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。

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...

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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 ...

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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 ...

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 ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

使用超声波血脑屏障破坏和锰增强MRI的脑功能成像

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Design and Assembly of an Ultra-light Motorized Microdrive for Chronic Neural Recordings in Small Animals

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of neural circuits underlying a variety of natural behaviors, including navigation1 , decision making 2,3, and the generation of complex motor sequences4,5,6. Advances in precision machining has allowed for the fabrication of light-weight devices suitable for chronic recordings in small animals, such as mice and songbirds. The ability to adjust the electrode position with small remotely controlled motors has further increased the recording yield in various behavioral contexts by reducing animal handling.6,7
 Here we describe a protocol to build an ultra-light motorized microdrive for long-term chronic recordings in small animals. Our design evolved from an earlier published version7, and has been adapted for ease-of use and cost-effectiveness to be more practical and accessible to a wide array of researchers. This proven design 8,9,10,11 allows for fine, remote positioning of electrodes over a range of ~ 5 mm and weighs less than 750 mg when fully assembled. We present the complete protocol for how to build and assemble these drives, including 3D CAD drawings for all custom microdrive components.

The design, fabrication and assembly of an ultra-light motorized microdrive is described. The device provides a cost-effective and easy-to-use solution for chronic recordings of single units in small behaving animals.

设计和组装的超轻型电动微型硬盘（Microdrive）慢性神经录音的小动物

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

双突触诱发的星形胶质细胞和神经元的反应，在急性海马脑片电生理记录

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Simultaneous Whole-cell Recordings from Photoreceptors and Second-order Neurons in an Amphibian Retinal Slice Preparation

One of the central tasks in retinal neuroscience is to understand the circuitry of retinal neurons and how those connections are responsible for shaping the signals transmitted to the brain. Photons are detected in the retina by rod and cone photoreceptors, which convert that energy into an electrical signal, transmitting it to other retinal neurons, where it is processed and communicated to central targets in the brain via the optic nerve. Important early insights into retinal circuitry and visual processing came from the histological studies of Cajal1,2 and, later, from electrophysiological recordings of the spiking activity of retinal ganglion cells - the output cells of the retina3,4.
A detailed understanding of visual processing in the retina requires an understanding of the signaling at each step in the pathway from photoreceptor to retinal ganglion cell. However, many retinal cell types are buried deep in the tissue and therefore relatively inaccessible for electrophysiological recording. This limitation can be overcome by working with vertical slices, in which cells residing within each of the retinal layers are clearly visible and accessible for electrophysiological recording.
Here, we describe a method for making vertical sections of retinas from larval tiger salamanders (Ambystoma tigrinum). While this preparation was originally developed for recordings with sharp microelectrodes5,6, we describe a method for dual whole-cell voltage clamp recordings from photoreceptors and second-order horizontal and bipolar cells in which we manipulate the photoreceptor's membrane potential while simultaneously recording post-synaptic responses in horizontal or bipolar cells. The photoreceptors of the tiger salamander are considerably larger than those of mammalian species, making this an ideal preparation in which to undertake this technically challenging experimental approach. These experiments are described with an eye toward probing the signaling properties of the synaptic ribbon - a specialized synaptic structure found in a only a handful of neurons, including rod and cone photoreceptors, that is well suited for maintaining a high rate of tonic neurotransmitter release7,8 - and how it contributes to the unique signaling properties of this first retinal synapse.

We describe the preparation of thin retinal slices from aquatic tiger salamanders (Ambystoma tigrinum) and explain how we use these slices to study synaptic processing in the retina by obtaining dual whole-cell voltage clamp recordings from photoreceptors and second-order horizontal and bipolar cells.

同时光感受器和二阶神经元在两栖类动物的视网膜切片制备全细胞记录

One of the central tasks in retinal neuroscience  is to understand the circuitry of retinal neurons and how those connections are  responsible for shaping ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

测定冷灵敏度和适应小鼠一种简单廉价的方法

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

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

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

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

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

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

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

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.