nNOS and Neurological, Neuropsychiatric Disorders: A 20-Year Story / 神经科学通报·英文版

In the central nervous system, nitric oxide (NO), a free gas with multitudinous bioactivities, is mainly produced from the oxidation of L-arginine by neuronal nitric oxide synthase (nNOS). In the past 20 years, the studies in our group and other laboratories have suggested a significant involvement of nNOS in a variety of neurological and neuropsychiatric disorders. In particular, the interactions between the PDZ domain of nNOS and its adaptor proteins, including post-synaptic density 95, the carboxy-terminal PDZ ligand of nNOS, and the serotonin transporter, significantly influence the subcellular localization and functions of nNOS in the brain. The nNOS-mediated protein-protein interactions provide new attractive targets and guide the discovery of therapeutic drugs for neurological and neuropsychiatric disorders. Here, we summarize the work on the roles of nNOS and its association with multiple adaptor proteins on neurological and neuropsychiatric disorders.

Immunohistochemical Overexpression of nNOS, S100 Protein, and α-Synuclein in Myenteric Plexus of AS/AGU Rats

Objective: There are four currently motor features characterizing Parkinson's disease (PD). These includerigidity of muscles, bradykinesia, tremors at rest, and instability of posture. Along the course of PD, theimpairment of motor functions is commonly preceded by nonmotor symptoms (NMS) such as olfactory deficit,difficult swallowing (dysphagia), drooling (sialorrhea), constipation, urinary bladder dysfunction, depression,and sleep disorder. It was suggested that the enteric nervous system could be the initial site for the pathologicalprocess leading to PD. Materials and Methods: Six male adult control AS rats (normal control) and six maleadults AS/AGU rats (model of PD) were sacrificed. A rectangular strip from the body of the stomach and across-section from the duodenum were dissected and processed for histological staining with hematoxylin andeosin, and immunohistochemical staining for detection of nNOS (neuronal NOS), S100 protein (astrocytemarker), and alpha-synuclein (α-synuclein). Results: The histological analysis of the stomach and duodenum ofAS/AGU rats demonstrated necrotic smooth muscle cells of muscularis externa. The immunohistochemicalanalysis of AS/AGU rats showed a statistically significant increase in the expression of nNOS, S100 protein, andα-synuclein expression of myenteric plexuses compared to the control strain AS rats. Conclusion:Gastroduodenal tract of AS/AGU rats showed marked histopathological changes and immunohistochemicaloverexpression of nNOS, S100, and α-synuclein.

Protection of spinal cord ischemia-reperfusion injury mediated by N-methyl-D-aspartate receptors / 中国胸心血管外科临床杂志

@#Objective    To analyze the protective mechanism of spinal cord ischemia-reperfusion injury mediated by N-methyl-D-aspartate (NMDA) receptor. Methods    A total of 42 SD rats were randomly assigned to 4 groups: a non-blocking group (n=6), a saline group (n=12), a NMDA receptor blocker K-1024 (25 mg/kg) group (n=12) and a voltage-gated Ca2+ channel blocker nimodipine (0.5 mg/kg) group (n=12). The medications were injected intraperitoneally 30 min before ischemia. The neural function was evaluated. The neuronal histologic change of spinal cord lumbar region, the release of neurotransmitter amino acids and expression of spinal cord neuronal nitric oxide synthase (nNOS) were compared. Results    At 8 h after reperfusion, the behavioral score of the K-1024 group was 2.00±0.00 points, which was statistically different from those of the saline group (5.83±0.41 points) and the nimodipine group (5.00±1.00 points, P<0.05). Compared with the saline group and nimodipine group, K-1024 group had more normal motor neurons (P<0.05). There was no significant difference in glutamic acid concentration in each group at 10 min after ischemia (P=0.731). The nNOS protein expression in the K-1024 group was significantly down-regulated compared with the saline group (P<0.01). After 8 h of reperfusion, the expression of nNOS protein in the K-1024 group was significantly up-regulated compared with the saline group (P<0.05). Conclusion    K-1024 plays a protective role in spinal cord ischemia by inhibiting NMDA receptor and down-regulating nNOS protein expression; during the reperfusion, K-1024 has a satisfactory protective effect on spinal cord function, structure and biological activity of nerve cells.

Study on the Mechanism of Improvement Effects of Gastrodin Injection on Methamphetamine Induced Neurotoxic Damage in Rats via nNOS Pathway / 中国药房

OBJECTIVE：To stu dy the mechanism of improvement effects of Gastrodin injection on methamphetamine induced neurotoxic damage in rats via nNOS pathway. METHODS ：SD rats were randomly divided into control group ，methamphetamine group，regular-dose of gastrodin group ，double-dose of gastrodin group ，negative control （NC）adenovirus group ，NC adenovirus+ methamphetamine group ，NC adenovirus+gastrodin group and nNOS adenovirus+gastrodin group ，with 10 rats in each group. Control group was given normal saline intraperitoneally ，twice a day. Methamphetamine group was given methamphetamine intraperitoneally（7.5 mg/kg），twice a day. Regular-dose and double-dose of gastrodin groups were respectively given different doses of Gastrodin injection （10，20 mg/kg）intraperitoneally 30 min earlier ，once a day ，and then given methamphetamine intraperitoneally by the same way as methamphetamine group. NC adenovirus group was given NC adenovirus （4.8×107 PFU）3 μL once in the striatum and normal saline intraperitoneally ，twice a day. NC adenovirus+methamphetamine group was given NC adenovirus by the same way and methamphetamine （7.5 mg/kg）intraperitoneally，twice a day. NC adenovirus+gastrodin group was given NC adenovirus+methamphetamine by the same way ，meanwhile given Gastrodin injection intraperitoneally （20 mg/kg）30 min before methamphetamine ，once a day. nNOS adenovirus+gastrodin group was given nNOS adenovirus and methamphetamine by the same way ，meanwhile given Gastrodin injection intraperitoneally （20 mg/kg）30 min before methamphetamine ，once a day. Each group was given relevant medicine intraperitoneally 1 mL/100 g，for consecutive 3 days. The stereotyped behavior of rats were observed and scored ；the apoptotic rate ，the protein expression of apoptotic factors （Bcl-2，Bax，Cleaved caspase- 3），the levels of oxidative stress factors （MDA，SOD，GPx） and NO ，the protein expression of nNOS were detected. RESULTS ： Compared with control group ，stereotyped behavior score ，cell apoptosis rate of striatum ，protein expression of Bax ，Cleaved caspase-3 and nNOS ，the levels of MDA and NO were increased significantly in methamphetamine group ；while the protein expression of Bcl- 2 and the levels of SOD and GPx were decreased significantly （P＜0.05 or P＜0.01）. Compared with methamphetamine group ，stereotyped behavior score ，cell apoptosis rate of striatum ，protein expression of Bax ，Cleaved caspase- 3 and nNOS ，the levels of MDA and NO were decreased significantly in regular-dose and double-dose of gastrodin groups ；while the protein expression of Bcl- 2，the levels of SOD and GPx were increased significantly ，and most above indexes in double-dose of gastradin group were better than regular-dose of gastrodin group （P＜0.05 or P＜0.01）. Compared with NC adenovirus group ，cell apoptosis rate of striatum ，protein expression of Bax ，Cleaved caspase- 3 and nNOS ，the levels of MDA and NO were increased significantly in NC adenovirus+methamphetamine group ；while the protein expression of Bcl- 2，the levels of SOD and GPx were decreased significantly （P＜0.01）. Compared with NC adenovirus+methamphetamine group ，cell apoptosis rate of striatum ，protein expression of Bax ，Cleaved caspase- 3 and nNOS ，the levels of MDA and NO were decreased significantly in NC adenovirus+ gastrodin group ；while the protein expression of Bcl- 2，the levels of SOD and GPx were increased significantly （P＜0.01）. Compared with NC adenovirus+gastrodin group ，cell apoptosis rate of striatum ，protein expression of Bax ，Cleaved caspase- 3 and nNOS，the levels of MDA and NO were increased significantly in nNOS adenovirus+gastrodin group ；while the protein expression of Bcl- 2，the levels of SOD and GPx were decreased significantly （P＜0.01）. CONCLUSIONS ：Gastrodin injection can protect rats against neurotoxic damage induced by methamphetamine ，and the effect is related to the inhibition of nNOS-mediated apoptosis and oxidative stress.

Establishment of a screening model for compounds uncoupling the interaction of nNOS with PSD-95 / 药学学报

In ischemic stroke, increased level of neuronal complex of nitric oxide synthase (nNOS)-postsynaptic density protein-95 (PSD-95) plays an important role in neuronal damage. We aimed to establish a screening model to identify compounds capable of uncoupling nNOS interaction with PSD-95. In this model, human embryonic kidney-293T (HEK-293T) cells were transfected with either pCDH-Flag-nNOS or pcDNA3.1-PSD-95 plasmid to obtain the protein of Flag-nNOS or PSD-95. Incubating Flag-nNOS with PSD-95 causes formation of the nNOS-PSD-95 complex. ZL006, a known uncoupler of nNOS-PSD-95 interaction, can disturb the interaction between Flag-nNOS and PSD-95, serving as a positive control. The method coupling antibodies to magnetic beads with glutaraldehyde was used to decrease the cost and increase the efficiency. To establish that our model is suitable for selecting nNOS-PSD-95 uncouplers, we evaluated the ability of IC87201, another reported uncoupler of nNOS-PSD-95 interaction, and structural analogs of ZL006. IC87201 and one structure analog of ZL006 showed uncoupling effect, supporting that our model can be used to select different types uncoupler blocking nNOS-PSD-95 interaction.

Efficient discovery and capturing of nNOS-PSD-95 uncouplers from Trifolium pratense / 中国中药杂志

Magnetic molecularly imprinted polymers(MMIPs) were prepared with ZL006 as template, acrylamide(AA) as the functional monomer, and acetonitrile as pore-forming agent; then Fourier transform infrared spectroscopy(FT-IR) and scanning electron microscopy(SEM) were used to characterize their forms and structures. Simultaneously, the MMIPs prepared previously were used as sorbents for dispersive magnetic solid phase extraction(DSPE) to capture and identify potential nNOS-PSD-95 uncouplers from extracts of Trifolium pratense and the the activities of the screened compounds were evaluated by the neuroprotective effect and co-immunoprecipitation test. The experiment revealed that the successfully synthesized MMIPs showed good dispersiveness, suitable particle size and good adsorption properties. Formononetin, prunetin and biochanin A were separated and enriched from Trifolium pratense by using the MMIPs as artificial antibodies and finally biochanin A was found to have higher cytoprotective action and uncoupling action according to the neuroprotective effect and co-immunoprecipitation test.

Moutan Cortex Radicis inhibits the nigrostriatal damage in a 6-OHDA-induced Parkinson's disease model / 中国天然药物

The traditionally used oriental herbal medicine Moutan Cortex Radicis [MCR; Paeonia Suffruticosa Andrews (Paeoniaceae)] exerts anti-inflammatory, anti-spasmodic, and analgesic effects. In the present study, we investigated the therapeutic effects of differently fractioned MCR extracts in a 6-hydroxydopamine (OHDA)-induced Parkinson's disease model and neuro-blastoma B65 cells. Ethanol-extracted MCR was fractionated by n-hexane, butanol, and distilled water. Adult Sprague-Dawley rats were treated first with 20 μg of 6-OHDA, followed by three MCR extract fractions (100 or 200 mg·kg) for 14 consecutive days. In the behavioral rotation experiment, the MCR extract-treated groups showed significantly decreased number of net turns compared with the 6-OHDA control group. The three fractions also significantly inhibited the reduction in tyrosine hydroxylase-positive cells in the substantia nigra pars compacta following 6-OHDA neurotoxicity. Western blotting analysis revealed significantly reduced tyrosine hydroxylase expression in the substantia nigra pars compacta in the 6-OHDA-treated group, which was significantly inhibited by the n-hexane or distilled water fractions of MCR. B65 cells were exposed to the extract fractions for 24 h prior to addition of 6-OHDA for 30 min; treatment with n-hexane or distilled water fractions of MCR reduced apoptotic cell death induced by 6-OHDA neurotoxicity and inhibited nitric oxide production and neuronal nitric oxide synthase expression. These results showed that n-hexane- and distilled water-fractioned MCR extracts inhibited 6-OHDA-induced neurotoxicity by suppressing nitric oxide production and neuronal nitric oxide synthase activity, suggesting that MCR extracts could serve as a novel candidate treatment for the patients with Parkinson's disease.

Moutan Cortex Radicis inhibits the nigrostriatal damage in a 6-OHDA-induced Parkinson's disease model / 中国天然药物

The traditionally used oriental herbal medicine Moutan Cortex Radicis [MCR; Paeonia Suffruticosa Andrews (Paeoniaceae)] exerts anti-inflammatory, anti-spasmodic, and analgesic effects. In the present study, we investigated the therapeutic effects of differently fractioned MCR extracts in a 6-hydroxydopamine (OHDA)-induced Parkinson's disease model and neuro-blastoma B65 cells. Ethanol-extracted MCR was fractionated by n-hexane, butanol, and distilled water. Adult Sprague-Dawley rats were treated first with 20 μg of 6-OHDA, followed by three MCR extract fractions (100 or 200 mg·kg) for 14 consecutive days. In the behavioral rotation experiment, the MCR extract-treated groups showed significantly decreased number of net turns compared with the 6-OHDA control group. The three fractions also significantly inhibited the reduction in tyrosine hydroxylase-positive cells in the substantia nigra pars compacta following 6-OHDA neurotoxicity. Western blotting analysis revealed significantly reduced tyrosine hydroxylase expression in the substantia nigra pars compacta in the 6-OHDA-treated group, which was significantly inhibited by the n-hexane or distilled water fractions of MCR. B65 cells were exposed to the extract fractions for 24 h prior to addition of 6-OHDA for 30 min; treatment with n-hexane or distilled water fractions of MCR reduced apoptotic cell death induced by 6-OHDA neurotoxicity and inhibited nitric oxide production and neuronal nitric oxide synthase expression. These results showed that n-hexane- and distilled water-fractioned MCR extracts inhibited 6-OHDA-induced neurotoxicity by suppressing nitric oxide production and neuronal nitric oxide synthase activity, suggesting that MCR extracts could serve as a novel candidate treatment for the patients with Parkinson's disease.

Effects of intracerebroventricular injection of 7-nitroindazole on depression-like behaviors and nNOS expression in hippocampal of rats / 中华行为医学与脑科学杂志

Objective To evaluate the effect of intracerebroventricular injection of 7-nitroindazole (7-NI) on the depression-like behaviors in normal rats.Methods According to body weights,48 SD rats were randomly divided into normal group,model group,sham-operation group and 7-NI groups at different concentrations (n=8).The model group was treated with chronic and unpredictable mild stress.The 7-NI groups received intracerebroventricular injection with 7-NI solutions at different concentrations,once every 3 days for 3 times in total.The sham-operation group was injected with DMSO of the same volume.The rat behaviors were then subjected to the open field test (OFT).The hippocampal nNOS protein levels were detected by Western blot.Results Compared with the normal group((132.47±31.72) m),the total movement distances of model group ((15.04±8.61) m) and 200 nmol/0.5 μl surgery group((18.18± 11.82) m) decreased significantly (P＜ 0.05).Compared with the model group,such distances of sham-operation group ((107.33±20.35)m) and 7-NI groups(50 nmol/0.5 μl:(138.40±56.85)m,(86.97±36.20)m);100 nmol/0.5 μl:(86.97±36.20)m) increased significantly (P＜ 0.05).The normal group entered the central area significantly more times(2.25±2.05) than model group (0.25±0.46)and 200nmol/0.5μl 7-NI group (0.25± 0.46) did (P＜0.05),and the number of times entering the central area of the model group (0.25±0.46)was significantly lower than that of the sham-operation group (1.00 ± 1.07,P＜ 0.05) and 50 nmol/0.5 μl group (0.75 ± 1.16).Compared with the normal group ((46.53 ±41.16) s),the durations of stay in the central area of model group ((1.27 ± 1.92) s) and 200 nmol/0.5 μl 7-NI group ((1.53 ± 2.90) s) were shortened significantly (P＜0.05).Compared with the model group,the durations of stay in the central area of 100 nmol/0.5μl group ((36.54±67.80) s) was lengthened significantly (P＜ 0.05).Western blotting showed that the hippocampal nNOS protein levels of model group (0.43±0.11) and 200 nmol/0.5μl 7-NI group(0.56±0.08) significantly exceeded that of the normal group (0.04±0.02,P＜0.05).The levels of nNOS in sham-operation group (0.04 ±0.02) and 50 nmol/0.5 μl 7-NI group (0.22± 0.08),which were significantly lower than that of the model group (0.43 ± 0.11,P＜ 0.05),were similar to that of the normal group (0.04 ± 0.02,P＞ 0.05).Conclusion Intracerebroventricular injection 200 nmol/0.5 μl 7-NI solution results in depression-like behaviors and increased the expression of nNOS protein reflexively in rats.

Tat-LK15-mediated delivery of nNOS siRNA in rats with chronic inflammatory pain / 实用医学杂志

Objective To investigate the potential application of a non-viral gene carrier , TAT-LK15 , for delivering nNOSsiRNAin vivo and to study whether TAT-LK15/siRNA can be a new treatment method for chronic inflammatory pain. Method TAT-LK15 was complexed with nNOSsiRNA or scrambled control siRNA. The expression of nNOS was determined in SCDH of chronic inflammatory pain rats by western-blot assay. Pain control efficacy was evaluated by mechanical withdrawal threshold (MWT) and thermal withdrawal duration (TWD) assays. Results nNOS protein expression was efficiently inhibited by intrathecal injection of TAT-LK15/siRNA complexes , with the reduction of nNOS protein by 52%. Moreover , injection of TAT-LK15/siRNA com-plexes significantly could decrease MWT , but increase TWD in rats with chronic inflammatory pain. Conclusions TAT-LK15 can efficiently deliver nNOSsiRNAin vivo and nNOSsiRNA can relieve chronic inflammatory pain in rats.

Delivery of therapeutic siRNA by Tat-LK15 targeting nNOS fusion gene in RGC-5 cells / 中国药理学通报

Aim To investigate the potential applica-tion of a non-viral gene carrier Tat-LK15 for delivering siRNA targeting nNOS in vitro, which provides evi-dence of Tat-LK15 mediating siRNA targeting nNOS in vivo for treatment of neuropathic pain. Methods 1. Tat-LK15 was mixed with siRNA, then the mixture was analyzed the best ratio by Gel retardation. The trans-fection efficiency of FAM-siRNA mediated by Tat-LK15 on RGC-5 cells was examined by Flow Cytome-try. The apoptosis ratio of RGC-5 was identified by Flow Cytometry 24 h after treated with the different do-ses of Tat-LK15 (1, 2. 5, 5, 10 and 20 μg). 2. The model of RGC-5 cell overexpression of nNOS protein was prepared. 3. RGC-5 cells were randomly divided into 5 groups:control group,model group, Tat-S group ( Tat-LK15 mediate nNOS/siRNA transfection model cell) , Lipo-S group ( LipofectamineTM RNAiMAX me-diate nNOS/siRNA transfection model cell) and Tat-N group ( Tat-LK15 mediate NCsiRNA transfection model cell) . Real-time Quantitative polymerase chain reac-tion( Q-PCR) and Western blot were used to evaluate nNOS expression level assay. Results It indicated that the Tat-LK15/siRNA complex completely formed at the weight ratio of 2∶ 1 (μg/μg) , and the transfec-tion efficiency was (84. 4 ± 3. 9)%. It caused cotytox-icity when Tat-LK15 dose was 20 μg ( 6. 1 μmol · L-1 ) , and the apoptosis rate more than control group [(10. 3 ± 1. 1)% vs (7. 4 ± 0. 9)%, P<0. 05]. The nNOS protein level of RGC-5 cells was significantly el-evated after modeling. Compared with that of model group, Tat-LK15/siRNA efficiently inhibited the ex-pression of nNOS at transcriptional level or protein leve1 of Tat-S group ( P <0. 05 ) , and there was no significant difference of the efficiency inhibited between Tat-S group and Lipo-S group. Conclusions Tat-LK15’ advantage is with high efficiency, low cytotox-icity. The Tat-LK15 can deliver siRNA targeting nNOS in vitro efficiently and safely.

Protective effects of Zhouluotong extract Z-6 on Schwann cells damaged by high-glucose and PI3 K/Akt/nNOS pathway / 中国病理生理杂志

AIM: To explore the role of PI3K/Akt/nNOS in Zhouluotong extract resisting diabetic peripheral neuropathy.METHODS:The Schwann cells were divided into normal group ( D-glucose 25 mmol/L) , model group ( D-glucose 100 mmol/L) , Zhouluotong extract Z-6 +high glucose group, Zhouluotong +high glucose group, mecobalamine+high glucose group.The viability, nitric oxide content and the Ca2+-ATPase activity in Schwann cells were determined by Cell Counting Kit-8 , nitric oxide assay kit and Ca2+-ATPase assay kit, respectively.The apoptosis of Schwann cells were analyzed by flow cytometry.The expression of Bcl-2, Bcl-xL, Bax, Bak and caspase-3, and the phosphorylation levels of nNOS and Akt were determined by Western blotting.The signal pathway of PI3K/Akt was explored by dominant negative PI3K and Akt (δp85 and DN-Akt) transient transfection assay.RESULTS:Under high-glucose culture, the cell viability, nitric oxide content in culture supernatant, the expression of Bcl-2 and Bcl-xL, and the phosphorylation levels of Akt and nNOS in the Schwann cells were significantly increased.The cell apoptosis, the expression of Bax, Bak and caspase 3 in the Schwann cells were significantly decreased by Zhouluotong extract Z-6, compared with model group.In-creased nitric oxide content and the up-regulation of nNOS were observed.However, the effects of blocking PI3K/Akt, the upstream pathway of nNOS , by transfection with DN-δp85 on Akt phosphorylation in the Schwann cells was still unclear. CONCLUSION:Zhouluotong extract Z-6 changes the phosphorylation of nNOS, and the expression of anti-apoptotic fac-tors , caspase-3 and pro-apoptotic factors in Schwann cells under high-glucose culture, thus reducing apoptosis and elevating viability.The relationship to PI3K/Akt/nNOS pathway needs further investigation.

Neurotoxicity induced by methamphetamine and the protective role of nNOS inhibitor in rats / 中国药理学通报

Aim Tostudytheprotectiveeffectsofin-hibition of neuronal nitric oxide synthase ( nNOS ) in methamphetamine ( METH ) induced neurotoxicity via oxidativestressinjuryinrats.Methods Ratmodelsof acute METH poisoning with/without 7 nitroindazole (7-NI) pretreatment were built to evaluate the protec-tive effects on the changes of ethology, nNOS, nitro-proteins, dopamine ( DA ) and apoptosis in rat stria-tum.Results ThenNOSexpression,nitroproteinex-pression and apoptosis increased significantly in the METH group( P0. 05 ) . Stereotyped behavior increased significantly in the METH group and the 7-NI combined group compared with the 7-NI group and the control group(P<0. 01). Conclusions 7-NIshowssignificantprotectiveeffects against the alterations of DA level, nNOS expression, nitroprotein expression and apoptosis in rat striatum caused by acute METH poisoning. However, there is no obvious protective effect on METH-induced stereo-typed behavior.

Neurobiological effect of 7-nitroindazole, a neuronal nitric oxide synthase inhibitor, in experimental paradigm of Alzheimer’s disease.

Nitric oxide plays a role in a series of neurobiological functions, underlying behaviour and memory. The functional role of nNOS derived nitric oxide in cognitive functions is elusive. The present study was designed to investigate the effect of specific neuronal nitric oxide synthase inhibitor, 7-nitroindazole, against intracerebroventricular streptozotocin-induced cognitive impairment in rats. Learning and memory behaviour was assessed using Morris water maze and elevated plus maze. 7-nitroindazole (25 mg/kg, ip) was administered as prophylactically (30 min before intracerebroventricular streptozotocin injection on day 1) and therapeutically (30 min before the assessment of memory by Morris water maze on day 15). Intracerebroventricular streptozotocin produced significant cognitive deficits coupled with alterations in biochemical indices.These behavioural and biochemical changes were significantly prevented by prophylactic treatment of 7-nitroindazole. However, therapeutic intervention of 7-nitroindazole did not show any significant reversal. The results suggests that 7-nitroindazole can be effective in the protection of dementiainduced by intracerebroventricular streptozotocin only when given prophylactically but not therapeutically.

Depressed nNOS expression during spine transition in the developing hippocampus of FMR1 KO mice

Nitric oxide (NO), synthesized as needed by NO synthase (NOS), is involved in spinogenesis and synaptogenesis. Immature spine morphology is characteristic of fragile X syndrome (FXS). The objective of this research was to investigate and compare changes of postnatal neuronal NOS (nNOS) expression in the hippocampus of male fragile X mental retardation 1 gene knockout mice (FMR1 KO mice, the animal model of FXS) and male wild-type mice (WT) at postnatal day 7 (P7), P14, P21, and P28. nNOS mRNA levels were analyzed by real-time quantitative PCR (N = 4-7) and nNOS protein was estimated by Western blot (N = 3) and immunohistochemistry (N = 1). In the PCR assessment, primers 5’-GTGGCCATCGTGTCCTACCATAC-3’ and 5’-GTTTCGAGGCAGGTGGAAGCTA-3’ were used for the detection of nNOS and primers 5’-CCGTTTCTCCTGGCTCAGTTTA-3’ and 5’-CCCCAATACCACATCATCCAT-3’ were used for the detection of β-actin. Compared to the WT group, nNOS mRNA expression was significantly decreased in FMR1 KO mice at P21 (KO: 0.2857 ± 0.0150, WT: 0.5646 ± 0.0657; P < 0.05). Consistently, nNOS immunoreactivity also revealed reduced staining intensity at P21 in the FMR1 KO group. Western blot analysis validated the immunostaining results by demonstrating a significant reduction in nNOS protein levels in the FMR1 KO group compared to the WT group at P21 (KO: 0.3015 ± 0.0897, WT: 1.7542 ± 0.5455; P < 0.05). These results suggest that nNOS was involved in the postnatal development of the hippocampus in FXS and impaired NO production may retard spine maturation in FXS.

Antinociceptive effects of gabapentin & its mechanism of action in experimental animal studies.

Background & objectives: Several studies have shown the possible analgesic effects of gabapentin, widely used as an antiepileptic. Thus, clinical studies have been carried out especially for neuropathic syndroms. This study was undertaken to investigate experimentally whether gabapentin has analgesic effects in mice and rats. Methods: The mice were divided into 10 groups (n=7) with various treatments to assess central and peripheral antinociceptive activity of gabapentin. Hot plate, tail clip and tail flick tests were applied for the investigation of central antinociceptive activity and the writhing test was applied for the investigation of peripheral antinociceptive activity. In addition, we also evaluated the levels of PGE2 and nNOS on perfused hippocampus slices of rats. Results: Gabapentin showed a peripheral antinociceptive effect at all doses and a central antinociceptive effect at 30mg/kg dose. While the L-NAME and cyproheptadine changed the central and peripheral effects of gabapentin, naloxone did not change these effects. In vitro studies showed that gabapentin significantly increased nNOS level. PGE2 and nNOS were found to have an important role in the antinociceptive effects of gabapentin at all doses and its combinations with L-NAME, cyproheptadine, indomethacine, and naloxone. As expected, PGE2 levels decreased in all groups, while nNOS levels increased, which is believed to be an adaptation mechanism. Interpretation & conclusions: Our findings indicate that arachidonate, nitrergic and serotonergic systems play an important role in the antinociceptive activity of gabapentin except for the opioidergic system. Additionally, this effect occured centrally and peripherally. These effects were also mediated by nNOS and PGE2.

Immunohistochemical study on the expression of calcium binding proteins (calbindin-D28k, calretinin, and parvalbumin) in the cerebral cortex and in the hippocampal region of nNOS knock-out(-/-) mice / 대한해부학회지

Nitric oxide (NO) modulates the activities of various channels and receptors to participate in the regulation of neuronal intracellular Ca2+ levels. Ca2+ binding protein (CaBP) expression may also be altered by NO. Accordingly, we examined expression changes in calbindin-D28k, calretinin, and parvalbumin in the cerebral cortex and hippocampal region of neuronal NO synthase knockout(-/-) (nNOS-/-) mice using immunohistochemistry. For the first time, we demonstrate that the expression of CaBPs is specifically altered in the cerebral cortex and hippocampal region of nNOS-/- mice and that their expression changed according to neuronal type. As changes in CaBP expression can influence temporal and spatial intracellular Ca2+ levels, it appears that NO may be involved in various functions, such as modulating neuronal Ca2+ homeostasis, regulating synaptic transmission, and neuroprotection, by influencing the expression of CaBPs. Therefore, these results suggest another mechanism by which NO participates in the regulation of neuronal Ca2+ homeostasis. However, the exact mechanisms of this regulation and its functional significance require further investigation.

Role of nitric oxide in the periaqueductal gray in defensive behavior in mice: influence of prior local N-methyl-D-aspartate receptor activation and aversive condition

Glutamate N-methyl-D-aspartate (NMDA) receptor activation within the dorsal column of the periaqueductal gray (dPAG) leads to antinociceptive, autonomic, and behavioral responses characterized as the fear reaction. Activation of NMDA receptors in the brain increases nitric oxide (NO) synthesis, and NO has been proposed to be a mediator of the aversive action of glutamate. This paper reviews a series of studies investigating the effects of neuronal NO synthase (nNOS) inhibition in the dPAG of mice in different aversive conditions. nNOS inhibition by infusion of Nω-propyl-L-arginine (NPLA) prevents fear-like reactions (e.g., jumping, running, freezing) induced by NMDA receptor stimulation within the dPAG and produces anti-aversive effects when injected into the same midbrain site in mice confronted with a predator. Interestingly, nNOS inhibition within the dPAG does not change anxiety-like behavior in mice exposed to the elevated plus maze (EPM), but it reverses the effect of an anxiogenic dose of NMDA injected into the same site in animals subjected to the EPM. Altogether, the results support a role for glutamate NMDA receptors and NO in the dPAG in the regulation of defensive behaviors in mice. However, dPAG nitrergic modulation of anxiety-like behavior appears to depend on the magnitude of the aversive stimulus.

The influence of zinc deficiency on chronic pain in Sprague Dawley rats.

Aim Zinc (Zn) defi ciency remains a problem in most developing countries, including Indonesia, especially in the East Nusa Tenggara (NTT) Islands. Zinc plays a major role in pain through the modulation process by the N-methyl-Daspartate (NMDA) receptors, which also includes neuronal nitric oxide synthase (nNOS) as a pain parameter. The purpose of this study is to reveal the effects of Zn towards pain response and modulation stage at the spinal cord level in rats. Methods Twenty Sprague Dawley (SD) rats were divided into two groups, a defi cient group and a normal group. The defi cient group was fed on an IRI-OB diet. Every group was further divided into two more groups, the acute pain group (transient noxious stimuli), and the chronic pain group (continuous noxious stimuli). The rats in chronic pain group were subjected to CCI Bennet operation. The pain thresholds in the defi cient group and normal group were measured clinically using a modifi ed Ugo Basille plantar test (thermal transient noxious stimuli). Measurement of chronic pain level was carried out by measuring the nNOS level by immunohistochemistry. Results Defi cient group showed an insignifi cant decrease in pain threshold (P= 0.251). However, there is a signifi cant increase in nNOS (P= 0.027) especially in the defi cient group with continuous noxious stimulation. Conclusions These results suggest that Zn defi ciency increases pain response, especially in chronic pain.

Increased Seizure Susceptibility and Up-regulation of nNOS Expression in Hippocampus Following Recurrent Early-life Seizures in Rats

This study aimed to determine the long-term change of seizure susceptibility and the role of nNOS on brain development following recurrent early-life seizures in rats. Video-EEG recordings were conducted between postnatal days 50 and 60. Alterations in seizure susceptibility were assayed on day 22 or 50 using the flurothyl method. Changes in nNOS expression were determined by quantitative immunoblotting on day 50. On average, rats had 8.4+/-2.7 seizures during 10 daily 1 hr behavioral monitoring sessions. As adults (days 50-60), all rats displayed interictal spikes in the hippocampus and/or overlying cortex. Brief electrographic seizures were recorded in only one of five animals. Rats appeared to progress from a period of marked seizure susceptibility (day 22) to one of lessened seizure susceptibility (day 50). Up-regulation of nNOS expression following early-life recurrent seizures was observed on day 50. In conclusion, these data suggested that recurrent early-life seizures had the long-term effects on seizure susceptibility late in life and up-regulatory nNOS expression on the hippocampus during brain development, and nNOS appeared to contribute to the persistent changes in seizure susceptibility, and epileptogenesis.