NMDA受体通道参与大鼠脊髓背角C纤维诱发电位LTP的表达

以往研究表明,激动NMDA受体是引起海马长时程增强(LTP)的必备条件,而LTP的表达主要与AMPA受体的磷酸化及其受体组装到突触后膜有关.但是,近年来有研究表明NMDA受体通道也参与了LTP的表达.为探讨NMDA受体通道是否参与了脊髓背角C纤维诱发电位LTP的表达,诱导LTP后,分别静脉或脊髓局部给予NMDA受体拮抗剂MK801或APV,观察其作用.发现静脉注射非竞争性NMDA受体MK801(0.1mg/kg)对脊髓LTP无影响,注射0.5mg/kg显著抑制LTP,但是当剂量增高到1.0mg/kg时,抑制作用并未进一步增大.脊髓局部给予MK801也能抑制脊髓背角LTP.为验证上述结果,使用了竞争性NMDA受体拮抗剂APⅤ.结果显示,脊髓局部给予50μmol/LAPⅤ对LTP无影响,100μmol/L对LTP有显著的抑制作用,当浓度升至200μmol/L时,抑制作用并未见进一步增强.因此认为,NMDA受体通道部分地参与了脊髓背角C纤维诱发电位LTP的表达.     

脊髓背角痛觉传递和调制的一些化学解剖学观察

本实验研究了脊髓背角内Ｃ纤维末梢的分布和突触学特征及其一些神经递质化学构筑;定量观察了急性痛引起背角的递质变化;显示了初级传入Ｃ纤维,抑制性中间神经元和背角伤害性感受神经元三者之间的突触关系,并探讨它们在痛觉信息传递和调制中的作用。     

Homocysteine induces COX-2 expression in macrophages through ROS generated by NMDA receptor-calcium signaling pathways

Abstract

Homocysteine (Hcy) at elevated levels is a putative risk factor for many cardiovascular disorders including atherosclerosis. In the present study, we investigated the effect of Hcy on the expression of cyclooxygenase (COX)-2 in murine macrophages and the mechanisms involved. Hcy increased the expression of COX-2 mRNA and protein in dose- and time-dependent manners, but did not affect COX-1 expression. Hcy-induced COX-2 expression was attenuated not only by the calcium chelators, EGTA and BAPTA-AM, but also by an antioxidant, N-acetylcysteine. Calcium chelators also attenuated Hcy-induced reactive oxygen species (ROS) production in macrophages, indicating that Hcy-induced COX-2 expression might be mediated through ROS generated by calcium-dependent signaling pathways. In another series of experiments, Hcy increased the intracellular concentration of calcium in a dose-dependent manner, which was attenuated by MK-801, an N-methyl-D-aspartate (NMDA) receptor inhibitor, but not by bicuculline, a gamma-aminobutyric acid receptor inhibitor. Molecular inhibition of NMDA receptor using small interfering RNA also attenuated Hcy-induced increases in intracellular calcium. Furthermore, both ROS production and Hcy-induced COX-2 expression were also inhibited by MK-801 as well as by molecular inhibition of NMDA receptor. Taken together, these findings suggest that Hcy enhances COX-2 expression in murine macrophages by ROS generated via NMDA receptor-mediated calcium signaling pathways.     

Extracellular Amino Acid Concentrations in the Dorsal Spinal Cord of Freely Moving Rats Following Veratridine and Nociceptive Stimulation

In vivo microdialysis was used to sample extracellular concentrations of amino acids in the dorsal lumbar spinal cord of freely moving rats. Changes in the extracellular concentrations of amino acids were measured in response to infusion of veratridine (180 microM), a sodium channel activator, as well as during acute noxious stimulation by an injection of 5% formalin into the metatarsal region of the hindleg. Veratridine produced a tetrodotoxin (TTX)-sensitive increase in the extracellular concentration of Glu. Concentrations of Asp, taurine, Ala, Asn, and Gly were not significantly elevated following veratridine stimulation. Intradermal injection of formalin produced a TTX-sensitive increase in Asp concentration and a non-TTX-sensitive increase in Glu concentration. These data support the hypothesis that Glu and Asp are dorsal horn neurotransmitters involved in nociception.     

钙结合蛋白CR在坐骨神经压榨模型脊髓内的表达

目的：研究大鼠坐骨神经压榨模型的钙结合蛋白Calretinin（CR）在脊髓的时空变化规律,为探讨其在神经再生中的作用提供实验依据。方法：36只SD大鼠随机分为假手术对照组和坐骨神经压榨组,实验组压榨后分别存活1d到21d,免疫组化结合图像分析技术观察CR在脊髓分布和含量的变化。结果：在对照组,CR样阳性神经元主要分布于腰髓背角Ⅰ,Ⅱ层,Ⅲ～Ⅵ层只观察到一些散在分布的CR样阳性神经元,脊髓前角Ⅷ层和Ⅸ层内也可见一些多极的中间型阳性神经元。坐骨神经压榨1d后,分布于腰髓背角H层内的CR样阳性神经元比对照组有轻微增加。3d后,CR样阳性神经元与对照组相比没有明显改变。7d后,CR样阳性神经元有轻微的减少;14d后,CR的表达显著下降;至21d,CR的表达有所恢复,但仍低于7d纽。脊髓后角CR免疫阳性产物灰度值测定结果显示：术后14d后角CR表达最低,与对侧和对照组相比有统计学意义（P〈0．05）。结论：坐骨神经压榨后CR表达变化呈现一定的时空模式,为进一步揭示CR在神经系统疾病中的作用提供实验依据。     

银杏内酯对大鼠急性脊髓损伤保护作用的实验研究

目的:探讨银杏内酯对大鼠急性脊髓损伤的保护作用。方法:选取健康成年正常SD大鼠54只,分正常组、损伤组和银杏内酯治疗组;采用改良Allen’s打击法制作脊髓损伤动物模型,分别在伤后6 h、12 h、24 h、72 h处死动物,采用免疫组织化学方法结合图像分析技术观测NF-κB和COX-2在脊髓腰段的表达情况。结果:脊髓神经功能评定显示银杏内酯治疗组大鼠神经功能较单纯损伤组有所改善;正常脊髓前角内NF-κB和COX-2有一定的基础表达。脊髓损伤后6 h脊髓神经元的胞浆及胞核内NF-κB和COX-2均先后表达上升,24 h达高峰,72 h仍维持在较高水平;而给予银杏内酯治疗后,各时间点NF-κB和COX-2的表达上调幅度均降低。结论:急性脊髓损伤后,银杏内酯可通过控制NF-κB和COX-2的表达上调的幅度而抑制炎症反应,对脊髓受损神经元起一定的保护作用。     

钙结合蛋白CR在坐骨神经压榨模型脊髓内的表达

目的:研究大鼠坐骨神经压榨模型的钙结合蛋白Calretinin(CR)在脊髓的时空变化规律,为探讨其在神经再生中的作用提供实验依据。方法:36只SD大鼠随机分为假手术对照组和坐骨神经压榨组,实验组压榨后分别存活1d到21d,免疫组化结合图像分析技术观察CR在脊髓分布和含量的变化。结果:在对照组,CR样阳性神经元主要分布于腰髓背角Ⅰ,Ⅱ层,Ⅲ～Ⅵ层只观察到一些散在分布的CR样阳性神经元,脊髓前角Ⅷ层和Ⅸ层内也可见一些多极的中间型阳性神经元。坐骨神经压榨1d后,分布于腰髓背角Ⅱ层内的CR样阳性神经元比对照组有轻微增加。3d后,CR样阳性神经元与对照组相比没有明显改变。7d后,CR样阳性神经元有轻微的减少;14d后,CR的表达显著下降;至21d,CR的表达有所恢复,但仍低于7d组。脊髓后角CR免疫阳性产物灰度值测定结果显示:术后14d后角CR表达最低,与对侧和对照组相比有统计学意义(P<0.05)。结论:坐骨神经压榨后CR表达变化呈现一定的时空模式,为进一步揭示CR在神经系统疾病中的作用提供实验依据。     

Fluorocitrate,an Inhibitor of Glial Metabolism,Inhibits the Up-Regulation of NOS Expression,Activity and NO Production in the Spinal Cord Induced by Formalin Test in Rats

Previous experiments have suggested that nitric oxide plays an important role in nociceptive transmission in the spinal cord. In order to explore the involvement of glia in the NO-mediated nociceptive transmission, the present study was undertaken to investigate the effect of fluorocitrate (FC), an inhibitor of glial metabolism, on NOS expression and activity and NO production in the spinal cord during the process of peripheral inflammatory pain and hyperalgesia induced by formalin test in rats. Sixty adult male Sprague–Dawley rats were randomly assigned into sham, formalin, formalin + normal saline (NS), and formalin + FC groups. The NOS expression, NOS activity and NO production was detected by NADPH-d histochemistry staining, NOS and NO assay kit, respectively. It was found that formalin test significantly up-regulated NOS expression and activity and NO production in the laminae I–II of the dorsal horn and the grey matter around the central canal in the lumbar spinal cord at 1 h after the formalin test. Selective inhibition of glia metabolism with intrathecal administration of FC (1 nmol) significantly inhibited the up-regulation in NOS expression and activity and NO production normally induced by the formalin test, which was represented with decreases in the number and density of the NADPH-d positive cells in the dorsal horn and grey matter around the central canal, and decrease in density of NADPH-d positive neuropil in the dorsal horn in formalin + FC group compared with formalin group. The results suggested that glia may be involved in the NO-mediated nociceptive transmission in the spinal cord. X.-C. Sun, W.-N. Chen and S.-Q. Li contributed equally to this work.     

In Vivo Electrochemical Monitoring of Serotonin in Spinal Dorsal Horn with Nafion-Coated Multi-Carbon Fiber Electrodes

Abstract: Biosensors sensitive for in vivo monitoring of serotonin (5-HT) in the CNS by differential normal pulse voltammetry were constructed by coating treated multi-carbon fiber electrodes (mCFEs) with Nafion (N-mCFE). In vitro sensitivities of mCFE and N-mCFE were compared in solutions ranging from 5 n M to 20 µ M of uric acid (UA), 5-hydroxyindoleacetic acid (5-HIAA), and 5-HT. The mCFEs were three to seven times less sensitive for 5-HIAA or UA than for 5-HT. Nafion treatment dramatically decreased sensitivity for 5-HIAA and UA of N-mCFEs (∼10³ times), whereas it remained in the nanomolar range for 5-HT. In vivo, in the dorsal horn of the lumbar spinal cord of anesthetized rats, the monoamine oxidase inhibitor clorgyline (10 mg/kg i.p.) produced a reduction (55 ± 3% at 180 min) of peak 3 of oxidation current (characteristic of 5-hydroxyindoles) monitored with mCFEs, but with N-mCFEs (in this latter case the peak was termed 3N) peak 3N increased to 135 ± 5% at 180 min. The 5-HT release-inducer p -chloroamphetamine (PCA; 6 mg/kg i.p.) induced a slight (12 ± 3% at 150 min) decrease in peak 3 measured with mCFEs, whereas with N-mCFEs PCA induced a rapid increase of peak 3N (137 ± 6% at 90 min). The xanthine oxidase inhibitor allopurinol (10 mg/kg i.p.) produced a decrease (30 ± 3% at 180 min) in peak 3 (mCFEs), but peak 3N (N-mCFEs) was not affected (106% at 180 min). After pretreatment with allopurinol, PCA also produced an increase (135 ± 6% at 90 min) in peak 3N. These in vitro and in vivo data provide evidence for a highly preferential detection of 5-HT versus 5-HIAA and UA by N-mCFEs, which could be used to follow the extracellular 5-HT concentration within very discrete structures throughout the CNS.     

Effect of Antipsychotic Drugs on the Gene Expression of NMDA Receptor Subunits in Rats

It has been hypothesized that glutamatergic neurons play an important role in clinical manifestations of schizophrenia and that the therapeutic effect of antipsychotic drugs is related to glutamatergic neurotransmission. To elucidate the effect of antipsychotic drugs on glutamatergic transmission, we examined gene expressions of NMDA receptor subunits Rl, R2A, R2B and R2C in the whole brains of rats after acute and chronic administrations of haloperidol and sulphide, using the Northern blot technique. The levels of NMDAR2B mRNAs decreased after the acute administration of haloperidol, but showed no change after the chronic administration. The levels of NMDAR2A and R2B mRNAs decreased after the acute administration of sulpiride, whereas the levels of R2A and R2B increased following the chronic administration. Neither haloperidol nor sulpiride influenced NMDAR1 mRNA levels. These data support differential expression of NMDA receptor subunits in rats upon treatment with haloperidol and sulpiride. The results imply that NMDAR2 subunits may be crucial in the regulation and modification of antipsychotic drugs.     

甲基苯丙胺中毒大鼠纹状体COX-2、PGE2、EP2 受体及小胶质细胞 表达的研究

目的:通过研究COX-2、PGE2、EP2受体及小胶质细胞在甲基苯丙胺中毒大鼠纹状体内的表达变化探讨甲基苯丙胺中毒大鼠纹状体中COX-2/PGE2系统与小胶质细胞活化之间的关系。方法:将40只健康成年雄性SD大鼠,随机分成对照组10只和实验组30只(实验组分成三个亚组,分为末次给药后1天组、2天组和3天组,n=10)。实验组给予10mg/kg的MA腹腔注射,对照组给予同样剂量的生理盐水,每天注射两次,注射时间为8:00、20:00,连续注射4天。分别于末次给药后的第1天、第2天、第3天处杀。用免疫组化技术对中毒大鼠纹状体(CPU)中COX-2、EP2受体及Iba1(钙离子接头蛋白,小胶质细胞内一种特异性标记物)的表达进行检测,并进行图像分析。另外,取大鼠的纹状体运用酶联免疫法检测PGE2的含量。结果:COX-2、PGE2、EP2受体及小胶质细胞在各组均有表达。与对照组相比,实验组中:COX-2、PGE2、EP2受体的1天组表达均不同程度下降;2天组中COX-2表达水平大幅度上升,PGE2、EP2受体表达仍低于正常水平;3天组COX-2表达水平继续升高,而PGE2、EP2受体表达趋于正常组水平。而小胶质细胞表达水平则是三个实验组均高于正常组,且3天组高于2天组,2天组高于1天组。对照组与实验组有显著性差异(P<0.05)。结论:COX-2/PGE2系统与甲基苯丙胺中毒大鼠纹状体内小胶质细胞活化无明显相关性;COX-2与甲基苯丙胺的神经毒性有关。     

Spatiotemporal Expression of Dexras1 After Spinal Cord Transection in Rats

Dexras1, a brain-enriched member of the Ras subfamily of GTPases, as a novel physiologic nitric oxide (NO) effector, anchor neuronal nitric oxide synthase (nNOS) that increased after spinal cord injury (SCI), to specific targets to enhance NO signaling, and is strongly and rapidly induced during treatment with dexamethasone. It is unknown how the central nervous system (CNS) trauma affects the expression of Dexras1. Here we used spinal cord transection (SCT) model to detect expression of Dexras1 at mRNA and protein level in spinal cord homogenates by real-time PCR and Western blot analysis. The results showed that Dexras1 mRNA upregulated at 3 day, 5 day, and 7 day significantly (P < 0.05) that was consistent with the protein level except at 7 day. Immunofluorescence revealed that both neurons and glial cells showed Dexras1 immunoreactivivty (IR) around SCT site, but the proportion is different. Importantly, injury-induced expression of Dexras1 was co-labeled by caspase-3 (apoptotic marker) and Tau-1 (marker for pathological oligodendrocyte). Furthermore, colocalization of Dexras1, carboxy-terminal PSD95/DLG/ZO-1 (PDZ) ligand of nNOS (CAPON) and nNOS was observed in neurons and glial cells, supporting the existence of ternary complexes in this model. Thus, the results that the transient high expression of Dexras1 which localized in apoptotic neurons and pathological oligodendrocytes might provide new insight into the secondary response after SCT. Xin Li, Chun Cheng, and Min Fei contributed equally to this work.     

Effects of Electro-acupuncture on PDGF Expression in Spared Dorsal Root Ganglion and Associated Dorsal Horn Subjected to Partial Dorsal Root Ganglionectomy in Cats

The effects of electro-acupuncture (EA) on the expression of platelet derived growth factor (PDGF) in spared dorsal root ganglion (DRG) and associated dorsal horns were evaluated in cats subjected to bilateral removal of L₁–L₅ and L₇–S₂ DRG, while sparing L₆ DRG and were demonstrated using Immunohistochemistry, Western blot and RT-PCR techniques. On the acupunctured side, there was a significant increase in the total number of PDGF positive neurons. Large neurons of the L₆ DRG at 7 days post operation (dpo), and small to medium-sized neurons at 14 dpo, as well as in the lamina II of the L₆ spinal cord at 14 dpo was observed. The expression of PDGF protein increased significantly in the L₆ DRG at 7 and 14 dpo and in the dorsal horn of the L₆ spinal cord at 14 dpo while the upregulation of PDGF mRNA was seen at 3 dpo in the L₆ DRG and the dorsal horn of the L₃ and L₆ spinal cord. These findings demonstrate that intrinsic PDGF has been upregulated in cats subjected to partial dorsal root ganglionectomy following EA, indicating endogenous PDGF is involved in promoting spinal plasticity following EA.     

NMDA and Non-NMDA Receptor Gene Expression Following Global Brain Ischemia in Rats: Effect of NMDA and Non-NMDA Receptor Antagonists

Abstract: Transient forebrain or global ischemia in rats induces selective and delayed damage of hippocampal CA1 neurons. In a previous sludy, we have shown that expression of GIuR2, the kainate/a-amino-3-hydroxy-5- methyl-4-isoxazolepropionic acid (AMPA) receptor subunit that governs Ca' permeability, is preferentially reduced in CA1 at a time point proceeding neuronal degeneration. Postischemic administration of the selective AMPA receptor antagonist, 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)quinoxaline (NBQX), protects CAI neurons against delayed death. In this study we examined the effects of NBQX (at a neuroprotective dose) and of MK-801 (a selective NMDA receptor anltagonist, not protective in this model) on kainate/AMPA receptor gene expression changes after global ischemia. We also examined the effects of transient forebrain ischemia on expression of the NMDA receptor subunit NMDARI. In ischemic rats treated with saline, GIuR2 and (31uR3 mRNAs were markedly reduced in CAI but were unchanged in CA3 or dentate gyrus. GluRl and NMDAR1 mRNAs were not significantly changed in any region examined. Administration of NBQX or MK-801 did not alter the ischemia-induced changes in kainate/AMPA receptor gene expression. These findings suggest that NBQX affords neuroprotection by a direct blockade of kainate/AMPA receptors, rather than by a modificatian of GIuR2 expression changes     

NMDA Receptor Heterogeneity During Postnatal Development of the Rat Brain: Differential Expression of the NR2A, NR2B, and NR2C Subunit Proteins

Abstract: Changes in the expression of the NMDA receptor subunits (NRs) NR2A, 2B, and 2C were investigated in histo blots of the developing rat brain with subunit-specific antisera. At birth, the NR2B subunit was detected almost ubiquitously, the NR2A subunit staining was faint and restricted to the hippocampus, cerebral cortex, and striatum, and no NR2C subunit immunoreactivity was detected. During the first 3 postnatal weeks, the NR2B subunit became confined to forebrain structures, whereas the NR2A immunoreactivity became abundantly expressed throughout the brain. The NR2C immunoreactivity emerged 5 days after birth in the olfactory bulb, thalamus, and vestibular nuclei and became very intense after 10 days in cerebellar granule cells, its primary site of expression in adulthood. After 3 weeks, NR2A and NR2B immunoreactivity decreased to adult levels, whereas NR2C immunoreactivity remained unchanged. The patterns of distribution of the subunit proteins were in agreement with those of their corresponding mRNAs, as monitored by in situ hybridization histochemistry, although the mRNA translation appeared to be delayed by several days in certain areas. Our results reveal a progressive increase in the heterogeneity of NMDA receptors due to the comparably late onset of NR2A and NR2C subunit expression and by the area-specific rearrangement of NR2B subunit expression following birth.     

福尔马林致痛对大鼠降钙素基因相关肽的影响

目的:研究福尔马林致痛后大鼠脊髓和背根神经节(dorsal root ganglion,DRG)内降钙素基因相关肽(calcitonin gene-related peptide,CGRP)的时空变化规律,为探讨CGRP在伤害性信息传递中的机制和作用提供实验依据。方法:选取健康成年正常SD大鼠54只随机分为生理盐水对照组和福尔马林实验组;实验组为右侧足底皮下给予0.1 mL 5%福尔马林后,分别存活15 min、30min、1 h、3 h、6 h、12h、24h和72h(n=6),免疫组化结合图像分析技术观测CGRP在脊髓腰段及L4~6DRG的表达变化。结果:正常DRG、脊髓前角和后角内CGRP有一定的基础表达。福尔马林致痛后3 h DRG的CGRP表达上升,12 h~24 h达峰值,72 h基本降低到正常;福尔马林致痛后6 h脊髓后角CGRP的表达上调,24 h达到高峰,72 h降低至正常;脊髓前角CGRP未见明显变化。结论:福尔马林致痛引起DRG和脊髓的CGRP的表达呈现一定的时空模式,可能是其参与伤害性信息传递的机制之一。     

Calcium/Calmodulin-Dependent Protein Kinase II Is Associated with NR2A/B Subunits of NMDA Receptor in Postsynaptic Densities

Abstract: NMDA receptors and Ca²⁺/calmodulin-dependent kinase II (CaMKII) have been reported to be highly concentrated in the postsynaptic density (PSD). Although the possibility that CaMKII in PSD might be associated with specific proteins has been put forward, the protein or proteins determining the targeting of the kinase in PSD have not yet been identified. Here we report that CaMKII binds to NR2A and NR2B subunits of NMDA receptors in PSD isolated from cortex and hippocampus. The association of NMDA receptor subunits and CaMKII was assessed by immunoprecipitating PSD proteins with antibodies specific for NR2A/B and CaMKII: CaMKII coprecipitated with NR2A/B and NR1 but not with other glutamate ionotropic receptor subunits, such as GluR1 and GluR2-3. A direct association between CaMKII and NR2A/B subunits was further confirmed by overlay experiments using either ³²P-autophosphorylated CaMKII or ³²P-NR2A/B and by evaluating the formation of a CaMKII-NR2A/B complex by means of the cross-linker disuccimidyl suberate. These data demonstrate an association between the NMDA receptor complex and CaMKII in the postsynaptic compartment, suggesting that this colocalization may be relevant for synaptic plasticity.     

Transient Global Ischemia Alters NMDA Receptor Expression in Rat Hippocampus: Correlation with Decreased Immunoreactive Protein Levels of the NR2A/2B Subunits, and an Altered NMDA Receptor Functionality

Abstract: We investigated the gene expression levels, the immunoreactive protein prevalence, and the functional activity of N -methyl- d -aspartate (NMDA) receptor complexes at early times after severe global ischemia challenge in rats. The mRNA expression levels for the NR2A and NR2B subunits of NMDA receptors changed to different degrees within different subregions of the hippocampus after reperfusion with respect to sham-operated control. No significant change in expression was observed in the vulnerable CA1 subfield at or before 6 h after challenge for either receptor subunit, although changes in expression in other hippocampal subfields were observed. At 12 and 24 h after challenge, significant decreases in expression for both subunits were found in the vulnerable CA1 subfield, as well as in other hippocampal regions. At the protein level, a significant decrease in the amount of NR2A/NR2B immunoreactivity in the total hippocampus was observed at both 6 and 24 h after reperfusion compared with sham control. Electrophysiological assessment of single-channel NMDA receptor activity in the CA1 subfield indicates that the main conductance state of NMDA receptor channels is maintained 6 h after challenge, although by 18–24 h after challenge, this main conductance state is rarely observed. The NMDA receptor component of the excitatory postsynaptic field potential was found to be significantly diminished from sham control 24 h after challenge, such that only ∼10% of the sham response remained, but was not significantly altered from sham control at 6 h after challenge. These results indicate that decreases in the expression levels, the immunoreactive protein prevalence, and that alterations in the functionality of NMDA receptors occur in the hippocampus at early times after severe transient global ischemia.