サミュエルL. Pfaffさんは、ソーク生物学研究所での遺伝子発現の研究​​室の教授、ハワードヒューズ医学研究所の研究者でもある。この作品は、クリフトファーリーブ麻痺財団によってサポートされていました。ジョーBelcovson、ソーク研究所のマルチメディアリソースのケントSchnoekerとマイクサリバンは写真撮影と編集との支援を行いました。

1。人工脳脊髄液（ACSF）1を準備します。まず、マグネシウムやカルシウムなしACSFの10X株式の2Lを用意。試薬はミリモルにリストされています。カタログ番号は、シグマ/アルドリッチを参照してください。 <table border="1" cellpadding="0"><tbody><tr><td colspan="4"> 2リットル10倍ACSF（マグネシウムまたはカルシウムなし） </td></tr><tr><td> 試薬 </td><td> mMの </td><td> g/2L </td><td> カタログ </td></tr><tr><td>塩化カリウム</td><td> 40 </td><td> 5.96 </td><td> P - 9333 </td></tr><tr><td> NaClの</td><td> 1280 </td><td> 149.61 </td><td> S - 7653 </td></tr><tr><td> NaHCO 3の </td><td> 210 </td><td> 35.28 </td><td> S - 6297 </td></tr><tr><td>のNaH 2 PO 4 </td><td> 5 </td><td> 1.38 </td><td> S - 9638 </td></tr><tr><td>グルコース</td><td> 300 </td><td> 108.12 </td><td> D - 9434 </td></tr><tr><td colspan="4"> 2Lに蒸留水を追加します。 </td></tr></tbody></table>我々はまた、実験間のモル濃度の調整を可能にするために50mLの水にし、MgSO 4とCaCl 2の1M溶液を調製し、カルシウムの溶液中に残っている保証する。 <table border="1" cellpadding="0"><tbody><tr><td colspan="4"> 1Mカルシウムとマグネシウムの株式 </td></tr><tr><td> 試薬 </td><td> M </td><td> g/50mL </td><td> カタログ </td></tr><tr><td> CaCl 2の </td><td> 1 </td><td> 7.35 </td><td> C - 5080 </td></tr><tr><td> MgSO 4を </td><td> 1 </td><td> 12.33 </td><td> M - 5921 </td></tr></tbody></table> ACSF液が沈殿するカルシウムやカルシウムを加える前にpHを下げるためにcarbogen（95％O2 / 5％CO2）でガス処刑されている必要があります。 <table border="1" cellpadding="0"><tbody><tr><td colspan="2"> 1リットルのACSF </td></tr><tr><td> 試薬 </td><td> ボリューム </td></tr><tr><td> 10X ACSF </td><td> 100mLの</td></tr><tr><td> 1MのCaCl 2 </td><td> 1 mLの（解剖）2mLの（記録） </td></tr><tr><td> 1M MgSO 4を </td><td> 1 mLの</td></tr><tr><td colspan="2">カルシウムを添加する前に2分間carbogenと〜800 mLの蒸留水、ガスを追加</td></tr><tr><td colspan="2"> 1Lに蒸留水を追加。 </td></tr></tbody></table>ポンプとチューブと解剖皿は使用前と使用後に洗い流してください。 解剖解剖しながら、1mMのカルシウムACSFを連続してcarbogenでガス処刑されるべきである。 ACSFは、ボトルから解剖皿にサイフォンと解剖皿からボトルにポンプまたは無駄に送ることができます。我々は、エラストマー（Sylgard、ダウコーニング）解剖を行うためにコートしたディッシュを使用してください。 新生児マウスは、急速に鋭いはさみと腹側胸部と腹部を介してはさみで作られた切開で斬首されています。動物は、その後evisceratedている。 evisceratedマウスは、ACSFでリンスする。動物は、前肢を通って尾の基部に挿入された昆虫ピンと解剖皿に固定されます。 脊柱は、腹側椎弓切除を行うことにより除去される。脊柱は、小型の鉗子で保持され、小さなハサミの一方のブレードは骨性脊柱にすぐに背側に挿入されます。そっと骨脊柱を持ち上げながら、ラミナは、片側と他にカットされます。これは、脊柱の長さのために行われている。 脊髄は、脊髄神経根を切断し、脊髄を取り巻く髄膜をカットする脊髄の両側に沿って切断により除去される。私たちは、左右両側にカットし、我々はそれを解放するために脊髄の背側に接続されている髄膜をカット。分離されたコードは、追加の動物が解剖されていなくても、適切な酸素化を確実にするACSFの入口近くに皿に固定されます。 分離された脊髄は、小さなスプーンやヘラを使って録音皿に移している。ここでは、酸素2mMのカルシウムACSFで準備を灌流。脊髄は、エラストマーコーティングされた皿や根の近くで細胞外、全ルートの吸引電極を移動するために使用されるマイクロマニピュレータに固定されます。電極の先端がフリールートの終わり近くになると、穏​​やかな吸引は吸引電極にルートを描画に適用されます。このプロセスは、同時にいくつかの根を記録するために繰り返すことができます。 <img src="/files/ftp_upload/1660/1660fig1.jpg" alt="図1" /> 図1。断頭と内臓摘出。A.急速に鋭いはさみでマウスを斬る。脳幹から多かれ少なかれ貢献カットのレベルに応じて達成することができます。断頭によって作成された胸腔の開口部にはさみのB.プレイスつのブレード。尾の付け根に腹胸部と腹部を開きます。内臓を取り出して、ACSFで洗い流してください。 <img src="/files/ftp_upload/1660/1660fig2.jpg" alt="図2"/&gt; 図2。脊柱の除去。A.コー​​ドに腹産卵の骨を介して脊髄やカットの右に脊柱と脊髄の間の空間にはさみの左のブレードを挿入します。その後、脊髄とカットの左に脊柱と脊髄の間の空間にはさみの右ブレードを挿入します。脊柱に穏やかなトラクションをかけながらB.はこのプロセスを繰り返します。低い角度でハサミを保持することによって穿刺脊髄にしないように注意してください。 <img src="/files/ftp_upload/1660/1660fig3.jpg" alt="図3" /> 図3。脊髄の除去。A.脊髄と脊髄への横方向敷設脊髄根と髄膜を介して脊髄やカットの右にある骨の間の空間にはさみの左のブレードを挿入します。その後、脊髄や脊髄への横方向敷設脊髄根と髄膜を介して脊髄やカットの左にある骨の間の空間にはさみの右のブレードを挿入します。 B.最後に、優しく吻側コードを持ち上げて、ラミナに背コードを接続する髄膜をカット。再び低角度でハサミを保持することによってではない穿刺に脊髄を注意してください。脊髄に近づきすぎて根を切断しないでください。

<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>

動物ケア<br /&gt;<br /動物の&gt;実験はAIACUCソーク生物学研究所が定めたガイドラインと規則に従って行った。

孤立した新生児脊髄は神経系のdevelopment1、2を勉強の扱いやすい方法が用意されています。新生児げっ歯類の腰部脊髄の中で、中枢パターン発生回路は、神経伝達物質の存在下で虚構の歩行運動を生成することができます。この架空の運動は、0.2から0.5 Hzで生成される活動のリズムが増加する、バースト、で構成されています。これらのバーストは、コードと屈筋 - 伸交替（同側のL2 L5からの?...

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

新生児マウスの脊髄には神経回路をして歩行運動の発展を研究するためのモデルです。我々は、脊髄の解剖と運動研究のために使用される記録のバスの人工脳脊髄液の準備を示しています。一度解剖、脊髄腹側神経根は、腰髄内の回路を生成する中枢パターンの電気生理学的信号を記録する記録電極に接続することができます。

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

Spinal Cord Electrophysiology

電気生理学的研究のための新生児マウス脊髄の分離のデモンストレーション。

脊髄電気生理学

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 ビュー.  Howard Hughes Medical Institute and Gene Expression Laboratory. 電気生理学的研究のための新生児マウス脊髄の分離のデモンストレーション。

ビデオ: 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.

電気生理学的にマイクロ流体の適用

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

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.

電気生理学的実験のための人工二重層の準備

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.

負傷ラット脊髄を修復するために末梢神経移植とマトリックスモジュレーションを組み合わせ

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.

スモールハーツの半自動光ビート解析

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.

パッチクランプ電気生理学および圧力筋運動記録法を用いた動脈平滑筋KV7カリウムチャネル機能を探る

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.

イメージングとパッチクランプ電気生理学用ジャイアントリポソーム製剤

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.

電気生理学基づき固体支持された膜の概要

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.

マウスにおける急速な非侵襲腰椎髄腔内注射を経由して生体内 siRNAトランスフェクションと脊髄における遺伝子ノックダウン

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.

注入カテーテル電極の繰り返し測定を可能にすると目がさめているラットの膀胱内圧測定と外部尿道括約筋の測定

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.

電気生理学測定の電気中立運送者の活動を研究への応用

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. ...