塞缪尔百福是在索尔克生物研究所的基因表达实验室教授和霍华德休斯医学研究所研究员。这项工作是由克里斯托弗和Dana里夫基金会的支持。乔Belcovson，肯特Schnoeker和迈克沙利文在索尔克研究所的多媒体资源提供援助，摄影和剪辑。

1。准备人工脑脊液（ACSF）1。首先，我们准备了学联的10倍的股票，没有一个2L的镁或钙。在毫上市的试剂。目录号是指西格玛/爱秩序。 <table border="1" cellpadding="0"><tbody><tr><td colspan="4"> 2升10X学联（无镁或钙） </td></tr><tr><td> 试剂 </td><td> mM的 </td><td> g/2L </td><td> 目录 </td></tr><tr><td>氯化钾</td><td> 40 </td><td> 5.96 </td><td> P - 9333 </td></tr><tr><td>氯化钠</td><td> 1280 </td><td> 149.61 </td><td> S - 7653 </td></tr><tr><td>碳酸氢钠3 </td><td> 210 </td><td> 35.28 </td><td> S - 6297 </td></tr><tr><td>的NaH 2 PO 4 </td><td> 5 </td><td> 1.38 </td><td> S - 9638 </td></tr><tr><td>血糖</td><td> 300 </td><td> 108.12 </td><td> D - 9434 </td></tr><tr><td colspan="4">加蒸馏水至2L </td></tr></tbody></table>我们还将准备在50ml水硫酸镁和氯化钙2 1M的解决方案，以便在摩尔的调整，从实验到实验，以保证钙的溶液中仍然。 <table border="1" cellpadding="0"><tbody><tr><td colspan="4"> 1M钙和镁的股票</td></tr><tr><td> 试剂 </td><td> 中号 </td><td> g/50mL </td><td> 目录 </td></tr><tr><td> 氯化钙 </td><td> 1 </td><td> 7.35 </td><td> C - 5080 </td></tr><tr><td> 硫酸镁 </td><td> 1 </td><td> 12.33 </td><td> M - 5921 </td></tr></tbody></table>学联的解决方案必须毒气carbogen（95％O2 / 5％CO2），较低的pH值前添加钙或钙会沉淀。 <table border="1" cellpadding="0"><tbody><tr><td colspan="2"> 1升学联 </td></tr><tr><td> 试剂 </td><td> 卷 </td></tr><tr><td> 10X学联</td><td> 100毫升</td></tr><tr><td> 1M氯化钙2 </td><td> 1毫升（解剖）2ML（录音） </td></tr><tr><td> 1M硫酸镁4 </td><td> 1毫升</td></tr><tr><td colspan="2"> 〜800 mL蒸馏水中，气体与carbogen 2分钟前加入钙添加</td></tr><tr><td colspan="2">加蒸馏水至1L。 </td></tr></tbody></table>前和使用后应冲洗泵和油管和解剖菜。 解剖在解剖，1mm的钙学联应不断毒气与carbogen。学联可以从一个瓶子被抽走解剖盘和泵瓶从解剖盘或发送到浪费。我们使用的弹性体（Sylgard，道康宁）涂菜进行解剖。 新生小鼠，迅速断头，用锋利的剪刀和剪刀通过腹胸部和腹部切口。动物是那么膛。净膛鼠标与ACSF冲洗。然后固定在动物与昆虫引脚通过前肢和尾巴基地插入解剖盘。 执行腹椎板切除脊柱删除。脊柱举行小镊子和小剪刀的刀片插入立即背脊柱骨。椎板切了一边，然后其他轻轻提起脊柱骨。这是开展了脊柱的长度。 沿着脊髓两侧切割，切断脊神经根和周围的脊髓切开脑膜，脊髓被删除。我们在左，右两侧切，然后我们切开脑膜脊髓背侧自由。隔离线，然后固定到附近的碟学联入口，以保证良好的充氧，如果其他动物解剖。 孤立的脊髓，然后传送到录音盘，用一个小的勺或锅铲。在这里，我们灌注含氧2MM钙学联的准备。脊髓是固定的弹性体涂层的菜和用于移动外，整根吸电极靠近根部的micromanipulators。当电极尖端附近免费的根端，温柔的吸力应用吸引到根吸电极。这个过程可能会重复出现，同时记录几个根源。 <img src="/files/ftp_upload/1660/1660fig1.jpg" alt="图1" /> 图1。断头和剜。快速斩首鼠标用锋利的剪刀。取决于切割的水平，可以实现从脑干或大或小的贡献。 B.将一个刀片，剪刀断头造成胸腔开放。打开腹胸部和腹部的尾巴的基地。取出内脏，冲洗学联。 <img src="/files/ftp_upload/1660/1660fig2.jpg" alt="图2"/&gt; 图2。脊柱：A.去除左剪刀刀片插入脊柱和脊髓的权利，并通过铺设到脊髓腹侧的骨头切脊髓之间的空间。然后将其插入正确的刀片，剪刀，脊柱和脊髓的脊髓和削减左侧之间的空间。 B.重复这一过程，而温柔牵引脊柱。采取以低角度拿着剪刀，小心不要穿刺脊髓。 <img src="/files/ftp_upload/1660/1660fig3.jpg" alt="图3" /> 图3。答去除脊髓。左剪刀刀片插入脊髓和削减铺设横向到电源线通过神经根和脊膜脊髓和权利的骨骼之间的空间。然后将其插入正确的剪刀刀片，脊髓和骨头之间的空间铺设横向到电源线通过神经根和脊膜脊髓左侧和削减。 B.最后，轻轻延髓脊髓和背线连接到椎板切开脑膜。同样要注意不要以低角度拿着剪刀穿刺脊髓。不要切断根部太近脊髓。

<ol>
	<li>Myers, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholinergic+input+is+required+during+embryonic+development+to+mediate+proper+assembly+of+spinal+locomotor+circuits.">Cholinergic input is required during embryonic development to mediate proper assembly of spinal locomotor circuits.</a> Neuron. 46, 37-49 (2005).</li><li>Goulding, M., Pfaff, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+circuits+that+generate+simple+rhythmic+behaviors+in+vertebrates.">Development of circuits that generate simple rhythmic behaviors in vertebrates.</a> Curr Opin Neurobiol. 15 (1), 14-20 (2005).</li><li>Gallarda, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Segregation+of+axial+motor+and+sensory+pathways+via+heterotypic+trans-axonal+signaling.">Segregation of axial motor and sensory pathways via heterotypic trans-axonal signaling.</a> Science. 320, (2008).</li><li>Gosgnach, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=V1+spinal+neurons+regulate+the+speed+of+vertebrate+locomotor+outputs.">V1 spinal neurons regulate the speed of vertebrate locomotor outputs.</a> Nature. 440 (7081), 215-219 (2006).</li><li>Lanuza, G. M., Gosgnach, S., Pierani, A., Jessell, T. M., Goulding, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+identification+of+spinal+interneurons+that+coordinate+left-right+locomotor+activity+necessary+for+walking+movements.">Genetic identification of spinal interneurons that coordinate left-right locomotor activity necessary for walking movements.</a> Neuron. 42 (3), 375-386 (2004).</li><li>Jiang, Z., Carlin, K. P., Brownstone, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro+functionally+mature+mouse+spinal+cord+preparation+for+the+study+of+spinal+motor+networks.">An in vitro functionally mature mouse spinal cord preparation for the study of spinal motor networks.</a> Brain Res. 816 (2), 493-499 (1999).</li><li>Ziskind-Conhaim, L., Gao, B. X., Hinckley, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethanol+dual+modulatory+actions+on+spontaneous+postsynaptic+currents+in+spinal+motoneurons.">Ethanol dual modulatory actions on spontaneous postsynaptic currents in spinal motoneurons.</a> J Neurophysiol. 89 (2), 806-813 (2003).</li><li>Tabak, J., Rinzel, J., O'Donovan, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+activity-dependent+network+depression+in+the+expression+and+self-regulation+of+spontaneous+activity+in+the+developing+spinal+cord.">The role of activity-dependent network depression in the expression and self-regulation of spontaneous activity in the developing spinal cord.</a> J Neurosci. 21 (22), 8966-8978 (2001).</li><li>Chub, N., Mentis, G. Z., O'Donovan, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chloride-sensitive+MEQ+fluorescence+in+chick+embryo+motoneurons+following+manipulations+of+chloride+and+during+spontaneous+network+activity.">Chloride-sensitive MEQ fluorescence in chick embryo motoneurons following manipulations of chloride and during spontaneous network activity.</a> J Neurophysiol. 95 (1), 323-330 (2006).</li></ol>

动物保健<br /&gt;<br /&gt;对动物的实验是在索尔克研究所的生物学研究AIACUC规定的准则和法规的规定执行。

隔离新生儿脊髓提供了一个研究神经系统development1，2的的易处理的方法。在新生儿啮齿类动物的腰椎脊髓中枢模式产生电路，可以产生神经递质的存在虚构的运动。这个虚构的运动节奏活动增加，阵阵，在0.2至0.5赫兹生产。这些爆发是有组织的生产线和屈，伸交替（同侧L2相到L5）的长度沿左，右交替。在小鼠体内的一些基因突变已被证明有异常行为3-5。了解如何在发展和遗传扰动改变的脊髓神?...

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		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>KCl</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>P-9333</td>
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<td>NaCl	</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>S-7653</td>
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<td>NaHCO3</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>S-6297</td>
<td>&nbsp;</td>
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<td>NaH2PO4</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>S-9638</td>
<td>&nbsp;</td>
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<td>Glucose</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>D-9434</td>
<td>&nbsp;</td>
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<td>CaCl2</td>
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<td>C-5080</td>
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<td>MgSO4</td>
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<td>M-5921</td>
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<td>Large Scissors</td>
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<td>Fine Science Tools</td>
<td>14070-12</td>
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<td>Forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11050-10</td>
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<td>Fine Scissors</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>15000-10</td>
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<td>Insect pins</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>26002-10</td>
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<td>Sylgard 184 (Dow-Corning)</td>
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<td>1L volumetric flask</td>
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<td>100mL volumetric flask</td>
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spinal cord electrophysiology

新生小鼠脊髓是一个模型，为研究神经设计电路和运动运动的发展。我们表明脊髓的解剖和准备录音浴人工脑脊液的运动研究中使用的液体。脊髓腹侧神经根的解剖，可以连接到记录电极记录的腰椎脊髓内中央格局产生电路的电生理信号。

Watch this Scientific Journal Video about Spinal Cord Electrophysiology at JoVE.com

Spinal Cord Electrophysiology

一个孤立的新生小鼠脊髓电生理研究的示范。

脊髓电

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord ...

Research

JoVE Journal

Biology

21.1K 次观看.  Howard Hughes Medical Institute and Gene Expression Laboratory. 一个孤立的新生小鼠脊髓电生理研究的示范。

视频: Spinal Cord Electrophysiology

Applying Microfluidics to Electrophysiology

Microfluidics can be integrated with standard electrophysiology techniques to allow new experimental modalities.  Specifically, the motivation for the microfluidic brain slice device is discussed including how the device docks to standard perfusion chambers and the technique of passive pumping which is used to deliver boluses of neuromodulators to the brain slice.  By simplifying the device design, we are able to achieve a practical solution to the current unmet electrophysiology need of applying multiple neuromodulators across multiple regions of the brain slice.  This is achieved by substituting the standard coverglass substrate of the perfusion chamber with a thin microfluidic device bonded to the coverglass substrate.  This was then attached to the perfusion chamber and small holes connect the open-well of the perfusion chamber to the microfluidic channels buried within the microfluidic substrate.  These microfluidic channels are interfaced with ports drilled into the edge of the perfusion chamber to access and deliver stimulants.  This project represents how the field of microfluidics is transitioning away from proof-of concept device demonstrations and into practical solutions for unmet experimental and clinical needs.

应用微流体电

Microfluidics can be integrated with standard electrophysiology techniques to allow new experimental modalities.  Specifically, the motivation for the ...

科研

生物学

Preparation of Artificial Bilayers for Electrophysiology Experiments

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. These bilayers can be used for many different studies, such as the characterization of membrane-active peptides, the reconstitution of ion channels or investigations on how changes in lipid bilayer properties alter the function of bilayer-spanning channels.  Here, we show how to form a planar bilayer and how to isolate small patches from the bilayer, and in a second video  will also demonstrate a procedure for using gramicidin channels to determine changes in lipid bilayer elastic properties.   We also demonstrate the individual steps needed to prepare the bilayer chamber, the electrodes and how to test that the bilayer is suitable for single-channel measurements.

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. Here, we demonstrate the individual steps needed to prepare the bilayer chamber, the electrodes and how to test that the bilayer is suitable for single-channel measurements.

电生理实验人工双层膜的制备

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. These bilayers can ...

Combining Peripheral Nerve Grafting and Matrix Modulation to Repair the Injured Rat Spinal Cord

Traumatic injury to the spinal cord (SCI) causes death of neurons, disruption of motor and sensory nerve fiber (axon) pathways and disruption of communication with the brain. One of the goals of our research is to promote axon regeneration to restore connectivity across the lesion site. To accomplish this we developed a peripheral nerve (PN) grafting technique where segments of sciatic nerve are either placed directly between the damaged ends of the spinal cord or are used to form a bridge across the lesion. There are several advantages to this approach compared to transplantation of other neural tissues; regenerating axons can be directed towards a specific target area, the number and source of regenerating axons is easily determined by tracing techniques, the graft can be used for electrophysiological experiments to measure functional recovery associated with axons in the graft, and it is possible to use an autologous nerve to reduce the possibility of graft rejection. In our lab we have performed both autologous (donor and recipient are the same animal) and heterologous (donor and recipient are different animals) grafts with comparable results. This approach has been used successfully in both acute and chronic injury situations. Regenerated axons that reach the distal end of the PN graft often fail to extend back into the spinal cord, so we use microinjections of chondroitinase to degrade inhibitory molecules associated with the scar tissue surrounding the area of SCI. At the same time we have found that providing exogenous growth and trophic molecules encourages longer distance axonal regrowth into the spinal cord. Several months after transplantation we perform a variety of anatomical, behavioral and electrophysiological tests to evaluate the recovery of function in our spinal cord injured animals. This experimental approach has been used successfully in several spinal cord injury models, at different levels of injury and in different species (mouse, rat and cat). Importantly, the peripheral nerve grafting approach is effective in promoting regeneration by acute and chronically injured neurons.

Traumatic injury to the spinal cord disrupts communication with the brain. To restore lost connectivity we utilize a peripheral nerve graft to provide a substratum for regenerating fibers in combination with neurotrophic factors and matrix-modulating enzymes to remove inhibitory molecules to promote long distance growth.

结合周边神经移植和矩阵调制修复受伤的大鼠脊髓

Traumatic injury to the spinal cord (SCI) causes death of neurons, disruption of motor and sensory nerve fiber (axon) pathways and disruption of ...

Semi-automated Optical Heartbeat Analysis of Small Hearts

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our Semi-automatic Optical Heartbeat Analysis (SOHA) uses a novel movement detection algorithm that is able to detect cardiac movements associated with individual contractile and relaxation events. The program provides a host of physiologically relevant readouts including systolic and diastolic intervals, heart rate, as well as qualitative and quantitative measures of heartbeat arrhythmicity. The program also calculates heart diameter measurements during both diastole and systole from which fractional shortening and fractional area changes are calculated. Output is provided as a digital file compatible with most spreadsheet programs. Measurements are made for every heartbeat in a record increasing the statistical power of the output. We demonstrate each of the steps where user input is required and show the application of our methodology to the analysis of heart function in all three genetically tractable heart models.

We have developed a Semi-automated Optical Heartbeat Analysis method (SOHA) for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts. We demonstrate the application of our methodology to the analysis of heart function in fruit fly and embryonic mouse hearts.

半自动小红桃光学心跳分析

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our ...

Exploring Arterial Smooth Muscle Kv7 Potassium Channel Function using Patch Clamp Electrophysiology and Pressure Myography

Contraction or relaxation of smooth muscle cells within the walls of resistance arteries determines the artery diameter and thereby controls flow of blood through the vessel and contributes to systemic blood pressure. The contraction process is regulated primarily by cytosolic calcium concentration ([Ca2+]cyt), which is in turn controlled by a variety of ion transporters and channels. Ion channels are common intermediates in signal transduction pathways activated by vasoactive hormones to effect vasoconstriction or vasodilation. And ion channels are often targeted by therapeutic agents either intentionally (e.g. calcium channel blockers used to induce vasodilation and lower blood pressure) or unintentionally (e.g. to induce unwanted cardiovascular side effects).
Kv7 (KCNQ) voltage-activated potassium channels have recently been implicated as important physiological and therapeutic targets for regulation of smooth muscle contraction. To elucidate the specific roles of Kv7 channels in both physiological signal transduction and in the actions of therapeutic agents, we need to study how their activity is modulated at the cellular level as well as evaluate their contribution in the context of the intact artery.
The rat mesenteric arteries provide a useful model system. The arteries can be easily dissected, cleaned of connective tissue, and used to prepare isolated arterial myocytes for patch clamp electrophysiology, or cannulated and pressurized for measurements of vasoconstrictor/vasodilator responses under relatively physiological conditions. Here we describe the methods used for both types of measurements and provide some examples of how the experimental design can be integrated to provide a clearer understanding of the roles of these ion channels in the regulation of vascular tone.

Measurements of Kv7 (KCNQ) potassium channel activity in isolated arterial myocytes (using patch clamp electrophysiological techniques) in parallel with measurements of constrictor/dilator responses (using pressure myography) can reveal important information about the roles of Kv7 channels in vascular smooth muscle physiology and pharmacology.

探讨动脉平滑肌的KV7钾离子通道功能使用膜片钳电生理和压力Myography

Contraction  or relaxation of smooth muscle cells within the walls of resistance arteries  determines the artery diameter and thereby controls flow of ...

Giant Liposome Preparation for Imaging and Patch-Clamp Electrophysiology

The reconstitution of ion channels into chemically defined lipid membranes for electrophysiological recording has been a powerful technique to identify and explore the function of these important proteins. However, classical preparations, such as planar bilayers, limit the manipulations and experiments that can be performed on the reconstituted channel and its membrane environment. The more cell-like structure of giant liposomes permits traditional patch-clamp experiments without sacrificing control of the lipid environment.
Electroformation is an efficient mean to produce giant liposomes &gt;10 &mu;m in diameter which relies on the application of alternating voltage to a thin, ordered lipid film deposited on an electrode surface. However, since the classical protocol calls for the lipids to be deposited from organic solvents, it is not compatible with less robust membrane proteins like ion channels and must be modified. Recently, protocols have been developed to electroform giant liposomes from partially dehydrated small liposomes, which we have adapted to protein-containing liposomes in our laboratory.
We present here the background, equipment, techniques, and pitfalls of electroformation of giant liposomes from small liposome dispersions. We begin with the classic protocol, which should be mastered first before attempting the more challenging protocols that follow. We demonstrate the process of controlled partial dehydration of small liposomes using vapor equilibrium with saturated salt solutions. Finally, we demonstrate the process of electroformation itself. We will describe simple, inexpensive equipment that can be made in-house to produce high-quality liposomes, and describe visual inspection of the preparation at each stage to ensure the best results.

Reconstituting functional membrane proteins into giant liposomes of defined composition is a powerful approach when combined with patch-clamp electrophysiology. However, conventional giant liposome production may be incompatible with protein stability. We describe protocols for producing giant liposomes from pure lipids or small liposomes containing ion channels.

巨脂质体制剂成像和膜片钳电

The reconstitution of ion channels into chemically defined lipid membranes for electrophysiological recording has been a powerful technique to identify ...

Introduction to Solid Supported Membrane Based Electrophysiology

The electrophysiological method we present is based on a solid supported membrane (SSM) composed of an octadecanethiol layer chemisorbed on a gold coated sensor chip and a phosphatidylcholine monolayer on top. This assembly is mounted into a cuvette system containing the reference electrode, a chlorinated silver wire.
After adsorption of membrane fragments or proteoliposomes containing the membrane protein of interest, a fast solution exchange is used to induce the transport activity of the membrane protein. In the single solution exchange protocol two solutions, one non-activating and one activating solution, are needed. The flow is controlled by pressurized air and a valve and tubing system within a faraday cage.
The kinetics of the electrogenic transport activity is obtained via capacitive coupling between the SSM and the proteoliposomes or membrane fragments. The method, therefore, yields only transient currents. The peak current represents the stationary transport activity. The time dependent transporter currents can be reconstructed by circuit analysis.
This method is especially suited for prokaryotic transporters or eukaryotic transporters from intracellular membranes, which cannot be investigated by patch clamp or voltage clamp methods.

Here we present an electrophysiological method based on solid supported membranes with focus on its applications for the characterization of electrogenic membrane transporters.

固体支持基于膜的电

The electrophysiological method we present is based on a solid supported membrane (SSM) composed of an octadecanethiol layer chemisorbed on a gold coated ...

In Vivo SiRNA Transfection and Gene Knockdown in Spinal Cord via Rapid Noninvasive Lumbar Intrathecal Injections in Mice

This report describes a step-by-step guide to the technique of acute intrathecal needle injections in a noninvasive manner, i.e. independent of catheter implantation. The technical limitation of this surgical technique lies in the finesse of the hands. The injection is rapid, especially for a trained experimenter, and since tissue disruption with this technique is minimal, repeated injections are possible; moreover immune reaction to foreign tools (e.g. catheter) does not occur, thereby giving a better and more specific read out of spinal cord modulation. Since the application of the substance is largely limited to the target region of the spinal cord, drugs do not need to be applied in large dosages, and more importantly unwanted effects on other tissue, as observed with a systemic delivery, could be circumvented1,2. Moreover, we combine this technique with in vivo transfection of nucleic acid with the help of polyethylenimine (PEI) reagent3, which provides tremendous versatility for studying spinal functions via delivery of pharmacological agents as well as gene, RNA, and protein modulators.

In Vivo SiRNA Transfection and Gene Knockdown in Spinal Cord via Rapid Noninvasive Lumbar Intrathecal Injections in Mice

This report describes a simple and rapid technique of intrathecal needle puncture for a localized transfection of siRNA in the lumbar spinal cord in mouse under short lasting light anesthesia.

体内转染和基因敲除在脊髓通过快速无创腰椎鞘内注射小鼠

This report describes a step-by-step guide to the technique of acute intrathecal needle injections in a noninvasive manner, i.e. independent of catheter ...

Cystometric and External Urethral Sphincter Measurements in Awake Rats with Implanted Catheter and Electrodes Allowing for Repeated Measurements

Lower urinary tract function is mainly assessed by means of cystometric bladder function analysis in rodents. Conventional cystometries are usually performed as terminal analysis under urethane anesthesia. It is well known that anesthetic drugs can influence bladder function. Hence, the aim of this technique is to perform cystometric measurements of the urinary bladder and external urethral sphincter in lightly restrained awake rats. For this purpose, a bladder catheter is implanted into the bladder dome. Subsequently, two electrodes are implanted bilateral to the external urethral sphincter and a ground electrode is sutured to a non-responsive skeletal muscle. The bladder catheter and the three electrodes are finally tunneled subcutaneously to the neck region and affixed to a harness. With this technique, the lower urinary tract can be measured at multiple time points in the same animal to assess lower urinary tract function. The main application of this technique is the follow-up of simultaneous urinary bladder and external urethral sphincter function in awake healthy rats and after induction of a disease or injury. Moreover, subsequent lower urinary tract monitoring can be performed during evaluation of the disease/injury and to monitor treatment efficacy.

This protocol first describes the surgical procedure of the permanent implantation of a urinary bladder catheter combined with external urethral sphincter electrodes, and second, the measurement of the function of the urinary bladder and external urethral sphincter in implanted awake animals.

压和外尿道括约肌测量在清醒大鼠植入导管和电极允许重复测量

Lower urinary tract function is mainly assessed by means of cystometric bladder function analysis in rodents. Conventional cystometries are usually ...

Application of Electrophysiology Measurement to Study the Activity of Electro-Neutral Transporters

The transport of ions through cell membranes ensures the fine control of ion content within and outside the cell that is indispensable for cell survival. These transport mechanisms are mediated by the activities of specialized transporter proteins. Specifically,pH dynamics are finely controlled by plasma membrane proton (H+) extrusion systems, such as the Na+/H+ exchanger (NHE) protein family. Despite extensive efforts to study the mechanisms underlying NHE regulation, our current understanding of the biophysical and molecular properties of the NHE family is inadequate because of the limited availability of methods to effectively measure NHE activity. In this manuscript, we used H+-selective electrodes during whole-cell patch clamping recording to measure NHE-induced H+ flux. We proposed this approach to overcome some limitations of typically used methods to measure NHE activity, such as radioactive uptake and fluorescent membrane permeants. Measurement of NHE activity using the described method enables high sensitivity and time resolution and more efficient control of intracellular H+ concentrations. H+-selective electrodes are based on the fact that transporter activity creates an ion gradient in close proximity to the cell membrane. An H+-selective electrode moving up to and away from the cell membrane in a repetitive, oscillatory fashion records a voltage difference that is dependent on H+ flux. While H+-selective electrodes are used to detect H+ flux moving out of the cell, the patch clamp method in the whole-cell configuration is used to control the intracellular ion composition. Moreover, application of the giant patch clamp technique allows modification of the intracellular composition of not only ions but also lipids. The transporter activity of NHE isoform 3 (NHE3) was measured using this technical approach to study the molecular basis of NHE3 regulation by phosphoinositides.

This manuscript describes the applications of proton-selective electrodes and patch clamping methods to measure the activity of proton transport systems. These methods overcome some limitations of techniques commonly used to study proton transport activity, such as moderate sensitivity, time resolution and insufficient intracellular milieu control.

电生理测量在中性转运体活性研究中的应用

The transport of ions through cell membranes ensures the fine control of ion content within and outside the cell that is indispensable for cell survival. ...