光学活性拟除虫菊酯对棉铃虫神经细胞钠通道电流的影响

用全细胞膜片钳技术对比分析了alpha体氯氰菊酯与theta体氯氰菊酯对棉铃虫Helicoverpa armigera幼虫离体培养中枢神经细胞Na⁺通道门控过程的影响。结果表明,alpha体氯氰菊酯作用后,神经细胞Na⁺通道电流（I_Na）先增大,同时通道的激活电压向负电位方向移动约10 mV,提示alpha体氯氰菊酯使通道激活电位降低,通道更容易被激活。药剂作用约10 min后,I_Na又迅速降低,表明alpha体氯氰菊酯对开放状态的Na⁺通道有抑制作用。另外,alpha体氯氰菊酯使I_Na到达峰值的时间缩短,但对失活时间无明显影响。Theta体氯氰菊酯也使I_Na激活电位左移,幅值降低,但降低速率较慢。总的结果表明alpha体氯氰菊酯与theta体氯氰菊酯对棉铃虫中枢神经细胞处于关闭和开放状态的钠通道均有作用,且alpha体氯氰菊酯对钠通道电流的抑制作用强于theta体氯氰菊酯。     

三氟氯氰菊酯对棉铃虫神经细胞钠及钙通道作用机理研究

用膜片钳技术对比分析了棉铃虫三氟氯氰菊脂抗性品系（R）及其同源对照品系（S）幼虫了体培养中枢神经细胞Na^2 通道的门控特性及杀虫剂对R和S神经细胞Na^ 、Ca^ 通道门控过程的影响。结果表明,S神经细胞Na^ 通道电流（S－INa）在－50－40mV激活,－20mV左右达峰值,R神经细胞Na^2 通道电流（R－INa）在－40mV左右激活,－10－0mV达峰值,即R－INa激活电压与峰值电压均向正电位方向移动约10mV,提示二者Na^ 通道控特性不同,R神经细胞Na^ 通道功能发生了变异。三氟氯氰菊酯作用后,S－INgn R-ISs的I－V曲线均向负电位方向移动的10mV,S－INa在20min后基本消失,而R－INa被阻断需时约90min,延长近5倍,其幅值有减小再增大的现象。对Ca^2 通道分析表明,杀虫剂作用后,R及S神经细胞Ca^2 通道电流的I－V曲线均向负电位移动10－20mV,提示三氟氯氰菊酯对Ca^2 通道的门控过程也有影响。与R－INa幅值起伏变化相联系,可推知杀虫剂对神经细胞的毒性作用中,Na^2 、Ca^2 通道均受影响。     

百日咳毒素对棉铃虫神经细胞电压门控钠、钙通道的调节作用

用膜片钳技术研究了百日咳毒素对离体培养的棉铃虫幼虫中枢神经细胞电压门控钠、钙通道的影响。结果表明,对照组细胞钠通道在-50～-40mV激活,在-20mV左右电流达到最大值,在记录的20min内、电流．电压关系曲线(I-V)和电流幅值未有明显变化;细胞与百日咳毒素预孵后,钠通道在-40mV左右激活,电流在0mV左右达到最大值,在记录过程中,激活电压和峰值电压继续向正方向移动约10mV、电流持续下降。对照组钙通道在-40～-30mV激活,在0mV左右电流达峰值;经百日咳毒素处理后,I-V曲线向负电位方向移动约10mV,在记录过程中,I-V曲线继续向负电位方向移动,电流的衰减(rundown)现象比对照组严重．此外,百日咳毒素处理引起钙电流达到峰值的时问显著延长。结果提示,百日咳毒素敏感的G蛋白(Gi)可能通过直接途径或间接途径调节棉铃虫神经细胞钠、钙通道的电压敏感性和开放几率以及钙通道由备用态向激活态转化的速度。同时,经百日咳毒素处理后钠通道的,I-V曲线与抗性棉铃虫I-V曲线非常相似,可能暗示Gi蛋白在棉铃虫抗药性形成中发挥作用。     

霍乱毒素对三氟氯氰菊酯抗性及敏感棉铃虫神经细胞L型钙通道的调节作用

霍乱毒素（CTX）可激活兴奋性异三聚体G蛋白（Gα_s）的α-亚基和刺激电压门控L-型钙通道,而昆虫的L-型钙通道可能是拟除虫菊酯类杀虫剂的作用靶点。为进一步探讨农业害虫对拟除虫菊酯类杀虫剂产生抗药性的作用机理,我们检测了CTX对三氟氯氰菊酯抗性及敏感棉铃虫Helicoverpa armigera中枢神经细胞电压门控L-型钙通道的调节作用。分别急性分离三氟氯氰菊酯抗性及敏感的3～4龄棉铃虫幼虫胸腹神经节细胞,并在改良的L15培养基（加入或未加入700 ng/mL的CTX）中培养12～16 h。钡离子为载流子,应用全细胞膜片钳技术记录电压门控L_型钙通道电流。结果显示,CTX可使敏感组棉铃虫神经细胞L-型钙通道的峰值电流密度增大36％、峰值电压左移5 mV,但对抗性组棉铃虫神经细胞L-型钙通道无上述作用。并且,CTX对敏感组及抗性组棉铃虫神经细胞L_型钙通道的激活电位、翻转电位、激活曲线和失活曲线等其他一些参数的影响也不明显。在无CTX作用时,所检测到的抗性组与敏感组棉铃虫神经细胞L_型钙通道的上述参数值间差异不显著。结果提示,棉铃虫神经细胞内存在Gs腺苷酸环化酶(AC)-cAMP-蛋白激酶A (PKA)-L-型钙通道信号调节系统;与敏感棉铃虫神经细胞L-型钙通道相比,三氟氯氰菊酯抗性棉铃虫神经细胞L-型钙通道的活性相对不易受到CTX调节,这可能与昆虫对拟除虫菊酯产生抗药性的机理有关。     

苦参碱对棉铃虫幼虫神经细胞钠通道的影响

用全细胞膜片钳技术研究了生物碱类植物杀虫剂苦参碱对棉铃虫Helicoverpa armigera幼虫离体培养中枢神经细胞钠离子通道门控过程的影响。结果表明： 苦参碱对棉铃虫幼虫神经细胞所表达的TTX （tetradotoxin, 河豚毒素)敏感钠通道具有浓度依赖性阻滞作用,1,10和100 μmol/L的苦参碱作用5 min后,分别使钠电流峰值较给药前下降（12.49±1.67）%、（18.79±2.16）%和（43.15±8.17）% (n=8, P<0.05)。苦参碱使钠电流的电流 电压关系曲线上移,但并不改变其激活电压、峰电压和电流电压关系曲线的形状。苦参碱对钠通道的阻滞作用可能是其具有某些毒理效应的离子基础。     

成年蜜蜂脑神经细胞的培养和电生理特征

为了研究杀虫剂等对蜜蜂毒性作用的神经机制,需在体外建立成年蜜蜂脑神经细胞的分离培养和电生理记录技术并研究其正常电生理特征,而对成年蜜蜂脑神经细胞的分离培养和电生理特性的研究报道甚少。我们采用酶解和机械吹打相结合的方法获得了数量较多且活力较好的成年意大利蜜蜂Apis mellifera脑神经细胞,并用全细胞膜片钳技术研究了成年意大利蜜蜂脑神经细胞对电流和电压刺激的反应,获得了成年意蜂脑神经细胞的基本电生理特征以及钠电流和钾电流的特性。全细胞电流钳的记录结果表明,在体外培养条件下,细胞无自发放电发生,注射电流后仅引起细胞单次放电,引起细胞放电的阈电流平均为60.8±63 pA; 细胞动作电位产生的阈电位平均为−27.4±2.3 mV。用全细胞电压钳记录了神经细胞的钠电流和钾电流。钠电流的分离是在电压刺激下通过阻断钾通道和钙通道实现。细胞的内向钠电流在指令电压为−40～−30 mV左右激活,−10 mV达峰值,钠通道的稳态失活电压V_1/2为−58.4 mV; 外向钾电流成份至少包括较小的快速失活钾电流和和较大的缓慢失活钾电流（占总钾电流的80%）,其半激活膜电位V_1/2为3.86 mV,无明显的稳态失活。结果提示缓慢失活钾电流的特征可能是细胞单次放电的机制之一。     

弱激光对大鼠海马神经元钠通道特性的影响

利用波长670nm、功率5mW的半导体激光器照射急性分离的大鼠海马CA3区锥体神经元,应用全细胞膜片钳技术研究其电压门控Na 通道的特性.实验发现:弱激光作用5min时,Na 通道激活电位和峰值电位开始向负电位方向移动,7min激光作用达稳定;激光照射对Na 通道电流峰值无影响,对照组和激光照射组峰值电流密度分别为(-383.51±26.93)pA/pF和(-368.36±33.14)pA/pF(n=8,P>0.05);激光作用降低了Na 通道的激活阈值电位和峰值电位,对照组通道电流在-40mV激活,-30mV达峰值,激光照射组通道电流在-60mV激活,-40mV达峰值;激光照射改变了Na 通道半数激活电压和斜率因子,对照组和激光照射组的半数激活电压分别为(-42.091±1.537)mV和(-54.971±1.846)mV(n=8,P<0.01),斜率因子分别为(1.529±0.667)mV和(2.634±0.519)mV(n=8,P<0.05).结果表明,弱激光照射海马神经元可改变Na 通道的激活特性,从而影响动作电位的去激化过程,进而会引起神经元细胞生理功能发生变化.     

河蟹眼柄神经分泌细胞离子通道的膜片钳研究

采用全细胞膜片钳技术对培养12－24小时不同形态河蟹眼柄视节端髓X器官（MTXO）神经分泌细胞离子通道进行了研究。结果表明,河蟹眼柄MTXO中分布的A、B、C三种类型神经分泌细胞均可记录到由向电流和外向电流组成的正常全细胞电流。内向电流由高电压激活钙离子通道电流（Lca)和对TTX敏感钠离子通道电流（INa）组成。ICa的激活电压为－30mV,在0－ 20mV电压下达到峰值,在－40mV和－70mV保持电压下记录的ICa激活阈值、初始峰值及I－V曲线无明显差别。外向电流明显,幅值较大,包括对4－AP敏感的快速激活、快速失活钾离子通道电流（IA）和对TEA敏感的缓慢激活、缓慢失活钾离子通道电流（IK）。正常蟹种、二龄成蟹和早熟蟹种MTXO神经分泌细胞均表达电压门控钠、钾、钙离子通道,通道电流和电压特征无明显区别.     

苦皮藤素Ⅳ麻醉机理的膜片钳研究

用膜片钳技术研究了植物杀虫剂苦皮藤素Ⅳ对棉铃虫幼虫离体培养中枢神经细胞钠通道门控过程的影响.结果表明,苦皮藤素Ⅳ对钠通道具有迅速的浓度依赖性阻滞作用,使电流-电压关系(I-V)曲线上移.0.1、1和10 μmol/L苦皮藤素Ⅳ作用3 min后,分别使钠电流峰值(INaMax)较给药前下降2735%±4.05%、62.72%±2.81%和88.53%±5.56%(P＜0.05),1和10μmol/L苦皮藤素Ⅳ还使钠通道的激活电压和峰电压分别向正电位方向移动了10 mV和20 mV左右.同时比较研究了利多卡因对棉铃虫幼虫神经细胞钠通道的影响,利多卡因对钠通道也具有阻滞作用,但有效作用浓度明显高于苦皮藤素Ⅳ.1、70 mmol/L的利多卡因注射液作用3 min后,使INaMax较用药前下降21.21%±2.52%和95.63%±2.10%(P＜0.05).苦皮藤素Ⅳ对钠通道门控过程的影响与利多卡因等局部麻醉剂非常相似,因此,对钠通道的阻滞作用可能是其发挥麻醉作用的重要机制.     

苦皮藤素IV麻醉机理的膜片钳研究

用膜片钳技术研究了植物杀虫剂苦皮藤素Ⅳ对棉铃虫幼虫离体培养中枢神经细胞钠通道门控过程的影响。结果表明,苦皮藤素Ⅳ对钠通道具有迅速的浓度依赖性阻滞作用,使电流-电压关系(I-V)曲线上移。0.1、1和10μmol/L苦皮藤素Ⅳ作用3min后,分别使钠电流峰值(INaMax)较给药前下降27.35%±4.05%、62.72%±2.81%和88.53%±5.56%(P<0.05),1和10μmol/L苦皮藤素Ⅳ还使钠通道的激活电压和峰电压分别向正电位方向移动了10mV和20mV左右。同时比较研究了利多卡因对棉铃虫幼虫神经细胞钠通道的影响,利多卡因对钠通道也具有阻滞作用,但有效作用浓度明显高于苦皮藤素Ⅳ。1、70mmol/L的利多卡因注射液作用3min后,使INaMax较用药前下降21.21%±2.52%和95.63%±2.10%(P<0.05)。苦皮藤素Ⅳ对钠通道门控过程的影响与利多卡因等局部麻醉剂非常相似,因此,对钠通道的阻滞作用可能是其发挥麻醉作用的重要机制。     

Effects of cyhalothrin on the transient outward potassium current in central neurons of Helicoverpa armigera

The effects of cyhalothrin on the transient outward potassium current in central neurons of Helicoverpa armigera were studied by using the patch clamp techniques. The results showed that before using cyhalothrin （10.5 mmol/L）, activation potential was approximately -40 mV, after application of the drug, the activation potential shifted roughly 10 mV to the negative potential direction, so channels can be activated more easily. Before and after cyhalothrin application, the change of current amplitude was insignificant. The value of V1/2 and k of activation curves did not change significantly, however, the V1/2 of the inactivation curves changed significantly. Inactivation curves significantly shifted to a negative direction, so that inactivation of the channels was hastened. It is indicated that there may exit a primary way in which cyhalothrin provides neurotoxicity to the nervous system through the regulation of activation potentials and inactivation state of IA channels.     

cAMP和cGMP对棉铃虫神经细胞高电压激活钙通道的调节作用

用全细胞膜片钳法研究了cAMP和cGMP对棉铃虫Helicoverpa armigera 3龄幼虫胸腹神经节细胞高电压激活钙通道的调节作用。细胞外液中加入腺苷酸环化酶（AC）激活剂福斯克林（forskolin） 0.1 mmol/L,对于Ba²⁺介导的钙通道电流激活电压、峰电压、峰电流变化以及通道激活和电流达到峰值的时间无影响。电极内液中加入1 mmol/L的cGMP则明显抑制峰电流,且抑制作用呈时间依赖性和浓度依赖性,而对激活电压、峰电压无影响。结果提示,棉铃虫神经细胞高电压激活钙通道的活动可能不受细胞内cAMP水平提高的影响,但被cGMP抑制。     

Voltage-dependent and odorant-regulated currents in isolated olfactory receptor neurons of the channel catfish.

The electrical properties of olfactory receptor neurons, enzymatically dissociated from the channel catfish (Ictalurus punctatus), were studied using the whole-cell patch-clamp technique. Six voltage-dependent ionic currents were isolated. Transient inward currents (0.1-1.7 nA) were observed in response to depolarizing voltage steps from a holding potential of -80 mV in all neurons examined. They activated between -70 and -50 mV and were blocked by addition of 1 microM tetrodotoxin (TTX) to the bath or by replacing Na+ in the bath with N-methyl-D-glucamine and were classified as Na+ currents. Sustained inward currents, observed in most neurons examined when Na+ inward currents were blocked with TTX and outward currents were blocked by replacing K+ in the pipette solution with Cs+ and by addition of 10 mM Ba2+ to the bath, activated between -40 and -30 mV, reached a peak at 0 mV, and were blocked by 5 microM nimodipine. These currents were classified as L-type Ca2+ currents. Large, slowly activating outward currents that were blocked by simultaneous replacement of K+ in the pipette with Cs+ and addition of Ba2+ to the bath were observed in all olfactory neurons examined. The outward K+ currents activated over approximately the same range as the Na+ currents (-60 to -50 mV), but the Na+ currents were larger at the normal resting potential of the neurons (-45 +/- 11 mV, mean +/- SD, n = 52). Four different types of K+ currents could be differentiated: a Ca(2+)-activated K+ current, a transient K+ current, a delayed rectifier K+ current, and an inward rectifier K+ current. Spontaneous action potentials of varying amplitude were sometimes observed in the cell-attached recording configuration. Action potentials were not observed in whole-cell recordings with normal internal solution (K+ = 100 mM) in the pipette, but frequently appeared when K+ was reduced to 85 mM. These observations suggest that the membrane potential and action potential amplitude of catfish olfactory neurons are significantly affected by the activity of single channels due to the high input resistance (6.6 +/- 5.2 G omega, n = 20) and low membrane capacitance (2.1 +/- 1.1 pF, n = 46) of the cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

GTP和GDP类似物对棉铃虫神经细胞高电压敏感钙通道的调节作用

以Ba²⁺为载流子,采用全细胞膜片钳法,研究了在电极液中分别加入G蛋白稳定 激活剂GTPγS（GTP类似物）和抑制剂GDPβS（GDP类似物）对棉铃虫Helicoverpaarmigera 3龄幼虫神经细胞高电压敏感钙通道的调节作用。Ba²⁺电流记录时间为20 min。对照组 和实验组的Ba²+电流在记录的初期均出现电流的增加现象,随后电流衰减,即“rundown ”。对照组峰电流在第20 min时降为初始值的(72.09±12.80)%。电极内液中加入2 mmol/L GTPγS可缓解电流的衰减现象,在第20 min时,峰电流为初始值的(95.99±7.93)%,明显大 于对照组的峰电流(P<0.01),而且电流 电压（I-V）关系曲线向正电压方向移动。相反 ,电极内液加入2 mmol/L GDPβS则导致峰电流衰减更加严重,第20 min时,峰电流仅为初始水 平的(41.95±9.32)%,显著小于对照组（P<0.01）,但未见电流 电压（I-V）关系曲 线的明显漂移。结果表明,棉铃虫神经细胞钙通道活动受G蛋白激活剂GTPγS和G蛋白抑制剂GDPβS的影响,提示G蛋白活动水平的改变调节钙通道的电流幅值和电压依赖性。     

Chronic sustained and intermittent hypoxia reduce function of ATP-sensitive potassium channels in nucleus of the solitary tract

Activation of neuronal ATP-sensitive potassium (K(ATP)) channels is an important mechanism that protects neurons and conserves neural function during hypoxia. We investigated hypoxia (bath gassed with 95% N(2)-5% CO(2) vs. 95% O(2)-5% CO(2) in control)-induced changes in K(ATP) current in second-order neurons of peripheral chemoreceptors in the nucleus of the solitary tract (NTS). Hypoxia-induced K(ATP) currents were compared between normoxic (Norm) rats and rats exposed to 1 wk of either chronic sustained hypoxia (CSH) or chronic intermittent hypoxia (CIH). Whole cell recordings of NTS second-order neurons identified after 4-(4-(dihexadecylamino)styryl)-N-methylpyridinium iodide (DiA) labeling of the carotid bodies were obtained in a brain stem slice. In Norm cells (n = 9), hypoxia (3 min) induced an outward current of 12.7 +/- 1.1 pA with a reversal potential of -73 +/- 2 mV. This current was completely blocked by the K(ATP) channel blocker tolbutamide (100 muM). Bath application of the K(ATP) channel opener diazoxide (200 muM, 3 min) evoked an outward current of 21.8 +/- 5.8 pA (n = 6). Hypoxia elicited a significantly smaller outward current in both CSH (5.9 +/- 1.4 pA, n = 11; P < 0.01) and CIH (6.8 +/- 1.7 pA, n = 6; P < 0.05) neurons. Diazoxide elicited a significantly smaller outward current in CSH (3.9 +/- 1.0 pA, n = 5; P < 0.05) and CIH (2.9 +/- 0.9 pA, n = 3; P < 0.05) neurons. Western blot analysis showed reduced levels of K(ATP) potassium channel subunits Kir6.1 and Kir6.2 in the NTS from CSH and CIH rats. These results suggest that hypoxia activates K(ATP) channels in NTS neurons receiving monosynaptic chemoreceptor afferent inputs. Chronic exposure to either sustained or intermittent hypoxia reduces K(ATP) channel function in NTS neurons. This may represent a neuronal adaptation that preserves neuronal excitability in crucial relay neurons in peripheral chemoreflex pathways.     

A new scorpion toxin with a very high affinity for sodium channels. An electrophysiological study

The neurotoxic action of toxin gamma from the venom of the Brazilian scorpion Tityus serrulatus (TiTx gamma) has been investigated in cultured mouse neuroblastoma cells (N1E115) using the suction pipette technique. Addition of 14 to 53 nM TiTx gamma to the external solution causes nerve cell membrane depolarization, membrane potential oscillations and spontaneous action potentials within 10 min. None of these effects were observed within 15 min after application of 1 microM toxin IV from Centruroides sculpturatus venom. Under voltage clamp the amplitude of the sodium current evoked by test pulses to potentials more positive than -30 mV is reversibly reduced by 50% after 17 to 105 nM TiTx gamma. On the other hand, a sodium current component appears after TiTx gamma at test pulse potentials between -70 and -40 mV, for which no sodium current is observed in the control experiment. The outward potassium current is not significantly affected by the highest TiTx gamma concentrations used. The potential-dependence of inactivation of the sodium current component that is induced by TiTx gamma is shifted by -30 mV with respect to control values. The local anaesthetic procain at 1 mM discriminates between the two populations of sodium channels observed in the presence of TiTx gamma.     

棉铃虫幼虫神经细胞的急性分离培养及其电压门控通道的膜片钳研究

探索了棉铃虫Helicoverpa armigera幼虫神经细胞的急性分离与体外培养的条件,并利用全细胞膜片钳技术首次对棉铃虫幼虫急性分离神经细胞的电压门控性钠、钾和钙通道的基本电生理学特性进行了研究。结果表明,棉铃虫幼虫中枢神经细胞在TC-100、L-15和Grace培养基中均可贴壁生长,在DMEM培养基中基本不能存活。在TC-100培养基分别与其它三种培养基按一定比例混合形成的培养液中,TC-100与L-15等量混合培养液更适合于神经细胞的生长。全细胞电压钳条件下,可分别记录到电压门控性钠、钾和钙通道电流。钙电流特征为高电压激活、缓慢失活;钠电流对河豚毒素敏感;钾电流可被细胞外液中的氯化四乙胺和4-氨基吡啶抑制。