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1.
实验用Fluo-3负载细胞,在激光扫描共聚焦显微镜下直接监测缺氧后分散培养的大鼠海马CA1区神经元内游离Ca^2+浓度([Ca^2+]i)的变化,观察腺苷对这种变化的影响并初步探讨其作用机制。结果发现,急性缺氧使海马神经元[Ca^2+]i显著升高;腺苷(100μmol/L)明显抑制缺氧引起的[Ca^2+]i增高,腺苷A1受体拮抗剂CPT以及K^+通道阻断剂4-AP和ATP敏感性K^+通道阻断剂gl 相似文献
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为了探讨糖皮质激素对海马兴奋性神经元和抑制性神经元的作用,本实验将地塞米松注入大白鼠侧脑室,2h 后经Nissl染色法、免疫组织化学方法和细胞计数法观察了海马谷氨酸免疫反应性(GluIR)神经元和γ氨基丁酸免疫反应性(GABAIR)神经元的变化。结果显示:(1)CA1、CA3 和SG区的GluIR神经元明显增多,特别是CA1 区。经细胞计数统计分析表明,与对照组相比CA1 有极显著性差异(P< 0001),CA3区有显著性差异(001< P< 005),SG处无明显差异(P> 005)。(2)与对照组相比,GABAIR神经元无明显变化。结果表明,糖皮质激素有增加海马谷氨酸能神经元的作用。尽管γ氨基丁酸能神经元无明显变化,并不表明糖皮质激素对其无影响 相似文献
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γ—氨基丁酸抑制缺氧所致神经元钙超载 总被引:4,自引:0,他引:4
本文以体外分散培养的新生大鼠海马CA1区神经细胞为标本,分别采用激光扫描共聚集显微镜动态监测单个细胞[Ca^2+]i和膜片箝全细胞记录的电生理技术检测细胞的NMDA电流和电压依赖性Ca^2+电流等方法,较为深入地研究了抑制性神经递质γ-氨基丁酸(GABA)及GABA-A受体激动剂蝇蕈醇对急性缺氧时海马CA1神经元[Ca^2+]i升高过程的影响方式及其作用机制。结果表明:对照组细胞缺氧后比缺氧前[C 相似文献
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垂体腺苷酸环化酶激活肽减轻谷氨酸对海马神经元的损伤并抑制胞内钙升高 总被引:6,自引:1,他引:6
对原代培养7~9d的海马神经元给予谷氨酸处理,24h后,神经元的存活率降低。预先给予垂体腺苷酸环化酶激活肽(PACAP)能显著减少谷氨酸引起的海马神经元死亡。谷氨酸呈剂量依赖性增加海马神经元细胞内钙离子含量,PACAP能抑制谷氨酸引起的海马神经元细胞内钙离子浓度的升高,特异性PACAP Ⅰ型受体拮抗剂PACAP 6-38能完全阻断PACAP减轻谷氨酸所致海马神经元损伤及降低谷氨酸所致神经元细胞内钙 相似文献
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一氧化氮对谷氨酸诱发的大鼠海马脑片CA1区神经元活动的抑制 总被引:5,自引:1,他引:4
用细胞外记录单位放电技术,在大鼠海马脑片上观察了L-精氨酸(L-arg)、N-硝基L-精氨酸(L-NNA)及SIN-1对谷氨酸(glutamate,Glu)诱导的CA1区神经元放电的影响。旨在了解L-精氨酸:NO通路在谷氨酸诱发的海马放电中的作用及其可能的机制。结果如下:(1)用GlU(0.5mmol/L)灌流海马脑片1min,12个放电单位放电频率明显增加,表现为癫痫样放电;(2)海马脑片2mi 相似文献
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大鼠海马CA1区锥体细胞的急性分离和鉴定 总被引:9,自引:0,他引:9
目的:建立一种适用膜片钳技术的大鼠海马CA1区锥体细胞的急性分离方法,并对其进行鉴定。方法:采用酶加机械分离法制备7~10d鼠龄的大鼠海马CA1区锥体细胞,用免疫荧光技术鉴定神经元,其电生理学特性测定用全细胞膜片钳技术。结果:分离的锥体细胞是海马CA1区锥体细胞,且具有正常的电生理学特性,并保存了某些主要的电压依赖性和受体依赖性离子通道特性。结论:成功地建立了一种简单、快速、高产的适用于膜片钳技术的大鼠海马CA1区锥体细胞的急性分离方法。 相似文献
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为了确定β-淀粉样蛋白(AβP)在影响神经元电生理特性并导致神经毒作用时的最短活性序列,实验采用膜片钳技术,在急性分离的大鼠海马CAI区锥体细胞的“内面向外”式膜片上,观察了AβPR的31-35和25-35片段对Ca2+激活大电导钾(BK)通道活动的影响。结果显示,浴液中给予 5μmol/L的AβP 31-35后,BK通道的平均开放概率(Po)和开放频率在1~3min内分别减少了85.8%(P<0.01)和72.1%(P<0.01);平均开放时间减少了41.1%(P<0.01);平均电流幅度则无明显改变(P>0.05);给子同样摩尔浓度的AβP 25-35后,BK通道平均Po减少了85.5%(P<0.01),平均开放时间减少了51.4%,(P<0.05)。结果提示,两种AβP片段对海马神经元BK通道具有抑制作用,这可能与AβP的神经毒性作用有关;AβP31-35片段可能是AβP分子中影响细胞电生理特性的最小活性序列。 相似文献
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采用全细胞膜片钳技术,研究了三氯化铝(AlCl3)对急性分离的大鼠海马CA1区神经元钾通道的影响。结果表明,AlCl3对钾电流有明显的抑制作用,具有一定的浓度依赖性,1000μmol/AlCl3可改变IA和IX激活曲线和失活曲线的Vb和k值,使钾电流激活曲线右移,使失活曲线左移。这些结果表明AlCl3对大鼠海马CA1区神经元K^ 通道有抑制作用,它可能是铝引起中枢神经系统损伤的机制之一。 相似文献
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5—羟色胺抑制谷氨酸对海马神经元的毒性作用 总被引:5,自引:1,他引:5
为探讨5-羟色胺(5-HT)对过量谷氨酸(glutamate,Glu)神经毒性的影响。观察了5-HT存在时,过量Glu对海马细胞存活率、海马脑片CA1区群锋电位(population spike,PS)及神经细胞膜Ga^2 电流的影响。结果发现:5-HT可明显提高过量Glu作用下海马神经细胞的存活率,减缓Glu对海马脑片CA1区PS的降低作用;在细胞膜上,5-HT可明显减弱Glu诱导的Ca^2 内向电流,推测,一定浓度的5-HT具有抑制过量Glu神经毒性的作用。在细胞膜上5-HT可明显减弱Glu诱导的Ca^2 内向电流,推测,一定浓度的5-HT具有抑制过量Glu神经毒性的作用,其机制可能在于5-HT与细胞膜上特定的受体结合,抑制了Glu诱导的Ca^2 内流。 相似文献
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The glial GLAST and GLT-1 glutamate transporters are transiently expressed in hippocampal neurons as shown by immunocytochemistry (Plachez et al., 2000. J. Neurosci. Res., 59, 587-593). In order to test if this transient expression is associated to a transient glutamate uptake activity, [3H]-glutamate uptake was studied during the in vitro development of embryonic hippocampal neurons cultured in a defined (serum free) medium. In these cultures, the ratio of the number of glial cells to the number of neurons increased from 1.7 to 11.3% during the first 10 days of culture, while 77% of the neurons died. The number of neurons then remains stable up to 23 days of culture. The initial glutamate uptake velocity at 20 and 200 microM [3H]-glutamate usually increased about five times between 1 and 10 days in vitro (DIV). Interestingly, at 2 microM [3H]-glutamate, the uptake initial velocity showed a biphasic pattern, with a transient peak between 1 and 6 DIV, the maximum being reached at 2 DIV and a delayed regular increase from 8 to 23 DIV. The concentration-dependent curves were best fitted with two saturable sites high and low affinities, at both 2 and 10 DIV. To pharmacologically characterize the transient increased glutamate uptake activity, four uptake inhibitors, L-threo-3-hydroxy-aspartic acid (THA), L-trans-pyrrolidine-2,4-dicarboxylic acid (L-trans-2,4-PDC), dihydrokainate (DHK), and DL-threo-beta-benzyloxyaspartate (TBOA) were tested. THA, L-trans-2,4-PDC and DL-TBOA inhibited glutamate uptake both at 2 and 10 DIV, while the GLT-1 selective uptake inhibitor DHK neither strongly affected the uptake at 2, nor at 10 DIV. These data indicated that, besides the regular increase in the glial-dependent glutamate uptake activity, a transient high-affinity, DHK insensitive, glutamate transport activity in hippocampal neurons in culture is present. This latter activity could potentially be related to the transient expression of the glial GLAST transporter in neurons. 相似文献
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Abstract: The effect of three metabolic inhibitors—iodoacetate, potassium cyanide, and potassium arsenate—on neuronal viability was studied in primary rat cortical and hippocampal CA1 neuronal cultures. Iodoacetate (0.1 m M ) applied for 5 min to 8-day-old cultures resulted in delayed neuronal death within 3–24 h in cortical and hippocampal CA1 neurons. Neuronal degeneration was preceded by transient inhibition of energy metabolism to ∼40% and a permanent inhibition of protein synthesis to ∼50%. The inhibition of protein synthesis and the neuronal death were prevented by the free radical scavenger vitamin E but not by the glutamate antagonist MK-801. Removal of calcium during iodoacetate exposure could not protect against toxicity, and there was no increase of intracellular calcium concentration during and shortly after iodoacetate treatment. Cyanide and arsenate produced only partial neuronal degeneration, even at a dose of 10 m M . These observations demonstrate that brief exposure of neurons to low concentrations of iodoacetate produces a delayed type of neuronal death that is not mediated by either calcium or glutamate. The therapeutic effect of vitamin E points to a free-radical mediated injury and suggests that this type of pathology may also be involved in delayed neuronal death after transient energy depletion in vivo. 相似文献
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We have focused on activation mechanisms of calcium/calmodulin-dependent protein kinase (CaM) kinase I in the hippocampal neurons and compared them with that of CaM kinase IV. Increased activation of CaM kinase I occurred by stimulation with glutamate and depolarization in cultured rat hippocampal neurons. Similar to CaM kinases II and IV, CaM kinase I was essentially activated by stimulation with the NMDA receptor. Although both CaM kinases I and IV seem to be activated by CaM kinase kinase, the activation of CaM kinase I was persistent during stimulation with glutamate in contrast to a transient activation of CaM kinase IV. In addition, CaM kinase I was activated in a lower concentration of glutamate than that of CaM kinase IV. Depolarization-induced activation of CaM kinase I was also evident in the cultured neurons and was largely blocked by nifedipine. In the experiment with 32P-labeled cells, phosphorylation of CaM kinase I was stimulated by glutamate treatment and depolarization. The glutamate- and depolarization-induced phosphorylation was inhibited by the NMDA receptor antagonist and nifedipine, respectively. These results suggest that, although CaM kinases I and IV are activated by the NMDA receptor and depolarization stimulation, these kinase activities are differently regulated in the hippocampal neurons. 相似文献
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Strukova SM Kiseleva EV Dugina TN Glusa E Storozhevykh TP Pinelis VG 《Rossi?skii fiziologicheski? zhurnal imeni I.M. Sechenova / Rossi?skaia akademiia nauk》2005,91(1):53-60
The effect of thrombin on the rat hippocampal neurons death in model of neurotoxicity induced by hemoglobin or glutamate, was studied. Thrombin (10 nM) was shown to inhibit 100-mkM glutamate--or 10-mkM hemoglobin-induced apoptosis of the rat hippocampal neurons. With the aid of PAR1 (protease-activated receptor1) agonist peptide and PAR1 antagonist, the PAR1 was found to be necessary for protective action of thrombin in hippocampal neurons in models of neurotoxicity induced by hemoglobin or glutamate. Because the prolonged elevation [Ca2+] ib neurons is a critical part of neurodestructive processes in CNS, the effect of thrombin on Ca2+-homeostatis of neurons after its injury by the inducer of neuronal apoptosis: a synthetic agonist of the NMDA receptors N-methyl-D-aspartate (NMDA), was studied. We hypothesized that thrombin via receptors PAR may prove to be neuroprotective for the hippocampus. Thrombin was shown to stimulate via PAR1 a transient increase in [Ca2+] in neurons in a concentration-dependent manner. Thrombin (1 nM) decreased the [Ca2+] signal induced by activation of the NMDA-subtype of glutamate receptors. This thrombin effect may be one of the reasons of the protective action of thrombin in hippocampal neurons. 相似文献
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高效氯氰菊酯对大鼠海马CA3区神经元电压门控钾通道的影响 总被引:5,自引:0,他引:5
利用全细胞膜片钳技术,在急性分离的新生大鼠海马CA3区锥体细胞上研究高效氯氰菊酯的两种组分高顺氯氰菊酯和高反氯氰菊酯对瞬时外向钾电流(transient outward potassiumcurrent,IA)和延迟整流钾电流(delayed rectifier potassiumcurrent,Ik)的影响。高顺氯氰菊酯使IA增大,而高反氯氰菊酯则使IA减小。高顺和高反氯氰菊酯均使IA激活曲线左移,反式结构还可促进IA的失活。高顺和高反氯氰菊酯均使IK减小,并使其激活曲线左移,而对IK的失活过程无影响,高反氯氰菊酯可使IK失活后恢复过程延长。结果表明,瞬时外向钾通道和延迟整流钾通道同样是高效氯氰菊酯的作用靶点,这可能是高效氯氰菊酯对哺乳动物产生毒性作用的原因之一。 相似文献
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Loss or "gain" of function mutations in voltage-gated ion channels often results in an adverse neurological phenotype. We have examined the electrical characteristics of hippocampal pyramidal cells in a transgenic mouse model to determine how overexpression of a Shaker-type potassium channel subunit during early postnatal development might alter excitability properties of developing neurons. We found that in CA3 neurons potassium channel overexpression led to a transient shortening in duration of single action potentials during the first two postnatal weeks. There was an increase in maximal repolarization rate, without significant effect on the rate of rise. Transgenic CA3 neurons also showed a decrease of firing frequency in response to sustained depolarizing current injection. In contrast, repolarization of action potentials in CA1 neurons was not significantly altered by trangene expression. Western Blot Analysis of membrane-associated transgene protein indicated that transgene protein levels decreased during development, in agreement with functional measures of spike width. Our data indicate that the functional consequences of potassium channel transgene expression are dependent on cellular environment and developmental stage. A transient period of hypoexcitability during a critical period of development for CA3 neurons may contribute to the hyperexcitable phenotype observed in adult animals. 相似文献
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The D-isomer of aspartate is efficiently transported by high-affinity Na(+)/K(+)-dependent glutamate transporters and is an effective ligand of N-methyl-d-aspartate (NMDA) receptors. To facilitate analysis of the regulation of these proteins in their native membranes, we synthesized a photolabile analogue of D-aspartate, 4-methoxy-7-nitroindolinyl-D-aspartate (MNI-D-aspartate). This compound was photolyzed with a quantum efficiency of 0.09 at pH 7.4. Photorelease of d-aspartate in acute hippocampal slices through brief (1 ms) UV laser illumination of MNI-d-aspartate triggered rapidly activating currents in astrocytes that were inhibited by the glutamate transporter antagonist DL-threo-beta-benzyloxyaspartic acid (TBOA), indicating that they resulted from electrogenic uptake of D-aspartate. These transporter currents exhibited a distinct tail component that was approximately 2% of the peak current, which may result from the release of K(+) into the extracellular space during counter transport. MNI-D-aspartate was neither an agonist nor an antagonist of glutamate transporters at concentrations up to 500 muM and was stable in aqueous solution for several days. Glutamate transporter currents were also elicited in Bergmann glial cells and Purkinje neurons of the cerebellum in response to photolysis of MNI-D-aspartate, indicating that this compound can be used for monitoring the occupancy and regulation of glutamate transporters in different brain regions. Photorelease of D-aspartate did not activate alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptors or metabotropic glutamate receptors (mGluRs) in neurons, but resulted in the selective, but transient, activation of NMDA receptors in hippocampal pyramidal neurons; MNI-D-aspartate was not an antagonist of NMDA receptors. These results indicate that MNI-D-aspartate also may be useful for studying the regulation of NMDA receptors at excitatory synapses. 相似文献
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Purinergic receptors mediate two distinct glutamate release pathways in hippocampal astrocytes 总被引:9,自引:0,他引:9
The purinergic P2X(7) receptor (P2X(7)R) can mediate glutamate release from cultured astrocytes. Using patch clamp recordings, we investigated whether P2X(7)Rs have the same action in hippocampal astrocytes in situ. We found that 2- and 3-O-(4-benzoylbenzoyl)ATP (BzATP), a potent, although unselective P2X(7)R agonist, triggers two different glutamate-mediated responses in CA1 pyramidal neurons; they are transient inward currents, which have the kinetic and pharmacological properties of previously described slow inward currents (SICs) due to Ca(2+)-dependent glutamate release from astrocytes, and a sustained tonic current. Although SICs were unaffected by P2X(7)Rs antagonists, the tonic current was inhibited, was amplified in low extracellular Ca(2+), and was insensitive to glutamate transporter and hemichannel inhibitors. BzATP triggered in astrocytes a large depolarization that was inhibited by P2X(7)R antagonists and amplified in low Ca(2+). In low Ca(2+) BzATP also induced lucifer yellow uptake into a subpopulation of astrocytes and CA3 neurons. Our results demonstrate that purinergic receptors other than the P2X(7)R mediate glutamate release that evokes SICs, whereas activation of a receptor that has features similar to the P2X(7)R, mediates a sustained glutamate efflux that generates a tonic current in CA1 neurons. This sustained glutamate efflux, which is potentiated under non-physiological conditions, may have important pathological actions in the brain. 相似文献