由美国国立卫生研究院（NS - 18205）。

1。配对录音鱼的制备<ol><li>程序开始之前，准备好五个关键的解决方案，其中包括:沐浴液，浴Tricaine（MS222），浴甲酰胺，神经元的内部解决方案和肌肉内部解决方案的解决方案解决方案。 </li><li>接下来，收集一个单一的幼虫斑马鱼，年龄72至96小时岁之间，在巴斯与Tricaine解决方案的地方。鱼在一分钟暴露的麻醉，不会响应触摸。 </li><li>放置一个塑料盘上的鱼，使用立体显微镜，具有双重的边缘刀片杀头。 </li><li>鱼转移到玻璃录音室，涂sylgard和充满槽液。法尔胜在每年年底通过脊索插入电解削尖的钨引脚。 </li><li>创建一个附近的鱼用别针将提供足够的抓地力了一双细镊子的喙皮瓣。抓住附近的喙针，一双细镊子取出覆皮肤。剥离尾方向慢慢消除皮肤。 </li><li>治疗浴3-5分钟，以块肌肉的收缩可能会干扰与录音甲酰胺溶液3毫升鱼。洗涤液与正常的5倍。取出轻轻取消钨针的一侧的浅层肌肉的5至6段的一部分。 </li><li>的鱼转移到一个堂堂正正的显微镜，在位于一个法拉第笼屏蔽电气噪声。显微镜配备有一个固定的阶段，工作距离长40倍的目标和0.5变焦4倍。该显微镜安装在机动XY翻译阶段，以编制高功率下的神经和肌肉之间的移动。利用微分干涉对比光学可视化的神经元。 </li><li>根据0.5倍变焦，使用一个大口径15微米吸管删除覆的肌肉和揭露脊髓。通过3毫升的一次性注射器和肌肉细胞被删除逐个应用负压。重复此过程骨骼肌背5至6段。相同的大口径吸管是用来同时删除两个高腹侧肌肉层，以获得目标快速骨骼肌。 </li><li>这样就完成了配对录音的准备和显微镜的放大是增加了约1.6倍。 </li></ol> 2。第电机神经元和目标肌肉细胞配对录音<ol><li>清洗段进行扫描，找到第神经元。每个半段脊髓包含一个大的，10微米直径，第电机神经元。这泪滴形SOMA位于脊髓硬膜下表面附近的段边界的最尾结束。一个CAP神经元是选择录制，并在相邻的尾鳍段腹目标肌肉扫描检查完整性。 </li><li>两个补丁电极定位录音，神经和肌肉的第二次。上电极有一个强硬脊髓硬膜和小费约2微米的开放渗透浅的锥度。下电极具有低电阻电压钳记录骨骼肌陡峭的锥形</li><li>神经元的电极定位在一个约30度，渗透到脊髓硬膜的角度，约一个半段到选定的第神经元延髓。提前使用轴驱动器的操纵者，而申请温柔的持续正压约60毫米汞电极。作为电极通过硬脑膜破裂，可能会流离失所第神经灌注电极，需要一个调整的重点。 </li><li>当电极接触的第神经元胞体的正压力释放，并立即形成一个gigaohm密封通常的结果。然而，在某些情况下，印章不能形成，就有必要申请一个温柔的负压。密封形成后，电极电位调整到具有80mV和应用负压破裂细胞膜。第神经元的特点是由140到180兆欧姆的输入电阻和一个动作电位，过冲40毫伏。这是由注入电流钳下逐步增加，直到引起动作电位的去极化步骤测试。 </li><li>接下来，在显微镜搬迁到腹侧肌，使用高功率下的XY的机动翻译。快肌细胞是选择目​​标字段。 </li><li>先进的肌肉电极对正压目标肌肉细胞，释放时，形成了一个gigaohm密封。电极电位调整到-50毫伏钠通道失活和温柔的吮吸下细胞膜破裂。一个在100至200兆欧姆电阻和突然出现的大电容瞬态信号输入的下降。 </li><li>为了测试配对记录，电机神经元去极化，直到动作电位引起的电流增量较大的注射。此时应引起肌肉终板电流。持续刺激motorneuron 1终板无故障电流的赫兹结果。由于刺激频率提高到100赫兹的终板电流波动幅度和接受刺激的10秒内功能枯竭。 </li></ol> 3。代表性的成果通常情况下，神经元的记录是稳定的，长达一小时，但肌肉细胞恶化反映作为一个串联电阻的增加。当发生这种情况的实验是终止或其他肌肉细胞是补丁夹住。所有记录应在一小时内完成。 <img src="/files/ftp_upload/2351/2351fig1.jpg" alt="图1" /> 图1。motorneuron动作电位（AP，顶部）和相关的肌肉终板电流（EPC，底部）的录音。过冲为第神经元动作电位+40 mV和EPC的上升在1毫秒的时间常数的指数时间当然沿300微秒内和腐烂。 <table border="1"><tbody><tr><td> 名称 </td><td> 配方 </td></tr><tr><td>沐浴液（毫米） </td><td> 134氯化钠，氯化钾2.9，2.1 1.2 10氯化镁2，葡萄糖，钠，HEPES 10，pH值7.8 2，氯化钙</td></tr><tr><td>液与Tricaine </td><td>液含0.02％tricaine </td></tr><tr><td>液与甲酰胺</td><td>沐浴液含2M甲酰胺</td></tr><tr><td>神经元的内部解决方案（毫米） </td><td> 115 K -葡萄糖，氯化钾，15 2 MgCl 2的 ，10 K-HEPES，5 K - EGTA，4毫克+ - ATP，pH值7.2 </td></tr><tr><td>肌肉内部解决方案（单位:毫米） </td><td> 120氯化钾，10 K-HEPES，5 BAPTA，pH值7.4 </td></tr></tbody></table> 表1。解决方案。

<ol>
	<li>Fetcho, J. R., O'Malley, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+neuronal+networks+in+behaving+animals.">Imaging neuronal networks in behaving animals.</a> Current Opinion in Neurobiology. 7, 832-838 (1997).</li><li>Fetcho, J. R., S, H. i. g. a. s. h. i. j. i. m. a. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+and+genetic+approaches+toward+understanding+neuronal+circuits+in+zebrafish.">Optical and genetic approaches toward understanding neuronal circuits in zebrafish.</a> Integr Comp Biol. 44, 57-70 (2004).</li><li>Iba&#241;ez-Tallon, I., Wen, H., Miwa, J., Xing, J., Aslantas-Tekinay, A., Ono, F., Brehm, P., Heintz, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tethering+naturally+occurring+peptide+toxins+for+cell+autonomous+modulation+of+ion+channels+and+receptors+in+vivo.">Tethering naturally occurring peptide toxins for cell autonomous modulation of ion channels and receptors in vivo.</a> Neuron. 43, 305-311 (2004).</li><li>Li, W., Ono, F., Brehm, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+measurements+of+presynaptic+release+in+mutant+zebrafish+lacking+postsynaptic+receptors.">Optical measurements of presynaptic release in mutant zebrafish lacking postsynaptic receptors.</a> J. Neuroscience. , 23-10467 (2003).</li><li>Ono, F., Mandel, G., Brehm, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acetylcholine+receptors+direct+rapsyn+clusters+to+the+neuromuscular+synapse+in+zebrafish.">Acetylcholine receptors direct rapsyn clusters to the neuromuscular synapse in zebrafish.</a> J. Neuroscience. 24, 475-5481 (2004).</li><li>Ono, F., Shcherbatko, A., Higashijima, S., Fetcho, J., Mandel, G., Brehm, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paralytic+zebrafish+lacking+ACh+receptors+fail+to+localize+rapsyn+clusters+to+the+synapse.">Paralytic zebrafish lacking ACh receptors fail to localize rapsyn clusters to the synapse.</a> J. Neuroscience. 21, 5439-5448 (2001).</li><li>Ono, F., Shcherbatko, A., Higashijima, S., Mandel, G., Brehm, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+motility+mutant+twitch+once+reveals+new+roles+for+rapsyn+in+synaptic+function.">The zebrafish motility mutant twitch once reveals new roles for rapsyn in synaptic function.</a> J. Neuroscience. 22, 6491-6498 (2002).</li><li>Wang, M., Wen, H., Brehm, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Function+of+neuromuscular+synapses+in+the+zebrafish+choline-acetyltransferase+mutant+bajan.">Function of neuromuscular synapses in the zebrafish choline-acetyltransferase mutant bajan.</a> J Neurophysiol. , 100-104 (2008).</li><li>Wen, H., Brehm, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paired+motor-neuron+muscle+recordings+in+zebrafish+test+the+receptor+blockade+model+for+shaping+synaptic+current.">Paired motor-neuron muscle recordings in zebrafish test the receptor blockade model for shaping synaptic current.</a> J. Neuroscience. 25, 8104-8111 (2005).</li></ol>

我们一直在用斑马鱼配对录音"（温家宝和布雷姆，2005年），主要研究在高频刺激囊泡的释放和回收的过程。这个过程被打乱在蠕动，其中正常的突触传递的突变体被破坏，由于点突变在突触的关键组件。例如，基因突变，破坏突触后受体聚合（Ono 等人，2001年，2002年，2004年）和合成突触前发射机（王等人，2008年）在联合国协调游泳的结果。配对录音提供的信息，可以精确定位功能缺陷的根源，从而连接行为，遗传学和生理学。它现在应该有可能进一步解剖的基本瞬时表达的方式，在使用的第明智的促销员电机神经元突触传递的过程。显性阴性或变异的基因，这样可以明确表示，在这些神经元的行为和电生理后果确定。整个单元的配置提供额外的好处让与代理商的第SOMA透析可能改变囊泡循环，如梭菌毒素和钙缓冲区。由于SOMA和肌肉之间的短距离，扩散录音时限内应该发生。该制剂的潜力可挖才刚刚开始。鱼在这种突触的年龄和位置表浅的透明度，也便于使用光学工具（Fetcho和奥马利，1997年; Fetcho和Higashijima，2004年）。远藤和使用FM染料和遗传指标，如Synaptophluorin（李等人 ，2003年）的鱼类生活的胞外分泌的测量，我们一直非常成功。此外，荧光标记的毒素可用于标签活鱼（伊瓦涅斯，塔伦等 ，2004）的成像突触蛋白。鉴于这种准备的简单，它认为未来研究的巨大潜力。

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<td>Pneumatic transducer tester</td>
<td>&nbsp;</td>
<td>Fluke Biomedical Instruments</td>
<td>DPM1B</td>
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<td>0.002 x 3 inch tungsten rod</td>
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<td>A-M Systems Inc</td>
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<td>Sylgard 184 Elastomer</td>
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<td>Dow Corning Corp.</td>
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<td>The thickness of application will affect the DIC optics</td>
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paired patch clamp recordings from motor-neuron and target skeletal muscle in zebrafish

幼虫斑马鱼第一脊椎动物的模型系统，以便从脊髓运动神经元和目标肌肉同时膜片钳记录。这是两种类型的细胞和能力，从视觉上分辨单节段第电机神经元的形态和位置的基础上无障碍的直接后果。本视频演示了用于识别一个CAP电机的神经元和目标肌肉细胞，以及从不同的细胞类型的录音方法的微观方法。第电机神经元类型的鉴定也证实了两种染料充填的生物物理特性，如动作电位的波形和细胞输入电阻。电机神经元录音例行的最后一小时，允许从多个不同的目标肌肉细胞长期的录音。在电机神经元发射模式的控制使神经肌肉接头处突触传递的频率依赖的测量。由于一个大型的量子尺寸和低噪音，全细胞电压钳提供，可以解决所有的单一事件的肌肉。此功能允许的释放特性，囊泡循环，以及突触抑郁症和便利，如基本突触性能的研究。 提供体内准备日食的神经肌肉模型系统的优势，研究了其中电机神经元通常是由外电极刺激的肌肉全细胞膜片钳过大。斑马鱼的准备工作是经得起一个范围广泛，包括转基因株系，吗啉击倒，药物干预，并在体内成像的方法相结合的电生理分析。这些方法，加上越来越多的神经肌肉疾病模型提供的斑马鱼突变体，打开门，为人类的神经肌肉疾病的新理解。

Watch this Scientific Journal Video about Paired Patch Clamp Recordings from Motor-neuron and Target Skeletal Muscle in Zebrafish at JoVE.com

Paired Patch Clamp Recordings from Motor-neuron and Target Skeletal Muscle in Zebrafish

幼虫斑马鱼第一脊椎动物的模型系统，让从脊髓运动神经元和目标骨骼肌同时膜片钳记录。本视频演示了用于识别段第电机神经元和目标肌肉细胞以及从不同的细胞类型的录音方法的微观方法。

配对补丁钳从电机神经元和斑马鱼的目标骨骼肌

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle.  ...

paired-patch-clamp-recordings-from-motor-neuron-target-skeletal

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

17.1K 次观看.  Oregon Health and Sciences University. 幼虫斑马鱼第一脊椎动物的模型系统，让从脊髓运动神经元和目标骨骼肌同时膜片钳记录。本视频演示了用于识别段第电机神经元和目标肌肉细胞以及从不同的细胞类型的录音方法的微观方法。

视频: Paired Patch Clamp Recordings from Motor-neuron and Target Skeletal Muscle in Zebrafish

Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of intracellular events that lead to closure of cyclic-nucleotide-gated channels in the cell membrane. The resulting change in membrane potential leads in turn to reductions in the amount of neurotransmitter release from both rod and cone synaptic terminals. To measure how the light-evoked change in photoreceptor membrane potential leads to downstream activity in the retina, scientists have made electrophysiological recordings from retinal slice preparations for decades1,2. In the past these slices have been cut manually with a razor blade on retinal tissue that is attached to filter paper; in recent years another method of slicing has been developed whereby retinal tissue is embedded in low gelling temperature agar and sliced in cool solution with a vibrating microtome3,4. This preparation produces retinal slices with less surface damage and very robust light-evoked responses. Here we document how this procedure can be done under infrared light to avoid bleaching the visual pigment.

Here we describe a procedure for generating dark-adapted slices of the mouse retina for electrophysiological recordings.

从一个暗适应切片制备小鼠视网膜神经细胞的膜片钳录音

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of ...

科研

神经科学

Patch Clamp Recordings in Inner Ear Hair Cells Isolated from Zebrafish

Patch clamp analyses of the voltage-gated channels in sensory hair cells isolated from a variety of species have been described previously1-4 but this video represents the first application of those techniques to hair cells from zebrafish. Here we demonstrate a method to isolate healthy, intact hair cells from all of the inner ear end-organs: saccule, lagena, utricle and semicircular canals. Further, we demonstrate the diversity in hair cell size and morphology and give an example of the kinds of patch clamp recordings that can be obtained. The advantage of the use of this zebrafish model system over others stems from the availability of zebrafish mutants that affect both hearing and balance. In combination with the use of transgenic lines and other techniques that utilize genetic analysis and manipulation, the cell isolation and electrophysiological methods introduced here should facilitate greater insight into the roles hair cells play in mediating these sensory modalities.

The purpose of this video is to demonstrate procedures for obtaining healthy, intact hair cells from the inner ear organs of adult zebrafish and then using them for patch clamp studies aimed at characterizing the biophysical properties of their voltage-gated channels.

膜片钳记录在内耳毛细胞的分离斑马鱼

Patch  clamp analyses of the voltage-gated channels in sensory hair cells isolated  from a variety of species have been described previously1-4 but this  ...

F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes

Mitochondria are involved in many important cellular functions including metabolism, survival1, development and, calcium signaling2. Two of the most important mitochondrial functions are related to the efficient production of ATP, the energy currency of the cell, by oxidative phosphorylation, and the mediation of signals for programmed cell death3.
The enzyme primarily responsible for the production of ATP is the F1FO-ATP synthase, also called ATP synthase4-5. In recent years, the role of mitochondria in apoptotic and necrotic cell death has received considerable attention. In apoptotic cell death, BCL-2 family proteins such as Bax enter the mitochondrial outer membrane, oligomerize and permeabilize the outer membrane, releasing pro-apoptotic factors into the cytosol6. In classic necrotic cell death, such as that produced by ischemia or excitotoxicity in neurons, a large, poorly regulated increase in matrix calcium contributes to the opening of an inner membrane pore, the mitochondrial permeability transition pore or mPTP. This depolarizes the inner membrane and causes osmotic shifts, contributing to outer membrane rupture, release of pro-apoptotic factors, and metabolic dysfunction. Many proteins including Bcl-xL7 interact with F1FO ATP synthase, modulating its function. Bcl-xL interacts directly with the beta subunit of F1FO ATP synthase, and this interaction decreases a leak conductance within the F1FOATPasecomplex, increasing the net transport of H+ by F1FO during F1FO ATPase activity8 and thereby increasing mitochondrial efficiency. To study the activity and modulation of the ATP synthase, we isolated from rodent brain submitochondrial vesicles (SMVs) containing F1FO ATPase. The SMVs retain the structural and functional integrity of the F1FO ATPase as shown in Alavian et al. Here, we describe a method that we have used successfully for the isolation of SMVs from rat brain and we delineate the patch clamp technique to analyze channel activity (ion leak conductance) of the SMVs.

F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes

A method to isolate submitochondrial vesicles enriched in F1FO ATP synthase complexes from rat brain is described. These vesicles allow the study of the activity of F1FO ATPase complex and its modulation using the technique of patch clamp recording.

<sub&gt; 1</sub&gt; F<sub&gt; O</subATP酶微囊的制备和表演膜片钳记录的Submitochondrial泡膜技术

Mitochondria are involved in many important cellular  functions including metabolism, survival1, development and, calcium signaling2. Two of the most ...

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

The marine gastropod mollusk Aplysia californica has a venerable history as a model of nervous system function, with particular significance in studies of learning and memory. The typical preparations for such studies are ones in which the sensory and motoneurons are left intact in a minimally dissected animal, or a technically elaborate neuronal co-culture of individual sensory and motoneurons. Less common is the isolated neuronal preparation in which small clusters of nominally homogeneous neurons are dissociated into single cells in short term culture. Such isolated cells are useful for the biophysical characterization of ion currents using patch clamp techniques, and targeted modulation of these conductances. A protocol for preparing such cultures is described. The protocol takes advantage of the easily identifiable glutamatergic sensory neurons of the pleural and buccal ganglia, and describes their dissociation and minimal maintenance in culture for several days without serum.

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

We describe the dissection of the nervous system of the marine sea hare Aplysia after anesthesia, the isolation of neurons for short term-tissue culture, and recordings of single cell ion currents via the patch clamp technique.

隔离的感觉神经元中<em&gt;海兔丫</em谷氨酸能电流膜片钳记录

The marine gastropod mollusk Aplysia californica has a venerable  history as a model of nervous system function, with particular significance in  studies ...

Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells

Mauthner cells (M-cells) are large reticulospinal neurons located in the hindbrain of teleost fish. They are key neurons involved in a characteristic behavior known as the C-start or escape response that occurs when the organism perceives a threat. The M-cell has been extensively studied in adult goldfish where it has been shown to receive a wide range of excitatory, inhibitory and neuromodulatory signals1. We have been examining M-cell activity in embryonic zebrafish in order to study aspects of synaptic development in a vertebrate preparation. In the late 1990s Ali and colleagues developed a preparation for patch clamp recording from M-cells in zebrafish embryos, in which the CNS was largely intact2,3,4. The objective at that time was to record synaptic activity from hindbrain neurons, spinal cord neurons and trunk skeletal muscle while maintaining functional synaptic connections within an intact brain-spinal cord preparation. This preparation is still used in our laboratory today. To examine the mechanisms underlying developmental synaptic plasticity, we record excitatory (AMPA and NMDA-mediated)5,6 and inhibitory (GABA and glycine) synaptic currents from developing M-cells. Importantly, this unique preparation allows us to return to the same cell (M-cell) from preparation to preparation to carefully examine synaptic plasticity and neuro-development in an embryonic organism. The benefits provided by this preparation include 1) intact, functional synaptic connections onto the M-cell, 2) relatively inexpensive preparations, 3) a large supply of readily available embryos 4) the ability to return to the same cell type (i.e. M-cell) in every preparation, so that synaptic development at the level of an individual cell can be examined from fish to fish, and 5) imaging of whole preparations due to the transparent nature of the embryos.

We have developed an intact brain-spinal cord preparation to record and monitor electrical activity via patch clamp recording from the Mauthner neurons and other reticulospinal cells in zebrafish embryos. Thus, we are able to record excitatory and inhibitory synaptic currents, voltage-gated channel activity and action potentials from key neurons in a developing embryo.

从胚胎斑马鱼的毛特纳细胞膜片钳记录

Mauthner cells (M-cells) are large  reticulospinal neurons located in the hindbrain of teleost fish. They are key  neurons involved in a characteristic ...

Whole-cell Patch-clamp Recordings from Morphologically- and Neurochemically-identified Hippocampal Interneurons

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal population (10-20%), but show a high level of physiological, morphological, and neurochemical heterogeneity, reflecting their diverse functions. Therefore, investigation of interneurons provides important insights into the organization principles and function of neuronal circuits. This, however, requires an integrated physiological and neuroanatomical approach for the selection and identification of individual interneuron types. Whole-cell patch-clamp recording from acute brain slices of transgenic animals, expressing fluorescent proteins under the promoters of interneuron-specific markers, provides an efficient method to target and electrophysiologically characterize intrinsic and synaptic properties of specific interneuron types. Combined with intracellular dye labeling, this approach can be extended with post-hoc morphological and immunocytochemical analysis, enabling systematic identification of recorded neurons. These methods can be tailored to suit a broad range of scientific questions regarding functional properties of diverse types of cortical neurons.

Cortical networks are controlled by a small, but diverse set of inhibitory interneurons. Functional investigation of interneurons therefore requires targeted recording and rigorous identification. Described here is a combined approach involving whole-cell recordings from single or synaptically-coupled pairs of neurons with intracellular labeling, post-hoc morphological and immunocytochemical analysis.

从Morphologically-和确定的Neurochemically海马的interneurons全细胞膜片钳记录

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal ...

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron. When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased. This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue. A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation. Paired whole cell recordings are often perceived as too challenging to perform. While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed. The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings. While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation. In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology.

Pair recordings are simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling precise electrophysiological and pharmacological characterization of the synapses between individual neurons. Here we describe the detailed methodology and requirements for establishing this technique in organotypic hippocampal slice cultures in any laboratory equipped for electrophysiology.

在器官型海马脑片搭配全细胞记录

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. Structural changes at the NMJ can result in neurotransmission failure, resulting in weakness, atrophy and even death of the muscle fiber. Many studies have investigated how genetic modifications or disease can alter the structure of the mouse NMJ. Unfortunately, it can be difficult to directly compare findings from these studies because they often employed different parameters and analytical methods. Three protocols are described here. The first uses maximum intensity projection confocal images to measure the area of acetylcholine receptor (AChR)-rich postsynaptic membrane domains at the endplate and the area of synaptic vesicle staining in the overlying presynaptic nerve terminal. The second protocol compares the relative intensities of immunostaining for synaptic proteins in the postsynaptic membrane. The third protocol uses Fluorescence Resonance Energy Transfer (FRET) to detect changes in the packing of postsynaptic AChRs at the endplate. The protocols have been developed and refined over a series of studies. Factors that influence the quality and consistency of results are discussed and normative data are provided for NMJs in healthy young adult mice.

The neuromuscular junction (NMJ) is altered in a variety of conditions that can sometimes culminate in synaptic failure. This report describes fluorescence microscope-based methods to quantify such structural changes.

神经肌肉接头处:测量突触大小，碎片和改变突触蛋白密度利用共聚焦荧光显微镜

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. ...

Electrophysiological and Morphological Characterization of Neuronal Microcircuits in Acute Brain Slices Using Paired Patch-Clamp Recordings

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with simultaneous intracellular biocytin filling allows a correlated analysis of their structural and functional properties. With this method it is possible to identify and characterize both pre- and postsynaptic neurons by their morphology and electrophysiological response pattern. Paired recordings allow studying the connectivity patterns between these neurons as well as the properties of both chemical and electrical synaptic transmission. Here, we give a step-by-step description of the procedures required to obtain reliable paired recordings together with an optimal recovery of the neuron morphology. We will describe how pairs of neurons connected via chemical synapses or gap junctions are identified in brain slice preparations. We will outline how neurons are reconstructed to obtain their 3D morphology of the dendritic and axonal domain and how synaptic contacts are identified and localized. We will also discuss the caveats and limitations of the paired recording technique, in particular those associated with dendritic and axonal truncations during the preparation of brain slices because these strongly affect connectivity estimates. However, because of the versatility of the paired recording approach it will remain a valuable tool in characterizing different aspects of synaptic transmission at identified neuronal microcircuits in the brain.

Patch-clamp recordings and simultaneous intracellular biocytin filling of synaptically coupled neurons in acute brain slices allow a correlated analysis of their structural and functional properties. The aim of this protocol is to describe the essential technical steps of electrophysiological recording from neuronal microcircuits and their subsequent morphological analysis.

神经微电路急性脑片电生理和形态特征采用配对膜片钳记录

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with ...

Whole-cell Patch-clamp Recordings in Brain Slices

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the neuron. In this configuration, the micropipette is in tight contact with the cell membrane, which prevents current leakage and thereby provides more accurate ionic current measurements than the previously used intracellular sharp electrode recording method. Classically, whole-cell recording can be performed on neurons in various types of preparations, including cell culture models, dissociated neurons, neurons in brain slices, and in intact anesthetized or awake animals. In summary, this technique has immensely contributed to the understanding of passive and active biophysical properties of excitable cells. A major advantage of this technique is that it provides information on how specific manipulations (e.g., pharmacological, experimenter-induced plasticity) may alter specific neuronal functions or channels in real-time. Additionally, significant opening of the plasma membrane allows the internal pipette solution to freely diffuse into the cytoplasm, providing means for introducing drugs, e.g., agonists or antagonists of specific intracellular proteins, and manipulating these targets without altering their functions in neighboring cells. This article will focus on whole-cell recording performed on neurons in brain slices,&#160;a preparation that has the advantage of recording neurons in relatively well preserved brain circuits, i.e., in a physiologically relevant context. In particular,&#160;when combined with appropriate pharmacology, this technique is a powerful tool allowing identification of specific neuroadaptations that occurred following any type of experiences, such as learning, exposure to drugs of abuse, and stress. In summary, whole-cell patch-clamp recordings in brain slices provide means to measure in ex vivo preparation long-lasting changes in neuronal functions that have developed in intact awake animals.

This protocol describes basic procedural steps for performing whole-cell patch-clamp recordings. This technique allows the study of&#160;the electrical behavior of neurons, and when performed in brain slices, allows the assessment of various neuronal functions from neurons that are still integrated in relatively well preserved brain circuits.

在脑片全细胞膜片钳记录

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the ...