立式自控微电极拉制器

玻璃微电极是进行电生理研究工作最常用的一种工具。近年来,应用多管微电极开展的微电泳技术,更促进了细胞学、生理学、药理学、病理学等领域的基础理论的发展。为了拉制合格率高的各种玻璃微电极,我们设制成功了立式自控微电极拉制器。一、一般介绍单管微电极拉制器在国内外已有多种设计,但由于微电极多为一次拉成,电极的锥部一般较短,不适于深部组织用。拉制长锥部单管及多管     

介绍一种活体在位实时监测脑内化学物质变化的新方法

介绍了一种新颖的在位实时监测脑内化学物质变化的新方法,在探头－透析电极制作中运用了酶化学,电化学和微透析技术,能连续测定行为动物脑人神经化学物质浓度的变化,并且不需要借助高效液相仪作测定^[1],同时,扼要地介绍了探头的结构以及该技术的应用。     

一种简便的多管玻璃微电极的制备方法

本文介绍一种简便的制备多管玻璃微电极方法,它包括毛坯制备、电极拉制和电解质灌注。     

胞内抗体及其研究进展

胞内抗体是指在细胞内表达并被定位于亚细胞区室的一类新的工程抗体。目前胞内抗体的研究主要集中于ScFv,ScFv基因结构简单,易导入细胞内表达且便于体外重组操作。胞内抗体作为一种新的基因治疗工具,在肿瘤基因治疗、人艾滋病基因治疗的实验研究及潜在的临床治疗方面展示了广泛的应用前景。同时,胞内抗体可以用作分析靶蛋白功能的研究工具,是对传统的“基因剔除”转基因动物的一种有效补充。现从胞内抗体的设计及载体选择、肿瘤基因治疗、人艾滋病基因治疗等方面对胞内抗体的研究进展进行综述。     

利用膜片钳技术标记脑片神经元的方法

目的:介绍一种利用膜片钳技术标记脑片神经元形态的方法.方法:利用振动切片机切好实验目标部位的脑片,用含有NeurobiotinTM Tracer的电极内液灌注玻璃微电极,并进行全细胞膜片钳记录;实验结束后将脑片先用4％多聚甲醛固定、漂洗,再用含有Streptavidin-Texas Red和Triton X-100的P...     

昆虫神经生物学研究技术:细胞内记录

细胞内记录是昆虫神经生物学研究中的常用技术。它用来获得神经元兴奋和抑制过程及神经脉冲产生机制的信息。该技术的特点是把一根微电极的顶尖插入到神经细胞内进行电生理记录 ,这根电极还能用于向膜内输入电流。作者以对蝗虫Schistocercagregaria后胸神经节内的 2个运动神经元的活性记录为例介绍了这一技术     

类固醇激素受体复合物的装配与核内转运

类固醇激素受体 (ＳＲ)包括糖皮质激素受体 (ＧＲ)、孕激素受体 (ＰＲ)、雌激素受体(ＥＲ)、雄激素受体 (ＡＲ)等 ,其中以前两者的研究较多。ＳＲ主要存在于类固醇激素的靶细胞胞质和胞核中 ,当细胞外液中类固醇激素通过细胞膜进入胞质后 ,它能与胞质中ＳＲ结合 ,通过胞质和胞核中ＳＲ的穿梭 ,从而调节核基因组相关产物的转录、翻译及分泌一些生物活性物质 ,以发挥类固醇激素的作用。ＳＲ在细胞内有游离形式和复合物形式 ,而且存在几种不同的复合物形式 ,它们是怎样形成以及形成后如何转运到核内的 ?本文将对此作一综述。1 .ＳＲ复合物的组…     

电刺激和低镁引起的大鼠内嗅皮层游离钙和钾离子浓度的动态变化

电刺激或降低细胞外的镁离子浓度,会引起Ｎ－甲基－Ｄ－天门冬氨酸受体通道的开放,造成胞内外离子浓度失平衡．使用离子选择性微电极结合脑片技术,对电刺激和低镁溶液引起的大鼠内嗅皮层游离钙和钾离子浓度及电位的动态变化的规律进行了研究。实验结果表明,电刺激和低镁溶液引起的内嗅皮层游离钙和钾离子浓度的改变,在细胞层Ⅳ－Ⅴ（皮层表面下９００－１１００μｍ）变化最大。低镁溶液引起游离钙离子浓度下降,同时钾离子浓度呈双相变化,即先增加后减少。低镁溶液灌流内嗅皮层脑片数小时后,胞外钙离子浓度持续地停留在低浓度水平,而钾离子浓度受影响较小．     

布鲁氏菌胞内生存机制研究进展

布鲁氏菌是一种胞内寄生菌,可以在专业和非专业吞噬细胞内生存和复制。当布鲁氏菌与细胞接触时,细菌可以通过受体分子进入细胞。布鲁氏菌在细胞内首先定位于早期吞噬体,然后,在胞内改变其运输方向,最终抵达其胞内复制部位内质网,开始大量复制。这种复制既不影响细胞的基本功能,也不诱导细胞的损伤。主要综述了布鲁氏菌对细胞的侵袭、胞内运输和复制的相关研究进展。     

一种新型金属微电极的制备方法

在利用微电极技术进行动物脑功能研究的急性和慢性实验中,常用环氧树酯绝缘漆作钨丝微电极的绝缘。此方法程序繁琐,电极绝缘性能较差,重复使用率低。我们结合自己的经验进行了绝缘的金属微电极探索,现扼要介绍在钨丝尖端紧包一薄层玻璃的微电极制备步骤。 1.准备直径为200～250μm、长10cm的挺直钨丝及长7cm、外径2mm、内径0.6mm的GG17厚壁玻璃毛细管。 2.把多根钨丝的一端排齐夹好,成排地浸入75％     

A pressure-polishing set-up to fabricate patch pipettes that seal on virtually any membrane,yielding low access resistance and efficient intracellular perfusion

When performing whole-cell configuration recordings, it is important to minimize series resistance to reduce the time constant of charging the cell membrane capacitance and to reduce error in membrane potential control. To this end, an existing method was improved by widening the patch pipette shank through the calibrated combination of heat and air pressure. The heat was produced by passing current through a filament that was shaped appropriately to ensure a homogeneous heating of the pipette shank. Pressurized air was applied to the lumen of a pipette, pulled from a borosilicate glass microcap, via the pressure port of a modified commercial holder. The pipette reshaping was viewed on an LCD monitor connected to a contrast-intensified CCD camera and coupled to a modified bright-field stereomicroscope. By appropriately regulating the timing of air pressure and the application of heating, the pipette shank and, independently, the tip opening diameter were widened as desired. The methods illustrated here to fabricate and use the patch pipettes, using just one glass type, allowed the sealing of a wide variety of cell types isolated from different amphibian, reptilian, fish, and mammalian tissues as well as a variety of artificial membranes made with many different lipid mixtures. The access resistance yielded by pressure-polished pipettes was approximately one-fourth the size of the one attained with conventional pipettes; besides improving the electrical recordings, this minimized intracellular ion accumulation or depletion as well. Enlarged shank geometry allowed for fast intracellular perfusion as shown by fluorescence imaging, also via pulled quartz or plastic tubes, which could be inserted very close to the pipette tip.     

介绍一种方便的细胞内灌流装置

本文研制了一种一体化的细胞内灌流换液装置,能使电极内液在４ｓ内全部被更换,同时又有效地避免了噪音干扰。我们用该装置观察了豚鼠心室肌ｃＡＭＰ－依赖性氯离子通道的调控,表明该装置操作简便、用液节省、适用有效。     

Fabrication of glass micropipettes: a semi-automatic approach for trimming the pipette tip.

Micropipettes as research instruments are well established in cell biology, including blood rheology. However, the experimental results are, to some extent, dependent on the quality of the pipette itself; it is usually critical to have the desired pipette internal diameter and a perpendicular tip. Pipette fabrication is a two-step procedure involving: a) the pulling of the pipette from a glass capillary; b) the trimming of the pipette tip. A common method to trim and fracture the pipette tip is the use of a melted glass bead on a heated tungsten wire. Previous devices using this method were often associated with problems because the heated wire varied in length with temperature. As a result, the bead together with the attached pipette tip moved markedly and thus hampered the possibility to obtain a perpendicularly cut pipette tip. An improved design, based on the same principle with a melted glass bead, is thus suggested; it eliminates the problem with a moving glass bead and, in addition, allows semi-automatic pipette trimming by utilizing the heat-induced elongation/retraction of the heated wire to fracture the tip without requiring manual assistance. Furthermore, a simple pipette storing technique is suggested, based on standard laboratory utensils, in order to more easily handle fragile pipettes without risk of breakage.     

Limitations of the whole cell patch clamp technique in the control of intracellular concentrations.

Recent experimental studies (Pusch and Neher, 1988) and theoretical studies (Oliva et al., 1988) have found that the pipette tip is a significant barrier to diffusion in the whole cell patch clamp configuration. In this paper, we extend the theoretical analysis of fluxes between the pipette and cell to include transmembrane fluxes. The general conclusions are: (a) within the pipette, ion fluxes are driven primarily by diffusion rather than voltage gradients. (b) At steady state there is a concentration difference between the bulk pipette and intracellular solution that is described by delta c = jRp/Dp, where delta c = 1 mM for a flux, j = 1 fmol/s, through a pipette of resistance, Rp = 1 M omega, filled with a solution of resistivity, p = 100 omega --cm, given a solute diffusion coefficient, D = 10(-5) cm2/s. (c) The time to steady state is always accelerated by membrane transport, regardless of the direction of transport. We apply our analysis to the measurement of transport by the Na/K pump and Na/Ca exchanger in cells from the ventricles of mammalian heart. We find that the binding curve for intracellular Na+ to the Na/K pump will appear significantly less steep and more linear if one does not correct for the concentration difference between intracellular and pipette Na+. Similar shifts in the binding curve for extracellular Na+ to the Na/Ca exchanger can occur due to depletion of intracellular Ca(+)+ when the exchanger is stimulated. Lastly, in Appendix we analyze the effects of mobile and fixed intracellular buffers on the movement of Ca(+)+ between the pipette and cell. Fixed buffers greatly slow the time for equilibration of pipette and intracellular Ca(+)+. Mobile buffers act like a shuttle system, as they carry Ca(+)+ from pipette to cell then diffuse back when they are empty. Vigorous transport by the Na/Ca exchanger depletes mobile buffered calcium, thus stimulating diffusion from the pipette to match the rate of Ca(+)+ transport. Moreover, we find that binding of Ca(+)+ to the exchanger can be affected by the mobile buffer.     

Potential clamp of isolated dialyzed neuron: minimalization of the effect of series resistance

A modification of the technique of intracellular dialysis of isolated single excitable cells, such as rat spinal ganglion neuron, suitable for potential clamping of its somatic membrane is described. The advantage of the new modification is the substantial reduction of the effect of inherent resistance in series (RS) to the membrane resistance (RM) on precision of potential clamping. This is attained by reversal of cell position in the perfusion pipette resulting in an approximately tenfold reduction in the area of active membrane. The resistance of this area proportionally increased while RS remained unchanged. Hence the error in potential fixation, which is inversely proportional to the ratio RM/RS, is by approximately one order smaller with the new technique than with the original one. An essential step in the new technique is the osmotic expansion of the cell to improve the contact of the cell with the perfusion pipette in the pore and to facilitate disruption of the appropriate part of the membrane. All features and advantages of the technique of intracellular dialysis, such as simplicity, the possibility to easily change ionic composition of media, and/or to apply drugs to any side of the membrane in the same cell, etc., have been maintained.     

Metabolism of hybridoma cells and antibody secretion at high cell densities in dialysis tubing.

The experimental setup, consisting of a bundle of dialysis tubing 2.5 mm in diameter [10-15 kD cutoff, mean pore size 25 A, 20 microns (dry) and 40 microns (wet) wall thickness] inserted into a 1-l glass bioreactor supplied with oxygen and pH electrodes, a porous gas distributor, a sampling tube, and a holder for the eight pieces of dialysis tubing, was developed to investigate the properties and the microenvironment of hybridoma cells enclosed in the tubing during their batch cultivation. The concentrations of low-molecular-weight medium components were the same inside and outside the tubing, and it was possible to control the microenvironment of the cells in the tubing easily. The cell damage caused by mechanical stress was less in the dialysis tubing than in stirred spinner flasks. The influence of the initial cell density in the range from 4 X 10(5) to 1 X 10(8) cells ml-1 and the cultivation time were evaluated according to the total and viable cell concentrations and the cell/cell fragment size distributions. Furthermore, the cell membrane properties, glucose consumption rate, lactate, ammonia and lipid storage material, and the monoclonal antibody production rates as well as intracellular enzyme activities in the culture medium were measured and compared to those in reference cultures in spinner flasks with the same inoculum at low initial cell densities. In dialysis tubing in a concentration range of 5 X 10(6) to 10(8) cells ml-1, the total and viable concentrations of cells remained the same during cultivation.(ABSTRACT TRUNCATED AT 250 WORDS)     

The ultrastructure of patch-clamped membranes: a study using high voltage electron microscopy

We have developed techniques for studying patch-clamped membranes inside glass pipettes using high voltage electron microscopy (HVEM). To preserve the patch structure with the least possible distortion, we rapidly froze and freeze dried the pipette tip. The pipette is transparent for more than 50 microns from the tip. HVEM images of patches confirm light microscopy observations that the patch is not a bare bilayer, but a membrane-covered bleb of cytoplasm that may include organelles and cytoskeleton. The membrane that spans the pipette is commonly tens of micrometers from the tip of the pipette and occasionally as far as 100 microns. The structure of patches taken from a single cell type is variable but there are consistent differences between patches made from different cell types. With suction applied to the pipette before seal formation, we have seen in the light microscope vesicles swept from the plasmalemma up the pipette. These vesicles are visible in electron micrographs, particularly those made from chick cardiac muscle. Colloidal gold labeling of the patch permitted identification of lectin-binding sites and acetylcholine receptors. In young cultures of Xenopus myocytes, the receptors were diffuse. In 1-wk-old cultures, the receptors formed densely packed arrays. The patch pipette can serve, not only as a recording device, but as a tool for sampling discrete regions of the cell surface. Because the pipette has a constant path length for axial rotation, it is a unique specimen holder for microtomography. We have made preliminary tomographic reconstructions of a patch from Xenopus oocyte.     

Intracellular Ice Formation Is Affected by Cell Interactions

Cell-to-cell and cell-to-surface interactions are important to the structure and function of tissues. These interactions are also important determinants of low-temperature responses in tissues. Four in vitro models using hamster fibroblast cells in tissue culture were used to investigate the influence of cell-cell and cell-surface interactions on intracellular ice formation in these systems. The four models were: (a) single cells in suspension; (b) cells individually attached to glass with only cell-to-surface adhesion; (c) colonies of cells attached to glass with both cell-cell and cell-surface interactions; and (d) multicellular spheroids with extensive cell-cell contacts. Cryomicroscopy was used to monitor the prevalence and kinetics of intracellular ice formation after ice nucleation in the extracellular solution. The temperature for intracellular freezing in 50% of the cells was significantly affected by both cell-cell and cell-surface interactions. There was also evidence of intercellular nucleation through cell-cell interactions. The results indicate that cell-cell and cell-surface interactions play a significant role in the low-temperature response of tissue systems.     

Membrane tension directly shifts voltage dependence of outer hair cell motility and associated gating charge.

The unique electromotility of the outer hair cell (OHC) is believed to promote sharpening of the passive mechanical vibration of the mammalian basilar membrane. The cell also presents a voltage-dependent capacitance, or equivalently, a nonlinear gating current, which correlates well with its mechanical activity, suggesting that membrane-bound voltage sensor-motor elements control OHC length. We report that the voltage dependence of the gating charge and motility are directly related to membrane stress induced by intracellular pressure. A tracking procedure was devised to continuously monitor the voltage at peak capacitance (VpkCm) after obtaining whole cell voltage clamp configuration. In addition, nonlinear capacitance was more fully evaluated with a stair step voltage protocol. Upon whole cell configuration, VpkCm was typically near -20 mV. Negative patch pipette pressure caused a negative shift in VpkCm, which obtained a limiting value near the normal resting potential of the OHC (approximately -70 mV) at the point of cell collapse. Positive pressure in the pipette caused a positive shift that could reach values greater than 0 mV. Measures of the mechanical activity of the OHC mirrored those of charge movement. Similar membrane-tension dependent peak shifts were observed after the cortical cytoskeletal network was disrupted by intracellular dialysis of trypsin from the patch pipette. We conclude that unlike stretch receptors, which may sense tension through elastic cytoskeletal elements, the OHC motor senses tension directly. Furthermore, since the voltage dependence of the OHC nonlinear capacitance and motility is directly regulated by intracellular turgor pressure, we speculate that modification of intracellular pressure in vivo provides a mechanism for controlling the gain of the mammalian "cochlear amplifier".     

Critical intracellular Ca2+ dependence of transient receptor potential melastatin 2 (TRPM2) cation channel activation

TRPM2 is a member of the melastatin-related TRP (transient receptor potential) subfamily. It is expressed in brain and lymphocytes and forms a cation channel that is activated by intracellular ADP-ribose and associated with cell death. In this study we investigated the calcium dependence of human TRPM2 expressed under a tetracycline-dependent promoter in HEK-293 cells. TRPM2 expression was associated with enhanced hydrogen peroxide-evoked intracellular calcium signals. In whole-cell patch clamp recordings, switching from barium- to calcium-containing extracellular solution markedly activated TRPM2 as long as ADP-ribose was in the patch pipette and exogenous intracellular calcium buffering was minimal. We suggest this effect reveals a critical dependence of TRPM2 channel activity on intracellular calcium. In the absence of extracellular calcium we observed concentration-dependent activation of TRPM2 channels by calcium delivered from the patch pipette (EC(50) 340 nM, slope 4.9); the maximum effect was at least as large as that evoked by extracellular calcium. Intracellular dialysis of cells with high concentrations of EGTA or 1,2-bis(o-Aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) strongly reduced the amplitude of the extracellular calcium response, and the residual response was abolished by a mixture of high and low affinity calcium buffers. TRPM2 channel currents in inside-out patches showed a strong requirement for Ca(2+) at the intracellular face of the membrane. We suggest that calcium entering via TRPM2 proteins acts at an intracellular calcium sensor closely associated with the channel, providing essential positive feedback for channel activation.