사무엘 L. 파프는 생물 학적 연구 솔크 연구소에서 유전자 발현 연구소에서 교수와 하워드 휴즈 의학 연구소의 탐정이다. 이 작품은 크리스토퍼와 다나 리브 재단​​에 의해 지원되었다. 조 Belcovson, 켄트 Schnoeker와 솔크 연구소에서 멀티미디어 자원에 마이크 설리반은 사진 촬영 및 편집 지원을 제공했습니다.

1. 인공 뇌척수 (aCSF 1)를 준비합니다. 우리가 먼저 마그네슘 또는 칼슘없이 aCSF의 10X 주식 2L를 준비합니다. 시약은 millimolar에 나열된. 카탈로그 번호 시그마 / 알드리치를 참조하십시오. <table border="1" cellpadding="0"><tbody><tr><td colspan="4"> 2 리터는 10X aCSF (MG 또는 칼슘 제외) </td></tr><tr><td> 시약 </td><td> MM </td><td> g/2L </td><td> 목록 </td></tr><tr><td> KCl </td><td> 40 </td><td> 5.96 </td><td> P - 9333 </td></tr><tr><td> NaCl </td><td> 1280 </td><td> 149.61 </td><td> S - 7653 </td></tr><tr><td> NaHCO 3 </td><td> 210 </td><td> 35.28 </td><td> S - 6297 </td></tr><tr><td> 아니 2 PO 4 </td><td> 5 </td><td> 1.38 </td><td> S - 9638 </td></tr><tr><td> 포도당 </td><td> 300 </td><td> 108.12 </td><td> D - 9434 </td></tr><tr><td colspan="4"> 2L에 증류수를 추가 </td></tr></tbody></table> 우리는 또한 실험에서 실험 molarity을 조정 수 있도록 50mL 물에 MgSO 4 CaCl 2 1M 솔루션을 준비하고 칼슘 용액에 남아 확신합니다. <table border="1" cellpadding="0"><tbody><tr><td colspan="4"> 1M 칼슘과 마그네슘 주식 </td></tr><tr><td> 시약 </td><td> M </td><td> g/50mL </td><td> 목록 </td></tr><tr><td> CaCl 2 </td><td> 1 </td><td> 7.35 </td><td> C - 5080 </td></tr><tr><td> MgSO 4 </td><td> 1 </td><td> 12.33 </td><td> M - 5921 </td></tr></tbody></table> aCSF 솔루션은 침전됩니다 칼슘이나 칼슘을 추가하기 전에 산도를 낮게 carbogen (95% O2 / 5 % CO2)와 기름해야합니다. <table border="1" cellpadding="0"><tbody><tr><td colspan="2"> 1 리터 aCSF </td></tr><tr><td> 시약 </td><td> 음량 </td></tr><tr><td> 10X aCSF </td><td> 100mL </td></tr><tr><td> 1M CaCl 2 </td><td> 1 ML (해부) 2mL (녹음) </td></tr><tr><td> 1M MgSO 4 </td><td> 1 ML </td></tr><tr><td colspan="2"> 칼슘을 추가하기 전에 ~ 800 ML 증류수, 2 분 동안 carbogen와 가스 추가 </td></tr><tr><td colspan="2"> 1L에 증류수를 추가합니다. </td></tr></tbody></table> 펌프와 관 그리고 해부 접시는 사용 전후에 씻어서해야합니다. 해부 해부 동안 1mM 칼슘 aCSF는 지속적으로 carbogen와 기름해야합니다. aCSF은 해부 접시에 한 병에서 siphoned 및 병에 대한 해부 접시에서 펌프 또는 폐기물로 보낼 수 있습니다. 우리는 탄성체 (Sylgard, 다우 코닝) 절개를 수행하기 위해 코팅 접시를 사용합니다. 신생아 마우스는 급속하게 날카로운 가위와 복부 흉부와 복부를 통해 가위로 만든 절개와 목이있다. 동물 그러면 eviscerated됩니다. eviscerated 마우스 aCSF로 씻어서 있습니다. 동물은 다음 앞발 통해 꼬리의 기지에 삽입 곤충 핀으로 해부 접시에 박혀있다. 척추는 복부 laminectomy를 수행하여 제거됩니다. 척추 열은 작은 집게와 함께 개최되는 작은 가위 하나 블레이드는 뼈에 척수 컬럼에 즉시 지느러미 삽입됩니다. 부드럽게 뼈다귀 척추 컬럼을 리프팅하는 동안 라미나 그런 다음 한쪽과 다른 한편으로는 상처입니다. 이것은 척추의 길이에 대해 수행됩니다. 척수는 척수 신경 뿌리를 끊어와 척수를 둘러싼 meninges를 잘라 척수의 양쪽을 따라 커팅에 의해 제거됩니다. 우리는 왼쪽과 오른쪽 양쪽에 상처, 우리는 그것을 무료로 척수의 지느러미 측면에 부착된 meninges를 잘라. 절연 코드는 다음 추가 동물 해부하는 경우 좋은 산소를 보장하기 위해 aCSF 입구 근처에있는 접시에 박혀있다. 분리된 척수는 다음 작은 숟가락이나 주걱을 사용하여 녹음 요리로 전송됩니다. 여기 산소 2mM 칼슘 aCSF으로 준비를 perfuse. 척수는 탄성체 코팅 접시와 뿌리 근처 세포, 전체 루트 흡입 전극을 이동하는 데 사용 micromanipulators에 박혀있다. 전극 팁은 무료 루트 끝 가까이하면, 부드러운 흡입은 흡입 전극에 뿌리를 그릴에 적용됩니다. 이 과정은 동시에 여러 개의 뿌리를 기록하는 반복될 수 있습니다. <img src="/files/ftp_upload/1660/1660fig1.jpg" alt="그림 1" /> 그림 1. 잘린과 내장 적출. A. 신속하게 날카로운 가위로 마우스를 목을 벨. 뇌간에서 크거나 낮은 기여 컷의 수준에 따라 형성될 수 있습니다. 잘린 만든 흉강의 개통에 가위 B. 플레이스 한 블레이드. 꼬리의 기본으로 복부 근육과 복부를 엽니다. 내장을 제거하고 aCSF와 린스. <img src="/files/ftp_upload/1660/1660fig2.jpg" alt="그림 2"/&gt; 그림 2. 척추. A. 제거 코드에 누워 복부 뼈를 통해 척수 및 절단의 오른쪽에있는 척추와 척수 사이의 공간에 가위의 왼쪽 날개를 삽입합니다. 다음 척수 및 절단의 왼쪽에있는 척추와 척수 사이의 공간에 가위의 오른쪽 날개를 삽입합니다. 척추 열 부드러운 견인을 적용하는 동안 B.이 과정을 반복합니다. 낮은 각도에서 가위를 들고하여 주사 척수 안됩니다. <img src="/files/ftp_upload/1660/1660fig3.jpg" alt="그림 3" /> 그림 3. 척수의 제거. A. 척수와 코드에 측면 누워 척추 뿌리와 meninges 통해 척수 및 절단의 오른쪽에있는 뼈 사이의 공간에 가위의 왼쪽 날개를 삽입합니다. 다음 척수의 왼쪽에있는 척수와 뼈 사이의 공간에 가위의 오른쪽 날개를 삽입하고 코드에 측면 누워 척추 뿌리와 meninges 통해 했네요. B. 마지막으로, 부드럽게 주동이의 코드를 채취해서 laminae에 지느러미 코드를 연결 meninges를 잘라. 다시 낮은 각도에서 가위를 들고하여 주사 척수에 신경을 안 받아. 척수에 너무 가까이 뿌리를 잘라하지 마십시오.

<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>

애니멀 케어<br /&gt;<br /동물&gt; 실험은 생물 학적 연구 AIACUC의 솔크 연구소에서 정한 지침과 규정에 따라 수행되었다.

절연 신생아 척수는 신경계의 development1, 2를 공부의 취급하기 쉬운 방법을 제공합니다. 신생아 설치류의 요추 척수 내에 중앙 패턴 생성 회로는 신경 전달 물질의 존재에 상상의 운동력을 생성하실 수 있습니다. 이 상상의 운동은 0.2-0.5 Hz에서에서 생산 활동에 리듬 증가, 파열로 이루어져 있습니다. 이러한 파열은 코드와 flexor - 신근 교대 (L5에 ipsilateral L2 상대)의 길이를 따라 왼쪽에서 오른쪽으?...

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<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>
		</div>

spinal cord electrophysiology

신생아 마우스 척수는 신경 circuitries과 전위의 운동의 발전을위한 연구 모델입니다. 우리는 척수의 해부와 전위의 연구에 사용되는 기록 목욕 인공 뇌척수의 준비를 보여줍니다. 일단 해부, 척수 복부 신경 뿌리는 허리 코드 내에서 중앙 패턴 생성 회로의 electrophysiologic 신호를 기록하는 기록 전극을 부착하실 수 있습니다.

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

Spinal Cord Electrophysiology

electrophysiologic 연구 신생아 마우스 척수의 고립의 데모.

척수 전기 생리학

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 조회수.  Howard Hughes Medical Institute and Gene Expression Laboratory. electrophysiologic 연구 신생아 마우스 척수의 고립의 데모.

비디오: 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.

전기 생리학에 Microfluidics 적용

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

연구

생물학

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.

전기 생리학 실험을위한 인공 Bilayers의 준비

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.

부상당한 쥐 척수를 복구하는 주변 신경 이식과 매트릭스 변조를 결합

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.

작은 하트의 세미 - 자동 광학 도면 분석

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.

패치 클램프 전기 생리학 및 압력 Myography를 사용하여 간선 평활근 Kv7 칼륨 채널 기능을 탐색

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.

이미징 및 패치 클램프 전기 생리학에 대한 거대한 리포좀 준비

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.

전기 생리학을 바탕으로 솔리드 지원 막 소개

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.

마우스의 빠른 비 침습 요추 경 막내 주사를 통해 생체 siRNA의 형질과 척수에있는 유전자 넉다운에

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.

반복된 측정에 대 한 수 있도록 전극 이식된 카 테 터와 깨어 있는 쥐에 Cystometric 및 외부 요도 괄약근 측정

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.

전기 중립 전송기의 활동을 연구 전기 생리학 측정의 응용

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. ...