In vivo patch clamp recording technique in the study of neurophysiology

The patch clamp recording technique in vivois a blind patch clamp recording methods to record the current of the spinal or cereral neurons of anaes：hesia （ or awake） animals. This technique can be used to study the synaptic function and plasticity in central nervous system in vivoin order to understand the physiological properties of the ion channels from an integrated point of view. The advantage of this technique have already presented itself in the study of the synaptic transmission and nervous network. Nowadays, in vivo patch whole-cell recording technique in combination with other techniques is becoming a common method in the research fields.

A patch-clamp study on human sperm Cl^- channel reassembled into giant liposome

Aim: To record the single-channel currents and characterize the electrophysiological properties of the Cl^- channels inhuman sperm membrane. Methods: The membrane proteins extracted from the human sperm were reassembled intoliposome bilayer, and the liposomes were fused into giant liposomes with a diameter more than 10μm by dehydration-rehydration procedure. The giant liposomes were used to study the Cl^- channel activities by patch-clamp technique.Results: By patch clamping the giant liposome in an asymmetric NMDG (N-methyl-D-glucamine)-Cl (bath 100//pipette 200 mmol/L) solution system, three kinds of single-channel events with unit conductances of (74.1 ± 8.3) pS,(117.0±5.7) pS and (144.7±4.5) pS, respectively, were detected. Their activities were voltage-dependent and allwere blocked by SITS (4-acetamido-4'-isothiocyanato-stilbene-2', 2'-disulfonic acid) in a concentration-dependentmanner. By constructing the open and close dwell time distribution histograms and then fitting them with exponentialfunction, two time constants were obtained in both the open and the close states. The burst activity and conductancesubstate of the channels were observed. Conclusion; There exist three kinds of Cl^- channels with different conduc-tance in human sperm membrane at least. (Asian J Androl 2001 Sep; 3: 185 - 191)

Induction of pancreatic duct cells of neonatal rats into insulin-producing cells with fetal bovine serum: A natural protocol and its use for patch clamp experiments

AIM： To induce the pancreatic duct cells into endocrine cells with a new natural protocol for electrophysiological study. METHODS： The pancreatic duct cells of neonatal rats were isolated, cultured and induced into endocrine ceils with 15% fetal bovine serum for a period of 20 d. During this period, insulin secretion, MTT value, and morphological change of neonatal and adult pancreatic islet cells were comparatively investigated. Pancreatic β-cells were identified by morphological and electrophysiological characteristics, while ATP sensitive potassium channels （KATP）, voltage-dependent potassium channels （Kv）, and voltage-dependent calcium channels （KcA） in β-cells were identified by patch clamp technique. RESULTS： After incubation with fetal bovine serum, the neonatal duct cells budded out, changed from duct-like cells into islet clusters. In the first 4 d, MTT value and insulin secretion increased slowly （MTT value from 0.024 ±0.003 to 3.028±0.003, insulin secretion from 2.6±0.6 to 3.1±0.8 mIU/L）. Then MTT value and insulin secretion increased quickly from d 5 to d 10 （MTT value from 0.028 ±0.003 to 0.052±0.008, insulin secretion from 3.1±0.8 to 18.3±2.6 mIU/L）, then reached high plateau （MTT value 〉0.052±0.008, insulin secretion 〉18.3±2.6 mIU/L）. In contrast, for the isolated adult pancreatic islet cells, both insulin release and MTT value were stable in the first 4 d （MTT value from 0.029±0.01 to 0.031±0.011, insulin secretion from 13.9±3.1 to 14.3±3.3 mIU/L）, but afterwards they reduced gradually （MTT value 〈0.031 ±0.011, insulin secretion 〈8.2±1.5 mIU/L）, and the pancreatic islet cells became dispersed, broken or atrophied correspondingly. The differentiated neonatal cells were identified as pancreatic islet cells by dithizone staining method, and pancreatic β-cells were further identified by both morphological features and electrophysiological characteristics, i.e. the existence of recording currents from KATP, Kv, and KCA. CONCLUSION： Islet cells differentiated from neonatal pancreatic duct cells with the new natural protocol are more advantageous in performing patch clamp study over the isolated adult pancreatic islet cells.

Action Mechanism Research of Lanthanons to Slow Vacuolar Ion Channels in Raphanus Satirus L.(Xinlimei) Radish by Patch-Clamp

We used whole-vacuolar patch-clamp recording mode to study the action mechanism of La3+ to Slow Vacuolar (SV) channels for the first time. We recorded SV channel currents of Xinlimei (Raphanus satirus L.) vacuolars. The minimum activation potentials of voltage-dependent SV channels tied in 25+/-5 mV. The increase in cytoplasmic Ca2+ led to enhancement of SV-type currents. It was found that the threshold potential of activation shifted towards more depolarized values whenever cytoplasmic Ca2+ was increased. When 10(-10) mol/L free La3+ was added to the bath, SV-type current was suppressed by 60 similar to 75%. These data showed La3+ reduced ion permeabilities of Xinlimei root vacuolar membrane.

Feasibility and limitation of patch-clamp recordings on neonatal rat cardiac ventricular slices

To attempt to the feasibility of whole-cell patch-clamp recordings on the cardiac ventricular slices of newborn (P3-P7) Sprague-Dawley (S-D) rats and find a good substitute of the single cardiac myocytes with enzymatic treatment. High resistance seals (】1 GΩ) could be obtained in the cardiac ventricle tissue on this preparation without enzymatic treatment. Then, the cell-attached and whole-cell patch-clamp techniques can be achieved in the thin (200μm) cardiac slices. Averaged sodium current (n=ll cells) was recorded in cell-attached mode, displayed similar features to that previously reported from isolated rat ventricular myocytes. The outward potassium current, Hyperpolarization-activated cation channel or If channel (HCN channel) and action potential (AP) were recorded in whole-cell configuration (n=2 cells) and the similar properties can be seen from the cardiac slices. Cell-attached mode is a stable and reliable mode to record the ion treatment. Resting potential for cardiac slice, measured from Current-clamp recording in whole-cell mode, is about -50 - - 70 mV. The resting potential value from the cardiac slice has similar property except that it is positive to the isolated cardiomyocytes by enzymes. Application of patch-clamp techniques to cardiac slices allows single channel recordings without complicated procedures of cell isolation. Moreover, possible alteration of channel properties caused by proteolytic enzymes can be avoided. In this paper, whole-cell patch-clamp recordings could be achieved on the cardiac slice and we affirmed the feasibility and values of the both recording modes on it. At the same time, there is difficulty and limitation of the application of whole-cell patch-clamp on the cardiac slice for the existence of large mount of connective tissue even in the newborn rats.

The Reconstructing of Low Signal-noise Ratio Single Ion Channel Signal from Patch-clamp Recordings Sampled in the Colored Background Noise

The single ion channel signal is an ionic current that can be recorded by the patch clamp technique. Hidden Markov model (HMM) algorithm has been used to convert the low signal noise ratio (SNR) noisy recording into an idealized quantal one in the case of white background noise. The traditional HMM algorithm is extended and adapted to the colored background noise. A new algorithm called EHMM (Extended HMM) algorithm is proposed, and mainly validated by simulation. Results show that it’s effective.

Depression and Insulin Resistance in Non-Diabetic Subjects: An Intervention Study with Insulin Clamp Technique

Aims: To clarify the relationship between depression and glucose metabolism using sensitive measures of insulin resistance, and to assess if remission of depression results in enhanced insulin sensitivity. Methods: An intervention study to quantify changes in insulin sensitivity before and after treatment of depression was carried out. Twenty six Pakistani women with newly diagnosed depression underwent euglycemic insulin clamp to measure insulin sensitivity at inclusion and again after treatment of depression 6 - 8 weeks later. Twenty-three individuals completed both tests. Results: Significant improvement of insulin sensitivity was observed following the treatment of depression. The improved insulin sensitivity remained statistically significant after controlling for confounding factors. Conclusions: This study establishes a relationship between depression and insulin resistance. It demonstrated that insulin sensitivity can be improved by treating depression.

Study of Oligonucleotide Fixation on Bilayer Lipid Membranes by Patch-clamp Pipette Support

One kind of novel BLMs was fabricated by patch-clamp pipette technology characterized in considerably sensitive to changes of electrochemical parameters.Detectiye currents and voltage presented linear relationship when BLMs was formed and it could be confirmed by Gramicidin method.Ion current was increased by dihexyl (C_ (12)) modified ssDNA fixed on the BLMs and also indicated linear relationship to ssDNA's concentration due to the interaction of (C_ 12)-ssDNA and BLMs.Further more,the regression equations were different from BLMs fixed with ssDNA probe and a blank control BLM in the same experimental conditions.The ssDNA probe was successfully fixed on patch-clamp pipette supported-BLMs.Based on our studies,a biosensor with reactive element of patch-clamp pipette-supported BLMs has been established.

马钱子碱抑制豚鼠心室肌细胞钠电流的作用研究

目的本研究旨在分析马钱子碱(brucine)对分离的单个豚鼠心室肌细胞钠电流(INa+)的作用,并探索其抗心律失常的可能机制。方法共选取24只健康的成年豚鼠作为实验对象,雌雄不拘,随机分为4组,每组6只:第1组是未接受任何处理的对照组,之后通过微量加样器使培养皿中brucine药物的终浓度分别达到3μmol/L、10μmol/L、30μmol/L。采用急性酶解法分离获得单个豚鼠心室肌细胞,通过全细胞膜片钳技术测量离子通道电流,以检测不同浓度的brucine对豚鼠心室肌细胞INa+的影响。结果正常的对照组没有明显的INa+电流峰值的变化,而brucine 3μmol/L组给药前后基本不影响INa+电流峰值,当brucine的浓度增加到10μmol/L时,给药前后INa+电流峰值显著下降(P<0.05),在30μmol/L组给药前后INa+电流峰值大幅度减少(P<0.01),并且这种抑制作用呈浓度依赖性。正常对照组及brucine 3μmol/L剂量组中,电流-电压曲线(Ⅰ~Ⅴ曲线)并未受到显著的影响;然而,10μmol/L、30μmol/L剂量组与对照组比较,INa+的Ⅰ~Ⅴ曲线上移,无平行移动,且曲线形状不变。结论Brucine能够通过抑制心室肌细胞钠通道电流发挥抗心律失常作用。

无镁诱导仓鼠原代皮质神经元电生理学特性

目的无镁细胞外液建立癫痫放电模型,利用全细胞膜片钳技术,检测仓鼠原代皮质神经元的电生理学特性。方法采用生后1~2 d新生叙利亚仓鼠,分离大脑皮质培养原代神经元培养至第12天,分别予有镁细胞外液(有镁组)和无镁细胞外液(无镁组)孵育3 h,3 h后均更换为正常孵育液继续培养24 h。利用全细胞膜片钳技术,电压钳模式下记录神经元兴奋性突触后电流(excitatory postsynaptic currents,EPSC),电流钳模式下记录神经元兴奋性突触后电位(excitatory postsynaptic potentials,EPSP)。结果与有镁组相比,无镁组仓鼠原代皮质神经元EPSC[(124.38±75.15)Hz vs.(33.93±22.32)Hz,P<0.001]及EPSP[(37.05±38.37)Hz vs.(5.63±9.52)Hz,P<0.01]的频率升高,且差异有统计学意义;而两组之间EPSC及EPSP的振幅、曲线下面积和半宽度的差异无统计学意义(P>0.05)。结论无镁处理后仓鼠原代皮质神经元兴奋性升高,仓鼠原代皮质神经元可用于构建癫痫细胞模型。

环状RNA mmu_circ_0005019影响小鼠心肌细胞钙激活钾通道电流及动作电位

目的 探索环状RNA mmu_circ_0005019调控小电导钙激活钾(small-conductance calcium-activated potassium, SK)通道蛋白亚基SK3编码基因Kcnn3的表达,以及对小电导钙激活钾通道电流(IK,Ca)和动作电位时程(action potential duration, APD)的影响。方法 在小鼠HL-1细胞中分别构建mmu_circ_0005019过表达和干扰模型,分为过表达组(n=3)、空质粒组(n=3)、干扰1组(n=3)、干扰2组(n=3)、对照组(n=3),通过RT-qPCR、Western blot分析mmu_circ_0005019调节Kcnn3表达的分子机制,应用膜片钳技术电流钳模式记录全细胞的IK,Ca,并用电压钳模式记录APD。结果 成功构建了环状RNA mmu_circ_0005019过表达和干扰的HL-1细胞模型。过表达组的Kcnn3基因表达与空质粒组相比明显上调(P<0.05);干扰1组和干扰2组的Kcnn3基因表达与对照组相比均明显下调(P<0.05)。电生理发现过表达mmu_circ_0005019增加了HL-1细胞IK,Ca电流密度,APD显著缩短;相反,干扰mmu_circ_0005019减少了IK,Ca电流密度,APD显著延长。结论 mmu_circ_0005019可以上调小鼠HL-1细胞Kcnn3的表达水平,从而改变IK,Ca和APD,提示环状RNA mmu_circ_0005019可能在房颤发生中起到促进作用。

神经示踪染料DiI影响溃疡性结肠炎小鼠背根神经节神经元电学特性

目的:探究在神经示踪结合膜片钳全细胞记录的实验中,示踪染料DiI是否会对小鼠背根神经节(dorsal root ganglion,DRG)神经元兴奋性产生影响。方法:采用SPF级雄性C57BL/6J小鼠,经5%葡聚糖硫酸钠(dextran sulfate sodium,DSS)诱导溃疡性结肠炎(ulcerative colitis,UC)模型,在T12~L2皮节多点注射DiI逆向示踪躯体感觉神经元,分离全组织DRG并进行全细胞膜片钳记录。结果:HE染色结果显示模型诱导成功。膜片钳记录未经标记的小鼠DRG神经元,模型组小鼠膜电位兴奋性高于正常组小鼠。两组小鼠示踪后,正常示踪组和模型示踪组DiI+神经元兴奋性均低于本组DiI-神经元;与两未示踪组的神经元相比较,两示踪组的DiI-神经元兴奋性分别升高。结论:本实验结果明确了示踪剂DiI会降低DRG神经元的兴奋性,提示相关实验设计中应注意示踪剂的应用对神经元兴奋性的影响,以保证实验结果的可靠性。

膜片箝pClamp采样软件中的P/N漏减分析

本文采用大鼠海马脑片盲法的全细胞记录技术 ,研究了Axon公司pClamp采样软件Clampex中P/N漏减的功能意义和作用机制 ,对P/N漏减脉冲电流的采集以及漏减脉冲的时间、极性、箝位电压、数目、位置等参数的设置进行了详细分析。结果表明 ,在采集电压门控性离子通道电流时 。

Loose-patch方法及其诱发的钙火花

目的 :采用Loose patch方法在心室肌单细胞上诱发出钙火花并探讨其主要特征 ,并与以往有关的研究结果作比较分析 .方法 :Loose patch方法 ,即非紧密封接的细胞贴附式低阻抗膜片钳制技术和共聚焦显微镜钙成像技术相结合的方法 .结果 :Loose patch方法可以较高的概率 (72 .2 % )成功地诱发钙火花 ;其诱发的平均钙火花的幅度为 (1 .0 7± 0 .0 6 )(单位为△F/F0 ,其中F0 为静息时的钙荧光强度 ) ;空间范围为 (1 .4 4± 0 .0 3) μm ;n =1 2 4 .结论 :Loose patch方法可在保证膜上的L型钙通道与胞内相邻近的Ryanodine受体之间正常的信息耦联关系的前提下 ,定点诱发钙火花 ,并通过共聚焦显微镜钙成像技术实现实时准确记录 .这对于明确钙火花的特征 ,准确理解兴奋

基于PiggyBac转座子优化稳定表达细胞系的新型瞬时受体电位M2通道抑制剂筛选

目的:使用PiggyBac(PB)转座系统建立稳定表达瞬时受体电位M2(TRPM2)通道的人胚胎肾细胞HEK293T,并进行TRPM2通道抑制剂筛选,为治疗脑缺血等疾病寻找潜在的药物。方法:根据PB转座原理,构建pPB-hTRPM2真核表达载体。将重组质粒和辅助质粒共同转染至HEK293T细胞中,利用系统携带的荧光与膜片钳鉴定TRPM2基因表达,通过Z'因子评估细胞模型是否适用于钙成像法的高通量筛选。利用钙成像法和膜片钳对11个先导化合物分子进行初步活性评价,测定化合物分子对TRPM2通道的抑制活性。利用氧糖剥夺/再灌注(OGD/R)损伤模型和细胞计数试剂盒8(CCK-8)方法验证筛选得到的化合物分子对损伤细胞的保护作用。利用流式细胞术检测细胞活性氧水平。利用小鼠短暂性大脑中动脉栓塞(tMCAO)模型评价化合物的神经保护作用。结果:成功构建pPB-hTRPM2真核表达载体,构建出可高效表达TRPM2基因的HEK293T细胞系,并同时表达增强型绿色荧光蛋白(EGFP)。在筛选获得的嘌呤霉素抗性细胞中,所有细胞荧光明亮可见,诱导后细胞表现出经典的TRPM2通道电流特征,TRPM2通道电流值与瞬时转染对照组差异无统计学意义(P>0.05)。该筛选系统进行钙成像实验的Z'因子为0.5416,表明其适用于高通量筛选。通过钙成像法结合膜片钳筛选出化合物6,其具有显著的TRPM2通道抑制作用,且1.0μmol/L的化合物6能够显著提高OGD/R后SH-SY5Y细胞的存活率(P<0.05),降低活性氧水平(P<0.05),0.3、1.0 mg/kg的化合物6能降低tMCAO小鼠脑梗死体积百分比(均P<0.05)。结论:利用PiggyBac基因编辑技术成功构建了TRPM2基因稳定表达细胞系,利用钙成像法和膜片钳在候选化合物中成功筛选出一个高亲和力的新的TRPM2通道抑制剂。该抑制剂可以通过降低细胞活性氧水平缓解OGD/R后细胞损伤,并对小鼠脑缺血再灌注损伤具有保护作用。

紫云英苷对SD大鼠心肌细胞瞬时外向钾通道电流的影响

为探究紫云英苷(astragalin,AG)对心肌细胞瞬时外向钾通道电流Ito的作用,用langendorff主动脉逆向灌流法急性分离出单个SD大鼠心肌细胞,进行全细胞膜片钳试验,记录给药前后Ito的变化及激活、失活和复活过程中的动力学特征。结果表明:不同浓度的AG均表现出对Ito的抑制作用,在50、100、200μmol·L^(-1)浓度范围内,随着药物浓度的增加,抑制作用逐渐增强。以给药前Ito幅值为参照,给予各浓度AG后,电流分别降至原来的(84.06±2.75)%、(66.09±4.65)%、(48.79±5.96)%,后2个浓度的效应与给药前相比具有统计学意义。在动力学特征方面,50、100、200μmol·L^(-1)浓度的AG使瞬时外向钾通道激活阈值上升,失活加速,从失活状态到活化状态的转换延长。综上,AG对SD大鼠心肌细胞上的Ito有抑制作用,此效应的产生与通道的激活、失活和失活后恢复动力学过程改变有关。

超极化激活的环核苷酸门控通道1参与七氟烷诱导的顺行性遗忘作用的机制研究

目的旨在探讨超极化激活的环核苷酸门控通道1(hyperpolarization-activated cyclic nucleotide-gated cation channel 1,HCN1)在七氟烷诱导的顺行性遗忘中的作用机制。方法采用雄性SPF级C57BL/6J野生型小鼠(WT组,n=60)和HCN1基因全身敲除(HCN1-/-)小鼠(HCN1-/-组,n=60)。将两组小鼠分别暴露于不同浓度的七氟烷(0%、0.1%、0.2%、0.4%、0.6%和0.8%)下,每个浓度10只小鼠。随后进行条件性恐惧实验,计算两组小鼠七氟烷抑制条件性恐惧记忆的半数效应浓度(median effective concentration,用EC50表示)。采用全细胞膜片钳技术记录0.125mmol/L七氟烷浓度灌流液对转染HCN1的人胚肾293细胞(HEK293-HCN1)上HCN1电流的影响,比较七氟烷处理前(Control组)和处理后(Sevoflurane组)HCN1电流的变化。结果与-/-WT组小鼠相比,HCN1组小鼠在七氟烷抑制场景恐惧记忆和声音恐惧记忆中的EC50值升高,差异有统计学意义(场景恐惧记忆:0.18%比0.26%,P<0.001;声音恐惧记忆:0.47%比0.49%,P<0.001)。全细胞电生理记录显示,与基础值相比,0.125mmol/L七氟烷抑制HEK293-HCN1细胞在﹣90~﹣140mV范围内的电流幅度(P值均<0.05),并增加了半最大激活电压(V1/2a)值[(﹣108±2.9)mV比(﹣100.1±3.0)mV,(P<0.001)]。结论HCN1电流的抑制可能参与了七氟烷诱导的顺行性遗忘作用。

基于声辐射模态的双面Patch近场声全息技术

针对近场声全息技术在应用过程中遇到的外界干扰和测量孔径效应的双重问题,提出了基于源强密度声辐射模态理论的双面Patch近场声全息技术。经过理论分析和公式推导,建立了由声辐射模态叠加表达的声场声压数学模型。首先,采用双面声场分离技术清除测量面背向干扰源的影响;然后,对分离数据进行内插与外推,等效增加声源在全息面处的声压数据,减小了测量间隔;最后,获得了声源在非自由声场环境下具有高空间分辨率的全息图像。与传统的分离方法和Patch技术相比,所提方法综合考虑了相关参数的优化选择与控制,便于应用与推广。为了验证该方法,在理论工作的基础上,以刚性脉动球为对象进行了仿真研究,以音箱为对象进行了试验测试,相关结果证明了该方法的有效性与正确性。

手动膜片钳检测盐酸罗哌卡因及其右旋异构体对HEK293细胞hERG电流的影响

目的研究比较盐酸罗哌卡因和盐酸罗哌卡因右旋异构体对高表达hERG钾通道的HEK293细胞hERG电流的影响。方法用手动膜片钳检测转染后hERG钾通道稳定表达的HEK293细胞电流,多菲莱德做阳性药,将盐酸罗哌卡因和盐酸罗哌卡因右旋异构体依次稀释成30.00、10.00、3.33、1.11、0.37μmol·L^(-1),依次作用于细胞,记录电流变化,计算抑制率。结果盐酸罗哌卡因0.37、1.11、3.33、10、30μmol·L^(-1)对电流Iherg-tail的抑制率分别为(6.12±0.30)%、(13.04±1.20)%、(19.21±0.33)%、(35.56±0.66)%、(65.37±4.17)%,IC_(50)为19.482μmol·L^(-1)(n=15)。盐酸罗哌卡因右旋异构体0.37、1.11、3.33、10.00、30.00μmol·L^(-1)对电流Iherg-tail的抑制率分别为(4.13±3.43)%、(7.34±5.60)%、(9.49±2.75)%、(16.60±0.87)%、(31.36±1.45)%,IC_(50)>30μmol·L^(-1)(n=15)。阳性对照药品多菲莱德0.00185、0.00556、0.01667、0.05000、0.15000μmol·L^(-1)对电流Iherg-tail的抑制率分别为(7.81±2.77)%、(19.67±1.88)%、(57.16±4.39)%、(89.71±3.55)%、(99.66±0.89)%、IC_(50)为0.015μmol·L^(-1)(n=15)。结论和阳性对照药品多菲莱德比较,盐酸罗哌卡因对hERG通道为弱抑制作用,盐酸罗哌卡因右旋异构体对hERG通道为无明显抑制作用。

冬凌草甲素对海马CA1星形胶质细胞P2X7受体电流的影响

目的:探索冬凌草甲素是否对海马CA1星形胶质细胞P2X7受体电流有影响。方法:采用Autodock软件对冬凌草甲素与P2X7蛋白进行分子对接;采用全细胞膜片钳记录冬凌草甲素对海马CA1星形胶质细胞P2X7受体激动剂Bz-ATP激发电流的影响。结果:冬凌草甲素与P2X7蛋白的结合能为-5.86 kcal/mol,可与P2X7蛋白的Lys592残基形成氢键,与Tyr550残基形成疏水作用;Bz-ATP能激发海马CA1区星形胶质细胞的电流反应,且1000μmol/L Bz-ATP激发的电流反应较强;10μmol/L冬凌草甲素溶液孵育1 h后,海马CA1区星形胶质细胞对谷氨酸能受体激动剂NMDA和AMPA溶液的电流反应无明显变化;对Bz-ATP的电流反应降低,差异具有统计学意义(P<0.05)。结论:冬凌草甲素可与P2X7蛋白对接形成稳定的复合物,具有较好的亲和力;冬凌草甲素可抑制海马CA1星形胶质细胞P2X7受体电流。