大鼠全脑缺血后海马CA1区锥体神经元L型钙通道电流的改变(英文)

已有研究表明在脑缺血期间及再灌流后早期,海马CA1锥体神经元细胞内钙浓度明显升高,这一钙超载被认为是缺血性脑损伤的重要机制之一.电压依赖性钙通道是介导正常CA1神经元钙内流的主要途径.实验观察了脑缺血再灌流后早期海马CA1锥体神经元电压依赖性L型钙通道的变化.以改良的四血管闭塞法制作大鼠 15min前脑缺血模型,在急性分离的海马CA1神经元上,采用膜片钳细胞贴附式记录L型电压依赖性钙通道电流.脑缺血后CA1神经元L型钙通道的总体平均电流明显增大,这是由于通道的开放概率增加所致.进一步分析单通道动力学显示,脑缺血后通道的开放时间变长,通道的开放频率增大.研究结果提示L型钙通道功能活动增强可能参与了缺血后海马CA1锥体神经元的细胞内钙浓度升高     

海马脑片锥体神经元钙离子通道的盲法膜片钳研究

目的和方法 :采用大鼠海马脑片盲法膜片钳全细胞记录技术研究CA1区锥体神经元电压门控性Ca2 通道的动力学特征。结果 :大鼠海马脑片CA1区锥体神经元电压门控性Ca2 通道电流具有如下特点 :①激活的阈电位偏低 ,为 (- 4 9.3± 8.6 )mV ,范围为 - 6 5～ - 30mV(n =2 3)。②衰减时间常数τ值较大 ,且变化范围大 (10 0～ 70 0ms) (n =12 ) ,并且衰减具有Ca2 电流幅值的依赖性 ,③稳态失活呈现电压依赖性 ,半失活电压为 (- 5 5 .4± 9.7)mV ,斜率因子为 (5 .3± 0 .9)mV(n =10 )。④当细胞外Ca2 浓度为 2 .5mmol/L时 ,Ca2 通道的反转电位为 (5 5±13)mV(n =10 )。⑤尾电流成分较为单一 ,不表现电压依赖性。另外 ,Ca2 电流对戊脉胺及双氢吡啶类化合物硝苯地平均不敏感。结论 :根据上述Ca2 电流特征 ,海马脑片CA1区锥体神经元上的Ca2 通道主要以N型为主     

琥珀酸对大鼠海马CA1区神经元电压依赖性钙电流的影响

目的：观察不同浓度的琥珀酸对大鼠海马CA1区神经元电压依赖性钙通道（voltage—dependent calcium channels,VDCC）电流的作用,初步探讨琥珀酸对神经元保护的电生理学基础。方法：采用传统全细胞膜片钳技术和制霉菌素（nystatin）穿孔膜片钳技术观察琥珀酸对海马CA1区神经元VDCC电流的影响。结果：不同浓度的琥珀酸（10^-6、10^-5、10^-4、10^-3、10^-2和10^-1mol·L^-1）在海马CA1区对低电压激活（low—voltage activated,LVA）钙通道电流未见任何影响,而对高电压激活（high—voltage activated,HVA）钙通道电流的抑制呈浓度依赖性。对照组HVA钙电流为580．05±17．32pA,分别给予10^-6、10^-5、10^-4、10^-3、10^-2和10^-1mol·L^-1。的琥珀酸后,HVA钙电流依次为563．74±16．65,517．99±15．24,444．66±13．26,405．32±19．11,269．03±9．96和86．41±3．25pA,同对照组相比差异有统计学意义（n=8,P〈0．01）。结论：琥珀酸能浓度依赖性地抑制HVA钙电流,而对LVA钙电流无影响。由此推测琥珀酸可能通过抑制HVA钙电流减少Ca^2＋内流而影响海马CA1区神经元的兴奋性,从而抑制癫痫的形成,其脑保护作用可能与此有关。     

二氧化硫衍生物对大鼠海马CA3区神经元瞬间外向钾电流的影响

为研究二氧化硫(SO2)衍生物——NaHSO3和Na2SO3(二者分子比为1：3)对大鼠海马CA3区神经元瞬间外向钾电流(IA)的影响,利用全细胞膜片钳技术,根据动力学和药理学特性分离鉴定大鼠海马CA3区神经元IA,观察SO2衍生物对IA的效应。发现SO2代谢衍生物可浓度依赖性地增大IA,使IA增大50％的剂量为25μmol/L。此外还与电压呈依赖关系,但不具有频率依赖性。10μmol/L的SO2代谢衍生物不影响IA电流的激活过程,但升高了A-通道稳态失活电压,延长了A-电流失活时间。说明SO2代谢衍生物可增大大鼠海马CA3区神经元IA电流,延长A-电流的失活时间,从而影响海马神经元的膜生理感应,这可能是SO2影响神经细胞功能的机理之一。     

产前应激对子代大鼠海马CA3区神经元Ca2+和K+通道的影响

本研究的目的在于探讨产前应激对子代大鼠海马CA3神经元高电压激活(HVA)钙通道、延迟整流钾电流(delayedrectifierpotassiumcurrents,IKD)的影响。产前应激(prenatalstress,PNS)组孕鼠孕晚期给予束缚应激,应用全细胞膜片钳技术进行研究。结果显示产前应激增加了子代海马CA3神经元HVA钙通道峰电流幅值,对照组和产前应激组子代CA3神经元平均最大HVA钙电流峰值分别为-576.52±7.03pA和-702.05±6.82pA(P<0.01)。同时未改变其电导-电压关系,也未改变延迟整流钾通道电流-电压关系、电导-电压关系。结果提示,在胎儿发育的关键时期,给予母体产前应激,引起子代海马神经元HVA钙电流增加,其机制一方面PNS导致皮质酮升高,从而可能增加HVA钙通道mRNA表达;另一方面PNS所致反应性氧化产物(reactiveoxygenspecies,ROS)增多,后者可能通过磷酸化HVACa2 通道亚单位,从而提高HVA钙电流幅值。     

白细胞介素-6对新生大鼠海马神经元电压依赖离子通道和NMDA电流的影响

目的: 研究白细胞介素-6对海马神经元电压依赖离子通道和NMDA电流的影响.方法: 应用全细胞膜片钳技术观察IL-6对电压依赖性钠通道电流(INa),延迟整流性钾通道电流(IK),电压依赖性钙通道电流(ICa),NMDA(N-methyl-D-aspartate)受体通道电流的影响.结果: 50 ng/ml IL-6作用24 h后IK 和ICa明显减小,Cm明显增大.50,500 ng/ml时减小NMDA电流.结论: IL-6通过作用于电压依赖钾通道,钙离子通道及NMDA通道影响神经元功能.     

琥珀酸对大鼠海马CAI区神经元电压依赖性钙电流的影响

目的:观察不同浓度的琥珀酸对大鼠海马CAI区神经元电压依赖性钙通道(voltage-dependent calcium channels,VDCC)流的作用,初步探讨琥珀酸对神经元保护的电生理学基础.方法:采用传统全细胞膜片钳技术和制霉菌素(nystatin)穿孔膜片钳技术观察琥珀酸对海马CAI区神经元VDCC电流的影响.结果:不同浓度的琉角酸(10-6、10-5、10-4、10-3、10-2和10-1mol·L-1)在海马CAI区对低电压激活(low-voltage activated,LVA)钙通道电流未见任何影响,而对高电压激活(high-voltage activated,HVA)钙通道电流的抑制呈浓度依赖性.对照组HVA钙电流为580.051±7.32pA,分别给予10-6、10-5、10-4、10-3、10-2和10-1 mol·L-1的琥珀酸后HVA钙电流依次为563.74±16.65,517.99±15.24,444.66±13.26,405.32±19.11,269.03±9.96和86.41±3.25pA,同对照组相比差异有统计学意义(n=8,P<0.01).结论:琥珀酸能浓度依赖性地抑制HVA钙电流,而对LVA钙电流无影响.由此推测琥珀酸可能通过抑制HVA钙电流减少Ca2+内流而影响海马CAI区神经元的兴奋性,从而抑制癫痫的形成,其脑保护作用可能与此有关.     

白细胞介素-6对新生大鼠海马神经元电压依赖离子通道和NMDA电流的影响

目的 :研究白细胞介素 6对海马神经元电压依赖离子通道和NMDA电流的影响。方法 :应用全细胞膜片钳技术观察IL 6对电压依赖性钠通道电流 (INa) ,延迟整流性钾通道电流 (IK) ,电压依赖性钙通道电流 (ICa) ,NMDA(N methyl D aspartate)受体通道电流的影响。结果 :5 0ng/mlIL 6作用 2 4h后IK和ICa明显减小 ,Cm明显增大。 5 0 ,5 0 0ng/ml时减小NMDA电流。结论 :IL 6通过作用于电压依赖钾通道 ,钙离子通道及NMDA通道影响神经元功能。     

成年大鼠海马CA1区锥体细胞KATP通道的特性

为了解成年大鼠海马CA1区锥体细胞KATP通道的特性,实验采用膜片钳技术的内面向外式记录法,在急性分离的CA1区锥体神经元上,研究了可被胞浆侧ATP所抑制的钾离子单通道的特性,当细胞膜内外两侧的K^ 浓度均为140mmol/L时,通道的电导为63pS,翻转电位为1．71mV,通道呈弱向内向整流性,在负钳制电位时,通道开放时常被短时的关闭所打断,而在正钳制电位时,这种短时程的关闭状态明显少于负钳制电位时,但通道开放概率未见明显的电压依赖性,ATP对通道活动的抑制作用呈浓度依赖性,抑制通道活动50％的ATP浓度为0．1mmol/L.KATP通道的特异性阻断剂tolbutamide(甲糖宁,1mmol/L)可完全阻断通道的活动,而KATP通道开放剂diazoxide(二氮嗪,1mmol/L）则不增强通道的活动。     

肢体缺血预处理减轻大鼠海马缺血/再灌注损伤

目的:探讨肢体缺血预处理(LIP)对大鼠全脑缺血/再灌注损伤的影响.方法: 36只大鼠椎动脉凝闭后随机分为假手术(Control)组、脑缺血组、肢体缺血组、LIP 0 d组(LIP后即刻行脑缺血)、LIP 1 d组(LIP后1 d行脑缺血)和LIP 2 d组(LIP后2 d行脑缺血).重复夹闭大鼠双侧股动脉3次(每次10 min,间隔10 min)作为LIP,夹闭颈总动脉进行全脑缺血8 min后再灌注.硫堇染色观察海马CA1区组织学分级及锥体神经元密度以判断海马损伤程度.结果:脑缺血组海马CA1区锥体神经元损伤严重,与Control组比较,组织学分级明显升高,神经元密度明显降低(P<0.01).LIP 0 d组海马CA1区神经元损伤较脑缺血组明显减轻,组织学分级明显降低,神经元密度明显升高(P<0.01).而LIP 1 d组和LIP 2 d组大鼠海马CA1区锥体细胞缺失较多,仍有明显的组织损伤.结论:LIP可减轻随后立即发生的脑缺血/再灌注损伤,但对间隔1 d后的脑缺血/再灌注损伤无显著对抗作用.     

Ischemic shortening of action potential duration as a result of KATP channel opening attenuates myocardial stunning by reducing calcium influx

Action potential duration (APD) shortening due to opening of sarcolemmal ATP-dependent potassium (KATP) channels has been postulated to protect the myocardium against postischemic damage by reducing Ca²⁺ influx. This hypothesis was assessed, assuming that increased postischemic stunning due to KATP channel inhibition with glibenclamide could be reverted by the addition of the Ca²⁺ channel blocker diltiazem. Percent wall thickening fraction (% WTh, conscious sheep) and APD (open-chest sheep) were obtained from the following groups: control: 12 min ischemia by anterior descending coronary artery occlusion followed by 2 h reperfusion; glibenclamide: same as control, with glibenclamide (0.4 mg/kg) infused 30 min before ischemia; diltiazem: same as control, with diltiazem (100 g/kg) administered prior to ischemia; glibenclamide+diltiazem: both drugs infused as in glibenclamide and diltiazem groups. APD was reduced in control ischemia. Conversely, KATP-channel blockade by glibenclamide lengthened APD and increased postischemic stunning (p < 0.01 vs. control); glibenclamide+diltiazem did not shorten APD but enhanced functional recovery (p < 0.01 vs. glibenclamide). Ca²⁺ channel blockade improvement of increased stunning provoked by KATP channel inhibition supports the hypothesis that APD shortening due to opening of KATP channels protects against postischemic stunning by limiting Ca²⁺ influx.     

Activation of BKCa Channels Mediates Hippocampal Neuronal Death After Reoxygenation and Reperfusion

Excessive K⁺ efflux promotes central neuronal apoptosis; however, the type of potassium channel that mediates K⁺ efflux in response to different apoptosis-inducing stimuli is still unknown. It is hypothesized that the activation of large-conductance Ca²⁺-activated K⁺ channels (BK_Ca) mediates hypoxia/reoxygenation (H/R)- and ischemia/reperfusion (I/R)-induced neuronal apoptosis. Rat hippocampal neuronal cultures underwent apoptosis after reoxygenation, as assessed by morphologic observation, terminal deoxynucleotidyl transferase dUTP nick end labeling staining, and caspase-3 activation. Single-channel recordings revealed upregulation of BK_Ca channel activity 6 h after reoxygenation, which might be caused by elevated cytosolic Ca²⁺. The K⁺ ionophore valinomycin and the BK_Ca channel opener NS1619 induced neuronal apoptosis. Transfection of the BK_Ca channel α subunit into Chinese hamster ovary (CHO-K1) cells, which do not express endogenous K⁺ channels, or into neurons will induce cell apoptosis, indicating that the opening of the BK_Ca channel serves as a pivotal event in mediating cell apoptosis. The specific BK_Ca channel blockers charybdotoxin and iberiotoxin and the nonselective K⁺ channel blocker tetraethylammonium at concentrations more specific to the BK_Ca channel were neuroprotective. The A-type potassium channel blocker 4-aminopyridine and apamin, a small-conductance Ca²⁺-activated K⁺ channel blocker, were not protective. This result suggests the involvement of the BK_Ca channel in H/R-induced apoptosis. Similarly, specific BK_Ca channel blockers also showed neuroprotection in neurons subjected to oxygen-glucose deprivation/reoxygenation or animals subjected to forebrain ischemia–reperfusion. These results demonstrate that the over-activity of BK_Ca channels mediates hippocampal neuronal damage induced by H/R in vitro and I/R in vivo.     

Raised Intracellular Calcium Contributes to Ischemia-Induced Depression of Evoked Synaptic Transmission

Oxygen-glucose deprivation (OGD) leads to depression of evoked synaptic transmission, for which the mechanisms remain unclear. We hypothesized that increased presynaptic [Ca²⁺]_i during transient OGD contributes to the depression of evoked field excitatory postsynaptic potentials (fEPSPs). Additionally, we hypothesized that increased buffering of intracellular calcium would shorten electrophysiological recovery after transient ischemia. Mouse hippocampal slices were exposed to 2 to 8 min of OGD. fEPSPs evoked by Schaffer collateral stimulation were recorded in the stratum radiatum, and whole cell current or voltage clamp recordings were performed in CA1 neurons. Transient ischemia led to increased presynaptic [Ca²⁺]_i, (shown by calcium imaging), increased spontaneous miniature EPSP/Cs, and depressed evoked fEPSPs, partially mediated by adenosine. Buffering of intracellular Ca²⁺ during OGD by membrane-permeant chelators (BAPTA-AM or EGTA-AM) partially prevented fEPSP depression and promoted faster electrophysiological recovery when the OGD challenge was stopped. The blocker of BK channels, charybdotoxin (ChTX), also prevented fEPSP depression, but did not accelerate post-ischemic recovery. These results suggest that OGD leads to elevated presynaptic [Ca²⁺]_i, which reduces evoked transmitter release; this effect can be reversed by increased intracellular Ca²⁺ buffering which also speeds recovery.     

短暂脑缺血大鼠海马CA1区锥体细胞大电导Ca2+依赖K+通道活动降低(英)

短暂脑缺血可对随后的损伤性脑缺血表现出明显的耐受.有研究表明大电导Ca²⁺依赖K⁺（BK_Ca）通道活动增强参与了缺血性脑损伤.采用膜片钳的内面向外式,观察了3 min短暂脑缺血后6 h、24 h以及48 h大鼠海马CA1区锥体细胞上BK_Ca通道活动的动态变化.短暂脑缺血后BK_Ca通道的单通道电导和翻转电位均未见明显变化,但通道的开放概率则在缺血预处理后的前24 h内显著降低.通道动力学分析显示通道关闭时间变长是短暂脑缺血后通道活动降低的主要原因,因为通道的开放时间未发生明显变化.结果提示短暂脑缺血所致的BK_Ca通道活动降低可能与缺血耐受的产生有关.     

The Reduction of EPSC Amplitude in CA1 Pyramidal Neurons by the Peroxynitrite Donor SIN-1 Requires Ca2+ Influx Via Postsynaptic Non-L-Type Voltage Gated Calcium Channels

The peroxynitrite free radical (ONOO^?) modulation of miniature excitatory postsynaptic currents (mEPSCs) and spontaneous excitatory postsynaptic currents (sEPSCs) was investigated in rat CA1 pyramidal neurons using the whole-cell patch clamp technique. SIN-1(3-morpholino-sydnonimine), which can lead the simultaneous generation of superoxide anion and nitric oxide, and then form the highly reactive species ONOO^?, induced dose-dependent inhibition in amplitudes of both mEPSCs and sEPSCs. The SIN-1 action on mEPSC amplitude was completely blocked by U0126, a selective MEK inhibitor, suggesting that MEK contributed to the action of ONOO^? on mEPSCs. The effect of SIN-1 was completely occluded either in the presence of the calcium chelator EGTA or the non-selective calcium channel antagonist Cd²⁺. Furthermore, the application of nifedipine (20 μM), the L-type calcium channel blocker, had no effect on the ONOO^?-induced decrease in mEPSC amplitude, excluding a role for L-type voltage-gated Ca²⁺ channels in this process. SIN-1 inhibited the frequency of sEPSCs but had no effect on mEPSC frequency, which suggested a presynaptic action potential-dependent the action of ONOO^? at CA1 pyramidal neuron synapses. The best-known glutamatergic input to CA1 pyramidal neurons is via Schaffer collaterals from CA3 area. However, no changes were observed in slices treated with SIN-1 on the spontaneous firing rates of CA3 pyramidal neurons. These findings suggested that SIN-1 inhibited glutamatergic synaptic transmission of CA1 pyramidal neurons by a postsynaptic non-L-type voltage gated calcium channel-dependent mechanism.     

Carbonyl stress: malondialdehyde induces damage on rat hippocampal neurons by disturbance of Ca<Superscript>2+</Superscript> homeostasis

The objective of this study was to investigate the influences of carbonyl stress induced by malondialdehyde (MDA), a typical intermediate of lipid peroxidation, on intracellular free Ca²⁺ concentration ([Ca²⁺]_i) alterations in cultured hippocampal neurons of rat. The microphotographic study clearly demonstrated that the hippocampal neurons became gradually damaged following exposure to different concentrations of MDA. Further study indicated that the plasma membrane Ca²⁺-ATPase (PMCA) activity was inhibited by MDA in a concentration- and time-dependent manner. The supplementation of 100 μM MDA was found to cause a notable early phase increase of [Ca²⁺]_i in hippocampal neuron cultures followed by a more pronounced late-phase elevation of [Ca²⁺]_i. Such effect of MDA was prevented by the addition of nimodipine, an inhibitor of L-type calcium channel or by an extracellular Ca²⁺ chelator EGTA. The identification of the calcium signalling pathways were studied by applying U73122, an inhibitor of PL-C, and H-89, an inhibitor of protein kinase A (PKA), showing the involvement of PL-C/IP3 pathway but not the PKA/cAMP pathway. These results suggested that MDA-related carbonyl stress caused damages of rat hippocampal neurons by triggering Ca²⁺ influx and influencing Ca²⁺ homeostasis in cultured neurons, and also MDA may act as a signalling molecule regulating Ca²⁺ release from intracellular stores.     

Protective effects of Lycium barbarum polysaccharide on neonatal rat primary cultured hippocampal neurons injured by oxygen-glucose deprivation and reperfusion

This study investigated the protective effects of Lycium barbarum polysaccharide (LBP) on alleviating injury from oxygen-glucose deprivation/reperfusion (OGD/RP) in primary cultured rat hippocampal neurons. Cultured hippocampal neurons were exposed to oxygen-glucose deprivation (OGD) for 2?h followed by a 24?h re-oxygenation. The MTT assay and the lactate dehydrogenase (LDH) release were used to determine the neuron viability. Superoxide dismutase (SOD), Glutathione peroxidase (GSH-PX), malondialdehyde (MDA) were determined by spectrophotometry using commercial kits. Mitochondrial membrane potential (MMP) and the intracellular free calcium concentration ([Ca²⁺]_i) in hippocampal neurons were measured using the confocal laser scanning microscope (CLSM). Treatment with LBP (10–40?mg/l) significantly attenuated neuronal damage and inhibited LDH release in a dose-dependent manner. Furthermore, LBP enhanced activities of SOD and GSH-PX but it decreased their MDA content, inhibited [Ca²⁺]_i elevation and decrease of MMP in ischemia–reperfusion treated hippocampal neurons. These findings suggested that LBP may be a potential neuroprotective agent for cerebral?ischemia–reperfusion injury.     

Imaging calcium entering the cytosol through a single opening of plasma membrane ion channels: SCCaFTs—fundamental calcium events

Recently, it has become possible to record the localized fluorescence transient associated with the opening of a single plasma membrane Ca²⁺ permeable ion channel using Ca²⁺ indicators like fluo-3. These Single Channel Ca²⁺ Fluorescence Transients (SCCaFTs) share some of the characteristics of such elementary events as Ca²⁺ sparks and Ca²⁺ puffs caused by Ca²⁺ release from intracellular stores (due to the opening of ryanodine receptors and IP₃ receptors, respectively). In contrast to intracellular Ca²⁺ release events, SCCaFTs can be observed while simultaneously recording the unitary channel currents using patch-clamp techniques to verify the channel openings. Imaging SCCaFTs provides a way to examine localized Ca²⁺ handling in the vicinity of a channel with a known Ca²⁺ influx, to obtain the Ca²⁺ current passing through plasma membrane cation channels in near physiological solutions, to localize Ca²⁺ permeable ion channels on the plasma membrane, and to estimate the Ca²⁺ currents underlying those elementary events where the Ca²⁺ currents cannot be recorded. Here we review studies of these fluorescence transients associated with caffeine-activated channels, L-type Ca²⁺ channels, and stretch-activated channels. For the L-type Ca²⁺ channel, SCCaFTs have been termed sparklets. In addition, we discuss how SCCaFTs have been used to estimate Ca²⁺ currents using the rate of rise of the fluorescence transient as well as the signal mass associated with the total fluorescence increase.