谷氨酸影响培养的海马神经元内钙变化机制

目的:研究谷氨酸对培养的大鼠海马神经元内钙信号的影响及作用机制。方法:用显微荧光测量技术监测神经元内钙信号的动态变化和谷氨酸对其的影响;阻断NMDA、AMPA受体后,观察谷氨酸对神经元内钙信号影响的变化。结果:谷氨酸使神经元内游离钙浓度明显升高,NMDA和AMPA受体拮抗剂、胞外钙离子浓度的降低均可不同程度地降低胞内游离钙离子升高幅度。结论:谷氨酸通过多种途径影响大鼠海马神经元内钙信号,激活NMDA和AMPA受体是其中的重要机制之一。     

Lead Can Inhibit NMDA^—,K^＋—,QA／KA—Induced Increases in Intracellular Free Ca^2＋ in Cultured at Hippocampal Neurons

Objective To examine the effects of Pb2+ on N-methyl-D-aspartate (NMDA)-, K+- and quisqualate(QA)/kainite(KA)-induced increases in intracellular free calcium concentration ([Ca2+]i) in cultured fetal rat hippocampal neurons in order to explain the cognitive and learning deficits produced by this heavy metal. Methods Laser scanning confocal microscopy was used. Results The results clearly demonstrated that adding Pb2+ before or after NMDA/glycine stimulation selectively inhibited the stimulated increases in [Ca2+]i in a concentration-dependent manner. In contrast, Pb2+ treatment did not markedly affect increases in [Ca2+]i induced by an admixture of QA and KA. The minimal inhibitory effect of Pb2+ occurred at 1 μ mol/L, and more than seventy percent abolition of the NMDA-stimulated increase in [Ca2+]iwas observed at 100 μmol/L Pb2+. Evaluation of pb2+-induced increase in [Ca2+]i response to elevating extracellular concentrations of NMDA, glycine or calcium revealed that Pb2+ was a noncompetitive antagonist of both NMDA and glycine, and a competitive antagonist of Ca2+ at NMDA receptor channels. In addition, Pb2+ inhibited depolarization-evoked increases in [Ca2+]i mediated by K+ stimulation (30 μmol/L), indicating that Pb2+ also depressed the voltage-dependent calcium channels. Also, the results showed that Pb2+ appeared to be able to elevate the resting levels of [Ca2+]i in cultured neurons, implying a reason for pb2+-enhanced spontaneous release of several neurotransmitters reported in several previous studies. Conclusion Lead can inhibit NMDA-, K+-, QA/KA-inducod increases in intracellular [Ca2+]i in cultured hippocampal neurons.     

Real-time Microwave Exposure Induces Calcium Efflux in Primary Hippocampal Neurons and Primary Cardiomyocytes

Objective To detect the effects of microwave on calcium levels in primary hippocampal neurons and primary cardiomyocytes by the real-time microwave exposure combined with laser scanning confocal microscopy. Methods The primary hippocampal neurons and primary cardiomyocytes were cultured and labeled with probes, including Fluo-4 AM, Mag-Fluo-AM, and Rhod-2, to reflect the levels of whole calcium [Ca~(2+)], endoplasmic reticulum calcium [Ca~(2+)]ER, and mitochondrial calcium [Ca~(2+)]MIT, respectively. Then, the cells were exposed to a pulsed microwave of 2.856 GHz with specific absorption rate(SAR) values of 0, 4, and 40 W/kg for 6 min to observe the changes in calcium levels. Results The results showed that the 4 and 40 W/kg microwave radiation caused a significant decrease in the levels of [Ca~(2+)], [Ca~(2+)]ER, and [Ca~(2+)]MIT in primary hippocampal neurons. In the primary cardiomyocytes, only the 40 W/kg microwave radiation caused the decrease in the levels of [Ca~(2+)], [Ca~(2+)]ER, and [Ca~(2+)]MIT. Primary hippocampal neurons were more sensitive to microwave exposure than primary cardiomyocytes. The mitochondria were more sensitive to microwave exposure than the endoplasmic reticulum. Conclusion The calcium efflux was occurred during microwave exposure in primary hippocampal neurons and primary cardiomyocytes. Additionally, neurons and mitochondria were sensitive cells and organelle respectively.     

共聚焦激光扫描显微镜技术在研究大鼠海马神经元胞内钙超载中的应用

目的 观察抑制性氨基酸牛磺酸对兴奋性氨基酸谷氨酸所致胞内钙超载的影响。方法 选择Fluo-3／ AM对培养的海马神经元进行着色后,用共聚焦激光扫描显微镜(confocal laser scanning microscope,CLSM)实时 扫描,动态显示海马神经元二维图像,以计算机相应软件对胞内钙的荧光强度变化进行分析统计。结果 在 0.5 mmol／L谷氨酸作用下,胞内钙荧光强度迅速升高(P<0.001);0.5 mmol／L谷氨酸与3 mmol／L牛磺酸共同 作用于海马神经元时,胞内钙荧光强度明显降低(P<0.001);灌流液中去除牛磺酸后,胞内钙荧光强度上升(P <0.001)。结论 抑制性氨基酸牛磺酸可抑制由兴奋性氨基酸谷氨酸所致神经元的胞内钙超载;CLSM以其独 特的优势,在高清晰度地显示海马神经元形态的同时,也可动态地观察活细胞在不同环境下胞内钙的快速变化 以及定量分析。该技术在动态观察和二维图像的高分辨率上比传统测钙方法有更大的优越性。     

慢性VNS对点燃大鼠大脑皮质及海马神经元兴奋性的影响

目的：探讨迷神经刺激（VNS）抗癫痫作用的细胞机制。方法：用戊四氮（PTZ）行大鼠腹空注射点燃制作癫痫模型,行迷走神经刺激抗癫痫,应用荧光分光光度法测定不同浓度谷氨酸（Glu）对正常、PTZ点燃及VNS治疗动物大脑皮质及海马神经元胞内游离钙升高的影响,同时观察VNS对动物癫痫行为的影响。结果：VNS组动物胞内游离钙浓度较PTZ点燃组明显降低,并对外源性Glu的升谪胞内游离钙有明显抑制作用,行为观察显示VNS能明显抑制癫痫发作的严重程度,延长癫痫的发作的潜伏期。结论：VNS能明显抑制大鼠PTZ的点燃效应,降低Glu对神经元胞内游离钙的升高作用。VNS可能通过调节神经元兴奋性／抑制性受体的活性来发挥抗癫痫作用。     

Rapid Inhibition of the Glutamate-induced Increase of Intracellular Free Calcium by Magnesium in Rat Hippocampal Neurons

Inrecentyears ,largenumbersofrelativestud iesonthemagnesium (Mg2 )andneurologicaldis easesverifiedinmanyneurologicaldiseases ,suchascerebrovasculardiseases,chronicalcoholpoisoning ,Wilsondiseaseetc .,wererelatedwiththeMg2 defi ciency .Magnesiumreagentsc…     

乙醇对颈上神经元细胞内外钙信号的双重作用

目的:探讨乙醇对原代培养颈上神经节神经元(SCGs)细胞质钙稳态的影响。方法:分散、培养新生12 h内大鼠SCGs,应用激光共聚焦显微技术,观察不同浓度乙醇对SCGs细胞质Ca2+浓度([Ca2+]i)的作用。结果:KCl及钙离子载体A23187所诱发的[Ca2+]i增高,可被乙醇(100 mmol/L)显著抑制,但是乙醇(30,100,600 mmol/L)自身却可浓度依赖性增加[Ca2+]i,并且使用无Ca2+外液和氯化镉对该增加作用无影响。结论:乙醇可抑制细胞外Ca2+内流,同时增加细胞质[Ca2+]i,来源可能是胞内钙库的释放。     

硒对大鼠缺血心肌Ca~(2+)转运功能的影响

目的探讨硒对急性缺血心肌保护作用的机制。方法采用结扎大鼠心肌左冠状动脉复制心肌缺血模型。ＳＤ大鼠１００只，分为正常对照组（Ｃ）、缺血组（ＭＩ）及硒组（ＭＩ＋Ｓｅ）。硒组按每日补硒８０μｇ·ｋｇ－１，连续７ｄ。观察硒对心肌缺血后３０、６０、９０ｍｉｎ肌浆网（ＳＲ）、线粒体（Ｍｉｔ）Ｃａ２＋－ＡＴＰ酶活性动态变化及缺血６０ｍｉｎ肌膜（ＳＬ）Ｎａ＋－Ｋ＋－ＡＴＰ酶活性和线粒体膜磷脂含量变化的影响。结果与正常对照组比较，心肌缺血不同时点，Ｍｉｔ、ＳＲ、Ｃａ２＋－ＡＴＰ酶活性均有不同程度降低，且呈随缺血时间延长进行性下降的趋势；缺血６０ｍｉｎＭｉｔ磷脂含量和ＳＬＮａ＋－Ｋ＋－ＡＴＰ酶活性也明显降低。但无论任何时点，硒组Ｍｉｔ、ＳＲＣａ２＋－ＡＴＰ酶、ＳＬＮａ＋－Ｋ＋－ＡＴＰ酶活性和Ｍｉｔ磷脂含量均显著高于缺血组。结论硒在一定程度上能减轻或延缓缺血对心肌细胞Ｃａ２＋转运功能的损害作用，可能是硒保护急性缺血心肌作用机制之一。     

Experimental Study of Effect of Corticosterone on Primary Cultured Hippocampal Neurons and Their Ca2+/CaMK Ⅱ Expression

To explore the effect of different concentrations of corticosterone (CORT) on primary cultured hippocampal neurons and their Ca2+/CaMK Ⅱ expression and possible mechanism, the changes of hippocampal neurons were observed in terms of morphology, activity of cells, cell death,concentrations of cytosolic free calcium, and the expression of CaMK Ⅱ by using MTT assay, flow cytometry, fluorescent labeling of Fura-2/AM and Western blotting after 10-7, 10-6 and 10-5 mol/L of CORT was added to culture medium, The evident effect of 10-6 and 10-5 mol/L of CORT on the morphology of hippocampal neuron was found. Compared with control neurons, the activity of the cells was markedly decreased and [Ca2+]i increased in the neurons treated with 10-6 and 10-5mol/L of CORT, but no change was observed in the neuron treated with 10-7 mol/L of CORT.The death was either by way of apoptosis or necrosis in the cells treated with 10-6 and 10-5 mol/L of CORT respectively. The correlation analysis showed that a reverse correlation existed between[Ca2 ]i and the expression of CaMK Ⅱ. Either apoptosis or necrosis occurs in the hippocampal neurons treated with CORT. The increased hippocampal [Ca2+ ]i is both the result of CORT impairing the hippocampal neurons and the cause of the apoptosis of hippocampal neurons and the decreased CaMK Ⅱ expression.     

用荧光钙离子指示剂fura—2AM检测心肌细胞内游离钙

本实验采用游离钙离子荧光指示剂fura－2AM检测大鼠离体心室肌细胞内游离钙的浓度。其结果如下；（1）KC1可以使心肌细胞内游离钙浓度升高，去掉细胞外液中钙离子，KC1的升钙作用则消失。（2）甲氧胺和咖啡因可使心肌细胞内游离钙浓度升高。二者的作用分别被哌唑嗪和普鲁卡因所对抗。提示该法切实可行。     

Nerve Growth Factor Inhibits Gd^3＋ -sensitive Calcium Influx and Reduces Chemical Anoxic Neuronal Death

To investigate whether glutamate and voltage-gated calcium channels-independent calcium influx exists during acute anoxic neuronal damage and its possible relationship to neuronal protective function of NGF. In in vitro model of acute anoxia, hippocampal cultures from newborn rats were exposed to 3 mmol/L KCN. Changes of intracellular Ca^2＋ concentration （[Ca^2＋]i） were monitored by con-focal imaging and cell viability was assayed by PI and cFDA staining. The results showed that after treatment with primary hippocampal cultures with 3 mmol/L KCN for 15 min, [Ca^2＋]i was significantly increased 6.27-fold compared to pre-anoxia level and 73.3% of the cells died. When combination of 20 μmol/L MK-801 （glutamate receptor antagonist）, 40 μmol/L CNQX （AMPA receptor antagonist） and 5 μmol/L nimodipine （voltage-gated calcium channel antagonist） （hereafter denoted as MCN） were administrated to hippocampal cultures, levels of [Ca^2＋]i and cell death rate induced by KCN were partially reduced by 35.9% and 47.5% respectively. However, Gd^3＋ （10 μmol/L） almost completely blocked KCN-mediated [Ca^2＋]i elevation by 81.9% and reduced neuronal death by 88.8% in the presence of MCN. It is noteworthy that NGF, used in combination with MCN, inhibited KCN-induced [Ca^2＋]i increase by 77.4% and reduced cell death by 87.1%. Only PLC in- hibitor U73122 （10 μmol/L） abolished NGF effects. It is concluded that Gd^3＋-sensitive calcium influx, which is NMDA （glutamate receptor） and voltage-gated calcium channels-independent, is responsible for acute anoxic neuronal death. NGF can inhibit Gd^3＋-sensitive calcium influx and reduce anoxic neuronal death through activating PLC pathway.     

Experimental Study of Effect of Corticosterone on Primary Cultured Hippocampal Neurons and Their Ca^2＋／CaMK Ⅱ Expression

Summary: To explore the effect of different concentrations of corticosterone (CORT) on primary cultured hippocampal neurons and their Ca^2 /CaMK Ⅱ expression and possible mechanism, the changes of hippocampal neurons were observed in terms of morphology, activity of cells, cell death.concentrations of cytosolic free calcium, and the expression of CaMK Ⅱ by using MTT assay, flow cytometry, fluorescent labeling of Fura-2/AM and Western hlotting after 10^-7. 10^-8 and 10^-5 mol/L of CORT was added to culture medium. The evident effect of 10^-6 and 10^-5 mol/L of CORT on the morphology of hippocampal neuron was found. Compared with control neurons, the activity of the cells was markedly decreased and [Ca^2 ], increased in the neurons treated with 10^-6 and 10^-7mol/L of CORT. but no change was observed in the neuron treated with 10^-7 mol/L of CORT.The death was either by way of apoptosisor necrosis in the cells treated with 10^-4 and 10^-5mol/L of CORT respectively. The correlation analysis showed that a reverse correlation existed between [Ca^2 ];and the expression of CaMKⅡ. Either apoptosis or necrosis occurs in the hippocampal neurons treated with CORT. The increased hippocampal [Ca^2 ], is both the result of CORT impairing the hippocampal neurons and the cause of the apoptosis of hippocampal neurons and the decreased CaMKⅡ expression.     

睫状神经营养因子对N-甲基-D-天冬氨酸引起海马神经元内钙离子浓度升高的影响

①目的　研究睫状神经营养因子 (CNTF)对谷氨酸 (Glu)受体激动剂N 甲基 D 天冬氨酸 (NMDA)引起的海马神经元内游离Ca2 + 浓度 ([Ca2 + ]i)升高的影响 ,并探讨G蛋白是否参与此作用。②方法　原代培养海马神经元 ,用活细胞内荧光探针Fura 2 /AM实时检测应用CNTF和G蛋白抑制剂百日咳毒素 (PTX)后由NMDA引起的海马神经元 [Ca2 + ]i 的变化。③结果　用CNTF孵育经NMDA刺激的海马神经元后 ,细胞内 [Ca2 + ]i 升高的程度较单独NMDA刺激细胞引起的 [Ca2 + ]i 升高程度低 (t=2 .97～ 4 .86 ,P <0 .0 5 ) ,加入PTX可减弱CNTF的抑制作用。④结论　G蛋白可能参与CNTF调节NMDA引起的海马神经元内 [Ca2 + ]i 升高的快速作用途径     

吡咯喹啉醌对谷氨酸损伤大鼠海马神经元Bcl-2、Bax表达的影响

目的:观察吡咯喹啉醌(PQQ)对谷氨酸损伤海马神经元存活和Bcl-2、Bax表达的影响。方法:原代培养胚鼠海马神经元,谷氨酸损伤15min后加入不同浓度的PQQ(50、100、200μmol/L)孵育24小时,采用MTT法检测细胞活性;Western blot法检测Bcl-2、Bax的表达。结果:不同浓度PQQ均可明显改善谷氨酸损伤后细胞的活力,增加Bcl-2/Bax的比值,且随浓度增加比值增高。结论:PQQ对谷氨酸损伤的海马神经元具有一定保护作用。     

大鼠海马神经元大电导钙激活钾通道的全细胞电流记录

目的 探讨记录全细胞的大电导钙激活钾通道的技术方法。方法 首先酶解急性分离大鼠海马神经元；利用全细胞膜片钳技术记录大电导钙激活钾电流。结果 可记录到一系列快速的外向钾离子电流。结论 所记录到的外向钾离子电流中，快速出现并很快减小的呈尖峰样的瞬变外向电流主要由大电导的钙激活钾电流组成。     

老年大鼠脑细胞内钙的变化

目的:观察老年大鼠脑细胞内钙浓度([Ca2 ]i)的变化.方法:以Fura/AM为荧光指示剂,采用荧光分光光度计测定急性分离大鼠脑[Ca2 ]i.结果:静息状态下老年大鼠脑[Ca2 ]i及对高钾除极化的反应均明显低于青年对照组(P＜0.01),但其增强作用在老年大鼠与青年大鼠间无明显差异(P＞0.05).结论:老年大鼠静息脑[Ca2 ]i降低,但在相同情况下老年大鼠脑细胞内钙超载更加明显,从而较青年大鼠更易发生脑细胞的损害.     

抑郁大鼠海马神经元损伤及其机制

目的探讨抑郁大鼠海马神经元损伤的机制.方法采用不可预测的应激建立大鼠抑郁模型,open field法观察动物的抑郁行为,组织化学观察和计数海马神经元数量,Fara-2荧光法测海马突触体内游离钙浓度.结果与对照组相比,抑郁大鼠水平运动和垂直活动次数显著减少,体重增幅和糖水消耗量显著下降,海马神经元大量丢失,同时海马突触体内游离钙浓度显著增高.结论抑郁大鼠表现的抑郁行为可能与海马神经元内钙离子浓度异常升高从而引起细胞大量丢失有关.     

CNTF对AMPA和NMDA引起海马神经元Ca2+浓度升高的作用

①目的探讨睫状神经营养因子(CNTF)对两种谷氨酸(Glu)离子型受体激动剂(α-氨基羧甲基异哑恶唑丙酸,AMPA;N-甲基-D-天冬氨酸,NMDA)激发的海马神经元内游离Ca2+([Ca2+]i)升高的作用.②方法原代培养海马神经元,在有或无CNTF条件下用活细胞内荧光探针Fura-2-AM实时检测AMPA和NMDA引起海马神经元内[Ca2+]i变化的情况.③结果 AMPA和NMDA均可引起细胞内[Ca2+]i升高,并呈量效依赖性;CNTF可快速抑制NMDA引起的海马神经元内[Ca2+]i升高(t=2.97～4.86,P＜0.05),而对AMPA的作用无影响(t=0.22～0.74,P＞0.05).④结论 CNTF可能通过与NMDA型受体相互作用而快速启动Ca2+信号的途径,保护因Glu引起的海马神经元损伤.AMPA型受体可能与CNTF的快速作用无关.     

睾酮对自由基损伤的海马神经元神经保护作用中细胞内Ca2+变化

目的：观测睾酮对自由基损伤的海马神经元神经保护用中是否有细胞内Ca2＋变化。方法：原代培养海马神经元,暴露于自由基并损伤后,分为对照组、不同浓度过氧化氢组、睾酮组;利用荧光探针进行标记,激光共聚焦显微镜下观察加入睾酮前后细胞内Ca2＋变化。结果：以100μM和200μM H2O2组引起细胞内Ca2＋增加最为明显;与对照组相比,Fi值变化差异有统计学意义（P〈0.05）;预先加入睾酮后再暴露于100μM H2O2与单独暴露于相同浓度H2O2组相比,细胞内Ca2＋荧光比值明显降低,二者差异有统计学意义（P〈0.05）。结论：自由基损伤能引起原代培养海马神经元内Ca2＋含量升高,睾酮能明显减轻由于自由基损伤引起的细胞内Ca2＋变化。     

Protective effects of Ginkgo biloba extract on rats during cerebral ischemia/rep erfusion

Objective To study the effect of Ginkgo biloba extract on rats during ischemia/reperfusion and its influence on intracellular calcium in hippocampal neurons.Methods Model of intraluminal occlusion of the middle cerebral artery (MCAO) was used to prepare the ischemia/reperfusion cortex tissue.Concentration of MDA was determined by measuring thiobarbituric acid-reactive substance.GSH-PX was quantified using the thiobarbituric acid (TBA) technique.SOD was assayed througha xanthine method.Endogenous amino acids were quantified by high performance liquid chromatographic (HPLC) analysis.Primary culturs of hippocampal neurons were prepared for a free intracellular calcium (［Ca(2+)］I ) assay by Fura-2 based single cell microfluoremetric technique.Results Comparing control and treatment groups, the concentration of SOD and GSH-PX were higher, whereas that of MDA was much lower; the concentration of glutamate and aspartate decreased and that of GABA increased markedly at all time point (P&lt;0.01), Gly also decreased at some time points (P&lt;0.05). The differences were significant between the groups of 10 mg/kg, 15 mg/kg and the groups of 5 mg/kg.When 1×10(-5) mol/L glutamate was applied with 25 μg/ml ginkgo biloba extract to cultured neurons, the increase in ［Ca(2+)］I was lower than that caused by applying glutamate alone.Its peak value was much lower and increased phase was longer, its declining phase was shorter.After returning to baseline, the application of 1×10(-5) mol/L glutamate could induce the reaction to recover.Conclusions Ginkgo biloba extract could protect damaged neurons by keeping the balance of inhibitory/excitatory aminoacids, enhancing the free radical scavengers system, and inhibiting the effect of glutamate on ［Ca(2+)］I.