我们感谢已故的鲁迪·林堡制造的万向臂周长，金佰利贾米森，马修麦克亨利和拉朱Metherate借给我们设备和Almut Kelber和有川太郎，以资鼓励。这项工作是由美国国家科学基金会（NSF）研究生研究奖学金给KJM和美国国家科学基金会资助IOS-1257627亚行支持

所有动物的人道待遇越好。昆虫被运来自哥斯达黎加昆虫供应，哥斯达黎加蛹。 1. 袖蝶属蛹护理<ol><li>杭所有蛹用昆虫针湿盒间隔2-3厘米。 </li><li>羽化后，让翅膀晾干然后继续蝴蝶存活至少1天在湿盒和记录每天前喂稀释的蜂蜜水溶液。 <ol><li>加水稀释蜂蜜体积约20％的蜂蜜水溶液，倒入浅培养皿。 </li><li>把个人的蝴蝶培养皿中，一个接一个。在触摸与它们的前跗溶液，蝴蝶将自动延长他们proboscides和从培养皿饮用。如果他们的长鼻不自动扩展，使用产钳拉出来长鼻，并介绍给蜂蜜的解决方案。 </li></ol></li></ol> 2.光学跟踪，校准和测量电实验光照条件的键相<ol><li>将带有外壳和通用电源和至少一米长，提供明亮的白色光的表的一个端部连接的聚光透镜组件150W的氙弧灯。 注意:氙弧灯产生极其明亮的光与强烈的紫外线的强度。防护眼镜应该始终佩戴并指示由制造商以防止因与大气中的氧气的UV光相互作用的臭氧累积应使用灯。 </li><li>设置在长度的光道一米离开壳体组件通过（ 图1）的光。 <ol><li>在光道与相距近似距离上按以下顺序发生:1）一个凸二氧化硅或石英透镜从冷凝器组件40厘米，2）的中性密度滤光轮（没有在光路进一步沿着当前滤波器）22厘米赛道上，3）驱动单元快门从ND滤镜14厘米S，4）的凹二氧化硅或石英透镜紧邻快门以下，和5）的准直光束探针还6厘米沿着轨道的远端。 </li><li>贴上直径600微米光缆准直光束探测器。 注意:根据光强度，5-10毫米直径的光纤电缆可能需要足够的光传送到其他棉片和可以取代此。 </li><li>调整的距离，高度和每个光学元件的角度，使光束离开组件是在最高强度可能。 </li><li>作为光学跟踪元素可与不同的应用程序略有不同，请确保所有元素在UVA和可见光范围（315-700纳米）的光。 </li></ol></li><li>一旦光道被组装时，测量通过设置使用分光计传递的光。校准分光计第一使用校准灯与已​​知的光谱和制造商的软件。 注意:我们描述了以下设置使用海洋光学的产品为清楚起见，但其他厂商（ 例如 ，Avantes公司）销售的产品相媲美。 <ol><li>开始测量前打开钨校准灯至少45分钟。 </li><li>为了校准，通过USB连接光谱仪到计算机上安装了相关软件。然后通过紫外可见发射余弦校正连接分光计钨校准灯。 </li><li>从"文件"选项卡中选择"新建绝对辐射测量"，然后选择谱仪"源"。 </li><li>按照提示创建一个新的校准"CAL"文件。出现提示时，加载提供的数据文件钨校准灯在可见光范围（300-800纳米）到软件，它可以自动计算从光谱仪输出校正频谱已知的频谱。 </li><li>保存校准文件。当initi加载此文件alizing使用光谱仪光光谱以后的测试软件。 </li></ol></li><li>一旦光谱仪校准，使用此记录从实验装置的光的光谱。此后被打开软件时，选择"新建绝对辐射测量"并加载以前保存的校准文件。接下来所有遮光到光谱仪拍摄暗光谱。 <ol><li>与目前的测量所需的试验光条件的光谱仪，调整积分时间（4毫秒），平均扫描（5），和棚车宽度（5），所以频谱正确缩放和平滑。保留设置同样为所有光谱测量，因此，从不同的测量的光强度可比拟。 </li></ol></li><li>测量为未衰减白色光光谱，对于在实验过程中所使用的所有中性密度过滤器，并为每个带通干涉滤光器（ 图2）。 <ol><li>中号通过从步骤2.2.2至分光计固定用光缆的自由端easure而不在光路中的任何过滤器的白光光谱。与来自步骤2.3加载的校准文件，使用光谱仪的软件作为文本文件保存白光光谱。 注意:光谱保存为文本文件列出的波长（X坐标）中的一列和光在第二列中的强度（y坐标），使得数据可以被加载到电子表格用于步骤2.6。 </li><li>使用相同的设置作为步骤2.5.1，由旋转中性密度（ND）滤光轮中的光道在实验过程中使用的每个光密度（OD）（0-3.5 OD）记录的光谱，并保存为文本文件每个OD。 </li><li>使用相同的设置步骤2.5.1，将10纳米半带宽干扰滤波器逐个放入光路，并记录每个滤光片观察频谱。重复此过程为每个41种不同的干扰滤波器W¯¯第i个的峰值透射率间隔每10纳米从300至700纳米。间隔更远（20纳米）过滤器是大多数应用上可接受的（对于光谱，参见图2）。 </li></ol></li><li>正确对中的光的强度差时，干涉滤光器被放置在光路中。每个干涉滤光器允许光子不同总数来传递，和一些过滤器的低传输使得难以进一步衰减强度，使所有过滤器允许光子数目相等。 <ol><li>计算每个10nm的带宽干涉滤光器的相对强度（I），在表达式I = T /秒，其中T是各为10nm干涉滤光器的光谱曲线下面积（从2.5.3）求解我和s为最大绝对辐射白色光中的每一个过滤器（参见图2为在520纳米的示例）的峰值波长（γ保存的文本文件的自2.5.1值）。 </li><li>所有除以计算intensit通过在2.6.1计算出的最大强度值独立实体正常化为一个，并采取用作施加于各波长的原始灵敏度的校正因子的相对归一化值的倒数（参见步骤6.4）。 </li></ol></li><li>一组实验之前只有一次执行步骤2.1至2.6。以上的实验过程中周期性地记录在明亮的光线和中性密度滤光片的氙弧灯的绝对辐射，以确保光刺激的强度不发生变化。 </li><li>在实验的过程中，如果通过干涉滤光片的透射光的任何细胞反应接近最大响应振幅，使用ND滤镜来衰减信号。如果实验期间使用ND滤镜，占光谱灵敏度的计算过程中强度的相应下降。 </li><li>设置光道，标定和过滤器天或数周的实验开始之前。保持器覆盖，防止灰尘堆积。 </li></ol> 3.录音设备安装<ol><li>通过法拉第笼喂养用于校准的相同光纤电缆和装入一个测角器上，如万向臂周长（参见图3为图）。电缆将是从检体的眼约10厘米的距离。 </li><li>放置一个金属舞台上的振动隔离表一个电极固定器直接安装在一个显微控制舞台上方（ 图4）。放置万向臂使试样的头部是在由臂的旋转运动产生的球面的中心。 </li><li>使用细胞内的前置放大器系统，它包括一个放大器（法拉第笼外）和前置放大器（探头，法拉第笼内部的制备附近）安装在其上放置样品将被放置在金属级的探头。 <ol><li>通过同轴电缆连接到探头BNC连接。拆分在同轴电缆的另一端开放只有针尖和电缆的外金属护套从内部线分开。 </li><li>焊接外鞘（保持在地电位）到一个绝缘铜导线的一端与另一端的鳄鱼夹。这鳄鱼夹会附着在试样平台（步骤5.1.4）在金属参比电极。 </li><li>焊接同轴电缆的内部钢丝，以薄的银线，以作为记录电极。这个导线应足够薄在步骤5.2.3至被送入溶液填充的玻璃电极。 </li></ol></li><li>放置连接于摆臂和碱法拉第笼外的板凳立体显微镜，因此，它可以摆动到降低电极进入眼睛，进出再次一旦电极是在眼转回。 </li><li>确保所有金属里面的法拉第笼正确接地。 </li><li>外法拉第笼，装上preamplif呃到50-60 Hz的噪声减速机（可选）的输入和输出连接到使用BNC T-适配器示波器的一个通道。 </li><li>使用T-适配器的另一端，连接通过示波器传递到硬件中的一个信道的信号。通过USB电缆，这将允许记录有前置放大器响应由软件的计算机上读取附加这个硬件的计算机。 </li><li>附上从光道的快门驱动到使用另一种T型适配器示波器的第二信道和这个连接到脉冲发生器，将控制传递到眼睛（步骤5.5）的光闪烁的频率和持续时间。 注:该钻机本身的设置应该只需要进行一次。打破这里，直到准备开始录音。 </li></ol> 4.准备上记录的天<ol><li>实验前打开氙气灯至少45分钟，然后打开玻璃微电极拉马至少30分钟前PUL凌玻璃电极。 </li><li>打开所有录音设备（快门，放大器，噪声消除器，脉冲发生器，示波器，和数据采集硬件），确保快门被默认所以没有光通过光纤电缆封闭。 </li><li>拉细硼硅（或铝）的玻璃微电极（100-250兆欧电阻是理想的）用玻璃微电极拉马。只有被拉到几个小时内使用玻璃电极。 </li><li>回填用3M氯化钾（KCl）中的电极。请注意，此溶液可以根据研究人员的需要进行修改， 例如 ，染料注入。 </li></ol> 5.标本准备和录制过程<ol><li>准备标本<ol><li>所以头部是不动，并从该管的一端突出贴上的小塑料管与热蜡内的个体蝴蝶。蜡向下长鼻，天线，和翼（ 图5）。 </li><li>按住ŧ他用一块干一块蜡的腹部和保持放置湿纸巾腹部背后的管内加湿管。确保标本是完全不动。 </li><li>用一小块蜡到一个小平台，一个连接到一个磁性底座一个球窝关节安装在管。 </li><li>在解剖显微镜下，插入直径0.125毫米的银线成可经由口器的头部被用作参考电极。实验前，永久修复线到平台以这样的方式，一旦平台放置在台上用于记录在步骤3.3.2铜导线可以夹在到它。 </li><li>一旦参考电极是在一个合适的位置时，它可以通过快速熔化，然后在导线周围冷却蜡被保持在适当位置。 </li><li>使用易碎的碳钢刀片，用刀片保持器的刀片的柄部和折断的小片用于切割角膜。 </li><li>切一小口（〜10 ommatidi在直径）在左角膜使用剃刀刃和封孔用凡士林以防止干燥。 </li></ol></li><li>一旦角膜被切断，插入记录电极到眼睛中尽快因为血淋巴中的眼睛会很快硬化，使之无法插入的电极。如果可能的话在钻机在记录将于执行清扫。 注意:凡士林不应在眼睛的其余部分涂因为这将散焦光学系统。 <ol><li>如果尚未在舞台上，安装的检体和平台放置到在记录钻机阶段。从步骤3.3.2使用鳄鱼夹将试样平台上的参考电极连接探头地线。 注意:如果可能使用红色滤光器照亮的动物。 </li><li>使用带有鹅颈附件短暂点亮一个立体镜下检体，同时降低了记录电极到眼睛中的光源。 </li><li>在SERT连接到探头从步骤3.3.3成在玻璃微电极的背面的氯化钾溶液中的银线。安装电极支架上的玻璃电极。 </li><li>调整电极支架使微电极正上方先前切割角膜，对角膜上方一毫米的洞。降低使用显微直到一个电路被完成，如由在电位（mV）在示波器上的大变化的微电极插入眼睛。 </li></ol></li><li>一旦进入眼睛，挥动法拉第笼外的体视镜，并关闭光源照射样品。所以眼睛变得暗适应的房间应避光保存。 </li><li>通过从放大器施加1 nA的电流，并注意到在电压的变化检查电极的电阻。电阻通常应该在100-250兆欧之间的范围内。较高电阻指示堵塞或电极的弯曲，和ELECTR的低电阻的颂破损。 </li><li>激活脉冲发生器，以便在快门打开允许光的具有50毫秒的持续时间的闪光每0.5秒，并允许它继续闪烁在实验的持续时间。 <ol><li>调整脉冲发生器所以允许多达50毫秒持续时间的闪烁。闪烁之间的持续时间和0.5秒的停顿保持标本尽可能接近在实验过程中适应尽可能暗。五十毫秒靠近最短闪光时间，这将引起相同的幅度响应更长闪光持续时间。 </li><li>在开始时和在实验（步骤5.16）的端部都重新测量响应。在约二十分钟实验的过程中，这些闪光灯设置不随时间降解的响应。不同的棉片可能需要调整这些闪光灯设置。 </li></ol></li><li>使光缆朝向眼睛的万向臂的位置。 </li><li>检查每个灯闪电压变化的示波器。在电压的负变化表示该电极还没有进入的小区。 </li><li>移动万向臂检体周围，直到它被定位成一角度在其中有一个最大的电压响应的眼睛。 </li><li>旋转显微来回，导致电极的非常小的垂直运动在两个方向上同时轻敲该电极座的碱或使用的前置放大器蜂鸣音功能。继续进行微调，直到出现在示波器（ 图6）去极化光响应。 </li><li>再次调整万向臂找到发病的，其中一道闪光产生最大的去极化信号的角度。使用显微小的调整，并使用巴斯函数的放大器作为需要确保该电极稳定地记录该小区和它会留在整个实验的细胞（见步骤5.11）。 </li><li>一旦建立稳定，开始RECO录制。一个稳定的记录应该不大，以静息电位，低背景噪声，并且始终如一大去极化应答没有变化（至少10:1的信噪比）。 <ol><li>运行计算机上的软件，并开始了"新实验"，这将打开一个弹出窗口有四个通道。 </li><li>调整电压规模的软件窗口500毫伏的右上角。第一通道将显示来自实时电极记录的响应，而第二个信道将记录由函数发生器产生的方波，如果信号通过示波器馈送到数据采集硬件，表示当快门打开。另外两个通道是不必要的。 </li><li>点击"开始"的右下角开始记录，并且允许软件在实验的持续时间运行。调节的X（时间）和Y（电压）的变焦轴，使得响应是清楚的。 </li></ol></li><li>首先，白光，最多记录用ND滤镜轮10个人响应为3.5 OD（约5-10秒）。 </li><li>下一个记录相同数量的反应在3.3的OD，然后3.1，3.0，2.5，2.3，2.1，等等，每一组合，直到0.0的OD。这些反应振幅为ND过滤器系列将在第6节提供响应登陆强度曲线如果出现脱色，在实验过程中，使用明亮的刺激较少闪烁。 </li><li>记录单元在所有波长下的响应，使用干涉滤光器。 <ol><li>首先找到峰值波长。没有在光路（0.0 OD）ND滤镜，放置一个紫外线发射滤波器在光路上，并简要观察响应幅度。带有蓝色发射器，绿色发射滤波器，和一个红色发射滤波器，这应该得到在峰值响应将是一些想法重复。 </li><li>在大约使用过滤器350，450，550，650纳米找到峰值灵敏度的一般地区步5.14.1。确切的波长并不在这个初始搜索阶段重要，因为所有波长将被记录在下一个步骤。如果估计存在峰值敏感性，或以前已记录的，使用一种称为波长快速识别峰值响应。 </li><li>一旦峰值响应或接近它是确定的，在该波长为10回应（大约5秒）的记录。 </li><li>在10纳米的步骤在峰值响应，记录与其他干扰滤波器的波长记录，从300-700纳米之后。从峰值开始，通过由一个从光路径中的一个交换过滤器出来（ 例如 ，如果峰值响应是在520nm，在该波长，然后再为510nm记录响应，随后530走出朝向两个短和更长的波长纳米，500纳米，540纳米，490纳米，550纳米，依此类推，直到没有没有响应）。 </li><li>允许每个滤波器多达10反应（每5秒）。当交换干扰滤波器，使细胞RESPOND 1-2闪烁白光而不在光路中，有利于监测峰值响应是否随时间降解的任何过滤器。减少的响应的数量，或者如果发生漂白增加OD值。 </li></ol></li><li>如果在任何干涉滤光器的响应过于接近，在0的OD在白光下的最大响应，然后用ND滤镜衰减。干涉滤光器，并在该试验中使用的光缆的尺寸大大衰减的光的强度，因此，通常不需要ND滤镜。 </li><li>如果记录仍然围绕峰值响应，其用作用于确认之前的响应幅度的pseudoreplicate并有助于确保的反应不会随时间劣化稳定，重新记录波长。一旦所有的波长都记录下来，再记录在ND系列下的反应，如在步骤5.12。 </li><li>在录像过程中对软件完成后点击"停止"，并保存记录进行分析。 </li><li>实验后，通过冷冻或冷却几分钟，随后通过快速切断的头部和破碎胸廓牺牲个人。 </li><li>关闭所有设备。如果需要做分析之前在这里休息。 </li></ol> 6.光谱灵敏度分析<ol><li>与用于记录的原始响应的软件，计算的10个体反应的平均响应振幅在ND串联，并为每个干涉滤光器的每个滤光器。 </li><li>创建响应对数强度（VlogI）从ND滤光器系列记录在步骤5.12-5.13（ 图7）的功能。通过绘制在X轴上强度（OD值），和响应的对数单位来在y轴上的每个强度做到这一点。 <ol><li>以导出在不同波长的细胞的光谱灵敏度，通常适合中落拉什顿方程从步骤6.2中的数据，并使用这个方程涉及不同波长吨实验获得的光谱响应以引发一个恒定波长根据该响应（在这种情况下，白光）需要O相对光子。 注:中落拉什顿方程为:V / V 最大 = I N /（I N + K n），其中我是刺激强度，V是响应振幅，V max是最大响应幅值，K为刺激强度给予半2V 以下 ，且n是指数斜率。各种方法可用于以适合该方程的VlogI数据，包括曲线拟合软件，或基于代码的统计软件包。 </li><li>为了适应使用简单的计算和电子表格程序纳卡-拉什顿方程，变换VlogI响应数据的每个刺激强度:登录[（V 最大 / V） - 1]。然后对变换后的数据进行线性回归以获得最佳拟合的直线的方程。 注:v MAX必须比任何测量的响应越大;保持这种一致性，这种方法估计2V 以下为1％GRE亚特比最高测量的响应。 </li><li>从回归直线的方程，估算通过采取负斜率的指数（n），并登录（K）= y截距/ N。 </li></ol></li><li>一旦参数为2V 以下 ，正和K已经估计，确定由在给定波长的测量的光谱响应堵塞如（Ⅴ）以引发在各波长下的细胞的光谱响应需要的光子和解决的相对数对于一</li><li>通过对每个干涉滤光器（来自步骤2.4.3）在各波长下的校正因子相乘，从步骤6.3所计算的刺激强度（I）。 </li><li>为了得到灵敏度，使他们可以比较所有的强度必须与在V日志-I曲线。通过与每个波长强度½2V 以下或K做到这一点，在步骤6.2.3计算。 <ol><li>减去每个波长校正强度（步骤6.4）从K. </li><li>然后，对每个波长强度，添加此"距离K"值，K和乘以（-1）。 </li><li>下一个带由在该系列的每个波长增加的最低数据点的绝对值阳性的所有数据点。 </li></ol></li><li>通过采取所有新计算强度的倒数从步骤6.5.1查找在每个波长的灵敏度。转换数据，以使灵敏度光谱落在0和1之间。 </li><li>从相同类型的平均一个以上的小区记录的最后应答和情节与标准误差条或95％置信区间（ 图8）之后。 </li></ol>

作者宣称，他们没有竞争的经济利益。

细胞内记录可以是一个很难掌握的技术，由于涉及很多技术步骤。对于成功的试验几个要点必须加以考虑。首先，对具有在其上进行的实验的合适的振动隔离表是重要的。许多研究人员使用的空气的表，从而彻底桌面从基座分离，得到优异的振动隔离。我们设置包括在上面沙箱，在其中放置在显微/电极架/样品台设备厚厚的大理石桌子。这是一种有效的和更经济的替代空气表，特别是如果进入内?...

细胞如神经元的电性能通过测量穿过细胞膜的离子流在电压或电流的变化观察到的。各种电生理技术已经开发了用于测量细胞的生物电活动。在动物的眼睛中发现的神经元接触，而其电路是较少复杂比在大脑中，使这些细胞的良好候选为电生理研究。在眼电生理常见的应用包括电图（ERG）1,2和微电极细胞内记录。 ERG涉及将一个电极或动物的眼，施加光刺激，并测量作为所有附近小区3-6的响应的总和在电压的变化。如果一个人在描述个人感光细胞的光谱灵敏度特别感兴趣，经常多种细胞类型同时在不同的优势，一个给定的刺激作出反应;因此可能难以确定来自ERG数据特定细胞类型的敏感性，特别是如果有几种不同类型的眼睛的光谱相似感光细胞。一个可能的解决办法是建立转基因果蝇与在大多数R1-6细胞的眼睛表示有兴趣感光（视蛋白）基因，然后执行尔格7。该方法的潜在缺点包括不向感光体蛋白8的低表达，并用于转基因动物的产生和筛选时间长帧。与种类较少光谱不同的光感受器的眼睛，用彩色滤光片的眼睛的适应可与降低一些细胞类型的ERG的贡献，从而使光谱灵敏度最大值9的估计帮助。 细胞内记录是另一种技术，其中一个精细电极刺入细胞和施加的刺激。电极仅记录逐张idual细胞的反应，使得从记录和分析多个单个细胞可以产生生理不同的细胞类型10-14的具体的灵敏度。虽然我们的协议侧重于光谱灵敏度的分析，细胞内的尖锐的电极记录的基本原理是修改其他应用程序。使用不同的制备试样，例如，和使用锋利的石英电极，可以在大脑从视叶或其他区域更深记录，这取决于所提出的问题。例如，从个人感光细胞15的响应时间，在视神经细胞活性裂片16（椎板，髓质或小叶17），脑18或其他神经节19还可以记录有类似的技术，或颜色刺激可以与偏振20来代替-22或运动刺激23,24。 光转导，该方法通过使光能量被吸收并转换成电化学信号，是共同的几乎所有现今动物门25一个古老的性状。在感光细胞发现，负责发起可视光转视色素视紫红质是。在所有动物中视紫红质是由视蛋白的蛋白质，所述7次跨膜G蛋白偶联受体家族的成员，和相关联的发色团是从视网膜或类似的分子26,27衍生的。视蛋白的氨基酸序列和发色团结构影响视紫红质，以不同波长的光的吸光度。当光子被发色团吸收的视紫红质被激活，启动了在最终导致膜结合离子通道28的开口的细胞的G蛋白级联。不像大多数神经元，感光细胞经历，可以作为一个相对变化响应的幅度不断变化的光刺激来衡量分级的潜在变化。通常，一个给定的感光器类型表明只有一个视蛋白基因（虽然存在例外8,10,29-31）。复杂的色觉，在许多脊椎动物和节肢动物发现的那种，与感光细胞的数百或数千各表达一种或偶尔多个视紫红质类型的复合眼实现。可视信息通过经由复杂的下游神经信号在眼睛和大脑比较在感光镶嵌反应，导致完全的颜色和运动图像的感知捕获。 测定感光细胞通过细胞内记录不同波长的光的原始应答后，就可以计算出其光谱感光度。此计算是基于Univariance的原理，其中指出，感光细胞的反应是依赖于它吸收的光子的数量，但不能在它吸收32的光子的特定性质。任何光子是ABSO通过视紫红质rbed会诱发同一种反应。在实践中，这意味着，一个细胞的原始响应振幅由于或者增加光强度（更多的光子吸收）增加， 或者在波长朝其峰值灵敏度的移位（视紫质的较高的概率吸收该波长）。我们使在公知的强度和相同波长的响应在不同波长和相同的强度，但未知相对感光度与细胞反应利用这一原理。细胞类型通常由波长在哪些其灵敏度峰识别。 在这里，我们显示了细胞内记录和蝴蝶的眼睛光感受器的光谱灵敏度分析的一种方法，其重点是使这种方法更广泛的研究团体更容易获得。虽然细胞内记录在文献中仍然普遍，尤其是相对于昆虫的色觉，我们已经发现塔的材料和方法吨描述通常太短，以允许该技术的再现。我们目前在视频格式的方法，允许其更容易复制的目的。我们还使用容易获得和负担得起的设备描述技术。我们解决经常不报共同注意事项，优化一个新的和复杂的技术，当它慢下来研究。

<table><tbody><tr><td>Butterfly pupae</td><td></td><td></td><td>Several local species available, need USDA permits for shipping. Carolina Bio Supply has several insect species that may be ordered within the U.S. without the need for additional permits</td></tr><tr><td>Large plastic cylinder</td><td></td><td></td><td>Any chamber that remains humidified will work</td></tr><tr><td>Insect pins, size 2</td><td>BioQuip</td><td>1208B2</td><td></td></tr><tr><td>100% Desert Mesquite Honey</td><td>Trader Joe&#039;s</td><td></td><td>Any honey or sucrose solution will work</td></tr><tr><td>Xenon Arc Lamp</td><td>Oriel Instruments</td><td>66003</td><td>Oriel is now a part of Newport Corporation</td></tr><tr><td>Universal Power Supply</td><td>Oriel Instruments</td><td>68805</td><td>Oriel is now a part of Newport Corporation</td></tr><tr><td>Optical Track</td><td>Oriel Instruments</td><td>11190</td><td>Oriel is now a part of Newport Corporation</td></tr><tr><td>Rail Carrier, Large (2x)</td><td>Oriel Instruments</td><td>11641</td><td>Oriel is now a part of Newport Corporation</td></tr><tr><td>Rail Carrier, Small (4x)</td><td>Oriel Instruments</td><td>11647</td><td>Oriel is now a part of Newport Corporation</td></tr><tr><td>Thread Adaptor, 8-32 Male to 1/4-20 Male, pack of 10</td><td>Newport Corporation</td><td>TA-8Q20-10</td><td></td></tr><tr><td>Optical Mounting Post, 1.0 in., 0.5 in. Dia. Stainless, 8-32 &amp;amp; 1/4-20 (5x)</td><td>Newport Corporation</td><td>SP-1</td><td></td></tr><tr><td>No Slip Optical Post Holder, 2 in., 0.5 in. Diameter Posts, 1/4-20 (5x)</td><td>Newport Corporation</td><td>VPH-2</td><td></td></tr><tr><td>Fixed lens mount, 50.8 mm</td><td>Newport Corporation</td><td>LH-2</td><td></td></tr><tr><td>Fixed lens mount, 25.4 mm</td><td>Newport Corporation</td><td>LH-1</td><td></td></tr><tr><td>Condenser lens assembly</td><td>Newport Corporation</td><td>60006</td><td></td></tr><tr><td>Convex silica lens, 50.8 mm</td><td>Newport Corporation</td><td>SPX055</td><td></td></tr><tr><td>Six Position Filter Wheel, x2</td><td>Newport Corporation</td><td>FW1X6</td><td></td></tr><tr><td>Filter Wheel Mount Hub</td><td>Newport Corporation</td><td>FWM</td><td></td></tr><tr><td>Concave silica lens, 25.4 mm</td><td>Newport Corporation</td><td>SPC034</td><td></td></tr><tr><td>Collimator holder</td><td>Newport Corporation</td><td>77612</td><td></td></tr><tr><td>Collimating beam probe</td><td>Newport Corporation</td><td>77644</td><td></td></tr><tr><td>Ferrule Converter, SMA Termination to 11 mm Standard Ferrule</td><td>Newport Corporation</td><td>77670</td><td>This adapter allows the fiber optic to fit into the collimator holder&amp;nbsp;</td></tr><tr><td>600 &amp;mu;m diameter UV-vis fiber obtic cable</td><td>Oriel Instruments</td><td>78367</td><td>Oriel is now a part of Newport Corporation</td></tr><tr><td>Shutter with drive unit</td><td>Uniblitz</td><td>100-2B</td><td></td></tr><tr><td>UV Fused Silica Metallic ND Filter, 0.1 OD</td><td>Newport</td><td>FRQ-ND01</td><td></td></tr><tr><td>UV Fused Silica Metallic ND Filter, 0.3 OD</td><td>Newport</td><td>FRQ-ND03</td><td></td></tr><tr><td>UV Fused Silica Metallic ND Filter, 0.5 OD</td><td>Newport</td><td>FRQ-ND05</td><td></td></tr><tr><td>UV Fused Silica Metallic ND Filter, 1.0 OD</td><td>Newport</td><td>FRQ-ND10</td><td></td></tr><tr><td>UV Fused Silica Metallic ND Filter, 2.0 OD</td><td>Newport</td><td>FRQ-ND30</td><td></td></tr><tr><td>UV Fused Silica Metallic ND Filter, 3.0 OD</td><td>Newport</td><td>FRQ-ND50</td><td></td></tr><tr><td>LS-1-Cal lamp</td><td>Ocean Optics</td><td>LS-1-Cal</td><td></td></tr><tr><td>Spectrometer</td><td>Ocean Optics</td><td>USB-2000</td><td></td></tr><tr><td>SpectraSuite Software</td><td>Ocean Optics</td><td></td><td></td></tr><tr><td>Interference bandpass filter, 300 nm&amp;nbsp;</td><td>Edmund Optics</td><td>67749</td><td></td></tr><tr><td>Interference bandpass filter, 310 nm&amp;nbsp;</td><td>Edmund Optics</td><td>67752</td><td></td></tr><tr><td>Interference bandpass filter, 320 nm&amp;nbsp;</td><td>Edmund Optics</td><td>67754</td><td></td></tr><tr><td>Interference bandpass filter, 330 nm&amp;nbsp;</td><td>Edmund Optics</td><td>67756</td><td></td></tr><tr><td>Interference bandpass filter, 340 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65614</td><td></td></tr><tr><td>Interference bandpass filter, 350 nm&amp;nbsp;</td><td>Edmund Optics</td><td>67757</td><td></td></tr><tr><td>Interference bandpass filter, 360 nm&amp;nbsp;</td><td>Edmund Optics</td><td>67760</td><td></td></tr><tr><td>Interference bandpass filter, 370 nm&amp;nbsp;</td><td>Edmund Optics</td><td>67761</td><td></td></tr><tr><td>Interference bandpass filter, 380 nm&amp;nbsp;</td><td>Edmund Optics</td><td>67762</td><td></td></tr><tr><td>Interference bandpass filter, 390 nm&amp;nbsp;</td><td>Edmund Optics</td><td>67763</td><td></td></tr><tr><td>Interference bandpass filter, 400 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65732</td><td></td></tr><tr><td>Interference bandpass filter, 410 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65619</td><td></td></tr><tr><td>Interference bandpass filter, 420 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65621</td><td></td></tr><tr><td>Interference bandpass filter, 430 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65622</td><td></td></tr><tr><td>Interference bandpass filter, 440 nm&amp;nbsp;</td><td>Edmund Optics</td><td>67764</td><td></td></tr><tr><td>Interference bandpass filter, 450 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65625</td><td></td></tr><tr><td>Interference bandpass filter, 460 nm&amp;nbsp;</td><td>Edmund Optics</td><td>67765</td><td></td></tr><tr><td>Interference bandpass filter, 470 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65629</td><td></td></tr><tr><td>Interference bandpass filter, 480 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65630</td><td></td></tr><tr><td>Interference bandpass filter, 492 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65633</td><td></td></tr><tr><td>Interference bandpass filter, 500 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65634</td><td></td></tr><tr><td>Interference bandpass filter, 510 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65637</td><td></td></tr><tr><td>Interference bandpass filter, 520 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65639</td><td></td></tr><tr><td>Interference bandpass filter, 532 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65640</td><td></td></tr><tr><td>Interference bandpass filter, 540 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65642</td><td></td></tr><tr><td>Interference bandpass filter, 550 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65644</td><td></td></tr><tr><td>Interference bandpass filter, 560 nm&amp;nbsp;</td><td>Edmund Optics</td><td>67766</td><td></td></tr><tr><td>Interference bandpass filter, 570 nm&amp;nbsp;</td><td>Edmund Optics</td><td>67767</td><td></td></tr><tr><td>Interference bandpass filter, 580 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65646</td><td></td></tr><tr><td>Interference bandpass filter, 589 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65647</td><td></td></tr><tr><td>Interference bandpass filter, 600 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65648</td><td></td></tr><tr><td>Interference bandpass filter, 610 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65649</td><td></td></tr><tr><td>Interference bandpass filter, 620 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65650</td><td></td></tr><tr><td>Interference bandpass filter, 632 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65651</td><td></td></tr><tr><td>Interference bandpass filter, 640 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65653</td><td></td></tr><tr><td>Interference bandpass filter, 650 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65655</td><td></td></tr><tr><td>Interference bandpass filter, 660 nm&amp;nbsp;</td><td>Edmund Optics</td><td>67769</td><td></td></tr><tr><td>Interference bandpass filter, 671 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65657</td><td></td></tr><tr><td>Interference bandpass filter, 680 nm&amp;nbsp;</td><td>Edmund Optics</td><td>67770</td><td></td></tr><tr><td>Interference bandpass filter, 690 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65659</td><td></td></tr><tr><td>Interference bandpass filter, 700 nm&amp;nbsp;</td><td>Edmund Optics</td><td>67771</td><td></td></tr><tr><td>Faraday cage</td><td></td><td></td><td>Any metal structure will work that can be grounded and that fits the experimental setup.</td></tr><tr><td>Stereomicroscope, 6X, 12X, 25X, 50X magnification</td><td>Wild Heerbrugg</td><td>Wild M5</td><td>Any Stereomicroscope will do</td></tr><tr><td>Microscope stand with swinging arm and heavy base</td><td>McBain Instruments</td><td></td><td>Any heavy base with arm will do</td></tr><tr><td>Cardan arm</td><td></td><td></td><td>Custom built, See &lt;strong&gt;Figure 4&lt;/strong&gt;</td></tr><tr><td>Fiber-lite high intensity illuminator</td><td>Dolan-Jenner</td><td>MI-150</td><td>For lighting specimen</td></tr><tr><td>Fiber-lite goose-neck light guide</td><td>Dolan-Jenner</td><td>EEG 2823</td><td>Any goose-neck light guide will do</td></tr><tr><td>Marble table</td><td></td><td></td><td></td></tr><tr><td>Raised wooden table</td><td></td><td></td><td>Hole should be cut through this table so that the sandbox can rest on the marble table underneath</td></tr><tr><td>Wooden box filled with sand</td><td></td><td></td><td>custom built, any box with sand</td></tr><tr><td>Manipulator</td><td>Carl Zeiss - Jena</td><td></td><td></td></tr><tr><td>Electrode holder</td><td></td><td></td><td></td></tr><tr><td>Specimen stage</td><td></td><td></td><td></td></tr><tr><td>Alligator clip wires for grounding</td><td></td><td></td><td></td></tr><tr><td>Insulated copper wire</td><td></td><td></td><td></td></tr><tr><td>Silver wire, 0.125 mm diameter</td><td>World Precision Instruments</td><td>AGW0510</td><td></td></tr><tr><td>BNC cables</td><td></td><td></td><td></td></tr><tr><td>Preamplifier with headstage</td><td>Dagan Corporation</td><td>IX2-700</td><td></td></tr><tr><td>Humbug Noise reducer</td><td>Quest Scientific</td><td>Humbug</td><td></td></tr><tr><td>Oscilloscope, 30 MHz, 2 CH, Dual Trace, Alt-triggering, without probe</td><td>EZ Digital</td><td>os-5030</td><td></td></tr><tr><td>BNC T-adapter</td><td></td><td></td><td></td></tr><tr><td>Powerlab hardware 2/20</td><td>ADI instruments</td><td>ML820</td><td></td></tr><tr><td>Labchart software</td><td>ADI instruments</td><td>Chart 5</td><td></td></tr><tr><td>10 MHz Pulse Generator</td><td>BK Precision</td><td>4030</td><td></td></tr><tr><td>Glass pipette puller</td><td>Sutter Instruments</td><td>P-87</td><td></td></tr><tr><td>Borosillicate glass capillaries with filament</td><td>World Precision Instruments</td><td>1B120F-4</td><td></td></tr><tr><td>Potassium chloride, 3 M</td><td></td><td></td><td></td></tr><tr><td>Slotted plastic tube</td><td></td><td></td><td></td></tr><tr><td>Low melting temperature wax</td><td></td><td></td><td></td></tr><tr><td>Soldering Iron</td><td>Weller</td><td></td><td></td></tr><tr><td>Platform with ball-and-socket magnetic base</td><td>Hama photo and video</td><td></td><td></td></tr><tr><td>Double edge carbon steel, breakable razor blade</td><td>Electron Microscopy Sciences</td><td>72004</td><td></td></tr><tr><td>Vaseline</td><td></td><td></td><td></td></tr><tr><td>Microsoft Excel</td><td>Microsoft</td><td></td><td></td></tr></tbody></table>

determination of photoreceptor cell spectral sensitivity in an insect model from in vivo intracellular recordings

细胞内记录是用于确定如何单个小区可以对给定的刺激响应的强大技术。在视觉研究，细胞内记录在历史上一直用于单个感光细胞的敏感性研究到至今仍在使用的不同的光刺激的常用技术。然而，仍详细方法在文献中为希望的眼睛来复制细胞内记录实验研究缺乏。在这里，我们目前的昆虫作为检查眼睛的生理更普遍的模式。昆虫感光细胞位于眼睛的表面附近，因此容易达到，而且许多涉及视力的机制跨越动物门保守的。我们描述了一个蝴蝶的眼睛在体内的感光细胞内录制的基本步骤，使得用这种方法更容易被研究人员在电子很少以往的经验的目标lectrophysiology。我们需要引入，如何准备活蝴蝶记录，如何插入玻璃微电极到一个单元格，最后录制过程本身的基本设备。我们还说明原始响应数据的基本分析，用于确定个体细胞类型的光谱灵敏度。虽然我们的协议的重点是确定的光谱的灵敏度，其他刺激（ 例如 ，偏振光），并且该方法的变型也适用于这种设置。

用于记录设置的许多元素，书面说明不提供足够的细节。 图1是涉及完整记录设置的组件的示意图。在图2中，光谱被绘制为白光和各干涉滤光器，得到为什么需要校正因子的感觉和所需要的计算这个校正。 图3显示了照片，并且用于这些的万向臂的图实验。 图4是示出在记录阶段和显微操作的合成图像。 图5示出了在实验蝴蝶的...

Watch this Scientific Journal Video about Determination of Photoreceptor Cell Spectral Sensitivity in an Insect Model from In Vivo Intracellular Recordings at JoVE.com

Determination of Photoreceptor Cell Spectral Sensitivity in an Insect Model from In Vivo Intracellular Recordings

细胞内记录的电生理学技术证明和用于确定在一个蝴蝶的复眼单一的感光细胞的光谱灵敏度。

光感受器细胞光谱灵敏度在从昆虫模型决定<em&gt;在体内</em&gt;细胞内记录

Intracellular recording is a powerful technique used to determine how a single cell may respond to a given stimulus. In vision research, intracellular ...

determination-photoreceptor-cell-spectral-sensitivity-an-insect-model

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

Determination of Photoreceptor Cell Spectral Sensitivity in an Insect Model from In Vivo Intracellular Recordings

11.3K 次观看.  University of California, Irvine. 细胞内记录的电生理学技术证明和用于确定在一个蝴蝶的复眼单一的感光细胞的光谱灵敏度。 

视频: Determination of Photoreceptor Cell Spectral Sensitivity in an Insect Model from In Vivo Intracellular Recordings

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

从胚胎脊髓​​连合神经的解剖和文化

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

科研

神经科学

Single-unit In vivo Recordings from the Optic Chiasm of Rat

Information about the visual world is transmitted to the brain in sequences of action potentials in retinal ganglion cell axons that make up the optic nerve. In vivo recordings of ganglion cell spike trains in several animal models have revealed much of what is known about how the early visual system processes and encodes visual information. However, such recordings have been rare in one of the most common animal models, the rat, possibly owing to difficulty in detecting spikes fired by small diameter axons. The many retinal disease models involving rats motivate a need for characterizing the functional properties of ganglion cells without disturbing the eye, as with intraocular or in vitro recordings. Here, we demonstrate a method for recording ganglion cell spike trains from the optic chiasm of the anesthetized rat. We first show how to fabricate tungsten-in-glass electrodes that can pick up electrical activity from single ganglion cell axons in rat. The electrodes outperform all commercial ones that we have tried. We then illustrate our custom-designed stereotaxic system for in vivo visual neurophysiology experiments and our procedures for animal preparation and reliable and stable electrode placement in the optic chiasm.

Single-unit In vivo Recordings from the Optic Chiasm of Rat

Retinal ganglion cells transmit visual information from the eye to the brain with sequences of action potentials. Here, we demonstrate how to record the action potentials of single ganglion cells in vivo from anesthetized rats.

单机在体内</em从大鼠视交叉>录音

Information about the visual world is transmitted to the brain in sequences of action potentials in retinal ganglion cell axons that make up the optic ...

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 ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

逆行荧光染料标记允许有针对性的外单细胞记录技术鉴定神经元<em&gt;在体内</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

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 Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, enabling the use of D. melanogaster molecular genetics to study inositol-lipid signaling and Transient Receptor Potential (TRP) channels at the single-molecule level. A handful of labs have mastered this powerful technique, which enables the analysis of the physiological responses to light under highly controlled conditions. This technique allows control over the intracellular and extracellular media; the membrane voltage; and the fast application of pharmacological compounds, such as a variety of ionic or pH indicators, to the intra- and extracellular media. With an exceptionally high signal-to-noise ratio, this method enables the measurement of dark spontaneous and light-induced unitary currents (i.e.&#160;spontaneous and quantum bumps) and macroscopic Light-induced Currents (LIC) from single D. melanogaster photoreceptors. This protocol outlines, in great detail, all the key steps necessary to perform this technique, which includes both electrophysiological and optical recordings. The fly retina dissection procedure for the attainment of intact and viable ex vivo isolated ommatidia in the bath chamber is described. The equipment needed to perform whole-cell and fluorescence imaging measurements are also detailed. Finally, the pitfalls in using this delicate preparation during extended experiments are explained.

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

Whole-cell recordings from Drosophila melanogaster photoreceptors enable the measurement of spontaneous dark bumps, quantum bumps, macroscopic responses to light, and current-voltage relationships under various conditions. In combination with D. melanogaster genetic manipulation tools, this method enables the study of the ubiquitous inositol-lipid signaling pathway and its target, the TRP channel.

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, ...

Desensitization and Recovery of Crayfish Photoreceptors Upon Delivery of a Light Stimulus

A method to study desensitization and recovery of crayfish photoreceptors is presented. We performed intracellular electrical recordings of photoreceptor cells in isolated eyestalks using the discontinuous single electrode-switched voltage-clamp configuration. First, with a razor blade we made an opening in the dorsal cornea to get access to the retina. Thereafter, we inserted a glass electrode through the opening, and penetrated a cell as reported by the recording of a negative potential. Membrane potential was clamped at the photoreceptor's resting potential and a light-pulse was applied to activate currents. Finally, the two light-flash protocol was employed to measure current desensitization and recovery. The first light-flash triggers, after a lag period, the transduction ionic current, which after reaching a peak amplitude decays towards a desensitized state; the second flash, applied at varying time intervals, assesses the state of the light-activated conductance. To characterize the light-elicited current, three parameters were measured: 1) latency (the time elapsed between light flash delivery and the moment in which current achieves 10% of its maximum value); 2) peak current; and 3) desensitization time constant (exponential time constant of the current decay phase). All parameters are affected by the first pulse.
To quantify recovery from desensitization, the ratio p2/p1 was employed versus time between pulses. p1 is the peak current evoked by the first light-pulse, and p2 is the peak current evoked by the second pulse. These data were fitted to a sum of exponential functions. Finally, these measurements were carried out as function of circadian time.

A protocol for the study of desensitization and sensitivity recovery of crayfish photoreceptors as a function of circadian time is presented.

光刺激后小龙虾光受体的脱敏和回收

A method to study desensitization and recovery of crayfish photoreceptors is presented. We performed intracellular electrical recordings of photoreceptor ...

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 ...

In Vivo Intracellular Recording of Photoreceptor Cell Spectral Sensitivity in a Butterfly

Source: McCulloch, K. J., et al.. <a href="https://appjove.unicartagenaproxy.elogim.com/v/53829">Determination of Photoreceptor Cell Spectral Sensitivity in an Insect Model from In Vivo Intracellular Recordings.</a> J. Vis. Exp. (2016)
This video demonstrates the procedure for in vivo intracellular recording of photoreceptor cells in the eye of a butterfly and determining the spectral sensitivity of individual cell types. This technique enables the study of sensitivities of individual photoreceptor cells to different light stimuli.

蝴蝶体内感光细胞光谱敏感性的细胞内记录

1. Specimen Prep and Recording Procedure

	Prepare the Specimen
	
		Affix an individual butterfly inside a small plastic tube with hot wax so the head is ...

JoVE Encyclopedia of Experiments

Neurophysiology

Imaging and Optical Techniques

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

Source: Juusola, M., et al. <a href="https://appjove.unicartagenaproxy.elogim.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 ...

光感受器细胞光谱灵敏度在从昆虫模型决定

摘要

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此视频中的章节

从胚胎脊髓连合神经的解剖和文化

单机在体内录音

在完整的视网膜电生理特性表达GFP的细胞群

逆行荧光染料标记允许有针对性的外单细胞记录技术鉴定神经元

为记录细胞内电压响应电法

全细胞电压钳记录的电生理方法

光刺激后小龙虾光受体的脱敏和回收

测量果蝇光感受器中光色素水平的电生理学方法

蝴蝶体内感光细胞光谱敏感性的细胞内记录

果蝇视网膜光感受器对光刺激的电压响应的电生理记录

光感受器细胞光谱灵敏度在从昆虫模型决定

摘要

探索更多视频

此视频中的章节

从胚胎脊髓​​连合神经的解剖和文化

单机在体内录音

在完整的视网膜电生理特性表达GFP的细胞群

逆行荧光染料标记允许有针对性的外单细胞记录技术鉴定神经元

为记录细胞内电压响应电法

全细胞电压钳记录的电生理方法

光刺激后小龙虾光受体的脱敏和回收

测量 果蝇 光感受器中光色素水平的电生理学方法

蝴蝶体内感光细胞光谱敏感性的细胞内记录

果蝇视网膜光感受器对光刺激的电压响应的电生理记录

从胚胎脊髓连合神经的解剖和文化

测量果蝇光感受器中光色素水平的电生理学方法