苯妥英对缺氧大鼠大脑皮质神经元钙激活钾通道的影响

目的 :研究苯妥英对缺氧大鼠大脑皮质神经元钙激活钾 [K(Ca) ]通道的影响。方法 :应用膜片钳单通道技术记录苯妥英对缺氧大鼠大脑皮质神经元上K(Ca)通道电流活动。电流信号经放大、滤波及转换后输入微机进行采样、储存和分析。结果 :苯妥英对缺氧大脑皮质神经元K(Ca)通道具有明显的激活作用 ,主要是增加通道的开放概率 (openprobability,Po)。结论 :苯妥英激活大脑皮质神经元K(Ca)通道 ,产生超极化电位 ,从而稳定细胞膜 ,降低细胞兴奋性 ,延缓缺氧除极的发生 ,这可能是苯妥英抗缺血脑损伤的另一个重要机制     

缺氧对大鼠大脑皮层神经元钙激活性钾通道的影响

目的　研究缺氧对大鼠大脑皮层神经元钙激活性钾 (Kca)通道的影响 ,以揭示神经元抗缺血损伤的电生理机制。方法　在不同缺氧条件下 ,应用膜片钳技术记录大脑皮层神经元上Kca通道电流活动。电流信号经放大、滤波及A/D、D/A转换后输入微机进行采样和储存。实验数据应用PClamp(6 .0 .2 )软件进行分析处理。结果　缺氧对通道的开放概率 (Po)及平均开放时间 (To)有明显影响 ,在缺氧实验早期通道Po明显增加 ,其中 10 μmol·L-1NaCN缺氧组其增加程度大于 2 0 μmol·L-1和 30 μmol·L-1NaCN缺氧组 (P <0 .0 5 )。而在缺氧实验后期通道Po和To明显降低 ,其中 30 μmol·L-1NaCN缺氧组其降低程度大于 2 0 μmol·L-1和 10 μmol·L-1NaCN缺氧组 (P <0 .0 5 )。结论　缺氧早期大脑皮层神经元Kca通道激活 ,产生超极化电位 ,从而稳定细胞膜 ,降低细胞兴奋性 ,延缓缺氧除极的发生 ,这可能是神经元自身的一种代偿作用     

缺氧对大鼠大脑皮层神经元外激活性钾通道的影响

目的：研究缺氧对大鼠大脑皮层神经元钙激活性钾（Kca)通道的影响，以揭示神经元抗缺血损伤的电生理机制。方法：在不同缺氧条件下，应用膜片钳技术记录大脑皮层神经元上Kca通道电流活动，电流信号经放，滤波及A／D、D／A转换后输入微机进行采样和储存。实验数据应用PClamp(6.0.2)软件进行分析处理。结果：缺氧对通道的开放概率（Po)及平均开放时间（To)有明显影响，在缺氧实验早期通道Po明显增加，其中10μmol.L^-1NaCN缺氧组其增加程度大于20μmol.L^-1和30μmol.L^-1NaCN缺氧组（P＜0．05）。而在缺氧实验后期通道Po和To明显增降低，其中30μmol.L^-1NaCN缺氧组其降低程度大于20μmol.L^-1和10μmol.L^-1NaCN缺氧组（P＜0．05）。结论：缺氧早期大脑皮层神经元Kca通道激活，产生超极化电位，从而稳定细胞膜，降低细胞兴奋性，延缓缺氧除极的发生，这可能是神经元自身的一种代偿作用。     

GABA_A受体激动剂对AD模型配体门控氯通道的作用

目的观察并分析GABA_A受体激动剂蝇蕈醇对GABA激活的配体门控氯通道的作用,为寻找新的抗阿尔茨海默病(AD)药物提供实验依据。方法 Aβ_(1-42)诱导海马神经细胞作为AD细胞模型;通过膜片钳(Patch clamp)技术在大鼠海马神经元上记录配体门控氯通道瞬时外向电流,采用非特异性氯通道阻断剂(NFA)和无GABA的细胞外液确定该电流成份为瞬时外向氯电流;通过膜片钳技术观察GABA_A受体激动剂蝇蕈醇对于在大鼠海马神经元上记录瞬时外向电流的作用;向AD细胞模型中加入GABA_A受体激动剂,采用MTT观察各组细胞活力。结果加入GABA_A受体激动剂蝇蕈醇后GABA激活氯电流强度增加;加入浓度为1 mmol/L蝇蕈醇的AD模型细胞组存活率为(91.32±3.73)%,略高于未加入蝇蕈醇的模型细胞组,两组对比P0.05。结论 GABA_A受体激动剂蝇蕈醇对于AD细胞模型具有保护作用。     

酮体抑制大鼠海马神经元酸敏感通道的开放

目的 探讨酮体对酸敏感通道的影响。方法 原代培养大鼠海马神经元,利用膜片钳技术检测乙酰乙酸、β羟丁酸和丙酮三种酮体对海马神经元酸敏感通道开放状态的影响。结果 在pH6.0条件下细胞外液中分别加入乙酰乙酸、β羟丁酸和丙酮,酸敏感通道电流均明显减弱。三种酮体均可显著抑制海马神经元表面酸敏感通道的开放,抑制率分别为92、47和77%。结论 酮体可以显著抑制酸敏感通道的开放。生酮饮食治疗难治性癫痫有可能是通过酮体抑制酸敏感通道来实现。     

化学预处理对大鼠海马脑片缺氧耐受性的影响及其与线粒体ATP敏感性钾通道的关系

目的:探讨3-硝基丙酸(3-NPA)预处理对海马脑片缺氧损伤的保护作用及线粒体内膜ATP敏感性钾(Mi-toK_(ATP))通道在预处理脑保护机制中的作用。方法:采用Nissl染色和透射电镜观察缺氧后大鼠海马CA_1区神经元密度、锥体细胞和亚细胞结构在预处理前后的变化,以及MitoK_(ATP)拮抗剂5-羟基癸酸盐(5-HD)孵育海马脑片对预处理效果的影响。结果:3-NPA组大鼠缺氧后海马CA_1区神经元密度(86.69±4.87)高于对照组(53.85±3.13,P<0.05),锥体细胞超微结构受损程度较对照组明显减轻。预处理大鼠海马脑片经100μmol/L 5-HD孵育后CA_1区神经元密度(57.31±7.89)较未孵育脑片降低(P<0.05),超微结构受损加重。结论:3-NPA预处理可提高海马脑片缺氧耐受能力,这种保护作用可能与MitoK_(ATP)通道开放有关。     

胞内乳酸对急性分离大鼠新皮层神经元KATP通道的影响

采用膜片钳技术内面向外式记录法，研究了胞内乳酸对急性分离大鼠皮层神经元上ＡＴＰ敏感钾通道（ＫＡＴＰ通道）的影响。结果显示：浴槽液中加入５～２０ ｍ ｍ ｏｌ／Ｌ乳酸，通道电流幅度增大，电导由（２０２±１１）ｐＳ增到大（２５３±１３）ｐＳ；乳酸浓度＞ ２０ ｍ ｍ ｏｌ／Ｌ时，部分通道出现多级开放。当膜超极化时，乳酸可增加通道平均开放时间及开放概率并随其浓度的增大而增加。浴槽液中乳酸浓度为２０ ｍ ｍ ｏｌ／Ｌ时，ＡＴＰ阻断通道电流活动的半数有效浓度（ＩＣ５０）为１ ｍ ｍ ｏｌ／Ｌ，较无乳酸时ＩＣ５０（０．０５ ｍ ｍ ｏｌ／Ｌ）明显增高，即通道对ＡＴＰ敏感性明显降低（Ｐ＜ ０．０１）。上述结果表明：胞内乳酸可通过增大电导、增加开放概率、诱导多通道开放及降低通道的ＡＴＰ敏感性参与调节皮层神经元上ＫＡＴＰ通道。提示：脑在缺氧情况下，胞内无氧代谢所产生的乳酸可先于ＡＴＰ耗竭激活ＫＡＴＰ通道，降低神经元兴奋性，从而起到保护作用。     

海马电刺激对耐药性颞叶癫痫大鼠CA1区神经元钠通道电流的影响

目的 建立多药耐药性颞叶癫痫模型,以海马CA1区锥体细胞钠通道电流的变化为观察指标,探讨海马电刺激治疗耐药性颞叶癫痫的可能机制.方法 选用Wistar大鼠80只制作慢性杏仁核点燃癫痫模型,制作成功后用经典抗癫痫药苯妥英钠和苯巴比妥进行筛选,根据癫痫大鼠对药物的反应区别出耐药癫痫大鼠及药物敏感大鼠,将筛选出的耐药性癫痫大鼠分为海马刺激组(n=6)及耐药对照组(n=6),用膜片钳全细胞记录模式观察海马电刺激后脑细胞钠通道电流的变化.结果 进行海马电刺激2周后,刺激杏仁核诱发的癫痫发作明显减轻,海马刺激组与耐药对照组Racine分级分别为(2.32±0.38)级和(4.45±0.42)级,差异具有统计学意义(t=84.600,P=0.000),后放电各项参数也明显改善,膜片钳全细胞记录结果表明,海马刺激组钠通道电流峰值及激活曲线向去极化方向偏移,失活曲线向超极化方向偏移,海马刺激组钠通道失活后恢复时间[(17.9±0.6)s]较耐药对照组[(16.3±0.3)s]明显延长(t=-25.420,P=0.000).结论 海马电刺激可以抑制钠通道电流,其治疗耐药性癫痫的作用可能是通过抑制钠通道电流而降低脑细胞兴奋性,从而减少癫痫性电活动的产生而实现的.     

应用脑片红外可视膜片钳技术观察大鼠海马神经元癫痫样放电

目的 应用脑片红外可视膜片钳技术观察大鼠海马神经元癫痫样放电。方法 应用红外微分干涉相差技术和膜片钳技术实时观察40只SD大鼠脑片海马CA1锥体神经元形态（与生物素染色对照）,记录全细胞电流;通过青霉素、藜芦碱、无钙人工脑脊液、无镁人工脑脊液、甲基四乙胺等不同致痫药物胞外灌流,观察其诱发的癫痫活动。结果 红外可视条件下,海马CA1锥体神经元的胞体和树突清晰可见,与生物素染色的细胞形态一致。青霉素、藜芦碱、无钙人工脑脊液、无镁人工脑脊液、甲基四乙胺胞外灌流均可诱发大鼠海马CA1锥体神经元产生癫痫样活动。结论 红外可视膜片钳技术可实时观察神经元形态,有效提高其封接成功率,同时可实时观察不同药物诱发的神经元癫痫样活动。     

ATP敏感性K^＋通道在海马缺氧损伤中的作用

目的研究ATP敏感性K 通道阻断剂glipizide（GLI）对缺氧后海马脑片损伤以及海马神经元[Ca(2 )]i变化的影响。方法以大鼠离体海马脑片和体外分散培养的海马神经元为标本，分别采用电生理微电极记录技术以及激光扫描共聚焦显微镜监测神经元[Ca(2 )]i的方法。结果预先用GLI（20μmol／L）灌流的海马脑片缺氧后PV持续时间较对照组显著缩短，提示其加重了海马不可逆缺氧损伤的发生；另外急性缺氧可诱导海马神经元[Ca(2 )]i迅速升高，而预先加入GLI（20μmol／L）能显著加剧[Ca(2 )]i的升高程度。结论ATP敏感性K 通道在缺氧过程中的开放对大鼠海马脑区具有重要的保护作用，它可显著降低缺氧所致神经元[Ca(2 )]i升高，提高海马脑片的抗缺氧能力。这可能是其对抗海马缺氧损伤的主要作用机制之一。     

氢气预处理对乳鼠海马神经元细胞缺氧/复氧损伤后细胞活力的影响

目的探讨氢气对乳鼠海马神经元细胞缺氧/复氧损伤后细胞活力的影响。方法原代培养的SD乳鼠海马神经元细胞,随机分成对照组,缺氧/复氧组和氢气预处理组。对照组常规培养;缺氧/复氧组在100%N2中培养15min后复氧30min;氢气预处理组在2%H2+98%N2中培养15min后复氧30min。采用MTT法检测乳鼠海马神经元细胞活力。结果氢气预处理能够增强细胞活力(P<0.05)。结论氢气预处理对海马神经元细胞缺氧/复氧损伤具有保护作用,可能与提高细胞活力有关。     

镁对海马神经元钙激活钾通道的作用

目的研究Mg     

快速老化小鼠海马神经元电压门控离子通道特点

目的：观察快速老化小鼠（Senescence-accelerated mouse,SAM)海马神经元的基本离子通道特点，并对抗快速老化亚系（SAM－resistance/1,SAMR1)与快速老化亚系（SAM－prone/8,SMAP8)的基本离子通道特点进行了比较，探讨了离子通道变化在衰老中的可能角度，方法：应用全细胞记录方式，观察并比较原代培养SAMR1和SAMP8海马神经元的电压门控离子通道及膜参数。结果：原代培养SAMR1和SAMP8海马神经元电压门控Na^2 通道电流（INa)和电压门控延迟整流K^ 通道电流（IK）的电学特点和幅度基本一致。SAMP8的电压门控Ca^2 通道电流（ICa)和瞬时外向K^ 通道电流（IA）的幅值则大于相同培养天数的SAMR1。经膜电容校正所得的ICa电流密度也表现出增大的变化规律。结论：SAMP8与SAMR1神经元间IA和ICa的差异可能与其神经系统变异而产生的学习记忆功能下降有关。     

降钙素基因相关肽对缺氧时大鼠海马培养神经元c—fos表达的影响

采用免疫组化（ABC）方法，观察缺氧诱导体外培养大鼠海马神经元c-fos的表达及降钙素基因相关肽（CGRP）的影响。结果显示，缺氧后海马神经元中Fos-免疫反应（Fos—IR）阳性胞核百分率随缺氧时间的延长而逐渐增多，图像分析的结果显示，缺氧后Fos—IR阳性胞核的平均失密度亦随缺氧时间的延长而逐渐增强。经CGRP孵育的海马神经元缺氧后Fos-IR阳性胞核的百分率和Fos-IR阳性胞核的平均光密度均明显低于非CGRP孵育组。本结果表明，缺氧能诱导体外培养海马神经元。c-fos的表达，神经肽CGRP能抑制缺氧海马神经元c-fos的表达。提示CGRP对海马神经元缺氧损伤可能具有一定保护作用。     

Inhibition of major cationic inward currents prevents spreading depression-like hypoxic depolarization in rat hippocampal tissue slices

Hypoxia-induced spreading depression-like depolarization (hypoxic SD, or anoxic depolarization) is accompanied by the near-loss of membrane potential, severe reduction of membrane resistance, and influx of Na⁺, Ca²⁺, Cl⁻ and water into neurons. The biophysical nature of these membrane changes is incompletely understood. In the present study we applied a pharmacological mixture (10 μM DNQX, 10 μM CPP, 1 μM TTX, and 2 mM Ni²⁺) to rat hippocampal tissue slices to inhibit major Na⁺ and Ca²⁺ inward currents. This inhibitory cocktail slightly depolarized CA1 pyramidal neurons and completely blocked all evoked potentials. In its presence severe hypoxia of up to 20 min duration failed to induce hypoxic SD and the accompanying intrinsic optical signal. Instead, only moderate, very slow negative shifts of the extracellular DC potential were observed. Following 10 min hypoxia and 1 hour wash-out of the inhibitors antidromic and orthodromic responses were still blocked but hypoxic SD with markedly delayed onset could be induced in most slices. In current-clamped CA1 pyramidal cells hypoxia induced a rapid, near-complete depolarization and decreased the input resistance by 89%. In the presence of the cocktail, however, hypoxia caused a gradual, partial depolarization, to about −40 mV; the membrane resistance decreased by only 37%. We conclude that simultaneous blockade of the known major Na⁺ and Ca²⁺ channels consistently prevents hypoxic SD. The hypothesis that SD initiation is the consequence of general loss of selective permeability or general membrane breakdown becomes unlikely. Instead, influx of Na⁺ and Ca²⁺ might play a crucial role in the generation of the rapid SD-like depolarization.     

Increased neuronal excitability after long-term O(2) deprivation is mediated mainly by sodium channels

We have previously observed that prolonged O(2) deprivation alters membrane protein expression and membrane properties in the central nervous system. In this work, we studied the effect of prolonged O(2) deprivation on the electrical activity of rat cortical and hippocampal neurons during postnatal development and its relationship to Na(+) channels. Rats were raised in low O(2) environment (inspired O(2) concentration = 9.5+/-0.5%) for 3-4 weeks, starting at an early age (2-3 days old). Using electrophysiologic recordings in brain slices, RNA analysis (northern and slot blots) and saxitoxin (a specific ligand for Na(+) channels) binding autoradiography, we addressed two questions: (1) does long-term O(2) deprivation alter neuronal excitability in the neocortical and hippocampal neurons during postnatal development? and (2) if so, what are the main mechanisms responsible for the change in excitability in the exposed brain? Our results show that (i) baseline membrane properties of cortical and hippocampal CA1 neurons from rats chronically exposed to hypoxia were not substantially different from those of naive neurons; (ii) acute stress (e.g., hypoxia) elicited a markedly exaggerated response in the exposed neurons as compared to naive ones; (iii) chronic hypoxia tended to increase Na(+) channel mRNA and saxitoxin binding density in the cortex and hippocampus as compared to control ones; and (iv) the enhanced neuronal response to acute hypoxia in the exposed cortical and CA1 neurons was considerably attenuated by applying tetrodotoxin, a voltage-sensitive Na(+) channel blocker, in a dose-dependent manner. We conclude that prolonged O(2) deprivation can lead to major electrophysiological disturbances, especially when exposed neurons are stressed acutely, which renders the chronically exposed neurons more vulnerable to subsequent micro-environmental stress. We suggest that this Na(+) channel-related over-excitability is likely to constitute a molecular mechanism for some neurological sequelae, such as epilepsy, resulting from perinatal hypoxic encephalopathy.     

Properties and rundown of sodium-activated potassium channels in rat olfactory bulb neurons.

We have used single-channel recording techniques to investigate the properties of sodium-activated potassium channels (KNa channels) in cultured rat olfactory bulb neurons, and in large neurons in the mitral cell layer of thin slices of olfactory bulb. Ion channels highly selective for potassium over sodium and chloride, and requiring 10-180 mM internal sodium (Nai) for their activation, were present in approximately 75% of inside-out membrane patches detached from cultured olfactory bulb neurons. Most of these patches contained several KNa channels. KNa channels were seen in cell-attached patches only when Nai was raised by including veratridine in the extracellular medium. Preincubation of the cell in TTX or removal of extracellular sodium prevented this effect of veratridine, confirming that the channels observed under these conditions were indeed KNa channels. Lithium did not substitute for Nai in activating these channels. With 150 mM potassium on both sides of the membrane, KNa channels had a single-channel conductance of 172 pS, and at least two subconducting states were observed in addition to this fully open state. Under these ionic conditions, the channels exhibited linear fully open channel current-voltage curves over the potential range of -100 to 0 mV. At voltages more positive than the potassium equilibrium potential, the single-channel currents exhibited inward rectification as a result of sodium block of outward potassium current. The channels opened in bursts, during which they fluctuated between the fully open and closed states, and the substates. Between bursts they sometimes entered a long-lived inactive state that could last for up to several minutes. In addition, KNa channels in the detached patches exhibited rundown, a progressive irreversible loss in activity, over a time course that varied from less than 1 min to longer than 1 hr. Rundown of KNa channel activity in cell-attached patches (in the presence of veratridine) did not occur, suggesting that some intracellular factor necessary for KNa channel activity is lost when the membrane patch is detached from the cell.     

低氧预适应小鼠脑匀浆液提取液对大鼠鼠胚海马神经元缺氧复氧后神经细胞的影响

目的 探讨低氧预适应小鼠脑匀浆液提取液对大鼠鼠胚海马神经元缺氧复氧后神经细胞活性和凋亡的影响. 方法在96孔培养板中将大鼠鼠胚海马神经细胞原代培养至8 d,将培养细胞按照处理的不同分为以下5组:(1)正常对照组(仅加入PBS)、(2)H<,4>R<,48>组(缺氧4 h/复氧48h后加PBS)、(3)H0组(缺氧4 h/复氧48 h前加正常小鼠脑匀浆提取液)、(4)H1组(缺氧4h/复氧48h前加急性低氧对照小鼠脑匀浆提取液)、(5)H4组(缺氧4 h/复氧48 h前加低氧预适应小鼠脑匀浆提取液),分别用酶标仪和流式细胞仪测定神经细胞活性和凋亡情况.结果 正常对照组细胞活性明显高于H<,4>R<,48>组,H0、H1和H4组分别与H<,4>R<,48>组相比,细胞活性明显增加,且H4组细胞活性又明显高于H0、H1组;正常对照组仅有极少量的凋亡细胞,而H<,4>R<,48>组凋亡细胞显著增多,H0、H1和H4组分别与H<,4>R<,48>组相比凋亡细胞明显减少,且H4组又分别明显少于H0、H1组.结论低氧预适应小鼠脑匀浆液提取液可能通过增加大鼠鼠胚海马神经元缺氧复氧后神经细胞活性和减少神经细胞凋亡起到抗缺氧性损伤作用.