Stargazin在切口痛大鼠脊髓背角GluR1-AMPA受体胞浆至胞膜转运中的作用

目的 评价Stargazin在切口痛大鼠脊髓背角含谷氨酸受体1亚基的使君子酸(GluR1-AMPA)受体胞浆至胞膜转运中的作用.方法 成年雄性清洁级SD大鼠45只,体重280 ～ 300 g,6～8周龄,采用随机数字表法,将其分为5组:正常对照组(C组)、假手术组(S组)、切口痛+生理盐水组(P组)、切口痛+ Stargazin小干扰RNA(siRNA)组(I组)和切口痛+Stargazin无意义siRNA组(N组).P组、I组和N组分别鞘内注射生理盐水10μl、20 μmol/L siRNA、20μmol/L无意义siRNA 10μl,2次/d,连续3d,4d后制备右足底切口痛模型.切口痛术后3h时测定大鼠累计痛评分(CPS)和机械缩足阈(PWT).然后处死大鼠,取L3-6节段脊髓背角,采用Westem blot法检测胞浆和胞膜GluR1和GluR2亚基表达,采用免疫共沉淀技术检测脊髓背角Stargazin与GluR1或GluR2亚基的共表达.结果 与C组比较,P组和N组CPS评分升高,PWT降低,脊髓背角胞浆GluR1表达下调,脊髓背角胞膜GluR1表达上调,I组CPS评分升高(P＜0.05或0.01);与P组比较,I组CPS评分降低,PWT升高,脊髓背角胞浆GluR1表达上调,脊髓背角胞膜GluR1表达下调,脊髓背角Stargazin和Stargazin与GluR1共表达下调(P＜ 0.05或0.01).结论 Stargazin介导了切口痛大鼠GluR1-AMPA受体从胞浆至胞膜转运.     

切口痛-瑞芬太尼痛觉过敏大鼠脊髓背角含GluR1和GluR2亚基AMPA受体表达的变化

目的 探讨切口痛-瑞芬太尼痛觉过敏大鼠脊髓背角含GluR1和GluR2亚基AMPA受体表达的变化.方法 雄性SD大鼠32只,体重240 ～ 260 g,2～3月龄,采用随机数字表法,将其分为4组(n=8):对照组(C组)、瑞芬太尼组(R组)、切口痛组(P组)和瑞芬太尼+切口痛组(R+I组).制备足底切口痛模型的同时开始静脉输注瑞芬太尼1.0 μg·kg-1·min-1,输注1h.分别瑞芬太尼输注前、输注停止后2、6、24和48 h时测定机械刺激缩足阈(PWT)和热刺激缩足潜伏期(PWL).最后一次行为学测试后处死大鼠,取脊髓背角L4-6节段,采用Western blot法测定总蛋白及膜蛋白AMPA受体GluR1和GluR2亚基表达,并计算膜蛋白二者表达的比值(GluR1/GluR2).结果 与C组比较,I组、R组和R+I组PWL缩短,PWT降低,总蛋白及膜蛋白GluR1表达上调,总蛋白及膜蛋白GluR2表达下调,膜蛋白GluR1/GluR2增加(P＜0.05).与R组和I组比较,R+I组PWL缩短,PWT降低,总蛋白及膜蛋白GluR1表达上调,总蛋白及膜蛋白GluR2表达下调,膜蛋白GluR1/GluR2增加(P＜0.01).结论 大鼠切口痛-瑞芬太尼痛觉过敏的形成与脊髓背角总蛋白及膜蛋白含GluR1亚基AMPA受体表达上调和含GluR2亚基AMPA受体表达下调,导致膜蛋白含GluR1亚基AMPA受体组成比例增加有关.     

异丙酚对内脏痛大鼠脊髓背角使君子酸受体GluR1亚基磷酸化水平的影响

目的 探讨异丙酚对内脏痛大鼠脊髓背角使君子酸(AMPA)受体谷氨酸受体1(GluRl)亚基831和845位点丝氨酸磷酸化水平的影响.方法 成年雄性SD大鼠,体重200～300 g,6～8周龄,行鞘内置管术,5d后选取鞘内置管成功的大鼠30只,采用随机数字表法,将其随机分为3组(n=1 0):假手术组(Ⅰ组)、内脏痛组(Ⅱ组)和异丙酚组(Ⅲ组).Ⅱ组和Ⅲ组采用经直肠注射50μl10％辣椒素的方法制备内脏痛模型,Ⅰ组给予等容量生理盐水,Ⅲ组于给予辣椒素前10 min鞘内注射异丙酚20μg,Ⅰ组和Ⅱ组给予等容量二甲基亚砜.记录给予辣椒素后30 min内大鼠累计痛评分,然后处死大鼠,取L3-6腰骶段脊髓背角组织,采用免疫印迹法检测AMPA受体GluR1亚基及其831和845位点丝氨酸磷酸化水平.结果与Ⅰ组比较,Ⅱ组和Ⅲ组累计痛评分和脊髓背角GluR1亚基831和845位点丝氨酸磷酸化水平升高(P ＜0.05或0.01),而GluR1亚基表差异无统计学意义(P＞0.05);与Ⅱ组比较,Ⅲ组累计痛评分和脊髓背角GluR1亚基831和845位点丝氨酸磷酸化水平降低(P＜0.05或0.01).结论 异丙酚可减轻大鼠内脏痛,其机制与抑制脊髓背角AMPA受体GluR1亚基831和845位点丝氨酸磷酸化有关.     

Stargazin调节使君子酸受体亚基转运和突触靶向——疼痛治疗的新靶点

背景 使君子酸(α-amino-3 -hydroxy-5 -methy-4-isoxazole propionate,AMPA)受体是中介中枢神经系统兴奋性突触传递的主要受体,参与疼痛信号传递.Stargazin蛋白是一种AMPA受体调节蛋白,在AMPA受体中介的疼痛信号传递中扮演重要角色.目的 对Stargazin蛋白调节AMPA受体亚基在胞浆胞膜中的转运作用及与疼痛的关系作用进行回顾与总结.内容 Stargazin蛋白可调节AMPA受体不同亚基在胞浆胞膜转运,并通过与突触后膜致密蛋白-95 (postsynaptic density-95,PSD-95)的相互作用,促进AMPA受体亚基突触靶向；Stargazin还通过C末端自身磷酸化修饰改变与PSD-95蛋白相互作用的强度,控制AMPA受体的突触靶向.Stargazin通过调节AMPA受体的转运,间接调控AMPA受体中介的疼痛信号传递.趋向 下调Stargazin的表达或干扰其与兴奋性突触后PSD-95蛋白的相互作用,可间接抑制AMPA受体的功能,是未来疼痛治疗研究的新靶点.     

糖原合成酶激酶-3β在切口痛-瑞芬太尼痛觉过敏大鼠脊髓含NR1及NR2B亚基的NMDA受体转运中的作用

目的 探讨糖原合成酶激酶-3β(GSK-3β)在切口痛-瑞芬太尼痛觉过敏大鼠脊髓含NR1及NR2B亚基的NMDA受体转运中的作用.方法 雄性SD大鼠24只,体重240～260 g,2～3月龄,采用随机数字表法,将大鼠随机分为3组(n=8):对照组(C组)尾静脉注射二甲基亚砜(DMSO)2ml/kg,随后输注与瑞芬太尼等容量生理盐水60min;瑞芬太尼组(R组)尾静脉注射DMSO 2 ml/kg后制备切口痛模型,同时静脉输注瑞芬太尼1.2 μg· kg-1·min-1 60 min;GSK-3β抑制剂组(TDZD-8组)尾静脉注射GSK-3β抑制剂(TDZD-8)1 mg/kg后制备切口痛模型,同时静脉输注瑞芬太尼1.2μg·kg-1·min-1 60 min.于静脉输注生理盐水或瑞芬太尼前24h、输注后2、6、24和48 h (T0-4)时测定机械刺激缩足阈值(PWT)和热刺激缩足潜伏期(PWL),最后1次测定痛阈后处死,取脊髓L4-6节段,采用Western blot法测定脊髓细胞膜(s)及胞浆内(i) NMDA受体NR1及NR2B亚基的表达,计算sNR1/iNR1及sNR2B/iNR2B比值.结果 与C组比较,R组和TDZD-8组PWT降低,PWL缩短,sNR1和sNR2B表达上调,iNR1和iNR2B表达下调,sNR1/iNR1及sNR2B/iNR2B升高(P＜0.05).与R组比较,TDZD-8组PWT升高,PWL延长,sNR1和sNR2B表达下调,iNR1和iNB2B表达上调,sNR1/iNR1及sNR2B/iNR2B降低(P＜0.05).结论 GSK-3β参与切口痛-瑞芬太尼痛觉过敏大鼠脊髓含NR1及NR2B亚基的NMDA受体从胞浆向胞膜的转运.     

丙泊酚后处理对脑缺血-再灌注大鼠ADAR2-AMPA受体GluR2通路的作用

目的研究丙泊酚后处理对脑缺血-再灌注大鼠腺苷脱氨酶(adenosine deaminase acting on RNA2,ADAR2)-α-氨基-3-羟基-5-甲基-4-异恶唑丙酸(α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid,AMPA)受体GluR2亚基(ADAR2-AMPA受体GluR2)通路的作用。方法健康雄性SD大鼠120只,体重260~280g,采用随机数字表法均分为三组:假手术组(S组)、缺血-再灌注组(IR组)和丙泊酚后处理组(P组)。IR组和P组采用大脑中动脉阻塞1h的方法制备大鼠脑缺血-再灌注损伤模型,P组于再灌注即刻经股静脉输注20mg·kg-1·h-1丙泊酚2h,S组和IR组给予等容量生理盐水。于处理后24h行改良神经行为学评分(mNSS);2,3,5-氯化三苯基四氮唑染色(TTC)测定脑梗死体积;RT-PCR检测缺血侧海马ADAR2mRNA表达;Western blot法检测缺血侧海马ADAR2胞核及总蛋白表达;巢式RT-PCR和特异性限制性内切酶BbV1检测ADAR2受体GluR2mRNA Q/R位点编辑比例。结果三组ADAR2mRNA和总蛋白表达差异无统计学意义。与S组比较,IR组和P组mNSS评分明显升高,脑梗死体积明显增加,ADAR2胞核/总蛋白表达比例明显降低,GluR2mRNA Q/R位点编辑比例明显下降(P0.05);与IR组比较,P组mNSS评分明显降低,脑梗死体积明显减少,ADAR2胞核/总蛋白表达比例明显增加,GluR2mRNA Q/R位点编辑比例明显升高(P0.05)。结论丙泊酚后处理对脑缺血-再灌注大鼠有急性脑保护作用,该作用可能与丙泊酚促进ADAR2蛋白核转位,进而增加AMPA受体GluR2亚基mRNA编辑有关。     

切口痛-瑞芬太尼痛觉过敏大鼠脊髓总蛋白及膜蛋白NMDA受体NRl 、NR2A及NR2B亚基表达的变化

目的 评价切口痛-瑞芬太尼痛觉过敏大鼠脊髓总蛋白及膜蛋白NMDA受体NR1、NR2A及NR2B亚基表达的变化.方法 尾静脉置管成功的雄性SD大鼠32只,体重240～260 g,月龄2～3月,采用随机数字表法,将大鼠随机分为4组(n=8):对照组(C组)静脉输注等容量生理盐水60 min,瑞芬太尼组(R组)静脉输注瑞芬太尼1.2μg·kg-1·min-1 60 min;切口痛组(I组)建立切口痛模型,同时静脉输注等容量生理盐水60 min;瑞芬太尼+切口痛组(R+I组)建立切口痛模型,同时静脉输注瑞芬太尼1.2 μg· kg-1 ·min-1 60 min.于生理盐水或瑞芬太尼给药前24h、给药后2、6、24和48 h时测定机械刺激缩足阈值(PWT)和热刺激缩足潜伏期(PWL),最后一次测定痛阈后处死取脊髓L4～6节段,采用Western blot法测定脊髓总蛋白及膜蛋白NMDA受体NR1、NR2A及NR2B亚基的表达,并计算膜蛋白中NR2B/NR2A比值.结果 与C组比较,I组、R组和R+I组PWT降低,PWL缩短,总蛋白及膜蛋白NMDA受体NR1和NR2B亚基表达上调,膜蛋白中NR2B/NR2A比值增加(P＜0.05).与R组和I组比较,R+I组PWT降低,PWL缩短,总蛋白及膜蛋白NMDA受体NR1和NR2B亚基表达上调,膜蛋白中NR2B/NR2A比值增加(P＜0.05).各组总蛋白及膜蛋白NMDA受体NR2A亚基表达差异无统计学意义(P＞0.05).结论 大鼠切口痛-瑞芬太尼痛觉过敏的形成可能与脊髓总蛋白及膜蛋白NMDA受体NR1和NR2B亚基表达上调和膜蛋白中NMDA受体NR2B亚基组成比例的增加有关.     

异丙酚后处理对大鼠脑缺血再灌注损伤的影响:长期观察

目的 评价异丙酚后处理对大鼠脑缺血再灌注损伤的影响:长期观察.方法 健康雄性SD大鼠144只,体重250～280g,7～8周龄,采用随机数字表法,将其随机分为4组(n=36):假手术组(S组)、缺血再灌注组(I/R组)、异丙酚后处理组(P组)和溶媒对照组(I组).I/R组、P组和I组 采用线栓法阻塞大脑中动脉60min进行再灌注的方法制备局灶性脑缺血再灌注损伤模型.P组在再灌注即刻开始静脉输注异丙酚20 mg·kg-1·h-1 2h,S组和I/R组给予等容量生理盐水,I组给予等容量10％脂肪乳.4组分别于术后1、14、28d取5只大鼠,进行神经功能评分及脑梗死体积测定；4组各取6只大鼠,分别于术后9、23d开始进行Morris水迷宫测试,连续6 d;4组分别于术后1、14、28 d取5只大鼠,取海马组织,测定使君子酸( AMPA)受体GluR1亚基及其在胞膜上表达水平,计算二者比值(胞膜GluR1亚基/GluR1亚基).结果 与S组比较,I/R组神经功能评分和跨越平台次数降低,脑梗死体积增加,逃避潜伏期延长,胞膜GluR1亚基/GluR1亚基增加(P＜0.05),而海马组织GluR1亚基表达差异无统计学意义(P＞0.05)；异丙酚后处理可抑制脑缺血再灌注诱导的上述改变(P＜0.05).结论 异丙酚后处理减轻大鼠脑缺血再灌注损伤的效应可连续到术后28d,其部分机制与抑制含GluR1亚基AMPA受体向细胞膜转运有关.     

脊髓小胶质细胞抑制对神经病理性痛大鼠脊髓背角GABAB受体表达的影响

目的 通过观察鞘内注射特异性小胶质细胞抑制剂米诺环素对神经病理性痛大鼠脊髓背角GABAB受体表达的影响,探讨脊髓小胶质细胞活化介导神经病理性痛发生的作用机制.方法 雄性SD大鼠48只,体重220～260 g,结扎L5神经根制备神经病理性痛模型,随机分为4组(n=12):Ⅰ组仅暴露L5神经根但不结扎,鞘内注射生理盐水10 μl;Ⅱ组暴露并结扎L5神经根,鞘内注射生理盐水10 μl;Ⅲ组仅暴露L5神经根但不结扎,鞘内注射米诺环素50 μg(10μl);Ⅳ组暴露并结扎L5神经根,鞘内注射米诺环素50 μg(10 μl).于术前1 d～术后18 d,每日鞘内注射生理盐水或米诺环素,2次/d.于术前1 d(基础状态)、术后1、2、4、6、8、10、12、14、16、18 d各组取6只大鼠测定机械痛阈,并确定机械痛周最低点,然后在机械痛阈最低点时各组另取6只大鼠测定脊髓背角GABABR2的表达.结果 与Ⅰ组相比,Ⅱ组机械痛阈降低,术侧脊髓背角GABABR2表达下调(P＜0.05或0.01);与Ⅱ组和Ⅲ组比较,Ⅳ组机械痛阈升高,术侧脊髓背角GABABR2表达上调(P＜0.05或0.01).结论 脊髓小胶质细胞活化介导神经病理性痛发生的作用机制可能与抑制GABAB受体的激活有关.     

炎性痛大鼠脊髓背角γ-氨基丁酸及其受体的表达

目的 观察福尔马林致痛大鼠腰膨大段脊髓背角γ-氨基丁酸(GABA)及其受体的表达。方法 12只SD大鼠随机分为两组(n=6):A组,空白对照组;B组,福尔马林炎性致痛组,24h后分别应用免疫组化和原位杂交方法检测大鼠腰膨大段两侧脊髓背角GABA免疫阳性细胞、GABA_(Aβ3)和GABA_(B1)受体mRNA的表达。结果 GABA免疫阳性细胞、GABA_(Aβ3)和GABA_(B1)受体mRNA在大鼠脊髓背角均有表达,且GABA免疫阳性细胞和GABA_(B1)受体mRNA在背角Ⅰ～Ⅲ层分布密度最高,GABA_(Aβ3)受体mRNA在脊髓各层面分布较均匀。GABA免疫阳性细胞在B组大鼠致痛侧腰段脊髓背角表达与非致痛侧和A组两侧相比明显增加(P<0.05),非致痛侧和A组两侧间的表达差异无显著性(P>0.05),脊髓背角两侧GABAA_(Aβ3)和GABA_(B1)受体mRNA的表达与A组相比明显增加(P<0.05),两侧间的表达差异无显著性(P>0.05)。结论 炎性痛时脊髓GABA神经递质的释放增加,同时受体基因的表达上调,抑制系统功能增强。     

Selective postsynaptic co-localization of MCT2 with AMPA receptor GluR2/3 subunits at excitatory synapses exhibiting AMPA receptor trafficking

MCT2 is the main neuronal monocarboxylate transporter needed by neurons if they are to use lactate as an additional energy substrate. Previous evidence suggested that some MCT2 could be located in postsynaptic elements of glutamatergic synapses. Using post-embedding electron microscopic immunocytochemistry, it is demonstrated that MCT2 is present at postsynaptic density of asymmetric synapses, in the stratum radiatum of both rat hippocampal CA1 and CA3 regions, as well as at parallel fibre-Purkinje cell synapses in mouse cerebellum. MCT2 levels were significantly lower at mossy fibre synapses on CA3 neurons, and MCT2 was almost absent from symmetric synapses on CA1 pyramidal cells. It could also be demonstrated using quantitative double-labeling immunogold cytochemistry that MCT2 and AMPA receptor GluR2/3 subunits have a similar postsynaptic distribution at asymmetric synapses with high levels expressed within the postsynaptic density. In addition, as for AMPA receptors, a significant proportion of MCT2 is located on vesicular membranes within the postsynaptic spine, forming an intracellular pool available for a putative postsynaptic endo/exocytotic trafficking at these excitatory synapses. Altogether, the data presented provide evidence for MCT2 expression in the postsynaptic density area at specific subsets of glutamatergic synapses, and also suggest that MCT2, like AMPA receptors, could undergo membrane trafficking.     

Modulation of AMPA receptor GluR1 subunit phosphorylation in neurons by the intravenous anaesthetic propofol

Background: The ionotropic glutamate receptor is a potential molecular sitein the central nervous system that general anaesthetics mayinteract with to produce some of their biological actions. Proteinphosphorylation has been well documented to occur in the intracellularC-terminal domain of -amino-3-hydroxy-5-methylisoxazole-4-propionicacid (AMPA) subtype of glutamate receptors, which representsa pivotal mechanism for the post-translational modulation ofAMPA receptor functions. In this study, we investigated a possibleinfluence of an i.v. anaesthetic agent propofol on the phosphorylationof AMPA receptor GluR1 subunits in cultured neurons. Methods: The effect of propofol on phosphorylation of GluR1 subunitsat serine 831 and 845 was assayed in cultured rat striatal andcortical neurons by western blot with phospho- and site-specificantibodies. Results: Propofol consistently elevated phosphorylation of GluR1 subunitsat the C-terminal serine 845 site in both striatal and corticalneurons. The elevation in phosphorylation was concentration-dependentand started at a low concentration (3 µM). This increasein serine 845 phosphorylation was rapid and sustained duringthe entire course of propofol exposure. In contrast to serine845, phosphorylation of GluR1 at serine 831 was not alteredby propofol in striatal and cortical neurons. Total GluR1 abundanceremained unchanged in response to propofol incubation. Conclusions: These data indicate that propofol possesses the ability to upregulateAMPA receptor GluR1 subunit phosphorylation at a specific serine845 site in neurons and provide evidence supporting the AMPAreceptor as a molecular target for general anaesthetics.     

AMPA receptor competitive antagonism reduces halothane MAC in rats.

Various subtypes of receptors have been identified for glutamate, an excitatory neurotransmitter. Previous studies have shown that antagonism of glutamate at the NMDA receptors reduces minimum alveolar concentration (MAC) for volatile anesthetics. NBQX (2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline) is a selective antagonist at the glutamatergic AMPA receptor. The purpose of this experiment was to determine whether AMPA receptor antagonism influences halothane MAC in the rat. Sprague-Dawley rats were anesthetized with halothane in 50% O2/balance N2, tracheally intubated and the lungs were mechanically ventilated. Increasing doses of NBQX were intravenously infused in three groups while the control group was infused with vehicle (D5W). Halothane MAC was then determined by the tail-clamp method. Halothane MAC was log-linearly related to plasma NBQX concentrations (MAC = 0.125 (In plasma concentration NBQX) + 1.035, r2 = 0.77). A maximal 58% reduction of halothane MAC was achieved with an NBQX loading dose of 42 mg/kg followed by a continuous infusion rate of 36 mg x kg-1 x h-1 (control = 1.02 +/- 0.07%; NBQX = 0.43 +/- 0.12%; P < .01). Larger doses of NBQX were not possible because of the poor aqueous solubility of this compound. In a separate experiment, awake rats were randomly assigned to groups based on the dose of NBQX infused. Pa(CO2) and mean arterial pressure were measured at time 0 and at 5 and 30 min after start of NBQX infusion. The infusion was then stopped. Time until recovery of the righting reflex was recorded.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanical injury modulates AMPA receptor kinetics via an NMDA receptor-dependent pathway

Alterations in glutamatergic transmission are thought to contribute to secondary neuronal damage following traumatic brain injury. Using an in vitro cell injury model, we previously demonstrated an apparent reduction in AMPA receptor desensitization and resultant potentiation of AMPA-evoked currents after stretch injury of cultured neonatal rat cortical neurons. In the present study, we sought to further characterize injury-induced enhancement of AMPA current and elucidate the mechanisms responsible for this pathological process. Using the patch-clamp technique, agonist-activated currents were recorded from control and injured neurons. Potentiation of AMPA-mediated currents occurred quickly, within 15-30 min following injury, and persisted for at least 24 h. Stretch-injury slowed the activation and desensitization of AMPA mediated currents recorded from excised outside-out patches. The co-application of 100 microM AMPA and 20 microM thiocyanate enhanced AMPA receptor desensitization in control neurons and restored desensitization in injured neurons. The potentiation of AMPA-elicited current was prevented by the NMDA receptor antagonist D-APV (20 microM) or the CaMKII inhibitor KN93 (10 microM). These results suggest that mechanical injury initiates a biochemical cascade that involves NMDA receptor and CaMKII activation and produces a long-lasting reduction of AMPA receptor desensitization, which may contribute to the pathophysiology of traumatic brain injury.     

Synergistic analgesic effects of intrathecal midazolam and NMDA or AMPA receptor antagonists in rats

PURPOSE: To investigate the interaction of midazolam and N-methyl-D-aspartate (NMDA) receptor or -amino-3-hydroxy-5-methyl isoxazole-4-propionic acid (AMPA) receptor antagonist on the effects of persistent inflammatory nociceptive activation. METHODS: Male Sprague-Dawley rats were implanted with lumbar intrathecal catheters and were tested for their responses to subcutaneous formalin injection into the hindpaw. Saline, midazolam (1 to 100 microg), AP-5 (I to 30 microg), a NMDA receptor antagonist, or YM872 (0.3 to 30 microg), an AMPA receptor antagonist was injected intrathecally 10 min before formalin injection. The combinations of midazolam and AP-5 or YM872 in a constant dose ratio based on the 50% effective dose (ED50) were also tested and were analysed with an isobologram. RESULTS: Dose-dependent effects were observed with midazolam (ED50 was 1.34 microg and 1.21 microg in phase 1 and 2 of the formalin test, respectively), AP-5 (7.64 microg and 1.4 microg) and YM872 (0.24 microg and 0.21 microg). Synergistic effects in both phases were obtained when combining midazolam with AP-5 or YM872. The ED50 of midazolam decreased to 0.012 microg (phase 1) and 0.27 microg (phase 2) with AP-5 and to 0.09 microg (phase 1) and 0.35 microg (phase 2) with YM872 (P < 0.01). CONCLUSIONS: These results suggest a functional coupling of benzodiazepine-aminobutyric acid (GABA)A receptor with NMDA and AMPA receptors in acute and persistent inflammatory nociceptive mechanisms in the spinal cord.     

Blockade of glutamate receptors and barbiturate anesthesia: increased sensitivity to pentobarbital-induced anesthesia despite reduced inhibition of AMPA receptors in GluR2 null mutant mice.

BACKGROUND: Barbiturates enhance gamma-aminobutyric acid type A (GABA(A)) receptor function and also inhibit the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) subtype of glutamate receptor. The relative contribution of these actions to the behavioral properties of barbiturates is not certain. Because AMPA receptor complexes that lack the GluR2 subunit are relatively insensitive to pentobarbital inhibition, GluR2 null mutant mice provide a novel tool to investigate the importance of AMPA receptor inhibition to the anesthetic effects of barbiturates. METHODS: GluR2 null allele (-/-), heterozygous (+/-), and wild-type (+/+) mice were injected with pentobarbital (30 and 35 mg/kg intraperitoneally). Sensitivity to anesthetics was assessed by measuring the latency to loss of righting reflex, sleep time, and the loss of corneal, pineal, and toe-pinch withdrawal reflexes. In addition, patch-clamp recordings of acutely dissociated CA1 hippocampal pyramidal neurons from (-/-) and (+/+) mice were undertaken to investigate the effects of barbiturates on kainate-activated AMPA receptors and GABA-activated GABA(A) receptors. RESULTS: Behavioral tests indicate that sensitivity to pentobarbital was increased in (-/-) mice. In contrast, AMPA receptors from (-/-) neurons were less sensitive to inhibition by pentobarbital (concentrations that produced 50% of the maximal inhibition [IC50], 301 vs. 51 microM), thiopental (IC50, 153 vs. 34 microM), and phenobarbital (IC50, 930 vs. 205 microM) compared with wild-type controls, respectively. In addition, the potency of kainate was greater in (-/-) neurons, whereas no differences were observed for the potentiation of GABA(A) receptors by pentobarbital. CONCLUSIONS: The GluR2 null mutant mice were more sensitive to pentobarbital anesthesia despite a reduced sensitivity of GluR2-deficient AMPA receptors to barbiturate blockade. Our results indicate that the inhibition of AMPA receptors does not correlate with the anesthetic effects of barbiturates in this animal model. We postulate that the increase in the sensitivity to anesthetics results from a global suppression of excitatory neurotransmission in GluR2-deficient mice.     

The effect of three inhaled anesthetics in mice harboring mutations in the GluR6 (kainate) receptor gene

Combinations of GluR5-GluR7, KA1, and KA2 subunits form kainate receptors, a subtype of excitatory ionotropic glutamate receptors. Isoflurane enhances the action of kainate receptors comprising GluR6 subunits expressed in oocytes. To test whether alterations of the GluR6 subunit gene affect the actions of inhaled anesthetics in vivo, we measured the minimum alveolar concentration of desflurane, isoflurane, and halothane in mice lacking the kainate receptor subunit GluR6 (GluR6 knockout mice) and mice with a dominant negative glutamine/arginine (Q/R) editing mutation in membrane domain 2 of the GluR6 receptor (GluR6 editing mutants), which increases the calcium permeability of kainate receptors containing GluR6Q. We also measured the capacity of isoflurane to interfere with Pavlovian fear conditioning to a tone and to context. Absence of the GluR6 subunit did not change the minimum alveolar concentration of isoflurane, desflurane, or halothane. Possibly, kainate receptors assembled from the remaining kainate receptor subunits compensate for the absent subunits and thereby produce a normal minimum alveolar concentration. A Q/R mutation that dominantly affects kainate receptors containing the GluR6 subunit in mice increased isoflurane minimum alveolar concentration (by 12%; P < 0.01), decreased desflurane minimum alveolar concentration (by 18%; P < 0.001), and did not change halothane minimum alveolar concentration (P = 0.25). These data may indicate that kainate receptors containing GluR6Q subunits differently modulate, directly or indirectly, the mechanism by which inhaled anesthetics cause immobility. The mutations of GluR6 that were studied did not affect the capacity of isoflurane to interfere with fear conditioning.