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。     

Kv4.2通道电流与大鼠海马神经元瞬间外向钾电流特性的比较

本研究比较了转染的Kv4.2钾电流与原代培养大鼠海马神经元上瞬间外向钾电流(IA)动力学特征。实验采用瞬时转染,细胞培养和全细胞膜片钳记录等方法。结果表明:转染的Kv4.2通道电流和海马神经元上IA均具有明显的A型电流特征。海马神经元IA的半数最大激活电位和斜率因子分别为-10.0±3.3 mV和13.9±2.6 mV;半数最大失活电位和斜率因子分别为-93.0±11.4 mV和-9.0±1.5 mV;失活后再激活恢复时间常数(T)为27.9±14.1 ms。Kv4.2的半数最大激活电位和斜率因子分别为-9.7±4.1 mV和15.8±5.7 mV;半数最大失活电位和斜率因子分别为-59.4±12.2 mV和8.0±3.1 mV;Kv4.2的灭活后再激活的恢复时间常数τ为172.8±10.0 ms。结果提示:Kv4.2通道电流可能是海马神经元上的IA电流的主要成分,但不是唯一成分。     

丙泊酚拮抗戊四氮对大鼠海马CA1区动作电位和突触传递的影响

目的：观察戊四氮对大鼠海马CA1区动作电位（action potential,AP）和兴奋性突触后电流（excitatory postsynaptic current,EPSC）的影响和丙泊酚的拮抗作用。方法：断头法分离Wistar大鼠海马半脑,切片机切出400μm厚度的海马脑片,全细胞电流钳记录CA1区锥体神经元动作电位发放情况,全细胞电压钳记录电刺激Schaeffer侧支／联合纤维诱发的CA1区锥体神经元EPSC的变化。结果：戊四氮使动作电位发放频率增加,EPSC值降低;丙泊酚拮抗戊四氮的作用,使动作电位发放减少甚至消失,EPSC值上升至加入丙泊酚前的2倍左右。结论：丙泊酚拮抗戊四氮对动作电位和EPSC的作用,所以临床上可用于抗癫痫治疗。     

神经元钙激活钾电流的特征和功能

钙激活钾电流是由动作电位激活的一类外向钾电流,它是由不同类型的钙激活钾通道所介导。钙激活钾电流参与动作电位复极化和后超极化的电位的形成,并通过调节神经元的放电频率和影响神经元的放电类型参与神经元的多种上生理功能。     

CRISPR/Cas9高效敲除突触结合蛋白Ⅰ的电生理学研究

研究一种蛋白质在神经元中的功能,最有效的方法之一是在该基因敲除动物的神经元中确认其表型.传统的用胚胎干细胞建立基因敲除动物模型的方法虽然稳定,但是复杂、耗时.近几年来,一种新型基因组编辑技术——CRISPR/Cas9,能够在不分裂的神经元中高效特异地敲除目的基因.本文研究了用CRISPR/Cas9系统敲除突触结合蛋白Ⅰ(synaptotagminⅠ,Syt1)基因后的小鼠海马培养神经元的电生理学特性.我们设计并构建了Syt1单导向RNA(Syt1 sgRNA)的慢病毒载体质粒,并用编码Cas9和Syt1 sgRNA的慢病毒感染培养的小鼠海马神经元,急性敲除神经元中Syt1基因(Syt1 sgRNA组),并用不靶向任何基因的Scramble sgRNA感染神经元作为阴性对照(Scramble组).通过全细胞膜片钳的方法检测单动作电位诱发的兴奋性突触后电流(single AP-eEPSC)、微小兴奋性突触后电流(mEPSCs)、高糖反应测量的即刻可释放囊泡池(RRP)以及10 Hz串刺激测量的囊泡释放概率(P_r).结果显示,Syt1 sgRNA组神经元丧失了Syt1的功能,并且与Syt1敲除(Syt1 KO)小鼠神经元的突触传递表型相似,而Scramble组神经元的各参数和野生型(WT)小鼠神经元相比没有显著性差异.本文为CRISPR/Cas9技术应用于神经元中基因的急性修饰提供了依据.     

吗啡对大鼠海马神经元突触传递的作用及机制探讨

目的 :从离子通道角度研究吗啡对中枢神经系统兴奋性及抑制性突触传递的作用并探讨其机制。方法 :　原代培养新生Wistar大鼠的海马神经元。采用膜片钳技术研究吗啡对其兴奋性及抑制性突触后电流及谷氨酸诱发电流的影响。结果 :①吗啡可明显增强海马神经元兴奋性突触传递 ,加吗啡后自发兴奋性突触后电流 (sEPSC)的发放频率增加了 ( 2 0 7.8± 2 0 .9) %。此作用可被阿片受体阻断剂纳洛酮阻断 (P <0 .0 1) ;②吗啡对微小兴奋性突触后电流 (mEPSC)的发放频率及谷氨酸诱发电流的幅度没有明显影响 (P >0 .0 5 ) ;③吗啡可明显抑制神经元自发抑制性突触后电流 (sIPSC) ,纳洛酮可拮抗吗啡作用 (n =13 ,P <0 .0 1)。结论 :实验结果提示吗啡对海马神经元的兴奋作用不是由于吗啡直接作用于兴奋性氨基酸—谷氨酸突触传递过程 ,而是可能由于抑制了抑制性中间神经元 ,间接产生的兴奋作用。     

突触前α7烟碱受体对海马神经元兴奋性突触传递的调控

采用盲法膜片钳技术观察突触前烟碱受体(nicotinic acetylcholinel receptors,nAChRs)对海马脑片CAl区锥体神经元兴奋性突触传递的调控作用。结果显示,nAChRs激动剂碘化二甲基苯基哌嗪(dimethylphenyl—piperazinium iodide,DMPP)不能在CAl区锥体神经元上诱发出烟碱电流。DMPP对CAl区锥体神经元自发兴奋性突触后电流(spontaneous excitatory postsynaptic current,sEPSC)具有明显的增频和增幅作用,并呈现明显的浓度依赖关系。DMPP对微小兴奋性突触后电流(miniature excitatory postsynaptic current,mEPSC)具有增频作用,但不具有增幅作用。上述DMPP增强突触传递的作用不能被nAChRs拮抗剂美加明、六烃季铵和双氢-β-刺桐丁所阻断,但可被α-银环蛇毒素阻断。上述结果提示,海马脑片CAl区锥体神经元兴奋性突触前nAChRs含有对α-银环蛇毒素敏感的胡亚单位,其激活可增强海马CAl区锥体神经元突触前递质谷氨酸的释放,从而对兴奋性突触传递发挥调控作用。     

海马锥体神经元树突中电压依赖性钾通道研究进展

海马锥体神经元树突上分布着多种电压依赖性钾离子通道,但这些通道在胞体和树突不同部位的分布密度以及在突触电活动中的功能意义各不相同。倒传递动作电位（b-AP）和兴奋性突触后电位（EPSP）是树突中常见的功能电信号。本文简要介绍了近年来海马锥体神经元树突上这些钾离子通道及其电活动的生理和病理学研究成果。     

AGS3蛋白对吗啡慢性处理大鼠前额叶皮层神经元瞬时外向钾电流的影响

目的:研究吗啡对大鼠皮层神经元瞬时外向钾电流(IA)的影响,并在此基础上,用G蛋白信号转导激活因子3(AGS3)的抗体阻断AGS3作用,观察其对瞬时外向钾电流(IA)的影响,从而探讨AGS3蛋白在吗啡成瘾中的机制。方法:采用全细胞膜片钳技术记录IA;吗啡对神经元IA电流密度-电压曲线(I-V曲线)的影响;在全细胞构型下,观察三种不同浓度AGS3抗体对吗啡处理大鼠前额叶皮层神经元IA的影响。结果:吗啡能引起大鼠皮质神经元IA增强;当膜电位+55mV时,10-3μg/L、10-2μg/L、10-1μg/L三种不同浓度的AGS3抗体作用于吗啡处理的神经元,10-3μg/L对电流密度的抑制没有显著差异;10-2μg/L、10-1μg/L的抗体能显著抑制吗啡引起的电流密度升高,有统计学差异。结论:吗啡能引起神经元I的增强,AGS3蛋白在成瘾机体中参与了对I通道进行调节的信号转导通路。     

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

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。     

Homeostatic control of presynaptic release is triggered by postsynaptic membrane depolarization.

Homeostatic mechanisms regulate synaptic function to maintain nerve and muscle excitation within reasonable physiological limits. The mechanisms that initiate homeostasic changes to synaptic function are not known. We specifically impaired cellular depolarization by expressing the Kir2.1 potassium channel in Drosophila muscle. In Kir2.1-expressing muscle there is a persistent outward potassium current ( approximately 10 nA), decreased muscle input resistance (50-fold), and a hyperpolarized resting potential. Despite impaired muscle excitability, synaptic depolarization of muscle achieves wild-type levels. A quantal analysis demonstrates that increased presynaptic release (quantal content), without a change in quantal size (mEPSC amplitude), compensates for altered muscle excitation. Because morphological synaptic growth is normal, we conclude that a homeostatic increase in presynaptic release compensates for impaired muscle excitability. These data demonstrate that a monitor of muscle membrane depolarization is sufficient to initiate synaptic homeostatic compensation.     

Plasmalemmal,voltage-dependent ionic currents from excitable pulvinar motor cells ofMimosa pudica

Summary Plasmalemmal ionic currents from excitable motor cells of the primary pulvinus ofMimosa pudica were investigated by patch-clamp techniques. In almost all of the enzymatically isolated protoplasts, a delayed rectifier potassium current was activated by depolarization, while no currents were detected upon hyperpolarization. This sustained outward current was reversibly blocked by Ba and TEA and serves to repolarize the membrane potential. Outward single channel currents that very likely underly the macroscopic outward potassium current had an elementary conductance of 20 pS. In addition, in a few protoplasts held at hyperpolarized potentials, depolarization-activated transient inward currents were observed, and under current clamp, action potential-like responses were triggered by depolarizing current injections or by mechanical perturbations. The activation characteristics of both inward currents and spikes showed striking similarities compared to those of action potentialsin situ.     

海马神经细胞胆固醇含量降低对其电压依赖钾电流的上调作用

胆固醇普遍存在于细胞膜中,其含量在细胞增殖、生长及各种疾病条件下会发生改变,这暗示胆固醇对细胞功能的调节起着重要的作用。运用全细胞膜片钳技术研究了胆固醇含量变化对海马神经细胞电压依赖钾电流的影响。实验观察到神经细胞经胆固醇去除剂β-甲基环化糊精(MβCD)处理后,胆固醇含量的减少促进了延迟整流钾电流IK的增加,且延缓了瞬间失活钾电流IA的失活。更进一步,延迟整流钾电流IK和瞬间失活钾电流IA分别经TEA和4-AP阻断后,MβCD对两种电流成分的影响显著降低。这一结果进一步表明胆固醇去除剂对电压依赖钾电流的上调是通过作用于IK和IA电流而共同实现的。基于电压依赖钾通道在神经细胞功能中的重要作用,实验结果暗示神经细胞胆固醇含量变化可对神经细胞的兴奋性起调节作用。     

Neurotoxic action of some antiarrhythmic agents: comparative effects of propafenone,lidocaine and amiodarone on leech Retzius nerve cell

A series of antiarrhythmic drugs was studied on spontaneous spike activity and depolarizing outward potassium current in leech Retzius nerve cells. Propafenone (0.7 μM/ml) produced a cardiac-like action potential with a rapid depolarization followed by a sustained depolarization or plateau, which is terminated after 250 msec by a rapid repolarization. The effect of lidocaine (0.7 μM/ml) on spontaneous spike activity was less pronounced, and early afterdepolarization has been recorded. Amiodarone at the same and much higher concentrations (3 μM/ml) did not generate either a cardiac-like action potential or an early afterdepolarization. In the voltage clamp experiments, fast and slow calcium-activated outward potassium currents were suppressed with propafenone and lidocaine but not with amiodarone. These results suggest that the antiarrhythmic drugs, propafenone and lidocaine modulate calcium-activated potassium channels in leech Retzius nerve cells.     

中华大蟾蜍卵母细胞质膜的外向整流型钾离子通道

我们用电压箝方法研究了中华大蟾蜍卵母细胞的膜生理特性。发现卵母细胞膜去极化至-30mV及更偏正时,有一持续的外向电流出现,该电流与去极化程度约呈正比增加,当膜电位箝在20mV时其峰值达3.7±1.4μA。该电流被钾离子通道拮抗剂TEA和4-AP抑制,TEA半抑制浓度为2.6mmol/L。氯通道拮抗剂9-AC(2.5mmol/L)无抑制作用。无钙的或钙离子浓度增加三倍的胞外灌流液均对该电流无影响、该外向电流的逆转电位随胞外钾离子浓度的改变而变化。胞外钾离子浓度增加十倍,逆转电位约增加47.3mV,而胞外钠、钙或氯离子浓度的改变对逆转电位基本上无影响,因此该电流可被认为主要是电压依赖性钾离子流。取自冬眠蟾蜍的卵母细胞经孕酮诱发成熟后,电压依赖性钾离子流减小,仅为原来的1/20-1/30,而取自全年在高温饲养的蟾蜍的卵母细胞经孕酮处理后未见成熟,其电压依赖性钾离子流仅减小至原来的三分之一。     

Dynamics of the inward rectifier K+ current during the action potential of guinea pig ventricular myocytes.

The potassium selective, inward rectifier current (IK1) is known to be responsible for maintaining the resting membrane potential of quiescent ventricular myocytes. However, the contribution of this current to the different phases of the cardiac action potential has not been adequately established. In the present study, we have used the action potential clamp (APC) technique to characterize the dynamic changes of a cesium-sensitive (i.e., Ik1) current which occur during the action potential. Our results show that (a) Ik1 is present during depolarization, as well as in the final phase of repolarization of the cardiac action potential. (b) The current reaches the zone of inward-going rectification before the regenerative action potential ensues. (c) The maximal outward current amplitude during repolarization is significantly lower than during depolarization, which supports the hypothesis that in adult guinea pig ventricular myocytes, Ik1 rectification is accentuated during the action potential plateau. Our results stress the importance of Ik1 in the modulation of cell excitability in the ventricular myocyte.     

Modelling action potentials and membrane currents of mammalian skeletal muscle fibres in coherence with potassium concentration changes in the T-tubular system

During prolonged activity the action potentials of skeletal muscle fibres change their shape. A model study was made as to whether potassium accumulation and removal in the tubular space is important with respect to those variations. Classical Hodgkin-Huxley type sodium and (potassium) delayed rectifier currents were used to determine the sarcolemmal and tubular action potentials. The resting membrane potential was described with a chloride conductance, a potassium conductance (inward rather than outward rectifier) and a sodium conductance (minor influence) in both sarcolemmal and tubular membranes. The two potassium conductances, the Na-K pump and the potassium diffusion between tubular compartments and to the external medium contributed to the settlement of the potassium concentration in the tubular space. This space was divided into 20 coupled concentric compartments. In the longitudinal direction the fibre was a cable series of 56 short segments. All the results are concerned with one of the middle segments. During action potentials, potassium accumulates in the tubular space by outward current through both the delayed and inward rectifier potassium conductances. In between the action potentials the potassium concentration decreases in all compartments owing to potassium removal processes. In the outer tubular compartment the diffusion-driven potassium export to the bathing solution is the main process. In the inner tubular compartment, potassium removal is mainly effected by re-uptake into the sarcoplasm by means of the inward rectifier and the Na-K pump. This inward transport of potassium strongly reduces the positive shift of the tubular resting membrane potential and the consequent decrease of the action potential amplitude caused by inactivation of the sodium channels. Therefore, both potassium removal processes maintain excitability of the tubular membrane in the centre of the fibre, promote excitation-contraction coupling and contribute to the prevention of fatigue. Received: 5 May 1998 / Revised version: 27 October 1998 / Accepted: 19 January 1999     

The role of slow potassium current in phenomena of voltage-dependent action of 4-aminopyridine in amphibian myelinated nerve fibres

The mechanism underlying the voltage-dependent action of 4-aminopyridine (4-AP) is investigated in experiments on amphibian myelinated nerve fibres (Rana ridibunda Pallas) by way of extracellular recording of electrical activity and using activators of potassium current (potassium-free solution and nitric oxide NO) and inhibitors of sodium current (tetrodotoxin). Measurement of action potential (AP) areas was used to evaluate the extent of general membrane depolarization during the activity of nerve fibres. Tetrodotoxin-induced decrease in general membrane depolarization (when the action potential amplitude was reduced by less than 20%) leads to an increase in the duration of depolarizing after-potential (DAP). This supports the dependence of time course of DAP in the presence of 4-AP on ratio of fast and slow potassium channels. In the absence of 4-AP, potassium-free solution and NO increase the potassium current through fast potassium channels (decreasing AP duration, reducing DAP and sometimes producing fast hyperpolarizing after-potential (HAP) after shortened AP), and in the presence of 4-AP these activators increase potassium current through unblocked slow potassium channels (making the development of slow HAP induced by 4-AP more rapid). The increase of slow HAP induced by 4-AP under the influence of potassium-free solution with NO supports the idea that slow HAP is due to activation of slow potassium channels and argues against the notion of removal of block of fast potassium channels. All analyzed phenomena of voltage-dependent action of 4-AP in amphibian myelinated nerve fibers can be accounted for by the activation of slow potassium current produced by membrane depolarization and a decrease of the amount of fast potassium channels involved in the membrane repolarization.     

The effect of pentylenetetrazol on the metacerebral neuron of Helix pomatia

The effects of Pentylenetetrazol (PTZ) on the metacerebral giant cell (MCC) of the snail, Helix pomatia were studied. Actions on membrane resistance, time constant, resting and action potentials, outward and inward ionic currents were examined. Superfusion with PTZ in concentrations of 25 to 50 mmol/l, induced a gradually evolving convulsive state, which could be studied by intracellular recording from the MCCs. In the pre-convulsive state an acceleration of the spontaneous activity developed and was followed by paroxysmal depolarization shifts (PDSs), in the convulsive phase. PTZ prolonged the membrane time constant by about 10 percent, but this could not be traced back to alterations in membrane resistance or capacity. The resting membrane potential was not significantly altered; the action potentials were prolonged by slowing down of both the rising and decaying phases. The outward potassium currents were repressed by PTZ in a voltage dependent manner. The decrease of the IA current became more pronounced at increasingly positive command pulses, while IK was relieved from depression especially at longer pulse durations. Inward currents were isolated with the aid of suppression of outward currents by 50 mmol/l TEA. Under these conditions sodium currents, measured in calcium deficient Ringer solution were moderately depressed, while the calcium currents, examined during sodium-free superfusion, were mildly enhanced by PTZ. It is concluded that PTZ effects on ionic conductances, on membrane parameters, on the resting potential and ionic currents explain only modifications of spike potentials occurring in the convulsive state and do not account for the PDS, the central phenomenon of the convulsive electrographic activity, at least in this thoroughly examined type of neuron.