive neurons revealed significant optical density increases in the dorsal LGN and ventral LGN of aged rats; however, no significant changes were observed in the number of neurons with age. 3-Nitrotyrosine immunoreactivity was increased in the dorsal LGN and ventral LGN of aged rats. These results indicate that increased nitric oxide production and peroxynitrite may be associated with alterations in visual function during aging.
|BMP2 promotes differentiation of nitrergic and catecholaminergic enteric neurons through a Smad1-dependent pathway. |
Anitha, Mallappa, et al.
Am. J. Physiol. Gastrointest. Liver Physiol., 298: G375-83 (2010) 2010
The bone morphogenetic protein (BMP) family is a class of transforming growth factor (TGF-beta) superfamily molecules that have been implicated in neuronal differentiation. We studied the effects of BMP2 and glial cell line-derived neurotrophic factor (GDNF) on inducing differentiation of enteric neurons and the signal transduction pathways involved. Studies were performed using a novel murine fetal enteric neuronal cell line (IM-FEN) and primary enteric neurons. Enteric neurons were cultured in the presence of vehicle, GDNF (100 ng/ml), BMP2 (10 ng/ml), or both (GDNF + BMP2), and differentiation was assessed by neurite length, markers of neuronal differentiation (neurofilament medium polypeptide and beta-III-tubulin), and neurotransmitter expression [neuropeptide Y (NPY), neuronal nitric oxide synthase (nNOS), tyrosine hydroxylase (TH), choline acetyltransferase (ChAT) and Substance P]. BMP2 increased the differentiation of enteric neurons compared with vehicle and GDNF-treated neurons (P < 0.001). BMP2 increased the expression of the mature neuronal markers (P < 0.05). BMP2 promoted differentiation of NPY-, nNOS-, and TH-expressing neurons (P < 0.001) but had no effect on the expression of cholinergic neurons (ChAT, Substance P). Neurons cultured in the presence of BMP2 have higher numbers of TH-expressing neurons after exposure to 1-methyl 4-phenylpyridinium (MPP(+)) compared with those cultured with MPP(+) alone (P < 0.01). The Smad signal transduction pathway has been implicated in TGF-beta signaling. BMP2 induced phosphorylation of Smad1, and the effects of BMP2 on differentiation of enteric neurons were significantly reduced in the presence of Smad1 siRNA, implicating the role of Smad1 in BMP2-induced differentiation. The effects of BMP2 on catecholaminergic neurons may have therapeutic implications in gastrointestinal motility disturbances.
|Rosuvastatin preconditioning provides neuroprotection against spinal cord ischemia in rats through modulating nitric oxide synthase expressions. |
Die J, Wang K, Fan L, Jiang Y, Shi Z
Brain Res 1346 251-61. Epub 2010 May 31. 2010
The study was aimed to investigate the protective effects of rosuvastatin on spinal cord ischemia in rats and to determine the effects of this agent on the expressions of nitric oxide synthase (NOS). Spinal cord ischemia was induced in male Sprague-Dawley rats by occluding the descending thoracic aorta. Experimental groups (n=30 per group) were as follows: sham operation, control (receiving only normal saline), rosuvastatin (5 mg/kg/day for 10 days before occlusion), and rosuvastatin-mevalonate (5 mg/kg/day rosuvastatin and 5 mg/kg/day mevalonate for 10 days before occlusion). Neurological function was assessed at 6, 12, 24, 48, and 72 h after reperfusion. After 72 h reperfusion, spinal cords were harvested for 2,3,5,-triphenyltetrazolium chloride (TTC) staining, TUNEL staining, and nitric oxide (NO) assay. Immunohistochemistry, reverse transcription polymerase chain reaction and western blot were performed to determine the expressions of inducible, endothelial, and neuronal NOS (iNOS, eNOS, and nNOS) in rats with spinal cord ischemia. Spinal cord ischemia thus induced was marked by neurological dysfunction, spinal infarction, and neural cell apoptosis in animals. The results show that rosuvastatin significantly reduced the motor disturbance and the volume of infarctions and attenuated apoptotic neural cells death in the treated rats. Treatment with rosuvastatin remarkably decreased the NO level in spinal cord tissue. In addition, rosuvastatin inhibited iNOS mRNA and protein expression and increased eNOS mRNA and protein expression. However, rosuvastatin had no influence on nNOS mRNA and protein expression. Administrations of mevalonate completely reversed the changes caused by rosuvastatin. These results indicate that rosuvastatin can protect rat spinal cord against ischemia injury by modulation of NOS expressions. Copyright 2010 Elsevier B.V. All rights reserved.
|Peripheral purinergic receptor modulation influences the trigeminal ganglia nitroxidergic system in an experimental murine model of inflammatory orofacial pain. |
Borsani, Elisa, et al.
J. Neurosci. Res., 88: 2715-26 (2010) 2010
ATP plays an important role as an endogenous pain mediator generating and/or modulating pain signaling from the periphery to the central nervous system. The aim of this study was to analyze the role of peripheral purinergic receptors in modulation of the nitroxidergic system at a trigeminal ganglia level by monitoring changes in nitric oxide synthase isoforms. We also evaluated Fos-positive neurons in brainstem (spinal trigeminal nucleus) and pain-related behavior. We found that local administration of the P2 purinergic receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) decreased face-rubbing activity, nitric oxide synthase isoform expression in trigeminal ganglia, and Fos expression in spinal trigeminal nucleus after subcutaneous injection of formalin. These results suggest a role for peripheral P2 purinergic receptors in orofacial pain transmission through modulation of the nitroxidergic system. .
|Phenotypic diversity and expression of GABAergic inhibitory interneurons during postnatal development in lumbar spinal cord of glutamic acid decarboxylase 67-green fluorescent protein mice. |
Dougherty, KJ; Sawchuk, MA; Hochman, S
Neuroscience 163 909-19 2009
The synthesis enzyme glutamic acid decarboxylase (GAD65 or GAD67) identifies neurons as GABAergic. Recent studies have characterized the physiological properties of spinal cord GABAergic interneurons using lines of GAD67-green fluorescent protein (GFP) transgenic mice. A more complete characterization of their phenotype is required to better understand the role of this population of inhibitory neurons in spinal cord function. Here, we characterize the distribution of lumbar spinal cord GAD67-GFP neurons at postnatal days (P) 0, 7, and 14, and adult based on their co-expression with GABA and determine the molecular phenotype of GAD67-GFP neurons at P14 based on the expression of various neuropeptides, calcium binding proteins, and other markers. At all ages greater than 67% of GFP(+) neurons were also GABA(+). With increasing age; (i) GFP(+) and GABA(+) cell numbers declined, (ii) ventral horn GFP(+) and GABA(+) neurons vanished, and (iii) somatic labeling was reduced while terminal labeling increased. At P14, vasoactive intestinal peptide and bombesin were expressed in approximately 63% and approximately 35% of GFP(+) cells, respectively. Somatostatin was found in a small number of neurons, whereas calcitonin gene-related peptide never co-localized with GFP. Moderate co-expression was found for all the Ca(2+) binding proteins examined. Notably, most laminae I-II parvalbumin(+) neurons were also GFP(+). Neurogranin, a protein kinase C substrate, was found in approximately 1/2 of GFP(+) cells. Lastly, while only 7% of GFP(+) cells contain nitric oxide synthase (NOS), these cells represent a large fraction of all NOS(+) cells. We conclude that GAD67-GFP neurons represent the majority of spinal GABAergic neurons and that mouse dorsal horn GAD67-GFP(+) neurons comprise a phenotypically diverse population.Full Text Article
|Oestrogen and progestins differently prevent glutamate toxicity in cortical neurons depending on prior hormonal exposure via the induction of neural nitric oxide synthase. |
Paolo Mannella, Angel Matias Sanchez, Maria Silvia Giretti, Andrea Riccardo Genazzani, Tommaso Simoncini
Steroids 74 650-6 2009
Sex steroids are important for brain function and protection. However, growing evidence suggests that these actions might depend on the timing of exposure to steroids. We have studied the effects of steroid administration on the survival of neural cells and we have partially characterized the possible mechanisms. The effect of a 24h pre-treatment with 17beta-estradiol or 17beta-estradiol plus progesterone or medroxyprogesterone acetate on the toxic action of l-glutamate was used to test the experimental hypothesis. Pre-exposure to either steroid combinations turned in enhanced cell survival. Instead, addition of sex steroids together with l-glutamate, in the absence of a pre-exposure had no protective effect. Pre-treatment with the steroid combinations resulted in increased neural NOS expression and activity and blockade of NOS abolished the cytoprotective effects of steroids. These results suggest that NOS induction might be involved in sex steroid-induced neuroprotection. Furthermore, these data supports the hypothesis that prolonged and continued exposure to oestrogen and progesterone, leading to changes in gene expression, is necessary to obtain neuroprotection induced by sex steroids.
|Distribution of NADPH-diaphorase and nitric oxide synthase reactivity in the central nervous system of the goldfish (Carassius auratus). |
Rosa M Giraldez-Perez, Susana P Gaytan, Diego Ruano, Blas Torres, Rosario Pasaro
Journal of chemical neuroanatomy 35 12-32 2008
The nitrergic system has been inferred from cells positive to nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry and/or to the neuronal isoform of nitric oxide synthase (nNOS) immunohistochemistry in different species of vertebrates. The aim of the present work was to systematically study the distribution of cell producing nitric oxide in the goldfish (Carassius auratus) brain. To reach this goal, we firstly studied co-localization for NADPHd and nNOS techniques and demonstrated an extensive double labeling. Then, we studied the distribution through the brain by the two separate methods and found labeled cells widely distributed in brain and spinal cord. In the telencephalon, such cells were in both dorsal and ventral areas. In the diencephalon, the cells were found in some nuclei of the preoptic area and hypothalamus, habenula, pretectum, and dorsal and ventral thalamic regions. In the midbrain, cells were observed in the optic tectum, torus longitudinalis, and tegmental nuclei. In the rhombencephalon, cells were found in the cerebellum, the reticular formation, the locus coeruleus, the raphe nuclei, and the nuclei of the cranial nerves. Labeled cells were also observed in the gray area of the spinal cord. Cognizing that a direct comparison of the present results with those reported in other vertebrates is not clear-cut because of homologies; we conclude that the nitrergic system is roughly similar from fish to mammals.
|Intrinsic differences among spatially distinct neural crest stem cells in terms of migratory properties, fate determination, and ability to colonize the enteric nervous system. |
Jack T Mosher, Kelly J Yeager, Genevieve M Kruger, Nancy M Joseph, Mark E Hutchin, Andrzej A Dlugosz, Sean J Morrison, Jack T Mosher, Kelly J Yeager, Genevieve M Kruger, Nancy M Joseph, Mark E Hutchin, Andrzej A Dlugosz, Sean J Morrison
Developmental biology 303 1-15 2007
We have systematically examined the developmental potential of neural crest stem cells from the enteric nervous system (gut NCSCs) in vivo to evaluate their potential use in cellular therapy for Hirschsprung disease and to assess differences in the properties of postmigratory NCSCs from different regions of the developing peripheral nervous system (PNS). When transplanted into developing chicks, flow-cytometrically purified gut NCSCs and sciatic nerve NCSCs exhibited intrinsic differences in migratory potential and neurogenic capacity throughout the developing PNS. Most strikingly, gut NCSCs migrated into the developing gut and formed enteric neurons, while sciatic nerve NCSCs failed to migrate into the gut or to make enteric neurons, even when transplanted into the gut wall. Enteric potential is therefore not a general property of NCSCs. Gut NCSCs also formed cholinergic neurons in parasympathetic ganglia, but rarely formed noradrenergic sympathetic neurons or sensory neurons. Supporting the potential for autologous transplants in Hirschsprung disease, we observed that Endothelin receptor B (Ednrb)-deficient gut NCSCs engrafted and formed neurons as efficiently in the Ednrb-deficient hindgut as did wild-type NCSCs. These results demonstrate intrinsic differences in the migratory properties and developmental potentials of regionally distinct NCSCs, indicating that it is critical to match the physiological properties of neural stem cells to the goals of proposed cell therapies.Full Text Article
|The expression of nNOS, iNOS and nitrotyrosine is increased in the rat cerebral cortex in experimental hepatic encephalopathy. |
Suárez, I, et al.
Neuropathol. Appl. Neurobiol., 32: 594-604 (2006) 2006
|An association of multiple endocrine neoplasia 2B, a RET mutation; constipation; and low substance P-nerve fiber density in colonic circular muscle. |
Sebastian K King, Bridget R Southwell, John M Hutson
Journal of pediatric surgery 41 437-42 2006
BACKGROUND: Multiple endocrine neoplasia (MEN) 2B is a rare hereditary syndrome that results from an activating mutation of the RET proto-oncogene. The RET gene is involved in the development of the enteric nervous system. Patients with MEN 2B have enlarged enteric ganglia and may be affected by gastrointestinal dysmotility. A deficiency of the neurotransmitter substance P (SP) has been identified in both pediatric and adult patients with chronic constipation. METHODS: Three patients, in whom constipation was the presenting symptom and MEN 2B had been provisionally diagnosed, underwent genetic analysis. Seromuscular colonic biopsies were taken for immunofluorescence imaging in all 3 patients. A retrospective review of the patient notes was undertaken. RESULTS: All 3 patients had constipation refractory to conservative treatment. Genetic analyses in the 3 patients confirmed an identical RET mutation (Met918Thr). Immunofluorescence imaging in all 3 patients identified grossly enlarged myenteric plexus ganglia but surprisingly a low density of SP-labeled nerve fibers in the colonic circular muscle. Nitric oxide synthase and vasoactive intestinal peptide labeling were not reduced. CONCLUSION: The results show an association between MEN 2B and its most common RET mutation, colonic dysmotility, and low density of SP in the colonic circular muscle. Larger numbers of patients need to be studied to investigate whether low SP is primarily associated with the constipation or RET mutation and if it is a common feature of MEN 2B.
|Platelet-activating factor in the enteric nervous system of the guinea pig small intestine. |
Wang, GD; Wang, XY; Hu, HZ; Fang, XC; Liu, S; Gao, N; Xia, Y
American journal of physiology. Gastrointestinal and liver physiology 291 G928-37 2006
Platelet-activating factor (PAF) is a proinflammatory mediator that may influence neuronal activity in the enteric nervous system (ENS). Electrophysiology, immunofluorescence, Western blot analysis, and RT-PCR were used to study the action of PAF and the expression of PAF receptor (PAFR) in the ENS. PAFR immunoreactivity (IR) was expressed by 6.9% of the neurons in the myenteric plexus and 14.5% of the neurons in the submucosal plexus in all segments of the guinea pig intestinal tract as determined by double staining with anti-human neuronal protein antibody. PAFR IR was found in 6.1% of the neurons with IR for calbindin, 35.8% of the neurons with IR for neuropeptide Y (NPY), 30.6% of the neurons with IR for choline acetyltransferase (ChAT), and 1.96% of the neurons with IR for vasoactive intestinal peptide (VIP) in the submucosal plexus. PAFR IR was also found in 1.5% of the neurons with IR for calbindin, 51.1% of the neurons with IR for NPY, and 32.9% of the neurons with IR for ChAT in the myenteric plexus. In the submucosal plexus, exposure to PAF (200-600 nM) evoked depolarizing responses (8.2 +/- 3.8 mV) in 12.4% of the neurons with S-type electrophysiological behavior and uniaxonal morphology and in 12.5% of the neurons with AH-type electrophysiological behavior and Dogiel II morphology, whereas in the myenteric preparations, depolarizing responses were elicited by a similar concentration of PAF in 9.5% of the neurons with S-type electrophysiological behavior and uniaxonal morphology and in 12.0% of the neurons with AH-type electrophysiological behavior and Dogiel II morphology. The results suggest that subgroups of secreto- and musculomotor neurons in the submucosal and myenteric plexuses express PAFR. Coexpression of PAFR IR with ChAT IR in the myenteric plexus and ChAT IR and VIP IR in the submucosal plexus suggests that PAF, after release in the inflamed bowel, might act to elevate the excitability of submucosal secretomotor and myenteric musculomotor neurons. Enhanced excitability of motor neurons might lead to a state of neurogenic secretory diarrhea.
|Transcriptional dysregulation in striatal projection- and interneurons in a mouse model of Huntington's disease: neuronal selectivity and potential neuroprotective role of HAP1. |
Zucker, B; Luthi-Carter, R; Kama, JA; Dunah, AW; Stern, EA; Fox, JH; Standaert, DG; Young, AB; Augood, SJ
Human molecular genetics 14 179-89 2005
Transcriptional dysregulation has been described as a central mechanism in the pathogenesis of Huntington's disease (HD), in which medium spiny projection neurons (MSN) selectively degenerate whereas neuronal nitric-oxide-synthase-positive interneurons (nNOS-IN) survive. In order to begin to understand this differential vulnerability we compared mRNA levels of selected genes involved in N-methyl-D-aspartate (NMDA) glutamate receptor and calcium (Ca2+) signaling pathways in MSN and nNOS-IN from 12-week-old R6/2 mice, a transgenic mouse model of HD and wild-type littermates. We undertook a laser capture microdissection (LCM) study to examine the contribution of transcriptional dysregulation in candidate genes involved in these two signaling pathways in discrete populations of striatal neurons. The use of LCM in combination with quantitative real-time polymerase chain reaction (Q-PCR) allowed us to quantify the neuronal abundance of candidate mRNAs. We found different transcriptional alterations in R6/2 neurons for both MSN and nNOS-IN, indicating that global transcriptional dysregulation alone does not account for selective vulnerability. Further, we observed a striking enrichment of several mRNAs in the nNOS-IN population, including that for the NMDA receptor subunit NR2D, the postsynaptic density protein 95 (PSD-95) and the huntingtin-associated protein 1 (HAP1) as well as nitric-oxide-synthase (nNOS) mRNA itself. The higher expression levels of these molecules in nNOS-IN when compared with MSN together with an association of nNOS, NR2D and HAP1 in a protein complex with PSD-95 suggest that these proteins may be involved in protective pathways that contribute to the resistance of this interneuron population to neurodegeneration in HD.
|Expression of superoxide dismutase in hyperglycemic focal cerebral ischemia in the rat. |
Chantal Bémeur, Line Ste-Marie, Paul Desjardins, Roger F Butterworth, Luc Vachon, Jane Montgomery, Alan S Hazell
Neurochemistry international 45 1167-74 2004
This study investigated the possibility that hyperglycemia induces early expression of various superoxide dismutases (SOD) and nitric oxide synthases (NOS) following focal cerebral ischemia in the rat. MnSOD, CuZnSOD, nNOS and eNOS mRNA and protein expression were examined 3 h after permanent middle cerebral artery occlusion under acute hyperglycemic or normoglycemic conditions. 2,3,5-triphenyltetrazolium chloride (TTC) treatment post-mortem revealed a significant area at risk of infarction following ischemia in hyperglycemic compared to normoglycemic rats. Although no changes in MnSOD, CuZnSOD, nNOS and eNOS mRNA expression were detected, Western blots of ischemic cortex revealed an increase in MnSOD and CuZnSOD protein expression in hyperglycemic compared to normoglycemic rats. Pre-treatment of hyperglycemic rats with the NOS inhibitors L-nitroarginine methyl ester (L-NAME) and 7-nitroindazole (7-NI) or dehydroascorbic acid (DHA), a superoxide scavenger, significantly reduced the TTC delineated zone. The hyperglycemia-induced post-transcriptional upregulation of MnSOD and CuZnSOD levels suggest a response to increased superoxide production which, in the presence of increased nitric oxide production, may play a major role in the increased risk of damage following hyperglycemic stroke.
|In vivo excitation of GABA interneurons in the medial prefrontal cortex through 5-HT3 receptors. |
M Victoria Puig,Noemí Santana,Pau Celada,Guadalupe Mengod,Francesc Artigas
Cerebral cortex (New York, N.Y. : 1991) 14 2004
Serotonin (5-hydroxytryptamine, 5-HT) controls pyramidal cell activity in prefrontal cortex (PFC) through various receptors, in particular, 5-HT1A and 5-HT2A receptors. Here we report that the physiological stimulation of the raphe nuclei excites local, putatively GABAergic neurons in the prelimbic and cingulate areas of the rat PFC in vivo. These excitations had a latency of 36 +/- 4 ms and a duration of 69 +/- 9 ms and were blocked by the i.v. administration of the 5-HT3 receptor antagonists ondansetron and tropisetron. The latency and duration were shorter than those elicited through 5-HT2A receptors in pyramidal neurons of the same areas. Double in situ hybridization histochemistry showed the presence of GABAergic neurons expressing 5-HT3 receptor mRNA in PFC. These cells were more abundant in the cingulate, prelimbic and infralimbic areas, particularly in superficial layers. The percentages of GAD mRNA-positive neurons expressing 5-HT3 receptor mRNA in prelimbic cortex were 40, 18, 6 and 8% in layers I, II-III, V and VI, respectively, a distribution complementary to that of cells expressing 5-HT2A receptors. Overall, these results support an important role of 5-HT in the control of the excitability of apical dendrites of pyramidal neurons in the medial PFC through the activation of 5-HT3 receptors in GABAergic interneurons.
|Estrogen induces nitric oxide production via activation of constitutive nitric oxide synthases in human neuroblastoma cells. |
Xia, Y; Krukoff, TL
Endocrinology 145 4550-7 2004
Although it is becoming increasingly evident that nitric oxide (NO) mediates some of estrogen's actions in the brain, the effects of estrogen on NO production through NO synthases (NOS) in neuronal cells have not yet been identified. Here we assessed changes in NO production induced by 17beta-estradiol (E2) in cells of neuronal origin using human SK-N-SH neuroblastoma cells, which we show express all three isoforms of NOS. Involvement of NOS isoforms in E2-induced NO production was examined using isoform-specific NOS inhibitors. E2 (10(-10)-10(-6) m) induced rapid increases in NO release and changes in endothelial NOS (eNOS) expression, which were blocked by ICI 182,780, an antagonist of estrogen receptors. Increased levels of NO release and NOS activity induced by E2 were blocked by N5-(1-Imino-3-butenyl)-L-ornithine, a neuronal NOS inhibitor, and N(5)-(1-Iminoethyl)-L-ornithine, an eNOS inhibitor, but not by 1400W, an inducible NOS inhibitor. These results demonstrate that E2-stimulated NO production occurs via estrogen receptor-mediated activation of the constitutive NOSs, neuronal NOS and eNOS. The E2-induced NO increase was abolished when extracellular Ca2+ was removed from the medium or after the addition of nifedipine, an L-type channel blocker, and was partially inhibited using 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester, an intracellular Ca2+ chelator. However, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester itself also caused an increase in NO release that was blocked by 1400W, suggesting that inducible NOS mediates this response. Together these data reveal that constitutive NOS activities are responsible for E2-induced NO production in neuroblastoma cells and that differential activation of NOS isoforms in these cells occurs in response to different treatments.
|Satellite cells of dorsal root ganglia are multipotential glial precursors. |
Svenningsen, Asa Fex, et al.
Neuron Glia Biol., 1: 85-93 (2004) 2004
|Neural crest stem cells persist in the adult gut but undergo changes in self-renewal, neuronal subtype potential, and factor responsiveness. |
Kruger, Genevieve M, et al.
Neuron, 35: 657-69 (2002) 2002
We found neural crest stem cells (NCSCs) in the adult gut. Postnatal gut NCSCs were isolated by flow-cytometry and compared to fetal gut NCSCs. They self-renewed extensively in culture but less than fetal gut NCSCs. Postnatal gut NCSCs made neurons that expressed a variety of neurotransmitters but lost the ability to make certain subtypes of neurons that are generated during fetal development. Postnatal gut NCSCs also differed in their responsiveness to lineage determination factors, affecting cell fate determination in vivo and possibly explaining their reduced neuronal subtype potential. These perinatal changes in gut NCSCs parallel perinatal changes in hematopoietic stem cells, suggesting that stem cells in different tissues undergo similar developmental transitions. The persistence of NCSCs in the adult PNS opens up new possibilities for regeneration after injury or disease.
|Pathways and Biomarkers of Oxidative Stress (EMD)|
|White Paper- Modern Methods in Oxidative Stress Research (EMD)|
|Anti-Nitric Oxide Synthase I - Data Sheet|