Name: simultaneous ex vivo functional testing of two retinas by in vivo electroretinogram system
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这项工作是由美国国立卫生研究院资助EY019312和EY021126（VJK），EY002687到眼科及视觉科学系在华盛顿大学的支持，以及防盲研究。

所有的实验方案符合指南实验动物的护理和使用，被批准的机构动物研究委员会在华盛顿大学。 1.设置灌注和标本持有人<ol><li>制备用于视网膜灌注新鲜的实验当天溶液。使用蒸馏水和去离子水。使用以下三种解决方案之一。 <ol><li>制备碳酸氢盐的含艾姆斯溶液（1L）:1瓶艾姆斯的"媒介和1.9克碳酸氢钠的， </li><li>制备洛克溶液（以mM计）:112.5氯化钠，3.6氯化钾，2.4的MgCl 2，1.2氯化钙2，10.0 HEPES，20.0 碳酸氢钠 ，3.0钠琥珀酸盐，0.5钠谷氨酸，0.02 EDTA和10.0葡萄糖，0.1％MEM维生素和氨基酸酸</li><li>制备HEPES缓冲林格氏溶液（以mM计）:133.3氯化钠，3.3氯化钾，2.0的MgCl 2，1.0氯化钙2，10.0葡萄糖，0.01 EDTA，12.0 HEPES，pH值调节至7.5以〜5.8毫升的1M氢氧化钠，加0.72克/升的Leibovitz培养基的L-15。使用20 - 50μMDL-AP4和50 - 100μM氯化钡2隔离带任何媒体灌注感光响应。 </li></ol></li><li>制备用于电极28（以mM计）溶液:140.0氯化钠，3.6氯化钾，2.4的MgCl 2，1.2氯化钙2，3 HEPES，0.01 EDTA和调节pH值至7.4 - 7.5用NaOH。电极溶液可以储存在RT几个月。 </li><li>准备和测试样品支架。 <ol><li>胶在试样保持器的底部的圆顶顶部黑/灰滤纸（ 见图1A）。小心地双组分5分钟环氧树脂胶围绕圆顶的平坦顶部的边缘。如果有必要，这样做解剖显微镜下。 </li><li>等到胶是几乎干燥（约4分钟），并通过使用一个扁平项按下圆顶的滤纸。胶滤纸至少一天实验前。该滤纸可多次使用，但在一个月的录音后应更换。用70％乙醇安装更换纸张之前，小心地从任何残胶清洁的圆顶。 </li><li>与电极液试图避 ​​免任何气泡和螺杆封闭带螺纹适配器插入电极通道粒料电极（ 见图1A和B）填充电极通道。连接的顶部和底部件的试样保持器的四个螺钉和填充灌注线与电极液。 </li><li>测量用万用表（ 图1B），每个电极对的导线之间的电阻和电压。电阻应该低于100Ωk和低于10毫伏的电压，如果信道是无气泡和电极是在良好的条件。 </li></ol></li><li>倾400 - 700毫升灌注介质中的玻璃瓶中。分开在视网膜和s的解剖中使用另外300ml的在冰箱撕毁了它。设置在热交换器区块灌注管，并放置在加热板上将预热块（参见图1D）。 </li><li>封闭瓶上的帽具有连接到CO 2 / O 2的气体（如果艾姆斯或洛克的使用）和灌注管（参见图1D）。预热瓶媒体37℃，并将其放置在加热板上或强光刺激单元的水浴设为37顶- 39℃下17。 <ol><li>总理灌注线用灌注填充媒体向他们发起重力驱动的流动。 <ol><li>在LKC系统，将瓶上的刺激器单元17的顶部，以提供一个显著重力流动，然后由流速调节器，而不会受到在实验过程中，在瓶子中灌注溶液的降低水平进行调整。 </li><li>在Ocuscience系统，放置在p法拉第笼内部erfusion瓶以减少噪声和放置从所述试样保持长期灌注输出线（见步骤2.8）大大低于试样保持器（和灌注瓶）的水平，以使溶液的重力驱动。这些屏蔽输出线和屏蔽连接到功放的地面，以防止电磁噪声的ERG信号的耦合。 </li></ol></li></ol></li><li>通过使用流速调节器5毫升/分钟 - 调节灌注到3。连接5％CO 2/95％O 2的气体从适当的调节器的气缸，并调整流量以确保介质在瓶子的稳定鼓泡通过空气石。 </li></ol> 2.样品制备<ol><li>组装清洁和锋利清扫工具包括直刀刃microscissors，一个或两个45°镊子，刀片和一块长方形的滤纸。 </li><li>倒入约200毫升冷perfu的在一个大的陪替氏培养皿锡永溶液中，使所述试样保持器（包括圆顶）的整个底部部分可以浸入到该溶液中。安装在圆顶的视网膜时，这一步就变得很重要（见步骤2.6）。虽然一些解决方案被设计成与卡波金（5％CO 2/95％O 2）是饱和的，这是不适合的夹层目的必不可少，并没有在这里描述的实验中进行。 </li><li>对于一个典型的实验中，让动物们在12/12小时黑暗/光照周期和暗适应他们6 - 录音前12小时。安乐死的动物通过CO 2吸入，随后在昏暗的红灯颈椎脱位，并做下暗红色或红外光所有的下列程序（ 如使用红色过滤器显微镜光源的前面）。 <ol><li>拉的眼睛通过使用镊子，把他们的报道。将一只眼睛在一个时间上的一小片纸（ 例如，一些正规的过滤纸），并作为点燃大约在锯齿缘的水平，同时保持与镊子眼睛（这是解决方案之外在这里完成）。 </li></ol></li><li>沿着锯齿缘切（或接近眼睛的赤道）配microscissors并去除角膜和晶状体。将眼罩在冷介质在大陪替氏培养皿，并用另一只眼睛重复相同的过程。 </li><li>通过保持视网膜和巩膜之间的剪刀，以保持视网膜作为完整尽可能削减从眼罩朝着视神经顶部的小切口。握从切口的两侧通过使用两个镊子和拉镊子远离彼此分离的视网膜巩膜。 <ol><li>切视神经和尝试隔离视网膜与身体接触的远端表面的最小量。视网膜色素上皮将自动从视网膜中的解剖过程大多分离。以避免视网膜的机械干扰比来执行更重要的是夹层很快，一般视网膜可以培养在溶液至少30分钟而对响应特性显著影响。 </li></ol></li><li>安装在试样保持器（ 图1C）的圆顶的视网膜。沉浸在培养皿与解剖视网膜的试样保持器的底部。滑动视网膜，感光体一侧（隔离视网膜的凸面）向上，在圆顶上并提起试样保持器，以使视网膜附着在滤纸上。重复上述步骤为其他视网膜。 </li><li>仔细擦干保持架板，以防止视网膜之间的电串扰，以及噪声和信号分流。试样保持器具有围绕底部圆顶O型环，以防止夹持器的底部和顶部部分之间溶液溢出以及帮助个体视网膜的光感受器和神经节细胞侧的电隔离。用四个螺丝安装支架的顶部部分（参见图1B），并通过用注射器和针填充灌注通道灌注溶液。 </li><li>传送旁边的热交换器区块的试样保持器，并连接在输入和输出灌注线连接到试样保持器（ 图1D）。连接电极以ERG的放大器（顶部电极连接到接地/减销中的放大器），并通过使用一个适配器附着在试样保持器的刺激器/控制单元或滑动加热垫成取决于所述刺激器单元在其上的体内 ERG系统被使用。 </li></ol> 3.录音<ol><li>通过使用在体内系统的软件配置放大器和刺激设置。采集频率设置为1至10千赫和低通值滤波，以300赫兹。不要使用高通滤波。使用60赫兹（或在欧洲50赫兹）如果必要的话陷波滤波器。 </li><li>没有光刺激和机智记录基线ħ暗淡刺激（ 例如 ，绿光的-35 dB或0.3 MCD平方米-2）来测试电极和视网膜样品之间良好的电连接。等待10 - 20分钟在开始收集数据之前，使视网膜温度和功能达到一个稳定的状态。 <ol><li>根据任何具体的实验选择的刺激参数，并开始数据收集。 例如，用绿光从-40到0 dB或从0.1上升到1000 MCD SM -2为暗反应的家庭。使用约2到3个对数单位亮闪烁明视录音哪里棒必须由背景光被抑制（3 - 30 CDM -2）或探针闪光灯（约1.3镉平方米-2 = 1分贝参见图3）。 </li><li>但是，请记住确切的光强值可能会一定程度上取决于你的系统校准和实验条件。作为一个经验法则，规模强度下降了5 - 10倍相比， 在体内 </eM&gt;使用绿灯时17录音。黑色覆盖有孔上面的视网膜（包含在商用适配器系统）可用于以减少光散射在样品支架和由商业体内系统的Ganzfeld球体同时促进视网膜的均匀和相等的刺激。 注:对于记录从暗适应视网膜中，在典型的实验漂白颜料只有微不足道的分数使用的测试闪烁的强度以使缺少RPE驱动再生的是不是一个问题。此外，先前已显示，锥体颜料再生仍然可以通过米勒细胞视觉周期20发生在隔离视网膜。 </li></ol></li><li>作为实验持续典型地从30分钟到几个小时，监测基线漂移，噪声水平和响应稳定性试验期间的变化在这些可能表示的技术问题。 注:噪音的增加或减少D响应幅度可以指示气泡在灌注或电极通道，过低或过高的温度，溶液泄漏/溢出或视网膜的位移。 </li></ol> 4.清洗<ol><li>从灌注线取下样品支架，打开它，并从滤纸冲洗的视网膜。取出电极，并用蒸馏水（不要使用乙醇）冲洗。清洁试样保持器（包括灌注通道）与乙醇和/或蒸馏水。同花顺灌注管小心地用&gt; 70％的乙醇。 </li><li>冲洗瓶灌注用蒸馏水（不要使用清洁剂）。乙醇也可以用来清洗瓶子。 </li></ol>

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圣路易斯华盛顿大学与Xenotec，Inc.的许可协议，并可以从出售体外适配器收到版税。

<html>我们在这里表明，用于通过使用在体内的ERG系统组件与离体的ERG适配器一起同时获得高品质的离体的ERG记录从两个分离的小鼠视网膜的关键步骤。在此研究中，我们灌注既视网膜从具有相同的溶液（或者艾姆斯，洛克的或林格氏）的动物，但它也可以将灌注每个视网膜与不同的溶液如用于药物测试的目的。为了获得高质量的数据，最重要的步骤是从电磁噪声，小心剥离视网膜，平稳较快灌注流量通过使用先进的定制试样架，并进行下昏暗的红色（或IR）所有样品制备程序屏蔽光。这里介绍的方法可以立即使用这两个视网膜和双重用途的体内 ERG设置的在体内和体外 ERG录音执行。 _content"&gt;的定制体外标本的ERG最近由美国17设计的，而现在市售的持有者，提高信噪比的近端和视网膜的远端部分的有效电气隔离和优化上面的视网膜（高解汇率灌注流量）。在离体的ERG信号慢频率噪声分量缺失允许定量分析即使从非常小的反应然而，我们发现， 体外记录更容易发生交流电源线噪声（60赫兹，在美国的干扰; 50赫兹在欧洲）比体内实验。这种噪声耦合到信号主要通过灌注线，并且它可以通过屏蔽（和接地）所有灌注部件（瓶，管）驻留在法拉第笼或Ganzfeld球体之外大多除去此外，有时60 Hz的噪声耦合到离体的ERG信号通过热交换器和该噪声还可以通过接地除去。&lt;/ P&gt; 我们展示如何通过添加药理学阻断剂进入灌注实验中，允许不同类型的细胞在相同的视网膜/实验的功能解剖有三个不同的生理灌注介质（ 图2）中以除去特定的ERG信号分量。最近的一项研究表明，灌注介质的选择会影响在单细胞记录32感光生理学。在这里，我们使用艾姆斯，洛克的和"的HEPES-林格氏介质来记录的情况下，并打算隔离ERG的信号（ 图2）的感光体组件的药理学试剂的存在下暗适应闪光响应。碳酸氢盐缓冲溶液，得到更大的a-和b-波振幅，高达1毫伏。在洛克的介质阻滞剂感光昏暗闪光答复中包含复杂的恢复波形（ 见图2D的插图），这是没有看到与AmesR17;或林格灌注。当使用离体的ERG的变得由多个实验室适于这将是有帮助的，以使用相同的灌注介质和标准方法来分离不同的信号分量。在这一点上看来，最通用的选择是艾姆斯媒体，因为它提供了稳定的大A和B波振幅。另外，感光体的反应，分离的药理学上在该溶液中，似乎有一个简单的波形让人联想到，从单个光感受器（ 图2E）记录。然而，一些开放的问题仍然在体内条件下观察到的其他ERG信号成分的存在。例如，在我们的体外拍摄条件，我们没有看到突出振荡电位，100 -这通常是在b波的体内的ERG响应的上升阶段观察到150赫兹振荡波。因此，有可能在我们的体外视网膜内层功能</eM&gt;的条件被破坏，虽然大型体外 B-波牵连可行的双极细胞的功能。今后的研究应解决是否在此处显示的实验方案的修改（夹层，灌注等）将允许我们记录体外条件下振荡电位。 锥的功能是我们的愿景是至关重要的。然而，锥调查是由它们的小尺寸和稀缺性特别是在小鼠视网膜受阻33。锥功能的分离更加复杂的鼠标ERG的录音，因为他们的M-锥和棒具有几乎相同的光谱灵敏度30。标准协议利用杆和视锥敏感的几个日志单位的差异用杆抑制背景光。然而，稳定的背景光被称为脱敏杆34,35以及通过杆和视锥细胞之间的缝隙连接的耦合调制可能影响锥功能&lt;SUP&gt; 36,37。因此，很难找到一个背景光的强度是足够明亮，保持在不影响锥饱和棒。在这里，我们证明替代双闪方法，它的两个较低的灵敏度和视锥细胞19,29,30的恢复快动力学优势。以这种方式很容易分离真正暗适应锥介导的应答。我们注意到，在艾姆斯或洛克的解决方案，没有任何阻滞，恢复波形的细节有些实验通过使用明亮的探头和闪烁的测试过程中受到影响。这个复杂的减法分析以分离锥的响应。但是，除去由钡的胶质成分有助于稳定，表明变性是由于米勒细胞成分的响应的尾部。以这种方式，有可能得到在野生型小鼠（ 图3）暗适应锥驱动的响应。锥反应分离双闪相比，这些通过使用背景光以抑制杆活动17,37来自WT小鼠的记录，来自WT小鼠的技术出现小。这种差异可以通过背景光的良好表征的效果，即慢慢（10分钟内）提高锥体响应的振幅可能由于移除锥体响应37-39的杆介导的抑制的进行说明。 总之，这里展示的方法使得有可能离体的电生理记录，研究视网膜的功能。在未来，我们希望有更多的实验室将适应这一研究动物和人类视网膜的生理和病理有效的方法，并推动我们的视网膜功能的理解和致盲疾病开发出更好的治疗方法。

电图（ERG）是一种公认​​的技术，可以用来记录由光引发的视网膜的电活动。 ERG的信号由引起径向电流的电压变化（沿感光细胞和双极细胞的轴）中流动的视网膜的电阻外空间，主要产生的。第一ERG信号是从的鱼眼1表面记录在1865年霍姆格伦。艾因特霍芬和乔利1908除以2的ERG响应于光的发作分为三个不同的波，称为A，B，和c波，现在已知以反映感光体的主要的活性，ON双极细胞，和色素上皮细胞，分别3-8。 ERG可以从麻醉动物或人类（ 体内 ）的眼睛被记录时，从分离的眼制剂9，穿过分离完整视网膜（ 离体 ）3,10-15或跨越特定视网膜层与微电极（本地ERG）4,16。在这些中， 在体内 ERG是目前最广泛使用的方法，以评估视网膜的功能。它是一种非侵入性的技术，可用于诊断目的或跟随在动物或患者的视网膜疾病的进展。然而， 在体内 ERG录音制作一个复杂的信号，层峦叠成分，往往是由生理性眼外噪音（ 如呼吸和心脏活动）污染。 本地ERG可用于记录整个视网膜的特定层的信号，但它是最侵入性和具有最低信噪比（SNR），为相对于其他的ERG记录的配置。本地ERG也是技术要求高，需要昂贵的设备（ 如，显微镜和显微操作）。 Transretinal ERG从完整的，孤立的视网膜（ 体外 ERG）提供了在体内和局部ERG方法之间的妥协，允许稳定HIG^ h SNR录音来自动物或人17完整的视网膜。最近，这种方法已成功地用于研究哺乳动物，灵长类动物和人类视网膜18-20杆和视锥感光功能。另外，由于不存在在体外视网膜色素上皮，视网膜电图信号的正C波分量被除去，一个突出的负慢PIII成分显露在体外的录音。缓慢PIII组件已经显示从米勒胶质细胞的活性，以发起在视网膜21-23。因此，也可以采用离体的ERG的方法来研究Müller细胞在完整视网膜。几项研究也表明， 体外 ERG记录可以用来测量药剂浓度周围视网膜24和 ​​测试药物25-27的安全性和有效性。 在体内系统中的多个商业可用和许多实验室不一定具有广泛的背景电使用。相比之下， 体外设备还没有着落直到最近17并因此只有极少数的实验室目前正在这个强大的技术优势。这将是有益的，使提供给更多的实验室体外 ERG录音，以推进我们对视网膜的生理和病理知识，并开发新的疗法致盲疾病。我们在这里展示了一个简单而经济实惠的体外 ERG装置17，并显示如何可以组合使用几种市售体内的ERG系统来记录棒状和锥形介导的信号（a-和b-波）和功能Müller细胞（慢PIII）从完整的野生型小鼠视网膜。

<table><tbody><tr><td>&lt;i&gt;In vivo&lt;/i&gt; ERG system</td><td>OcuScience</td><td>HMsERG</td><td>&lt;a href=&quot;http://www.ocuscience.us/id77.html&quot;&gt;www.ocuscience.us/id77.html&lt;/a&gt;</td></tr><tr><td>&lt;i&gt;In vivo&lt;/i&gt; ERG system</td><td>LKC Technologies</td><td>UTAS-E 3000</td><td>&lt;a href=&quot;http://www.lkc.com/products/UTAS/bigshot.html&quot;&gt;www.lkc.com/products/UTAS/bigshot.html&lt;/a&gt;</td></tr><tr><td>&lt;i&gt;Ex vivo&lt;/i&gt; adapter</td><td>OcuScience</td><td>Ex VIVO ERG adapter</td><td>&lt;a href=&quot;http://www.ocuscience.us/id107.html&quot;&gt;www.ocuscience.us/id107.html&lt;/a&gt;</td></tr><tr><td>Dissection microscope</td><td>North Central Instruments</td><td>Leica M80</td><td>&lt;a href=&quot;http://www.ncimicro.com/store/ListProduct.asp?bSub=T&amp;amp;idCategory=27&quot;&gt;May use any brand&lt;/a&gt;</td></tr><tr><td>IR emitter</td><td>Opto Diode Corp.</td><td>OD-50L</td><td>&lt;a href=&quot;http://www.optodiode.com/&quot;&gt;www.optodiode.com&lt;/a&gt;</td></tr><tr><td>Prowler Night Vision Scopes</td><td>B.E. Meyers Electro Optics</td><td>D4300-I</td><td>Military grade product.</td></tr><tr><td>Red filter</td><td>Rosco Laboratories</td><td>Roscolux #27 Medium Red</td><td>May be used instead of IR system</td></tr><tr><td>Red head light</td><td>OcuScience</td><td>ERGX011</td><td>&lt;a href=&quot;http://www.ocuscience.us/catalog/i29.html&quot;&gt;www.ocuscience.us/catalog/i29.html&lt;/a&gt;</td></tr><tr><td>Microscissors</td><td>WPI, Inc.</td><td>500086</td><td>&lt;a href=&quot;http://www.wpiinc.com/&quot;&gt;www.wpiinc.com/&lt;/a&gt;</td></tr><tr><td>Dumont tweezers #5</td><td>WPI, Inc.</td><td>14101</td><td></td></tr><tr><td>Razor blades</td><td>Electron Microscopy Sciences</td><td>72000</td><td>&lt;a href=&quot;http://www.emsdiasum.com/&quot;&gt;www.emsdiasum.com&lt;/a&gt;</td></tr><tr><td>Scale</td><td>Metler Toledo</td><td>AB54-S/FACT</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/&quot;&gt;May use any brand&lt;/a&gt;</td></tr><tr><td>pH meter and electrode</td><td>Beckman Coulter</td><td>pHI 350</td><td>May use any brand</td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S7653</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/&quot;&gt;May use any brand&lt;/a&gt;</td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>60129</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/&quot;&gt;May use any brand&lt;/a&gt;</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>63020</td><td>1.0 M solution</td></tr><tr><td>CaCl2</td><td>Sigma-Aldrich</td><td>21114</td><td>1.0 M solution</td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>431788</td><td>May use any brand</td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td>May use any brand</td></tr><tr><td>Sodium Bicarbonate</td><td>Sigma-Aldrich</td><td>S6297</td><td>May use any brand</td></tr><tr><td>Ames medium</td><td>Sigma-Aldrich</td><td>A1420</td><td>May use any brand</td></tr><tr><td>BaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>B0750</td><td>May use any brand</td></tr><tr><td>DL-AP4</td><td>Tocris Bioscience</td><td>101</td><td>May use any brand</td></tr><tr><td>Succinic acid disodium salt</td><td>Sigma-Aldrich</td><td>224731</td><td>May use any brand</td></tr><tr><td>L-Glutamic acid</td><td>Sigma-Aldrich</td><td>G2834</td><td>May use any brand</td></tr><tr><td>D-(+)-Glucose</td><td>Sigma-Aldrich</td><td>G7528</td><td>May use any brand</td></tr><tr><td>Leibovitz culture medium L-15</td><td>Sigma-Aldrich</td><td>L4386</td><td>May use any brand</td></tr><tr><td>MEM vitamins</td><td>Sigma-Aldrich</td><td>M6895</td><td></td></tr><tr><td>MEM amino acids</td><td>Sigma-Aldrich</td><td>M5550</td><td></td></tr><tr><td>Carbogen</td><td>Airgas</td><td>UN3156</td><td>5% CO&lt;sub&gt;2&lt;/sub&gt;</td></tr></tbody></table>

simultaneous ex vivo functional testing of two retinas by in vivo electroretinogram system

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

我们记录从暗适应的野生型闪光灯反应（WT）的C57BL / 6小鼠的视网膜通过以下通过使用不同的标准灌注液（ 图2）如上所述和在图1中所示的实验方案。的响应波形和动力学以及视杆细胞的灵敏度出现在埃姆斯&#39;和洛克的媒体（ 图2A和B）类似。另一方面，在HEPES缓冲的Ringer溶液（无碳酸氢盐或5％的CO 2/95％O 2）的反应振幅均显著小。我们还发现，在这些条件下b波稳定性受到损害。加入40μMAPB（DL-AP4）在所有三个媒体（ 图2D-F）有效去除积极b波。去除b波透露了大量慢负波（ 图2D），已被归结为米勒细胞活性21。添加100＃956;钡并购废除了这个组件，露出体外 ERG信号（ 图2D-F）的感光响应。我们可以记录长达埃姆斯与洛克的1 mV的饱和感光反应，而最大的反应是典型的下林格灌注大约200μV。 锥感光响应先前已经分离，其中一个明亮的闪光探测器饱和杆之后是测试闪光的时候锥已经恢复其暗适应状态，但仍然棒饱和19,29所谓的双闪技术。在这里，我们分离锥介导的ERG响应（同时含有α-和b波）在补充有100μM的钡，但不DL-AP4艾姆斯媒体通过使用双闪技术（ 图3）。钡被用来除去这似乎使反应更加可变的后期部分，尤其是在重复使用brigh缓慢胶质成分牛逼闪烁。我们用一个恒定的探头闪300毫秒探头后闪光饱和棒和可变测试闪烁，引起锥响应。减去从&#39;探针+测试闪光的反应探测闪光响应得到的锥体响应家庭示于图3B。 <img alt="图1" src="/files/ftp_upload/52855/52855fig1.jpg" /> 电极通道的图1，使用离体的ERG样品架。（A）的填充和组装的试样保持器的清扫通过测量电极对之间的电阻和电压之前安装的电极。（B）测试。（℃）安装连接到灌注线和ERG放大器在商业ERG学年的试样保持器的对中的试样保持器的滤纸视网膜。（D）的干。灌注流动路径由蓝色箭头表示。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52855/52855fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/52855/52855fig2.jpg" /> 从灌注洛克的（A）暗适应WT小鼠视网膜，艾姆斯（B），和HEPES缓冲林格（C）中记录图2.闪存响应的家庭。该闪烁交付3，40，130，390 1,400光子微米-2（从-36至-9 dB或-3.6 -0.9到日志（CD SM -2）绿光（530纳米））。黑色的痕迹，除了40微米APB和100μM钡从后在视网膜（AC）记录（DF）显示响应。从视网膜的记录（D）显示红色的反应痕迹（A）<html>灌注洛克的添加40μM建业，但不是钡。插图显示的响应三个最暗的闪烁含有APB和钡洛克的媒体。该闪烁7〜14000光子微米-2（从-32到1dB绿灯）（D）不等，从3至1400光子微米-2（从-36〜-9分贝绿光）的（E），并从7至1400光子微米-2（从-32至-9 dB的绿光）中（F）。见Vinberg 等人2014 17和Lyubarsky 等人 1999年第30和2004年31转换镉SM给明视光能量-2光子微米-2的详细信息。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52855/52855fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 负载/ 52855 / 52855fig3.jpg"/&gt; 图3.隔离与WT鼠标双击闪光法锥响应。（A）响应探测闪光灯（14000光子微米-21分贝绿灯，黑色）和响应探测闪光随后进行测试闪光（81000光子微米-2或9分贝绿色光，红色）。（B）的锥闪光灯的响应以测试分离减去探针闪光响应闪烁范围从360至81000的光子微米-2（-14至10dB绿光）"探头+测试闪光"的回应。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52855/52855fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System at JoVE.com

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

同时<em&gt;体外</em两个由视网膜&gt;功能测试<em&gt;在体内</em&gt;电图系统

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

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Universidad San Sebastian

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JoVE Journal

Neuroscience

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

9.1K 次观看.  Washington University in St. Louis. Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

视频: Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Multifocal Electroretinograms

A limitation of traditional full-field electroretinograms (ERG) for the diagnosis of retinopathy is lack of sensitivity. Generally, ERG results are normal unless more than approximately 20% of the retina is affected. In practical terms, a patient might be legally blind as a result of macular degeneration or other scotomas and still appear normal, according to traditional full field ERG. An important development in ERGs is the multifocal ERG (mfERG). Erich Sutter adapted the mathematical sequences called binary m-sequences enabling the isolation from a single electrical signal an electroretinogram representing less than each square millimeter of retina in response to a visual stimulus1.
Results that are generated by mfERG appear similar to those generated by flash ERG. In contrast to flash ERG, which best generates data appropriate for whole-eye disorders. The basic mfERG result is based on the calculated mathematical average of an approximation of the positive deflection component of traditional ERG response, known as the b-wave1. Multifocal ERG programs measure electrical activity from more than a hundred retinal areas per eye, in a few minutes. The enhanced spatial resolution enables scotomas and retinal dysfunction to be mapped and quantified.
In the protocol below, we describe the recording of mfERGs using a bipolar speculum contact lens.
Components of mfERG systems vary between manufacturers. For the presentation of visible stimulus, some suitable CRT monitors are available but most systems have adopted the use of flat-panel liquid crystal displays (LCD). The visual stimuli depicted here, were produced by a LCD microdisplay subtending 35 - 40 degrees horizontally and 30 - 35 degrees vertically of visual field, and calibrated to produce multifocal flash intensities of 2.7 cd s m-2. Amplification was 50K. Lower and upper bandpass limits were 10 and 300 Hz. The software packages used were VERIS versions 5 and 6.

The development of the multifocal electroretinogram (mfERG) is an important advance in the diagnosis and characterization of retinopathy. Multifocal electroretinograms are a mathematical average of an approximation of a b-wave. Software programs can derive ERGs from more than a hundred retinal areas in a few minutes per eye. Scotomas and retinal dysfunction can be mapped and quantified.

多焦视网膜电

A limitation of traditional full-field electroretinograms (ERG) for the diagnosis of retinopathy is lack of sensitivity.  Generally, ERG results are ...

科研

医学

Transretinal ERG Recordings from Mouse Retina: Rod and Cone Photoresponses

There are two distinct classes of image-forming photoreceptors in the vertebrate retina: rods and cones. Rods are able to detect single photons of light whereas cones operate continuously under rapidly changing bright light conditions. Absorption of light by rod- and cone-specific visual pigments in the outer segments of photoreceptors triggers a phototransduction cascade that eventually leads to closure of cyclic nucleotide-gated channels on the plasma membrane and cell hyperpolarization. This light-induced change in membrane current and potential can be registered as a photoresponse, by either classical suction electrode recording technique1,2 or by transretinal electroretinogram recordings (ERG) from isolated retinas with pharmacologically blocked postsynaptic response components3-5. The latter method allows drug-accessible long-lasting recordings from mouse photoreceptors and is particularly useful for obtaining stable photoresponses from the scarce and fragile mouse cones. In the case of cones, such experiments can be performed both in dark-adapted conditions and following intense illumination that bleaches essentially all visual pigment, to monitor the process of cone photosensitivity recovery during dark adaptation6,7. In this video, we will show how to perform rod- and M/L-cone-driven transretinal recordings from dark-adapted mouse retina. Rod recordings will be carried out using retina of wild type (C57Bl/6) mice. For simplicity, cone recordings will be obtained from genetically modified rod transducin &alpha;-subunit knockout (T&alpha;-/-) mice which lack rod signaling8.

We describe a relatively simple method of transretinal electroretinogram (ERG) recordings for obtaining rod and cone photoresponses from intact mouse retina. This approach takes advantage of the block of synaptic transmission from photoreceptors to isolate their light responses and record them using field electrodes placed across the isolated flat-mounted retina.

transretinal ERG的录音，从小鼠视网膜杆和锥光反应

There are two distinct classes of image-forming photoreceptors in the vertebrate retina: rods and cones. Rods are able to detect single photons of light ...

神经科学

Electrophysiological Characterization of GFP-Expressing Cell Populations in the Intact Retina

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack conspicuous morphological features or show a low population density. This applies particularly to retinal amacrine cells, an exceptionally multiform class of interneurons that comprise roughly 30 subtypes in mammals1. Though being a crucial part of the visual processing by shaping the retinal output2, most of these subtypes have not been studied up to now in a functional context because encountering these cells with a recording electrode is a rare event.
Recently, a multitude of transgenic mouse lines is available that express fluorescent markers like green fluorescent protein (GFP) under the control of promoters for membrane receptors or enzymes that are specific to only a subset of neurons in a given tissue3,4. These pre-labeled cells are therefore accessible to directed microelectrode targeting under microscopic control, permitting the systematic study of their physiological properties in situ. However, excitation of fluorescent markers is accompanied by the risk of phototoxicity for the living tissue. In the retina, this approach is additionally hampered by the problem that excitation light causes appropriate stimulation of the photoreceptors, thus inflicting photopigment bleaching and transferring the retinal circuits into a light-adapted condition. These drawbacks are overcome by using infrared excitation delivered by a mode-locked laser in short pulses of the femtosecond range. Two-photon excitation provides energy sufficient for fluorophore excitation and at the same time restricts the excitation to a small tissue volume minimizing the hazards of photodamage5. Also, it leaves the retina responsive to visual stimuli since infrared light (&gt;850 nm) is only poorly absorbed by photopigments6.
In this article we demonstrate the use of a transgenic mouse retina to attain electrophysiological in situ recordings from GFP-expressing cells that are visually targeted by two-photon excitation. The retina is prepared and maintained in darkness and can be subjected to optical stimuli which are projected through the condenser of the microscope (Figure 1). Patch-clamp recording of light responses can be combined with dye filling to reveal the morphology and to check for gap junction-mediated dye coupling to neighboring cells, so that the target cell can by studied on different experimental levels.

This article depicts the recording of individual cells from fluorescently tagged neuronal populations in the intact mouse retina. By using two-photon infrared excitation transgenetically labeled cells were targeted for patch-clamp recording to study their light responses, receptive field properties, and morphology.

在完整的视网膜电生理特性表达GFP的细胞群

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack ...

Direct-Coupled Electroretinogram (DC-ERG) for Recording the Light-Evoked Electrical Responses of the Mouse Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is a specialized monolayer of cells strategically located between the retina and the choriocapillaris that maintain the overall health and structural integrity of the photoreceptors. The RPE is polarized, exhibiting apically and basally located receptors or channels, and performs vectoral transport of water, ions, metabolites, and secretes several cytokines.
In vivo noninvasive measurements of RPE function can be made using direct-coupled ERGs (DC-ERGs). The methodology behind the DC-ERG was pioneered by Marmorstein, Peachey, and colleagues using a custom-built stimulation recording system and later demonstrated using a commercially available system. The DC-ERG technique uses glass capillaries filled with Hank&#8217;s buffered salt solution (HBSS) to measure the slower electrical responses of the RPE elicited from light-evoked concentration changes in the subretinal space due to photoreceptor activity. The prolonged light stimulus and length of the DC-ERG recording make it vulnerable to drift and noise resulting in a low yield of useable recordings. Here, we present a fast, reliable method for improving the stability of the recordings while reducing noise by using vacuum pressure to reduce/eliminate bubbles that result from outgassing of the HBSS and electrode holder. Additionally, power line artifacts are attenuated using a voltage regulator/power conditioner. We include the necessary light stimulation protocols for a commercially available ERG system as well as scripts for analysis of the DC-ERG components: c-wave, fast oscillation, light peak, and off response. Due to the improved ease of recordings and rapid analysis workflow, this simplified protocol is particularly useful in measuring age-related changes in RPE function, disease progression, and in the assessment of pharmacological intervention.

Direct-Coupled Electroretinogram DC-ERG for Recording the Light-Evoked Electrical Responses of the Mouse Retinal Pigment Epithelium

Here, we present a method for recording light-evoked electrical responses of the retinal pigment epithelium (RPE) in mice using a technique known as DC-ERGs first described by Marmorstein, Peachey, and colleagues in the early 2000s.

直接耦合电雷电图（DC-ERG），用于记录小鼠视网膜颜料皮皮的光唤起电响应

The retinal pigment epithelium (RPE) is a specialized monolayer of cells strategically located between the retina and the choriocapillaris that maintain ...

化学

Using the Electroretinogram to Assess Function in the Rodent Retina and the Protective Effects of Remote Limb Ischemic Preconditioning

The ERG is the sum of all retinal activity. The ERG is usually recorded from the cornea, which acts as an antenna that collects and sums signals from the retina. The ERG is a sensitive measure of changes in retinal function that are pan-retinal, but is less effective for detecting damage confined to a small area of retina. In the present work we describe how to record the &#8216;flash&#8217; ERG, which is the potential generated when the retina is exposed to a brief light flash. We describe methods of anaesthesia, mydriasis and corneal management during recording; how to keep the retina dark adapted; electrode materials and placement; the range and calibration of stimulus energy; recording parameters and the extraction of data. We also describe a method of inducing ischemia in one limb, and how to use the ERG to assess the effects of this remote-from-the-retina ischemia on retinal function after light damage. A two-flash protocol is described which allows isolation of the cone-driven component of the dark-adapted ERG, and thereby the separation of the rod and cone components. Because it can be recorded with techniques that are minimally invasive, the ERG has been widely used in studies of the physiology, pharmacology and toxicology of the retina. We describe one example of this usefulness, in which the ERG is used to assess the function of the light-damaged retina, with and without a neuroprotective intervention; preconditioning by remote ischemia.

The electroretinogram (ERG) is an electrical potential generated by the retina in response to light. This paper describes how to use the ERG to assess retinal function, in dark-adapted rats, and how it can be can be used to assess a neuroprotective intervention, in the present case remote ischemic preconditioning.

使用电生理评估功能，在啮齿动物的视网膜和远程肢体缺血预适应的保护作用

The ERG is the sum of all retinal activity. The ERG is usually recorded from the cornea, which acts as an antenna that collects and sums signals from the ...

Simultaneous Recording of Electroretinography and Visual Evoked Potentials in Anesthetized Rats

The electroretinogram (ERG) and visual evoked potential (VEP) are commonly used to assess the integrity of the visual pathway. The ERG measures the electrical responses of the retina to light stimulation, while the VEP measures the corresponding functional integrity of the visual pathways from the retina to the primary visual cortex following the same light event. The ERG waveform can be broken down into components that reflect responses from different retinal neuronal and glial cell classes. The early components of the VEP waveform represent the integrity of the optic nerve and higher cortical centers. These recordings can be conducted in isolation or together, depending on the application. The methodology described in this paper allows simultaneous assessment of retinal and cortical visual evoked electrophysiology from both eyes and both hemispheres. This is a useful way to more comprehensively assess retinal function and the upstream effects that changes in retinal function can have on visual evoked cortical function.

This protocol describes simultaneous measurement of electroretinogram and visual evoked potentials in anesthetized rats.

电图同时记录和麻醉大鼠视觉诱发电位

The electroretinogram (ERG) and visual evoked potential (VEP) are commonly used to assess the integrity of the visual pathway. The ERG measures the ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

小鼠视网膜的经瞳双光子体内成像

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...

Optimizing the Setup and Conditions for Ex Vivo Electroretinogram to Study Retina Function in Small and Large Eyes

Measurements of retinal neuronal light responses are critical to investigating the physiology of the healthy retina, determining pathological changes in retinal diseases, and testing therapeutic interventions. The ex vivo electroretinogram (ERG) allows the quantification of contributions from individual cell types in the isolated retina by addition of specific pharmacological agents and evaluation of tissue-intrinsic changes independently of systemic influences. Retinal light responses can be measured using a specialized ex vivo ERG specimen holder and recording setup, modified from existing patch clamp or microelectrode array equipment. Particularly, the study of ON-bipolar cells, but also of photoreceptors, has been hampered by the slow but progressive decline of light responses in the ex vivo ERG over time. Increased perfusion speed and adjustment of the perfusate temperature improve ex vivo retinal function and maximize response amplitude and stability. The ex vivo ERG uniquely allows the study of individual retinal neuronal cell types. In addition, improvements to maximize response amplitudes and stability allow the investigation of light responses in retina samples from large animals, as well as human donor eyes, making the ex vivo ERG a valuable addition to the repertoire of techniques used to investigate retinal function.

Optimizing the Setup and Conditions for Ex Vivo Electroretinogram to Study Retina Function in Small and Large Eyes

Modification of existing multielectrode array or patch clamp equipment makes the ex vivo electroretinogram more widely accessible. Improved methods to record and maintain ex vivo light responses facilitate the study of photoreceptor and ON-bipolar cell function in the healthy retina, animal models of eye diseases, and human donor retinas.

优化离 体 视网膜电图的设置和条件，以研究小眼睛和大眼睛的视网膜功能

Measurements of retinal neuronal light responses are critical to investigating the physiology of the healthy retina, determining pathological changes in ...

Retinal Explant of the Adult Mouse Retina as an Ex Vivo Model for Studying Retinal Neurovascular Diseases

One of the challenges in retina research is studying the cross-talk between different retinal cells such as retinal neurons, glial cells, and vascular cells. Isolating, culturing, and sustaining retinal neurons in vitro have technical and biological limitations. Culturing retinal explants may overcome these limitations and offer a unique ex vivo model to study the cross-talk between various retinal cells with well-controlled biochemical parameters and independent of the vascular system. Moreover, retinal explants are an effective screening tool for studying novel pharmacological interventions in various retinal vascular and neurodegenerative diseases such as diabetic retinopathy. Here, we describe a detailed protocol for retinal explants' isolation and culture for an extended period. The manuscript also presents some of the technical problems during this procedure that may affect the desired outcomes and reproducibility of the retinal explant culture. The immunostaining of the retinal vessels, glial cells, and neurons demonstrated intact retinal capillaries and neuroglial cells after 2 weeks from the beginning of the retinal explant culture. This establishes retinal explants as a reliable tool for studying changes in the retinal vasculature and neuroglial cells under conditions that mimic retinal diseases such as diabetic retinopathy.

Retinal Explant of the Adult Mouse Retina as an Ex Vivo Model for Studying Retinal Neurovascular Diseases

This protocol presents and describes steps for the isolation, dissection, culturing, and staining of retinal explants obtained from an adult mouse. This method is beneficial as an ex vivo model for studying different retinal neurovascular diseases such as diabetic retinopathy.

成年小鼠视网膜的视网膜外植体作为研究视网膜神经血管疾病的离 体 模型

One of the challenges in retina research is studying the cross-talk between different retinal cells such as retinal neurons, glial cells, and vascular ...

Two-Photon In Vivo Imaging of the Mouse Retina

<a href='https://app-jove-com.bdigitaluss.remotexs.co/v/61970/transpupillary-two-photon-in-vivo-imaging-of-the-mouse-retina'>Source: Wang, Z., et al.&#160;Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina. J. Vis. Exp.&#160;(2021).</a>This video demonstrates a technique for in vivo retinal imaging using two-photon microscopy. Anesthetized mice are prepared for imaging by securing their heads, Applying eye lubricant, and placing a coverslip over the eye. Retinal ganglion cells (RGCs) expressing fluorescent proteins are first visualized using epifluorescence for alignment. Finally, two-photon imaging is employed, allowing for high-resolution visualization of RGCs.

Source: Wang, Z., et al.&#160;Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina. J. Vis. Exp.&#160;(2021).This video demonstrates a technique ...

同时

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探索更多视频

此视频中的章节

多焦视网膜电

transretinal ERG的录音，从小鼠视网膜杆和锥光反应

在完整的视网膜电生理特性表达GFP的细胞群

直接耦合电雷电图（DC-ERG），用于记录小鼠视网膜颜料皮皮的光唤起电响应

使用电生理评估功能，在啮齿动物的视网膜和远程肢体缺血预适应的保护作用

电图同时记录和麻醉大鼠视觉诱发电位

小鼠视网膜的经瞳双光子体内成像

优化离体视网膜电图的设置和条件，以研究小眼睛和大眼睛的视网膜功能

成年小鼠视网膜的视网膜外植体作为研究视网膜神经血管疾病的离体模型

Two-Photon In Vivo Imaging of the Mouse Retina

同时

摘要

探索更多视频

此视频中的章节

多焦视网膜电

transretinal ERG的录音，从小鼠视网膜杆和锥光反应

在完整的视网膜电生理特性表达GFP的细胞群

直接耦合电雷电图（DC-ERG），用于记录小鼠视网膜颜料皮皮的光唤起电响应

使用电生理评估功能，在啮齿动物的视网膜和远程肢体缺血预适应的保护作用

电图同时记录和麻醉大鼠视觉诱发电位

小鼠视网膜的经瞳双光子体内成像

优化离 体 视网膜电图的设置和条件，以研究小眼睛和大眼睛的视网膜功能

成年小鼠视网膜的视网膜外植体作为研究视网膜神经血管疾病的离 体 模型

Two-Photon In Vivo Imaging of the Mouse Retina

优化离体视网膜电图的设置和条件，以研究小眼睛和大眼睛的视网膜功能

成年小鼠视网膜的视网膜外植体作为研究视网膜神经血管疾病的离体模型