这项研究的实验部分得到美国以色列双国科学基金会（BM和IL），以色列科学基金会（ISF），德语以色列项目（DIP）（BM）和生物技术和生物科学研究委员会（BBSRC授权号:BB / M007006 / 1和BB / D007585 / 1）

试剂准备注意:按照表1-4中的说明准备所有解决方案。 <ol><li>向细胞外溶液（ES或ES-0Ca 2+ ;根据需要）加入10 mL注射器， 见表1 ，并保存在冰上。 </li><li>准备一瓶研磨溶液（TS; 参见表2 ; 即 ES或ES-0Ca 2+ + FBS和蔗糖），并将其保存在冰上。 </li><li>为实验准备足够的ES，并将其保存在冰上，直到需要。 注意:不需要连续浴灌注，因此ES的体积不需要超过几十mL。 </li><li>使用22μm的PVDF过滤器，将细胞内溶液（ 见表3或表4 ）装入1 mL注射器，电极填充细长的尖端。保持在冰上。 </li></ol> 2.解剖工具的一般设置 图1:制备分离的Ommatidia制备所需的工具和设备。照片显示了创建孤立的ommatidia制剂所需的各种装置，如上述详细协议所述。两个烧杯，一个充满水（ A ），另一个填充有乙醇（ B ），用于清洁连接到管道（ C ）的研磨移液管（ D ）。解剖工具有:2对精细和1对粗镊子（ F ）和视网膜扫描器（ E ）。为了准备视网膜扫描器，在用作凹部（ G ）的两个车刀之间按压微型解剖针（ H ）以使针（ I ）的顶部变平。然后连接到一个细长的pi<html>使用胶水（ J ）并安装在针架（ E ）上。剃刀刀片刀片和刀柄:使用剃须刀刀架（ K ）将剃刀刀片的小三角形刀片拆下并安装。解剖工作区域由具有两个光导（ N ）的双目（ L ）和冷红光源（（ M） ）组成。包括镊子（ F ），视网膜刮刀（ E ），烧杯（ A和B ），带有红色过滤器（ Q ）的手电筒和精致擦拭器（ R ））的夹层工具放置在双目镜的两侧。具有ES，FBS-ES，细胞内溶液注射器，60mm培养皿（ S ）和电极夹（ P ）的冰桶也放置在桌子上。使用水平拉线器（ T ）拉出记录电极。e.com/files/ftp_upload/55627/55627fig1large.jpg"target ="_ blank"&gt;请点击此处查看此图的较大版本。 <ol><li> 构建视网膜扫描仪隔离视网膜。 <ol><li>插入两个虎钳钳口之间的微型解剖针（昆虫针，长12毫米，直径0.1毫米）的点（1-4毫米），并用小锤敲打将其平坦化。 </li><li>将扁平针安装在微解剖针架上（参见材料表 ）。 </li><li>使用一对镊子，使针的扁平端弯曲形成约2.5毫米曲率的钩。 </li></ol></li><li> 创建用于恶臭分离的研磨移液器。 <ol><li>将1.2 x 0.68 mm（OD x ID）玻璃毛细管放置在明火上，并对其进行火焰抛光，以减少其开口。 </li><li>在显微镜下测量毛细管孔的尺寸。将创建的ommatidial研磨移液器排序成seven组根据其开口尺寸（ 即 0.2-0.5毫米）。将它们储存在单独的合适容器（ 例如试管）中。 </li><li>将一个小烧杯用双蒸水（DDW）和70％乙醇的另一个小烧杯填充。用石蜡膜盖住每个烧杯。 </li><li>在覆盖DDW烧杯的石蜡膜片上打一个小孔（ 见图1A ）。 </li><li>在覆盖乙醇烧杯的石蜡膜片上打出七个小孔，并将孔从0到6（ 见图1B ）。 </li><li>在每个石蜡膜孔中放置一个来自每个大小组的小型研磨移液管，使得具有最大开口的移液管位于第0 个孔中，并且具有最小开口的移液管位于第6 个孔中。 </li><li>将塑料200μL移液管接头连接到35厘米长，1.57 x 1.14毫米（OD x ID）的聚乙烯管上。 CONNEC将管道的另一端连接到具有最大开口（ 即 0.46-0.5mm）的一个奥氏体研磨移液管中，确保研磨移液管的尖端不面向管道。 </li></ol></li><li>通过从含有玻璃毛细管的1 x 0.58 mm（OD x ID）硼硅酸盐细丝中拉出膜片钳移液器来准备全细胞记录移液器。 注意:使用葡萄糖酸钾的细胞内溶液（IS1）时，移液器的电阻应为8-15MΩ。可以使用任何合适的补片吸管拔出器（例如，参见材料表 ）。防火抛光不是必需的。 </li><li>通过使用熔融石蜡或高真空硅胶将盖玻片（参见材料表 ）固定到浴室底部来准备记录室（参见图2 ）。使用任何合适的自制或商业室，允许电极进入和灌注。 </li><li>将沐浴放在倒置显微镜的舞台上。将灌注系统管，抽吸系统和研磨（ 即 Ag-AgCl丝/丸）置于浴中（参见材料表和图2 ）。 </li><li>在工作表面放置两对精细＃5镊子（ 图1 ），以便在视网膜剥离和ommatidia分离步骤期间使用。 </li></ol> D.黑腹果蝇饲养<ol><li>在19-24℃下，含有标准玉米面食的瓶子中，高密度的黑麦角酱以低人口密度（ 即 6盎司瓶中约20只苍蝇）飞行。 注意:最好在黑暗适应的苍蝇上工作。为了保持对光的高度敏感性，减少多样性并防止突变体蝇中的视网膜变性。 </li><li>在黑暗中将苍蝇放在实验前至少24小时。 注意:用于实验的苍蝇应该是接收ntly eclosed（&lt;2 h），仍然柔软，苍白，并显示胎粪。蛹虫草也可以很容易地从蛹制备，尽管它们对光的敏感性随后依赖于10岁。 </li></ol>视网膜切除术和Ommatidia隔离:选项1 注意:在立体放大显微镜下，使用适合正确观察准备的放大图像，执行以下所有步骤（参见图1 ）。 <ol><li>将四滴ES-0Ca 2+和一滴TS溶液置于已经翻转的60-mm培养皿上。 </li><li>使用粗糙的镊子，抓住一个新近的羽毛球（&lt;2小时后），翅膀或身体飞行。从这一点开始，在20±1°C的昏暗的红色照明下快速执行所有程序。 </li><li>在用粗镊子抓住飞行的同时，使用第一对细镊子将飞头从身体上分离出来。次亚头部在第一个ES-0Ca 2+下降。 </li><li>使用第二对精细镊子将头部沿着矢状面解剖一半。确保在此步骤结束时，双眼仍然完好无损。 </li><li>将头部的一半转移到第二个ES-0Ca 2+下降，另一半转移到第三个ES-0Ca 2+下降。 </li><li>使用精细的镊子，尽可能多地去除眼睛周围的组织，并确保不会对视网膜造成伤害。 </li><li>用精细的镊子牢牢抓住一个角膜的边缘，并用扫帚舀出视网膜。 注意:完成此步骤后，角膜将保持空白且完整，与完整的视网膜分离。 </li><li>用DDW冲洗连接到管道的研磨移液管，并从第四滴中用少量的ES-0Ca 2+填充移液管。 注意:每次使用新的分离移液器时，必须执行此步骤乙醇烧杯（填充移液器的溶液应与视网膜浸没的溶液相匹配）。 </li><li>通过口腔轻轻抽出将分离的视网膜吸入移液管。非常小心，不要将气泡吸入移液器。 </li><li>将分离的视网膜转移到TS的一滴。在第二只眼睛执行步骤4.6-4.10。 </li><li>使用精致的抹布擦掉ES-0Ca 2+的滴液，只留下含有两种视网膜的TS滴在培养皿上。再添加六滴TS到培养皿的顶部。将两个视网膜转移到其他TS滴液中。 </li><li>用小直径开口的移液管更换研磨移液器。按照步骤4.8中所述进行冲洗，使用TS作为溶液填充移液器。 </li><li>快速，反复地抽吸并使溶液中的视网膜到期，从而开始从整个视网膜中分离脱色的色素细胞。 注意:孤立的麻将Tidia在TS滴中是可见的，并且随着隔离过程的进行，TS降低变得不透明。 </li><li>将剩余的视网膜转移到下一个TS下降。将整个原始TS液滴（含有隔离的ommatidia）填充到移液管中，并将液体滴入浴室。 </li><li>重复步骤4.12-4.14以实现最大的孤立的ommatidia。等待大约1分钟，以使孤立的凶手沉没并固定在浴室的底部。 </li><li>使用灌注系统，开始使用1.5mM Ca 2+的ES-0Ca 2+流入浴室。确保腔室完全充满溶液，从底部到顶部，并且地面完全浸没在溶液中。继续洗澡4-5x。 </li></ol> 5.视网膜解剖和Ommatidia隔离:选项2 注意:使用放大镜在立体变焦显微镜下执行以下所有步骤适用于正确观察制剂（ 见图1 ）。 <ol><li>准备一个剃须刀片和保持架。使用剃须刀刀架拆下并安装剃刀刀片的小三角形刀片（ 图1 ）。 </li><li>根据制造商的说明准备硅胶夹层/块（见材料表 ）。 </li><li>为了解剖，在硅胶剥离块上产生大量的（0.5mL）ES溶液。向60 mm培养皿中加入TS溶液的两个"储存"液滴（约50μL） </li><li>将冰封上的新近放眼（&lt;2小时后）飞行在玻璃管中，并用镊子将其从翅膀上取下。从这一点开始，在20±1°C的昏暗的红色照明下快速执行所有程序。 </li><li>用镊子握住飞行物，使用安装在支架上的剃刀刀片芯片切断飞头。拿起昆虫针（12毫米长，直径为0.1毫米）与镊子刺穿眼睛之间的头部。 </li><li>将头部淹没在70％乙醇中;这防止在头/眼表面上形成气泡。将头部插入有机硅夹层皿下的ES滴下。 </li><li>通过使用沿着眼睛正面边缘线的锯切运动，使用剃刀刀片切割双眼。 </li><li>用精细的镊子牢牢抓住一个角膜的边缘。 </li><li>使用洗手盆舀出视网膜。 注意:完成此步骤后，角膜将保持空白且完整，与完整的视网膜分离。 </li><li>不破坏视网膜，使用镊子和洗手液轻轻去除附着的气囊和多余的脑组织。 注意:分离的视网膜的制备也可用于非特异性视网膜蛋白11 ，全部组织学和全视网膜成像的Western印迹分析。贴片夹记录也可以在ph整个视网膜的受体12 。 </li><li>取最大直径的研磨移液管，将其连接到管道上，并通过温和吸入（ 即通过口）从培养皿中的一个储液罐中的少量TS回填移液管。每次使用新的ommatidia研磨移液器时，请执行此步骤。 </li><li>轻轻地将TS穿过两个视网膜，然后用温和的抽吸将孤立的视网膜吸入移液管。请注意确保没有气泡进入移液器。 </li><li>将分离的视网膜转移到培养皿中，形成小滴（约20μL），并从其中一个水滴中用TS洗涤一次或两次。 </li><li>在黑暗中孵育视网膜20-25分钟。 </li><li>用小直径开口的移液管（使用来自其中一个储液罐的新鲜TS，如在步骤4.6中回填移液管）更换奥米那迪研磨移液管。 </li><li>急速吸出并以小滴（〜20μL）的方式将视网膜排除，以开始分离孤立的恶心。 注意:在第一阶段，周围的着色胶质细胞会分解，留下可见的小碎屑溶解在溶液中。 </li><li>在积累了大量小碎屑之后，但是在许多口臭分离之前，从其中一个水库滴下使用新鲜的TS，并将视网膜转移到新的小滴。 </li><li>选择较小直径的研磨移液器，回填，并继续研磨。 注意:由于ommatidia现在开始分离，它们的细长形状应该在立体显微镜的高功率下清晰可见。如有必要，请继续将造粒移液器更换成更小的直径，直到看到良好的产品质量</li><li>一旦合理产量的恶臭是可见的，并且液滴不再是半透明的，则将整个滴液装入移液管中，并将其放入包含隔离的油腻剂中，并将液滴轻轻地放入浴室底部预填充ES。 </li><li>等待大约1分钟，让孤立的恶魔堕入沉浴池底部。 </li><li>使用灌注系统，启动ES流入浴室。确保室完全充满溶液，从底部到顶部，并且地面完全浸没在溶液中。继续洗澡4-5x。 注意:此后，不需要连续灌注，尽管在引入新的补片移液器之前，应短暂洗净浴液。 </li></ol> 6.全细胞记录<img alt="图2" src="/files/ftp_upload/55627/55627fig2.jpg" /> 图2:电生理和光学设置概述。该装置包含一个铁黑色法拉第笼（ F ）。前面用黑色的窗帘覆盖着铜网。这个配置使得制剂完全可以与任何杂光隔离，并适合于记录黑暗的自发性隆起物。倒置荧光显微镜（ A ）固定在防振台（ E ）上。感光记录装置由具有灌注输入（ Q ）的自制丙烯酸玻璃浴室（ O ），吸移移液管（ S ）和银 - 氯化银粉底（ P ）组成。浴室用自制适配器安装在显微镜台（ T ）上。记录移液管通过银 - 氯化银线连接到丙烯酸玻璃保持器，其连接到放大器头部台阶（ C ）。然后将头阶段安装在安装在精细XYZ机械显微操纵器（ B ）上的粗糙显微操纵器上。灌注系统（ D ）由注射器组成，而液体流动d由夹紧阀（ H ）控制。机架电连接到与法拉第笼内所有设备连接的相同的中心地，由片钳钳放大器（ N ），示波器（ J ），脉冲/功能发生器（ K ），A至D转换器（ L ），灌注控制器（ I ）以及滤光轮和快门控制器（ M ）。对于成像实验，冷却（-110°C，参见表格材料 ）CCD摄像机通过侧面端口（ G ）连接。 <a href="http://ecsource.jove.com/files/ftp_upload/55627/55627fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> 注意:在以下所有步骤中，只能使用昏暗的红光照明，并确保轻度的光线暴露极少（ 即快速工作和在执行不需要观察小眼痣的任务时，关闭用于观察恶心的解剖光源和室红色照明。另外，根据标准电生理方案执行以下所有步骤。 <ol><li> 在倒置显微镜（40X物镜）下，仔细检查浴缸中的所有o虫，并选择适合的小卵泡进行实验。 <ol><li>确保小眼the的外膜是光滑和完整的，长轴相对于电极接近方向大致成直角（ 如图3A所示 ），并且小眼the的远端部分不被任何多余的组织。将所选的小眼放在物镜的光轴（视野的中心）），以确保均匀的照明。 </li></ol></li><li>用细胞内溶液（IS1或IS2）填充补片移液管。 注意:对于mea确定强度响应和碰撞分析，使用IS1。要测量光敏通道的反向电位，请使用IS2。 </li><li>将补片移液器安装到电极支架上。 </li><li>通过嘴巴吹入移液管，通过连接到电极支架的管道，使其以正压填充。关闭管阀以保持压力。 </li><li>使用显微操纵器将电极插入浴室。 </li><li>引导电极靠近小眼的远端部分，使得电极和小痣之间不存在接触，直到在小痣中可以观察到小的凹坑（由于贴片移液管中的正压力）。 </li><li>打开录音软件（参见表格材料 ）。打开"膜测试模块"，以100 Hz的速率施加2 mV的连续平方电压脉冲。 </li><li>通过调整适当的kno将结电位设置为"零"b在贴片钳放大器中将平方脉冲的基极设置为"零"电流 注意:电生理设置包括连接到放大器（ 即第二级放大）的头阶段（ 即第一级放大）。放大的模拟信号使用A / D转换器转换为数字信号，该转换器由安装在PC计算机上的软件控制。 </li><li>通过打开连接到电极夹的管的阀，释放移液管中的正压。通过从管吸出来轻轻地在移液管中产生负压，导致移液管与细胞膜的结合。关闭管阀以保持压力。 </li><li>确保在电脑屏幕上看到的电极电阻升高到100 - 150MΩ。通过手动打开连接到电极夹的管的阀，释放移液管中的负压。 </li><li>确保el电极电阻升高至至少1-2GΩ。 注意:此时，已经在电极和感光体之间形成了密封。 </li><li>通过调整贴片钳放大器中的适当旋钮来抵消移液器的电容电流。 </li><li>在电极中产生快速，短暂和有力的负压开环，通过口连接到电极夹持器的管嘴吸入"破裂"到感光膜中并产生全细胞构型。或者，使用"Zap按钮"来应用短的矩形电脉冲，以"0.1 ms"的持续时间开始，或应用两种方法的组合。 注意:全细胞配置的产生由移液管电容的突然增加（通常为野生型R1-6感光体的约60pF）显示;仅〜20pF的电容表示来自R7感光体的记录;上述电容〜90 pF表示从两个记录感光器）。 </li><li>使用贴片钳放大器中的适当旋钮手动将感光体的保持电位设置为所需电压（通常为-70 mV）。 注意:在获得密封（步骤6.11）之后，在完成整个单元配置之前，可以执行此步骤。 </li><li>抵消电容电流和串联电阻（测量的串联电阻值大于25MΩ表示电极移液器已堵塞），如果需要（ 即 ，对于较大的电流），应使用片钳钳位放大器中适当的旋钮进行串联电阻补偿。 </li><li>关闭法拉第笼的黑色前帘，以获得最大的黑暗和电气隔离。 </li><li>使用软件开始记录过程，并根据所需的实验程序施用光刺激和/或药物物质。 </li></ol> 7.同步Wh油细胞记录和Ca 2+成像<ol><li>对于遗传编码的Ca 2+指示剂，使用表达GCaMP6f 13的 黑腹果蝇蝇分离上述的恶臭。使用CCD相机（参见材料表 ）进行荧光测量，并确保显微镜配备有适当的激发和发射滤光片和分色镜（参见材料表 ） 4 。 </li><li>对于使用外源性Ca 2+指示剂（ 图4 ;参见材料表 ），如上所述分离出恶臭。此外，确保移液管溶液包含20-100μM钙指示剂。 </li><li>使用成像软件（参见材料表 ）以40赫兹的速率获取图像。在10秒的黑暗时期内进行图像采集，然后进行强烈的2秒光照mulation。 </li><li>使用成像软件并定义感兴趣区域（ROI）。测量ROI中的荧光强度。平均暗荧光（F D ），并从光（F L ）刺激（F L -F D ）中的荧光记录中减去它。根据光刺激开始时的荧光强度（F L 0 ）对这些测量进行归一化。 </li></ol>

<ol>
	<li>Hardie, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-cell+recordings+of+the+light+induced+current+in+dissociated+Drosophila+photoreceptors:+evidence+for+feedback+by+calcium+permeating+the+light-sensitive+channels.">Whole-cell recordings of the light induced current in dissociated Drosophila photoreceptors: evidence for feedback by calcium permeating the light-sensitive channels.</a> Proc. R. Soc. Lond. B. 245, 203-210 (1991).</li><li>Ranganathan, R., Harris, G. L., Stevens, C. F., Zuker, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Drosophila+mutant+defective+in+extracellular+calcium-+dependent+photoreceptor+deactivation+and+rapid+desensitization.">A Drosophila mutant defective in extracellular calcium- dependent photoreceptor deactivation and rapid desensitization.</a> Nature. 354, 230-232 (1991).</li><li>Martin, J. H., Benzer, S., Rudnicka, M., Miller, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calphotin:+a+Drosophila+photoreceptor+cell+calcium-binding+protein.">Calphotin: a Drosophila photoreceptor cell calcium-binding protein.</a> Proc. Natl. Acad. Sci. U.S.A. 90 (4), 1531-1535 (1993).</li><li>Weiss, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compartmentalization+and+Ca2++buffering+are+essential+for+prevention+of+light-induced+retinal+degeneration.">Compartmentalization and Ca2+ buffering are essential for prevention of light-induced retinal degeneration.</a> J Neurosci. 32 (42), 14696-14708 (2012).</li><li>Wang, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+activation,+adaptation,+and+cell+survival+functions+of+the+Na+++/Ca+2++exchanger+CalX.">Light activation, adaptation, and cell survival functions of the Na + /Ca 2+ exchanger CalX.</a> Neuron. 45 (3), 367-378 (2005).</li><li>Weckstrom, M., Hardie, R. C., Laughlin, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltage-activated+potassium+channels+in+blowfly+photoreceptors+and+their+role+in+light+adaptation.">Voltage-activated potassium channels in blowfly photoreceptors and their role in light adaptation.</a> J. Physiol. Lond. 440, (1991).</li><li>Frolov, R. V., Immonen, E. V., Weckstr&#246;m, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performance+of+blue-+and+green-sensitive+photoreceptors+of+the+cricket+Gryllus+bimaculatus.">Performance of blue- and green-sensitive photoreceptors of the cricket Gryllus bimaculatus.</a> J Comp Physiol A Neuroethol Sens Neural Behav Physiol. 200 (3), 209-219 (2014).</li><li>Nasi, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-cell+clamp+of+dissociated+photoreceptors+from+the+eye+of+Lima+scabra.">Whole-cell clamp of dissociated photoreceptors from the eye of Lima scabra.</a> J Gen Physiol. 97 (1), 35-54 (1991).</li><li>Nasi, E., Gomez, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-activated+ion+channels+in+solitary+photoreceptors+of+the+scallop+Pecten+irradians.">Light-activated ion channels in solitary photoreceptors of the scallop Pecten irradians.</a> J. Gen. Physiol. 99, 747-769 (1992).</li><li>Hardie, R. C., Peretz, A., Pollock, J. A., Minke, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca+2++limits+the+development+of+the+light+response+in+Drosophila+photoreceptors.">Ca 2+ limits the development of the light response in Drosophila photoreceptors.</a> Proc. R. Soc. Lond. B. Biol. Sci. 252, 223-229 (1993).</li><li>Kohn, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+Cooperation+between+the+IP3+Receptor+and+Phospholipase+C+Secures+the+High+Sensitivity+to+Light+of+Drosophila+Photoreceptors+In+Vivo.">Functional Cooperation between the IP3 Receptor and Phospholipase C Secures the High Sensitivity to Light of Drosophila Photoreceptors In Vivo.</a> J Neurosci. 35 (6), 2530-2546 (2015).</li><li>Hevers, W., Hardie, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serotonin+modulates+the+voltage+dependence+of+delayed+rectifier+and+Shaker+potassium+channels+in+Drosophila+photoreceptors.">Serotonin modulates the voltage dependence of delayed rectifier and Shaker potassium channels in Drosophila photoreceptors.</a> Neuron. 14 (4), 845-856 (1995).</li><li>Chen, T. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasensitive+fluorescent+proteins+for+imaging+neuronal+activity.">Ultrasensitive fluorescent proteins for imaging neuronal activity.</a> Nature. 499 (7458), 295-300 (2013).</li><li>Henderson, S. R., Reuss, H., Hardie, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+photon+responses+in+Drosophila+photoreceptors+and+their+regulation+by+Ca+2.">Single photon responses in Drosophila photoreceptors and their regulation by Ca 2.</a> J. Physiol. Lond. 524 (Pt 1), 179-194 (2000).</li><li>Scott, K., Zuker, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+of+the+Drosophila+phototransduction+cascade+into+a+signalling+complex+shapes+elementary+responses.">Assembly of the Drosophila phototransduction cascade into a signalling complex shapes elementary responses.</a> Nature. 395 (6704), 805-808 (1998).</li><li>Elia, N., Frechter, S., Gedi, Y., Minke, B., Selinger, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excess+of+G+betae+over+G+qalphae+in+vivo+prevents+dark,+spontaneous+activity+of+Drosophila+photoreceptors.">Excess of G betae over G qalphae in vivo prevents dark, spontaneous activity of Drosophila photoreceptors.</a> J. Cell Biol. 171 (3), 517-526 (2005).</li><li>Katz, B., Minke, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phospholipase+C-Mediated+Suppression+of+Dark+Noise+Enables+Single-Photon+Detection+in+Drosophila+Photoreceptors.">Phospholipase C-Mediated Suppression of Dark Noise Enables Single-Photon Detection in Drosophila Photoreceptors.</a> J. Neurosci. 32 (8), 2722-2733 (2012).</li><li>Hardie, R. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+basis+of+amplification+in+Drosophila+phototransduction.+Roles+for+G+protein,+phospholipase+C,+and+diacylglycerol+kinase.">Molecular basis of amplification in Drosophila phototransduction. Roles for G protein, phospholipase C, and diacylglycerol kinase.</a> Neuron. 36 (4), 689-701 (2002).</li><li>Reuss, H., Mojet, M. H., Chyb, S., Hardie, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+analysis+of+the+Drosophila+light-sensitive+channels,+TRP+and+TRPL.">In vivo analysis of the Drosophila light-sensitive channels, TRP and TRPL.</a> Neuron. 19, 1249-1259 (1997).</li><li>Liu, C. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+identification+and+manipulation+of+the+Ca+2++selectivity+filter+in+the+Drosophila+transient+receptor+potential+channel.">In vivo identification and manipulation of the Ca 2+ selectivity filter in the Drosophila transient receptor potential channel.</a> J. Neurosci. 27 (3), 604-615 (2007).</li><li>Ahmad, S. T., Natochin, M., Barren, B., Artemyev, N. O., O'Tousa, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterologous+expression+of+bovine+rhodopsin+in+Drosophila+photoreceptor+cells.">Heterologous expression of bovine rhodopsin in Drosophila photoreceptor cells.</a> Invest Ophthalmol Vis Sci. 47 (9), 3722-3728 (2006).</li><li>Scott, K., Sun, Y., Beckingham, K., Zuker, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calmodulin+regulation+of+Drosophila+light-activated+channels+and+receptor+function+mediates+termination+of+the+light+response+in+vivo.">Calmodulin regulation of Drosophila light-activated channels and receptor function mediates termination of the light response in vivo.</a> Cell. 91 (3), 375-383 (1997).</li><li>Liu, C. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca+2++-dependent+metarhodopsin+inactivation+mediated+by+calmodulin+and+NINAC+myosin+III.">Ca 2+ -dependent metarhodopsin inactivation mediated by calmodulin and NINAC myosin III.</a> Neuron. 59 (5), 778-789 (2008).</li><li>Cook, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phospholipase+C+and+termination+of+G-protein-mediated+signalling+in+vivo.">Phospholipase C and termination of G-protein-mediated signalling in vivo.</a> Nat. Cell Biol. 2 (5), 296-301 (2000).</li><li>Hardie, R. C., Franze, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photomechanical+responses+in+Drosophila+photoreceptors.">Photomechanical responses in Drosophila photoreceptors.</a> Science. 338 (6104), 260-263 (2012).</li><li>Huang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+TRP+channels+by+protons+and+phosphoinositide+depletion+in+Drosophila+photoreceptors.">Activation of TRP channels by protons and phosphoinositide depletion in Drosophila photoreceptors.</a> Curr. Biol. 20 (3), 189-197 (2010).</li><li>Minke, B., Wu, C. F., Pak, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+light-induce+response+of+the+central+retinular+cells+from+the+electroretinogram+of+Drosophila.">Isolation of light-induce response of the central retinular cells from the electroretinogram of Drosophila.</a> J. Comp. Physiol. 98, 345-355 (1975).</li><li>Hardie, R. C., Minke, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+trp+gene+is+essential+for+a+light-activated+Ca2++channel+in+Drosophila+photoreceptors.">The trp gene is essential for a light-activated Ca2+ channel in Drosophila photoreceptors.</a> Neuron. 8, 643-651 (1992).</li><li>Niemeyer, B. A., Suzuki, E., Scott, K., Jalink, K., Zuker, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+light-activated+conductance+is+composed+of+the+two+channels+TRP+and+TRPL.">The Drosophila light-activated conductance is composed of the two channels TRP and TRPL.</a> Cell. 85 (5), 651-659 (1996).</li><li>Huber, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transient+receptor+potential+protein+(Trp),+a+putative+store-+operated+Ca+2++channel+essential+for+phosphoinositide-mediated+photoreception,+forms+a+signaling+complex+with+NorpA,+InaC+and+InaD.">The transient receptor potential protein (Trp), a putative store- operated Ca 2+ channel essential for phosphoinositide-mediated photoreception, forms a signaling complex with NorpA, InaC and InaD.</a> EMBO J. 15 (24), 7036-7045 (1996).</li><li>Shieh, B. H., Niemeyer, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+protein+encoded+by+the+InaD+gene+regulates+recovery+of+visual+transduction+in+Drosophila.">A novel protein encoded by the InaD gene regulates recovery of visual transduction in Drosophila.</a> Neuron. 14 (1), 201-210 (1995).</li><li>Tsunoda, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multivalent+PDZ-domain+protein+assembles+signalling+complexes+in+a+G-protein-coupled+cascade.">A multivalent PDZ-domain protein assembles signalling complexes in a G-protein-coupled cascade.</a> Nature. 388 (6639), 243-249 (1997).</li><li>Tsacopoulos, M., Veuthey, A. L., Saravelos, S. G., Perrottet, P., Tsoupras, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+cells+transform+glucose+to+alanine,+which+fuels+the+neurons+in+the+honeybee+retina.">Glial cells transform glucose to alanine, which fuels the neurons in the honeybee retina.</a> J. Neurosci. 14 (3 Pt 1), 1339-1351 (1994).</li><li>Hardie, R. C., Minke, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+activation+of+light-sensitive+channels+in+Drosophila+photoreceptors.">Spontaneous activation of light-sensitive channels in Drosophila photoreceptors.</a> J. Gen. Physiol. 103, 389-407 (1994).</li><li>Agam, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+stress+reversibly+activates+the+Drosophila+light-sensitive+channels+TRP+and+TRPL+in+vivo.">Metabolic stress reversibly activates the Drosophila light-sensitive channels TRP and TRPL in vivo.</a> J Neurosci. 20 (15), 5748-5755 (2000).</li><li>Agam, K., Frechter, S., Minke, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+the+Drosophila+TRP+and+TRPL+channels+requires+both+Ca2++and+protein+dephosphorylation.">Activation of the Drosophila TRP and TRPL channels requires both Ca2+ and protein dephosphorylation.</a> Cell Calcium. 35 (2), 87-105 (2004).</li><li>Delgado, R., Mu&#241;oz, Y., Pe&#241;a-Cort&#233;s, H., Giavalisco, P., Bacigalupo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diacylglycerol+activates+the+light-dependent+channel+TRP+in+the+photosensitive+microvilli+of+Drosophila+melanogaster+photoreceptors.">Diacylglycerol activates the light-dependent channel TRP in the photosensitive microvilli of Drosophila melanogaster photoreceptors.</a> J Neurosci. 34 (19), 6679-6686 (2014).</li><li>Parnas, M., Katz, B., Minke, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Open+channel+block+by+Ca2++underlies+the+voltage+dependence+of+Drosophila+TRPL+channel.">Open channel block by Ca2+ underlies the voltage dependence of Drosophila TRPL channel.</a> J. Gen. Physiol. 129 (1), 17-28 (2007).</li></ol>

作者没有什么可以披露的。

将全细胞记录应用于黑腹果蝇光感受器允许新型信号蛋白如TRP通道27,28,29和INAD 30,31,32框架蛋白的发现和功能性阐明。自从这种技术的初步介绍以来，它能够解决关于光响应的离子机制和电压依赖性的长期基本问题。这是因为赋予了精确控制膜电压和胞外和细胞内离子组合物19,28的能力。 黑腹果蝇斑块夹紧技术的主要障碍是孤立的ommatidia prepa的脆弱性配给。详细研究表明，光转导机制的完整性主要取决于ATP的连续供应，特别是在光照下，导致ATP消耗量大。不幸的是，用贴片移液管到达感光膜所需的颜料（ 即胶质细胞）的机械条纹消除了ATP生产所需的主要代谢源33 。外源ATP在记录移液器中的应用仅部分满足大量ATP的需求。短暂的ATP供应导致TRP通道的自发激活和光激活通道的光转导机制的解离，导致细胞Ca 2+的大量增加和废除对光的正常反应34,35 。这个事件的顺序不是由于损坏的光感受器通过解剖程序，而是ATP的细胞衰竭。为了防止发生这种事件序列并保持正常的光反应，光感受器不应该暴露于消耗大量ATP的强光下。此外，NAD必须包括在记录移液器中，大概是为了促进线粒体中的ATP生成18,36。对于自发和量子碰撞的测量，上述困难是最小的，因为只使用昏暗的灯。实际上，稳定的全细胞记录可以保持约20-25分钟，尽管在这一时期内有反应动力学减慢的倾向。单一的解离的恶臭药物的制备可以保持存活长达2小时。 孤立的ommatidia制剂的另一个缺点是微绒毛的不可接近性，这转化为TRP的不可接近性，TRPL通道到记录移液器，防止单声道录音。 Bacigalupo和他的同事使用他们开发的方法，成功地直接记录了横纹肌的单通道活动。然而，该通道活性不同于在组织培养细胞38中异源表达的TRPL通道的通道活性和来自从孤立的ommatidia 34获得的喷射噪声分析的TRP通道活性。推测使用这种方法时，解剖程序会大大损伤感光细胞。

果蝇的深入遗传研究，果蝇黑腹果蝇（ D. melanogaster） ，在100多年前开始，已经建立了黑腹果蝇，作为复杂生物过程的遗传解剖非常有用的实验模型。下面描述的方法将黑腹果蝇分子遗传学的积累能力与全细胞膜片钳记录的高信噪比相结合。这种组合允许研究黑腹果蝇光转移作为肌醇 - 脂质信号和TRP通道调节和活化的模型，无论是在天然环境中还是在单分子的最高分辨率下。 全细胞记录方法对黑腹果蝇光感受器的应用已经彻底改变了无脊椎动物光转导的研究。这种方法是由Hardie 1和Indep开发的由Ranganathan及其同事2 〜26年前提出，旨在利用黑腹果蝇广泛的遗传操作工具，利用它们发现光转导和肌醇 - 脂质信号传导机制。首先，这种技术在解剖过程中光敏感性快速降低，恶心病的产量低，这阻碍了详细的定量研究。之后，将ATP和NAD添加到贴片移液器中，显着提高了长时间定量记录制剂的适用性。此后，实现了分子水平信号转导机制的广泛表征。 目前， 黑腹果蝇光转移是在单分子分辨率下离体研究磷酸肌醇信号和TRP通道的少数系统之一。这使得黑腹果蝇的光转导和我研究这种机制的方法学是一个高度敏感的模型系统。该方案描述了如何解剖黑腹果蝇视网膜并机械剥离孤立的ommatidia与周围的颜料（胶质细胞）细胞。这使得能够在感光体细胞体上形成千兆密封和全细胞膜片钳。幸运的是，大多数信号蛋白被限制在横纹肌上，不扩散。此外，还有一种称为钙调蛋白的固定Ca 2+缓冲液，位于信号传导室和细胞体3,4之间，在微绒毛5中有一个高表达量的Na + / Ca 2+交换体（CalX）。在一起，对横纹肌，钙镁缓冲液的蛋白质限制和CalX的高表达允许相对延长（ 即高达〜20分钟）全细胞记录，而不损失必需组分的光转换过程，同时保持对光的高灵敏度。以下协议描述了如何获得孤立的ommatidia并执行看起来保持光转录级联的天然特性的全细胞记录。对于分离的蟑螂（ 美洲大蠊 ） 6和板球（ Gryllus bimaculatus ） 7的 o虫进行全细胞膜片钳实验，与对黑腹果蝇类似地进行。此外，以与黑腹果蝇进行的方式稍微不同的方式进行文件蛤（ Lima scabra ）和扇贝（ Pecten radians ）的离解光感受器的膜片钳实验 ，允许全细胞8和单通道测量9 。在这里，描述了使用这种技术在黑腹果蝇获得的主要成就。讨论我包括对这种技术的一些陷阱和限制的描述。

<table><tbody><tr><td>10 mL syringe</td><td></td><td></td><td></td></tr><tr><td>5 mL syringe</td><td></td><td></td><td></td></tr><tr><td>1 mL syringe with elongated tip</td><td></td><td></td><td></td></tr><tr><td>Petri dish</td><td></td><td></td><td>60 mm</td></tr><tr><td>Syringe filters</td><td>Millex</td><td></td><td>22 &amp;micro;m PVDF filter&amp;nbsp;</td></tr><tr><td>Capillaries (for omatidia separation)</td><td></td><td></td><td>Glass, 1.2 x 0.68 mm (~7.5 cm each)</td></tr><tr><td>Polyethylene Tubing</td><td>Becton Dickinson</td><td></td><td>1.57 x 1.14 mm (35 cm)</td></tr><tr><td>&amp;nbsp;2 small beakers</td><td></td><td></td><td>50 mL or less</td></tr><tr><td>2 paraffin film sheets</td><td>Parafilm M</td><td></td><td>~5 x 5 cm</td></tr><tr><td>Bath chamber</td><td>home-made</td><td></td><td></td></tr><tr><td>Cover slips</td><td></td><td></td><td>22 x 22 mm No. 0</td></tr><tr><td>Paraplast Plus</td><td>Sigma</td><td>Paraffin &amp;ndash; polyisobutylene mixture</td><td>To glue the coverslip onto the bottom of the bath chamber</td></tr><tr><td>Ground</td><td>Warner Instruments&amp;nbsp;</td><td>64-1288</td><td>Hybrid Assembly Ag-AgCl Wire Assembly</td></tr><tr><td>Headless micro dissection needle</td><td></td><td></td><td>Entomology, 12 mm</td></tr><tr><td>Micro dissecting needle holder</td><td></td><td></td><td></td></tr><tr><td>Vise</td><td></td><td></td><td></td></tr><tr><td>2 fine tweezers + 1 rough tweezers</td><td></td><td>Dumont #5, Biology&amp;nbsp;</td><td>0.05 x 0.02 mm, length 110 mm, Inox</td></tr><tr><td>Stereoscopic zoom Microscope&amp;nbsp;</td><td>Nikon</td><td>SMZ-2B</td><td></td></tr><tr><td>Cold light source</td><td>Schott</td><td>KL1500 LCD</td><td></td></tr><tr><td>Filter (Color) for cold light source</td><td>Schott</td><td>RG620</td><td></td></tr><tr><td>Delicate wipers</td><td>Kimtech</td><td></td><td>Kimwipes</td></tr><tr><td>Electrode holder</td><td>Warner Instruments</td><td>QSW-T10P</td><td>Q series Holders compatible with Axon amplifiers, straight body style</td></tr><tr><td>Silver Wire</td><td>Warner Instruments</td><td></td><td>0.25 mm diameter, needs to be chloridized</td></tr><tr><td>Micromanipulator</td><td>Sutter Instruments</td><td>MP 85</td><td>Huxley-Wall Style Micromanipulator</td></tr><tr><td>Faraday cage</td><td>home made</td><td></td><td>Electromagnetic noise shielding and black front curtain</td></tr><tr><td>Anti-vibration Table</td><td>Newport</td><td>VW-3036-OPT-01</td><td></td></tr><tr><td>Osilloscope</td><td>GW</td><td>GOS-622G</td><td></td></tr><tr><td>Perfusion system</td><td>Warner Instruments</td><td>VC-8P</td><td>Pinch valve control system</td></tr><tr><td>Perfusion valve controller</td><td>Scientific instruments</td><td>BPS-8</td><td></td></tr><tr><td>Suction system</td><td></td><td></td><td></td></tr><tr><td>Amplifier</td><td>Molecular Device</td><td>Axopatch-1D</td><td></td></tr><tr><td>Head-stage</td><td>Molecular Device</td><td>CV - 4</td><td>Gain: x 1/100</td></tr><tr><td>A/D converter</td><td>Molecular Device</td><td>Digidata 1440A</td><td></td></tr><tr><td>Clampex</td><td>Molecular Device</td><td>10</td><td>software</td></tr><tr><td>pCLAMP</td><td>Molecular Device</td><td>10</td><td>software</td></tr><tr><td>Light source (Xenon Arc lamp)</td><td>Sutter Instruments</td><td>Lambda LS</td><td></td></tr><tr><td>Light detector</td><td>home made</td><td></td><td>phototransistor</td></tr><tr><td>Filter wheel and shutter controller</td><td>Sutter Instruments</td><td>Lambda 10-2 with a Uniblitz shutter</td><td></td></tr><tr><td>Filters (Natural density filter)</td><td>Chroma</td><td>6,5,4,3,2,1,0.5,0.3</td><td></td></tr><tr><td>Filter (Color)</td><td>Schott</td><td>OG590, Edge filter</td><td></td></tr><tr><td>Xenon Flash Lamp system</td><td>Dr. Rapp OptoElecftronic</td><td>JML-C2</td><td></td></tr><tr><td>Light guide</td><td></td><td></td><td>Quartz&amp;nbsp;</td></tr><tr><td>Pulse generator</td><td>AMPI</td><td>Master 8</td><td></td></tr><tr><td>Microscope</td><td>Olympus</td><td>IX71, Inverted</td><td></td></tr><tr><td>Red illumination filter (Microscope)</td><td></td><td></td><td>RG630 / RG645 &amp;oslash;45mm</td></tr><tr><td>Microscope objective</td><td>Olympus</td><td>X60/0.9 UplanFL N air or X60/1.25 UplanFI oil</td><td></td></tr><tr><td>CCD Camera</td><td>Andor</td><td>iXon DU885K</td><td></td></tr><tr><td>NIS Element&amp;nbsp;</td><td>Nikon</td><td>AR</td><td>software</td></tr><tr><td>Ca&lt;sup&gt;+2&lt;/sup&gt; indicator</td><td>Invitrogen</td><td>Calcium green 5N</td><td></td></tr><tr><td>Excitation &amp;amp; emission filters and dichroic mirror</td><td>Chroma</td><td>19002 - AT - GFP/FITC Longpass set</td><td></td></tr><tr><td>Vertical pipette puller</td><td>Narishige&amp;nbsp;</td><td>Model PP83</td><td>Use either vertical or horizontal puller, as preferred.</td></tr><tr><td>Horizontal pipette puller</td><td>Sutter Instrument&amp;nbsp;</td><td>Model P-1000 Flaming/Brown Micropipette Puller</td><td></td></tr><tr><td>Filament</td><td>Sutter Instrument</td><td></td><td>3 mm trough or square box</td></tr><tr><td>Capillaries</td><td>Harvard Apparatus</td><td>borosilicate glass capillaries</td><td>1 x 0.58 mm</td></tr></tbody></table>

electrophysiological method for whole-cell voltage clamp recordings from drosophila photoreceptors

从黑腹果蝇光感受器的全细胞电压钳记录已经彻底改变了无脊椎动物视觉转导的领域，使得能够使用黑腹果蝇分子遗传学在单分子水平上研究肌醇 - 脂质信号传导和瞬时受体电位（TRP）通道。一些实验室掌握了这种强大的技术，可以在高度受控条件下分析对光的生理反应。这种技术允许控制细胞内和细胞外介质;膜电压;以及药物化合物如各种离子或pH指示剂快速应用于细胞外和细胞外培养基。具有非常高的信噪比，该方法能够测量暗自发和光诱导的单一电流（ 即自发和量子隆起）和宏观光诱导电流（LIC）从single D. melanogaster光感受器。该协议非常详细地概述了执行此技术所需的所有关键步骤，包括电生理和光学记录。描述了在浴室中获得完整和可行的离体体外分离的眼药水的蝇眼视网膜解剖程序。还需要进行全细胞和荧光成像测量所需的设备。最后，解释了在扩展实验期间使用这种精致准备的陷阱。

所描述的方法使得能够精确地记录产生自发和光诱发量子隆起的基本单一电流，其在规定的条件下相加以产生对光的宏观响应。它还允许在关键信号分子中具有缺陷的野生型和突变体蝇之间的比较（ 图3和图5 ）14,15,16,17,18。此外，在双离子条件下测量反转电位的能力揭示了TRP和TRP样（TRPL）通道18,19的基本生物物理性质。它还能够测量修饰其Ca 2+的TRP细胞区域中氨基酸取代的作用渗透性20 。 通过膜片钳全细胞记录获得的光响应线性依赖于光强度至少4个数量级。这不能通过使用ERG和细胞内记录方法来解决。因此，对强度增加的短暂闪光和强度响应函数的一系列响应揭示了随着光强度的增加，闪光响应的严格线性。严格的线性度可以达到至少几百pA，但无论是线性还是钳位控制分解，都是有争议的（ 图6 ）。这些结果表明，对光的宏观反应是对光的单一响应（ 即量子隆起）的线性求和。 已经很好地建立了使用暗淡光刺激的电压记录大多数无脊椎动物物种的离散电压波动（ 即量子隆起）。 黑腹果蝇的量子隆起来自于隆起18峰顶的〜15个TRP通道和〜2个TRPL通道的协调开启。每个凸起由单个光子的吸收产生，而对更强烈的光的宏观响应是这些基本响应14,21的总和。即使在刺激条件相同的情况下，颠簸在潜伏期，时间过程和幅度上也会有很大差异。凹凸生成是由泊松统计所描述的随机过程，其中每个有效吸收的光子仅引起一个凹凸。单光子单碰撞关系要求级联中的每个步骤不仅包括有效的"开启"机制，而且包括同样有效的"关闭"机制。功能优势是生产a具有快速瞬态响应的非常灵敏的光子计数器非常适合视觉系统所需的灵敏度和时间分辨率。当光活化色素（ 即异视紫质，M）或其目标，G qα，在光被关闭后长时间不能失活并导致连续产生隆起时，显示出有效的关断机制的要求（ 图3 ） 15,22,23,24 。 该突起代表微生物中TRP / TRPL通道的合作活性。因此，信道激活的任何假设也应该解释协作信道激活。最近，Hardie和他的同事已经证明光引起光感受器的快速收缩，这表明光敏感e通道（TRP / TRPL）可以机械门控25 。这种机械活化以及通过PLC介导的PIP 2水解释放的观察到的质子促进了TRP / TRPL通道的开放，并解释了凸块生产的合作性质26 。目前， 黑腹果蝇光感受器是其中可以在体内研究磷酸肌醇信号和TRP通道的少数系统之一，从而使得黑腹果蝇光转换和开发研究这种机制的方法是一个非常有价值的模型系统。 <img alt="图3" src="/files/ftp_upload/55627/55627fig3.jpg" /> 图3:inaC P209和inaD P215突变体显示对光和单量子轰击的宏观响应的缓慢响应终止。 （ A ）分离的小燕麦准备在全细胞记录期间呈现填充有荧光Lucifer Yellow CH染料（激发:430nm;发射:540nm）的补片移液器的配给。注意，荧光染料扩散并标记了单个光感受器细胞体，并且感光细胞体从其细长的轴突脱离，但是仍保持活力。该制剂适用于同时进行全细胞记录和成像实验。 （ BD ）上图:WT中的连续暗光（开放条）的全细胞电压钳位量子隆起反应，P209和P2a突变体蝇。在相对于WT苍蝇的P209和P2a突变体中观察到颠簸的缓慢终止。下面的插图显示了单个凸块的放大形状。底图:归一化的全细胞记录对上述野生型500毫秒光脉冲（每秒1.5×10 5个光子）的宏观反应<html>和突变体蝇。 （EG）上图:全细胞电压钳位的量子隆起对短暂（1 ms）的反应，在野生型， arr2 3和ninaC P235突变体蝇中引发单光子反应的暗光。注意在arr2 3和ninaC P235突变体中观察到的颠簸火花响应单光子吸收。底板:在相应的突变体中，全细胞电压钳位对500ms光脉冲（1.5×10 4光子/ s）的归一化响应。注意在arr2 3中观察到的宏观反应的缓慢终止 和ninaC P235突变体相对于WT而飞行。 <a href="http://ecsource.jove.com/files/ftp_upload/55627/55627fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> d / 55627 / 55627fig4.jpg"/&gt; 图4:细胞Ca 2+动力学信号诱导的Ca 2+流入受钙镁光合作用影响。野生型和Cpn 1％苍蝇的感光体图像的时间序列显示光刺激期间Ca 2+指示剂的荧光。使用假色编码（bar =10μm;箭头表示移液管）绘制原始强度图像。图片经Weiss 等人转载。 4 。 <a href="http://ecsource.jove.com/files/ftp_upload/55627/55627fig4large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图5" src="/files/ftp_upload/55627/55627fig5.jpg" /> 图5:WT，trp和trpl突变体的电生理特性。 （ A ）全电池电压钳响应于WT，trpl 302和trp P343无效突变体蝇中连续暗光（开放条）的量子隆起的反应。观察到高度降低的trp P34 3个隆起的振幅。插图:显示野生型和trp P343无效突变体蝇的放大单量子隆起。 （ B ）响应野生型3s光脉冲和相应突变体的全细胞电压钳记录。观察到trp P343突变体的瞬时稳态反应。插图:显示WT和trp P343突变体的放大光反应。 （ C ）以反转电位（E rev ）周围测量的在3mV的电压阶跃响应于20ms的光脉冲引起的上述蝇应变的叠加的光诱导电流族。 （ D ） 绘制野生型和各种变异体的平均E转换的直方图TS。误差是SEM SEM的逆转电位（E rev ）在仅表达TRP的trpl 302的正E 转和仅表达TRPL的trp P343无效突变体的E rev之间。 <a href="http://ecsource.jove.com/files/ftp_upload/55627/55627fig5large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图6" src="/files/ftp_upload/55627/55627fig6.jpg" /> 图6:闪光响应与光强增加呈严格线性关系。 一系列对光强增加的短暂闪烁的反应，以及光响应的峰值振幅对短暂闪光强度增加的依赖性的曲线。这种关系揭示了闪光响应和光强度增加之间的严格线性关系。这严格线性持有至少几百pA，光强度超过4个数量级，而无论是线性还是钳夹控制在此之后发生故障，都是有争议的。 <a href="http://ecsource.jove.com/files/ftp_upload/55627/55627fig6large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> pH值</td><td> 7.15（用NaOH调节） </td></tr><tr><td> 试剂 </td><td> 浓度（mM） </td></tr><tr><td>氯化钠</td><td> 120 </td></tr><tr><td>氯化钾</td><td>五</td></tr><tr><td> MgCl 2 </td><td> 4 </td></tr><tr><td> TES </td><td> 10 </td></tr><tr><td>脯氨酸</td><td> 25 </td></tr><tr><td>丙氨酸</td><td>五</td></tr><tr><td></td><td>储存于-20°C。 </td></tr><tr><td colspan="2"&gt;注意:该溶液名义上不含Ca 2+，但没有添加Ca 2+缓冲液，因此将具有大约5 - 10μM的痕量Ca 2+ 。细胞外溶液（ES）=具有1.5mM CaCl 2的 ES-0Ca 2+ ，通过将CaCl 2从0.5或1M储备溶液加入到ES-0Ca 2+中而制备。 </td></tr></tbody></table> 表1:不含Ca +2的细胞外溶液（ES）。化学品描述和生产不含Ca +2的 ES所需的具体数量。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 试剂 </td><td> 量 </td></tr><tr><td> FBS </td><td> 15 mL </td></tr><tr><td>蔗糖</td><td> 1.5克</td></tr><tr><td colspan="2">在1.5 mL小瓶中分成150μL等分试样，储存于-20℃ 76℃。 </td></tr><tr><td>研磨溶液（TS） </td><td>用1,350mL ES或ES-0Ca 2+将1mL的150mL储备溶液填充以与在解剖过程中使用的溶液相匹配。 </td></tr></tbody></table> 表2:胎牛血清（FBS）+蔗糖 - 储备溶液。产生胎牛血清（FBS）+蔗糖 - 储备液所需的化学说明和特定量。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> pH值</td><td> 7.15（用KOH调节） </td></tr><tr><td> 试剂 </td><td> 浓度（mM） </td></tr><tr><td>葡萄糖酸钾（Kglu） </td><td> 140 </td></tr><tr><td> MgCl 2 </td><td> 2 </td></tr><tr><td> TES </td><td> 10 </td></tr><tr><td> ATP镁盐（MgATP） </td><td> 4 </td></tr><tr><td> GTP钠盐（Na 2 GTP） </td><td> 0.4 </td></tr><tr><td> β-烟酰胺腺嘌呤二核苷酸水合物（NAD） </td><td> 1 </td></tr><tr><td></td><td>储存于-20°C。 </td></tr></tbody></table> 表3:细胞内溶液（IS1）。化学描述和生产IS1所需的具体数量，主要用于强度响应和量子碰撞测量。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> pH值</td><td> 7.15（用CsOH调节） </td></tr><tr><td> 试剂 </td><td> 浓度（mM） </td></tr><tr><td>氯化铯</td><td> 120 </td></tr><tr><td> MgCl 2 </td><td> 2 </td></tr><tr><td> TES </td><td> 10 </td></tr><tr><td> ATP镁盐（MgATP） </td><td> 4 </td></tr><tr><td> GTP钠盐（Na 2 GTP） </td><td> 0.4 </td></tr><tr><td> β-烟酰胺腺嘌呤二核苷酸水合物（NAD） </td><td> 1 </td></tr><tr><td>四乙基氯化铵（TAE） </td><td> 15 </td></tr><tr><td></td><td>储存于-20°C。 </td></tr></tbody></table> 表4:细胞内溶液（IS2）。化学描述和产生细胞内溶液IS2所需的具体数量，其主要用于光诱导电流的反向电位测量。

Watch this Scientific Journal Video about Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors at JoVE.com

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

从黑腹果蝇光感受器的全细胞记录使得能够测量自发的黑色凸起，量子隆起，对光的宏观反应以及在各种条件下的电流 - 电压关系。与黑腹果蝇遗传操作工具结合使用，该方法能够研究普遍存在的肌醇脂质信号通路及其靶标TRP通道。

全细胞电压钳记录的电生理方法<em&gt;果蝇</em&gt;光感受器

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, ...

electrophysiological-method-for-whole-cell-voltage-clamp-recordings

Research

JoVE Journal

Neuroscience

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

14.9K 次观看.  Hebrew University. 从黑腹果蝇光感受器的全细胞记录使得能够测量自发的黑色凸起，量子隆起，对光的宏观反应以及在各种条件下的电流 - 电压关系。与黑腹果蝇遗传操作工具结合使用，该方法能够研究普遍存在的肌醇脂质信号通路及其靶标TRP通道。 

视频: Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole Cell Recordings from Brain of Adult Drosophila

In this video, we demonstrate the procedure for isolating whole brains from adult Drosophila in preparation for recording from single neurons. We begin by describing the dissecting solution and capture of the adult females used in our studies. The procedure for removing the whole brain intact, including both optic lobes, is illustrated. Dissection of the overlying trachea is also shown.  The isolated brain is not only small but needs special care in handling at this stage to prevent damage to the neurons, many of which are close to the outer surface of the tissue. We show how a special holder we developed is used to stabilize the brain in the recording chamber. A standard electrophysiology set up is used for recording from single neurons or pairs of neurons. A fluorescent image, viewed through the recording microscope, from a GAL4 line driving GFP expression (GH146) illustrates how projection neurons (PNs) are identified in the live brain. A high power Nomarski image shows a view of a single neuron that is being targeted for whole cell recording. When the brain is successfully removed without damage, the majority of the neurons are spontaneously active, firing action potentials and/or exhibiting spontaneous synaptic input. This in situ preparation, in which whole cell recording of identified neurons in the whole brain can be combined with genetic and pharmacological manipulations, is a useful model for exploring cellular physiology and plasticity in the adult CNS.

This video demonstrates the procedure for isolating whole brains from adult Drosophila in preparation for recording from single neurons using standard whole cell technology. It includes images of GFP labeled cells and neurons viewed during recording.

从整个成年果蝇大脑细胞记录

In this video, we demonstrate the procedure for isolating whole brains from adult Drosophila in preparation for recording from single neurons. We begin by ...

科研

生物学

Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster

When startled adult D. melanogaster react by jumping into the air and flying away. In many invertebrate species, including D. melanogaster, the "escape" (or "startle") response during the adult stage is mediated by the multi-component neuronal circuit called the Giant Fiber System (GFS). The comparative large size of the neurons, their distinctive morphology and simple connectivity make the GFS an attractive model system for studying neuronal circuitry. The GFS pathway is composed of two bilaterally symmetrical Giant Fiber (GF) interneurons whose axons descend from the brain along the midline into the thoracic ganglion via the cervical connective. In the mesothoracic neuromere (T2) of the ventral ganglia the GFs form electro-chemical synapses with 1) the large medial dendrite of the ipsilateral motorneuron (TTMn) which drives the tergotrochanteral muscle (TTM), the main extensor for the mesothoracic femur/leg, and 2) the contralateral peripherally synapsing interneuron (PSI) which in turn forms chemical (cholinergic) synapses with the motorneurons (DLMns) of the dorsal longitudinal muscles (DLMs), the wing depressors. The neuronal pathway(s) to the dorsovental muscles (DVMs), the wing elevators, has not yet been worked out (the DLMs and DVMs are known jointly as indirect flight muscles - they are not attached directly to the wings, but rather move the wings indirectly by distorting the nearby thoracic cuticle) (King and Wyman, 1980; Allen et al., 2006). The di-synaptic activation of the DLMs (via PSI) causes a small but important delay in the timing of the contraction of these muscles relative to the monosynaptic activation of TTM (~0.5 ms) allowing the TTMs to first extend the femur and propel the fly off the ground. The TTMs simultaneously stretch-activate the DLMs which in turn mutually stretch-activate the DVMs for the duration of the flight. The GF pathway can be activated either indirectly by applying a sensory (e.g."air-puff" or "lights-off") stimulus, or directly by a supra-threshold electrical stimulus to the brain (described here). In both cases, an action potential reaches the TTMs and DLMs solely via the GFs, PSIs, and TTM/DLM motoneurons, although the TTMns and DLMns do have other, as yet unidentified, sensory inputs. Measuring "latency response" (the time between the stimulation and muscle depolarization) and the "following to high frequency stimulation" (the number of successful responses to a certain number of high frequency stimuli) provides a way to reproducibly and quantitatively assess the functional status of the GFS components, including both central synapses (GF-TTMn, GF-PSI, PSI-DLMn) and the chemical (glutamatergic) neuromuscular junctions (TTMn-TTM and DLMn-DLM). It has been used to identify genes involved in central synapse formation and to assess CNS function.

Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster

The Giant Fiber System is a simple neuronal circuit of adult Drosophila melanogaster containing the largest neurons in the fly. We describe the protocol for monitoring synaptic transmission through this pathway by recording post synaptic potentials in dorsal longitudinal (DLM) and tergotrochanteral (TTM) muscles following direct stimulation of the Giant Fiber interneurons.

从巨纤维通路的电生理记录 D。果蝇</em

When startled adult D. melanogaster react by jumping into the air and flying away. In many invertebrate species, including D. melanogaster, the "escape" ...

神经科学

Electrophysiological Methods for Recording Synaptic Potentials from the NMJ of Drosophila Larvae

In this video, we describe the electrophysiological methods for recording synaptic transmission at the neuromuscular junction (NMJ) of Drosophila larva. The larval neuromuscular system is a model synapse for the study of synaptic physiology and neurotransmission, and is a valuable research tool that has defined genetics and is accessible to experimental manipulation. Larvae can be dissected to expose the body wall musculature, central nervous system, and peripheral nerves. The muscles of Drosophila and their innervation pattern are well characterized and muscles are easy to access for intracellular recording. Individual muscles can be identified by their location and orientation within the 8 abdominal segments, each with 30 muscles arranged in a pattern that is repeated in segments A2 - A7. Dissected drosophila larvae are thin and individual muscles and bundles of motor neuron axons can be visualized by transillumination1. Transgenic constructs can be used to label target cells for visual identification or for manipulating gene products in specific tissues. In larvae, excitatory junction potentials (EJP&#8217;s) are generated in response to vesicular release of glutamate from the motoneurons at the synapse. In dissected larvae, the EJP can be recorded in the muscle with an intracellular electrode. Action potentials can be artificially evoked in motor neurons that have been cut posterior to the ventral ganglion, drawn into a glass pipette by gentle suction and stimulated with an electrode. These motor neurons have distinct firing thresholds when stimulated, and when they fire simultaneously, they generate a response in the muscle. Signals transmitted across the NMJ synapse can be recorded in the muscles that the motor neurons innervate. The EJP&#8217;s and minature excitatory junction potentials (mEJP&#8217;s) are seen as changes in membrane potential. Electrophysiological responses are recorded at room temperature in modified minimal hemolymph-like solution2 (HL3) that contains 5 mM Mg2+ and 1.5 mM Ca2+. Changes in the amplitude of evoked EJP&#8217;s can indicate differences in synaptic function and structure. Digitized recordings are analyzed for EJP amplitude, mEJP frequency and amplitude, and quantal content.

Here we describe electrophysiological methods for measuring synaptic transmission at the neuromuscular junction of Drosophila larva. Evoked release is initiated artificially by stimulating the motor neuron axons, and transmission through the NMJ can be measured by the postsynaptic response evoked in the muscle.

电生理方法记录从果蝇幼虫NMJ突触潜力

In this video, we describe the electrophysiological methods for recording synaptic transmission at the neuromuscular junction (NMJ) of Drosophila larva. ...

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  ...

Electrophysiological Recording in the Drosophila Embryo

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite isolated from each other, with largely independent histories and scientific communities. However, the interface between these usually disparate fields is the developmental programs underlying acquisition of functional electrical signaling properties and differentiation of functional chemical synapses during the final phases of neural circuit formation. This interface is a critically important area for investigation. In Drosophila, these phases of functional development occur during a period of &lt;8 hours (at 25&deg;C) during the last third of embryogenesis. This late developmental period was long considered intractable to investigation owing to the deposition of a tough, impermeable epidermal cuticle. A breakthrough advance was the application of water-polymerizing surgical glue that can be locally applied to the cuticle to enable controlled dissection of late-stage embryos. With a dorsal longitudinal incision, the embryo can be laid flat, exposing the ventral nerve cord and body wall musculature to experimental investigation. Whole-cell patch-clamp techniques can then be employed to record from individually-identifiable neurons and somatic muscles. These recording configurations have been used to track the appearance and maturation of ionic currents and action potential propagation in both neurons and muscles. Genetic mutants affecting these electrical properties have been characterized to reveal the molecular composition of ion channels and associated signaling complexes, and to begin exploration of the molecular mechanisms of functional differentiation. A particular focus has been the assembly of synaptic connections, both in the central nervous system and periphery. The glutamatergic neuromuscular junction (NMJ) is most accessible to a combination of optical imaging and electrophysiological recording. A glass suction electrode is used to stimulate the peripheral nerve, with excitatory junction current (EJC) recordings made in the voltage-clamped muscle. This recording configuration has been used to chart the functional differentiation of the synapse, and track the appearance and maturation of presynaptic glutamate release properties. In addition, postsynaptic properties can be assayed independently via iontophoretic or pressure application of glutamate directly to the muscle surface, to measure the appearance and maturation of the glutamate receptor fields. Thus, both pre- and postsynaptic elements can be monitored separately or in combination during embryonic synaptogenesis. This system has been heavily used to isolate and characterize genetic mutants that impair embryonic synapse formation, and thus reveal the molecular mechanisms governing the specification and differentiation of synapse connections and functional synaptic signaling properties.

Electrophysiological Recording in the Drosophila Embryo

Electrophysiological recordings from Drosophila embryos allow analyses of developing muscle and neuron electrical properties, as well as characterization of functional synaptogenesis at the glutamatergic neuromuscular junction and central cholinergic and GABAergic synapses.

在电生理记录果蝇胚胎

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

为记录细胞内电压响应电法<em&gt;果蝇</em&gt;光感受器和中间对光刺激<em&gt;在体内</em

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

Electrophysiological Methods for Measuring Photopigment Levels in Drosophila Photoreceptors

The Drosophila G-protein-coupled photopigment rhodopsin (R) is composed of a protein (opsin) and a chromophore. The activation process of rhodopsin is initiated by photon absorption-inducing isomerization of the chromophore, promoting conformational changes of the opsin and resulting in a second dark-stable photopigment state (metarhodopsin, M). Investigation of this bi-stable photopigment using random mutagenesis requires simple and robust methods for screening mutant flies. Therefore, several methods for measuring reductions in functional photopigment levels have been designed. One such method exploits the charge displacements within the photopigment following photon absorption and the huge amounts of photopigment molecules expressed in the photoreceptors. This electrical signal, named the early receptor potential (or early receptor current), is measured by a variety of electrophysiological methods (e.g., electroretinogram and whole-cell recordings) and is linearly proportional to functional photopigment levels. The advantages of this method are the high signal-to-noise ratio, direct linear measurement of photopigment levels, and independence of phototransduction mechanisms downstream to rhodopsin or metarhodopsin activation. An additional electrophysiological method called prolonged depolarizing afterpotential (PDA) exploits the bi-stability of Drosophila photopigment and the absorption-spectral differences of fly R and M pigment states. The PDA is induced by intense blue light, converting saturating amounts of rhodopsin to metarhodopsin, resulting in the failure of light-response termination for an extended time in darkness, but it can be terminated by metarhodopsin to rhodopsin conversion using intense orange light. Since the PDA is a robust signal that requires massive photopigment conversion, even small defects in the biogenesis of the photopigment lead to readily detected abnormal PDA. Indeed, defective PDA mutants led to the identification of novel signaling proteins important for phototransduction.

Electrophysiological Methods for Measuring Photopigment Levels in Drosophila Photoreceptors

We present a protocol to electrophysiologically characterize bi-stable photopigments: (i) exploiting the charge displacements within the photopigment molecules following photon-absorption and their huge amount in the photoreceptors, and (ii) exploiting the absorption-spectra differences of rhodopsin and metarhodopsin photopigment states. These protocols are useful to screen for mutations affecting bi-stable photopigment systems.&#160;

测量 果蝇 光感受器中光色素水平的电生理学方法

The Drosophila G-protein-coupled photopigment rhodopsin (R) is composed of a protein (opsin) and a chromophore. The activation process of rhodopsin is ...

Electrophysiological Recording of Voltage Responses of Drosophila Retinal Photoreceptors to Light Stimuli

Source: Juusola, M., et al. <a href="https://app.jove.com/v/54142">Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo.</a> J. Vis. Exp. (2016).
This video outlines the protocol for electrophysiological recording of photoreceptor activity in the eye of an immobilized Drosophila, enabling precise measurement of voltage changes in response to light stimuli.

果蝇视网膜光感受器对光刺激的电压响应的电生理记录

1. Recording from R1-R6 Photoreceptors 

	Always be grounded when operating the microelectrode amplifier (for example by touching the metal surface of the ...

JoVE Encyclopedia of Experiments

Neurophysiology

Electrophysiology Techniques

Investigating Prolonged Depolarizing Afterpotential (PDA) in Drosophila Photoreceptors

Source: Gutorov, R., et al. <a href="https://app.jove.com/v/63514">Electrophysiological Methods for Measuring Photopigment Levels in Drosophila Photoreceptors.</a> J. Vis. Exp. (2022).
This video demonstrates an experimental protocol to investigate the prolonged depolarizing afterpotential (PDA) in white-eyed Drosophila. The fly&#39;s eye is exposed to intense blue light pulses to convert the photopigment rhodopsin to metarhodopsin, initiating a signaling cascade that opens positive ion channels and causes membrane depolarization. Because blue light prevents the reconversion of metarhodopsin to rhodopsin, the continuous influx of positive ions results in sustained depolarization, known as the prolonged depolarizing afterpotential (PDA).

研究果蝇光感受器中的延长去极化后电位 （PDA）

1. Measuring the prolonged depolarizing afterpotential (PDA) using the electroretinogram

	Suitable rearing conditions for Drosophila melanogaster ...

Stimulation and Modulation Techniques

Whole-Cell Voltage Clamp Recordings from Drosophila Photoreceptors

Source:&#160; Katz, B., et al., <a href="https://app.jove.com/v/55627">Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors.</a> J. Vis. Exp. (2017)
The video demonstrates recording electrical activity in the photoreceptor neurons of Drosophila&#39;s ommatidium by establishing a whole-cell configuration. It highlights the activation of transient receptor potential channels by light pulses, resulting in measurable quantum bumps.

来自果蝇光感受器的全细胞电压钳记录

NOTE: During all of the following steps, use only dim red light illumination and ensure that the ommatidia&#39;s exposure to light is minimal (i.e., work ...

全细胞电压钳记录的电生理方法

摘要

探索更多视频

此视频中的章节

从整个成年果蝇大脑细胞记录

从巨纤维通路的电生理记录 D。果蝇

电生理方法记录从果蝇幼虫NMJ突触潜力

膜片钳记录在内耳毛细胞的分离斑马鱼

在电生理记录果蝇胚胎

为记录细胞内电压响应电法

测量果蝇光感受器中光色素水平的电生理学方法

果蝇视网膜光感受器对光刺激的电压响应的电生理记录

研究果蝇光感受器中的延长去极化后电位（PDA）

来自果蝇光感受器的全细胞电压钳记录

全细胞电压钳记录的电生理方法

摘要

探索更多视频

此视频中的章节

从整个成年果蝇大脑细胞记录

从巨纤维通路的电生理记录 D。果蝇

电生理方法记录从果蝇幼虫NMJ突触潜力

膜片钳记录在内耳毛细胞的分离斑马鱼

在电生理记录果蝇胚胎

为记录细胞内电压响应电法

测量 果蝇 光感受器中光色素水平的电生理学方法

果蝇视网膜光感受器对光刺激的电压响应的电生理记录

研究果蝇光感受器中的延长去极化后电位 （PDA）

来自果蝇光感受器的全细胞电压钳记录

测量果蝇光感受器中光色素水平的电生理学方法

研究果蝇光感受器中的延长去极化后电位（PDA）