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;雷基，2001年）和切片的解决方案组合。热琼脂溶液，在微波1-2分钟，或直到它完全溶解，在水浴（30-33℃）孵化琼脂，以防止快速凝固（凝胶化转变温度:26 ± 2 ° C） </li><li>准备录制解决方案（正常钙）从10倍原液，每天添加2，氯化钙，氯化镁和D -葡萄糖。最后1X解决方案consisTS（毫米）:100氯化钠，2.5氯化钾，氯化镁2 1，25碳酸氢钠3 0.2 2.5 L -抗坏血酸， 氯化钙 ，12 D -葡萄糖。冒泡的解决方案，在95％Ø 2 / 5％的CO 2（carbogen）5-10分钟后，设置pH值7.4（用NaOH），并调整渗透压260 mOsm（H 2 O和10倍原液）。 </li><li>等分（1毫升）的内部移液器解决方案，提前做好准备，并储存在冷冻室（-20℃）。两个内部移液器解决方案通常用于隔离双极细胞钙电流（毫米）: <ol><li> 40 CS -葡萄糖，CSCL，60 10四乙基胺（TEA）- CL，28 HEPES，镁ATP钠GTP，EGTA 2 3。调整pH值至7.2（与CsOH）和渗透压设置为250 mOsm。这种高氯内部解决方案通常会提供一个双极细胞静息膜电位为-60 mV的低串联电阻录音（更好的电压钳条件）和较大的抑制性突触后电流（iPS细胞）。 </li><li> 95 CS -葡萄糖，10茶- CL，25 HEPES，3毫克，ATP，0.5钠GTP，EGTA 0.5。调整pH值至7.2（用CsOH），并设置渗透压250 mOsm 22。这种低EGTA的内部解决方案通常会导致去极化步脉冲引起较高的膜电容的变化，从而使小泡池枯竭和短期的经济大萧条的研究。 </li></ol></li></ol> 2。膜片钳吸管电极<ol><li>准备厚壁（外直径1.5毫米），高硼硅玻璃（1B150F - 4;世界精密仪器，萨拉索塔，佛罗里达州）和拉补丁移液器使用垂直拉马（Narishige，PP830，东京，日本）。打开提示补丁录音解决方案移液器电阻7-8MΩ的移液器充满内部液＃1。 </li><li>大衣补丁吸管均匀，从尖轴，达到吸管人，牙科用蜡，（CAVEX，西切斯特，PA）的水平。这将最大限度地减少吸管电容和电气噪声，并启用更准确，低噪音电容测量。吸管低电容也有助于电子EPC - 9（或EPC - 10）膜片钳放大器的C快（快速电容）的补偿。 </li></ol> 3。琼脂包埋的视网膜切片的制备<ol><li>选择金鱼（ 鲫鱼 ; 8-16厘米），在一个黑暗的房间在暗适应30分钟的覆盖斗和地点。麻醉后，安​​乐死的镇静剂金鱼快速断头和脊髓和脑干双pithing。然后取出弯尖的剪刀和弯曲的尖镊子的眼睛。在俄勒冈卫生科学大学的机构动物护理和使用委员会（IACUC）已经批准了这些程序。 </li><li> Hemisect每只眼睛由围绕眼睛的前面，均匀地切割剪刀弹簧（15003-08，精细的科学工具;发起削减剪刀提示穿刺），并放置在冷冻切片的解决方案（低钙）的眼罩。如果necessary，取出镊子镜头（镜头通常会分离与眼睛的前面）。取出的视网膜色素上皮细胞使用的是一双45 °角细尖镊子（11251-35，精细科学的工具）视网膜剥离远离眼罩，进展缓慢周围360 °连接。切断或削减或者罚款钳或弹簧剪刀视神经。轻轻取出视网膜结合的角度的精尖钳和修改后的巴斯德吸管或移液管（大尖）应用的吸。使用角度的精尖钳取出视网膜色素上皮剩余。表面的清洁和健康的视网膜的，应该出现暗，光滑，红色。对于所有深色的色素上皮细胞的完整切除暗适应1 小时 （8，1992）在视网膜上。 </li><li>治疗孤立视网膜〜0.03％（WT /卷;片〜0.36 mg / ml的1X解决方案）;在室温15-30分钟的透明质酸（H6254， 西格玛 3 ）清除玻璃体的温度（20-23℃）。在此潜伏期，你可以准备冰冷片解决方案。 </li><li>通过消除与刀片的一小部分（Personna，一把双刃剑，用70％乙醇和H 2 O清洗）连接到弯曲的边缘剪下一块长方形的视网膜（〜2 × 2毫米，含视网膜全层） 1毫升的塑料注射器的身体，和转移视网膜一块充满琼脂溶液（1.2）准备一个小容器。尽量减少周围视网膜的解决方案，因为它是放置到液体培养基。直接浸泡在冰冷的片解决方案（准备（3.3）），以巩固琼脂20，18，9。我们用一个小的，手工制作的容器。简单地说，一个小圆管（Fisherbrand，聚乙烯样品瓶2.5毫升，03 - 338 - 1B）在两端被切断，并用封口膜（PM - 996，佩希内塑料包装）修补密封。 </li><li>固体琼脂块切成1x1x1厘米的立方体包含一块长方形的视网膜。 </li><li>块转移到片室和胶切片板的表面。片室在冷冻室（-20℃）预冷。 200-250微米厚的薄片，在冰冷的片解决方案使用一个Vibratome切片机（VT1000S或VT 1200S，徕卡），切成块。可以得到总共5至10片，这是可行的约5至6小时。 </li><li>切片传输到录音室。将一个网格片上粘一个U形的铂金帧19的尼龙线，并连续灌注与95％O 2 / 5％CO 2（carbogen冒泡的录音解决方案（1-2毫升每分钟的速度） ）。 </li></ol>故障排除: 如果视网膜一块镶嵌在琼脂块分离或在切片的琼脂，可以增加层厚200微米至300微米20微米的增量。此外，尽量减少周围视网膜的解决方案，因为它是转移和放置到液体培养基卷"Kimwipe"纸尖吸干多余的解决方案。为了避免这个问题的另一种方法是减少最初放在琼脂视网膜片的大小。由于玻璃体禁止琼脂完全附着在视网膜一块，可能有必要做出新的透明质酸或调整孵化时间（步骤（3.3））。 4。在视网膜切片的双极细胞的突触终端的识别<ol><li>在直立显微镜（如BX51WI，奥林巴斯）下视网膜切片的位置。我们认为与红外微分干涉对比（IR - DIC的）通过一个60倍的水浸泡的目的，奥林巴斯（NA 0.90）和CCD相机（XC - 75，索尼）光学视网膜切片。 CCD相机的输出被发送到对比度增强的可视化模拟的索尼13"双语法例咨询委员会前一个摄像机控制器（C2400，滨松）K和黑白显示器。显微镜是装在一个XY平移台，录音室是放置在一个固定的阶段14。 </li><li>完整和axotomized（轴突切）双极细胞终端，可以通过它们的大小，形状，切片（ 图1）中的位置确定。杰出Axotomized和完整的终端也可以通过检查其库容的瞬态电流衰减时间（单指数与双指数衰减，分别） 和 Ca 2 +电流（图 2，12，14，15） 。 MB码头位于sublamina b最近端层（ON型层;相邻的神经节细胞层）在金鱼视网膜内丛状层。 MB终端，与他们的沉闷的表面和平面外观，可以很容易地区别于节和流离失所的无长突胞体，从而出现相光明和球形。此外，MB终端的优势是覆盖高密度的突触联系，并显得粗糙和不规则的，比较干净的表面光滑，神经节和无长突细胞（图1A - C ）。 Axotomized MB终端出现圆形，近圆形（ 图1c），而完整的终端出现椭圆形和延伸，往往带有捏指着走向内心的视网膜核层（图1A，B）结束。 </li><li>损坏或不健康的终端往往显得肿胀，表面上显示几个小颗粒结构。它可能会获得这些终端GΩ的印章，但他们可能破裂后不久突破。其他不健康的终端迹象包括一个中空的外观，对比度和/或亮度周围切片，有时在片表面的位置相同。这是健康端子切片中的深（优点:更完整的突触电路）记录和片表面附近（ADVantage:更快地访问灌注药物在外部的解决方案;更容易密封）。 </li></ol> 5。电生理记录和Ca 2 +成像<ol><li>使用一个0.2微米的过滤嘴（NALGENE）的注射器，填补内部解决方案的水平1-2厘米移液器背面的玻璃修补吸管。取出后的提示气泡，保护持有人的吸管，申请正压（1.2-1.6 PSI），并朝着目标使用一个显微的终端（MPC - 200，萨特仪器）。同时通过片尖向下，在横向席卷方向移动吸管，以确保切片是不拉用刀尖。 </li><li>移动终端周围的吸头，清洁膜表面。向下推终端创建一个酒窝（缩进），并释放正压边缘的一角。如果GΩ的密封并不构成立即的，适用于轻微的负压力。 </李&gt; <li>一个GΩ的印章后，切换到EPC - 9膜片钳放大器的细胞记录模式，并保持电位变化到-60或-70毫伏。应用的C -快速（快速电容）校正，等待为5-10GΩ的稳定之间的密封，然后破裂的犀利，温柔的吸口应用，建立了全细胞记录配置的膜。如果整个单元配置已正确建立，将串联电阻之间14-30MΩ。输入电阻将完整的终端和14 axotomized端子1-3GΩ的200-500MΩ。 </li><li>观察电容电流瞬变（ 图2A和2C），区分完整和axotomized双极细胞终端。完整和axotomized MB终端有一个基线膜9-16 pF的电容和3-8 pF的分别。 </li><li>申请一个200毫秒从-70至0 mV至axotomized MB终端电压钳一步。这将使大（〜200-600 PA），隔离外来的Ca 2 +激活电流电压依赖性L -型钙2开放+通道（ 图2b）的直接测量。与此同时，一个是能够监控突触囊泡的胞吐作用所造成的电容跳，并迅速，pH介导的抑制的 Ca 2 +电流如下胞吐15，和GABA能相互反馈抑制 （ 图 3C; 22）。如果一个补丁一个完整的MB双极细胞终端的结果将显示在图2C和2D。没有明显的Ca 2 +电流是因为大电流由于"泄漏"差距MB的双极细胞树突的交界处耦合观察。 </li><li>膜电容的变化，可以使用"正弦+直流"6时间分辨的膜电容测量方法，从保持电位-70 mV的0 mV时的一个步骤去极化诱发。膜电容的跳跃表明突触囊泡与质膜融合。 2 kHz的正弦电压钳位命令（30 mV峰 - 峰）添加到-70 mV的电位。记录目前是由EPC - 9膜片钳放大器的软件仿真的两个正交相位角分析一个锁相放大器（HEKA，Lambrecht，德国）。在图2d，高频率（2 kHz）的正弦刺激范围进行分析，以终端的神经末梢和轴突，其中有一个基线电容厘米〜6.8 PF的膜电容的完整的终端。双极细胞的胞体和树突膜，从而过滤掉高频电压钳正弦波 13 。 </li><li>对Ca 2 +成像实验，彻底混合的Ca 2 +敏感的荧光染料与内部的补丁吸管解决方案（如俄勒冈绿488 BAPTA - 1（100-200μM，O - 6806，Invitrogen公司））和重复的协议50.1到5.5。这将允许一个结合荧光成像和电生理记录。例如，它可以形象的Ca 2 +的瞬态一个200 ms的步长从-70至0 MV MB终端（图3A，B）在小telodendria附属物。我们整合了我们直立的奥林巴斯显微镜，旋转盘激光共聚焦显微镜系统（CSU - X1，横河电机）。共聚焦显微镜使用488 nm和561 nm激光线，是由一个声光可调谐滤波器调制。数据采集​​使用Slidebook软件（3I;智能成像仪器）。对于使用金鱼视网膜神经细胞钙成像技术的进一步详细信息，请参阅24，17，3 。 </li></ol>故障排除: 如果一个人经常未能建立一个全细胞配置，甚至形成一个稳定的GΩ的印章后，它可能是有益的，以减少使用负压穿刺终端我mbrane。这也可能是建立全细胞配置最佳的吸气压力作为膜曲率（SOMA&gt;终端）的功能变化的情况下。它也可以使用ZAP协议（幅度:400 mV时，持续时间:100微秒），单独或结合一个尖脉冲负压进入全细胞配置。我们已经找到了ZAP协议一起使用时，在超过12MΩ浴电阻一个枪头，要突破特别有用。 6。代表性的成果<img alt="图1" src="/files/ftp_upload/3345/3345fig1.jpg" /> 图1。MB双极终端在金鱼视网膜片细胞的鉴定。 （AC）IR - DIC的MB双极细胞终端的图像。我们可以找出可能axotomized（轴突切割）和完整​​的IR - DIC的形象终端。 axotomized终端出现圆形和圆形， 在 C所示一个完整的终端，而更可能出现椭圆形，在A和 B所示。这种分类可以确认瞬态电容测量（见图2），或由染料充填法（D - F） 。红色箭头表示在A和 B的交点之间的轴突和轴突终端的完整的终端的位置。红色虚线表示MB双极细胞码头的轮廓。 （DF）。 MB双极终端与一个完整的轴突（D），一个部分切断的轴突（E），或完全拆除的轴突（F）细胞荧光图像。 Alexa的555（A20501MP Invitrogen公司）荧光染料充满了内部解决方案之前录制。视网膜切片膜片钳记录后，立即被转移到4％（WT /卷）多聚甲醛磷酸盐缓冲液（P6148，Sigma公司）（70013，GIBCO公司）孵育30分钟。片安装到Superfrost幻灯片（Fisher Scientific这）抗衰落代理（Biomeda公司）在水溶液中的安装介质。 Alexa的555 MB双极细胞终端被视为与555 nm激光线（红色）使用共聚焦激光扫描显微镜（LSM的710卡尔蔡司）一个40倍的水浸泡目标。注意telodendria从轴突终末（蓝色箭头）凸出的小附属物的存在。比例尺显示10微米（A - F）。 INL:内核层，彩光:内网状层，GCL:神经节细胞层。 <img alt="图2" src="/files/ftp_upload/3345/3345fig2.jpg" /> 图2。axotomized和完整MB双极细胞终端的电气性能。 （A，C）的瞬态电流响应从-60 mV的控股潜力-70毫伏的电压钳步超极化激活。短路电流-10 mV的电压钳一步可能会导致:1）单一快速指数电流衰减，高输入电阻在-70毫伏（axotomized终端指示;面板），或2）与低输入阻抗的双指数-70毫伏（指示性的衰变一个完整的终端; C组，另见12，14，15）。 （B，D）的Ca 2 +电流和膜电容跳一步的去极化从-70至0 mV时引起。时间分辨的膜电容测量使用一个2 kHz的正弦波叠加刺激休息控股潜力-70 毫伏 6 。红色迹线是电容数据点的平均值。请注意，在图2B和2D电容跳都等于200 FF。 <img alt="图3" src="/files/ftp_upload/3345/3345fig3.jpg" /> 图3。直接测量的Ca 2 +成像信号，胞吐作用，并在M的抑制反馈b双极细胞终端。 （ 一 ）瞬态崛起中的Ca 2 +浓度在一个MB终端telodendria由电压钳一步的去极化从-70至0 mV时为200毫秒（箭头）被激活。俄勒冈州绿488 BAPTA - 1（100μM）， 钙离子指 ​​示剂，包括在补丁吸管。 （ 二 ）通讯MB telodendria（红色虚线表示有兴趣的地区）的荧光图像。 （三）短期突触抑郁症，神经递质的释放（胞吐） 。一双20 ms的去极化到0 MV（上跟踪; 200毫秒脉冲间间隔）诱发的Ca 2 +电流（中间曲线）和膜电容（低跟踪）。箭头表示exocytosed质子的作用的 Ca 2 +电流也从邻近的无长突细胞15，21 GABA能相互反馈抑制。请注意，质子介导抑制的Ca 2 +电流也GAB目前，仅在第一次去极化反应，这也有一个电容跳由于突触囊泡的胞吐Aergic互惠反馈抑制。

<ol>
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我们没有竞争的利益披露。

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 ...

patch-clamp-capacitance-measurements-ca2-imaging-at-single-nerve

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

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

18.1K 次观看.  Oregon Health and Science University. 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。

视频: 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

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

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

Four-dimensional (4D) light imaging has been used to study behavior of small structures within motor nerve terminals of the thin transversus abdominis muscle of the garter snake. Raw data comprises time-lapse sequences of 3D z-stacks. Each stack contains 4-20 images acquired with epifluorescence optics at focal planes separated by 400-1,500 nm. Steps in the acquisition of image stacks, such as adjustment of focus, switching of excitation wavelengths, and operation of the digital camera, are automated as much as possible to maximize image rate and minimize tissue damage from light exposure. After acquisition, a set of image stacks is deconvolved to improve spatial resolution, converted to the desired 3D format, and used to create a 4D "movie" that is suitable for variety of computer-based analyses, depending upon the experimental data sought. One application is study of the dynamic behavior of two classes of endosomes found in nerve terminals-macroendosomes (MEs) and acidic endosomes (AEs)-whose sizes (200-800 nm for both types) are at or near the diffraction limit. Access to 3D information at each time point provides several advantages over conventional time-lapse imaging. In particular, size and velocity of movement of structures can be quantified over time without loss of sharp focus. Examples of data from 4D imaging reveal that MEs approach the plasma membrane and disappear, suggesting that they are exocytosed rather than simply moving vertically away from a single plane of focus. Also revealed is putative fusion of MEs and AEs, by visualization of overlap between the two dye-containing structures as viewed in each three orthogonal projections.

Four-dimensional (4D) imaging is utilized to study the behavior and interactions among two types of endosomes in living vertebrate nerve terminals. Movement of these small structures is characterized in three dimensions, permitting confirmation of events such as endosome fusion and exocytosis.

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

Neuronal transmission is an integral part of cellular communication within the brain. Depolarization of the presynaptic membrane leads to vesicle fusion known as exocytosis that mediates synaptic transmission. Subsequent retrieval of synaptic vesicles is necessary to generate new neurotransmitter-filled vesicles in a process identified as endocytosis. During exocytosis, fusing vesicle membranes will result in an increase in surface area and subsequent endocytosis results in a decrease in the surface area. Here, our lab demonstrates a basic introduction to cell-attached capacitance recordings of single endocytic events in the mouse adrenal chromaffin cell. This type of electrical recording is useful for high-resolution recordings of exocytosis and endocytosis at the single vesicle level. While this technique can detect both vesicle exocytosis and endocytosis, the focus of our lab is vesicle endocytosis. Moreover, this technique allows us to analyze the kinetics of single endocytic events. Here the methods for mouse adrenal gland tissue dissection, chromaffin cell culture, basic cell-attached techniques, and subsequent examples of individual traces measuring singular endocytic event are described.

After exocytosis, fused plasma membrane is retrieved through a process known as endocytosis. This mechanism reforms new synaptic vesicles for the next round of release. Individual endocytic events are captured and analyzed through the use of the cell-attached capacitance recordings in mouse adrenal chromaffin cells.

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

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 ...

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

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. Retinal neurons in the network comprise of numerous subtypes. More than 10 subtypes of bipolar cells, ganglion cells, and amacrine cells have been identified by morphological studies. Multiple subtypes of retinal neurons are thought to encode distinct features of visual signaling, such as motion and color, and form multiple neural pathways. However, the functional roles of each neuron in visual signal processing are not fully understood. The patch clamp method is useful to address this fundamental question. Here, a protocol to record light-evoked synaptic responses in mouse retinal neurons using patch clamp recordings in dark-adapted conditions is provided. The mouse eyes are dark-adapted O/N, and retinal slice preparations are dissected in a dark room using infrared illumination and viewers. Infrared light does not activate mouse photoreceptors and thus preserves their light responsiveness. Patch clamp is used to record light-evoked responses in retinal neurons. A fluorescent dye is injected during recordings to characterize neuronal morphological subtypes. This procedure enables us to determine the physiological functions of each neuron in the mouse retina.

We will demonstrate how to prepare retinal slices from the mouse eye and record light responses in retinal neurons. The entire procedure is conducted in dark-adapted conditions.

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

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to blindness in many retinal diseases. Calcium (Ca2+), a key second messenger in photoreceptor signaling and metabolism, has been proposed to be indirectly linked with photoreceptor degeneration in various animal models. Systematically studying these aspects of cone physiology and pathophysiology has been hampered by the difficulties of electrically recording from these small cells, in particular in the mouse where the retina is dominated by rod photoreceptors. To circumvent this issue, we established a two-photon Ca2+ imaging protocol using a transgenic mouse line that expresses the genetically encoded Ca2+ biosensor TN-XL exclusively in cones and can be crossbred with mouse models for photoreceptor degeneration. The protocol described here involves preparing vertical sections (&#8220;slices&#8221;) of retinas from mice and optical imaging of light stimulus-evoked changes in cone Ca2+ level. The protocol also allows &#8220;in-slice measurement&#8221; of absolute Ca2+ concentrations; as the recordings can be followed by calibration. This protocol enables studies into functional cone properties and is expected to contribute to the understanding of cone Ca2+ signaling as well as the potential involvement of Ca2+ in photoreceptor death and retinal degeneration.

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

We describe a protocol to monitor Ca2+ dynamics in the axon terminals of cone photoreceptors using an ex-vivo&#160;slice preparation of the mouse retina. This protocol allows comprehensive studies of cone Ca2+ signaling in an important mammalian model system, the mouse.

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 ...

High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA receptors (NMDARs) and AMPA receptors (AMPARs). Physiological data have indicated that glutamate receptors at RGCs are expressed not only in postsynaptic but also in perisynaptic or extrasynaptic membrane compartments. However, precise anatomical locations for glutamate receptors at RGC synapses have not been determined. Although a high-resolution quantitative analysis of glutamate receptors at central synapses is widely employed, this approach has had only limited success in the retina. We developed a postembedding immunogold method for analysis of membrane receptors, making it possible to estimate the number, density and variability of these receptors at retinal ribbon synapses. Here we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of retinal fixation, 2) freeze-substitution, 3) postembedding immunogold electron microscope (EM) immunocytochemistry and, 4) quantitative visualization of glutamate receptors at ribbon synapses.

The postembedding immunogold method is one of the most effective ways to provide high-resolution analyses of the subcellular localization of specific molecules. Here we describe a protocol to quantitatively analyze glutamate receptors at retinal ribbon synapses.

高分辨率定量免疫膜受体在视网膜带状突触分析

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA ...

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 ...

Slice Patch Clamp Technique for Analyzing Learning-Induced Plasticity

The slice patch clamp technique is a powerful tool for investigating learning-induced neural plasticity in specific brain regions. To analyze motor-learning induced plasticity, we trained rats using an accelerated rotor rod task. Rats performed the task 10 times at 30-s intervals for 1 or 2 days. Performance was significantly improved on the training days compared to the first trial. We then prepared acute brain slices of the primary motor cortex (M1) in untrained and trained rats. Current-clamp analysis showed dynamic changes in resting membrane potential, spike threshold, afterhyperpolarization, and membrane resistance in layer II/III pyramidal neurons. Current injection induced many more spikes in 2-day trained rats than in untrained controls.
To analyze contextual-learning induced plasticity, we trained rats using an inhibitory avoidance (IA) task. After experiencing foot-shock in the dark side of a box, the rats learned to avoid it, staying in the lighted side. We prepared acute hippocampal slices from untrained, IA-trained, unpaired, and walk-through rats. Voltage-clamp analysis was used to sequentially record miniature excitatory and inhibitory postsynaptic currents (mEPSCs and mIPSCs) from the same CA1 neuron. We found different mean mEPSC and mIPSC amplitudes in each CA1 neuron, suggesting that each neuron had different postsynaptic strengths at its excitatory and inhibitory synapses. Moreover, compared with untrained controls, IA-trained rats had higher mEPSC and mIPSC amplitudes, with broad diversity. These results suggested that contextual learning creates postsynaptic diversity in both excitatory and inhibitory synapses at each CA1 neuron.
AMPA or GABAA receptors seemed to mediate the postsynaptic currents, since bath treatment with CNQX or bicuculline blocked the mEPSC or mIPSC events, respectively. This technique can be used to study different types of learning in other regions, such as the sensory cortex and amygdala.

The slice patch clamp technique is an effective method for analyzing learning-induced changes in the intrinsic properties and plasticity of excitatory or inhibitory synapses.

分层膜片钳技术在学习诱导可塑性分析中的应用

The slice patch clamp technique is a powerful tool for investigating learning-induced neural plasticity in specific brain regions. To analyze ...

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

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 challenging and low-throughput technique due to its complexity and high reliance on user operation and control. This manuscript demonstrates an image-guided automatic patch clamp system for in vitro whole-cell patch clamp experiments in acute brain slices. Our system implements a computer vision-based algorithm to detect fluorescently labeled cells and to target them for fully automatic patching using a micromanipulator and internal pipette pressure control. The entire process is highly automated, with minimal requirements for human intervention. Real-time experimental information, including electrical resistance and internal pipette pressure, are documented electronically for future analysis and for optimization to different cell types. Although our system is described in the context of acute brain slice recordings, it can also be applied to the automated image-guided patch clamp of dissociated neurons, organotypic slice cultures, and other non-neuronal cell types.

This protocol describes how to conduct automatic image-guided patch-clamp experiments using a system recently developed for standard in vitro electrophysiology equipment.

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 ...

膜片钳电容测量和Ca^{2 +}单神经末梢成像在视网膜切片

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