Identification and Pathogen Stimulation Patterns of Neuronal Nitric Oxide Synthase(nNOS)in Black Rockfish(Sebastes schlegelii)

Neuronal nitric oxide synthase(nNOS)was the producer of nitric oxide(NO)which played important gas messenger molecules in biological process.It also can take effect as immune regulation molecule in organism.Black rockfish(Sebastes schlegelii)is an important economic fish which were widely farmed in East Asia countries.Meanwhile,the pathogenic bacteria such as the Edwardsiella tarda and Vibrio anguillarum in seawater always brought serious obstacles to their healthy growth.In order to explore the expression pattern of n NOS gene under the pathogen stimulation and predict its immune function,the n NOS gene in black rockfish named Ssn NOS was identified.It was 3780 bp in length,located on chromosome 6,and contained 27 coding domain sequence(CDs).According to the phylogenetic analysis,the Ssn NOS showed closest relative to the counterpart gene of swamp eel(Monopterus albus).Meanwhile,analysis of Ssn NOS expression in various healthy tissues showed that Ssn NOS expression level was highest in healthy brain tissues,followed by intestinal tissues.In addition,Ssn NOS showed significant expression changes in response to stimulation by two pathogens.Particular in gill,the expression of Ssn NOS after pathogenic stimulation increased significantly.The Elisa analysis showed the Ssn NOS content in gills was much higher than that in other tissues at all time points.Moreover,the expression patterns of Ssn NOS in brain,intestine and kidney after stimulation by pathogens showed a distinct expression pattern which first down-regulated and then up-regulated.Therefore,the Ssn NOS may be an important signaling molecule for fish to respond rapidly in immune stimulation.

Role of brain-derived neurotrophic factor and neuronal nitric oxide synthase in stress-induced depression

BACKGROUND： Accumulated evidence indicates an important role for hippocampal dendrite atrophy in development of depression, while brain-derived neurotrophic factor （BDNF） participates in hippocampal dendrite growth. OBJECTIVE： To discuss the role of BDNF and neuronal nitric oxide synthase （nNOS） in chronic and unpredictable stress-induced depression and the pathogenesis of depression. DESIGN, TIME AND SETTING： Randomized, controlled animal experiment. The experiment was carded out from October 2006 to May 2007 at the Department of Animal Physiology, College of Life Science, Shaanxi Normal University. MATERIALS： Thirty-seven male Sprague-Dawley rats weighing 250-300 g at the beginning of the experiment were obtained from Shaanxi Provincial Institute of Traditional Chinese Medicine （Xi＇an, China）. BDNF antibody and nNOS antibody were provided by Santa Cruz （USA）. K252a （BDNF inhibitor） and 7-NI （nNOS inhibitor） were provided by Sigma （USA）. METHODS： Animals were randomly divided into five groups： Control group, chronic unpredicted mild stress （CUMS） group, K252a group, K252a＋7-NI group and 7-NI＋CUMS group. While the Control, K252a and K252a＋7-NI groups of rats not subjected to stress had free access to food and water, other groups of rats were subjected to nine stressors randomly applied for 21 days, with each stressor applied 2-3 times. On days 1, 7, 14 and 21 during CUMS, rats received microinjection of 1 μL of physiological saline in the Control and CUMS groups, 1 ~ L of K252a in the K252a group, 1 μL of K252a and 7-NI in the K252a＋7-NI group, and 1 μL of 7-NI in the 7-NI＋CUMS group. We observed a variety of alterations in sucrose preference, body weight change, open field test and forced swimming test, and observed the expression of BDNF and nNOS in rat hippocampus by immunohistochemistry; MAIN OUTCOME MEASURES： ① A variety.of behavioral alterations of rats; ② The expression of BDNF and nNOS in rat hippocampus. RESULTS： Compared with the Control group, the behavior of the CUMS rats was significantly depressed, the expression of BDNF decreased （P 〈 0.01） but the expression of nNOS increased （P 〈 0.01）. The behavior of rats given intra-hippocampal injection of BDNF inhibitor was significantly depressed and the expression of nNOS was significantly increased （P 〈 0.01）. Intra-hippocampal injections of an nNOS inhibitor reversed the depression-like behavioral changes induced by CUMS or intra-hippocampal injection of BDNF inhibitor. CONCLUSION： CUMS induced a decrease in expression of BDNF and an increase in expression of NO in the hippocampus, which may lead to depression.

Targeting neuronal nitric oxide synthase as a valuable strategy for the therapy of neurological disorders

The management of neurological disorders have huge and increasing human and economic costs. Despite this, there is a scarcity of effective therapeutics, and there is an extreme urgency for new and real treatments. In this short review we analyze some promising advancements in the search of new bioactive molecules targeting neuronal nitric oxide synthase （nNOS）, an enzyme deputed to the biosynthesis of nitric oxide （NO）. In different conditions of neuronal damages, this molecule is overproduced, contributing to the pathogenesis and progression of neuronal diseases. Two main approaches to modulate nNOS are discussed： a first one consisting in the direct inhibition of the enzyme by means of small organic molecules, which can be also active against other different targets involved in such diseases. A second section is dedicated to molecules able to prevent the formation of the ternary complex N-methyl-D-aspartate （NMDA）type glutamate receptors, postsynaptic density-95 （PSD95） protein-nNOS, which is necessary to activate the latter for the biosynthesis of NO.

Changes of learning and memory ability associated with neuronal nitric oxide synthase in brain tissues of rats with acute alcoholism

BACKGROUD： Ethanol can influence neural development and the ability of leaming and memory, but its mechanism of the neural toxicity is not clear till now. Endogenous nitric oxide （NO） as a gaseous messenger is proved to play an important role in the formation of synaptic plasticity, transference of neuronal information and the neural development, but excessive nitro oxide can result in neurotoxicity. OBJECTIVE ： To observe the effects of acute alcoholism on the learning and memory ability and the content of neuronal nitric oxide synthase （nNOS） in brain tissue of rats. DESIGN ： A randomized controlled animal experiment. SETTING ： Department of Physiology, Xinxiang Medical College MATERIALS： Eighteen male clean-degree SD rats of 18-22 weeks were raised adaptively for 2 days, and then randomly divided into control group （n = 8） and experimental group （n = 10）. The nNOS immunohistochemical reagent was provided by Beijing Zhongshan Golden Bridge Biotechnology Co.,Ltd. Y-maze was produced by Suixi Zhenghua Apparatus Plant. METHODS ： The experiment was carded out in the laboratory of the Department of Physiology, Xinxiang Medical College from June to October in 2005. ① Rats in the experimental group were intraperitoneally injected with ethanol （2.5 g/kg） which was dissolved in normal saline （20%）. The loss of righting reflex and ataxia within 5 minutes indicated the successful model. Whereas rats in the control group were given saline of the same volume. ② Examinations of learning and memory ability： The Y-maze tests for learning and memory ability were performed at 6 hours after the models establishment. The rats were put into the Y-maze separately. The test was performed in a quiet and dark room. There was a lamp at the end of each of three pathways in Y-maze and the base of maze had electric net. All the lamps of the three pathways were turned on for 3 minutes and then turned off. One lamp was turned on randomly, and the other two delayed automatically. In 5 seconds after alternation, pulsating electric current presented in the base of unsafe area to stimulate rat＇s feet to run to the safe area. The lighting lasted for 15 seconds as one test. Running from unsafe area to safe area at one time in 10 seconds was justified as successful. Such test was repeated for 10 times for each rat and the successful frequency was recorded. The qualified standard of maze test was that the rat ardved in the safe area g times during 10 experiments. The number of trainings for the qualified standard was used to represent the result of spatial learning. ③ Determination of the content of nNOS in brain tissue： After the Y-maze test, the rats were anaesthetized, and blood was let from the incision on right auricle, transcardially perfused via the left ventricle with about 200 mL saline, then fixed by perfusion of 40 g/L paraformaldehyde. Hippocampal CA1 region, corpus striatum and cerebellum were taken to prepare serial freezing coronal sections. The nNOS contents in the brain regions were determined with the immunohistochemical methods to reflect the changes of nitdc oxide in brain tissue. MAIN OUTCOME MEASURES ： The changes of learning and memory ability and the changes of the nNOS contents in the brain tissue of rats with acute alcoholism were observed. RESULTS ： One rat in the experimental group was excluded due to its slow reaction to electdc stimulation in the Y-maze test, and the other 17 rats were involved in the analysis of results. ① The training times to reach qualifying standards of Y-maze in the expedmental group was more than that in the control group [（34.33 ±13.04）, （27.50±8.79） times, P〈 0.05]. ② Forms and numbers of nNOS positive neurons in brain tissue： It could be observed under light microscope that in the hippocampal CA1 region, there were fewer nNOS positive neurons, which were lightly stained, and the processes were not clear enough; But the numbers of the positive neurons which were deeply stained as huffy were obviously increased in the experimental group, the cell body and cyloplasm of process were evenly stained, but the nucleus was not stained. The nNOS positive neurons in corpus stdatum had similar forms and size in the experimental group and control group. The form of the nNOS positive neurons in cerebellum were similar between the two groups. The numbers of nNOS positive neurons in hippocampal CA1 region and corpus striatum in the expedmental group [（18.22±7.47）, （11.38±5.00） cells/high power field] were obviously higher than those in the control group [（10.15±4.24）, （6.15±3.69） cells/high power field. The number of nNOS positive neurons in cerebellum had no significant difference between the two groups [（49.56±18.84）, （44.43±15.42） cells/high power field, P〉 0.05]. CONCLUSION ： Acute alcoholism may impair learning and memory ability, and nitric oxide may be involved in mediating the neurotoxic role of ethanol.

The role of nitric oxide and neuronal nitric oxide synthase in zebrafish(Danio rerio)shoaling

Nitric oxide(NO)-the product of arginine metabolism catalyzed by nitric oxide synthases(NOS)-is a well-known neurotransmitter which plays an important role in metabolism and amino acid transportation in the nervous system.In particular,it can inhibit monoamine neurotransmitter transportation which affects animal behavior,especially social behavior.Shoaling-is a one kind of social behavior.It is a behavior that individual fish choose to join with their group within two factors;food and predation risk.Shoaling fish has quickly responded to predator and increased the change in feeding competition.In addition,shoaling also effect to stress response on stock density of aquaculture system.The effect of NO molecular signaling on the dopamine pathway was investigated using zebrafish(Danio rerio)as a model organism.Our aim was to understand the role of NOS and NO in shoaling behavior,which is typical of zebrafish.The concentration of NO in the zebrafish brain was modulated using a knockout for the neuronal NOS gene,and NO production was induced through treatment with L-arginine.The existence of NO in the zebrafish brain was confirmed by using a fluorescent probe.Dopamine concentration in the brain was measured by UPLC tandem mass spectrometer.We measured shoaling cohesion of all individual fish of D.rerio,using average distance between all pairs of fish(nearest neighbor distance)and analyzed tracking by Zebralab ViewPoint software.Collectively,our results suggest that a lower level of NO was associated with a higher level of dopamine,which in turn leads to the shoaling behavior.

Effects of Repetitive Transcranial Magnetic Stimulation on Astrocytes Proliferation and nNOS Expression in Neuropathic Pain Rats

This study investigated the effects of different frequencies of repetitive transcranial magnetic stimulation （rTMS） on chronic neuropathic pain in rats. The behavior of rats with experimental chronic neuropathic pain was observed, and the expression of neuronal nitric oxide synthase （nNOS） in the ipsilateral dorsal root ganglions （DRGs） and the activation and proliferation of astrocytes in the ipsilateral spinal dorsal horn were detected. Thirty-two male Sprague-Dawley rats were randomly divided into four groups： sham-operated group, sham-rTMS group, 1 Hz group and 20 Hz group （8 rats in each group）. Chronic constriction nerve injury induced by sciatic nerve ligation was made to establish the models of the chronic neuropathic pain in rats except those in the sham-operated group. Then we applied different frequencies of rTMS to the primary motor cortex （M1） contralateral to the pain side once daily for 10 consecutive days. Pain behavior scores were observed before and after treatment. Western blot analysis was used to detect the expression of nNOS in ipsilateral L4-6 DRGs. Double immunofluorescent labeling for glial fibrillary acidic protein （GFAP） and 5-bromo-2- deoxyuridine （BrdU） was employed to observe the activation and proliferation of astrocytes in the ipsilateral L4-6 spinal dorsal horn. After rTMS treatment, the spontaneous pain behavior scores were significantly lower in the 20 Hz group than those in the sham-rTMS group （P〈0.05）. Moreover, the brush-evoked pain behavior scores were significantly lower in the 20 Hz group than those in the sham-rTMS and 1 Hz group （P〈0.05）, suggesting that the spontaneous pain and brush-evoked pain in the 20 Hz group were significantly alleviated. Western blot analysis revealed that the expression of nNOS in ipsilateral L4-6 DRGs was significantly decreased in the 20 Hz group as compared with the sham-rTMS group and the 1 Hz group （P〈0.01） after rTMS treatment. Double immunofluorescence suggested that the expression of GFAP and the co-localization with BrdU in astrocytes were less in the sham-operated group than those in the sham-rTMS group and the 1 Hz group in L4-6 spinal dorsal horn ipsilateral to the neuropathic pain. After rTMS treatment, the expression of GFAP and the co-localization with BrdU decreased in the 20 Hz group as compared with the sham-rTMS group and the 1 Hz group （P〈0.05）. In addition, the alleviation degree of spontaneous pain and brush-evoked pain in the 20 Hz group was negatively correlated with the expression of nNOS in ipsilateral DRGs and the number of GFAP/BrdU co-labelled astrocytes in L4-6 spinal dorsal horn ipsilateral to the neuropathic pain （P〈0.05）. It was suggested that high-frequency rTMS may relieve neuropathic pain through down-regulating the overexpression of nNOS in ipsilateral DRGs and inhibiting the activity and proliferation of astrocytes in L4-6 spinal dorsal horn ipsilateral to the neuropathic pain.

Glucagon-like peptide-2 modulates the nitrergic neurotransmission in strips from the mouse gastric fundus

AIM To investigate whether glucagon-like peptide-2(GLP-2) influences the neurally-induced responses in gastric strips from mice, since no data are available. METHODS For functional experiments, gastric fundal strips were mounted in organ baths containing Krebs-Henseleit solution. Mechanical responses were recorded via forcedisplacement transducers, which were coupled to a polygraph for continuous recording of isometric tension. Electrical field stimulation(EFS) was applied via two platinum wire rings through which the preparationwas threaded. The effects of GLP-2(2 and 20 nmol/L) were evaluated on the neurally-induced contractile and relaxant responses elicited by EFS. Neuronal nitric oxide synthase(n NOS) enzyme was evaluated by immunohistochemistry.RESULTS In the functional experiments, electrical field stimulation(EFS, 4-16 Hz) induced tetrodotoxin(TTX)-sensitive contractile responses, which were reduced in amplitude by GLP-2(P < 0.05). In the presence of the nitric oxide(NO) synthesis inhibitor L-NNA, GLP-2 no longer influenced the neurally-evoked contractile responses(P > 0.05). The direct smooth muscle response to methacholine was not influenced by GLP-2(P > 0.05). In the presence of guanethidine and carbachol, the addition of GLP-2 to the bath medium evoked TTX-sensitive relaxant responses that were unaffected by L-NNA(P > 0.05). EFS induced a fast NO-mediated relaxation, whose amplitude was enhanced in the presence of the hormone(P < 0.05). Immunohistochemical experiments showed a significant increase(P < 0.05) in n NOS immunoreactivity in the nerve structures after GLP-2 exposure. CONCLUSION The results demonstrate that in gastric fundal strips, GLP-2 influences the amplitude of neurally-induced responses through the modulation of the nitrergic neurotransmission and increases n NOS expression.

Penile erectile dysfunction after brachial plexus root avulsion injury in rats

Our previous studies have demonstrated that some male patients suffering from brachial plexus injury, particularly brachial plexus root avulsion, show erectile dysfunction to varying degrees. However, the underlying mechanism remains poorly understood. In this study, we evaluated the erectile function after establishing brachial plexus root avulsion models with or without spinal cord injury in rats. After these models were established, we administered apomorphine （via a sub- cutaneous injection in the neck） to observe changes in erectile function. Rats subjected to simple brachial plexus root avulsion or those subjected to brachial plexus root avulsion combined with spinal cord injury had significantly fewer erections than those subjected to the sham operation. Expression of neuronal nitric oxide synthase did not change in brachial plexus root avulsion rats. However, neuronal nitric oxide synthase expression was significantly decreased in brachial plexus root avulsion ＋ spinal cord injury rats. These findings suggest that a decrease in neuronal nitric oxide synthase expression in the penis may play a role in erectile dysfunction caused by the combi- nation of brachial plexus root avulsion and spinal cord injury.