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.

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.

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)

Effect of Cu^2＋ on K^＋ Current in Acutely Isolated Rat Hippocampal Neurons by Whole Cell Patch Clamp Technique

Using the whole cell patch clamp technique, the effect of Cu^2＋on transient outward K^＋current （/to） and delayed rectifier K^＋ current （Idr） was studied in acutely isolated rat hippocampal neurons.Ito and Idr were increased when the concentration of Cu^2＋ was lower than 2 × 10^-5 and 10^-5 tool/L, respectively, and increased ratio was decreased with increasing Cu^2＋concentration in the bath solutions. When the concentration continued to increase to 5× 10^-5 and 2 × 10^- 5 mol/L, the currents were hardly changed, while the concentration was more than 10^-4 and 5 × 10^-5 mol/L, the currents were inhibited remarkably. Cu^2＋ （10^-5 mol/L） did not affect the activation and inactivation process of Ito. The activation curve of Idr was shifted toward positive potential, but 10^-5 mol/L Cu^2＋did not affect slope factor. According to these results, it was considered that Cu^2＋at low concentration in the bath solution could promote Ito and Idr while at high concentration could inhibit them, and change of amplitude was different with different membrane voltage. Conclusion was drawn： Cu^2＋may be involved in the pathophysiologic mechanism of diseases with neuropathological components.

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.

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.

Study of transmembrane La^(3+) movement in rat ventricular myocytes by the patch-clamp technique

We have studied transmembrane La3+ movement in rat ventricular myocytes for the first time by using the whole-cell patch-clamp recording mode. La3+ (0.01-5.0 mmol/L) could not bring out inward currents through the L-type calcium channel in rat ventricular myocytes, while it could enter the cells by the same way carried by 1μmo1/L ionomycin. When the outward Na+ concentration gradient is formed, La3+ can enter the cells via Na-Ca exchange, and the exchange currents increase with the increase of external La3+ concentrations. But compared with Na-Ca exchange currents in the same concentration, the former is only 14%-38% of the latter. The patch-clamp experiment indicates that La3+ normally can not enter ventricular myocytes through L-type calcium channel, but it can enter the cells via Na-Ca exchange.

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.

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。

Effect of La^(3+) on myocardiac potassium channels revealed by patch-clamp technique

The effect of La3+ on potassium channels in rat ventricular myocytes was investigated using the whole-cell patch-clamp recording mode. The Ca2+-independent voltage- activated outward K+ current was activated by the depolar- izing pulse in enzymatically isolated rat ventricular myocytes. After addition of different concentrations La3+ to the bath solution, the outward K+ current was depressed gradually. The inhibition effect was in a concentration-dependent man- ner. The phenomena of the outward K+ current, being the main repolarizing current suppressed by La3+, suggest that the effect of lanthanides on myocardial function should be exploited further.

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

Effect of temperature on the activation of myocardial K_(ATP) channel in guinea pig ventricular myocytes: a pilot study by whole cell patch clamp recording

Background The myocardial ATP sensitive potassium channel （KATP channel） has been known for more than two decades, the properties of this channel have been intensively investigated, especially the myocardial protection effect by opening this channel. Numerous studies, including hypothermic, using KATP agonists to achieve a hyperpolarizing cardioplegic arrest, have shown a better myocardial protection than potassium arrest. However, there is no evidence showing that KATP channel could be opened by its agonists under profound hypothermia. We investigated the effect of temperature on activation of myocardial KATP channel by nicorandil.Methods Isolated ventricular myocytes were obtained by collagenase digestion of the hearts of guinea pigs and stored in KB solution at 4℃. With a steady ground current, the myocytes were perfused with 1 mmol/L nicorandil until a steady IKATP occurred. Then the cells were perfused with 1 mmol/L nicorandil plus 1 μmol/L glybenclamide. Currents signals were recorded on whole cells using patch clamp technique at several temperatures. The temperature of the bath solution around myocytes was monitored and was controlled at 4℃, 10℃, 20℃, 25℃ and 35℃ respectively. About 10 cells were tested at each temperature, the cells were considered useful only when the outward current could be induced by nicorandil and blocked by glybenclamide. All data were analyzed using Graphpad PRISM 3.0 （Graphpad, San Diego, CA, USA）. Nonlinear curve fitting was done in Clampfit （Axon） or Sigmaplot （SPSS）.Results At 4℃, 10℃, 20℃, 25℃ and 35℃, the time needed to open the myocardial KATP channel was （81.0±0） minutes, （50.54±11.7） minutes, （28.84±2.3） minutes, （9.4± 10.2） minutes and （2.3± 1.0） minutes respectively （P=0.003）. The linear relationship between temperature and time needed to open the channel was y （min） = （4348.790±124.277x）/60, where y （min） is time needed to open KATP channel, x is temperature, correlation coefficient r =-0.942 （P=0.00）, regression coefficient b =-124.277 （P=0.00）. The current densities among different temperatures were statistically different （P=0.022）, the current density was greater after the activation of KATP channel at higher temperatures. The lower the temperature, the fewer cells in which KATP channels could be opened. At 4℃, only one cell in which the KATP channel could be opened, took a quite long time （81 minutes）and the Ⅰ-Ⅴ curve was quite untypical.Conclusions KATP channel activated by nicorandil is temperature dependent and the temperature linearly related to time needed to open KATP channel; the lower the temperature, the longer the time needed to open channel and the smaller the current density. At profound hypothermia, it is difficult to activate KATP channels.

THE ELECTROPHYSIOLOGIC EFFECTS OF ENDOTHELIN A PATCH CLAMP STUDY IN GUINEA PIG VENTRICULAR MYOCYTES

To study the electrophysiologic effects of endothelin-1 (ET-1), we used patch clamp and glass microelectrode techniques to investigate the effects of ET-1 on cardiac L-Ica, Ik and Ik2 in guinea pig ventricular myocytes. The prolongation of APD50, was induced and EADs was triggered by 50 nM ET-1 perfusion. L-lea and Ik were enhanced by various ET-1 concentration from 1 to 50 nM with dose-dependence. Their steady-state activations of L-Iea and Ik shifted left with ET-1 concentration increments. ET-1 elicited a kind of GTP- dependent inward rectifier K current having a mean conductance of 82.36±1.27 pS. The open time and close time ( both interburst intervals and burst durations ) abbreviated with ET-1 concentration increase. The results suggested that EADs -ET evoked was ascribed to the prolongation on the plateau level, which resulted from L-Ica inhancement. The ET- evoked inward rectifier K+ current should be further studied.

Left ventricular reconstruction with no-patch technique：early and late clinical outcomes

Background Few studies have evaluated late clinical outcome of no-patch technique in patients with large left ventricular aneurysms. The objectives of this study were to evaluate a no-patch surgical technique to reconstruct the left ventricle in patients with left ventricular aneurysm and to assess early and late clinical outcomes.Methods In 1995, we began using a no-patch technique in patients with dyskinetic left ventricular aneurysms. A total of 145 patients underwent left ventricular reconstruction with this technique and were followed up for （59±29） months （range,1-127 months）. Risk factors for early mortality were analyzed by bivariate analyses. Cox＇s proportional hazards model was used to calculate risk factors for all-cause mortality and hospital readmission. Kaplan-Meier methodology was used to analyze late survival.Results One week after operation, left ventricular end-diastolic diameter had decreased from （61±8） mm to （55±8）mm, and geometry of the left ventricle was restored to a more normal conical shape. Early mortality was 3% and late mortality 11%. Over a 5-year follow-up period, hospital readmission was 28%. One-, 5-, and 10-year survival estimates were 95% （95% confidence interval （CI） 91%-99%）, 86% （95% CI 78%-94%）, and 74% （95% CI 60%-88%）.Readmission-free survival at 1 and 5 years after operation was 87% （95% CI81%-93%） and 60% （95% CI50%-70%）,respectively.Conclusion The no-patch technique for left ventricular reconstruction is an effective and simple procedure that can achieve satisfactory early and late clinical outcomes in patients with left ventricular aneurysms.

Design and fabrication of a planar patch-clamp substrate using a silicon-on-insulator wafer

The planar patch-clamp technique has been applied to high throughput screening in drug discovery. The key feature of this technique is the fabrication of a planar patch-clamp substrate using appropriate materials. In this study, a planar patch-clamp substrate was designed and fabricated using a silicon-on-insulator （SOI） wafer. The access resistance and capacitance of SOl-based planar patch-clamp substrates are smaller than those of bulk silicon-based planar substrates, which will reduce the distributed RC noise.

Effects of BaCl_2 on slow vacuolar ion channels on radish by patch-clamp

The effects of BaCl_2 on slow vacuolar (SV) currents of radish are studied byusing the whole-vacuolar patch-clamp recording mode. The Ca^(2+)-dependent SV channel can beactivated by cytosolic Ca^(2+). When 1 mmol/L BaCI_2 is added into pipette solution, SV currents aresuppressed remarkably. Then adding BaCI_2 of different concentrations into the bath solution, SVcurrents reflect different effects. The results show that BaCl_2 with a lower concentration (< 3mmol/L) promotes the channel currents and the currents are saturated when BaCl_2 concentrations arebetween 1 μmol/L and 1 mmol/L, but BaCl_2 with higher concentration (≥ 3 mmol/L) inhibits SVcurrents.

膜片钳技术10年发展:基于CiteSpace和VOSviewer的可视化分析

背景:膜片钳技术作为研究离子通道的“金标准”,已有40多年的发展历史。然而,科研机构的研究内容相对独立,没有对现有研究成果进行系统总结,导致现有研究存在重复性高、创新性弱的现象。因此,急需对膜片钳技术做一个全面的回顾,以明晰现今的研究现状、热点和未来发展方向。目的:总结近10年膜片钳技术领域的研究现状和发展趋势。方法:使用Web of Science核心合集数据库收集了2013-2023年关于膜片钳技术的出版物。采用CiteSpace和VOSviewer软件对出版物数量进行量化分析,并分析文献条目网络,包括国家、机构、期刊、作者、关键词、高被引文献和共被引参考文献。结果与结论:①近10年间,膜片钳技术领域研究已逐步进入稳定发展阶段。②中国和美国是这方面的领先国家,中国科学院是具有核心影响力的机构,《Journal of Neuroscience》是主要出版刊物,PARK,WON SUN团队(韩国全北国立大学)和CHU,LI团队(中国河北省心脑血管病中医药防治研究重点实验室)在该领域作出了杰出的贡献,但团队之间的协作与交流较少,尚未形成网络合作模式。③膜片钳技术主要应用在神经系统的电生理特性及其疾病的病理机制方面,是研究人员持续关注的焦点。④在心血管系统电生理特性及其疾病病理机制的研究方面,对原代心肌细胞、诱导多能干细胞衍生的心肌细胞的电生理特性和心房颤动、心脏毒性、心源性猝死和高血压等心血管疾病的病理机制方面的研究,是近几年来研究的热点。⑤在膜片钳技术与其他生物技术的结合应用方面,关注的是与光遗传学、双光子钙成像等技术的交叉融合,将是一个重要的研究方向。⑥在药物筛选及治疗靶点的识别研究方面,尤其对于膜片钳技术和中药复方的研究,将成为未来组分中药研究中的一大助力。

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 。