Name: patch-clamp capacitance measurements and ca2+ imaging at single nerve terminals in retinal slices
Uploaded: 2012-01-19T13:00:00
Description: Watch this Scientific Journal Video about Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices at JoVE.com

我们感谢他的弗雷德雷基博士琼脂包埋的视网膜切片制备的一种解释，当我们开始在我们的实验室中使用的协议。我们也感谢洛瑞Vaskalis的示意图和DRS的插图。 Veeramuthu维文和Soyoun赵上的文字和视频的有益的意见。这项工作是支持由聂NIH RO1的授予，由韩国政府资助的韩国研究基金会格兰特也部分支持KRF - 2008 - 357 - E00032]。

1。外部和内部的解决方案<ol><li>准备从10倍原液切片溶液（低钙）和每天添加2，氯化钙 ，氯化镁和D -葡萄糖。最后1X解决方案，包括（毫米）：119氯化钠，氯化钾2.5，3.2，0.25 MgCl 2的氯化钙，12个D -葡萄糖，0.2 L -抗坏血酸，12 HEPES 。设置pH值至7.4（用NaOH），并调节渗透压260 mOsm（使用H 2 O和10倍原液） 。 </li><li>称取3％的低胶凝温度琼脂（琼脂糖类型VII - A，A0701，Sigma公司...

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一个关键和艰难的一步，在我们的协议转让的视网膜上成的琼脂溶液（3.4协议） 。这是必要的，小心地取出玻璃体和视网膜片残余片解决方案，传输不失真或弯曲。为了做到这一点，我们使用了一个尖弯曲90 °角，沿直角尖镊子（11251-35，精细科学的工具），位置视网膜的小抹刀（21 - 401 - 25B，Fisherbrand）片解决方案的整个传输过程中的一块。仔细涂抹表面与一个折叠或卷起Kimwipes（金...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>胶凝温度琼脂低</td><td>西格玛</td><td> A0701 </td><td>琼脂糖类型七 - </td></tr><tr><td>补丁移液器</td><td>世界精密仪器</td><td> 1B150F - 4 </td><td>厚壁（1.5毫米外径）的高硼硅玻璃</td></tr><tr><td>立式拉拔</td><td> Narishige </td><td> PP830 </td><td></td></tr><tr><td>牙科用蜡</td><td> CAVEX </td><td></td><td></td></tr><tr><td>春季剪刀</td><td>精细科学工具</td><td> 15003-08 </td><td></td></tr><tr><td> 45 °角的精尖钳</td><td>精细科学工具</td><td> 11251-35 </td><td></td></tr><tr><td>刀片</td><td> Personna </td><td></td><td>双刃，清洗用70％乙醇和H 2 O </td></tr><tr><td>圆管</td><td> Fisherbrand </td><td> 03 - 338 - 1B </td><td>聚乙烯样品瓶2.5毫升</td></tr><tr><td>透明质酸</td><td>西格玛</td><td> H6254 </td><td></td></tr><tr><td> Vibratome切片机</td><td>徕卡</td><td> VT1000S或VT1200S </td><td></td></tr><tr><td>立式显微镜</td><td>奥林巴斯</td><td> BX51WI </td><td></td></tr><tr><td> 60倍的水浸泡目标</td><td>奥林巴斯</td><td> LUMPlanFl </td><td>不适用0.90 </td></tr><tr><td> CCD相机</td><td>索尼</td><td> XC - 75 </td><td></td></tr><tr><td>摄像头控制器</td><td>滨松</td><td> C2400 </td><td></td></tr><tr><td>监视器&lt;/ TD&gt; <td>索尼</td><td></td><td> 13“黑白监视器</td></tr><tr><td>注射器过滤器</td><td> NALGENE </td><td></td><td> 0.2微米</td></tr><tr><td>微操作机器人</td><td>萨特仪器</td><td> MPC - 200 </td><td></td></tr><tr><td>锁相放大器</td><td> HEKA </td><td></td><td> EPC-9/10放大器具有软件仿真</td></tr><tr><td>旋转盘激光共聚焦显微镜</td><td>横河电机</td><td> CSU - X1 </td><td>修补后的活细胞成像钳全细胞记录</td></tr><tr><td> Slidebook软件</td><td>智能成像仪器（3I） </td><td></td><td>成像数据采集和分析</td></tr><tr><td>多聚甲醛</td><td>西格玛</td><td> P6148 </td><td></td></tr><tr><td>磷酸盐缓冲溶液</td><td>胃肠道BCO </td><td> 70013 </td><td></td></tr><tr><td> Superfrost幻灯片</td><td> Fisher Scientific则</td><td></td><td>玻璃片</td></tr><tr><td>抗褪色剂</td><td> Biomeda公司。 </td><td></td><td></td></tr><tr><td>共聚焦激光扫描显微镜</td><td>卡尔蔡司</td><td> LSM 710 </td><td>固定组织成像</td></tr><tr><td>抹刀</td><td> Fisherbrand </td><td> 21 - 401 - 25B </td><td></td></tr><tr><td>曼努埃尔垂直切片机</td><td> Narishige </td><td> ST - 20 </td><td></td></tr><tr><td>俄勒冈州绿488 BAPTA - 1 </td><td> Invitrogen公司</td><td> O - 6806 </td><td>的Ca 2 +敏感的荧光染料</td></tr><tr><td>的Alexa Fluor 555肼</td><td> Invitrogen公司</td><td> A - 20501MP </td><td>荧光染料</td></tr></tbody></table>

patch-clamp capacitance measurements and ca2+ imaging at single nerve terminals in retinal slices

发现视觉刺激，并转达了专门的神经元在脊椎动物视网膜的光线强度和频率的变化在很宽的动态范围。两个班的视网膜神经细胞，感光细胞和双极细胞，使用色带类型的活动区，从而使长的时间周期，持续和高吞吐量的神经递质的释放。型混合双极细胞终端（MB）在金鱼视网膜，从而去极化光刺激和接收混合杆和锥感光输入，适合的带状型突触都因他们的大尺寸（〜10-12的研究微米直径）和他们的许多横向和互惠的无长突细胞树突的突触联系。 MB双极细胞在金鱼视网膜切片膜片钳技术终端直接访问允许测量突触前的Ca 2 +电流，膜电容的变化，和互惠的反馈抑制GABA介导的突触&lt;分&gt; A和GABA 彗星受体表达上的终端。突触前膜电容测量胞吐允许一个短期的可塑性研究的兴奋性神经递质的释放14,15。此外，短期和长期的无长突细胞的抑制性神经递质释放的可塑性也可以抵达的21 MB终端的相互反馈抑制录音进行调查。 GABA能无长突细胞的相互反馈抑制经过短时间的时间（例如〜10秒），通过GABA的小泡池枯竭 11双脉冲抑郁症。在视网膜内丛状层视网膜微型突触动力学因此可以直接研究。 大脑切片技术引入超过40年前，但仍然是非常有用的神经元的电性能，无论是在单细胞胞体，树突或轴突单，调查，和Microcircuit突触水平19。组织太小，无法直接粘到切片室往往是先嵌入在琼脂（或放置到一个滤纸），然后切片20，23，18，9。在这段视频中，我们采用预嵌入琼脂技术，使用金鱼视网膜。在金鱼视网膜片的巨型双极细胞终端axotomized（轴突切割）在切片过程中。这允许我们分离单个突触前神经末梢输入，录音排除，因为从axotomized终端的信号从胞体树突状车厢。另外，也可以从完整的MB双极细胞记录，从没有减少在切片过程中的轴突连接到终端记录。总体而言，这个实验性的协议的使用将有助于在视网膜突触生理学，微电路功能分析，带状突触的突触传递的研究。

Watch this Scientific Journal Video about Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices at JoVE.com

Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

在这里，我们描述了一个协议，准备琼脂嵌入式适合电和Ca2 +成像视网膜切片。这种方法允许一个研究使用录音单突触前神经末梢的直接膜片钳视网膜微型带状型突触。

膜片钳电容测量和Ca 2 +单神经末梢成像在视网膜切片

Visual stimuli are detected and conveyed over a wide dynamic range of light intensities and frequency changes by specialized neurons in the vertebrate ...

Research

JoVE Journal

Neuroscience

Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

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

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

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

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.  ...

A Computer-assisted Multi-electrode Patch-clamp System

计算机辅助多电极膜片钳系统

The patch-clamp technique is today the most  well-established method for recording electrical activity from individual  neurons or their subcellular ...

Visualization of Endosome Dynamics in Living Nerve Terminals with Four-dimensional Fluorescence Imaging

内吞体动力学在生活神经末梢与四维荧光成像可视化

Four-dimensional (4D) light imaging has been used to study behavior of small structures within motor nerve terminals of the thin transversus abdominis ...

Methods for Cell-attached Capacitance Measurements in Mouse Adrenal Chromaffin Cell

方法在小鼠肾上腺嗜铬细胞细胞贴附电容测量

Neuronal transmission is an integral part of cellular communication within the brain. Depolarization of the presynaptic membrane leads to vesicle fusion ...

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

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 ...

Recording Light-evoked Postsynaptic Responses in Neurons in Dark-adapted, Mouse Retinal Slice Preparations Using Patch Clamp Techniques

记录光诱发神经元突触后反应在暗适应，小鼠视网膜切片准备利用膜片钳技术

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. ...

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

成像钙<sup&gt; 2+</sup&gt;在小鼠视网膜锥光感受器轴突终末动态

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to ...

Whole-cell Patch-clamp Recordings in Brain Slices

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 ...

Application of Automated Image-guided Patch Clamp for the Study of Neurons in Brain Slices

自动图像引导贴片夹在脑切片神经元研究中的应用

Whole-cell patch clamp is the gold-standard method to measure the electrical properties of single cells. However, the in vitro patch clamp remains a ...